Expression of a functional intrabody against hepatitis C virus core protein in Escherichia coli and silkworm pupae.

PubMed

Kato, Tatsuya; Hasegawa, Moeko; Yamamoto, Takeshi; Miyazaki, Takatsugu; Suzuki, Ryosuke; Wakita, Takaji; Suzuki, Tetsuro; Park, Enoch Y

2018-10-01

It has been shown that the single-domain intrabody 2H9-L against the hepatitis C virus (HCV) capsid (core) protein inhibits the viral propagation and NF-κB promoter activity induced by the HCV core. In this study, 2H9-L fused with the FLAG tag sequence was expressed in both Escherichia coli and silkworm pupae and then purified. In addition, the full-length and its C terminal deletions of the HCV core protein, i.e., 1-123 amino acid residues (C123), 1-152 amino acid residues (C152), 1-177 amino acid residues (C177) and 1-191 amino acid residues (C191), were expressed as fusion proteins with a 6 × His tag at their N-terminus in E. coli and then purified. Approximately 175 and 132 μg of the intrabody were purified from 100 ml of E. coli culture and 10 silkworm pupae, respectively, by affinity chromatography. The C123, C152, C177 and C191 HCV core protein variants were purified to approximately 152, 127, 103 and 155 μg, respectively, from 100 ml of E. coli culture. An ELISA in which the intrabodies were immobilized revealed that the intrabodies purified from both hosts were bound to all HCV core protein variants. However, their binding to the C191 appeared to be weak compared to their bindings to the other HCV core protein variants. When C152 was immobilized in the ELISA, the binding of each intrabody to the core protein was also observed. These purified intrabodies can be used in biochemical analyses of the inhibitory mechanism of HCV propagation and as protein interference reagents, thus providing a potential pathway to developing a new type of antiviral drug. Copyright © 2018 Elsevier Inc. All rights reserved.

Synonymous Mutations in the Core Gene Are Linked to Unusual Serological Profile in Hepatitis C Virus Infection

PubMed Central

Budkowska, Agata; Kakkanas, Athanassios; Nerrienet, Eric; Kalinina, Olga; Maillard, Patrick; Horm, Srey Viseth; Dalagiorgou, Geena; Vassilaki, Niki; Georgopoulou, Urania; Martinot, Michelle; Sall, Amadou Alpha; Mavromara, Penelope

2011-01-01

The biological role of the protein encoded by the alternative open reading frame (core+1/ARF) of the Hepatitis C virus (HCV) genome remains elusive, as does the significance of the production of corresponding antibodies in HCV infection. We investigated the prevalence of anti-core and anti-core+1/ARFP antibodies in HCV-positive blood donors from Cambodia, using peptide and recombinant protein-based ELISAs. We detected unusual serological profiles in 3 out of 58 HCV positive plasma of genotype 1a. These patients were negative for anti-core antibodies by commercial and peptide-based assays using C-terminal fragments of core but reacted by Western Blot with full-length core protein. All three patients had high levels of anti-core+1/ARFP antibodies. Cloning of the cDNA that corresponds to the core-coding region from these sera resulted in the expression of both core and core+1/ARFP in mammalian cells. The core protein exhibited high amino-acid homology with a consensus HCV1a sequence. However, 10 identical synonymous mutations were found, and 7 were located in the aa(99–124) region of core. All mutations concerned the third base of a codon, and 5/10 represented a T>C mutation. Prediction analyses of the RNA secondary structure revealed conformational changes within the stem-loop region that contains the core+1/ARFP internal AUG initiator at position 85/87. Using the luciferase tagging approach, we showed that core+1/ARFP expression is more efficient from such a sequence than from the prototype HCV1a RNA. We provide additional evidence of the existence of core+1/ARFP in vivo and new data concerning expression of HCV core protein. We show that HCV patients who do not produce normal anti-core antibodies have unusually high levels of antit-core+1/ARFP and harbour several identical synonymous mutations in the core and core+1/ARFP coding region that result in major changes in predicted RNA structure. Such HCV variants may favour core+1/ARFP production during HCV infection. PMID:21283512

PACAP38 Differentially Effects Genes and CRMP2 Protein Expression in Ischemic Core and Penumbra Regions of Permanent Middle Cerebral Artery Occlusion Model Mice Brain

PubMed Central

Hori, Motohide; Nakamachi, Tomoya; Shibato, Junko; Rakwal, Randeep; Tsuchida, Masachi; Shioda, Seiji; Numazawa, Satoshi

2014-01-01

Pituitary adenylate-cyclase activating polypeptide (PACAP) has neuroprotective and axonal guidance functions, but the mechanisms behind such actions remain unclear. Previously we examined effects of PACAP (PACAP38, 1 pmol) injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with control saline (0.9% NaCl) injection. Transcriptomic and proteomic approaches using ischemic (ipsilateral) brain hemisphere revealed differentially regulated genes and proteins by PACAP38 at 6 and 24 h post-treatment. However, as the ischemic hemisphere consisted of infarct core, penumbra, and non-ischemic regions, specificity of expression and localization of these identified molecular factors remained incomplete. This led us to devise a new experimental strategy wherein, ischemic core and penumbra were carefully sampled and compared to the corresponding contralateral (healthy) core and penumbra regions at 6 and 24 h post PACAP38 or saline injections. Both reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to examine targeted gene expressions and the collapsin response mediator protein 2 (CRMP2) protein profiles, respectively. Clear differences in expression of genes and CRMP2 protein abundance and degradation product/short isoform was observed between ischemic core and penumbra and also compared to the contralateral healthy tissues after PACAP38 or saline treatment. Results indicate the importance of region-specific analyses to further identify, localize and functionally analyse target molecular factors for clarifying the neuroprotective function of PACAP38. PMID:25257527

Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

PubMed

Shen, Xianjun; Yi, Li; Jiang, Xingpeng; He, Tingting; Yang, Jincai; Xie, Wei; Hu, Po; Hu, Xiaohua

2017-01-01

How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI) data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment). It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

In Vitro Assembly of Alphavirus Cores by Using Nucleocapsid Protein Expressed in Escherichia coli

PubMed Central

Tellinghuisen, Timothy L.; Hamburger, Agnes E.; Fisher, Bonnie R.; Ostendorp, Ralf; Kuhn, Richard J.

1999-01-01

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19–264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19–264) or could inhibit assembly if its molar ratio relative to CP(19–264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly. PMID:10364277

Hepatitis C virus core protein induces dysfunction of liver sinusoidal endothelial cell by down-regulation of silent information regulator 1.

PubMed

Sun, Li-Jie; Yu, Jian-Wu; Shi, Yu-Guang; Zhang, Xiao-Yu; Shu, Meng-Ni; Chen, Mo-Yang

2018-05-01

Hepatic fibrosis is a frequent feature of chronic hepatitis C virus (HCV) infection. Some evidence has suggested the potential role of silent information regulator 1 (SIRT1) in organ fibrosis. The aim of this study was to investigate the effect of HCV core protein on expression of SIRT1 of liver sinusoidal endothelial cell (LSEC) and function of LSEC. LSECs were co-cultured with HepG2 cells or HepG2 cells expressing HCV core protein and LSECs cultured alone were used as controls. After co-culture, the activity and expression levels of mRNA and protein of SIRT1 in LSEC were detected by a SIRT1 fluorometric assay kit, real time-PCR (RT-PCR), Western blot, respectively. The levels of adiponectin receptor 2 (AdipoR2), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by Western blot. Cluster of differentiation 31 (CD31), CD14, and von Willebrand factor (vWf) of LSECs was performed by flow cytometry. The level of reactive oxygen species (ROS) was assayed. Malondialdehyde (MDA), superoxide dismutase (SOD), adiponectin, nitric oxide (NO), and endothelin-1 (ET-1) levels in the co-culture supernatant were measured. The co-culture supernatant was then used to cultivate LX-2 cells. The levels of α-smooth muscle actin (ASMA) and transforming growth factor-β1 (TGF-β1) protein in LX-2 cells were measured by Western blot. Compared with LSEC co-cultured with HepG2 cells group, in LSEC co-cultured with HepG2-core cells group, the activity and expression level of mRNA and protein of SIRT1 reduced; the level of adiponectin reduced and the expression level of AdipoR2 protein decreased; ROS levels increased; the expression level of eNOS, VEGF protein decreased; and the expression level of CD14 decreased; the expression level of vWf and CD31 increased; NO and SOD levels decreased; whereas ET-1 and MDA levels increased; the levels of ASMA and TGF-β1 protein in LX-2 cells increased. SIRT1 activator improved the above-mentioned changes. HCV core protein may down-regulate the activity and the expression of SIRT1 of LSEC, then decreasing synthesis of adiponectin and the expression of AdipoR2, thus inducing contraction of LSEC and hepatic sinusoidal capillarization and increasing oxidative stress, ultimately cause hepatic stellate cell (HSC) activation. Treatment with SIRT1 activator restored the function of LSEC and inhibited the activation of HSC. © 2018 Wiley Periodicals, Inc.

A Concerted Action of Hepatitis C Virus P7 and Nonstructural Protein 2 Regulates Core Localization at the Endoplasmic Reticulum and Virus Assembly

PubMed Central

Boson, Bertrand; Granio, Ophélia; Bartenschlager, Ralf; Cosset, François-Loïc

2011-01-01

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly. PMID:21814513

The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts.

PubMed

Donker, Rogier B; Mouillet, Jean-François; Nelson, D Michael; Sadovsky, Yoel

2007-04-01

Endogenous microRNAs (miRNAs) post-transcriptionally regulate mRNA and protein expression during tissue development and function. Whereas adaptation to environmental insults are tightly regulated in human tissues, the role of miRNAs and miRNA biogenesis proteins in this context is inadequately explored. We sought to analyse the expression of the key RNAi enzyme Argonaute2 (Ago2) and other miRNA biogenesis proteins in human trophoblasts during differentiation and in hypoxic environment. Using an in vitro analysis of primary term human trophoblasts, we identified the expression of the core miRNA biogenesis proteins in human villous trophoblasts, with expression levels unaffected by cellular differentiation. We found that the miRNA biosynthetic pathway was functional and produced miRNAs, with miR-93 up-regulated and miR-424 down-regulated in hypoxic environment. In contrast, hypoxia did not alter the expression of key miRNA machinery proteins. The pivotal miRNA processing enzyme Ago2, along with its interacting protein DP103, were expressed in normal placentas as well as in placentas from pregnancies complicated by placental hypoperfusion that resulted in fetal growth restriction. Ago2 and DP103 co-immunoprecipitated, and did not limit trophoblast response to hypoxic stress. We concluded that the core miRNA machinery proteins are expressed and functional in human trophoblasts. The influence of hypoxia on the expression of a subset of placental miRNA species is unlikely to reflect altered expression of key miRNA biogenesis proteins.

The Native Form and Maturation Process of Hepatitis C Virus Core Protein

PubMed Central

Yasui, Kohichiroh; Wakita, Takaji; Tsukiyama-Kohara, Kyoko; Funahashi, Shin-Ichi; Ichikawa, Masumi; Kajita, Tadahiro; Moradpour, Darius; Wands, Jack R.; Kohara, Michinori

1998-01-01

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles. PMID:9621068

Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

NASA Technical Reports Server (NTRS)

Winchester, S. K.; Selvamurugan, N.; D'Alonzo, R. C.; Partridge, N. C.

2000-01-01

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

Role of HCV Core gene of genotype 1a and 3a and host gene Cox-2 in HCV-induced pathogenesis

PubMed Central

2011-01-01

Background Hepatitis C virus (HCV) Core protein is thought to trigger activation of multiple signaling pathways and play a significant role in the alteration of cellular gene expression responsible for HCV pathogenesis leading to hepatocellular carcinoma (HCC). However, the exact molecular mechanism of HCV genome specific pathogenesis remains unclear. We examined the in vitro effects of HCV Core protein of HCV genotype 3a and 1a on the cellular genes involved in oxidative stress and angiogenesis. We also studied the ability of HCV Core and Cox-2 siRNA either alone or in combination to inhibit viral replication and cell proliferation in HCV serum infected Huh-7 cells. Results Over expression of Core gene of HCV 3a genotype showed stronger effect in regulating RNA and protein levels of Cox-2, iNOS, VEGF, p-Akt as compared to HCV-1a Core in hepatocellular carcinoma cell line Huh-7 accompanied by enhanced PGE2 release and cell proliferation. We also observed higher expression levels of above genes in HCV 3a patient's blood and biopsy samples. Interestingly, the Core and Cox-2-specific siRNAs down regulated the Core 3a-enhanced expression of Cox-2, iNOS, VEGF, p-Akt. Furthermore, the combined siRNA treatment also showed a dramatic reduction in viral titer and expression of these genes in HCV serum-infected Huh-7 cells. Taken together, these results demonstrated a differential response by HCV 3a genotype in HCV-induced pathogenesis, which may be due to Core and host factor Cox-2 individually or in combination. Conclusions Collectively, these studies not only suggest a genotype-specific interaction between key players of HCV pathogenesis but also may represent combined viral and host gene silencing as a potential therapeutic strategy. PMID:21457561

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

PubMed Central

Shi, Shali; Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wu, Xinjiao; Wang, Xiaohong; Yang, Darong

2014-01-01

Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C. PMID:24307579

CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

PubMed Central

Aguado, Enrique; Garcia-Cozar, Francisco

2014-01-01

Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127lowPD-1highTIM-3high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein. PMID:24465502

Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, T.; Niepel, M.; McDermott, J. E.

It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundancemore » of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.« less

Expression of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) in the nucleus accumbens is critical for the acquisition, expression and reinstatement of morphine-induced conditioned place preference.

PubMed

Lv, Xiu-Fang; Xu, Ya; Han, Ji-Sheng; Cui, Cai-Lian

2011-09-30

Activity-regulated cytoskeleton-associated protein (Arc), also known as activity-regulated gene 3.1 (Arg3.1), is an immediate early gene whose mRNA is selectively targeted to recently activated synaptic sites, where it is translated and enriched. This unique feature suggests a role for Arc/Arg3.1 in coupling synaptic activity to protein synthesis, leading to synaptic plasticity. Although the Arc/Arg3.1 gene has been shown to be induced by a variety of abused drugs and its protein has been implicated in diverse forms of long-term memory, relatively little is known about its role in drug-induced reward memory. In this study, we investigated the potential role of Arc/Arg3.1 protein expression in reward-related associative learning and memory using morphine-induced conditioned place preference (CPP) in rats. We found that (1) intraperitoneal (i.p.) injection of morphine (10mg/kg) increased Arc/Arg3.1 protein levels after 2h in the NAc core but not in the NAc shell. (2) In CPP experiments, Arc/Arg3.1 protein was increased in the NAc shell of rats following both morphine conditioning and the CPP expression test compared to rats that received the conditioning without the test or those that did not receive morphine conditioning. (3) Microinjection of Arc/Arg3.1 antisense oligodeoxynucleotide (AS) into the NAc core inhibited the acquisition, expression and reinstatement of morphine CPP; however, intra-NAc shell infusions of the AS only blocked the expression of CPP. These findings suggest that expression of the Arc/Arg3.1 protein in the NAc core is required for the acquisition, context-induced retrieval and reinstatement of morphine-associated reward memory, whereas Arc/Arg3.1 protein expression in the NAc shell is only critical for the context-induced retrieval of memory. As a result, Arc/Arg3.1 may be a potential therapeutic target for the prevention of drug abuse or the relapse of drug use. Copyright © 2011 Elsevier B.V. All rights reserved.

Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly

PubMed Central

Ait-Goughoulte, Malika; Hourioux, Christophe; Patient, Romuald; Trassard, Sylvie; Brand, Denys; Roingeard, Philippe

2006-01-01

SUMMARY Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. We used this model to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. We designed several mutants as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. Our data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly. PMID:16528035

Hepatitis C virus core protein activates autophagy through EIF2AK3 and ATF6 UPR pathway-mediated MAP1LC3B and ATG12 expression

PubMed Central

Wang, Ji; Kang, Rongyan; Huang, He; Xi, Xueyan; Wang, Bei; Wang, Jianwei; Zhao, Zhendong

2014-01-01

HCV infection induces autophagy, but how this occurs is unclear. Here, we report the induction of autophagy by the structural HCV core protein and subsequent endoplasmic reticular (ER) stress in Huh7 hepatoma cells. During ER stress, both the EIF2AK3 and ATF6 pathways of the unfolded protein response (UPR) were activated by HCV core protein. Then, these pathways upregulated transcription factors ATF4 and DDIT3. The ERN1-XBP1 pathway was not activated. Through ATF4 in the EIF2AK3 pathway, the autophagy gene ATG12 was upregulated. DDIT3 upregulated the transcription of autophagy gene MAP1LC3B (LC3B) by directly binding to the –253 to –99 base region of the LC3B promoter, contributing to the development of autophagy. Collectively, these data suggest not only a novel role for the HCV core protein in autophagy but also offer new insight into detailed molecular mechanisms with respect to HCV-induced autophagy, specifically how downstream UPR molecules regulate key autophagic gene expression. PMID:24589849

Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

PubMed

Salehi, Nasrin; Peng, Ching-An

2016-07-08

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

Differential expression of syndecan isoforms during mouse incisor amelogenesis.

PubMed

Muto, Taro; Miyoshi, Keiko; Munesue, Seiichi; Nakada, Hiroshi; Okayama, Minoru; Matsuo, Takashi; Noma, Takafumi

2007-08-01

Syndecans are transmembranous heparan sulfate proteoglycans (HSPGs) with covalently attached glycosaminoglycan side-chains located on the cell surface. The mammalian syndecan family is composed of four types of syndecans (syndecan-1 to -4). Syndecans interact with the intracellular cytoskeleton through the cytoplasmic domains of their core proteins and membrane proteins, extracellular enzymes, growth factors, and matrix components, through their heparan-sulfate chains, to regulate developmental processes.Here, as a first step to assess the possible roles of syndecan proteins in amelogenesis, we examined the expression patterns of all syndecan isoforms in continuously growing mouse incisors, in which we can overview major differentiation stages of amelogenesis at a glance. Understanding the expression domain of each syndecan isoform during specific developmental stages seems useful for investigating their physiological roles in amelogenesis. Immunohistochemical analysis of syndecan core proteins in the lower incisors from postnatal day 1 mice revealed spatially and temporally specific expression patterns, with syndecan-1 expressed in undifferentiated epithelial and mesenchymal cells, and syndecan-2, -3, and -4 in more differentiated cells. These findings suggest that each syndecan isoform functions distinctly during the amelogenesis of the incisors of mice.

Comparative Bacterial Proteomics: Analysis of the Core Genome Concept

PubMed Central

Callister, Stephen J.; McCue, Lee Ann; Turse, Joshua E.; Monroe, Matthew E.; Auberry, Kenneth J.; Smith, Richard D.; Adkins, Joshua N.; Lipton, Mary S.

2008-01-01

While comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry, experimental validation of the existence of this core genome requires extensive measurement and is typically not undertaken. Enabled by an extensive proteome database developed over six years, we have experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. Although genomic studies can establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits. PMID:18253490

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

PubMed Central

Peyret, Hadrien; Gehin, Annick; Thuenemann, Eva C.; Blond, Donatienne; El Turabi, Aadil; Beales, Lucy; Clarke, Dean; Gilbert, Robert J. C.; Fry, Elizabeth E.; Stuart, David I.; Holmes, Kris; Stonehouse, Nicola J.; Whelan, Mike; Rosenberg, William; Lomonossoff, George P.; Rowlands, David J.

2015-01-01

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody. PMID:25830365

Expression of neuronal and signaling proteins in penumbra around a photothrombotic infarction core in rat cerebral cortex.

PubMed

Demyanenko, S V; Panchenko, S N; Uzdensky, A B

2015-06-01

Photodynamic impact on animal cerebral cortex using water-soluble Bengal Rose as a photosensitizer, which does not cross the blood-brain barrier and remains in blood vessels, induces platelet aggregation, vessel occlusion, and brain tissue infarction. This reproduces ischemic stroke. Irreversible cell damage within the infarction core propagates to adjacent tissue and forms a transition zone - the penumbra. Tissue necrosis in the infarction core is too fast (minutes) to be prevented, but much slower penumbral injury (hours) can be limited. We studied the changes in morphology and protein expression profile in penumbra 1 h after local photothrombotic infarction induced by laser irradiation of the cerebral cortex after Bengal Rose administration. Morphological study using standard hematoxylin/eosin staining showed a 3-mm infarct core surrounded by 1.5-2.0 mm penumbra. Morphological changes in the penumbra were lesser and decreased towards its periphery. Antibody microarrays against 224 neuronal and signaling proteins were used for proteomic study. The observed upregulation of penumbra proteins involved in maintaining neurite integrity and guidance (NAV3, MAP1, CRMP2, PMP22); intercellular interactions (N-cadherin); synaptic transmission (glutamate decarboxylase, tryptophan hydroxylase, Munc-18-1, Munc-18-3, and synphilin-1); mitochondria quality control and mitophagy (PINK1 and Parkin); ubiquitin-mediated proteolysis and tissue clearance (UCHL1, PINK1, Parkin, synphilin-1); and signaling proteins (PKBα and ERK5) could be associated with tissue recovery. Downregulation of PKC, PKCβ1/2, and TDP-43 could also reduce tissue injury. These changes in expression of some neuronal proteins were directed mainly to protection and tissue recovery in the penumbra. Some upregulated proteins might serve as markers of protection processes in a penumbra.

Evidence that the Dictyostelium STAT protein Dd-STATa plays a role in the differentiation of inner basal disc cells and identification of a promoter element essential for expression in these cells.

PubMed

Shimada, Nao; Maruo, Toshinari; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

2005-02-01

Dd-STATa, a Dictyostelium homolog of the metazoan STAT (signal transducers and activators of transcription) proteins, is necessary in the slug for correct entry into culmination. Dd-STATa-null mutant fails to culminate and its phenotype correlates with the loss of a funnel-shaped core region, the pstAB core region, which expresses both the ecmA and ecmB genes. To understand how the differentiation of pstAB core cells is regulated, we identified an EST that is expressed in the core cells of normal slugs but down-regulated in the Dd-STATa-null mutant. This EST, SSK348, encodes a close homolog of the Dictyostelium acetyl-CoA synthetase (ACS). A promoter fragment of the cognate gene, aslA (acetyl-CoA synthetase-like A), was fused to a lacZ reporter and the expression pattern determined. As expected from the behavior of the endogenous aslA gene, the aslA::lacZ fusion gene is not expressed in Dd-STATa-null slugs. In parental cells, the aslA promoter is first activated in the funnel-shaped core cells located at the slug anterior, the "pstAB core." During culmination, the pstAB core cells move down, through the prespore cells, to form the inner part of the basal disc. As the spore mass climbs the stalk, the aslA gene comes to be expressed in cells of the upper and lower cups, structures that cradle the spore head. Deletion and point mutation analyses of the promoter identified an AT-rich sequence that is necessary for expression in the pstAB core. This acts in combination with repressor regions that prevent ectopic aslA expression in the pre-stalk regions of slugs and the stalks of culminants. Thus, this study confirms that Dd-STATa is necessary for the differentiation of pstAB core cells, by showing that it is needed for the activation of the aslA gene. It also identifies aslA promoter elements that are likely to be regulated, directly or indirectly, by Dd-STATa.

HCV IRES-Mediated Core Expression in Zebrafish

PubMed Central

Zhang, Jing-Pu; Hu, Zhan-Ying; Tong, Jun-Wei; Ding, Cun-Bao; Peng, Zong-Gen; Zhao, Li-Xun; Song, Dan-Qing; Jiang, Jian-Dong

2013-01-01

The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism. PMID:23469178

The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System

PubMed Central

Wang, Chao-Wen; Cheng, Yun-Hsin; Irokawa, Hayato; Hwang, Gi-Wook; Naganuma, Akira; Kuge, Shusuke

2016-01-01

Chronic infection with the hepatitis C virus frequently induces steatosis, which is a significant risk factor for liver pathogenesis. Steatosis is characterized by the accumulation of lipid droplets in hepatocytes. The structural protein core of the virus induces lipid droplet formation and localizes on the surface of the lipid droplets. However, the precise molecular mechanisms for the core-induced formation of lipid droplets remain elusive. Recently, we showed that the expression of the core protein in yeast as a model system could induce lipid droplet formation. In this study, we probed the cellular factors responsible for the formation of core-induced lipid-droplets in yeast cells. We demonstrated that one of the enzymes responsible for triglyceride synthesis, a phospholipid:diacylglycerol acyltransferase (Lro1), is required for the core-induced lipid droplet formation. While core proteins inhibit Lro1 degradation and alter Lro1 localization, the characteristic localization of Lro1 adjacent to the lipid droplets appeared to be responsible for the core-induced lipid droplet formation. RNA virus genomes have evolved using high mutation rates to maintain their ability to replicate. Our observations suggest a functional relationship between the core protein with hepatocytes and yeast cells. The possible interactions between core proteins and the endoplasmic reticulum membrane affect the mobilization of specific proteins. PMID:27459103

Common and distinctive localization patterns of Crumbs polarity complex proteins in the mammalian eye.

PubMed

Kim, Jin Young; Song, Ji Yun; Karnam, Santi; Park, Jun Young; Lee, Jamie J H; Kim, Seonhee; Cho, Seo-Hee

2015-01-01

Crumbs polarity complex proteins are essential for cellular and tissue polarity, and for adhesion of epithelial cells. In epithelial tissues deletion of any of three core proteins disrupts localization of the other proteins, indicating structural and functional interdependence among core components. Despite previous studies of function and co-localization that illustrated the properties that these proteins share, it is not known whether an individual component of the complex plays a distinct role in a unique cellular and developmental context. In order to investigate this question, we primarily used confocal imaging to determine the expression and subcellular localization of the core Crumbs polarity complex proteins during ocular development. Here we show that in developing ocular tissues core Crumbs polarity complex proteins, Crb, Pals1 and Patj, generally appear in an overlapping pattern with some exceptions. All three core complex proteins localize to the apical junction of the retinal and lens epithelia. Pals1 is also localized in the Golgi of the retinal cells and Patj localizes to the nuclei of the apically located subset of progenitor cells. These findings suggest that core Crumbs polarity complex proteins exert common and independent functions depending on cellular context. Copyright © 2015 Elsevier B.V. All rights reserved.

Scrg1, a novel protein of the CNS is targeted to the large dense-core vesicles in neuronal cells.

PubMed

Dandoy-Dron, Françoise; Griffond, Bernadette; Mishal, Zohar; Tovey, Michael G; Dron, Michel

2003-11-01

Scrapie responsive gene one (Scrg1) is a novel transcript discovered through identification of the genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies. Scrg1 mRNA is distributed principally in the central nervous system and the cDNA sequence predicts a small cysteine-rich protein 98 amino acids in length, with a N-terminal signal peptide. In this study, we have generated antibodies against the predicted protein and revealed expression of a predominant immunoreactive protein of 10 kDa in mouse brain by Western blot analysis. We have established CAD neuronal cell lines stably expressing Scrg1 to determine its subcellular localization. Several lines of evidence show that the protein is targeted to dense-core vesicles in these cells. (i) Scrg1 is detected by immunocytochemistry as very punctate signals especially in the Golgi apparatus and tips of neurites, suggesting a vesicular localization for the protein. Moreover, Scrg1 exhibits a high degree of colocalization with secretogranin II, a dense-core vesicle marker and a very limited colocalization with markers for small synaptic vesicles. (ii) Scrg1 immunoreactivity is associated with large secretory granules/dense-core vesicles, as indicated by immuno-electron microscopy. (iii) Scrg1 is enriched in fractions of sucrose density gradient where synaptotagmin V, a dense-core vesicle-associated protein, is also enriched. The characteristic punctate immunostaining of Scrg1 is observed in N2A cells transfected with Scrg1 and for the endogenous protein in cultured primary neurons, attesting to the generality of the observations. Our findings strongly suggest that Scrg1 is associated with the secretory pathway of neuronal cells.

Protein degradation machinery is present broadly during early development in the sea urchin.

PubMed

Zazueta-Novoa, Vanesa; Wessel, Gary M

2014-07-01

Ubiquitin-dependent proteosome-mediated proteolysis is an important pathway of degradation that controls the timed destruction of cellular proteins in all tissues. All intracellular proteins and many extracellular proteins are continually being hydrolyzed to their constituent amino acids as a result of their recognition by E3 ligases for specific targeting of ubiquitination. Gustavus is a member of an ECS-type E3 ligase which interacts with Vasa, a DEAD-box RNA helicase, to regulate its localization during sea urchin embryonic development, and Gustavus mRNA accumulation is highly localized and dynamic during development. We tested if the core complex for Gustavus function was present in the embryo and if other SOCS box proteins also had restricted expression profiles that would inform future research. Expression patterns of the key members of the proteasomal function, such as the E3 core complex which interacts with Gustavus, and other E3-SOCS box proteins, are widely spread and dynamic in early development of the embryo suggesting broad core complex availability in the proteasome degradation pathway and temporal/spatial enrichments of various E3 ligase dependent targeting mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein degradation machinery is present broadly during early development in the sea urchin

PubMed Central

Zazueta-Novoa, Vanesa; Wessel, Gary M.

2014-01-01

Ubiquitin-dependent proteosome-mediated proteolysis is an important pathway of degradation that controls the timed destruction of cellular proteins in all tissues. All intracellular proteins and many extracellular proteins are continually being hydrolyzed to their constituent amino acids as a result of their recognition by E3 ligases for specific targeting of ubiquitination. Gustavus is a member of an ECS-type E3 ligase which interacts with Vasa, a DEAD-box RNA helicase, to regulate its localization during sea urchin embryonic development, and Gustavus mRNA accumulation is highly localized and dynamic during development. We tested if the core complex for Gustavus function was present in the embryo and if other SOCS box proteins also had restricted expression profiles that would inform future research. Expression patterns of the key members of the proteasomal function, such as the E3 core complex which interacts with Gustavus, and other E3-SOCS box proteins, are widely spread and dynamic in early development of the embryo suggesting broad core complex availability in the proteasome degradation pathway and temporal/spatial enrichments of various E3 ligase dependent targeting mechanisms. PMID:24963879

Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

NASA Technical Reports Server (NTRS)

Funderburgh, J. L.; Funderburgh, M. L.; Brown, S. J.; Vergnes, J. P.; Hassell, J. R.; Mann, M. M.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1993-01-01

Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

Impairment of interferon regulatory factor-3 activation by hepatitis C virus core protein basic amino acid region 1.

PubMed

Inoue, Kazuaki; Tsukiyama-Kohara, Kyoko; Matsuda, Chiho; Yoneyama, Mitsutoshi; Fujita, Takashi; Kuge, Shusuke; Yoshiba, Makoto; Kohara, Michinori

2012-11-30

Interferon regulatory factor-3 (IRF-3), a key transcriptional factor in the type I interferon system, is frequently impaired by hepatitis C virus (HCV), in order to establish persistent infection. However, the exact mechanism by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-β induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-β mRNA expression was quantified by real-time PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic:polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-β mRNA levels and IRF-3 dimerization, induced by NDV infection. Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection. Copyright © 2012 Elsevier Inc. All rights reserved.

Cytoscape: a software environment for integrated models of biomolecular interaction networks.

PubMed

Shannon, Paul; Markiel, Andrew; Ozier, Owen; Baliga, Nitin S; Wang, Jonathan T; Ramage, Daniel; Amin, Nada; Schwikowski, Benno; Ideker, Trey

2003-11-01

Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Although applicable to any system of molecular components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other molecular states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of additional computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined physical/functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.

Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

PubMed

Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

2018-02-15

Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this concept needs expansion as the functions are more diverse. We showed that capsid protein VI regulates the antiviral response by modulation of the transcription factor Daxx during infection. Moreover, core protein VII interacts with SPOC1 restriction factor, which is beneficial for efficient viral gene expression. Here, we were able to show that core protein V also represents a novel substrate of the host SUMOylation machinery and contains several conserved SCMs; mutation of these consensus motifs reduced SUMOylation of the protein. Unexpectedly, we observed that introducing these mutations into HAdV promotes adenoviral replication. In conclusion, we offer novel insights into adenovirus core proteins and provide evidence that SUMOylation of HAdV factors regulates replication efficiency. Copyright © 2018 American Society for Microbiology.

Ribosome profiling reveals the rhythmic liver translatome and circadian clock regulation by upstream open reading frames

PubMed Central

Janich, Peggy; Arpat, Alaaddin Bulak; Castelo-Szekely, Violeta; Lopes, Maykel; Gatfield, David

2015-01-01

Mammalian gene expression displays widespread circadian oscillations. Rhythmic transcription underlies the core clock mechanism, but it cannot explain numerous observations made at the level of protein rhythmicity. We have used ribosome profiling in mouse liver to measure the translation of mRNAs into protein around the clock and at high temporal and nucleotide resolution. We discovered, transcriptome-wide, extensive rhythms in ribosome occupancy and identified a core set of approximately 150 mRNAs subject to particularly robust daily changes in translation efficiency. Cycling proteins produced from nonoscillating transcripts revealed thus-far-unknown rhythmic regulation associated with specific pathways (notably in iron metabolism, through the rhythmic translation of transcripts containing iron responsive elements), and indicated feedback to the rhythmic transcriptome through novel rhythmic transcription factors. Moreover, estimates of relative levels of core clock protein biosynthesis that we deduced from the data explained known features of the circadian clock better than did mRNA expression alone. Finally, we identified uORF translation as a novel regulatory mechanism within the clock circuitry. Consistent with the occurrence of translated uORFs in several core clock transcripts, loss-of-function of Denr, a known regulator of reinitiation after uORF usage and of ribosome recycling, led to circadian period shortening in cells. In summary, our data offer a framework for understanding the dynamics of translational regulation, circadian gene expression, and metabolic control in a solid mammalian organ. PMID:26486724

Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

PubMed

Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

2015-05-01

Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the 30 sera of known genotypes. The antigens did not detect antibodies to genotype-3a, but detected antibodies to all genotypes and did not discriminate them genotype wise. A panel of 175 of HCV-suspected serum samples was subjected to comparative analysis with our in-house ELISA assay and with commercial HCV screening assays. After subjecting the results to the formulas for determining the quality parameters, immunoblot assay had 100% sensitivity and specificity, while the ELISA assay had 100% sensitivity and 98.8% specificity as compared to commercially available assays. This study indicates that a mixture of Core and E2 antigens are potentially valuable antigens and there is the possibility of developing serological assays for monitoring HCV infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Intrafocal heterogeneity of ERG protein expression and gene fusion pattern in prostate cancer.

PubMed

Suh, Ja Hee; Park, Jeong Hwan; Lee, Cheol; Moon, Kyung Chul

2017-10-01

Prostate cancer is considered to be highly heterogeneous, with various morphologic features and biologic behaviors. The TMPRSS2-ERG gene fusion is the most frequently observed genetic aberration in prostate cancer. The aim of this study was to elucidate the intrafocal heterogeneity of ERG gene fusion status. ERG immunohistochemistry (IHC) was performed in samples from 168 prostate cancer patients who had undergone radical prostatectomy, and 40 cases showing ERG-positive IHC staining were selected for tissue microarray (TMA) construction. Two to six representative cores were selected from each tumor focus. In the cases with heterogeneous ERG IHC staining intensity, the areas showing different intensities were separately selected. Using the TMA blocks, IHC and fluorescence in situ hybridization (FISH) were conducted to evaluate the heterogeneity of ERG protein expression and ERG fusion gene patterns, respectively, in a single tumor focus. Heterogeneity of ERG IHC staining was defined as the simultaneous presence of negative and positive cores in the same tumor focus. Heterogeneity of ERG FISH was defined by the presence of cores with positive and negative FISH signals or cores with break-apart and interstitial deletion FISH signals in the same tumor focus. A total of 202 TMA cores were isolated from 40 ERG-positive cases. Of the 202 total cores, 19 were negative for ERG IHC staining, and 46 showed 1+, 52 showed 2+, and 85 showed 3+ ERG staining intensity. Eleven cores were negative for ERG FISH signal, 119 cores showed ERG break-apart FISH signals, and the remaining 72 cores revealed interstitial deletion. Intrafocal heterogeneity of ERG IHC staining was found in 20% (8/40) of cases, and intrafocal heterogeneity of ERG gene fusion pattern was found in 32.5% (13/40) of cases. In summary, this study showed significantly frequent intrafocal heterogeneity of ERG protein expression, gene fusion status and fusion pattern. This heterogeneity can be caused by the development of subclones during cancer progression or the intermingling of different tumors. © 2017 Wiley Periodicals, Inc.

Bidirectional Lipid Droplet Velocities Are Controlled by Differential Binding Strengths of HCV Core DII Protein

PubMed Central

Lyn, Rodney K.; Hope, Graham; Sherratt, Allison R.; McLauchlan, John; Pezacki, John Paul

2013-01-01

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV. PMID:24223760

Utilization of fluorescent microspheres and a green fluorescent protein-marked strain for assessment of microbiological contamination of permafrost and ground ice core samples from the Canadian High Arctic.

PubMed

Juck, D F; Whissell, G; Steven, B; Pollard, W; McKay, C P; Greer, C W; Whyte, L G

2005-02-01

Fluorescent microspheres were applied in a novel fashion during subsurface drilling of permafrost and ground ice in the Canadian High Arctic to monitor the exogenous microbiological contamination of core samples obtained during the drilling process. Prior to each drill run, a concentrated fluorescent microsphere (0.5-microm diameter) solution was applied to the interior surfaces of the drill bit, core catcher, and core tube and allowed to dry. Macroscopic examination in the field demonstrated reliable transfer of the microspheres to core samples, while detailed microscopic examination revealed penetration levels of less than 1 cm from the core exterior. To monitor for microbial contamination during downstream processing of the permafrost and ground ice cores, a Pseudomonas strain expressing the green fluorescent protein (GFP) was painted on the core exterior prior to processing. Contamination of the processed core interiors with the GFP-expressing strain was not detected by culturing the samples or by PCR to detect the gfp marker gene. These methodologies were quick, were easy to apply, and should help to monitor the exogenous microbiological contamination of pristine permafrost and ground ice samples for downstream culture-dependent and culture-independent microbial analyses.

Utilization of Fluorescent Microspheres and a Green Fluorescent Protein-Marked Strain for Assessment of Microbiological Contamination of Permafrost and Ground Ice Core Samples from the Canadian High Arctic

PubMed Central

Juck, D. F.; Whissell, G.; Steven, B.; Pollard, W.; McKay, C. P.; Greer, C. W.; Whyte, L. G.

2005-01-01

Fluorescent microspheres were applied in a novel fashion during subsurface drilling of permafrost and ground ice in the Canadian High Arctic to monitor the exogenous microbiological contamination of core samples obtained during the drilling process. Prior to each drill run, a concentrated fluorescent microsphere (0.5-μm diameter) solution was applied to the interior surfaces of the drill bit, core catcher, and core tube and allowed to dry. Macroscopic examination in the field demonstrated reliable transfer of the microspheres to core samples, while detailed microscopic examination revealed penetration levels of less than 1 cm from the core exterior. To monitor for microbial contamination during downstream processing of the permafrost and ground ice cores, a Pseudomonas strain expressing the green fluorescent protein (GFP) was painted on the core exterior prior to processing. Contamination of the processed core interiors with the GFP-expressing strain was not detected by culturing the samples or by PCR to detect the gfp marker gene. These methodologies were quick, were easy to apply, and should help to monitor the exogenous microbiological contamination of pristine permafrost and ground ice samples for downstream culture-dependent and culture-independent microbial analyses. PMID:15691963

Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons

PubMed Central

Chen, Augustine; Kao, Y. F.; Brown, Chris M.

2005-01-01

The human hepatitis B virus (HBV) has a compact genome encoding four major overlapping coding regions: the core, polymerase, surface and X. The polymerase initiation codon is preceded by the partially overlapping core and four or more upstream initiation codons. There is evidence that several mechanisms are used to enable the synthesis of the polymerase protein, including leaky scanning and ribosome reinitiation. We have examined the first AUG in the pregenomic RNA, it precedes that of the core. It initiates an uncharacterized short upstream open reading frame (uORF), highly conserved in all HBV subtypes, we designated the C0 ORF. This arrangement suggested that expression of the core and polymerase may be affected by this uORF. Initiation at the C0 ORF was confirmed in reporter constructs in transfected cells. The C0 ORF had an inhibitory role in downstream expression from the core initiation site in HepG2 cells and in vitro, but also stimulated reinitiation at the polymerase start when in an optimal context. Our results indicate that the C0 ORF is a determinant in balancing the synthesis of the core and polymerase proteins. PMID:15731337

Suppression of grp78 core promoter element-mediated stress induction by the dbpA and dbpB (YB-1) cold shock domain proteins.

PubMed Central

Li, W W; Hsiung, Y; Wong, V; Galvin, K; Zhou, Y; Shi, Y; Lee, A S

1997-01-01

The highly conserved grp78 core promoter element plays an important role in the induction of grp78 under diverse stress signals. Previous studies have established a functional region in the 3' half of the core (stress-inducible change region [SICR]) which exhibits stress-inducible changes in stressed nuclei. The human transcription factor YY1 is shown to bind the SICR and transactivate the core element under stress conditions. Here we report that expression library screening with the core element has identified two new core binding proteins, YB-1 and dbpA. Both proteins belong to the Y-box family of proteins characterized by an evolutionarily conserved DNA binding motif, the cold shock domain (CSD). In contrast to YY1, which binds only double-stranded SICR, the Y-box/CSD proteins much prefer the lower strand of the SICR. The Y-box proteins can repress the inducibility of the grp78 core element mediated by treatment of cells with A23187, thapsigargin, and tunicamycin. In gel shift assays, YY1 binding to the core element is inhibited by either YB-1 or dbpA. A yeast interaction trap screen using LexA-YY1 as a bait and a HeLa cell cDNA-acid patch fusion library identified YB-1 as a YY1-interacting protein. In cotransfection experiments, the Y-box proteins antagonize the YY1-mediated enhancement of transcription directed by the grp78 core in stressed cells. Thus, the CSD proteins may be part of the stress signal transduction mechanism in the mammalian system. PMID:8972186

Recombinant Expression of Tandem-HBc Virus-Like Particles (VLPs).

PubMed

Stephen, Sam L; Beales, Lucy; Peyret, Hadrien; Roe, Amy; Stonehouse, Nicola J; Rowlands, David J

2018-01-01

The hepatitis B virus (HBV) core protein (HBc) has formed the building block for virus-like particle (VLP) production for more than 30 years. The ease of production of the protein, the robust ability of the core monomers to dimerize and assemble into intact core particles, and the strong immune responses they elicit when presenting antigenic epitopes all demonstrate its promise for vaccine development (reviewed in Pumpens and Grens (Intervirology 44: 98-114, 2001)). HBc has been modified in a number of ways in attempts to expand its potential as a novel vaccine platform. The HBc protein is predominantly α-helical in structure and folds to form an L-shaped molecule. The structural subunit of the HBc particle is a dimer of monomeric HBc proteins which together form an inverted T-shaped structure. In the assembled HBc particle the four-helix bundle formed at each dimer interface appears at the surface as a prominent "spike." The tips of the "spikes" are the preferred sites for the insertion of foreign sequences for vaccine purposes as they are the most highly exposed regions of the assembled particles. In the tandem-core modification two copies of the HBc protein are covalently linked by a flexible amino acid sequence which allows the fused dimer to fold correctly and assemble into HBc particles. The advantage of the modified structure is that the assembly of the dimeric subunits is defined and not formed by random association. This facilitates the introduction of single, larger sequences at the tip of each surface "spike," thus overcoming the conformational clashes contingent on insertion of large structures into monomeric HBc proteins.Differences in inserted sequences influence the assembly characteristics of the modified proteins, and it is important to optimize the design of each novel construct to maximize efficiency of assembly into regular VLPs. In addition to optimization of the construct, the expression system used can also influence the ability of recombinant structures to assemble into regular isometric particles. Here, we describe the production of recombinant tandem-core particles in bacterial, yeast and plant expression systems.

Adenovirus core protein VII contains distinct sequences that mediate targeting to the nucleus and nucleolus, and colocalization with human chromosomes.

PubMed

Lee, Tim W R; Blair, G Eric; Matthews, David A

2003-12-01

During adenovirus infection, following capsid dissociation, core protein VII enters the host cell nucleus complexed with adenovirus DNA. In order to determine whether protein VII may have an active role in this nuclear import, regions of the preVII gene were amplified by PCR, and further oligonucleotide mutants were designed with site-directed mutation of codons for the basic amino acids arginine and lysine. Fragments were cloned into a mammalian expression plasmid to express the peptides as N-terminal fusions to enhanced green fluorescent protein. Results demonstrate that preVII protein contains both nuclear and nucleolar targeting sequences. Such signals may be important in the delivery of adenovirus DNA to the host cell nucleus during adenovirus infection. Furthermore, the data suggest that protein VII may bind to human chromosomes by means of two distinct domains, one sharing homology with the N-terminal regulatory tail of histone H3.

Dissecting the Calcium-Induced Differentiation of Human Primary Keratinocytes Stem Cells by Integrative and Structural Network Analyses

PubMed Central

Toufighi, Kiana; Yang, Jae-Seong; Luis, Nuno Miguel; Aznar Benitah, Salvador; Lehner, Ben; Serrano, Luis; Kiel, Christina

2015-01-01

The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation. PMID:25946651

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay

2011-01-01

The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, wemore » detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.« less

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

PubMed

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-09-27

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

PubMed Central

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-01-01

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification. PMID:21952135

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans*

PubMed Central

Yang, Qiang; Wang, Lai-Xi

2016-01-01

Understanding the biosynthetic pathway of protein glycosylation in various expression cell lines is important for controlling and modulating the glycosylation profiles of recombinant glycoproteins. We found that expression of erythropoietin (EPO) in a HEK293S N-acetylglucosaminyltransferase I (GnT I)−/− cell line resulted in production of the Man5GlcNAc2 glycoforms, in which more than 50% were core-fucosylated, implicating a clear GnT I-independent core fucosylation pathway. Expression of GM-CSF and the ectodomain of FcγIIIA receptor led to ∼30% and 3% core fucosylation, suggesting that the level of core fucosylation also depends on the nature of the recombinant proteins. To elucidate the GnT I-independent core fucosylation pathway, we generated a stable HEK293S GnT I−/− cell line with either knockdown or overexpression of FUT8 by a highly efficient lentivirus-mediated gene transfer approach. We found that the EPO produced from the FUT8 knockdown cell line was the pure Man5GlcNAc2 glycoform, whereas that produced from the FUT8-overexpressing cell line was found to be fully core-fucosylated oligomannose glycan (Man5GlcNAc2Fuc). These results provide direct evidence that FUT8, the mammalian α1,6-fucosyltransferase, is the sole enzyme responsible for the GnT I-independent core fucosylation pathway. The production of the homogeneous core-fucosylated Man5GlcNAc2 glycoform of EPO in the FUT8-overexpressed HEK293S GnT I−/− cell line represents the first example of production of fully core-fucosylated high-mannose glycoforms. PMID:27008861

gC1qR expression in chimpanzees with resolved and chronic infection: Potential role of HCV core/gC1qR-mediated T cell suppression in the outcome of HCV infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao Zhiqang; Shata, Mohamed Tarek; Tricoche, Nancy

2006-03-15

Chimpanzee is a unique animal model for HCV infection, in which about 50% of infections resolve spontaneously. It has been reported that the magnitude of T cell responses to HCV core in recovered chimpanzees is greater than that in chronically infected ones. However, the mechanism(s) by which the chimpanzees with resolved infection overcome core-mediated immunosuppression remains unknown. In this study, we examined the effect of HCV core on T cell responsiveness in chimpanzees with resolved and chronic HCV infection. We found that core protein strongly inhibited T cell activation and proliferation in chimpanzees with chronic infection, while this inhibition wasmore » limited in chimpanzees with resolved infection. Notably, the level of gC1qR, as well as the binding of core protein, on the surface of T cells was lower in recovered chimpanzees when compared to chimpanzees with chronic HCV infection. Intriguingly, the observed differences in gC1qR expression levels and susceptibility to core-induced suppression amongst HCV-chronically infected and recovered chimpanzees were observed prior to HCV challenge, suggesting a possible genetic determination of the outcome of infection. These findings suggest that gC1qR expression on the surface of T cells is crucial for HCV core-mediated T cell suppression and viral clearance, and that represents a novel mechanism by which a virus usurps host machinery for persistence.« less

Loss of switch/sucrose non-fermenting complex protein expression is associated with dedifferentiation in endometrial carcinomas.

PubMed

Karnezis, Anthony N; Hoang, Lien N; Coatham, Mackenzie; Ravn, Sarah; Almadani, Noorah; Tessier-Cloutier, Basile; Irving, Julie; Meng, Bo; Li, Xiaodong; Chow, Christine; McAlpine, Jessica; Kuo, Kuan-Ting; Mao, Tsui-Lien; Djordjevic, Bojana; Soslow, Robert A; Huntsman, David G; Blake Gilks, C; Köbel, Martin; Lee, Cheng-Han

2016-03-01

Dedifferentiated endometrial carcinoma is an aggressive type of endometrial cancer that contains a mix of low-grade endometrioid and undifferentiated carcinoma components. We performed targeted sequencing of eight dedifferentiated carcinomas and identified somatic frameshift/nonsense mutations in SMARCA4, a core ATPase of the switch/sucrose non-fermenting (SWI/SNF) complex, in the undifferentiated components of four tumors. Immunohistochemical analysis confirmed the loss of SMARCA4 in the undifferentiated component of these four SMARCA4-mutated cases, whereas the corresponding low-grade endometrioid component showed retained SMARCA4 expression. An expanded survey of other members of the SWI/SNF complex showed SMARCB1 loss in the undifferentiated component of two SMARCA4-intact tumors, and all SMARCA4- or SMARCB1-deficient tumors showed concomitant loss of expression of SMARCA2. We subsequently examined the expression of SMARCA2, SMARCA4, and SMARCB1 in an additional set of 22 centrally reviewed dedifferentiated carcinomas and 31 grade 3 endometrioid carcinomas. Combining the results from the index and the expansion set, 15 of 30 (50%) of the dedifferentiated carcinomas examined showed either concurrent SMARCA4 and SMARCA2 loss (37%) or concurrent SMARCB1 and SMARCA2 loss (13%) in the undifferentiated component. The loss of SMARCA4 or SMARCB1 was mutually exclusive. All 31 grade 3 endometrioid carcinomas showed intact expression of these core SWI/SNF proteins. The majority (73%) of the SMARCA4/SMARCA2-deficient and half of SMARCB1/SMARCA2-deficient undifferentiated component developed in a mismatch repair-deficient molecular context. The observed spatial association between SWI/SNF protein loss and histologic dedifferentiation suggests that inactivation of these core SWI/SNF proteins may contribute to the development of dedifferentiated endometrial carcinoma.

Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Jian; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071; Zhang, Huaidong

2015-05-08

Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type Imore » viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.« less

Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro

NASA Technical Reports Server (NTRS)

Long, C. J.; Roth, M. R.; Tasheva, E. S.; Funderburgh, M.; Smit, R.; Conrad, G. W.; Funderburgh, J. L.

2000-01-01

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.

Diel pattern of circadian clock and storage protein gene expression in leaves and during seed filling in cowpea (Vigna unguiculata).

PubMed

Weiss, Julia; Terry, Marta I; Martos-Fuentes, Marina; Letourneux, Lisa; Ruiz-Hernández, Victoria; Fernández, Juan A; Egea-Cortines, Marcos

2018-02-14

Cowpea (Vigna unguiculata) is an important source of protein supply for animal and human nutrition. The major storage globulins VICILIN and LEGUMIN (LEG) are synthesized from several genes including LEGA, LEGB, LEGJ and CVC (CONVICILIN). The current hypothesis is that the plant circadian core clock genes are conserved in a wide array of species and that primary metabolism is to a large extent controlled by the plant circadian clock. Our aim was to investigate a possible link between gene expression of storage proteins and the circadian clock. We identified cowpea orthologues of the core clock genes VunLHY, VunTOC1, VunGI and VunELF3, the protein storage genes VunLEG, VunLEGJ, and VunCVC as well as nine candidate reference genes used in RT-PCR. ELONGATION FACTOR 1-A (ELF1A) resulted the most suitable reference gene. The clock genes VunELF3, VunGI, VunTOC1 and VunLHY showed a rhythmic expression profile in leaves with a typical evening/night and morning/midday phased expression. The diel patterns were not completely robust and only VungGI and VungELF3 retained a rhythmic pattern under free running conditions of darkness. Under field conditions, rhythmicity and phasing apparently faded during early pod and seed development and was regained in ripening pods for VunTOC1 and VunLHY. Mature seeds showed a rhythmic expression of VunGI resembling leaf tissue under controlled growth chamber conditions. Comparing time windows during developmental stages we found that VunCVC and VunLEG were significantly down regulated during the night in mature pods as compared to intermediate ripe pods, while changes in seeds were non-significant due to high variance. The rhythmic expression under field conditions was lost under growth chamber conditions. The core clock gene network is conserved in cowpea leaves showing a robust diel expression pattern except VunELF3 under growth chamber conditions. There appears to be a clock transcriptional reprogramming in pods and seeds compared to leaves. Storage protein deposition may be circadian regulated under field conditions but the strong environmental signals are not met under artificial growth conditions. Diel expression pattern in field conditions may result in better usage of energy for protein storage.

Differential assembly of alpha- and gamma-filagenins into thick filaments in Caenorhabditis elegans

NASA Technical Reports Server (NTRS)

Liu, F.; Ortiz, I.; Hutagalung, A.; Bauer, C. C.; Cook, R. G.; Epstein, H. F.

2000-01-01

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized alpha- and gamma-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that alpha-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas gamma-filagenin is localized in the flanking regions, and that alpha-filagenin is expressed in 1.5-twofold embryos while gamma-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of alpha- and gamma-filagenins into thick filaments of distinct lengths may be developmentally regulated.

Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses.

PubMed

Narusaka, Yoshihiro; Nakashima, Kazuo; Shinwari, Zabta K; Sakuma, Yoh; Furihata, Takashi; Abe, Hiroshi; Narusaka, Mari; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2003-04-01

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.

FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3.

PubMed

Wilson, J B; Yamamoto, K; Marriott, A S; Hussain, S; Sung, P; Hoatlin, M E; Mathew, C G; Takata, M; Thompson, L H; Kupfer, G M; Jones, N J

2008-06-12

Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2, which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2-FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2-FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.

Intrinsically disordered proteins--relation to general model expressing the active role of the water environment.

PubMed

Kalinowska, Barbara; Banach, Mateusz; Konieczny, Leszek; Marchewka, Damian; Roterman, Irena

2014-01-01

This work discusses the role of unstructured polypeptide chain fragments in shaping the protein's hydrophobic core. Based on the "fuzzy oil drop" model, which assumes an idealized distribution of hydrophobicity density described by the 3D Gaussian, we can determine which fragments make up the core and pinpoint residues whose location conflicts with theoretical predictions. We show that the structural influence of the water environment determines the positions of disordered fragments, leading to the formation of a hydrophobic core overlaid by a hydrophilic mantle. This phenomenon is further described by studying selected proteins which are known to be unstable and contain intrinsically disordered fragments. Their properties are established quantitatively, explaining the causative relation between the protein's structure and function and facilitating further comparative analyses of various structural models. © 2014 Elsevier Inc. All rights reserved.

Hepatitis B Virus Core Gene Mutations Which Block Nucleocapsid Envelopment

PubMed Central

Koschel, Matthias; Oed, Daniela; Gerelsaikhan, Tudevdagwa; Thomssen, Reiner; Bruss, Volker

2000-01-01

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment. PMID:10590084

T-bet-mediated Tim-3 expression dampens monocyte function during chronic hepatitis C virus infection.

PubMed

Yi, Wenjing; Zhang, Peixin; Liang, Yan; Zhou, Yun; Shen, Huanjun; Fan, Chao; Moorman, Jonathan P; Yao, Zhi Q; Jia, Zhansheng; Zhang, Ying

2017-03-01

Hepatitis C virus (HCV) induces a high rate of chronic infection via dysregulation of host immunity. We have previously shown that T-cell immunoglobulin and mucin domain protein-3 (Tim-3) is up-regulated on monocyte/macrophages (M/Mφ) during chronic HCV infection; little is known, however, about the transcription factor that controls its expression in these cells. In this study, we investigated the role of transcription factor, T-box expressed in T cells (T-bet), in Tim-3 expression in M/Mφ in the setting of HCV infection. We demonstrate that T-bet is constitutively expressed in resting CD14 + M/Mφ in the peripheral blood. M/Mφ from chronically HCV-infected individuals exhibit a significant increase in T-bet expression that positively correlates with an increased level of Tim-3 expression. Up-regulation of T-bet is also observed in CD14 + M/Mφ incubated with HCV + Huh7.5 cells, as well as in primary M/Mφ or monocytic THP-1 cells exposed to HCV core protein in vitro, which is reversible by blocking HCV core/gC1qR interactions. Moreover, the HCV core-induced up-regulation of T-bet and Tim-3 expression in M/Mφ can be abrogated by incubating the cells with SP600125 - an inhibitor for the c-Jun N-terminal kinase (JNK) signalling pathway. Importantly, silencing T-bet gene expression decreases Tim-3 expression and enhances interleukin-12 secretion as well as signal transducer and activator of transcription 1 phosphorylation. These data suggest that T-bet, induced by the HCV core/gC1qR interaction, enhances Tim-3 expression via the JNK pathway, leading to dampened M/Mφ function during HCV infection. These findings reveal a novel mechanism for Tim-3 regulation via T-bet during HCV infection, providing new targets to combat this global epidemic viral disease. © 2016 John Wiley & Sons Ltd.

A high-resolution transcriptome map of cell cycle reveals novel connections between periodic genes and cancer

PubMed Central

Dominguez, Daniel; Tsai, Yi-Hsuan; Gomez, Nicholas; Jha, Deepak Kumar; Davis, Ian; Wang, Zefeng

2016-01-01

Progression through the cell cycle is largely dependent on waves of periodic gene expression, and the regulatory networks for these transcriptome dynamics have emerged as critical points of vulnerability in various aspects of tumor biology. Through RNA-sequencing of human cells during two continuous cell cycles (>2.3 billion paired reads), we identified over 1 000 mRNAs, non-coding RNAs and pseudogenes with periodic expression. Periodic transcripts are enriched in functions related to DNA metabolism, mitosis, and DNA damage response, indicating these genes likely represent putative cell cycle regulators. Using our set of periodic genes, we developed a new approach termed “mitotic trait” that can classify primary tumors and normal tissues by their transcriptome similarity to different cell cycle stages. By analyzing >4 000 tumor samples in The Cancer Genome Atlas (TCGA) and other expression data sets, we found that mitotic trait significantly correlates with genetic alterations, tumor subtype and, notably, patient survival. We further defined a core set of 67 genes with robust periodic expression in multiple cell types. Proteins encoded by these genes function as major hubs of protein-protein interaction and are mostly required for cell cycle progression. The core genes also have unique chromatin features including increased levels of CTCF/RAD21 binding and H3K36me3. Loss of these features in uterine and kidney cancers is associated with altered expression of the core 67 genes. Our study suggests new chromatin-associated mechanisms for periodic gene regulation and offers a predictor of cancer patient outcomes. PMID:27364684

Hepatitis C virus Core overcomes all-trans retinoic acid-induced apoptosis in human hepatoma cells by inhibiting p14 expression via DNA methylation

PubMed Central

Kwak, Juri; Choi, Jung-Hye; Jang, Kyung Lib

2017-01-01

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to induce p14 expression via promoter hypomethylation to activate the p14-MDM2-p53 pathway, which leads to activation of the p53-dependent apoptotic pathway and subsequent induction of apoptosis in human hepatoma cells. In the present study, we found that hepatitis C virus (HCV) Core derived from ectopic expression or HCV infection overcomes ATRA-induced apoptosis in p53-positive hepatoma cells. For this effect, HCV Core upregulated both protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b and thereby repressed p14 expression via promoter hypermethylation, resulting in inactivation of the pathway leading to p53 accumulation in the presence of ATRA. As a result, HCV Core prevented ATRA from activating several apoptosis-related molecules, including Bax, p53 upregulated modulator of apoptosis, caspase-9, caspase-3, and poly (ADP-ribose) polymerase. In addition, complementation of p14 in the Core-expressing cells by either ectopic expression or treatment with 5-Aza-2′dC almost completely abolished the potential of HCV Core to suppress ATRA-induced apoptosis. Based on these observations, we conclude that HCV Core executes its oncogenic potential by suppressing the p53-dependent apoptosis induced by ATRA in human hepatoma cells. PMID:29156743

Logical Analysis of Regulation of Interleukin-12 Expression Pathway Regulation During HCV Infection.

PubMed

Farooqi, Zia-Ur-Rehman; Tareen, Samar H K; Ahmed, Jamil; Zaidi, Najam-Us-Sahar S

2016-01-01

Hepatitis C virus (HCV) triggers coordinated innate and adaptive response in host cell. HCV genome and proteins of the replicating virus are recognized as non-self-antigens by host cell to activate Toll Like Receptors (TLRs). Activated TLRs ultimately express cytokines, which can clear virus either by activating interferon (IFN), protein kinase C (PKC) and RNA Lase system or through activation of cytotoxic T-lymphocytes. Interleukin-12 (IL-12) is a potent antiviral cytokine, capable of clearing HCV by bridging both innate and adaptive antiviral immune response. Activation of TLR-4 on macrophages surface induces expression of IL-12 via NF-κB and AP-1 transcriptional pathway. After expression, IL- 12 releases IFN-γ, which activates anti-HCV cytotoxic lymphocytes. Conversely, in chronic HCV infection downregulation of IL-12 has been reported instead of by number of studies. Keeping in view of the above mentioned facts, this study was designed to evaluate HCV-core mediated down-regulation of IL-12 transcriptional pathway by employing a logical modeling approach based on the Ren´e Thomas formalism. The logical parameters of entities were estimated by using SMBioNet. The Logical model represents all possible dynamics of protein expression involved during course of HCV pathology. Results demonstrated that at chronic stage of infection, though TLR-4 was constantly active but yet it failed to express the NF-κB, AP-1, IL-12 and IFN-γ. This mechanism was indicative of incorporation of core mediated changes in IL-12 regulatory pathway. Moreover, results also indicate that HCV adopts different trajectories to accomplish the persistence of chronic phase of infection. It also implicated that human immune system tries to clear HCV but core is capable of inducing system oscillations to evade the immunity.

Determination of the core promoter regions of the Saccharomyces cerevisiae RPS3 gene.

PubMed

Joo, Yoo Jin; Kim, Jin-Ha; Baek, Joung Hee; Seong, Ki Moon; Lee, Jae Yung; Kim, Joon

2009-01-01

Ribosomal protein genes (RPG), which are scattered throughout the genomes of all eukaryotes, are subjected to coordinated expression. In yeast, the expression of RPGs is highly regulated, mainly at the transcriptional level. Recent research has found that many ribosomal proteins (RPs) function in multiple processes in addition to protein synthesis. Therefore, detailed knowledge of promoter architecture as well as gene regulation is important in understanding the multiple cellular processes mediated by RPGs. In this study, we investigated the functional architecture of the yeast RPS3 promoter and identified many putative cis-elements. Using beta-galactosidase reporter analysis and EMSA, the core promoter of RPS3 containing UASrpg and T-rich regions was corroborated. Moreover, the promoter occupancy of RPS3 by three transcription factors was confirmed. Taken together, our results further the current understanding of the promoter architecture and trans-elements of the Saccharomyces cerevisiae RPS3 gene.

First principles design of a core bioenergetic transmembrane electron-transfer protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goparaju, Geetha; Fry, Bryan A.; Chobot, Sarah E.

Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteinsmore » and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.« less

Studies of proteoglycan involvement in CPP-mediated delivery.

PubMed

Wittrup, Anders; Zhang, Si-He; Belting, Mattias

2011-01-01

Cell-penetrating peptides (CPPs) are widely used to deliver macromolecular cargoes to intracellular sites of action. Many CPPs have been demonstrated to rely on cell surface heparan sulfate proteoglycans (HSPGs) for efficient cellular entry and delivery. In this chapter, we describe methods for the study of PG involvement in CPP uptake. We provide descriptions of how to determine whether uptake of a CPP of interest is dependent on PGs. We also provide detailed protocols for the purification of PGs by anion-exchange chromatography as well as the characterization of the HSPG core protein composition of a cell line of interest. Finally, we present methods for modulating the expression level of specific HSPG core proteins as a means to determine the core protein specificity in the uptake of a particular CPP.

[Eukaryotic expression and application of HCV Hebei strain E2 extracellular core region].

PubMed

Ye, Chuantao; Bian, Peiyu; Weng, Daihui; Zhang, Hui; Yang, Jing; Zhang, Ying; Lei, Yingfeng; Jia, Zhansheng

2016-06-01

Objective To express core region of HCV1b (Hebei strain) E2 protein (E2c) by eukaryotic system, and establish the detection method of specific anti-HCV E2 antibody in the sera from hepatitis C patients. Methods Based on the literature, the E2c gene was modified from the HCV1b gene and synthesized via overlapping PCR. Thereafter, the E2c gene including tissue-type plasminogen activator (tPA) signal peptide was cloned into the pCI-neo eukaryotic expression vector, and the product was named pCI-tpa-1bE2c. After HEK293T cells were transfected with pCI-tpa-1bE2c, the supernatant was collected, condensed and purified. Its specificity was identified by Western blotting. Galanthus nivalis agglutinin (GNA)-based ELISA was used to detect the antibody against HCVE2 in the sera from hepatitis C patients. Results Modified HCV E2c protein was successfully expressed in HEK293T cells and the GNA-based ELISA was developed for detecting the antibody against HCV E2 in the sera from hepatitis C patients. Conclusion HCV-1bE2c protein can be effectively expressed in HEK293T cells and applied clinically.

Induction of Syndecan-4 by Organic-Inorganic Hybrid Molecules with a 1,10-Phenanthroline Structure in Cultured Vascular Endothelial Cells.

PubMed

Hara, Takato; Kojima, Takayuki; Matsuzaki, Hiroka; Nakamura, Takehiro; Yoshida, Eiko; Fujiwara, Yasuyuki; Yamamoto, Chika; Saito, Shinichi; Kaji, Toshiyuki

2017-02-08

Organic-inorganic hybrid molecules constitute analytical tools used in biological systems. Vascular endothelial cells synthesize and secrete proteoglycans, which are macromolecules consisting of a core protein and glycosaminoglycan side chains. Although the expression of endothelial proteoglycans is regulated by several cytokines/growth factors, there may be alternative pathways for proteoglycan synthesis aside from downstream pathways activated by these cytokines/growth factors. Here, we investigated organic-inorganic hybrid molecules to determine a variant capable of analyzing the expression of syndecan-4, a transmembrane heparan-sulfate proteoglycan, and identified 1,10-phenanthroline ( o -Phen) with or without zinc (Zn-Phen) or rhodium (Rh-Phen). Bovine aortic endothelial cells in culture were treated with these compounds, and the expression of syndecan-4 mRNA and core proteins was determined by real-time reverse transcription polymerase chain reaction and Western blot analysis, respectively. Our findings indicated that o -Phen and Zn-Phen specifically and strongly induced syndecan-4 expression in cultured vascular endothelial cells through activation of the hypoxia-inducible factor-1α/β pathway via inhibition of prolyl hydroxylase-domain-containing protein 2. These results demonstrated an alternative pathway involved in mediating induction of endothelial syndecan-4 expression and revealed organic-inorganic hybrid molecules as effective tools for analyzing biological systems.

Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/β-catenin pathway.

PubMed

Jiang, Xiao-Hua; Xie, Yu-Tao; Cai, Ya-Ping; Ren, Jing; Ma, Tao

2017-05-25

Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/β-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis. Compared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced β-catenin/Tcf-dependent transcriptional activity (F = 40.87, P < 0.01); increased nuclear translocation of β-catenin (F = 165.26, P < 0.01); upregulated nuclear β-catenin, cytoplasmic β-catenin, Wnt1, c-myc, and cyclin D1 protein expression (P < 0.01); and promoted proliferation of Huh7 cells (P < 0.01 or P < 0.05). Neither protein enhanced β-catenin/Tcf-dependent transcriptional activity in the LO2 cells (F = 0.65, P > 0.05), but they did significantly enhance Wnt3a-induced β-catenin/Tcf-dependent transcriptional activity (F = 64.25, P < 0.01), and promoted the nuclear translocation of β-catenin (F = 66.54, P < 0.01) and the Wnt3a-induced proliferation of LO2 cells (P < 0.01 or P < 0.05). Moreover, activation of the Wnt/β-catenin signaling pathway was greater with the core protein than with NS4B (P < 0.01 or P < 0.05). HCV core protein and NS4B directly activate the Wnt/β-catenin signaling pathway in Huh7 cells and LO2 cells induced by Wnt3a. These data suggest that HCV core protein and NS4B contribute to HCV-associated hepatocellular carcinogenesis.

The vaccinia virus E6 protein influences virion protein localization during virus assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Condit, Richard C., E-mail: condit@mgm.ufl.edu; Moussatche, Nissin

2015-08-15

Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulatemore » in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. - Highlights: • Mutation of E6 disrupts association of viral membranes with viral core proteins • Mutation of E6 does not perturb viral membrane biosynthesis • Mutation of E6 does not perturb localization of viral transcription enzymes • Mutation of E6 causes mis-localization and aggregation of viral core proteins • Vaccinia assembly uses three subassemblies: membranes, viroplasm, prenucleocapsid.« less

Decreased cathepsin K levels in human atherosclerotic plaques are associated with plaque instability.

PubMed

Zhao, Huiying; Qin, Xiujiao; Wang, Shuai; Sun, Xiwei; Dong, Bin

2017-10-01

Investigating the determinants and dynamics of atherosclerotic plaque instability is a key area of current cardiovascular research. Extracellular matrix degradation from excessive proteolysis induced by enzymes such as cathepsin K (Cat K) is implicated in the pathogenesis of unstable plaques. The current study assessed the expression of Cat K in human unstable atherosclerotic plaques. Specimens of popliteal arteries with atherosclerotic plaques were classified as stable (<40% lipid core plaque area; n=6) or unstable (≥40% lipid core plaque area; n=14) based on histopathological examinations of hematoxylin and eosin stained sections. The expression of Cat K and cystatin C (Cys C) were assessed by immunohistochemical examination and levels of Cat K mRNA were detected by semi-quantitative reverse transcriptase polymerase chain reaction. Morphological changes including a larger lipid core, endothelial proliferation with foam cells and destruction of internal elastic lamina were observed in unstable atherosclerotic plaques. In unstable plaques, the expression of Cat K protein and mRNA was upregulated, whereas Cys C protein expression was downregulated. The interplay between Cat K and Cys C may underlie the progression of plaques from stable to unstable and the current study indicated that Cat K and Cys C are potential targets for preventing and treating vulnerable atherosclerotic plaque ruptures.

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

A Critical Role for Protein Degradation in the Nucleus Accumbens Core in Cocaine Reward Memory

PubMed Central

Ren, Zhen-Yu; Liu, Meng-Meng; Xue, Yan-Xue; Ding, Zeng-Bo; Xue, Li-Fen; Zhai, Suo-Di; Lu, Lin

2013-01-01

The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli contribute to cocaine seeking and relapse. Previous studies have shown impairment in cocaine reward memories by manipulating a labile state induced by memory retrieval, but the mechanisms that underlie the destabilization of cocaine reward memory are unknown. In this study, using a Pavlovian cocaine-induced conditioned place preference (CPP) procedure in rats, we tested the contribution of ubiquitin-proteasome system-dependent protein degradation in destabilization of cocaine reward memory. First, we found that polyubiquitinated protein expression levels and polyubiquitinated N-ethylmaleimide-sensitive fusion (NSF) markedly increased 15 min after retrieval while NSF protein levels decreased 1 h after retrieval in the synaptosomal membrane fraction in the nucleus accumbens (NAc) core. We then found that infusion of the proteasome inhibitor lactacystin into the NAc core prevented the impairment of memory reconsolidation induced by the protein synthesis inhibitor anisomycin and reversed the effects of anisomycin on NSF and glutamate receptor 2 (GluR2) protein levels in the synaptosomal membrane fraction in the NAc core. We also found that lactacystin infusion into the NAc core but not into the shell immediately after extinction training sessions inhibited CPP extinction and reversed the extinction training-induced decrease in NSF and GluR2 in the synaptosomal membrane fraction in the NAc core. Finally, infusions of lactacystin by itself into the NAc core immediately after each training session or before the CPP retrieval test had no effect on the consolidation and retrieval of cocaine reward memory. These findings suggest that ubiquitin-proteasome system-dependent protein degradation is critical for retrieval-induced memory destabilization. PMID:23303053

s-core network decomposition: A generalization of k-core analysis to weighted networks

NASA Astrophysics Data System (ADS)

Eidsaa, Marius; Almaas, Eivind

2013-12-01

A broad range of systems spanning biology, technology, and social phenomena may be represented and analyzed as complex networks. Recent studies of such networks using k-core decomposition have uncovered groups of nodes that play important roles. Here, we present s-core analysis, a generalization of k-core (or k-shell) analysis to complex networks where the links have different strengths or weights. We demonstrate the s-core decomposition approach on two random networks (ER and configuration model with scale-free degree distribution) where the link weights are (i) random, (ii) correlated, and (iii) anticorrelated with the node degrees. Finally, we apply the s-core decomposition approach to the protein-interaction network of the yeast Saccharomyces cerevisiae in the context of two gene-expression experiments: oxidative stress in response to cumene hydroperoxide (CHP), and fermentation stress response (FSR). We find that the innermost s-cores are (i) different from innermost k-cores, (ii) different for the two stress conditions CHP and FSR, and (iii) enriched with proteins whose biological functions give insight into how yeast manages these specific stresses.

Identification and Targeting of an Interaction between a Tyrosine Motif within Hepatitis C Virus Core Protein and AP2M1 Essential for Viral Assembly

PubMed Central

Ziv-Av, Amotz; Gerber, Doron; Jacob, Yves; Einav, Shirit

2012-01-01

Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core's YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development. PMID:22916011

[Effect of Hepatitis C virus proteins on the production of proinflammatory and profibrotic cytokines in Huh7.5 human hepatoma cells].

PubMed

Masalova, O V; Lesnova, E I; Permyakova, K Yu; Samokhvalov, E I; Ivanov, A V; Kochetkov, S N; Kushch, A A

2016-01-01

Hepatitis C virus (HCV) is a widespread dangerous human pathogen. Up to 80% of HCV-infected individuals develop chronic infection, which is often accompanied by liver inflammation and fibrosis and, at terminal stages, liver cirrhosis and cancer. Treatment of patients with end-stage liver disease is often ineffective, and even patients with suppressed HCV replication have higher risk of death as compared with noninfected subjects. Therefore, investigating the mechanisms that underlie HCV pathogenesis and developing treatments for virus-associated liver dysfunction remain an important goal. The effect of individual HCV proteins on the production of proinflammatory and profibrotic cytokines in hepatocellular carcinoma Huh7.5 cells was analyzed in a systematic manner. Cells were transfected with plasmids encoding HCV proteins. Cytokine production and secretion was accessed by immunocytochemistry and ELISA of the culture medium, and transcription of the cytokine genes was assessed using reverse transcription and PCR. HCV proteins proved to differ in effect on cytokine production. Downregulation of interleukin 6 (IL-6) production was observed in cells expressing the HCV core, NS3, and NS5A proteins. Production of transforming growth factor β1 (TGF-β1) was lower in cells expressing the core proteins, NS3, or E1/E2 glycoproteins. A pronounced increase in production and secretion of tumor necrosis factor α (TNF-α) was observed in response to expression of the HCV E1/E2 glycoproteins. A higher biosynthesis, but a lower level in the cell culture medium, was detected for interleukin 1β (IL-1β) in cells harboring NS4 and IL-6 in cells expressing NS5В. The finding was possibly explained by protein-specific retention and consequent accumulation of the respective cytokines in the cell.

Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins

NASA Astrophysics Data System (ADS)

Faure, Guilhem; Koonin, Eugene V.

2015-05-01

Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

Identification of GRB2 and GAB1 Coexpression as an Unfavorable Prognostic Factor for Hepatocellular Carcinoma by a Combination of Expression Profile and Network Analysis

PubMed Central

Yang, Mei; Wang, Danhua; Yu, Lingxiang; Guo, Chaonan; Guo, Xiaodong; Lin, Na

2013-01-01

Aim To screen novel markers for hepatocellular carcinoma (HCC) by a combination of expression profile, interaction network analysis and clinical validation. Methods HCC significant molecules which are differentially expressed or had genetic variations in HCC tissues were obtained from five existing HCC related databases (OncoDB.HCC, HCC.net, dbHCCvar, EHCO and Liverome). Then, the protein-protein interaction (PPI) network of these molecules was constructed. Three topological features of the network ('Degree', 'Betweenness', and 'Closeness') and the k-core algorithm were used to screen candidate HCC markers which play crucial roles in tumorigenesis of HCC. Furthermore, the clinical significance of two candidate HCC markers growth factor receptor-bound 2 (GRB2) and GRB2-associated-binding protein 1 (GAB1) was validated. Results In total, 6179 HCC significant genes and 977 HCC significant proteins were collected from existing HCC related databases. After network analysis, 331 candidate HCC markers were identified. Especially, GAB1 has the highest k-coreness suggesting its central localization in HCC related network, and the interaction between GRB2 and GAB1 has the largest edge-betweenness implying it may be biologically important to the function of HCC related network. As the results of clinical validation, the expression levels of both GRB2 and GAB1 proteins were significantly higher in HCC tissues than those in their adjacent nonneoplastic tissues. More importantly, the combined GRB2 and GAB1 protein expression was significantly associated with aggressive tumor progression and poor prognosis in patients with HCC. Conclusion This study provided an integrative analysis by combining expression profile and interaction network analysis to identify a list of biologically significant HCC related markers and pathways. Further experimental validation indicated that the aberrant expression of GRB2 and GAB1 proteins may be strongly related to tumor progression and prognosis in patients with HCC. The overexpression of GRB2 in combination with upregulation of GAB1 may be an unfavorable prognostic factor for HCC. PMID:24391994

Efficient production of a folded and functional, highly disulfide-bonded beta-helix antifreeze protein in bacteria.

PubMed

Bar, Maya; Bar-Ziv, Roy; Scherf, Tali; Fass, Deborah

2006-08-01

The Tenebrio molitor thermal hysteresis protein has a cysteine content of 19%. This 84-residue protein folds as a compact beta-helix, with eight disulfide bonds buried in its core. Exposed on one face of the protein is an array of threonine residues, which constitutes the ice-binding face. Previous protocols for expression of this protein in recombinant expression systems resulted in inclusion bodies or soluble but largely inactive material. A long and laborious refolding procedure was performed to increase the fraction of active protein and isolate it from inactive fractions. We present a new protocol for production of fully folded and active T. molitor thermal hysteresis protein in bacteria, without the need for in vitro refolding. The protein coding sequence was fused to those of various carrier proteins and expressed at low temperature in a bacterial strain specially suited for production of disulfide-bonded proteins. The product, after a simple and robust purification procedure, was analyzed spectroscopically and functionally and was found to compare favorably to previously published data on refolded protein and protein obtained from its native source.

Prickle isoforms control the direction of tissue polarity by microtubule independent and dependent mechanisms.

PubMed

Sharp, Katherine A; Axelrod, Jeffrey D

2016-02-10

Planar cell polarity signaling directs the polarization of cells within the plane of many epithelia. While these tissues exhibit asymmetric localization of a set of core module proteins, in Drosophila, more than one mechanism links the direction of core module polarization to the tissue axes. One signaling system establishes a polarity bias in the parallel, apical microtubules upon which vesicles containing core proteins traffic. Swapping expression of the differentially expressed Prickle isoforms, Prickle and Spiny-legs, reverses the direction of core module polarization. Studies in the proximal wing and the anterior abdomen indicated that this results from their differential control of microtubule polarity. Prickle and Spiny-legs also control the direction of polarization in the distal wing (D-wing) and the posterior abdomen (P-abd). We report here that this occurs without affecting microtubule polarity in these tissues. The direction of polarity in the D-wing is therefore likely determined by a novel mechanism independent of microtubule polarity. In the P-abd, Prickle and Spiny-legs interpret at least two directional cues through a microtubule-polarity-independent mechanism. © 2016. Published by The Company of Biologists Ltd.

Regulation of myeloid leukemia factor-1 interacting protein (MLF1IP) expression in glioblastoma.

PubMed

Hanissian, Silva H; Teng, Bin; Akbar, Umar; Janjetovic, Zorica; Zhou, Qihong; Duntsch, Christopher; Robertson, Jon H

2005-06-14

The myelodysplasia/myeloid leukemia factor 1-interacting protein MLF1IP is a novel gene which encodes for a putative transcriptional repressor. It is localized to human chromosome 4q35.1 and is expressed in both the nuclei and cytoplasm of cells. Northern and Western blot analyses have revealed MLF1IP to be present at very low amounts in normal brain tissues, whereas a number of human and rat glioblastoma (GBM) cell lines demonstrated a high level expression of the MLF1IP protein. Immunohistochemical analysis of rat F98 and C6 GBM tumor models showed that MLF1IP was highly expressed in the tumor core where it was co-localized with MLF1 and nestin. Moreover, MLF1IP expression was elevated in the contralateral brain where no tumor cells were detected. These observations, together with previous data demonstrating a role for MLF1IP in erythroleukemias, suggest a possible function for this protein in glioma pathogenesis and potentially in other types of malignancies.

High-Yield Secretion of Multiple Client Proteins in Aspergillus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Segato, F.; Damasio, A. R. L.; Goncalves, T. A.

2012-07-15

Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degradingmore » enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.« less

An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems.

PubMed

Cheng, Hao-Wen; Chen, Kuan-Chun; Raja, Joseph A J; Li, Jian-Xian; Yeh, Shyi-Dong

2013-04-15

NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, "(109)KFTMHNQIF(117)", denoted as "nss", retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Expression of the highly conserved vaccinia virus E6 protein is required for virion morphogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Resch, Wolfgang; Weisberg, Andrea S.; Moss, Bernard, E-mail: bmoss@nih.go

2009-04-10

The vaccinia virus E6R gene (VACVWR062) is conserved in all members of the poxvirus family and encodes a protein associated with the mature virion. We confirmed this association and provided evidence for an internal location. An inducible mutant that conditionally expresses E6 was constructed. In the absence of inducer, plaque formation and virus production were severely inhibited in several cell lines, whereas some replication occurred in others. This difference could be due to variation in the stringency of repression, since we could not isolate a stable deletion mutant even in the more 'permissive' cells. Under non-permissive conditions, viral late proteinsmore » were synthesized but processing of core proteins was inefficient, indicative of an assembly block. Transmission electron microscopy of sections of cells infected with the mutant in the absence of inducer revealed morphogenetic defects with crescents and empty immature virions adjacent to dense inclusions of viroplasm. Mature virions were infrequent and cores appeared to have lucent centers.« less

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

PubMed

Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D; Nystul, Todd G; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E; Murphy, Terence D; Levis, Robert W; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A; Spradling, Allan C

2007-03-01

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

Gene transcripts selectively down-regulated in the shell of the nucleus accumbens long after heroin self-administration are up-regulated in the core independent of response contingency.

PubMed

Jacobs, Edwin H; de Vries, Taco J; Smit, August B; Schoffelmeer, Anton N M

2004-01-01

Long-term drug-induced alterations in neurotransmission within the nucleus accumbens (NAc) shell and core may underlie relapse to drug-seeking behavior and drug-taking upon re-exposure to drugs and drug-associated stimuli (cues) during abstinence. Using an open screening strategy, we recently identified 25 gene transcripts, encoding for proteins involved in neuronal functioning and structure that are down-regulated in rat NAc shell after contingent (active), but not after non-contingent (passive), heroin administration. Studying the expression of the same transcripts in the NAc core by means of quantitative PCR, we now demonstrate that most of these transcripts are up-regulated in that NAc subregion long (3 weeks) after heroin self-administration in rats. A similar up-regulation in gene expression was also apparent in the NAc core of animals with a history of non-contingent heroin administration (yoked controls). These data indicate that heroin self-administration differentially regulates genes in the NAc core as compared with the shell. Moreover, whereas cognitive processes involved in active drug self-administration (e.g., instrumental learning) seems to direct gene expression in the NAc shell, neuroplasticity in the NAc core may be due to the pharmacological effects of heroin (including Pavlovian conditioning), as expressed in rats upon contingent as well as non-contingent administration of heroin.

Regulator of G protein signaling 4 is a novel target of GATA-6 transcription factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yonggang; Li, Fang; Xiao, Xiao

GATA transcription factors regulate an array of genes important in cell proliferation and differentiation. Here we report the identification of regulator of G protein signaling 4 (RGS4) as a novel target for GATA-6 transcription factor. Although three sites (a, b, c) within the proximal region of rabbit RGS4 promoter for GATA transcription factors were predicted by bioinformatics analysis, only GATA-a site (16 bp from the core TATA box) is essential for RGS4 transcriptional regulation. RT-PCR analysis demonstrated that only GATA-6 was highly expressed in rabbit colonic smooth muscle cells but GATA-4/6 were expressed in cardiac myocytes and GATA-1/2/3 expressed inmore » blood cells. Adenovirus-mediated expression of GATA-6 but not GATA-1 significantly increased the constitutive and IL-1β-induced mRNA expression of the endogenous RGS4 in colonic smooth muscle cells. IL-1β stimulation induced GATA-6 nuclear translocation and increased GATA-6 binding to RGS4 promoter. These data suggest that GATA factor could affect G protein signaling through regulating RGS4 expression, and GATA signaling may develop as a future therapeutic target for RGS4-related diseases. - Highlights: • GATA-6 is highly expressed in colonic smooth muscle cells. • RGS4 is a novel target for GATA-6 transcription factor. • GATA-a response element is essential to regulate the core promoter of RGS4. • GATA-6 regulates IL-1β-induced RGS4 upregulation.« less

PML tumor suppressor protein is required for HCV production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuroki, Misao; Research Fellow of the Japan Society for the Promotion of Science; Center for AIDS Research, Kumamoto University, Kumamoto 860-0811

2013-01-11

Highlights: Black-Right-Pointing-Pointer PML tumor suppressor protein is required for HCV production. Black-Right-Pointing-Pointer PML is dispensable for HCV RNA replication. Black-Right-Pointing-Pointer HCV could not alter formation of PML-NBs. Black-Right-Pointing-Pointer INI1 and DDX5, PML-related proteins, are involved in HCV life cycle. -- Abstract: PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown.more » To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production.« less

Hepatitis B core protein promotes liver cancer metastasis through miR-382-5p/DLC-1 axis.

PubMed

Du, Juan; Bai, Fuxiang; Zhao, Peiqing; Li, Xiaoyan; Li, Xueen; Gao, Lifen; Ma, Chunhong; Liang, Xiaohong

2018-01-01

The hepatitis B virus core protein (HBc), also named core antigen, is well-known for its key role in viral capsid formation and virus replication. Recently, studies showed that HBc has the potential to control cell biology activity by regulating host gene expression. Here, we utilized miRNA microarray to identify 24 upregulated miRNAs and 21 downregulated miRNAs in HBc-expressed HCC cells, which were involved in multiple biological processes, including cell motility. Consistently, the in vitro transwell assay and the in vivo tail-vein injection model showed HBc promotion on HCC metastasis. Further, the miRNA-target gene network analysis displayed that the deleted in liver cancer (DLC-1) gene, an important negative regulator for cell motility, was potentially targeted by several differentially expressed miRNAs in HBc-introduced cells. Introduction of miRNAs mimics or inhibitors and 3'UTR luciferase activity assay proved that miR-382-5p efficiently suppressed DLC-1 expression and its 3'-UTR luciferase reporter activity. Importantly, cotransfection of miR-382-5p mimics/inhibitors and the DLC-1 expression vector almost abrogated HBc promotion on cell motility, indicating that the miR-382-5p/DLC-1 axis is important for mediating HBc-enhanced HCC motility. Clinical HCC samples also showed a negative correlation between miR-382-5p and DLC-1 expression level. Furthermore, HBc-positive HCC tissues showed high miR-382-5p level and reduced DLC-1 expression. In conclusion, our findings revealed that HBc promoted HCC motility by regulating the miR-382-5p/DLC-1 axis, which might provide a novel target for clinical diagnosis and treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

PubMed

Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

2011-12-01

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

Promyelocytic Leukemia (Pml) Nuclear Bodies Are Protein Structures That Do Not Accumulate RNA

PubMed Central

Boisvert, François-Michel; Hendzel, Michael J.; Bazett-Jones, David P.

2000-01-01

The promyelocytic leukemia (PML) nuclear body (also referred to as ND10, POD, and Kr body) is involved in oncogenesis and viral infection. This subnuclear domain has been reported to be rich in RNA and a site of nascent RNA synthesis, implicating its direct involvement in the regulation of gene expression. We used an analytical transmission electron microscopic method to determine the structure and composition of PML nuclear bodies and the surrounding nucleoplasm. Electron spectroscopic imaging (ESI) demonstrates that the core of the PML nuclear body is a dense, protein-based structure, 250 nm in diameter, which does not contain detectable nucleic acid. Although PML nuclear bodies contain neither chromatin nor nascent RNA, newly synthesized RNA is associated with the periphery of the PML nuclear body, and is found within the chromatin-depleted region of the nucleoplasm immediately surrounding the core of the PML nuclear body. We further show that the RNA does not accumulate in the protein core of the structure. Our results dismiss the hypothesis that the PML nuclear body is a site of transcription, but support the model in which the PML nuclear body may contribute to the formation of a favorable nuclear environment for the expression of specific genes. PMID:10648561

In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles

PubMed Central

Su, Xingfang; Fricke, Jennifer; Kavanagh, Daniel; Irvine, Darrell J.

2012-01-01

Biodegradable core-shell structured nanoparticles with a poly(β-amino-ester) (PBAE) core enveloped by a phospholipid bilayer shell were developed for in vivo mRNA delivery, with a view toward delivery of mRNA-based vaccines. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Messenger RNA was efficiently adsorbed via electrostatic interactions onto the surface of these net positively-charged nanoparticles. In vitro, mRNA-loaded particle uptake by dendritic cells (DCs) led to mRNA delivery into the cytosol with low cytotoxicity, followed by translation of the encoded protein in these difficult-to-transfect cells at a frequency of ~30%. Particles loaded with mRNA administered intranasally in mice led to the expression of the reporter protein luciferase in vivo as soon as 6 h after administration, a timepoint when naked mRNA given i.n. showed no expression. At later timepoints, luciferase expression was detected in naked mRNA-treated mice, but this group showed a wide variation in levels of transfection, compared to particle-treated mice. This system may thus be promising for non-invasive delivery of mRNA-based vaccines. PMID:21417235

Pro-hormone Secretogranin II Regulates Dense Core Secretory Granule Biogenesis in Catecholaminergic Cells*

PubMed Central

Courel, Maïté; Soler-Jover, Alex; Rodriguez-Flores, Juan L.; Mahata, Sushil K.; Elias, Salah; Montero-Hadjadje, Maïté; Anouar, Youssef; Giuly, Richard J.; O'Connor, Daniel T.; Taupenot, Laurent

2010-01-01

Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H+-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network. PMID:20061385

Pro-hormone secretogranin II regulates dense core secretory granule biogenesis in catecholaminergic cells.

PubMed

Courel, Maïté; Soler-Jover, Alex; Rodriguez-Flores, Juan L; Mahata, Sushil K; Elias, Salah; Montero-Hadjadje, Maïté; Anouar, Youssef; Giuly, Richard J; O'Connor, Daniel T; Taupenot, Laurent

2010-03-26

Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H(+)-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network.

[Anti-HBV effects of genetically engineered replication-defective HBV with combined expression of antisense RNA and dominant negative mutants of core protein and construction of first-generation packaging cell line for HBV vector].

PubMed

Sun, Dian Xing; Hu, Da Rong; Wu, Guang Hui; Hu, Xue Ling; Li, Juan; Fan, Gong Ren

2002-08-01

To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein. Full length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. The mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, P<0.01), 66.54% 4.45% (t=42.3, P<0.01), and 73.68% 5.07% (t=51.9, P<0.01) in group 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS, and 2.2.15-CPAS, respectively. The mean inhibitory rates of HBeAg were 4.46% 4.25%, 52.86% 1.32% (t=36.2, P<0.01), 26.36% 1.69% (t=22.3, P<0.01), and 59.28% 2.10% (t=39.0, P<0.01), respectively. The inhibitory rates of HBc related HBV DNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium. It has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual expression of them. With the help of wild-type HBV, the modified HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified. The packaging cell line can provide packaging for replication-defective HBV, but with low efficiency.

Unconventional secretory processing diversifies neuronal ion channel properties

PubMed Central

Hanus, Cyril; Geptin, Helene; Tushev, Georgi; Garg, Sakshi; Alvarez-Castelao, Beatriz; Sambandan, Sivakumar; Kochen, Lisa; Hafner, Anne-Sophie; Langer, Julian D; Schuman, Erin M

2016-01-01

N-glycosylation – the sequential addition of complex sugars to adhesion proteins, neurotransmitter receptors, ion channels and secreted trophic factors as they progress through the endoplasmic reticulum and the Golgi apparatus – is one of the most frequent protein modifications. In mammals, most organ-specific N-glycosylation events occur in the brain. Yet, little is known about the nature, function and regulation of N-glycosylation in neurons. Using imaging, quantitative immunoblotting and mass spectrometry, we show that hundreds of neuronal surface membrane proteins are core-glycosylated, resulting in the neuronal membrane displaying surprisingly high levels of glycosylation profiles that are classically associated with immature intracellular proteins. We report that while N-glycosylation is generally required for dendritic development and glutamate receptor surface expression, core-glycosylated proteins are sufficient to sustain these processes, and are thus functional. This atypical glycosylation of surface neuronal proteins can be attributed to a bypass or a hypo-function of the Golgi apparatus. Core-glycosylation is regulated by synaptic activity, modulates synaptic signaling and accelerates the turnover of GluA2-containing glutamate receptors, revealing a novel mechanism that controls the composition and sensing properties of the neuronal membrane. DOI: http://dx.doi.org/10.7554/eLife.20609.001 PMID:27677849

Heparan sulfate proteoglycans undergo differential expression alterations in right sided colorectal cancer, depending on their metastatic character.

PubMed

Fernández-Vega, Iván; García-Suárez, Olivia; García, Beatriz; Crespo, Ainara; Astudillo, Aurora; Quirós, Luis M

2015-10-20

Heparan sulfate proteoglycans (HSPGs) are complex molecules involved in the growth, invasion and metastatic properties of cancerous cells. This study analyses the alterations in the expression patterns of these molecules in right sided colorectal cancer (CRC), both metastatic and non-metastatic. Twenty right sided CRCs were studied. A transcriptomic approach was used, employing qPCR to analyze both the expression of the enzymes involved in heparan sulfate (HS) chains biosynthesis, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate (CS) chains, we include the study of the genes involved in the biosynthesis of these glycosaminoglycans. Immunohistochemical techniques were also used to analyze tissue expression of particular genes showing significant expression differences, of potential interest. Changes in proteoglycan core proteins differ depending on their location; those located intracellularly or in the extracellular matrix show very similar alteration patterns, while those located on the cell surface vary greatly depending on the nature of the tumor: glypicans 1, 3, 6 and betaglycan are affected in the non-metastatic tumors, whereas in the metastatic, only glypican-1 and syndecan-1 are modified, the latter showing opposing alterations in levels of RNA and of protein, suggesting post-transcriptional regulation in these tumors. Furthermore, in non-metastatic tumors, polymerization of glycosaminoglycan chains is modified, particularly affecting the synthesis of the tetrasaccharide linker and the initiation and elongation of CS chains, HS chains being less affected. Regarding the enzymes responsible for the modificaton of the HS chains, alterations were only found in non-metastatic tumors, affecting N-sulfation and the isoforms HS6ST1, HS3ST3B and HS3ST5. In contrast, synthesis of the CS chains suggests changes in epimerization and sulfation of the C4 and C2 in both types of tumor. Right sided CRCs show alterations in the expression of HSPGs, including the expression of the cell surface core proteins, many glycosiltransferases and some enzymes that modify the HS chains depending on the metastatic nature of the tumor, resulting more affected in non-metastatic ones. However, matrix proteoglycans and enzymes involved in CS fine structure synthesis are extensively modified independetly of the presence of lymph node metastasis.

Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

PubMed Central

Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Sun, Guochang

2012-01-01

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium. PMID:23166741

Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm.

PubMed

Oszvald, Maria; Kang, Tae-Jin; Tomoskozi, Sandor; Tamas, Cecilia; Tamas, Laszlo; Kim, Tae-Geum; Yang, Moon-Sik

2007-03-01

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.

Calpain-GRIP Signaling in Nucleus Accumbens Core Mediates the Reconsolidation of Drug Reward Memory.

PubMed

Liang, Jie; Li, Jia-Li; Han, Ying; Luo, Yi-Xiao; Xue, Yan-Xue; Zhang, Yàn; Zhang, Yán; Zhang, Li-Bo; Chen, Man-Li; Lu, Lin; Shi, Jie

2017-09-13

Exposure to drug-paired cues causes drug memories to be in a destabilized state and interfering with memory reconsolidation can inhibit relapse. Calpain, a calcium-dependent neutral cysteine protease, is involved in synaptic plasticity and the formation of long-term fear memory. However, the role of calpain in the reconsolidation of drug reward memory is still unknown. In the present study, using a conditioned place preference (CPP) model, we found that exposure to drug-paired contextual stimuli induced the activation of calpain and decreased the expression of glutamate receptor interacting protein 1 (GRIP1) in the nucleus accumbens (NAc) core, but not shell, of male rats. Infusions of calpain inhibitors in the NAc core immediately after retrieval disrupted the reconsolidation of cocaine/morphine cue memory and blocked retrieval-induced calpain activation and GRIP1 degradation. The suppressive effect of calpain inhibitors on the expression of drug-induced CPP lasted for at least 14 d. The inhibition of calpain without retrieval 6 h after retrieval or after exposure to an unpaired context had no effects on the expression of reward memory. Calpain inhibition after retrieval also decreased cocaine seeking in a self-administration model and this effect did not recover spontaneously after 28 d. Moreover, the knock-down of GRIP1 expression in the NAc core by lentivirus-mediated short-hairpin RNA blocked disruption of the reconsolidation of drug cue memories that was induced by calpain inhibitor treatment. These results suggest that calpain activity in the NAc core is crucial for the reconsolidation of drug reward memory via the regulation of GRIP1 expression. SIGNIFICANCE STATEMENT Calpain plays an important role in synaptic plasticity and long-term memory consolidation, however, its role in the reconsolidation of drug cue memory remains unknown. Using conditioned place preference and self-administration procedures, we found that exposure to drug-paired cues induced the activation of calpain and decreased glutamate receptor interacting protein 1 (GRIP1) expression in the nucleus accumbens (NAc) core. The inhibition of calpain activity in the NAc core immediately after retrieval disrupted the reconsolidation of cocaine/morphine cue memory that was blocked by prior GRIP1 knock-down. Our findings indicate that calpain-GRIP signaling is essential for the restabilization process that is associated with drug cue memory and the inhibition of calpain activity may be a novel strategy for the prevention of drug relapse. Copyright © 2017 the authors 0270-6474/17/378938-14$15.00/0.

Core histone genes of Giardia intestinalis: genomic organization, promoter structure, and expression

PubMed Central

Yee, Janet; Tang, Anita; Lau, Wei-Ling; Ritter, Heather; Delport, Dewald; Page, Melissa; Adam, Rodney D; Müller, Miklós; Wu, Gang

2007-01-01

Background Giardia intestinalis is a protist found in freshwaters worldwide, and is the most common cause of parasitic diarrhea in humans. The phylogenetic position of this parasite is still much debated. Histones are small, highly conserved proteins that associate tightly with DNA to form chromatin within the nucleus. There are two classes of core histone genes in higher eukaryotes: DNA replication-independent histones and DNA replication-dependent ones. Results We identified two copies each of the core histone H2a, H2b and H3 genes, and three copies of the H4 gene, at separate locations on chromosomes 3, 4 and 5 within the genome of Giardia intestinalis, but no gene encoding a H1 linker histone could be recognized. The copies of each gene share extensive DNA sequence identities throughout their coding and 5' noncoding regions, which suggests these copies have arisen from relatively recent gene duplications or gene conversions. The transcription start sites are at triplet A sequences 1–27 nucleotides upstream of the translation start codon for each gene. We determined that a 50 bp region upstream from the start of the histone H4 coding region is the minimal promoter, and a highly conserved 15 bp sequence called the histone motif (him) is essential for its activity. The Giardia core histone genes are constitutively expressed at approximately equivalent levels and their mRNAs are polyadenylated. Competition gel-shift experiments suggest that a factor within the protein complex that binds him may also be a part of the protein complexes that bind other promoter elements described previously in Giardia. Conclusion In contrast to other eukaryotes, the Giardia genome has only a single class of core histone genes that encode replication-independent histones. Our inability to locate a gene encoding the linker histone H1 leads us to speculate that the H1 protein may not be required for the compaction of Giardia's small and gene-rich genome. PMID:17425802

Hsp-72, a candidate prognostic indicator of heatstroke.

PubMed

Dehbi, Mohammed; Baturcam, Engin; Eldali, Abdelmoneim; Ahmed, Maqbool; Kwaasi, Aaron; Chishti, Muhammad Azhar; Bouchama, Abderrezak

2010-09-01

Exposure of rats to environmental heat enhances the expression of heat shock protein-72 (Hsp-72) in most of their organs proportionally to heat stress severity. Pre-induction or over-expression of Hsp-72 prevents organ damage and lethality, suggesting that heat shock proteins (Hsps) may have a pathogenic role in this condition. We investigated the expression profile of Hsps in baboons subjected to environmental heat stress until the core temperature attained 42.5 degrees C (moderate heatstroke) or occurrence of hypotension associated with core temperature > or = 43.5 degrees C (severe heatstroke). Western blot analysis demonstrated a differential induction of Hsp-72 among organs of heat-stressed animals with the highest induction in the liver and the lowest in lung. Hsp-60 and Hsc-70 expression was similar between control and heat-stressed animals. ELISA studies indicated a marked release of Hsp-72 into the circulation of baboons with severe heatstroke with a peak at 24 h post-heatstroke onset and remained sustained up to 72 h. Hsp-72 release was not associated with core temperature or systolic blood pressure, but correlated with markers of liver, myocardium, and skeletal muscle tissue necrosis. Non-survivors displayed significantly higher Hsp-72 levels than survivors. No Hsp-60 was detected in the circulation. These findings add further evidence that increased expression of Hsp-72 may be an important component of the host response to severe heatstroke. They also suggest that extracellular Hsp-72 is a marker of multiple organs tissue damage. Whether extracellular Hsp-72 plays a role in the host immune response to heat stress merits further studies.

The ubiquitin-proteasome system is required for African swine fever replication.

PubMed

Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

2017-01-01

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

Hypoperfusion induces overexpression of beta-amyloid precursor protein mRNA in a focal ischemic rodent model.

PubMed

Shi, J; Yang, S H; Stubley, L; Day, A L; Simpkins, J W

2000-01-17

Silent stroke is one of the risk factors of dementia. In the present study, we used a novel focal ischemic animal model to investigate the effects of comparatively small changes of cerebral blood flow (CBF) on the expression of beta-amyloid precursor protein (APP) mRNA. Focal ischemia was achieved by introducing a 4-0 monofilament to the bifurcation of anterior and middle cerebral arteries. Brain samples were harvested from ischemic core and penumbra of cortices at 1, 4 and 7 days following ischemia. The expression of APP mRNA was assessed by RT-PCR. The CBF was decreased to 50% for 1 day after stroke and recovered to 90% at the fourth day after stroke. The changes of CBF were accompanied by an increase in the expression of APP mRNA. APP mRNA increased to 208% and 152% in the penumbra and core ischemic regions, respectively, on the fourth day after MCAO and remained high through the seventh day of ischemia. This study suggests brain hypoperfusion enhances APP mRNA expression and may contribute to the progression of cognitive impairment after silent stroke.

The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental Studies

PubMed Central

Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D.; Nystul, Todd G.; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E.; Murphy, Terence D.; Levis, Robert W.; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A.; Spradling, Allan C.

2007-01-01

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions. PMID:17194782

Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cleveland, Thomas E.; He, Wei; Evans, Angela C.

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergentmore » removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.« less

Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles

DOE PAGES

Cleveland, Thomas E.; He, Wei; Evans, Angela C.; ...

2018-02-13

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergentmore » removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.« less

The minimal promoter region of the dense-core vesicle protein IA-2: transcriptional regulation by CREB.

PubMed

Cai, Tao; Hirai, Hiroki; Xu, Huanyu; Notkins, Abner L

2015-06-01

IA-2 is a transmembrane protein found in the dense-core vesicles (DCV) of neuroendocrine cells and one of the major autoantigens in type 1 diabetes. DCV are involved in the secretion of hormones (e.g., insulin) and neurotransmitters. Stimulation of pancreatic β cells with glucose upregulates the expression of IA-2 and an increase in IA-2 results in an increase in the number of DCV. Little is known, however, about the promoter region of IA-2 or the transcriptional factors that regulate the expression of this gene. In the present study, we constructed eight deletion fragments from the upstream region of the IA-2 transcription start site and linked them to a luciferase reporter. By this approach, we have identified a short bp region (-216 to +115) that has strong promoter activity. We also identified a transcription factor, cAMP responsive element-binding protein (CREB), which binds to two CREB-related binding sites located in this region. The binding of CREB to these sites enhanced IA-2 transcription by more than fivefold. We confirmed these findings by site-directed mutagenesis, chromatin immunoprecipitation assays and RNAi inhibition. Based on these findings, we conclude that the PKA pathway is a critical, but not the exclusive signaling pathway involved in IA-2 gene expression.

Promoting gene expression in plants by permissive histone lysine methylation

PubMed Central

Millar, Tony; Finnegan, E Jean

2009-01-01

Plants utilize sophisticated epigenetic regulatory mechanisms to coordinate changes in gene expression during development and in response to environmental stimuli. Epigenetics refers to the modification of DNA and chromatin associated proteins, which affect gene expression and cell function, without changing the DNA sequence. Such modifications are inherited through mitosis, and in rare instances through meiosis, although it can be reversible and thus regulatory. Epigenetic modifications are controlled by groups of proteins, such as the family of histone lysine methytransferases (HKMTs). The catalytic core known as the SET domain encodes HKMT activity and either promotes or represses gene expression. A large family of SET domain proteins is present in Arabidopsis where there is growing evidence that two classes of these genes are involved in promoting gene expression in a diverse range of developmental processes. This review will focus on the function of these two classes and the processes that they control, highlighting the huge potential this regulatory mechanism has in plants. PMID:19816124

A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells

PubMed Central

Komatsu, Tetsuro; Dacheux, Denis; Kreppel, Florian; Nagata, Kyosuke; Wodrich, Harald

2015-01-01

Inside the adenovirus virion, the genome forms a chromatin-like structure with viral basic core proteins. Core protein VII is the major DNA binding protein and was shown to remain associated with viral genomes upon virus entry even after nuclear delivery. It has been suggested that protein VII plays a regulatory role in viral gene expression and is a functional component of viral chromatin complexes in host cells. As such, protein VII could be used as a maker to track adenoviral chromatin complexes in vivo. In this study, we characterize a new monoclonal antibody against protein VII that stains incoming viral chromatin complexes following nuclear import. Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection. This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells. These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes. PMID:26332038

WFDC2 is differentially expressed in the mammary gland of the tammar wallaby and provides immune protection to the mammary gland and the developing pouch young.

PubMed

Watt, Ashalyn P; Sharp, Julie A; Lefevre, Christophe; Nicholas, Kevin R

2012-03-01

WAP four disulfide core domain 2 (WFDC2) is a four disulfide core (4-DSC) protein secreted in the milk of the tammar wallaby. It is comprised of two 4-DSC domains assigned domain III at the NH2-terminal end and domain II at the COOH-terminal end. The WFDC2 gene was expressed only during pregnancy, early lactation, towards the end of lactation and involution. The WFDC2 protein showed antibacterial activity against Staphylococcus aureus, Salmonella enterica and Pseudomonas aeruginosa and this activity resided with domain II. There was no antibacterial activity detected against Enterococcus faecalis. The observed expression pattern of tammar WFDC2 and its antibacterial activity suggests a role to either reduce mastitis in the mammary gland caused by S. aureus or to protect the gut of the young at a time when it is not immune-competent. The latter effect could be achieved without disturbing the balance of commensal gut flora such as E. faecalis. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Tau regulates the localization and function of End-binding proteins 1 and 3 in developing neuronal cells.

PubMed

Sayas, Carmen Laura; Tortosa, Elena; Bollati, Flavia; Ramírez-Ríos, Sacnicte; Arnal, Isabelle; Avila, Jesús

2015-06-01

The axonal microtubule-associated protein tau is a well-known regulator of microtubule stability in neurons. However, the putative interplay between tau and End-binding proteins 1 and 3 (EB1/3), the core microtubule plus-end tracking proteins, has not been elucidated yet. Here, we show that a cross-talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E-115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down-regulation leads to a reduction of EB1/3 comet length, as observed in shRNA-stably depleted neuroblastoma cells and TAU-/- neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over-expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E-115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non-neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull-down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross-talk between a classical microtubule-associated protein and a core microtubule plus-end tracking protein may contribute to the fine-tuned regulation of microtubule dynamics and stability during neuronal differentiation. We describe here a novel function for tau as a direct regulator of End binding (EB) proteins in differentiating neuronal cells. EB1/3 cellular mobility and localization in extending neurites and axons is modulated by tau levels and localization. We provide new evidence of the interplay between classical microtubule-associated proteins (MAPs) and "core" microtubule plus-end tracking proteins (+TIPs) during neuronal development. © 2015 International Society for Neurochemistry.

Blockade of PD-1/B7-H1 Interaction Restores Effector CD8+ T Cell Responses in a Hepatitis C Virus Core Murine Model1

PubMed Central

Lukens, John R.; Cruise, Michael W.; Lassen, Matthew G.; Hahn, Young S.

2010-01-01

The impaired function of CD8+ T cells is characteristic of hepatitis C virus (HCV) persistent infection. HCV core protein has been reported to inhibit CD8+ T cell responses. To determine the mechanism of the HCV core in suppressing Ag-specific CD8+ T cell responses, we generated a transgenic mouse, core(+) mice, where the expression of core protein is directed to the liver using the albumin promoter. Using a recombinant adenovirus to deliver Ag, we demonstrated that core(+) mice failed to clear adenovirus-LacZ (Ad-LacZ) infection in the liver. The effector function of LacZ-specific CD8+ T cells was particularly impaired in the livers of core(+) mice, with suppression of IFN-γ, TNF-α, and granzyme B production by CD8+ T cells. In addition, the impaired CD8+ T cell responses in core(+) mice were accompanied by the enhanced expression of the inhibitory receptor programmed death-1 (PD-1) by LacZ-specific CD8+ T cells and its ligand B7-H1 on liver dendritic cells following Ad-LacZ infection. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway (using a B7-H1 blocking antibody) in core(+) mice enhanced effector function of CD8+ T cells and cleared Ad-LacZ-infection as compared with that in mice treated with control Ab. This suggests that the regulation of the PD-1/B7-H1 inhibitory pathway is crucial for HCV core-mediated impaired T cell responses and viral persistence in the liver. This also suggests that manipulation of the PD-1/B7-H1 pathway may be a potential immunotherapy to enhance effector T cell responses during persistent HCV infection. PMID:18354211

Changes in proliferating and apoptotic markers of leiomyoma following treatment with a selective progesterone receptor modulator or gonadotropin-releasing hormone agonist.

PubMed

Yun, Bo Seong; Seong, Seok Ju; Cha, Dong Hyun; Kim, Ji Yeon; Kim, Mi-La; Shim, Jeong Yun; Park, Ji Eun

2015-08-01

To evaluate changes in proliferating and apoptotic markers of myoma tissue from patients treated with a selective progesterone receptor modulator (SPRM) or GnRH agonist by measuring expression of PDGF-A mRNA, IGF-1 mRNA, bcl-2 mRNA, and PCNA and caspase-3 protein. Between December 2013 and July 2014, women with symptomatic leiomyoma were divided into control (no treatment before surgery), SPRM (treatment with ulipristal acetate [SPRM] for 3 months before surgery), and GnRHa (treatment with leuprolide acetate [GnRH agonist] for 3 months before surgery) groups. Tissue specimens were collected from the myoma core and normal myometrium of all patients. The expression of mRNA and protein was assessed by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blot. A total of 38 patients were enrolled (control group, n=14; SPRM group, n=13; GnRHa group, n=11). PDGF-A mRNA expression was lower in both the myoma core and normal myometrium tissues of the SPRM compared with the control group, but there was no difference between the control and GnRHa group. There were also no group differences in bcl-2 mRNA or IGF-1 mRNA expression. Both PCNA and caspase-3 protein expression were higher in the leiomyoma tissue of the SPRM compared with the control group, but there was no difference between the control and GnRHa groups in the expression of either protein. Both proliferation and apoptosis were increased in the leiomyoma of patients after SPRM treatment, but there was no change following GnRH agonist treatment, in vivo. However, PDGF-A mRNA was decreased after SPRM treatment, indicating a dual effect of progesterone on the regulation of growth factors. Furthermore, there was an increase in caspase-3 protein, but not bcl-2 mRNA, expression in the SPRM group suggesting that SPRM may exert its effects in pathways other than the bcl-2 apoptotic pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

In Situ FTIR Microspectroscopy of Brain Tissue from a Transgenic Mouse Model of Alzheimer Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rak,M.; Del Bigio, M.; Mai, S.

2007-01-01

Plaques composed of the A{beta} peptide are the main pathological feature of Alzheimer's disease. Dense-core plaques are fibrillar deposits of A{beta}, showing all the classical properties of amyloid including {beta}-sheet secondary structure, while diffuse plaques are amorphous deposits. We studied both plaque types, using synchrotron infrared (IR) microspectroscopy, a technique that allows the chemical composition and average protein secondary structure to be investigated in situ. We examined plaques in hippocampal, cortical and caudal tissue from 5- to 21-month-old TgCRND8 mice, a transgenic model expressing doubly mutant amyloid precursor protein, and displaying impaired hippocampal function and robust pathology from an earlymore » age. Spectral analysis confirmed that the congophilic plaque cores were composed of protein in a {beta}-sheet conformation. The amide I maximum of plaque cores was at 1623 cm-1, and unlike for in vitro A{beta} fibrils, the high-frequency (1680-1690 cm-1) component attributed to antiparallel {beta}-sheet was not observed. A significant elevation in phospholipids was found around dense-core plaques in TgCRND8 mice ranging in age from 5 to 21 months. In contrast, diffuse plaques were not associated with IR detectable changes in protein secondary structure or relative concentrations of any other tissue components.« less

Integrative Identification of Arabidopsis Mitochondrial Proteome and Its Function Exploitation through Protein Interaction Network

PubMed Central

Cui, Jian; Liu, Jinghua; Li, Yuhua; Shi, Tieliu

2011-01-01

Mitochondria are major players on the production of energy, and host several key reactions involved in basic metabolism and biosynthesis of essential molecules. Currently, the majority of nucleus-encoded mitochondrial proteins are unknown even for model plant Arabidopsis. We reported a computational framework for predicting Arabidopsis mitochondrial proteins based on a probabilistic model, called Naive Bayesian Network, which integrates disparate genomic data generated from eight bioinformatics tools, multiple orthologous mappings, protein domain properties and co-expression patterns using 1,027 microarray profiles. Through this approach, we predicted 2,311 candidate mitochondrial proteins with 84.67% accuracy and 2.53% FPR performances. Together with those experimental confirmed proteins, 2,585 mitochondria proteins (named CoreMitoP) were identified, we explored those proteins with unknown functions based on protein-protein interaction network (PIN) and annotated novel functions for 26.65% CoreMitoP proteins. Moreover, we found newly predicted mitochondrial proteins embedded in particular subnetworks of the PIN, mainly functioning in response to diverse environmental stresses, like salt, draught, cold, and wound etc. Candidate mitochondrial proteins involved in those physiological acitivites provide useful targets for further investigation. Assigned functions also provide comprehensive information for Arabidopsis mitochondrial proteome. PMID:21297957

A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

PubMed

Rodgers, K K; Villey, I J; Ptaszek, L; Corbett, E; Schatz, D G; Coleman, J E

1999-07-15

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

THE LIVER OF WOODCHUCKS CHRONICALLY INFECTED WITH THE WOODCHUCK HEPATITIS VIRUS CONTAINS FOCI OF VIRUS CORE ANTIGEN NEGATIVE HEPATOCYTES WITH BOTH ALTERED AND NORMAL MORPHOLOGY

PubMed Central

Xu, Chunxiao; Yamamoto, Toshiki; Zhou, Tianlun; Aldrich, Carol E.; Frank, Katy; Cullen, John M.; Jilbert, Allison R.; Mason, William S.

2007-01-01

The livers of woodchucks chronically infected with woodchuck hepatitis virus (WHV) contain foci of morphologically altered hepatocytes (FAH) with “basophilic”, “amphophilic” and “clear cell” phenotypes, which are possibly pre-neoplastic in nature. Interestingly, most fail to express detectable levels of WHV proteins and nucleic acids. We studied sections of WHV-infected liver tissue to determine if all foci of hepatocytes that failed to express detectable levels of WHV, as assessed by immunoperoxidase staining for WHV core antigen, could be classified morphologically as FAH. We found that at least half of the foci of WHV core antigen negative hepatocytes did not show clear morphological differences in either H&E or PAS (periodic acid Schiff) stained sections from surrounding hepatocytes, and were therefore not designated as FAH. In the second approach, we assayed core antigen negative foci for the presence of fetuin B, a serum protein produced by normal hepatocytes, but not by neoplastic hepatocytes in hepatocellular carcinomas. Basophilic and amphophilic FAH had reduced levels of fetuin B compared to hepatocytes present in the surrounding liver; fetuin B staining was detected in clear cell FAH but the level could not be accurately assessed because of the displacement of fetuin B to the cell periphery by accumulated glycogen. The foci of morphologically normal WHV core antigen negative hepatocytes had similar levels of fetuin B to that of the surrounding hepatocytes. The co-existence of at least four types of WHV core antigen negative foci, including those with no obvious morphologic changes, raises the possibility that the different foci arise from distinct primary events. We hypothesize that a common event is loss of the ability to express WHV, allowing these hepatocytes to escape immune mediated cell death and to undergo clonal expansion to form distinct foci. PMID:17078989

Purification and characterization of human pancreatic polypeptide expressed in E. coli.

PubMed

Griko, Y V; Kapanadze, M D

1995-08-04

The region of cDNA encoding human pancreatic polypeptide (hPP) was obtained by polymerase chain reaction (PCR) and subcloned into an expression vector. The pancreatic polypeptide gene was expressed in Escherichia coli in two versions: as a cleavable fusion protein with IgG-binding synthetic ZZ domains of protein A from Staphylococcus aureus or with the 1-48 fragment of lambda Cro repressor. Site-specific hydrolysis by hydroxylamine was used to cleave the fusion protein, releasing the human polypeptide. The structure of the obtained hPP has been studied by scanning microcalorimetry and circular dichroism spectrometry. It has been shown that hPP in solutions close to neutral has a compact and unique spatial structure with an extended hydrophobic core. This structure is stable at 20 degrees C and co-operatively breaks down upon heating from this temperature.

Cell type-specific translational repression of Cyclin B during meiosis in males.

PubMed

Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T

2015-10-01

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I. © 2015. Published by The Company of Biologists Ltd.

Interaction of Hepatitis C Virus Core Protein with Janus Kinase Is Required for Efficient Production of Infectious Viruses

PubMed Central

Lee, Choongho

2013-01-01

Chronic hepatitis C virus (HCV) infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. HCV core protein plays not only a structural role in the virion morphogenesis by encapsidating a virus RNA genome but also a non-structural role in HCV-induced pathogenesis by blocking innate immunity. Especially, it has been shown to regulate JAK-STAT signaling pathway through its direct interaction with Janus kinase (JAK) via its proline-rich JAK-binding motif (79PGYPWP84). However, little is known about the physiological significance of this HCV core-JAK association in the context of the virus life cycle. In order to gain an insight, a mutant HCV genome (J6/JFH1-79A82A) was constructed to express the mutant core with a defective JAK-binding motif (79AGYAWP84) using an HCV genotype 2a infectious clone (J6/JFH1). When this mutant HCV genome was introduced into hepatocarcinoma cells, it was found to be severely impaired in its ability to produce infectious viruses in spite of its robust RNA genome replication. Taken together, all these results suggest an essential requirement of HCV core-JAK protein interaction for efficient production of infectious viruses and the potential of using core-JAK blockers as a new anti-HCV therapy. PMID:24009866

Adenovirus Core Protein VII Protects the Viral Genome from a DNA Damage Response at Early Times after Infection▿

PubMed Central

Karen, Kasey A.; Hearing, Patrick

2011-01-01

Adenovirus has a linear, double-stranded DNA genome that is perceived by the cellular Mre11-Rad50-Nbs1 (MRN) DNA repair complex as a double-strand break. If unabated, MRN elicits a double-strand break repair response that blocks viral DNA replication and ligates the viral genomes into concatemers. There are two sets of early viral proteins that inhibit the MRN complex. The E1B-55K/E4-ORF6 complex recruits an E3 ubiquitin ligase and targets MRN proteins for proteasome-dependent degradation. The E4-ORF3 protein inhibits MRN through sequestration. The mechanism that prevents MRN recognition of the viral genome prior to the expression of these early proteins was previously unknown. Here we show a temporal correlation between the loss of viral core protein VII from the adenovirus genome and a gain of checkpoint signaling due to the double-strand break repair response. While checkpoint signaling corresponds to the recognition of the viral genome, core protein VII binding to and checkpoint signaling at viral genomes are largely mutually exclusive. Transcription is known to release protein VII from the genome, and the inhibition of transcription shows a decrease in checkpoint signaling. Finally, we show that the nuclease activity of Mre11 is dispensable for the inhibition of viral DNA replication during a DNA damage response. These results support a model involving the protection of the incoming viral genome from checkpoint signaling by core protein VII and suggest that the induction of an MRN-dependent DNA damage response may inhibit adenovirus replication by physically masking the origins of DNA replication rather than altering their integrity. PMID:21345950

Circadian Timing in the Lung; A Specific Role for Bronchiolar Epithelial Cells

PubMed Central

Gibbs, J. E.; Beesley, S.; Plumb, J.; Singh, D.; Farrow, S.; Ray, D. W.; Loudon, A. S. I.

2015-01-01

In addition to the core circadian oscillator, located within the suprachiasmatic nucleus, numerous peripheral tissues possess self-sustaining circadian timers. In vivo these are entrained and temporally synchronized by signals conveyed from the core oscillator. In the present study, we examine circadian timing in the lung, determine the cellular localization of core clock proteins in both mouse and human lung tissue, and establish the effects of glucocorticoids (widely used in the treatment of asthma) on the pulmonary clock. Using organotypic lung slices prepared from transgenic mPER2::Luc mice, luciferase levels, which report PER2 expression, were measured over a number of days. We demonstrate a robust circadian rhythm in the mouse lung that is responsive to glucocorticoids. Immunohistochemical techniques were used to localize specific expression of core clock proteins, and the glucocorticoid receptor, to the epithelial cells lining the bronchioles in both mouse and human lung. In the mouse, these were established to be Clara cells. Murine Clara cells retained circadian rhythmicity when grown as a pure population in culture. Furthermore, selective ablation of Clara cells resulted in the loss of circadian rhythm in lung slices, demonstrating the importance of this cell type in maintaining overall pulmonary circadian rhythmicity. In summary, we demonstrate that Clara cells are critical for maintaining coherent circadian oscillations in lung tissue. Their coexpression of the glucocorticoid receptor and core clock components establishes them as a likely interface between humoral suprachiasmatic nucleus output and circadian lung physiology. PMID:18787022

Repacking the Core of T4 lysozyme by automated design.

PubMed

Mooers, Blaine H M; Datta, Deepshikha; Baase, Walter A; Zollars, Eric S; Mayo, Stephen L; Matthews, Brian W

2003-09-19

Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type. Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.

Purification, crystallization and preliminary X-ray diffraction study of human ribosomal protein L10 core domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimura, Mitsuhiro; Protein Research Group, RIKEN Yokohama Institute, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045; Kaminishi, Tatsuya

2007-11-01

A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution. Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to spacemore » group P3{sub 1}21 or P3{sub 2}21.« less

JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

PubMed Central

Licht, Alexander H.; Pein, Oliver T.; Florin, Lore; Hartenstein, Bettina; Reuter, Hendrik; Arnold, Bernd; Lichter, Peter; Angel, Peter; Schorpp-Kistner, Marina

2006-01-01

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis. PMID:17158955

Rab33B Controls Hepatitis B Virus Assembly by Regulating Core Membrane Association and Nucleocapsid Processing.

PubMed

Bartusch, Christina; Döring, Tatjana; Prange, Reinhild

2017-06-21

Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites.

Memory T-Cell-Mediated Immune Responses Specific to an Alternative Core Protein in Hepatitis C Virus Infection

PubMed Central

Bain, Christine; Parroche, Peggy; Lavergne, Jean Pierre; Duverger, Blandine; Vieux, Claude; Dubois, Valérie; Komurian-Pradel, Florence; Trépo, Christian; Gebuhrer, Lucette; Paranhos-Baccala, Glaucia; Penin, François; Inchauspé, Geneviève

2004-01-01

In vitro studies have described the synthesis of an alternative reading frame form of the hepatitis C virus (HCV) core protein that was named F protein or ARFP (alternative reading frame protein) and includes a domain coded by the +1 open reading frame of the RNA core coding region. The expression of this protein in HCV-infected patients remains controversial. We have analyzed peripheral blood from 47 chronically or previously HCV-infected patients for the presence of T lymphocytes and antibodies specific to the ARFP. Anti-ARFP antibodies were detected in 41.6% of the patients infected with various HCV genotypes. Using a specific ARFP 99-amino-acid polypeptide as well as four ARFP predicted class I-restricted 9-mer peptides, we show that 20% of the patients display specific lymphocytes capable of producing gamma interferon, interleukin-10, or both cytokines. Patients harboring three different viral genotypes (1a, 1b, and 3) carried T lymphocytes reactive to genotype 1b-derived peptides. In longitudinal analysis of patients receiving therapy, both core and ARFP-specific T-cell- and B-cell-mediated responses were documented. The magnitude and kinetics of the HCV antigen-specific responses differed and were not linked with viremia or therapy outcome. These observations provide strong and new arguments in favor of the synthesis, during natural HCV infection, of an ARFP derived from the core sequence. Moreover, the present data provide the first demonstration of the presence of T-cell-mediated immune responses directed to this novel HCV antigen. PMID:15367612

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

PubMed

Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E; Gertman, Or; Laurino, Paola; Ashani, Yacov; Dym, Orly; Unger, Tamar; Albeck, Shira; Prilusky, Jaime; Lieberman, Raquel L; Aharoni, Amir; Silman, Israel; Sussman, Joel L; Tawfik, Dan S; Fleishman, Sarel J

2016-07-21

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

H-NS-like nucleoid-associated proteins, mobile genetic elements and horizontal gene transfer in bacteria.

PubMed

Dorman, Charles J

2014-09-01

Horizontal gene transfer plays an important role in the evolution of bacterial species, conferring new genetic traits on the recipient bacterium that extend its range of phenotypes and plasmids make important contributions to this process. However, the inappropriate expression of newly acquired genes may lead to a loss of competitive fitness, resulting in the elimination of the new gene-bacterium combination. It is thought that transcriptional silencing of horizontally acquired genes offers a route out of this dilemma and that nucleoid-associated proteins, especially those related to the H-NS protein, play a particularly important role in the silencing process. The discovery that many plasmids express orthologues of nucleoid-associated proteins adds an interesting dimension to current models of regulatory integration following lateral transfer of DNA. Other horizontally acquired genetic elements, such as genomic islands, also express nucleoid-associated proteins of their own. Here the interactions of H-NS-like nucleoid-associated proteins encoded by the core genome, genomic islands and plasmids are described. Copyright © 2014 Elsevier Inc. All rights reserved.

A comprehensive glycome profiling of Huntington's disease transgenic mice.

PubMed

Gizaw, Solomon T; Koda, Toshiaki; Amano, Maho; Kamimura, Keiko; Ohashi, Tetsu; Hinou, Hiroshi; Nishimura, Shin-Ichiro

2015-09-01

Huntington's disease (HD) is an autosomal, dominantly inherited and progressive neurodegenerative disease, nosologically classified as the presence of intranuclear inclusion bodies and the loss of GABA-containing neurons in the neostriatum and subsequently in the cerebellar cortex. Abnormal processing of neuronal proteins can result in the misfolding of proteins and altered post-translational modification of newly synthesized proteins. Total glycomics, namely, N-glycomics, O-glycomics, and glycosphingolipidomics (GSL-omics) of HD transgenic mice would be a hallmark for central nervous system disorders in order to discover disease specific biomarkers. Glycoblotting method, a high throughput glycomic protocol, and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) were used to study the total glycome expression levels in the brain tissue (3 mice of each sex) and sera (5 mice of each sex) of HD transgenic and control mice. All experiments were performed twice and differences in the expression levels of major glycoforms were compared between HD transgenic and control mice. We estimated the structure and expression levels of 87 and 58N-glycans in brain tissue and sera, respectively, of HD transgenic and control mice. The present results clearly indicated that the brain glycome and their expression levels are significantly gender specific when compared with those of other tissues and serum. Core-fucosylated and bisecting-GlcNAc types of N-glycans were found in increased levels in the brain tissue HD transgenic mice. Accordingly, core-fucosylated and sialic acid (particularly N-glycolylneuraminic acid, NeuGc) for biantennary type glycans were found in increased amounts in the sera of HD transgenic mice compared to that of control mice. Core 3 type O-glycans were found in increased levels in male and in decreased levels in both the striatum and cortexes of female HD transgenic mice. Furthermore, serum levels of core 1 type O-glycans decreased and were undetected for core 2 type O-glycans for HD transgenic mice. In glycosphingolipids, GD1a in brain tissue and GM2-NeuGc serum levels were found to have increased and decreased, respectively, in HD transgenic mice compared to those of the control group mice. Total glycome expression levels are significantly different between HD transgenic and control group mice. Glycoblotting combined with MALDI-TOF/MS total glycomics warrants a comprehensive, effective, novel and versatile technique for qualitative and quantitative analysis of total glycome expression levels. Furthermore, glycome-focused studies of both environmentally and genetically rooted neurodegenerative diseases are promising candidates for the discovery of potential disease glyco-biomarkers in the post-genome era. Copyright © 2015 Elsevier B.V. All rights reserved.

[Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells].

PubMed

Masalova, O V; Lesnova, E I; Samokhvalov, E I; Permyakova, K Yu; Ivanov, A V; Kochetkov, S N; Kushch, A A

2017-01-01

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1β, TNF-α, and TGF-β) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-β in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1β in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.

p53 targets chromatin structure alteration to repress alpha-fetoprotein gene expression.

PubMed

Ogden, S K; Lee, K C; Wernke-Dollries, K; Stratton, S A; Aronow, B; Barton, M C

2001-11-09

Many of the functions ascribed to p53 tumor suppressor protein are mediated through transcription regulation. We have shown that p53 represses hepatic-specific alpha-fetoprotein (AFP) gene expression by direct interaction with a composite HNF-3/p53 DNA binding element. Using solid-phase, chromatin-assembled AFP DNA templates and analysis of chromatin structure and transcription in vitro, we find that p53 binds DNA and alters chromatin structure at the AFP core promoter to regulate transcription. Chromatin assembled in the presence of hepatoma extracts is activated for AFP transcription with an open, accessible core promoter structure. Distal (-850) binding of p53 during chromatin assembly, but not post-assembly, reverses transcription activation concomitant with promoter inaccessibility to restriction enzyme digestion. Inhibition of histone deacetylase activity by trichostatin-A (TSA) addition, prior to and during chromatin assembly, activated chromatin transcription in parallel with increased core promoter accessibility. Chromatin immunoprecipitation analyses showed increased H3 and H4 acetylated histones at the core promoter in the presence of TSA, while histone acetylation remained unchanged at the site of distal p53 binding. Our data reveal that p53 targets chromatin structure alteration at the core promoter, independently of effects on histone acetylation, to establish repressed AFP gene expression.

PhenomeExpress: a refined network analysis of expression datasets by inclusion of known disease phenotypes.

PubMed

Soul, Jamie; Hardingham, Timothy E; Boot-Handford, Raymond P; Schwartz, Jean-Marc

2015-01-29

We describe a new method, PhenomeExpress, for the analysis of transcriptomic datasets to identify pathogenic disease mechanisms. Our analysis method includes input from both protein-protein interaction and phenotype similarity networks. This introduces valuable information from disease relevant phenotypes, which aids the identification of sub-networks that are significantly enriched in differentially expressed genes and are related to the disease relevant phenotypes. This contrasts with many active sub-network detection methods, which rely solely on protein-protein interaction networks derived from compounded data of many unrelated biological conditions and which are therefore not specific to the context of the experiment. PhenomeExpress thus exploits readily available animal model and human disease phenotype information. It combines this prior evidence of disease phenotypes with the experimentally derived disease data sets to provide a more targeted analysis. Two case studies, in subchondral bone in osteoarthritis and in Pax5 in acute lymphoblastic leukaemia, demonstrate that PhenomeExpress identifies core disease pathways in both mouse and human disease expression datasets derived from different technologies. We also validate the approach by comparison to state-of-the-art active sub-network detection methods, which reveals how it may enhance the detection of molecular phenotypes and provide a more detailed context to those previously identified as possible candidates.

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway. PMID:21595909

Synthetic Core Promoters as Universal Parts for Fine-Tuning Expression in Different Yeast Species

PubMed Central

2016-01-01

Synthetic biology and metabolic engineering experiments frequently require the fine-tuning of gene expression to balance and optimize protein levels of regulators or metabolic enzymes. A key concept of synthetic biology is the development of modular parts that can be used in different contexts. Here, we have applied a computational multifactor design approach to generate de novo synthetic core promoters and 5′ untranslated regions (UTRs) for yeast cells. In contrast to upstream cis-regulatory modules (CRMs), core promoters are typically not subject to specific regulation, making them ideal engineering targets for gene expression fine-tuning. 112 synthetic core promoter sequences were designed on the basis of the sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs. The synthetic core promoters were fused to the Pichia pastoris AOX1 CRM, and the resulting activity spanned more than a 200-fold range (0.3% to 70.6% of the wild type AOX1 level). The top-ten synthetic core promoters with highest activity were fused to six additional CRMs (three in P. pastoris and three in Saccharomyces cerevisiae). Inducible CRM constructs showed significantly higher activity than constitutive CRMs, reaching up to 176% of natural core promoters. Comparing the activity of the same synthetic core promoters fused to different CRMs revealed high correlations only for CRMs within the same organism. These data suggest that modularity is maintained to some extent but only within the same organism. Due to the conserved role of eukaryotic core promoters, this rational design concept may be transferred to other organisms as a generic engineering tool. PMID:27973777

Localized surface plasmon mediated energy transfer in the vicinity of core-shell nanoparticle

NASA Astrophysics Data System (ADS)

Shishodia, Manmohan Singh; Juneja, Soniya

2016-05-01

Multipole spectral expansion based theory of energy transfer interactions between a donor and an acceptor molecule in the vicinity of a core-shell (nanoshell or core@shell) based plasmonic nanostructure is developed. In view of the diverse applications and rich plasmonic features such as tuning capability of surface plasmon (SP) frequencies, greater sensitivity to the change of dielectric environment, controllable redirection of electromagnetic radiation, closed form expressions for Energy Transfer Rate Enhancement Factor (ETREF) near core-shell particle are reported. The dependence of ETREF on different parameters is established through fitting equations, perceived to be of key importance for developing appropriate designs. The theoretical approach developed in the present work is capable of treating higher order multipoles, which, in turn, are also shown to play a crucial role in the present context. Moreover, closed form expressions derived in the present work can directly be used as formula, e.g., for designing SP based biosensors and estimating energy exchange between proteins and excitonic interactions in quantum dots.

Rate heterogeneity in six protein-coding genes from the holoparasite Balanophora (Balanophoraceae) and other taxa of Santalales

PubMed Central

Su, Huei-Jiun; Hu, Jer-Ming

2012-01-01

Background and Aims The holoparasitic flowering plant Balanophora displays extreme floral reduction and was previously found to have enormous rate acceleration in the nuclear 18S rDNA region. So far, it remains unclear whether non-ribosomal, protein-coding genes of Balanophora also evolve in an accelerated fashion and whether the genes with high substitution rates retain their functionality. To tackle these issues, six different genes were sequenced from two Balanophora species and their rate variation and expression patterns were examined. Methods Sequences including nuclear PI, euAP3, TM6, LFY and RPB2 and mitochondrial matR were determined from two Balanophora spp. and compared with selected hemiparasitic species of Santalales and autotrophic core eudicots. Gene expression was detected for the six protein-coding genes and the expression patterns of the three B-class genes (PI, AP3 and TM6) were further examined across different organs of B. laxiflora using RT-PCR. Key Results Balanophora mitochondrial matR is highly accelerated in both nonsynonymous (dN) and synonymous (dS) substitution rates, whereas the rate variation of nuclear genes LFY, PI, euAP3, TM6 and RPB2 are less dramatic. Significant dS increases were detected in Balanophora PI, TM6, RPB2 and dN accelerations in euAP3. All of the protein-coding genes are expressed in inflorescences, indicative of their functionality. PI is restrictively expressed in tepals, synandria and floral bracts, whereas AP3 and TM6 are widely expressed in both male and female inflorescences. Conclusions Despite the observation that rates of sequence evolution are generally higher in Balanophora than in hemiparasitic species of Santalales and autotrophic core eudicots, the five nuclear protein-coding genes are functional and are evolving at a much slower rate than 18S rDNA. The mechanism or mechanisms responsible for rapid sequence evolution and concomitant rate acceleration for 18S rDNA and matR are currently not well understood and require further study in Balanophora and other holoparasites. PMID:23041381

Expression, purification and crystallization of pecan (Carya illinoinensis) vicilin.

PubMed

Lee, BoRam; Zhang, Renhao; Du, Wen-Xian; Grauke, Larry J; McHugh, Tara H; Zhang, Yu-Zhu

2014-08-01

Tree nuts are responsible for many cases of severe food allergies. The 7S seed storage protein vicilin has been identified as a food allergen in many kinds of tree nuts. The vicilin protein consists of an N-terminal low-complexity region with antimicrobial activity and a C-terminal domain that forms a trimeric structure that belongs to the cupin superfamily. In this study, vicilin from pecan (Carya illinoinensis) was isolated and was expressed in bacteria for the first time. The cupin structural core of the protein, residues 369-792, was purified by metal-affinity and gel-filtration chromatography to high purity. Vicilin crystals were obtained and the best crystal diffracted to 2.65 Å resolution in space group P212121.

Expression, purification and crystallization of pecan (Carya illinoinensis) vicilin

PubMed Central

Lee, BoRam; Zhang, Renhao; Du, Wen-Xian; Grauke, Larry J.; McHugh, Tara H.; Zhang, Yu-Zhu

2014-01-01

Tree nuts are responsible for many cases of severe food allergies. The 7S seed storage protein vicilin has been identified as a food allergen in many kinds of tree nuts. The vicilin protein consists of an N-terminal low-complexity region with antimicrobial activity and a C-terminal domain that forms a trimeric structure that belongs to the cupin superfamily. In this study, vicilin from pecan (Carya illinoinensis) was isolated and was expressed in bacteria for the first time. The cupin structural core of the protein, residues 369–792, was purified by metal-affinity and gel-filtration chromatography to high purity. Vicilin crystals were obtained and the best crystal diffracted to 2.65 Å resolution in space group P212121. PMID:25084379

Chromatin Insulators: A Role in Nuclear Organization and Gene Expression

PubMed Central

Yang, Jingping; Corces, Victor G.

2011-01-01

Chromatin insulators are DNA-protein complexes with broad functions in nuclear biology. Based on the ability of insulator proteins to interact with each other, it was originally thought that insulators form loops that could constitute functional domains of co-regulated gene expression. Nevertheless, data from genome-wide localization studies indicate that insulator proteins can be present in intergenic regions as well as at the 5′, introns or 3′ of genes, suggesting a broader role in chromosome biology. Cells have developed mechanisms to control insulator activity by recruiting specialized proteins or by covalent modification of core components. Recent results suggest that insulators mediate intra- and inter-chromosomal interactions to affect transcription, imprinting and recombination. It is possible that these interactions set up cell-specific blueprints of nuclear organization that may contribute to the establishment of different patterns of gene expression during cell differentiation. As a consequence, disruption of insulator activity could result in the development of cancer or other disease states. PMID:21704228

In situ expression of eukaryotic ice-binding proteins in microbial communities of Arctic and Antarctic sea ice.

PubMed

Uhlig, Christiane; Kilpert, Fabian; Frickenhaus, Stephan; Kegel, Jessica U; Krell, Andreas; Mock, Thomas; Valentin, Klaus; Beszteri, Bánk

2015-11-01

Ice-binding proteins (IBPs) have been isolated from various sea-ice organisms. Their characterisation points to a crucial role in protecting the organisms in sub-zero environments. However, their in situ abundance and diversity in natural sea-ice microbial communities is largely unknown. In this study, we analysed the expression and phylogenetic diversity of eukaryotic IBP transcripts from microbial communities of Arctic and Antarctic sea ice. IBP transcripts were found in abundances similar to those of proteins involved in core cellular processes such as photosynthesis. Eighty-nine percent of the IBP transcripts grouped with known IBP sequences from diatoms, haptophytes and crustaceans, but the majority represented novel sequences not previously characterized in cultured organisms. The observed high eukaryotic IBP expression in natural eukaryotic sea ice communities underlines the essential role of IBPs for survival of many microorganisms in communities living under the extreme conditions of polar sea ice.

Spatial gradients of protein-level time delays set the pace of the traveling segmentation clock waves

PubMed Central

Ay, Ahmet; Holland, Jack; Sperlea, Adriana; Devakanmalai, Gnanapackiam Sheela; Knierer, Stephan; Sangervasi, Sebastian; Stevenson, Angel; Özbudak, Ertuğrul M.

2014-01-01

The vertebrate segmentation clock is a gene expression oscillator controlling rhythmic segmentation of the vertebral column during embryonic development. The period of oscillations becomes longer as cells are displaced along the posterior to anterior axis, which results in traveling waves of clock gene expression sweeping in the unsegmented tissue. Although various hypotheses necessitating the inclusion of additional regulatory genes into the core clock network at different spatial locations have been proposed, the mechanism underlying traveling waves has remained elusive. Here, we combined molecular-level computational modeling and quantitative experimentation to solve this puzzle. Our model predicts the existence of an increasing gradient of gene expression time delays along the posterior to anterior direction to recapitulate spatiotemporal profiles of the traveling segmentation clock waves in different genetic backgrounds in zebrafish. We validated this prediction by measuring an increased time delay of oscillatory Her1 protein production along the unsegmented tissue. Our results refuted the need for spatial expansion of the core feedback loop to explain the occurrence of traveling waves. Spatial regulation of gene expression time delays is a novel way of creating dynamic patterns; this is the first report demonstrating such a control mechanism in any tissue and future investigations will explore the presence of analogous examples in other biological systems. PMID:25336742

Regulation of the grapevine polygalacturonase-inhibiting protein encoding gene: expression pattern, induction profile and promoter analysis.

PubMed

Joubert, D Albert; de Lorenzo, Giulia; Vivier, Melané A

2013-03-01

Regulation of defense in plants is a complex process mediated by various signaling pathways. Promoter analysis of defense-related genes is useful to understand these signaling pathways involved in regulation. To this end, the regulation of the polygalacturonase-inhibiting protein encoding gene from Vitis vinifera L. (Vvpgip1) was analyzed with regard to expression pattern and induction profile as well as the promoter in terms of putative regulatory elements present, core promoter size and the start of transcription. Expression of Vvpgip1 is tissue-specific and developmentally regulated. Vvpgip1 expression was induced in response to auxin, salicylic acid and sugar treatment, wounding and pathogen infection. The start of transcription was mapped to 17 bp upstream of the ATG and the core promoter was mapped to the 137 bp upstream of the ATG. Fructose- and Botrytis responsiveness were identified in the region between positions -3.1 and -1.5 kb. The analyses showed induction in water when the leaves were submersed and this response and the response to wounding mapped to the region between positions -1.1 and -0.1 kb. In silico analyses revealed putative cis-acting elements in these areas that correspond well to the induction stimuli tested.

Age-Related Changes in the Expression of the Circadian Clock Protein PERIOD in Drosophila Glial Cells

PubMed Central

Long, Dani M.; Giebultowicz, Jadwiga M.

2018-01-01

Circadian clocks consist of molecular negative feedback loops that coordinate physiological, neurological, and behavioral variables into “circa” 24-h rhythms. Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Here, we conducted immunocytochemistry experiments to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. Our data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain. PMID:29375400

Plasma Hsp72 (HSPA1A) and Hsp27 (HSPB1) expression under heat stress: influence of exercise intensity.

PubMed

Périard, Julien D; Ruell, Patricia; Caillaud, Corinne; Thompson, Martin W

2012-05-01

Extracellular heat-shock protein 72 (eHsp72) expression during exercise-heat stress is suggested to increase with the level of hyperthermia attained, independent of the rate of heat storage. This study examined the influence of exercise at various intensities to elucidate this relationship, and investigated the association between eHsp72 and eHsp27. Sixteen male subjects cycled to exhaustion at 60% and 75% of maximal oxygen uptake in hot conditions (40°C, 50% RH). Core temperature, heart rate, oxidative stress, and blood lactate and glucose levels were measured to determine the predictor variables associated with eHsp expression. At exhaustion, heart rate exceeded 96% of maximum in both conditions. Core temperature reached 39.7°C in the 60% trial (58.9 min) and 39.0°C in the 75% trial (27.2 min) (P < 0.001). The rate of rise in core temperature was 2.1°C h(-1) greater in the 75% trial than in the 60% trial (P < 0.001). A significant increase and correlation was observed between eHsp72 and eHsp27 concentrations at exhaustion (P < 0.005). eHsp72 was highly correlated with the core temperature attained (60% trial) and the rate of increase in core temperature (75% trial; P < 0.05). However, no common predictor variable was associated with the expression of both eHsps. The similarity in expression of eHsp72 and eHsp27 during moderate- and high-intensity exercise may relate to the duration (i.e., core temperature attained) and intensity (i.e., rate of increase in core temperature) of exercise. Thus, the immuno-inflammatory release of eHsp72 and eHsp27 in response to exercise in the heat may be duration and intensity dependent.

Purification and characterization of a human pancreatic adenocarcinoma mucin.

PubMed

Khorrami, Ali M; Choudhury, Amit; Andrianifahanana, Mahefatiana; Varshney, Grish C; Bhattacharyya, Sambhu N; Hollingsworth, Michael A; Kaufman, Bernard; Batra, Surinder K

2002-01-01

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.

Alternate Reading Frame Protein (F Protein) of Hepatitis C Virus: Paradoxical Effects of Activation and Apoptosis on Human Dendritic Cells Lead to Stimulation of T Cells

PubMed Central

Samrat, Subodh Kumar; Li, Wen; Singh, Shakti; Kumar, Rakesh; Agrawal, Babita

2014-01-01

Hepatitis C virus (HCV) leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs) process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF) have been implicated in modulation of dendritic cells (DCs) and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F), whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a) were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans. PMID:24475147

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells.

PubMed

Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S

2005-10-14

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

Progesterone receptor expression during prostate cancer progression suggests a role of this receptor in stromal cell differentiation.

PubMed

Yu, Yue; Yang, Ou; Fazli, Ladan; Rennie, Paul S; Gleave, Martin E; Dong, Xuesen

2015-07-01

The progesterone receptor, like the androgen receptor, belongs to the steroid receptor superfamily. Our previous studies have reported that the PR is expressed specifically in prostate stroma. PR inhibits proliferation of, and regulates cytokine secretion by stromal cells. However, PR protein expression in cancer-associated stroma during prostate cancer progression has not been profiled. Since the phenotypes of prostate stromal cells change dynamically as tumors progress, whether the PR plays a role in regulating stromal cell differentiation needs to be investigated. Immunohistochemistry assays measured PR protein levels on human prostate tissue microarrays containing 367 tissue cores from benign prostate, prostate tumors with different Gleason scores, tumors under various durations of castration therapy, and tumors at the castration-resistant stage. Immunoblotting assays determined whether PR regulated the expression of alpha smooth muscle actin (α-SMA), vimentin, and fibroblast specific protein (FSP) in human prostate stromal cells. PR protein levels decreased in cancer-associated stroma when compared with that in benign prostate stroma. This reduction in PR expression was not correlated with Gleason scores. PR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors progressed to the castration-resistant stage. Enhanced PR expression in human prostate stromal cells increased α-SMA, but decreased vimentin and FSP protein levels ligand-independently. These results suggest that PR plays an active role in regulating stromal cell phenotypes during prostate cancer progression. © 2015 Wiley Periodicals, Inc.

Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

PubMed

Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

2016-06-01

Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2+ breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry

PubMed Central

Calderón-González, Karla Grisel; Valero Rustarazo, Ma Luz; Labra-Barrios, Maria Luisa; Bazán-Méndez, César Isaac; Tavera-Tapia, Alejandra; Herrera-Aguirre, Marí;aEsther; Sánchez del Pino, Manuel M.; Gallegos-Pérez, José Luis; González-Márquez, Humberto; Hernández-Hernández, Jose Manuel; León-Ávila, Gloria; Rodríguez-Cuevas, Sergio; Guisa-Hohenstein, Fernando; Luna-Arias, Juan Pedro

2015-01-01

Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article “Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry” (Calderón-González et al. [1] in press). PMID:26217805

Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly.

PubMed

Pham, Tu M; Tran, Si C; Lim, Yun-Sook; Hwang, Soon B

2017-02-01

Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were upregulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core protein and deposited core protein into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region, which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly. Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at the transcriptional level and altered the balance between GDP- and GTP-bound Rab32 toward production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core protein and enriched core in the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential for HCV particle assembly. Copyright © 2017 American Society for Microbiology.

Hepatitis C Virus Core Protein Promotes miR-122 Destabilization by Inhibiting GLD-2

PubMed Central

Kim, Geon-Woo; Lee, Seung-Hoon; Cho, Hee; Kim, Minwoo; Shin, Eui-Cheol; Oh, Jong-Won

2016-01-01

The liver-specific microRNA miR-122, which has essential roles in liver development and metabolism, is a key proviral factor for hepatitis C virus (HCV). Despite its crucial role in the liver and HCV life cycle, little is known about the molecular mechanism of miR-122 expression regulation by HCV infection. Here, we show that the HCV core protein downregulates the abundance of miR-122 by promoting its destabilization via the inhibition of GLD-2, a non-canonical cytoplasmic poly(A) polymerase. The decrease in miR-122 expression resulted in the dysregulation of the known functions of miR-122, including its proviral activity for HCV. By high-throughput sequencing of small RNAs from human liver biopsies, we found that the 22-nucleotide (nt) prototype miR-122 is modified at its 3′ end by 3′-terminal non-templated and templated nucleotide additions. Remarkably, the proportion of miR-122 isomers bearing a single nucleotide tail of any ribonucleotide decreased in liver specimens from patients with HCV. We found that these single-nucleotide-tailed miR-122 isomers display increased miRNA activity and stability over the 22-nt prototype miR-122 and that the 3′-terminal extension is catalyzed by the unique terminal nucleotidyl transferase activity of GLD-2, which is capable of adding any single ribonucleotide without preference of adenylate to the miR-122 3′ end. The HCV core protein specifically inhibited GLD-2, and its interaction with GLD-2 in the cytoplasm was found to be responsible for miR-122 downregulation. Collectively, our results provide new insights into the regulatory role of the HCV core protein in controlling viral RNA abundance and miR-122 functions through miR-122 stability modulation. PMID:27366906

Mice lacking GPR3 receptors display late-onset obese phenotype due to impaired thermogenic function in brown adipose tissue

PubMed Central

Godlewski, Grzegorz; Jourdan, Tony; Szanda, Gergő; Tam, Joseph; Resat Cinar; Harvey-White, Judith; Liu, Jie; Mukhopadhyay, Bani; Pacher, Pál; Ming Mo, Fong; Osei-Hyiaman, Douglas; George Kunos

2015-01-01

We report an unexpected link between aging, thermogenesis and weight gain via the orphan G protein-coupled receptor GPR3. Mice lacking GPR3 and maintained on normal chow had similar body weights during their first 5 months of life, but gained considerably more weight thereafter and displayed reduced total energy expenditure and lower core body temperature. By the age of 5 months GPR3 KO mice already had lower thermogenic gene expression and uncoupling protein 1 protein level and showed impaired glucose uptake into interscapular brown adipose tissue (iBAT) relative to WT littermates. These molecular deviations in iBAT of GPR3 KO mice preceded measurable differences in body weight and core body temperature at ambient conditions, but were coupled to a failure to maintain thermal homeostasis during acute cold challenge. At the same time, the same cold challenge caused a 17-fold increase in Gpr3 expression in iBAT of WT mice. Thus, GPR3 appears to have a key role in the thermogenic response of iBAT and may represent a new therapeutic target in age-related obesity. PMID:26455425

Structural characterization of core-bradavidin in complex with biotin

PubMed Central

Agrawal, Nitin; Määttä, Juha A. E.; Kulomaa, Markku S.; Hytönen, Vesa P.; Johnson, Mark S.; Airenne, Tomi T.

2017-01-01

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin. PMID:28426764

Associations between expression levels of nucleotide excision repair proteins in lymphoblastoid cells and risk of squamous cell carcinoma of the head and neck.

PubMed

Han, Peng; Liu, Hongliang; Shi, Qiong; Liu, Zhensheng; Troy, Jesse D; Lee, Walter T; Zevallos, Jose P; Li, Guojun; Sturgis, Erich M; Wei, Qingyi

2018-06-01

Squamous cell carcinoma of head and neck (SCCHN) is one of the most common malignancies worldwide, and nucleotide excision repair (NER) is involved in SCCHN susceptibility. In this analysis of 349 newly diagnosed SCCHN patients and 295 cancer-free controls, we investigated whether expression levels of eight core NER proteins were associated with risk of SCCHN. We quantified NER protein expression levels in cultured peripheral lymphocytes using a reverse-phase protein microarray. Compared with the controls, SCCHN patients had statistically significantly lower expression levels of ERCC3 and XPA (P = 0.001 and 0.001, respectively). After dividing the subjects by controls' median values of expression levels, we found a dose-dependent association between an increased risk of SCCHN and low expression levels of ERCC3 (adjusted OR, 1.75, and 95% CI: 1.26-2.42; P trend  = 0.008) and XPA (adjusted OR, 1.88; 95% CI, 1.35-2.60; P trend  = 0.001). We also identified a significant multiplicative interaction between smoking status and ERCC3 expression levels (P = 0.014). Finally, after integrating demographic and clinical variables, we found that the addition of ERCC3 and XPA expression levels to the model significantly improved the sensitivity of the expanded model on SCCHN risk. In conclusion, reduced protein expression levels of ERCC3 and XPA were associated with an increased risk of SCCHN. However, these results need to be confirmed in additional large studies. © 2018 Wiley Periodicals, Inc.

Genome Editing for Cancer Therapy: Delivery of Cas9 Protein/sgRNA Plasmid via a Gold Nanocluster/Lipid Core-Shell Nanocarrier.

PubMed

Wang, Peng; Zhang, Lingmin; Xie, Yangzhouyun; Wang, Nuoxin; Tang, Rongbing; Zheng, Wenfu; Jiang, Xingyu

2017-11-01

The type II bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (CRISPR-associated protein) system (CRISPR-Cas9) is a powerful toolbox for gene-editing, however, the nonviral delivery of CRISPR-Cas9 to cells or tissues remains a key challenge. This paper reports a strategy to deliver Cas9 protein and single guide RNA (sgRNA) plasmid by a nanocarrier with a core of gold nanoclusters (GNs) and a shell of lipids. By modifying the GNs with HIV-1-transactivator of transcription peptide, the cargo (Cas9/sgRNA) can be delivered into cell nuclei. This strategy is utilized to treat melanoma by designing sgRNA targeting Polo-like kinase-1 ( Plk1 ) of the tumor. The nanoparticle (polyethylene glycol-lipid/GNs/Cas9 protein/sgPlk1 plasmid, LGCP) leads to >70% down-regulation of Plk1 protein expression of A375 cells in vitro. Moreover, the LGCP suppresses melanoma progress by 75% on mice. Thus, this strategy can deliver protein-nucleic acid hybrid agents for gene therapy.

Twelve Transmembrane Helices Form the Functional Core of Mammalian MATE1 (Multidrug and Toxin Extruder 1) Protein*

PubMed Central

Zhang, Xiaohong; He, Xiao; Baker, Joseph; Tama, Florence; Chang, Geoffrey; Wright, Stephen H.

2012-01-01

The x-ray structure of the prototypic MATE family member, NorM from Vibrio cholerae, reveals a protein fold composed of 12 transmembrane helices (TMHs), confirming hydropathy analyses of the majority of (prokaryotic and plant) MATE transporters. However, the mammalian MATEs are generally predicted to have a 13th TMH and an extracellular C terminus. Here we affirm this prediction, showing that the C termini of epitope-tagged, full-length human, rabbit, and mouse MATE1 were accessible to antibodies from the extracellular face of the membrane. Truncation of these proteins at or near the predicted junction between the 13th TMH and the long cytoplasmic loop that precedes it resulted in proteins that (i) trafficked to the membrane and (ii) interacted with antibodies only after permeabilization of the plasma membrane. CHO cells expressing rbMate1 truncated at residue Gly-545 supported levels of pH-sensitive transport similar to that of cells expressing the full-length protein. Although the high transport rate of the Gly-545 truncation mutant was associated with higher levels of membrane expression (than full-length MATE1), suggesting the 13th TMH may influence substrate translocation, the selectivity profile of the mutant indicated that TMH13 has little impact on ligand binding. We conclude that the functional core of MATE1 consists of 12 (not 13) TMHs. Therefore, we used the x-ray structure of NorM to develop a homology model of the first 12 TMHs of MATE1. The model proved to be stable in molecular dynamic simulations and agreed with topology evident from preliminary cysteine scanning of intracellular versus extracellular loops. PMID:22722930

BAG3 is involved in neuronal differentiation and migration.

PubMed

Santoro, Antonietta; Nicolin, Vanessa; Florenzano, Fulvio; Rosati, Alessandra; Capunzo, Mario; Nori, Stefania L

2017-05-01

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6

NASA Astrophysics Data System (ADS)

Dai, Yawei; Seeger, Markus; Weng, Jingwei; Song, Song; Wang, Wenning; Tan, Yan-Wen

2016-08-01

Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view.

Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells.

PubMed

Yamamoto, Mioko; Kawashima, Nobuyuki; Takashino, Nami; Koizumi, Yu; Takimoto, Koyo; Suzuki, Noriyuki; Saito, Masahiro; Suda, Hideaki

2014-03-01

Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p<0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone. 3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling. Copyright © 2013 Elsevier Ltd. All rights reserved.

Immunization of Mice by BCG Formulated HCV Core Protein Elicited Higher Th1-Oriented Responses Compared to Pluronic-F127 Copolymer

PubMed Central

Yazdanian, Maryam; Memarnejadian, Arash; Mahdavi, Mehdi; Sadat, Seyed Mehdi; Motevali, Fatemeh; Vahabpour, Rouhollah; Khanahmad, Hossein; Siadat, Seyed Davar; Aghasadeghi, Mohammad Reza; Roohvand, Farzin

2013-01-01

Background A supreme vaccine for Hepatitis C virus (HCV) infection should elicit strong Th1-oriented cellular responses. In the absence of a Th1-specific adjuvant, immunizations by protein antigens generally induce Th2-type and weak cellular responses. Objectives To evaluate the adjuvant effect of BCG in comparison with nonionic copolymer-Pluronic F127 (F127) as a classic adjuvant in the formulation of HCV core protein (HCVcp) as a candidate vaccine for induction of Th1 immune responses. Materials and Methods Expression of N-terminally His-Tagged HCVcp (1-122) by pIVEX2.4a-core vector harboring the corresponding gene under the control of arabinose-inducible (araBAD) promoter was achieved in BL21-AI strain of E.coli and purified through application of nitrilotriacetic acid (Ni-NTA) chromatography. Mice were immunized subcutaneously (s.c.) in base of the tail with 100 μl of immunogen (F127+HCVcp or BCG+HCVcp; 5 μgHCVcp/mouse/dose) or control formulations (PBS, BCG, F127) at weeks 0, 3, 6. Total and subtypes of IgG, as well as cellular immune responses (Proliferation, In vivo CTL and IFN-γ/IL-4 ELISpot assays against a strong and dominant H2-d restricted, CD8+-epitopic peptide, core 39-48; RRGPRLGVRA of HCVcp) were compared in each group of immunized animals. Results Expression and purification of core protein around the expected size (21 kDa) was confirmed by Western blotting. The HCVcp + BCG vaccinated mice showed significantly higher lymphocyte proliferation and IFN-γ production but lower levels of cell lysis (45% versus 62% in CTL assay) than the HCVcp+F127 immunized animals. “Besides, total anti-core IgG and IgG1 levels were significantly higher in HCVcp + F127 immunized mice as compared to HCVcp + BCG vaccinated animals, indicating relatively higher efficacy of F127 for the stimulation of humoral and Th2-oriented immune responses”. Conclusions Results showed that HCVcp + BCG induced a moderate CTL and mixed Th1/Th2 immune responses with higher levels of cell proliferation and IFN-γ secretion, indicating that BCG may have a better outcome when formulated in HCVcp-based subunit vaccines. PMID:24348641

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)*

PubMed Central

Cao-Ehlker, Xiaochun; Zong, Xiangang; Hammelmann, Verena; Gruner, Christian; Fenske, Stefanie; Michalakis, Stylianos; Wahl-Schott, Christian; Biel, Martin

2013-01-01

Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1–4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes. PMID:23382386

Exposure time-dependent thermal effects of radiofrequency electromagnetic field exposure on the whole body of rats.

PubMed

Ohtani, Shin; Ushiyama, Akira; Maeda, Machiko; Hattori, Kenji; Kunugita, Naoki; Wang, Jianqing; Ishii, Kazuyuki

2016-01-01

We investigated the thermal effects of radiofrequency electromagnetic fields (RF-EMFs) on the variation in core temperature and gene expression of some stress markers in rats. Sprague-Dawley rats were exposed to 2.14 GHz wideband code division multiple access (W-CDMA) RF signals at a whole-body averaged specific absorption rate (WBA-SAR) of 4 W/kg, which causes behavioral disruption in laboratory animals, and 0.4 W/kg, which is the limit for the occupational exposure set by the International Commission on Non-Ionizing Radiation Protection guideline. It is important to understand the possible in vivo effects derived from RF-EMF exposures at these intensities. Because of inadequate data on real-time core temperature analyses using free-moving animal and the association between stress and thermal effects of RF-EMF exposure, we analyzed the core body temperature under nonanesthetic condition during RF-EMF exposure. The results revealed that the core temperature increased by approximately 1.5°C compared with the baseline and reached a plateau till the end of RF-EMF exposure. Furthermore, we analyzed the gene expression of heat-shock proteins (Hsp) and heat-shock transcription factors (Hsf) family after RF-EMF exposure. At WBA-SAR of 4 W/kg, some Hsp and Hsf gene expression levels were significantly upregulated in the cerebral cortex and cerebellum following exposure for 6 hr/day but were not upregulated after exposure for 3 hr/day. On the other hand, there was no significant change in the core temperature and gene expression at WBA-SAR of 0.4 W/kg. Thus, 2.14-GHz RF-EMF exposure at WBA-SAR of 4 W/kg induced increases in the core temperature and upregulation of some stress markers, particularly in the cerebellum.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis.

PubMed

Wang, Qiang; Ma, Xiaonan; Qian, ShaSha; Zhou, Xin; Sun, Kai; Chen, Xiaolan; Zhou, Xueping; Jackson, Andrew O; Li, Zhenghe

2015-10-01

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

PubMed Central

Zhou, Xin; Sun, Kai; Chen, Xiaolan; Zhou, Xueping; Jackson, Andrew O.; Li, Zhenghe

2015-01-01

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses. PMID:26484673

Comprehensive proteomic analysis of the human spliceosome

NASA Astrophysics Data System (ADS)

Zhou, Zhaolan; Licklider, Lawrence J.; Gygi, Steven P.; Reed, Robin

2002-09-01

The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome `core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify ~145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.

An unexpected way forward: towards a more accurate and rigorous protein-protein binding affinity scoring function by eliminating terms from an already simple scoring function.

PubMed

Swanson, Jon; Audie, Joseph

2018-01-01

A fundamental and unsolved problem in biophysical chemistry is the development of a computationally simple, physically intuitive, and generally applicable method for accurately predicting and physically explaining protein-protein binding affinities from protein-protein interaction (PPI) complex coordinates. Here, we propose that the simplification of a previously described six-term PPI scoring function to a four term function results in a simple expression of all physically and statistically meaningful terms that can be used to accurately predict and explain binding affinities for a well-defined subset of PPIs that are characterized by (1) crystallographic coordinates, (2) rigid-body association, (3) normal interface size, and hydrophobicity and hydrophilicity, and (4) high quality experimental binding affinity measurements. We further propose that the four-term scoring function could be regarded as a core expression for future development into a more general PPI scoring function. Our work has clear implications for PPI modeling and structure-based drug design.

Co-expression of hepatitis C virus polytope-HBsAg and p19-silencing suppressor protein in tobacco leaves.

PubMed

Mohammadzadeh, Sara; Roohvand, Farzin; Memarnejadian, Arash; Jafari, Anis; Ajdary, Soheila; Salmanian, Ali-Hatef; Ehsani, Parastoo

2016-01-01

Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition. We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine. A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector. Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants. The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

Body Temperature Cycles Control Rhythmic Alternative Splicing in Mammals.

PubMed

Preußner, Marco; Goldammer, Gesine; Neumann, Alexander; Haltenhof, Tom; Rautenstrauch, Pia; Müller-McNicoll, Michaela; Heyd, Florian

2017-08-03

The core body temperature of all mammals oscillates with the time of the day. However, direct molecular consequences of small, physiological changes in body temperature remain largely elusive. Here we show that body temperature cycles drive rhythmic SR protein phosphorylation to control an alternative splicing (AS) program. A temperature change of 1°C is sufficient to induce a concerted splicing switch in a large group of functionally related genes, rendering this splicing-based thermometer much more sensitive than previously described temperature-sensing mechanisms. AS of two exons in the 5' UTR of the TATA-box binding protein (Tbp) highlights the general impact of this mechanism, as it results in rhythmic TBP protein levels with implications for global gene expression in vivo. Together our data establish body temperature-driven AS as a core clock-independent oscillator in mammalian peripheral clocks. Copyright © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Deug-Nam; Park, Mi-Ryung; Park, Jong-Yi

Highlights: {yields} The sequences of -604 to -84 bp of the pUPII promoter contained the region of a putative negative cis-regulatory element. {yields} The core promoter was located in the 5F-1. {yields} Transcription factor HNF4 can directly bind in the pUPII core promoter region, which plays a critical role in controlling promoter activity. {yields} These features of the pUPII promoter are fundamental to development of a target-specific vector. -- Abstract: Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, butmore » little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells. We also identified a new core promoter region and putative negative cis-regulatory element within a minimal promoter region. In addition, we showed that hepatocyte nuclear factor 4 (HNF4) can directly bind in the pUPII core promoter (5F-1) region, which plays a critical role in controlling promoter activity. Transient cotransfection experiments showed that HNF4 positively regulates pUPII gene promoter activity. Thus, the binding element and its binding protein, HNF4 transcription factor, may be involved in the mechanism that specifically regulates pUPII gene transcription.« less

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

PubMed

Ansardi, D C; Porter, D C; Morrow, C D

1991-04-01

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.

Expression of Bmi-1, P16, and CD44v6 in Uterine Cervical Carcinoma and Its Clinical Significance.

PubMed

Weng, Mei-Ying; Li, Lin; Feng, Shu-Ying; Hong, Shun-Jia

2012-03-01

Bmi-1, a putative proto-oncogene, is a core member of the polycomb gene family, which is expressed in many human tumors. The p16 protein negatively regulated cell proliferation, whereas CD44v6 is associated with proliferation as an important protein. Additionally, CD44v6 is an important nuclear antigen closely correlated to tumor metastasis. The present study aims to investigate the expression and significance of Bmi-1, p16, and CD44v6 in uterine cervical carcinoma (UCC). A total of 62 UCC, 30 cervical neoplasic, and 20 normal cervical mucosal tissues were used in the current study. The expression of Bmi-1, p16, and CD44v6 in these tissues was determined using immunohistochemical assay. The relationships among the expression of these indices, the clinicopathologic features of UCC, and the survival rate of UCC patients were also discussed. The correlation between Bmi-1 protein expression and p16 or CD44v6 protein in UCC was analyzed. The expression of Bmi-1, p16, and CD44v6 was significantly high in cervical carcinoma compared with that in the cervical neoplasia and normal colorectal mucosa (P<0.05). The over-expression of Bmi-1 protein in UCC was apparently related to the distant metastasis (P<0.01) and the tumor, nodes and metastasis-classification, i.e. the TNM staging, World Health Organization (P<0.05). Nevertheless, the positive expression of p16 protein in UCC was not significantly associated with the clinicopathologic features (P>0.05). The Kaplan-Meier survival analysis showed that the over-expression of Bmi-1 significantly decreased the survival rate of UCC patients (P<0.05). A strong correlation indicated that there was statistical significance between the expression of Bmi-1 and CD44V6 proteins in UCC (r=0.419, P=0.001). The over-expression of Bmi-1 and CD44v6 protein closely correlate to the tumorigenesis, metastasis, and prognosis of UCC. Bmi-1 and CD44v6 may be used to predict the prognosis of cervical carcinoma. Bmi-1 may indirectly regulate the expression of CD44v6 in UCC patients. The positive expression of p16 protein is possibly associated with the tumorigenesis, but not with the metastasis or prognosis of UCC.

Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration

PubMed Central

Musiek, Erik S.; Lim, Miranda M.; Yang, Guangrui; Bauer, Adam Q.; Qi, Laura; Lee, Yool; Roh, Jee Hoon; Ortiz-Gonzalez, Xilma; Dearborn, Joshua T.; Culver, Joseph P.; Herzog, Erik D.; Hogenesch, John B.; Wozniak, David F.; Dikranian, Krikor; Giasson, Benoit I.; Weaver, David R.; Holtzman, David M.; FitzGerald, Garret A.

2013-01-01

Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator–like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration. PMID:24270424

Chronic intermittent ethanol exposure and withdrawal leads to adaptations in nucleus accumbens core postsynaptic density proteome and dendritic spines.

PubMed

Uys, Joachim D; McGuier, Natalie S; Gass, Justin T; Griffin, William C; Ball, Lauren E; Mulholland, Patrick J

2016-05-01

Alcohol use disorder is a chronic relapsing brain disease characterized by the loss of ability to control alcohol (ethanol) intake despite knowledge of detrimental health or personal consequences. Clinical and pre-clinical models provide strong evidence for chronic ethanol-associated alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc). However, the neural mechanisms that contribute to aberrant glutamatergic signaling in ethanol-dependent individuals in this critical brain structure remain unknown. Using an unbiased proteomic approach, we investigated the effects of chronic intermittent ethanol (CIE) exposure on neuroadaptations in postsynaptic density (PSD)-enriched proteins in the NAc of ethanol-dependent mice. Compared with controls, CIE exposure significantly changed expression levels of 50 proteins in the PSD-enriched fraction. Systems biology and functional annotation analyses demonstrated that the dysregulated proteins are expressed at tetrapartite synapses and critically regulate cellular morphology. To confirm this latter finding, the density and morphology of dendritic spines were examined in the NAc core of ethanol-dependent mice. We found that CIE exposure and withdrawal differentially altered dendrite diameter and dendritic spine density and morphology. Through the use of quantitative proteomics and functional annotation, these series of experiments demonstrate that ethanol dependence produces neuroadaptations in proteins that modify dendritic spine morphology. In addition, these studies identified novel PSD-related proteins that contribute to the neurobiological mechanisms of ethanol dependence that drive maladaptive structural plasticity of NAc neurons. © 2015 Society for the Study of Addiction.

Small glutamine-rich tetratricopeptide repeat-containing protein alpha is present in human ovaries but may not be differentially expressed in relation to polycystic ovary syndrome.

PubMed

Butler, Miriam S; Yang, Xing; Ricciardelli, Carmela; Liang, Xiaoyan; Norman, Robert J; Tilley, Wayne D; Hickey, Theresa E

2013-06-01

To evaluate the expression and function of small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), an androgen receptor (AR) molecular chaperone, in human ovarian tissues. Examine the effect of SGTA on AR subcellular localization in granulosa tumor cells (KGN) and SGTA expression in ovarian tissues. University-based research laboratory. Archived tissues from premenopausal women and granulosa cells from infertile women receiving assisted reproduction. None. AR subcellular localization and SGTA protein or mRNA levels. SGTA and AR proteins were expressed in the cytoplasm of KGN cells and exposure to androgen stimulated AR nuclear localization. SGTA protein knockdown increased AR nuclear localization at low (0-0.1 nmol/L) but not high (1-10 nmol/L) concentrations of androgen hormone. In ovarian tissues, SGTA was localized to the cytoplasm of granulosa cells at all stages of folliculogenesis and in thecal cells of antral follicles. SGTA protein levels were similar when comparing primordial and primary follicles within core biopsies (n = 40) from women with and without polycystic ovary syndrome (PCOS). Likewise, SGTA mRNA levels were not significantly different in granulosa cells from preovulatory follicles after hyperstimulation of women with and without PCOS. SGTA is present in human ovaries and has the potential to modulate AR signalling, but it may not be differentially expressed in PCOS. Copyright © 2013 American Society for Reproductive Medicine. All rights reserved.

Localized surface plasmon mediated energy transfer in the vicinity of core-shell nanoparticle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shishodia, Manmohan Singh, E-mail: manmohan@gbu.ac.in; Juneja, Soniya

2016-05-28

Multipole spectral expansion based theory of energy transfer interactions between a donor and an acceptor molecule in the vicinity of a core-shell (nanoshell or core@shell) based plasmonic nanostructure is developed. In view of the diverse applications and rich plasmonic features such as tuning capability of surface plasmon (SP) frequencies, greater sensitivity to the change of dielectric environment, controllable redirection of electromagnetic radiation, closed form expressions for Energy Transfer Rate Enhancement Factor (ETREF) near core-shell particle are reported. The dependence of ETREF on different parameters is established through fitting equations, perceived to be of key importance for developing appropriate designs. Themore » theoretical approach developed in the present work is capable of treating higher order multipoles, which, in turn, are also shown to play a crucial role in the present context. Moreover, closed form expressions derived in the present work can directly be used as formula, e.g., for designing SP based biosensors and estimating energy exchange between proteins and excitonic interactions in quantum dots.« less

Cloning and characterization of a mouse gene with homology to the human von Hippel-Lindau disease tumor suppressor gene: implications for the potential organization of the human von Hippel-Lindau disease gene.

PubMed

Gao, J; Naglich, J G; Laidlaw, J; Whaley, J M; Seizinger, B R; Kley, N

1995-02-15

The human von Hippel-Lindau disease (VHL) gene has recently been identified and, based on the nucleotide sequence of a partial cDNA clone, has been predicted to encode a novel protein with as yet unknown functions [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. The length of the encoded protein and the characteristics of the cellular expressed protein are as yet unclear. Here we report the cloning and characterization of a mouse gene (mVHLh1) that is widely expressed in different mouse tissues and shares high homology with the human VHL gene. It predicts a protein 181 residues long (and/or 162 amino acids, considering a potential alternative start codon), which across a core region of approximately 140 residues displays a high degree of sequence identity (98%) to the predicted human VHL protein. High stringency DNA and RNA hybridization experiments and protein expression analyses indicate that this gene is the most highly VHL-related mouse gene, suggesting that it represents the mouse VHL gene homologue rather than a related gene sharing a conserved functional domain. These findings provide new insights into the potential organization of the VHL gene and nature of its encoded protein.

PIOX, a new pathogen-induced oxygenase with homology to animal cyclooxygenase.

PubMed

Sanz, A; Moreno, J I; Castresana, C

1998-09-01

Changes in gene expression induced in tobacco leaves by the harpin HrpN protein elicitor were examined, and a new cDNA, piox (for pathogen-induced oxygenase), with homology to genes encoding cyclooxygenase or prostaglandin endoperoxide synthase (PGHS), was identified. In addition to the amino acid identity determined, the protein encoded by piox is predicted to have a structural core similar to that of ovine PGHS-1. Moreover, studies of protein functionality demonstrate that the PIOX recombinant protein possesses at least one of the two enzymatic activities of PGHSs, that of catalyzing the oxygenation of polyunsaturated fatty acids. piox transcripts accumulated after protein elicitor treatment or inoculation with bacteria. Expression of piox was induced in tissues responding to inoculation with both incompatible and compatible bacteria, but RNA and protein accumulation differed for both types of interactions. We show that expression of piox is rapidly induced in response to various cellular signals mediating plant responses to pathogen infection and that activation of piox expression is most likely related to the oxidative burst that takes place during the cell death processes examined. Cyclooxygenase catalyzes the first committed step in the formation of prostaglandins and thromboxanes, which are lipid-derived signal molecules that mediate many cellular processes, including the immune response in vertebrates. The finding of tobacco PIOX suggests that more similarities than hitherto expected will be found between the lipid-based responses for plant and animal systems.

Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli

PubMed Central

Ortiz-Soto, Maria Elena; Seibel, Jürgen

2016-01-01

Sialyltransferases (STs) are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs). Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications. We report the successful expression of active human sialyltransferases ST3Gal1 and ST6Gal1 in commercial Escherichia coli strains designed for production of disulfide-containing proteins. Fusion of hST3Gal1 with different solubility enhancers and substitution of exposed hydrophobic amino acids by negatively charged residues (supercharging-like approach) were performed to promote solubility and folding. Co-expression of sialyltransferases with the chaperon/foldases sulfhydryl oxidase, protein disulfide isomerase and disulfide isomerase C was explored to improve the formation of native disulfide bonds. Active sialyltransferases fused with maltose binding protein (MBP) were obtained in sufficient amounts for biochemical and structural studies when expressed under oxidative conditions and co-expression of folding factors increased the yields of active and properly folded sialyltransferases by 20%. Mutation of exposed hydrophobic amino acids increased recovery of active enzyme by 2.5-fold, yielding about 7 mg of purified protein per liter culture. Functionality of recombinant enzymes was evaluated in the synthesis of sialosides from the β-d-galactoside substrates lactose, N-acetyllactosamine and benzyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside. PMID:27166796

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase.

PubMed

Spencer, Michael L; Shao, Haipeng; Andres, Douglas A

2002-06-07

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.

The Histone Database: an integrated resource for histones and histone fold-containing proteins

PubMed Central

Mariño-Ramírez, Leonardo; Levine, Kevin M.; Morales, Mario; Zhang, Suiyuan; Moreland, R. Travis; Baxevanis, Andreas D.; Landsman, David

2011-01-01

Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins. Database URL: The Histone Sequence Database is freely available and can be accessed at http://research.nhgri.nih.gov/histones/. PMID:22025671

Characterization of Chlamydomonas reinhardtii Core Histones by Top-Down Mass Spectrometry Reveals Unique Algae-Specific Variants and Post-Translational Modifications.

PubMed

Khan, Aliyya; Eikani, Carlo K; Khan, Hana; Iavarone, Anthony T; Pesavento, James J

2018-01-05

The unicellular microalga Chlamydomonas reinhardtii has played an instrumental role in the development of many new fields (bioproducts, biofuels, etc.) as well as the advancement of basic science (photosynthetic apparati, flagellar function, etc.). Chlamydomonas' versatility ultimately derives from the genes encoded in its genome and the way that the expression of these genes is regulated, which is largely influenced by a family of DNA binding proteins called histones. We characterize C. reinhardtii core histones, both variants and their post-translational modifications, by chromatographic separation, followed by top-down mass spectrometry (TDMS). Because TDMS has not been previously used to study Chlamydomonas proteins, we show rampant artifactual protein oxidation using established nuclei purification and histone extraction methods. After addressing oxidation, both histones H3 and H4 are found to each have a single polypeptide sequence that is minimally acetylated and methylated. Surprisingly, we uncover a novel monomethylation at lysine 79 on histone H4 present on all observed molecules. Histone H2B and H2A are found to have two and three variants, respectively, and both are minimally modified. This study provides an updated assessment of the core histone proteins in the green alga C. reinhardtii by top-down mass spectrometry and lays the foundation for further investigation of these essential proteins.

Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chunmao; Yu, You; Yang, Maojun, E-mail: maojunyang@tsinghua.edu.cn

2015-10-23

Fhb is a surface virulence protein from Streptococcus suis, which could aid bacterial evasion of host innate immune defense by recruiting complement regulator factor H to inactivate C3b deposited on bacterial surface in blood. Here we successfully expressed and purified the N terminal domain of Fhb (N-Fhb) and obtained crystals of the N-Fhb by sitting-drop vapor diffusion method with a resolution of 1.50 Å. The crystals belong to space group C2 with unit cell parameters a = 127.1 Å, b = 77.3 Å, c = 131.6 Å, α = 90°, β = 115.9°, γ = 90°. The structure of N-Fhb was determined by SAD method and the core structure of N-Fhb is a β sandwich. Wemore » speculated that binding of Fhb to human factor H may be mainly mediated by surface amino acids with negative charges. - Highlights: • We expressed N-Fhb as the soluble protein in Escherichia coli. • Crystals of N-Fhb were grown by sitting drop vapor diffusion method. • Crystals of N-Fhb could diffracted to 1.5 Å. • The core structure of N-Fhb was a β sandwich. • A part of the surface of N-Fhb was rich with negative charges.« less

Conformation of Tax-response elements in the human T-cell leukemia virus type I promoter.

PubMed

Cox, J M; Sloan, L S; Schepartz, A

1995-12-01

HTLV-I Tax is believed to activate viral gene expression by binding bZIP proteins (such as CREB) and increasing their affinities for proviral TRE target sites. Each 21 bp TRE target site contains an imperfect copy of the intrinsically bent CRE target site (the TRE core) surrounded by highly conserved flanking sequences. These flanking sequences are essential for maximal increases in DNA affinity and transactivation, but they are not, apparently, contacted by protein. Here we employ non-denaturing gel electrophoresis to evaluate TRE conformation in the presence and absence of bZIP proteins, and to explore the role of DNA conformation in viral transactivation. Our results show that the TRE-1 flanking sequences modulate the structure and modestly increase the affinity of a CREB bZIP peptide for the TRE-1 core recognition sequence. These flanking sequences are also essential for a maximal increase in stability of the CREB-DNA complex in the presence of Tax. The CRE-like TRE core and the TRE flanking sequences are both essential for formation of stable CREB-TRE-1 and Tax-CREB-TRE-1 complexes. These two DNA segments may have co-evolved into a unique structure capable of recognizing Tax and a bZIP protein.

Reduction of N-linked xylose and fucose by expression of rat beta1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression.

PubMed

Karg, Saskia R; Frey, Alexander D; Kallio, Pauli T

2010-03-01

Plant-specific N-glycosylation, such as the introduction of core alpha1,3-fucose and beta1,2-xylose residues, is a major obstacle to the utilization of plant cell- or plant-derived recombinant therapeutic proteins. The beta1,4-N-acetylglucosaminyltransferase III (GnTIII) introduces a bisecting GlcNAc residue into N-glycans, which exerts a high level of substrate mediated control over subsequent modifications, for example inhibiting mammalian core fucosylation. Based on similar findings in plants, we used Nicotianatabacum BY-2 cells to study the effects of localization and expression levels of GnTIII in the remodeling of the plant N-glycosylation pathway. The N-glycans produced by the cells expressing GnTIII were partially bisected and practically devoid of the paucimannosidic type which is typical for N-glycans produced by wildtype BY-2 suspension cultured cells. The proportion of human-compatible N-glycans devoid of fucose and xylose could be increased from an average of 4% on secreted protein from wildtype cells to as high as 59% in cells expressing chimeric GnTIII, named GnTIII(A.th.) replacing its native localization domain with the cytoplasmic tail, transmembrane, and stem region of Arabidopsis thaliana mannosidase II. The changes in N-glycosylation observed were dependent on the catalytic activity of GnTIII, as the expression of catalytically inactive GnTIII mutants did not show a significant effect on N-glycosylation. Copyright 2010 Elsevier B.V. All rights reserved.

Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes

PubMed Central

van der Does, H. Charlotte; Schmidt, Sarah M.; Langereis, Léon; Hughes, Timothy R.

2016-01-01

Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called ‘effectors’. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the ‘pathogenicity’ chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol pathogenicity chromosome may be partially transcriptionally autonomous, but there are also extensive transcriptional connections between core and accessory chromosomes. PMID:27855160

Gene Networks and Functional Features of Gravitropic response in Rice Shoot Bases

NASA Astrophysics Data System (ADS)

Hu, Liwei; Zang, Aiping; Ai, Qianru; Chen, Haiying; Li, Lin; Li, Rui; Su, Feng; Chen, Xijiang; Rong, Hui; Dou, Xianying; Reinhold-Hurek, Barbara; Li, Qi; Cai, Weiming

To delineate key genes and the corresponding physiological functions as well as the coordina-tion of genes involved in the gravitropism of rice shoot bases, we used whole-genome microarray analysis of upper and lower parts of rice shoot bases at 0.5 h and 6 h after gravistimulation. And bio-information analysis was applied including GO-analysis, expression tendency and net-work analysis. In the lower shoot bases, auxin-mediated signaling pathway and glutathione transferase activity with the biggest enrichment were activated at 0.5 h, while cytokinin stimu-lus and photosynthesis were activated at 6 h. Meanwhile, several processes were suppressed in the lower shoot bases, including: xyloglucan:xyloglucosyl transferase activity, glucan metabolic processes, and ATPase activity at 0.5 h; and tRNA isopentenyltransferase activity, and chiti-nase activity, etc. at 6 h. Gene expression profile responding to gravistimulation suggested that the asymmetrically activation of several phytohormone signaling pathways including auxin, gib-berellin and cytokinin brassinolide ethylene and cytokinin-related genes were involved in the differentially growth between the upper and lower parts of rice shoot bases, and so do cell wall-related genes. Topological analysis of the coexpression networks revealed the core statue of AY177699.1(apetala3-like protein) and AK105103.1 at 0.5 h; AK062612.1 (ethylene response factor) and AK099932.1 (lectin-like receptor kinase 72) at 6 h. All the core factors have the function "response to endogenous stimulus". Additionally, AK108057.1(similar to germin-like protein precursor) was discovered as the most important core gene in the upper shoot bases in 6h after gravistimualtion while AK067424.1(cellulose synthase-like protein), AK120101.1 (Zinc finger, B-box domain containing protein) and CR278698 (ATPase associated with various cel-lular activities cellulose synthase-like protein) contribute equally to gravitropic response in the lower shoot bases.

Quantitative Proteomics of Sleep-Deprived Mouse Brains Reveals Global Changes in Mitochondrial Proteins

PubMed Central

Li, Tie-Mei; Zhang, Ju-en; Lin, Rui; Chen, She; Luo, Minmin; Dong, Meng-Qiu

2016-01-01

Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson’s, Huntington’s, and Alzheimer’s, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration. PMID:27684481

Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation.

PubMed

Zhao, Qiong; Hu, Zhanying; Cheng, Junjun; Wu, Shuo; Luo, Yue; Chang, Jinhong; Hu, Jianming; Guo, Ju-Tao

2018-07-01

Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly. IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids. Copyright © 2018 American Society for Microbiology.

Interactions between Early Life Stress, Nucleus Accumbens MeCP2 Expression, and Methamphetamine Self-Administration in Male Rats

PubMed Central

Lewis, Candace R; Bastle, Ryan M; Manning, Tawny B; Himes, Sarah M; Fennig, Paulette; Conrad, Phoebe R; Colwell, Jenna; Pagni, Broc A; Hess, Lyndsay A; Matekel, Caitlin G; Newbern, Jason M; Olive, M Foster

2016-01-01

Early life stress (ELS) is highly related to the development of psychiatric illnesses in adulthood, including substance use disorders. A recent body of literature suggests that long-lasting changes in the epigenome may be a mechanism by which experiences early in life can alter neurobiological and behavioral phenotypes in adulthood. In this study, we replicate our previous findings that ELS, in the form of prolonged maternal separation, increases adult methamphetamine self-administration (SA) in male rats as compared with handled controls. In addition, we show new evidence that both ELS and methamphetamine SA alter the expression of the epigenetic regulator methyl CpG-binding protein 2 (MeCP2) in key brain reward regions, particularly in the nucleus accumbens (NAc) core. In turn, viral-mediated knockdown of MeCP2 expression in the NAc core reduces methamphetamine SA, as well as saccharin intake. Furthermore, NAc core MeCP2 knockdown reduces methamphetamine, but not saccharin, SA on a progressive ratio schedule of reinforcement. These data suggest that NAc core MeCP2 may be recruited by both ELS and methamphetamine SA and promote the development of certain aspects of drug abuse-related behavior. Taken together, functional interactions between ELS, methamphetamine SA, and the expression of MeCP2 in the NAc may represent novel mechanisms that can ultimately be targeted for intervention in individuals with adverse early life experiences who are at risk for developing substance use disorders. PMID:27312406

A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence

PubMed Central

Connolly, James P. R.; Gabrielsen, Mads; Goldstone, Robert J.; Grinter, Rhys; Wang, Dai; Cogdell, Richard J.; Walker, Daniel; Smith, David G. E.; Roe, Andrew J.

2016-01-01

The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to “sense” levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host. PMID:26727373

Genetic manipulation of iron biomineralization enhances MR relaxivity in a ferritin-M6A chimeric complex.

PubMed

Radoul, Marina; Lewin, Limor; Cohen, Batya; Oren, Roni; Popov, Stanislav; Davidov, Geula; Vandsburger, Moriel H; Harmelin, Alon; Bitton, Ronit; Greneche, Jean-Marc; Neeman, Michal; Zarivach, Raz

2016-05-23

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene - ferritin-M6A - in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.

An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein

PubMed Central

Jaag, Hannah Miriam; Kawchuk, Lawrence; Rohde, Wolfgang; Fischer, Rainer; Emans, Neil; Prüfer, Dirk

2003-01-01

Potato leafroll polerovirus (PLRV) genomic RNA acts as a polycistronic mRNA for the production of proteins P0, P1, and P2 translated from the 5′-proximal half of the genome. Within the P1 coding region we identified a 5-kDa replication-associated protein 1 (Rap1) essential for viral multiplication. An internal ribosome entry site (IRES) with unusual structure and location was identified that regulates Rap1 translation. Core structural elements for internal ribosome entry include a conserved AUG codon and a downstream GGAGAGAGAGG motif with inverted symmetry. Reporter gene expression in potato protoplasts confirmed the internal ribosome entry function. Unlike known IRES motifs, the PLRV IRES is located completely within the coding region of Rap1 at the center of the PLRV genome. PMID:12835413

Arabidopsis CML38, a Calcium Sensor That Localizes to Ribonucleoprotein Complexes under Hypoxia Stress1[OPEN

PubMed Central

McClintock, Carlee; Li, Tian

2016-01-01

During waterlogging and the associated oxygen deprivation stress, plants respond by the induction of adaptive programs, including the redirected expression of gene networks toward the synthesis of core hypoxia-response proteins. Among these core response proteins in Arabidopsis (Arabidopsis thaliana) is the calcium sensor CML38, a protein related to regulator of gene silencing calmodulin-like proteins (rgsCaMs). CML38 transcripts are up-regulated more than 300-fold in roots within 6 h of hypoxia treatment. Transfer DNA insertional mutants of CML38 show an enhanced sensitivity to hypoxia stress, with lowered survival and more severe inhibition of root and shoot growth. By using yellow fluorescent protein (YFP) translational fusions, CML38 protein was found to be localized to cytosolic granule structures similar in morphology to hypoxia-induced stress granules. Immunoprecipitation of CML38 from the roots of hypoxia-challenged transgenic plants harboring CML38pro::CML38:YFP followed by liquid chromatography-tandem mass spectrometry analysis revealed the presence of protein targets associated with messenger RNA ribonucleoprotein (mRNP) complexes including stress granules, which are known to accumulate as messenger RNA storage and triage centers during hypoxia. This finding is further supported by the colocalization of CML38 with the mRNP stress granule marker RNA Binding Protein 47 (RBP47) upon cotransfection of Nicotiana benthamiana leaves. Ruthenium Red treatment results in the loss of CML38 signal in cytosolic granules, suggesting that calcium is necessary for stress granule association. These results confirm that CML38 is a core hypoxia response calcium sensor protein and suggest that it serves as a potential calcium signaling target within stress granules and other mRNPs that accumulate during flooding stress responses. PMID:26634999

The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis.

PubMed

Satchwell, Timothy J; Hawley, Bethan R; Bell, Amanda J; Ribeiro, M Leticia; Toye, Ashley M

2015-01-01

Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation. Copyright© Ferrata Storti Foundation.

The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

PubMed Central

Satchwell, Timothy J; Hawley, Bethan R; Bell, Amanda J; Ribeiro, M. Leticia; Toye, Ashley M

2015-01-01

Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation. PMID:25344524

Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes.

PubMed

Enjapoori, Ashwantha Kumar; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

2017-02-01

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians. Copyright © 2015 Elsevier Inc. All rights reserved.

Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6

PubMed Central

Dai, Yawei; Seeger, Markus; Weng, Jingwei; Song, Song; Wang, Wenning; Tan, Yan-Wen

2016-01-01

Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view. PMID:27493064

Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

PubMed

Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

2018-05-01

We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

The Citrus ABA signalosome: identification and transcriptional regulation during sweet orange fruit ripening and leaf dehydration.

PubMed

Romero, Paco; Lafuente, María T; Rodrigo, María J

2012-08-01

The abscisic acid (ABA) signalling core in plants include the cytosolic ABA receptors (PYR/PYL/RCARs), the clade-A type 2C protein phosphatases (PP2CAs), and the subclass III SNF1-related protein kinases 2 (SnRK2s). The aim of this work was to identify these ABA perception system components in sweet orange and to determine the influence of endogenous ABA on their transcriptional regulation during fruit development and ripening, taking advantage of the comparative analysis between a wild-type and a fruit-specific ABA-deficient mutant. Transcriptional changes in the ABA signalosome during leaf dehydration were also studied. Six PYR/PYL/RCAR, five PP2CA, and two subclass III SnRK2 genes, homologous to those of Arabidopsis, were identified in the Citrus genome. The high degree of homology and conserved motifs for protein folding and for functional activity suggested that these Citrus proteins are bona fide core elements of ABA perception in orange. Opposite expression patterns of CsPYL4 and CsPYL5 and ABA accumulation were found during ripening, although there were few differences between varieties. In contrast, changes in expression of CsPP2CA genes during ripening paralleled those of ABA content and agreeed with the relevant differences between wild-type and mutant fruit transcript accumulation. CsSnRK2 gene expression continuously decreased with ripening and no remarkable differences were found between cultivars. Overall, dehydration had a minor effect on CsPYR/PYL/RCAR and CsSnRK2 expression in vegetative tissue, whereas CsABI1, CsAHG1, and CsAHG3 were highly induced by water stress. The global results suggest that responsiveness to ABA changes during citrus fruit ripening, and leaf dehydration was higher in the CsPP2CA gene negative regulators than in the other ABA signalosome components.

The Citrus ABA signalosome: identification and transcriptional regulation during sweet orange fruit ripening and leaf dehydration

PubMed Central

Rodrigo, María J.

2012-01-01

The abscisic acid (ABA) signalling core in plants include the cytosolic ABA receptors (PYR/PYL/RCARs), the clade-A type 2C protein phosphatases (PP2CAs), and the subclass III SNF1-related protein kinases 2 (SnRK2s). The aim of this work was to identify these ABA perception system components in sweet orange and to determine the influence of endogenous ABA on their transcriptional regulation during fruit development and ripening, taking advantage of the comparative analysis between a wild-type and a fruit-specific ABA-deficient mutant. Transcriptional changes in the ABA signalosome during leaf dehydration were also studied. Six PYR/PYL/RCAR, five PP2CA, and two subclass III SnRK2 genes, homologous to those of Arabidopsis, were identified in the Citrus genome. The high degree of homology and conserved motifs for protein folding and for functional activity suggested that these Citrus proteins are bona fide core elements of ABA perception in orange. Opposite expression patterns of CsPYL4 and CsPYL5 and ABA accumulation were found during ripening, although there were few differences between varieties. In contrast, changes in expression of CsPP2CA genes during ripening paralleled those of ABA content and agreeed with the relevant differences between wild-type and mutant fruit transcript accumulation. CsSnRK2 gene expression continuously decreased with ripening and no remarkable differences were found between cultivars. Overall, dehydration had a minor effect on CsPYR/PYL/RCAR and CsSnRK2 expression in vegetative tissue, whereas CsABI1, CsAHG1, and CsAHG3 were highly induced by water stress. The global results suggest that responsiveness to ABA changes during citrus fruit ripening, and leaf dehydration was higher in the CsPP2CA gene negative regulators than in the other ABA signalosome components. PMID:22888124

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC).

PubMed

Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A; Schoenhard, Grant; Zemelman, Boris V; Mechref, Yehia; Raab-Graham, Kimberly F

2016-02-01

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder-neurological disorders that exhibit elevated mTORC1 activity. Through a protein-protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The influence of Argonaute proteins on alternative RNA splicing.

PubMed

Batsché, Eric; Ameyar-Zazoua, Maya

2015-01-01

Alternative splicing of precursor RNAs is an important process in multicellular species because it impacts several aspects of gene expression: from the increase of protein repertoire to the level of expression. A large body of evidences demonstrates that factors regulating chromatin and transcription impact the outcomes of alternative splicing. Argonaute (AGO) proteins were known to play key roles in the regulation of gene expression at the post-transcriptional level. More recently, their role in the nucleus of human somatic cells has emerged. Here, we will discuss some of the nuclear functions of AGO, with special emphasis on alternative splicing. The AGO-mediated modulation of alternative splicing is based on several properties of these proteins: their binding to transcripts on chromatin and their interactions with many proteins, especially histone tail-modifying enzymes, HP1γ and splicing factors. AGO proteins may favor a decrease in the RNA-polymerase II kinetics at actively transcribed genes leading to the modulation of alternative splicing decisions. They could also influence alternative splicing through their interaction with core components of the splicing machinery and several splicing factors. We will discuss the modes of AGO recruitment on chromatin at active genes. We suggest that long intragenic antisense transcripts (lincRNA) might be an important feature of genes containing splicing events regulated by AGO. © 2014 John Wiley & Sons, Ltd.

Sulforaphane suppresses lipopolysaccharide-induced cyclooxygenase-2 (COX-2) expression through the modulation of multiple targets in COX-2 gene promoter.

PubMed

Woo, Kyung Jin; Kwon, Taeg Kyu

2007-12-15

Sulforaphane is a natural, biologically active compound extracted from cruciferous vegetables such as broccoli and cabbage. It possesses potent anti-inflammation and anti-cancer properties. The mechanism by which sulforaphane suppresses COX-2 expression remains poorly understood. In the present report, we investigated the effect of sulforaphane on the expression of COX-2 in lipopolysaccharide (LPS)-activated Raw 264.7 cells. Sulforaphane significantly suppressed the LPS-induced COX-2 protein and mRNA expression in a dose-dependent manner. The ability of sulforaphane to suppress the expression of the COX-2 was investigated using luciferase reporters controlled by various cis-elements in COX-2 promoter region. Electrophoretic mobility shift assay (EMSA) verified that NF-kappaB, C/EBP, CREB and AP-1 were identified as responsible for the sulforaphane-mediated COX-2 down-regulation. In addition, we demonstrated the signal transduction pathway of mitogen-activated protein kinase (MAP kinase) in LPS-induced COX-2 expression. Taken together, these results demonstrate that sulforaphane effectively suppressed the LPS-induced COX-2 protein via modulation of multiple core promoter elements (NF-kappaB, C/EBP, CREB and AP-1) in the COX-2 transcriptional regulation. These results will provide new insights into the anti-inflammatory and anti-carcinogenic properties of sulforaphane.

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)*

PubMed Central

Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A.; Schoenhard, Grant; Zemelman, Boris V.; Mechref, Yehia; Raab-Graham, Kimberly F.

2016-01-01

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states. PMID:26419955

Transcriptome Analysis of Core Dinoflagellates Reveals a Universal Bias towards "GC" Rich Codons.

PubMed

Williams, Ernest; Place, Allen; Bachvaroff, Tsvetan

2017-04-27

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen "core" dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression.

Regulation of aromatase expression in the anterior amygdala of the developing mouse brain depends on ERβ and sex chromosome complement.

PubMed

Cisternas, Carla Daniela; Cabrera Zapata, Lucas Ezequiel; Arevalo, María Angeles; Garcia-Segura, Luis Miguel; Cambiasso, María Julia

2017-07-13

During development sex differences in aromatase expression in limbic regions of mouse brain depend on sex chromosome factors. Genes on the sex chromosomes may affect the hormonal regulation of aromatase expression and this study was undertaken to explore that possibility. Male E15 anterior amygdala neuronal cultures expressed higher levels of aromatase (mRNA and protein) than female cultures. Furthermore, treatment with oestradiol (E2) or dihydrotestosterone (DHT) increased Cyp19a1 expression and aromatase protein levels only in female neuronal cultures. The effect of E2 on aromatase expression was not imitated by oestrogen receptor (ER) α agonist PPT or the GPER agonist G1, but it was fully reproduced by DPN, a specific ligand of ERβ. By contrast, the effect of DHT on aromatase expression was not blocked by the anti-androgen flutamide, but completely abrogated by the ERβ antagonist PHTPP. Experiments using the four core genotype model showed a sex chromosome effect in ERβ expression (XY > XX) and regulation by E2 or DHT (only XX respond) in amygdala neurons. In conclusion, sex chromosome complement governs the hormonal regulation of aromatase expression through activation of ERβ in developing mouse brain.

A ‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase

PubMed Central

Segall-Shapiro, Thomas H; Meyer, Adam J; Ellington, Andrew D; Sontag, Eduardo D; Voigt, Christopher A

2014-01-01

Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co-expressed to function. The DNA-binding loop is encoded in a C-terminal 285-aa ‘σ fragment’, and fragments with different specificity can direct the remaining 601-aa ‘core fragment’ to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67-aa N-terminal ‘α fragment’ and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids. PMID:25080493

Machine Learning-based Classification of Diffuse Large B-cell Lymphoma Patients by Their Protein Expression Profiles.

PubMed

Deeb, Sally J; Tyanova, Stefka; Hummel, Michael; Schmidt-Supprian, Marc; Cox, Juergen; Mann, Matthias

2015-11-01

Characterization of tumors at the molecular level has improved our knowledge of cancer causation and progression. Proteomic analysis of their signaling pathways promises to enhance our understanding of cancer aberrations at the functional level, but this requires accurate and robust tools. Here, we develop a state of the art quantitative mass spectrometric pipeline to characterize formalin-fixed paraffin-embedded tissues of patients with closely related subtypes of diffuse large B-cell lymphoma. We combined a super-SILAC approach with label-free quantification (hybrid LFQ) to address situations where the protein is absent in the super-SILAC standard but present in the patient samples. Shotgun proteomic analysis on a quadrupole Orbitrap quantified almost 9,000 tumor proteins in 20 patients. The quantitative accuracy of our approach allowed the segregation of diffuse large B-cell lymphoma patients according to their cell of origin using both their global protein expression patterns and the 55-protein signature obtained previously from patient-derived cell lines (Deeb, S. J., D'Souza, R. C., Cox, J., Schmidt-Supprian, M., and Mann, M. (2012) Mol. Cell. Proteomics 11, 77-89). Expression levels of individual segregation-driving proteins as well as categories such as extracellular matrix proteins behaved consistently with known trends between the subtypes. We used machine learning (support vector machines) to extract candidate proteins with the highest segregating power. A panel of four proteins (PALD1, MME, TNFAIP8, and TBC1D4) is predicted to classify patients with low error rates. Highly ranked proteins from the support vector analysis revealed differential expression of core signaling molecules between the subtypes, elucidating aspects of their pathobiology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dysfunction of Iron Metabolism and Iron-Regulatory Proteins in the Rat Hippocampus After Heat Stroke.

PubMed

Liu, Jing; Wan, Shengming; Zhang, Yun; Zhang, Shu; Zhang, Hongying; Wu, Shiwen

2018-05-11

Heat stroke, the most serious type of heat illness, refers to the presence of hyperthermia (core temperature >40°C), accompanied by central nervous system dysfunction. The hippocampus is a particularly vulnerable region in the early stage of heat stroke. Increasing evidence suggests that dysregulation of brain iron metabolism is involved in many neurodegenerative diseases. However, whether heat stroke causes dysfunction of iron metabolism, as well as iron-regulatory proteins, in the hippocampus remains unknown. The present study was conducted to explore the effects on spatial learning and memory, as well as iron content, ferroportin 1 (Fpn1), and hepcidin expression in the hippocampus after heat stroke in rats. Compared with the Sham group, learning ability and memory declined in rats after heat stroke. Iron concentration was significantly increased in the hippocampus. Expression of Fpn1 protein significantly decreased in the hippocampus, while expression of hepcidin increased. Interestingly, Fpn1 mRNA expression in the hippocampus increased. Our data thereby indicate that heat stroke can decrease learning ability and memory in rats. The mechanism may be related to changes of iron levels, as well as Fpn1 and hepcidin expression, in the hippocampus. Furthermore, hepcidin may rapidly decrease cellular Fpn1 protein levels, even under conditions of iron loading, indicating that hepcidin is a more dominant regulator of Fpn1 than is iron.

Expression of Bmi-1, P16, and CD44v6 in Uterine Cervical Carcinoma and Its Clinical Significance

PubMed Central

Weng, Mei-ying; Li, Lin; Feng, Shu-ying; Hong, Shun-jia

2012-01-01

Objective Bmi-1, a putative proto-oncogene, is a core member of the polycomb gene family, which is expressed in many human tumors. The p16 protein negatively regulated cell proliferation, whereas CD44v6 is associated with proliferation as an important protein. Additionally, CD44v6 is an important nuclear antigen closely correlated to tumor metastasis. The present study aims to investigate the expression and significance of Bmi-1, p16, and CD44v6 in uterine cervical carcinoma (UCC). Methods A total of 62 UCC, 30 cervical neoplasic, and 20 normal cervical mucosal tissues were used in the current study. The expression of Bmi-1, p16, and CD44v6 in these tissues was determined using immunohistochemical assay. The relationships among the expression of these indices, the clinicopathologic features of UCC, and the survival rate of UCC patients were also discussed. The correlation between Bmi-1 protein expression and p16 or CD44v6 protein in UCC was analyzed. Results The expression of Bmi-1, p16, and CD44v6 was significantly high in cervical carcinoma compared with that in the cervical neoplasia and normal colorectal mucosa (P<0.05). The over-expression of Bmi-1 protein in UCC was apparently related to the distant metastasis (P<0.01) and the tumor, nodes and metastasis-classification, i.e. the TNM staging, World Health Organization (P<0.05). Nevertheless, the positive expression of p16 protein in UCC was not significantly associated with the clinicopathologic features (P>0.05). The Kaplan–Meier survival analysis showed that the over-expression of Bmi-1 significantly decreased the survival rate of UCC patients (P<0.05). A strong correlation indicated that there was statistical significance between the expression of Bmi-1 and CD44V6 proteins in UCC (r=0.419, P=0.001). Conclusions The over-expression of Bmi-1 and CD44v6 protein closely correlate to the tumorigenesis, metastasis, and prognosis of UCC. Bmi-1 and CD44v6 may be used to predict the prognosis of cervical carcinoma. Bmi-1 may indirectly regulate the expression of CD44v6 in UCC patients. The positive expression of p16 protein is possibly associated with the tumorigenesis, but not with the metastasis or prognosis of UCC. PMID:23691455

Bcs1p can rescue a large and productive cytochrome bc(1) complex assembly intermediate in the inner membrane of yeast mitochondria.

PubMed

Conte, Laura; Trumpower, Bernard L; Zara, Vincenzo

2011-01-01

The yeast cytochrome bc(1) complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc(1) complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc(1) assembly and the formation of a functionally inactive bc(1) core structure of about 500-kDa. This immature bc(1) core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc(1) core structure leading to the formation of the functional homodimeric bc(1) complex. Following Bcs1p expression, the mature bc(1) complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc(1) complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc(1) complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc(1) core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc(1) complex and gives new insights into the molecular mechanisms involved in the last steps of bc(1) assembly. Copyright © 2010 Elsevier B.V. All rights reserved.

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

PubMed Central

Cicek, Kader; Gulec, Burcu; Ungor, Rifat; Hasanova, Gulnara

2017-01-01

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes. PMID:28827815

Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients.

PubMed

Shehat, Michael G; Bahey-El-Din, Mohammed; Kassem, Mervat A; Farghaly, Faten A; Abdul-Rahman, Medhat H; Fanaki, Nourhan H

2015-08-01

HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.

The Schizosaccharomyces pombe HIRA-Like Protein Hip1 Is Required for the Periodic Expression of Histone Genes and Contributes to the Function of Complex Centromeres

PubMed Central

Blackwell, Chris; Martin, Kate A.; Greenall, Amanda; Pidoux, Alison; Allshire, Robin C.; Whitehall, Simon K.

2004-01-01

HIRA-like (Hir) proteins are evolutionarily conserved and are implicated in the assembly of repressive chromatin. In Saccharomyces cerevisiae, Hir proteins contribute to the function of centromeres. However, S. cerevisiae has point centromeres that are structurally different from the complex centromeres of metazoans. In contrast, Schizosaccharomyces pombe has complex centromeres whose domain structure is conserved with that of human centromeres. Therefore, we examined the functions of the fission yeast Hir proteins Slm9 and the previously uncharacterised protein Hip1. Deletion of hip1+ resulted in phenotypes that were similar to those described previously for slm9Δ cells: a cell cycle delay, synthetic lethality with cdc25-22, and poor recovery from nitrogen starvation. However, while it has previously been shown that Slm9 is not required for the periodic expression of histone H2A, we found that loss of Hip1 led to derepression of core histone genes expression outside of S phase. Importantly, we found that deletion of either hip1+ or slm9+ resulted in increased rates of chromosome loss, increased sensitivity to spindle damage, and reduced transcriptional silencing in the outer centromeric repeats. Thus, S. pombe Hir proteins contribute to pericentromeric heterochromatin, and our data thus suggest that Hir proteins may be required for the function of metazoan centromeres. PMID:15121850

The Schizosaccharomyces pombe HIRA-like protein Hip1 is required for the periodic expression of histone genes and contributes to the function of complex centromeres.

PubMed

Blackwell, Chris; Martin, Kate A; Greenall, Amanda; Pidoux, Alison; Allshire, Robin C; Whitehall, Simon K

2004-05-01

HIRA-like (Hir) proteins are evolutionarily conserved and are implicated in the assembly of repressive chromatin. In Saccharomyces cerevisiae, Hir proteins contribute to the function of centromeres. However, S. cerevisiae has point centromeres that are structurally different from the complex centromeres of metazoans. In contrast, Schizosaccharomyces pombe has complex centromeres whose domain structure is conserved with that of human centromeres. Therefore, we examined the functions of the fission yeast Hir proteins Slm9 and the previously uncharacterised protein Hip1. Deletion of hip1(+) resulted in phenotypes that were similar to those described previously for slm9 Delta cells: a cell cycle delay, synthetic lethality with cdc25-22, and poor recovery from nitrogen starvation. However, while it has previously been shown that Slm9 is not required for the periodic expression of histone H2A, we found that loss of Hip1 led to derepression of core histone genes expression outside of S phase. Importantly, we found that deletion of either hip1(+) or slm9(+) resulted in increased rates of chromosome loss, increased sensitivity to spindle damage, and reduced transcriptional silencing in the outer centromeric repeats. Thus, S. pombe Hir proteins contribute to pericentromeric heterochromatin, and our data thus suggest that Hir proteins may be required for the function of metazoan centromeres.

Definition of a core module for the nuclear retrograde response to altered organellar gene expression identifies GLK overexpressors as gun mutants.

PubMed

Leister, Dario; Kleine, Tatjana

2016-07-01

Retrograde signaling can be triggered by changes in organellar gene expression (OGE) induced by inhibitors such as lincomycin (LIN) or mutations that perturb OGE. Thus, an insufficiency of the organelle-targeted prolyl-tRNA synthetase PRORS1 in Arabidopsis thaliana activates retrograde signaling and reduces the expression of nuclear genes for photosynthetic proteins. Recently, we showed that mTERF6, a member of the so-called mitochondrial transcription termination factor (mTERF) family, is involved in the formation of chloroplast (cp) isoleucine-tRNA. To obtain further insights into its functions, co-expression analysis of MTERF6, PRORS1 and two other genes for organellar aminoacyl-tRNA synthetases was conducted. The results suggest a prominent role of mTERF6 in aminoacylation activity, light signaling and seed storage. Analysis of changes in whole-genome transcriptomes in the mterf6-1 mutant showed that levels of nuclear transcripts for cp OGE proteins were particularly affected. Comparison of the mterf6-1 transcriptome with that of prors1-2 showed that reduced aminoacylation of proline (prors1-2) and isoleucine (mterf6-1) tRNAs alters retrograde signaling in similar ways. Database analyses indicate that comparable gene expression changes are provoked by treatment with LIN, norflurazon or high light. A core OGE response module was defined by identifying genes that were differentially expressed under at least four of six conditions relevant to OGE signaling. Based on this module, overexpressors of the Golden2-like transcription factors GLK1 and GLK2 were identified as genomes uncoupled mutants. © 2016 Scandinavian Plant Physiology Society.

The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles

PubMed Central

Wang, Yun; Lin, Fu-xing; Zhao, Yu; Wang, Mo-zhen; Ge, Xue-wu; Gong, Zheng-xing; Bao, Dan-dan; Gu, Yu-fang

2014-01-01

Novel submicron core-shell-structured chitosan-based composite particles encapsulated with enhanced green fluorescent protein plasmids (pEGFP) were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS) and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC). pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection. PMID:25364253

The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles.

PubMed

Wang, Yun; Lin, Fu-xing; Zhao, Yu; Wang, Mo-zhen; Ge, Xue-wu; Gong, Zheng-xing; Bao, Dan-dan; Gu, Yu-fang

2014-01-01

Novel submicron core-shell-structured chitosan-based composite particles encapsulated with enhanced green fluorescent protein plasmids (pEGFP) were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS) and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC). pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Chun -Jun; Sun, Wei -Wen; Bruno, Kenneth S.

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPSlike genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas inmore » conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.« less

Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.

PubMed

Tu, Zhengkun; Pierce, Robert H; Kurtis, Jonathan; Kuroki, Yoshio; Crispe, I Nicholas; Orloff, Mark S

2010-01-01

Kupffer cells (KC) are important innate immune cells of the liver, functioning as scavenging sinusoidal phagocytes and transducers of pattern recognition signals, including those of toll-like receptors (TLRs). The hepatitis C virus core protein (HCVc) engages TLR2 on peripheral blood monocytes and induces production of multiple inflammatory cytokines. We examined the effects of HCVc on human primary KC functions. KC were isolated from living donor allografts and stimulated with HCVc and/or ligands for TLRs. KC were examined for production of cytokines, expression of programmed death-ligand 1 (PD-L1), secretion of type 1 interferons (IFNs), and expression of the apoptosis-inducing protein tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). HCVc acts as a ligand for TLR2 on human KC, inducing them to secrete interleukin (IL)-1beta, TNF-alpha, and IL-10 and up-regulate cell surface PD-L1. HCVc blocked TLR3-mediated secretion of IFN-alpha, IFN-beta, and cell surface expression of the cytotoxic molecule TRAIL. Inhibition of phosphoinositide 3 kinase with LY294002 blocked the up-regulation of PD-L1 by TLR ligands and the TLR3-specific induction of TRAIL and type 1 IFNs. KC are intravascular macrophages that are continuously exposed to, and tolerant of, bacterial TLR ligands, which are delivered via the portal circulation. By mimicking a bacterial TLR2 ligand and effectively blocking the TLR3-mediated, double-stranded RNA-induced antiviral response, HCVc might appear to exploit this unique aspect of immunity in the liver. Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii

PubMed Central

Onishi, Masayuki; Pringle, John R.

2016-01-01

The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins. PMID:27770025

Interaction of structural core protein of Classical Swine Fever Virus with endoplasmic reticulum-associated degradation pathway protein OS9

USDA-ARS?s Scientific Manuscript database

Classical Swine Fever Virus (CSFV) Core protein is involved in virus RNA protection, transcription regulation and virus virulence. To discover additional Core protein functions a yeast two-hybrid system was used to identify host proteins that interact with Core. Among the identified host proteins, t...

MiR-3613-3p affects cell proliferation and cell cycle in hepatocellular carcinoma

PubMed Central

Zhang, Donghui; Liu, Enqin; Kang, Jian; Yang, Xin; Liu, Hong

2017-01-01

Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumors with poor sensitivity to chemotherapy drugs and poor prognosis among patients. In the present study, we downloaded the original data from the Gene Expression Omnibus and compared gene expression profiles of liver cancer cells in patients with HCC with those of colon epithelial cells of healthy controls to identify differentially expressed genes (DEGs). After filtering target microRNAs (miRNA) from core DEGs, we cultured HepG2 cells in vitro, knocked down the miRNA and core mRNAs, and analyzed the effects. We found 228 differentially expressed genes between liver cancer tissue and healthy control tissue. We also integrated the protein-proteininteraction network and module analysis to screen 13 core genes, consisting of 12 up-regulated genes and 1 down-regulated gene. Five core genes were regulated hsa-miR-3613-3p, therefor we hypothesized that hsa-miR-3613-3p was a critical miRNA. After the transfection procedure, we found that changes in hsa-miR-3613-3p were the most obvious. Therefore, we speculated that hsa-miR-3613-3p was a main target miRNA. In addition, we transfected with si (BIRC5, CDK1, NUF2, ZWINT and SPC24), to target genes that can be targeted by miR-3613-3p. Our data shows that BIRC5, NUF2, and SPC24 may be promising liver cancer biomarkers that may not only predict disease occurrence but also potential personalized treatment options. PMID:29190974

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

PubMed Central

Schreiner, Sabrina; Bürck, Carolin; Glass, Mandy; Groitl, Peter; Wimmer, Peter; Kinkley, Sarah; Mund, Andreas; Everett, Roger D.; Dobner, Thomas

2013-01-01

Death domain–associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein–protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling. PMID:23396441

A Wnt5 Activity Asymmetry and Intercellular Signaling via PCP Proteins Polarize Node Cells for Left-Right Symmetry Breaking.

PubMed

Minegishi, Katsura; Hashimoto, Masakazu; Ajima, Rieko; Takaoka, Katsuyoshi; Shinohara, Kyosuke; Ikawa, Yayoi; Nishimura, Hiromi; McMahon, Andrew P; Willert, Karl; Okada, Yasushi; Sasaki, Hiroshi; Shi, Dongbo; Fujimori, Toshihiko; Ohtsuka, Toshihisa; Igarashi, Yasunobu; Yamaguchi, Terry P; Shimono, Akihiko; Shiratori, Hidetaka; Hamada, Hiroshi

2017-03-13

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a -/- Wnt5b -/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry. Copyright © 2017 Elsevier Inc. All rights reserved.

A Role for Timely Nuclear Translocation of Clock Repressor Proteins in Setting Circadian Clock Speed

PubMed Central

Lee, Euna

2014-01-01

By means of a circadian clock system, all the living organisms on earth including human beings can anticipate the environmental rhythmic changes such as light/dark and warm/cold periods in a daily as well as in a yearly manner. Anticipating such environmental changes provide organisms with survival benefits via manifesting behavior and physiology at an advantageous time of the day and year. Cell-autonomous circadian oscillators, governed by transcriptional feedback loop composed of positive and negative elements, are organized into a hierarchical system throughout the organisms and generate an oscillatory expression of a clock gene by itself as well as clock controlled genes (ccgs) with a 24 hr periodicity. In the feedback loop, hetero-dimeric transcription factor complex induces the expression of negative regulatory proteins, which in turn represses the activity of transcription factors to inhibit their own transcription. Thus, for robust oscillatory rhythms of the expression of clock genes as well as ccgs, the precise control of subcellular localization and/or timely translocation of core clock protein are crucial. Here, we discuss how sub-cellular localization and nuclear translocation are controlled in a time-specific manner focusing on the negative regulatory clock proteins. PMID:25258565

Functional Analysis of a Bacterial Antifreeze Protein Indicates a Cooperative Effect between Its Two Ice-Binding Domains.

PubMed

Wang, Chen; Oliver, Erin E; Christner, Brent C; Luo, Bing-Hao

2016-07-19

Antifreeze proteins make up a class of ice-binding proteins (IBPs) that are possessed and expressed by certain cold-adapted organisms to enhance their freezing tolerance. Here we report the biophysical and functional characterization of an IBP discovered in a bacterium recovered from a deep glacial ice core drilled at Vostok Station, Antarctica (IBPv). Our study showed that the recombinant protein rIBPv exhibited a thermal hysteresis of 2 °C at concentrations of >50 μM, effectively inhibited ice recrystallization, and enhanced bacterial viability during freeze-thaw cycling. Circular dichroism scans indicated that rIBPv mainly consists of β strands, and its denaturing temperature was 53.5 °C. Multiple-sequence alignment of homologous IBPs predicted that IBPv contains two ice-binding domains, a feature unique among known IBPs. To examine functional differences between the IBPv domains, each domain was cloned, expressed, and purified. The second domain (domain B) expressed greater ice binding activity. Data from thermal hysteresis and gel filtration assays supported the idea that the two domains cooperate to achieve a higher ice binding effect by forming heterodimers. However, physical linkage of the domains was not required for this effect.

SWI/SNF Subunits SMARCA4, SMARCD2 and DPF2 Collaborate in MLL-Rearranged Leukaemia Maintenance.

PubMed

Cruickshank, V Adam; Sroczynska, Patrycja; Sankar, Aditya; Miyagi, Satoru; Rundsten, Carsten Friis; Johansen, Jens Vilstrup; Helin, Kristian

2015-01-01

Alterations in chromatin structure caused by deregulated epigenetic mechanisms collaborate with underlying genetic lesions to promote cancer. SMARCA4/BRG1, a core component of the SWI/SNF ATP-dependent chromatin-remodelling complex, has been implicated by its mutational spectrum as exerting a tumour-suppressor function in many solid tumours; recently however, it has been reported to sustain leukaemogenic transformation in MLL-rearranged leukaemia in mice. Here we further explore the role of SMARCA4 and the two SWI/SNF subunits SMARCD2/BAF60B and DPF2/BAF45D in leukaemia. We observed the selective requirement for these proteins for leukaemic cell expansion and self-renewal in-vitro as well as in leukaemia. Gene expression profiling in human cells of each of these three factors suggests that they have overlapping functions in leukaemia. The gene expression changes induced by loss of the three proteins demonstrate that they are required for the expression of haematopoietic stem cell associated genes but in contrast to previous results obtained in mouse cells, the three proteins are not required for the expression of c-MYC regulated genes.

Suppression of survivin promoter activity by YM155 involves disruption of Sp1-DNA interaction in the survivin core promoter

PubMed Central

Cheng, Qiuying; Ling, Xiang; Haller, Andrew; Nakahara, Takahito; Yamanaka, Kentaro; Kita, Aya; Koutoku, Hiroshi; Takeuchi, Masahiro; Brattain, Michael G; Li, Fengzhi

2012-01-01

YM155, a novel survivin suppressant, shows potent antitumor activity against various human cancers and is currently in phase II clinical trials. In this study, we investigated whether YM155 selectively inhibits survivin transcription. We hypothesize that inhibition of survivin transcription plays a role in YM155-mediated survivin inhibition. We found that YM155 inhibited survivin promoter activity, while it showed minimal inhibitory effect on four control gene promoters in transfection and luciferase activity assay experiments, indicating its selectivity. Transfection of various survivin promoter-luciferase constructs followed by luciferase assays revealed that the survivin core promoter (269 bp) plays a major role in YM155-mediated inhibitory effects. However, flow cytometry analysis indicated that inhibition of survivin promoter activity by YM155 is cell cycle-independent without G1 cell arrests. Electrophoretic mobility shift assays (EMSA) identified that YM155 abrogates nuclear proteins binding to the region of -149 to -71, in which Sp1 is a major candidate, and that YM155 treatment induces Sp1 re-subcellular localization without inhibiting its expression. Forced expression of Sp1 neutralized YM155-mediated downregulation of survivin promoter activity. Consistently, mutation of the identified Sp1 sites in the oligonucleotide probe diminished DNA-protein interactions in EMSA experiments, and mutation of the Sp1 sites in the survivin promoter-luciferase construct diminished survivin promoter activity. These findings indicate that YM155 inhibition of survivin expression is at least in part through its inhibition of survivin transcription by disruption of Sp1 interaction with the region of -149 to -71 in the survivin core promoter. PMID:22773958

Reliable Entity Subtyping in Non-small Cell Lung Cancer by Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry on Formalin-fixed Paraffin-embedded Tissue Specimens*

PubMed Central

Kriegsmann, Mark; Casadonte, Rita; Kriegsmann, Jörg; Dienemann, Hendrik; Schirmacher, Peter; Hendrik Kobarg, Jan; Schwamborn, Kristina; Stenzinger, Albrecht; Warth, Arne; Weichert, Wilko

2016-01-01

Histopathological subtyping of non-small cell lung cancer (NSCLC) into adenocarcinoma (ADC), and squamous cell carcinoma (SqCC) is of utmost relevance for treatment stratification. However, current immunohistochemistry (IHC) based typing approaches on biopsies are imperfect, therefore novel analytical methods for reliable subtyping are needed. We analyzed formalin-fixed paraffin-embedded tissue cores of NSCLC by Matrix-assisted laser desorption/ionization (MALDI) imaging on tissue microarrays to identify and validate discriminating MALDI imaging profiles for NSCLC subtyping. 110 ADC and 98 SqCC were used to train a Linear Discriminant Analysis (LDA) model. Results were validated on a separate set of 58 ADC and 60 SqCC. Selected differentially expressed proteins were identified by tandem mass spectrometry and validated by IHC. The LDA classification model incorporated 339 m/z values. In the validation cohort, in 117 cases (99.1%) MALDI classification on tissue cores was in accordance with the pathological diagnosis made on resection specimen. Overall, three cases in the combined cohorts were discordant, after reevaluation two were initially misclassified by pathology whereas one was classified incorrectly by MALDI. Identification of differentially expressed peptides detected well-known IHC discriminators (CK5, CK7), but also less well known differentially expressed proteins (CK15, HSP27). In conclusion, MALDI imaging on NSCLC tissue cores as small biopsy equivalents is capable to discriminate lung ADC and SqCC with a very high accuracy. In addition, replacing multislide IHC by an one-slide MALDI approach may also save tissue for subsequent predictive molecular testing. We therefore advocate to pursue routine diagnostic implementation strategies for MALDI imaging in solid tumor typing. PMID:27473201

Reliable Entity Subtyping in Non-small Cell Lung Cancer by Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry on Formalin-fixed Paraffin-embedded Tissue Specimens.

PubMed

Kriegsmann, Mark; Casadonte, Rita; Kriegsmann, Jörg; Dienemann, Hendrik; Schirmacher, Peter; Hendrik Kobarg, Jan; Schwamborn, Kristina; Stenzinger, Albrecht; Warth, Arne; Weichert, Wilko

2016-10-01

Histopathological subtyping of non-small cell lung cancer (NSCLC) into adenocarcinoma (ADC), and squamous cell carcinoma (SqCC) is of utmost relevance for treatment stratification. However, current immunohistochemistry (IHC) based typing approaches on biopsies are imperfect, therefore novel analytical methods for reliable subtyping are needed. We analyzed formalin-fixed paraffin-embedded tissue cores of NSCLC by Matrix-assisted laser desorption/ionization (MALDI) imaging on tissue microarrays to identify and validate discriminating MALDI imaging profiles for NSCLC subtyping. 110 ADC and 98 SqCC were used to train a Linear Discriminant Analysis (LDA) model. Results were validated on a separate set of 58 ADC and 60 SqCC. Selected differentially expressed proteins were identified by tandem mass spectrometry and validated by IHC. The LDA classification model incorporated 339 m/z values. In the validation cohort, in 117 cases (99.1%) MALDI classification on tissue cores was in accordance with the pathological diagnosis made on resection specimen. Overall, three cases in the combined cohorts were discordant, after reevaluation two were initially misclassified by pathology whereas one was classified incorrectly by MALDI. Identification of differentially expressed peptides detected well-known IHC discriminators (CK5, CK7), but also less well known differentially expressed proteins (CK15, HSP27). In conclusion, MALDI imaging on NSCLC tissue cores as small biopsy equivalents is capable to discriminate lung ADC and SqCC with a very high accuracy. In addition, replacing multislide IHC by an one-slide MALDI approach may also save tissue for subsequent predictive molecular testing. We therefore advocate to pursue routine diagnostic implementation strategies for MALDI imaging in solid tumor typing. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Divergent N-Terminal Sequences Target an Inducible Testis Deubiquitinating Enzyme to Distinct Subcellular Structures

PubMed Central

Lin, Haijiang; Keriel, Anne; Morales, Carlos R.; Bedard, Nathalie; Zhao, Qing; Hingamp, Pascal; Lefrançois, Stephane; Combaret, Lydie; Wing, Simon S.

2000-01-01

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-γ-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action. PMID:10938131

Conformational Entropy from NMR Relaxation in Proteins: The SRLS Perspective.

PubMed

Tchaicheeyan, Oren; Meirovitch, Eva

2017-02-02

Conformational entropy changes associated with bond-vector motions in proteins contribute to the free energy of ligand-binding. To derive such contributions, we apply the slowly relaxing local structure (SRLS) approach to NMR relaxation from 15 N-H bonds or C-CDH 2 moieties of several proteins in free and ligand-bound form. The spatial restraints on probe motion, which determine the extent of local order, are expressed in SRLS by a well-defined potential, u(θ). The latter yields the orientational probability density, P eq  = exp(-u(θ)), and hence the related conformational entropy, Ŝ = -∫P eq (θ) ln[P eq (θ)] sin θ dθ (Ŝ is "entropy" in units of k B T, and θ represents the bond-vector orientation in the protein). SRLS is applied to 4-oxalocrotonate tautomerase (4-OT), the acyl-coenzyme A binding protein (ACBP), the C-terminal SH2 domain of phospholipase C γ 1 (PLC γ 1C SH2), the construct dihydrofolate reductase-E:folate (DHFR-E:folate), and their complexes with appropriate ligands, to determine ΔŜ. Eglin C and its V18A and V34A mutants are also studied. Finally, SRLS is applied to the structurally homologous proteins TNfn3 and FNfn10 to characterize within its scope the unusual "dynamics" of the TNfn3 core. Upon ligand-binding, the backbones of 4-OT, ACBP, and PLC γ 1C SH2 show limited, increased, and decreased order, respectively; the cores of DHFR-E:folate and PLC γ 1C SH2 become more ordered. The V18A (V34A) mutation increases (decreases) the order within the eglin C core. The core of TNfn3 is less ordered structurally and more mobile kinetically. Secondary structure versus loops, surface-binding versus core insertion, and ligand size emerged as being important in rationalizing ΔŜ. The consistent and general tool developed herein is expected to provide further insights in future work.

The peripheral clock regulates human pigmentation.

PubMed

Hardman, Jonathan A; Tobin, Desmond J; Haslam, Iain S; Farjo, Nilofer; Farjo, Bessam; Al-Nuaimi, Yusur; Grimaldi, Benedetto; Paus, Ralf

2015-04-01

Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

Regulation of nrf operon expression in pathogenic enteric bacteria: sequence divergence reveals new regulatory complexity

PubMed Central

Godfrey, Rita E.; Lee, David J.; Busby, Stephen J. W.

2017-01-01

Summary The Escherichia coli K‐12 nrf operon encodes a periplasmic nitrite reductase, the expression of which is driven from a single promoter, pnrf. Expression from pnrf is activated by the FNR transcription factor in response to anaerobiosis and further increased in response to nitrite by the response regulator proteins, NarL and NarP. FNR‐dependent transcription is suppressed by the binding of two nucleoid associated proteins, IHF and Fis. As Fis levels increase in cells grown in rich medium, the positioning of its binding site, overlapping the promoter −10 element, ensures that pnrf is sharply repressed. Here, we investigate the expression of the nrf operon promoter from various pathogenic enteric bacteria. We show that pnrf from enterohaemorrhagic E. coli is more active than its K‐12 counterpart, exhibits substantial FNR‐independent activity and is insensitive to nutrient quality, due to an improved −10 element. We also demonstrate that the Salmonella enterica serovar Typhimurium core promoter is more active than previously thought, due to differences around the transcription start site, and that its expression is repressed by downstream sequences. We identify the CsrA RNA binding protein as being responsible for this, and show that CsrA differentially regulates the E. coli K‐12 and Salmonella nrf operons. PMID:28211111

The High-Throughput Protein Sample Production Platform of the Northeast Structural Genomics Consortium

PubMed Central

Xiao, Rong; Anderson, Stephen; Aramini, James; Belote, Rachel; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John K.; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Jiang, Mei; Kornhaber, Gregory J.; Lee, Dong Yup; Locke, Jessica Y.; Ma, Li-Chung; Maglaqui, Melissa; Mao, Lei; Mitra, Saheli; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Sharma, Seema; Shastry, Ritu; Swapna, G.V.T.; Tong, Saichu N.; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.; Acton, Thomas B.

2014-01-01

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (> 97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as > 26,000 constructs. Over the past nine years, more than 16,000 of these expressed protein, and more than 4,400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last five years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities. PMID:20688167

The Role of Multiple Transcription Factors In Archaeal Gene Expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Charles J. Daniels

2008-09-23

Since the inception of this research program, the project has focused on two central questions: What is the relationship between the 'eukaryal-like' transcription machinery of archaeal cells and its counterparts in eukaryal cells? And, how does the archaeal cell control gene expression using its mosaic of eukaryal core transcription machinery and its bacterial-like transcription regulatory proteins? During the grant period we have addressed these questions using a variety of in vivo approaches and have sought to specifically define the roles of the multiple TATA binding protein (TBP) and TFIIB-like (TFB) proteins in controlling gene expression in Haloferax volcanii. H. volcaniimore » was initially chosen as a model for the Archaea based on the availability of suitable genetic tools; however, later studies showed that all haloarchaea possessed multiple tbp and tfb genes, which led to the proposal that multiple TBP and TFB proteins may function in a manner similar to alternative sigma factors in bacterial cells. In vivo transcription and promoter analysis established a clear relationship between the promoter requirements of haloarchaeal genes and those of the eukaryal RNA polymerase II promoter. Studies on heat shock gene promoters, and the demonstration that specific tfb genes were induced by heat shock, provided the first indication that TFB proteins may direct expression of specific gene families. The construction of strains lacking tbp or tfb genes, coupled with the finding that many of these genes are differentially expressed under varying growth conditions, provided further support for this model. Genetic tools were also developed that led to the construction of insertion and deletion mutants, and a novel gene expression scheme was designed that allowed the controlled expression of these genes in vivo. More recent studies have used a whole genome array to examine the expression of these genes and we have established a linkage between the expression of specific tfb genes and the regulation of nitrogen metabolism and other global cellular responses.« less

Crosstalk of clock gene expression and autophagy in aging

PubMed Central

Kalfalah, Faiza; Janke, Linda; Schiavi, Alfonso; Tigges, Julia; Ix, Alexander; Ventura, Natascia; Boege, Fritz; Reinke, Hans

2016-01-01

Autophagy and the circadian clock counteract tissue degeneration and support longevity in many organisms. Accumulating evidence indicates that aging compromises both the circadian clock and autophagy but the mechanisms involved are unknown. Here we show that the expression levels of transcriptional repressor components of the circadian oscillator, most prominently the human Period homologue PER2, are strongly reduced in primary dermal fibroblasts from aged humans, while raising the expression of PER2 in the same cells partially restores diminished autophagy levels. The link between clock gene expression and autophagy is corroborated by the finding that the circadian clock drives cell-autonomous, rhythmic autophagy levels in immortalized murine fibroblasts, and that siRNA-mediated downregulation of PER2 decreases autophagy levels while leaving core clock oscillations intact. Moreover, the Period homologue lin-42 regulates autophagy and life span in the nematode Caenorhabditis elegans, suggesting an evolutionarily conserved role for Period proteins in autophagy control and aging. Taken together, this study identifies circadian clock proteins as set-point regulators of autophagy and puts forward a model, in which age-related changes of clock gene expression promote declining autophagy levels. PMID:27574892

Crosstalk of clock gene expression and autophagy in aging.

PubMed

Kalfalah, Faiza; Janke, Linda; Schiavi, Alfonso; Tigges, Julia; Ix, Alexander; Ventura, Natascia; Boege, Fritz; Reinke, Hans

2016-08-28

Autophagy and the circadian clock counteract tissue degeneration and support longevity in many organisms. Accumulating evidence indicates that aging compromises both the circadian clock and autophagy but the mechanisms involved are unknown. Here we show that the expression levels of transcriptional repressor components of the circadian oscillator, most prominently the human Period homologue PER2 , are strongly reduced in primary dermal fibroblasts from aged humans, while raising the expression of PER2 in the same cells partially restores diminished autophagy levels. The link between clock gene expression and autophagy is corroborated by the finding that the circadian clock drives cell-autonomous, rhythmic autophagy levels in immortalized murine fibroblasts, and that siRNA-mediated downregulation of PER2 decreases autophagy levels while leaving core clock oscillations intact. Moreover, the Period homologue lin-42 regulates autophagy and life span in the nematode Caenorhabditis elegans , suggesting an evolutionarily conserved role for Period proteins in autophagy control and aging. Taken together, this study identifies circadian clock proteins as set-point regulators of autophagy and puts forward a model, in which age-related changes of clock gene expression promote declining autophagy levels.

Discovery of Colorectal Cancer Biomarker Candidates by Membrane Proteomic Analysis and Subsequent Verification using Selected Reaction Monitoring (SRM) and Tissue Microarray (TMA) Analysis*

PubMed Central

Kume, Hideaki; Muraoka, Satoshi; Kuga, Takahisa; Adachi, Jun; Narumi, Ryohei; Watanabe, Shio; Kuwano, Masayoshi; Kodera, Yoshio; Matsushita, Kazuyuki; Fukuoka, Junya; Masuda, Takeshi; Ishihama, Yasushi; Matsubara, Hisahiro; Nomura, Fumio; Tomonaga, Takeshi

2014-01-01

Recent advances in quantitative proteomic technology have enabled the large-scale validation of biomarkers. We here performed a quantitative proteomic analysis of membrane fractions from colorectal cancer tissue to discover biomarker candidates, and then extensively validated the candidate proteins identified. A total of 5566 proteins were identified in six tissue samples, each of which was obtained from polyps and cancer with and without metastasis. GO cellular component analysis predicted that 3087 of these proteins were membrane proteins, whereas TMHMM algorithm predicted that 1567 proteins had a transmembrane domain. Differences were observed in the expression of 159 membrane proteins and 55 extracellular proteins between polyps and cancer without metastasis, while the expression of 32 membrane proteins and 17 extracellular proteins differed between cancer with and without metastasis. A total of 105 of these biomarker candidates were quantitated using selected (or multiple) reaction monitoring (SRM/MRM) with stable synthetic isotope-labeled peptides as an internal control. The results obtained revealed differences in the expression of 69 of these proteins, and this was subsequently verified in an independent set of patient samples (polyps (n = 10), cancer without metastasis (n = 10), cancer with metastasis (n = 10)). Significant differences were observed in the expression of 44 of these proteins, including ITGA5, GPRC5A, PDGFRB, and TFRC, which have already been shown to be overexpressed in colorectal cancer, as well as proteins with unknown function, such as C8orf55. The expression of C8orf55 was also shown to be high not only in colorectal cancer, but also in several cancer tissues using a multicancer tissue microarray, which included 1150 cores from 14 cancer tissues. This is the largest verification study of biomarker candidate membrane proteins to date; our methods for biomarker discovery and subsequent validation using SRM/MRM will contribute to the identification of useful biomarker candidates for various cancers. Data are available via ProteomeXchange with identifier PXD000851. PMID:24687888

Discovery of colorectal cancer biomarker candidates by membrane proteomic analysis and subsequent verification using selected reaction monitoring (SRM) and tissue microarray (TMA) analysis.

PubMed

Kume, Hideaki; Muraoka, Satoshi; Kuga, Takahisa; Adachi, Jun; Narumi, Ryohei; Watanabe, Shio; Kuwano, Masayoshi; Kodera, Yoshio; Matsushita, Kazuyuki; Fukuoka, Junya; Masuda, Takeshi; Ishihama, Yasushi; Matsubara, Hisahiro; Nomura, Fumio; Tomonaga, Takeshi

2014-06-01

Recent advances in quantitative proteomic technology have enabled the large-scale validation of biomarkers. We here performed a quantitative proteomic analysis of membrane fractions from colorectal cancer tissue to discover biomarker candidates, and then extensively validated the candidate proteins identified. A total of 5566 proteins were identified in six tissue samples, each of which was obtained from polyps and cancer with and without metastasis. GO cellular component analysis predicted that 3087 of these proteins were membrane proteins, whereas TMHMM algorithm predicted that 1567 proteins had a transmembrane domain. Differences were observed in the expression of 159 membrane proteins and 55 extracellular proteins between polyps and cancer without metastasis, while the expression of 32 membrane proteins and 17 extracellular proteins differed between cancer with and without metastasis. A total of 105 of these biomarker candidates were quantitated using selected (or multiple) reaction monitoring (SRM/MRM) with stable synthetic isotope-labeled peptides as an internal control. The results obtained revealed differences in the expression of 69 of these proteins, and this was subsequently verified in an independent set of patient samples (polyps (n = 10), cancer without metastasis (n = 10), cancer with metastasis (n = 10)). Significant differences were observed in the expression of 44 of these proteins, including ITGA5, GPRC5A, PDGFRB, and TFRC, which have already been shown to be overexpressed in colorectal cancer, as well as proteins with unknown function, such as C8orf55. The expression of C8orf55 was also shown to be high not only in colorectal cancer, but also in several cancer tissues using a multicancer tissue microarray, which included 1150 cores from 14 cancer tissues. This is the largest verification study of biomarker candidate membrane proteins to date; our methods for biomarker discovery and subsequent validation using SRM/MRM will contribute to the identification of useful biomarker candidates for various cancers. Data are available via ProteomeXchange with identifier PXD000851. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger

PubMed Central

Schäpe, Paul; Müller-Hagen, Dirk; Ouedraogo, Jean-Paul; Heiderich, Caroline; Jedamzick, Johanna; van den Hondel, Cees A.; Ram, Arthur F.; Meyer, Vera

2016-01-01

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes. PMID:27835655

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger.

PubMed

Paege, Norman; Jung, Sascha; Schäpe, Paul; Müller-Hagen, Dirk; Ouedraogo, Jean-Paul; Heiderich, Caroline; Jedamzick, Johanna; Nitsche, Benjamin M; van den Hondel, Cees A; Ram, Arthur F; Meyer, Vera

2016-01-01

Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes.

The effects of HIV-1 regulatory TAT protein expression on brain reward function, response to psychostimulants and delay-dependent memory in mice.

PubMed

Kesby, James P; Markou, Athina; Semenova, Svetlana

2016-10-01

Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals. Copyright © 2016 Elsevier Ltd. All rights reserved.

FireProt: web server for automated design of thermostable proteins

PubMed Central

Musil, Milos; Stourac, Jan; Brezovsky, Jan; Prokop, Zbynek; Zendulka, Jaroslav; Martinek, Tomas

2017-01-01

Abstract There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot. PMID:28449074

Regulation of catalase expression in healthy and cancerous cells.

PubMed

Glorieux, Christophe; Zamocky, Marcel; Sandoval, Juan Marcelo; Verrax, Julien; Calderon, Pedro Buc

2015-10-01

Catalase is an important antioxidant enzyme that dismutates hydrogen peroxide into water and molecular oxygen. The catalase gene has all the characteristics of a housekeeping gene (no TATA box, no initiator element sequence, high GC content in promoter) and a core promoter that is highly conserved among species. We demonstrate in this review that within this core promoter, the presence of DNA binding sites for transcription factors, such as NF-Y and Sp1, plays an essential role in the positive regulation of catalase expression. Additional transcription factors, such as FoxO3a, are also involved in this regulatory process. There is strong evidence that the protein Akt/PKB in the PI3K signaling pathway plays a major role in the expression of catalase by modulating the activity of FoxO3a. Over the past decade, other transcription factors (PPARγ, Oct-1, etc.), as well as genetic, epigenetic, and posttranscriptional processes, have emerged as crucial contributors to the regulation of catalase expression. Altered expression levels of catalase have been reported in cancer tissues compared to their normal counterparts. Deciphering the molecular mechanisms that regulate catalase expression could, therefore, be of crucial importance for the future development of pro-oxidant cancer chemotherapy. Copyright © 2015. Published by Elsevier Inc.

Protease nexin-1 promotes secretory granule biogenesis by preventing granule protein degradation.

PubMed

Kim, Taeyoon; Loh, Y Peng

2006-02-01

Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation.

Protease Nexin-1 Promotes Secretory Granule Biogenesis by Preventing Granule Protein Degradation

PubMed Central

Kim, Taeyoon; Loh, Y. Peng

2006-01-01

Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation. PMID:16319172

Analysis of core-periphery organization in protein contact networks reveals groups of structurally and functionally critical residues.

PubMed

Isaac, Arnold Emerson; Sinha, Sitabhra

2015-10-01

The representation of proteins as networks of interacting amino acids, referred to as protein contact networks (PCN), and their subsequent analyses using graph theoretic tools, can provide novel insights into the key functional roles of specific groups of residues. We have characterized the networks corresponding to the native states of 66 proteins (belonging to different families) in terms of their core-periphery organization. The resulting hierarchical classification of the amino acid constituents of a protein arranges the residues into successive layers - having higher core order - with increasing connection density, ranging from a sparsely linked periphery to a densely intra-connected core (distinct from the earlier concept of protein core defined in terms of the three-dimensional geometry of the native state, which has least solvent accessibility). Our results show that residues in the inner cores are more conserved than those at the periphery. Underlining the functional importance of the network core, we see that the receptor sites for known ligand molecules of most proteins occur in the innermost core. Furthermore, the association of residues with structural pockets and cavities in binding or active sites increases with the core order. From mutation sensitivity analysis, we show that the probability of deleterious or intolerant mutations also increases with the core order. We also show that stabilization centre residues are in the innermost cores, suggesting that the network core is critically important in maintaining the structural stability of the protein. A publicly available Web resource for performing core-periphery analysis of any protein whose native state is known has been made available by us at http://www.imsc.res.in/ ~sitabhra/proteinKcore/index.html.

The cysteine-rich core domain of REIC/Dkk-3 is critical for its effect on monocyte differentiation and tumor regression.

PubMed

Kinoshita, Rie; Watanabe, Masami; Huang, Peng; Li, Shun-Ai; Sakaguchi, Masakiyo; Kumon, Hiromi; Futami, Junichiro

2015-06-01

Reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3 is a tumor-suppressor gene and has been studied as a promising therapeutic gene for cancer gene therapy. Intratumoral injection of an adenovirus vector carrying the human REIC/Dkk-3 gene (Ad-REIC) elicits cancer cell-specific apoptosis and anticancer immune responses. The cytokine-like effect of secretory REIC/Dkk-3 on the induction of dendritic cell (DC)-like cell differentiation from monocytes plays a role in systemic anticancer immunity. In the present study, we generated recombinant full-length and N-terminally truncated REIC/Dkk-3 to characterize the biological activity of the protein. During the purification procedure, we identified a 17 kDa cysteine-rich stable product (C17-REIC) showing limited degradation. Further analysis showed that the C17-REIC domain was sufficient for the induction of DC-like cell differentiation from monocytes. Concomitant with the differentiation of DCs, the REIC/Dkk-3 protein induced the phosphorylation of glycogen synthase kinase 3β (GSK-3β) and signal transducers and activators of transcription (STAT) at a level comparable to that of granulocyte/macrophage colony-stimulating factor. In a mouse model of subcutaneous renal adenocarcinoma, intraperitoneal injection of full-length and C17-REIC proteins exerted anticancer effects in parallel with the activation of immunocompetent cells such as DCs and cytotoxic T lymphocytes in peripheral blood. Taken together, our results indicate that the stable cysteine-rich core region of REIC/Dkk-3 is responsible for the induction of anticancer immune responses. Because REIC/Dkk-3 is a naturally circulating serum protein, the upregulation REIC/Dkk-3 protein expression could be a promising option for cancer therapy.

Expression of small leucine-rich proteoglycans in rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Syaidah, Rahimi; Fujiwara, Ken; Tsukada, Takehiro; Ramadhani, Dini; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

2013-01-01

Proteoglycans are components of the extracellular matrix and comprise a specific core protein substituted with covalently linked glycosaminoglycan chains. Small leucine-rich proteoglycans (SLRPs) are a major family of proteoglycans and have key roles as potent effectors in cellular signaling pathways. Research during the last two decades has shown that SLRPs regulate biological functions in many tissues such as skin, tendon, kidney, liver, and heart. However, little is known of the expression of SLRPs, or the characteristics of the cells that produce them, in the anterior pituitary gland. Therefore, we have determined whether SLRPs are present in rat anterior pituitary gland. We have used real-time reverse transcription with the polymerase chain reaction to analyze the expression of SLRP genes and have identified the cells that produce SLRPs by using in situ hybridization with a digoxigenin-labeled cRNA probe. We have clearly detected the mRNA expression of SLRP genes, and cells expressing decorin, biglycan, fibromodulin, lumican, proline/arginine-rich end leucine-rich repeat protein (PRELP), and osteoglycin are located in the anterior pituitary gland. We have also investigated the possible double-staining of SLRP mRNA and pituitary hormones, S100 protein (a marker of folliculostellate cells), desmin (a marker of capillary pericytes), and isolectin B4 (a marker of endothelial cells). Decorin, biglycan, fibromodulin, lumican, PRELP, and osteoglycin mRNA have been identified in S100-protein-positive and desmin-positive cells. Thus, we conclude that folliculostellate cells and pericytes produce SLRPs in rat anterior pituitary gland.

A core viral protein binds host nucleosomes to sequester immune danger signals

PubMed Central

Avgousti, Daphne C.; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J.; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C.; Blumenthal, Daniel; Paris, Andrew J.; Reyes, Emigdio D.; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H.; Worthen, G. Scott; Black, Ben E.; Garcia, Benjamin A.; Weitzman, Matthew D.

2016-01-01

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses1. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important role in innate immune responses2. Viral encoded core basic proteins compact viral genomes but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones3. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles4,5, it is unknown whether protein VII impacts cellular chromatin. Our observation that protein VII alters cellular chromatin led us to hypothesize that this impacts antiviral responses during adenovirus infection. We found that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in chromatin of members of the high-mobility group protein B family (HMGB1, HMGB2, and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses6,7. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling. PMID:27362237

Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells

PubMed Central

Finka, Andrija; Mattoo, Rayees U. H.

2010-01-01

Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases. Electronic supplementary material The online version of this article (doi:10.1007/s12192-010-0216-8) contains supplementary material, which is available to authorized users. PMID:20694844

Proteomics and bioinformatics analysis of altered protein expression in the placental villous tissue from early recurrent miscarriage patients.

PubMed

Pan, Hai-Tao; Ding, Hai-Gang; Fang, Min; Yu, Bin; Cheng, Yi; Tan, Ya-Jing; Fu, Qi-Qin; Lu, Bo; Cai, Hong-Guang; Jin, Xin; Xia, Xian-Qing; Zhang, Tao

2018-01-01

Recurrent miscarriage (RM) affects 5% of women, it has an adverse emotional impact on women. Because of the complexities of early development, the mechanism of recurrent miscarriage is still unclear. We hypothesized that abnormal placenta leads to early recurrent miscarriage (ERM). The aim of this study was to identify ERM associated factors in human placenta villous tissue using proteomics. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying recurrent miscarriage (RM). To gain more insight into mechanisms of recurrent miscarriage (RM), a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies was analyzed using iTRAQ technology and bioinformatics analysis used by Ingenuity Pathway Analysis (IPA) software. In this study, we employed an iTRAQ based proteomics analysis of four placental villous tissues from patients with early recurrent miscarriage (ERM) and four from normal pregnant women. Finally, we identified 2805 proteins and 79,998 peptides between patients with RM and normal matched group. Further analysis identified 314 differentially expressed proteins in placental villous tissue (≥1.3-fold, Student's t-test, p < 0.05); 209 proteins showed the increased expression while 105 proteins showed decreased expression. These 314 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the growth of embryo. Furthermore, network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development. We used another 8 independent samples (4 cases and 4 controls) to cross validation of the proteomic data. This study has identified several proteins that are associated with early development, these results may supply new insight into mechanisms behind recurrent miscarriage. Copyright © 2017 Elsevier Ltd. All rights reserved.

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus.

PubMed

Qian, Shasha; Chen, Xiaolan; Sun, Kai; Zhang, Yang; Li, Zhenghe

2017-06-13

Recovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus. In this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5' termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus. Optimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency. Our study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids.

Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

PubMed

Mattsson, J G; Ljunggren, E L; Bergström, K

2001-05-01

The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.

An intramolecular disulfide bridge between Cys-7 and Cys61 determines the structure of the secretory core gene product (e antigen) of hepatitis B virus.

PubMed Central

Nassal, M; Rieger, A

1993-01-01

Hepatitis B virus, the prototypic member of the Hepadnaviridae, is a small enveloped DNA virus that replicates via reverse transcription. Efficient usage of its compact 3.2-kb genome is exemplified by the pre-C/C gene from which two proteins with largely overlapping primary sequences but distinctly different properties are synthesized: the self-assembling core protein p21c (hepatitis B core antigen [HbcAg]) and the secretory, nonparticulate protein p17e (hepatitis B e antigen [HbeAg]). Mature p17e carries a 10-amino-acid N-terminal extension with a Cys residue (Cys-7). Using transient transfection of a human liver cell line with constructs expressing wild-type p17 or a series of Cys mutants of p17, we show that Cys-7 forms an intramolecular S-S bond to Cys61, which in assembly-competent core proteins is available for intermolecular disulfide bonds between two neighboring subunits. Removal of the Cys-7/Cys61 bond by mutating either residue has differential effects: in the absence of Cys-7, secretion is relatively efficient and independent of Cys61; however, the molecules are exported as homodimers exhibiting both HBe and HBc antigenicity. In the absence of Cys61, the nonpaired Cys-7 interferes with secretion efficiency. The amino acid sequence flanking Cys-7 also contributes to the formation of the proper intramolecular S-S bond. These results suggest that the Cys-7/Cys61 bond imposes on p17e a conformation that is critical for its secretion and distinct biophysical and antigenic properties. This mechanism adds selective disulfide formation to the repertoire of hepatitis B virus for efficient use of its tiny genome. Images PMID:8510224

The therapeutic effect of negative pressure in treating femoral head necrosis in rabbits.

PubMed

Zhang, Yin-gang; Wang, Xuezhi; Yang, Zhi; Zhang, Hong; Liu, Miao; Qiu, Yushen; Guo, Xiong

2013-01-01

Because negative pressure can stimulate vascular proliferation, improve blood circulation and promote osteogenic differentiation of bone marrow stromal cells, we investigated the therapeutic effect of negative pressure on femoral head necrosis (FHN) in a rabbit model. Animals were divided into four groups (n = 60/group): [1] model control, [2] core decompression, [3] negative pressure and [4] normal control groups. Histological investigation revealed that at 4 and 8 weeks postoperatively, improvements were observed in trabecular bone shape, empty lacunae and numbers of bone marrow hematopoietic cells and fat cells in the negative pressure group compared to the core decompression group. At week 8, there were no significant differences between the negative pressure and normal control groups. Immunohistochemistry staining revealed higher expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) in the femoral heads in the negative pressure group compared with the core decompression group. Transmission electron microscopy revealed that cell organelles were further developed in the negative pressure group compared with the core decompression group. Microvascular ink staining revealed an increased number of bone marrow ink-stained blood vessels, a thicker vascular lumen and increased microvascular density in the negative pressure group relative to the core decompression group. Real-time polymerase chain reaction revealed that expression levels of both VEGF and BMP-2 were higher in the negative pressure group compared with the core decompression group. In summary, negative pressure has a therapeutic effect on FHN. This effect is superior to core decompression, indicating that negative pressure is a potentially valuable method for treating early FHN.

The Therapeutic Effect of Negative Pressure in Treating Femoral Head Necrosis in Rabbits

PubMed Central

Zhang, Yin-gang; Wang, Xuezhi; Yang, Zhi; Zhang, Hong; Liu, Miao; Qiu, Yushen; Guo, Xiong

2013-01-01

Because negative pressure can stimulate vascular proliferation, improve blood circulation and promote osteogenic differentiation of bone marrow stromal cells, we investigated the therapeutic effect of negative pressure on femoral head necrosis (FHN) in a rabbit model. Animals were divided into four groups (n = 60/group): [1] model control, [2] core decompression, [3] negative pressure and [4] normal control groups. Histological investigation revealed that at 4 and 8 weeks postoperatively, improvements were observed in trabecular bone shape, empty lacunae and numbers of bone marrow hematopoietic cells and fat cells in the negative pressure group compared to the core decompression group. At week 8, there were no significant differences between the negative pressure and normal control groups. Immunohistochemistry staining revealed higher expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) in the femoral heads in the negative pressure group compared with the core decompression group. Transmission electron microscopy revealed that cell organelles were further developed in the negative pressure group compared with the core decompression group. Microvascular ink staining revealed an increased number of bone marrow ink-stained blood vessels, a thicker vascular lumen and increased microvascular density in the negative pressure group relative to the core decompression group. Real-time polymerase chain reaction revealed that expression levels of both VEGF and BMP-2 were higher in the negative pressure group compared with the core decompression group. In summary, negative pressure has a therapeutic effect on FHN. This effect is superior to core decompression, indicating that negative pressure is a potentially valuable method for treating early FHN. PMID:23383276

The Human Cytomegalovirus IE2 and UL112-113 Proteins Accumulate in Viral DNA Replication Compartments That Initiate from the Periphery of Promyelocytic Leukemia Protein-Associated Nuclear Bodies (PODs or ND10)

PubMed Central

Ahn, Jin-Hyun; Jang, Won-Jong; Hayward, Gary S.

1999-01-01

During human cytomegalovirus (HCMV) infection, the periphery of promyelocytic leukemia protein (PML)-associated nuclear bodies (also known as PML oncogenic domains [PODs] or ND10) are sites for both input viral genome deposition and immediate-early (IE) gene transcription. At very early times after infection, the IE1 protein localizes to and subsequently disrupts PODs, whereas the IE2 protein localizes within or adjacent to PODs. This process appears to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment formation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by confocal microscopy of human fibroblasts (HF cells) infected with both wild-type HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to disrupt PODs. In HCMV(Towne)-infected HF cells at 24 to 48 h, IE2 also accumulated in newly formed viral DNA replication compartments containing the polymerase processivity factor (UL44), the single-stranded DNA binding protein (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bromodeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiates within granular structures that bud from the periphery of some of the PODs and subsequently coalesce into larger structures that are flanked by PODs. In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the central region between them, were found to be necessary for both the punctate distribution of IE2 and its association with PODs. Like IE2, the UL112-113 accessory replication protein was also distributed in a POD-associated pattern in both DNA-transfected and virus-infected cells beginning at 6 h. Furthermore, when all six replication core machinery proteins (polymerase complex, SSB, and helicase-primase complex) were expressed together in the presence of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results show that (i) subsequent to accumulating at the periphery of PODs, IE2 is incorporated together with the core proteins into viral DNA replication compartments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to have a key role in assembling and recruiting the core replication machinery proteins in the initial stages of viral replication compartment formation. PMID:10559364

Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

PubMed

Rinker, Torri E; Philbrick, Brandon D; Temenoff, Johnna S

2017-07-01

Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation. Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic core-shell heparin-PEG microparticles presented here overcome this limitation by sequestering proteins through a PEG-based shell onto a protein-protective heparin core, temporarily isolating bound proteins from the cellular microenvironment, and re-delivering proteins only after degradation of the PEG-based shell. Thus, these core-shell microparticles have potential to be a novel tool to harness and isolate proteins produced in the cellular environment and then control when proteins are re-introduced for the most effective tissue regeneration and repair. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins.

PubMed

Wolffe, E J; Gause, W C; Pelfrey, C M; Holland, S M; Steinberg, A D; August, J T

1990-01-05

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins

PubMed Central

Hyun, Seong-In; Weisberg, Andrea

2017-01-01

ABSTRACT The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many aspects of cell biology. The I2 protein is conserved in all poxviruses that infect vertebrates, suggesting an important role. The present study revealed that this protein is essential for vaccinia virus morphogenesis and that its absence results in an accumulation of deformed virus particles retaining the scaffold protein and deficient in surface proteins needed for cell entry. PMID:28490596

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

PubMed

Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

2014-04-25

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity

DOE PAGES

Guo, Chun -Jun; Sun, Wei -Wen; Bruno, Kenneth S.; ...

2015-07-13

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPSlike genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas inmore » conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.« less

HCV Core Residues Critical for Infectivity Are Also Involved in Core-NS5A Complex Formation

PubMed Central

Gawlik, Katarzyna; Baugh, James; Chatterji, Udayan; Lim, Precious J.; Bobardt, Michael D.; Gallay, Philippe A.

2014-01-01

Hepatitis C virus (HCV) infection is a major cause of liver disease. The molecular machinery of HCV assembly and particle release remains obscure. A better understanding of the assembly events might reveal new potential antiviral strategies. It was suggested that the nonstructural protein 5A (NS5A), an attractive recent drug target, participates in the production of infectious particles as a result of its interaction with the HCV core protein. However, prior to the present study, the NS5A-binding site in the viral core remained unknown. We found that the D1 domain of core contains the NS5A-binding site with the strongest interacting capacity in the basic P38-K74 cluster. We also demonstrated that the N-terminal basic residues of core at positions 50, 51, 59 and 62 were required for NS5A binding. Analysis of all substitution combinations of R50A, K51A, R59A, and R62A, in the context of the HCVcc system, showed that single, double, triple, and quadruple mutants were fully competent for viral RNA replication, but deficient in secretion of viral particles. Furthermore, we found that the extracellular and intracellular infectivity of all the mutants was abolished, suggesting a defect in the formation of infectious particles. Importantly, we showed that the interaction between the single and quadruple core mutants and NS5A was impaired in cells expressing full-length HCV genome. Interestingly, mutations of the four basic residues of core did not alter the association of core or NS5A with lipid droplets. This study showed for the first time that basic residues in the D1 domain of core that are critical for the formation of infectious extracellular and intracellular particles also play a role in core-NS5A interactions. PMID:24533158

Vaccinia-related kinase 3 (VRK3) sets the circadian period and amplitude by affecting the subcellular localization of clock proteins in mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Nayoung; Department of Brain Science, Ajou University School of Medicine, 164 Worldcup-ro, Yeongtong-gu, Suwon, Kyunggi-do, 16499; Song, Jieun

In the eukaryotic circadian clock machinery, negative feedback repression of CLOCK (CLK) and BMAL1 transcriptional activity by PERIOD (PER) and CRYPTOCHROME (CRY) underlies the basis for 24 h rhythmic gene expression. Thus, precise regulation of the time-dependent nuclear entry of circadian repressors is crucial to generating normal circadian rhythms. Here, we sought to identify novel kinase(s) that regulate nuclear entry of mammalian CRY1 (mCRY1) with an unbiased screening using red fluorescent protein (RFP)-tagged human kinome expression plasmids in mammalian cells. Transient expression of human vaccinia-related kinase 3 (hVRK3) reduced the nuclear presence of mCRY1. hVRK3 expression also induced alterations in themore » subcellular localization of other core clock proteins, including mCRY2, mPER2, and BMAL1. In contrast, the subcellular localization of mCLK was not changed. Given that singly expressed mCLK mostly resides in the cytoplasm and that nuclear localization sequence (NLS) mutation of hVRK3 attenuated the effect of hVRK3 co-expression on subcellular localization, ectopically expressed hVRK3 presumably reduces the retention of proteins in the nucleus. Finally, downregulation of hvrk3 using siRNA reduced the amplitude and lengthened the period of the cellular bioluminescence rhythm. Taken together, these data suggest that VRK3 plays a role in setting the amplitude and period length of circadian rhythms in mammalian cells. - Highlights: • Screening was performed to identify kinases that regulate CRY1 subcellular localization. • VRK3 alters the subcellular localization of CRY1, CRY2, PER2, and BMAL1. • VRK3 knock-down alters the circadian bioluminescence rhythm in mammalian cells.« less

Single Cell Synchrotron FT-IR Microspectroscopy Reveals a Link between Neutral Lipid and Storage Carbohydrate Fluxes in S. cerevisiae

PubMed Central

Jamme, Frédéric; Vindigni, Jean-David; Méchin, Valérie; Cherifi, Tamazight; Chardot, Thierry; Froissard, Marine

2013-01-01

In most organisms, storage lipids are packaged into specialized structures called lipid droplets. These contain a core of neutral lipids surrounded by a monolayer of phospholipids, and various proteins which vary depending on the species. Hydrophobic structural proteins stabilize the interface between the lipid core and aqueous cellular environment (perilipin family of proteins, apolipoproteins, oleosins). We developed a genetic approach using heterologous expression in Saccharomyces cerevisiae of the Arabidopsis thaliana lipid droplet oleosin and caleosin proteins AtOle1 and AtClo1. These transformed yeasts overaccumulate lipid droplets, leading to a specific increase in storage lipids. The phenotype of these cells was explored using synchrotron FT-IR microspectroscopy to investigate the dynamics of lipid storage and cellular carbon fluxes reflected as changes in spectral fingerprints. Multivariate statistical analysis of the data showed a clear effect on storage carbohydrates and more specifically, a decrease in glycogen in our modified strains. These observations were confirmed by biochemical quantification of the storage carbohydrates glycogen and trehalose. Our results demonstrate that neutral lipid and storage carbohydrate fluxes are tightly connected and co-regulated. PMID:24040242

Impact of novel histone deacetylase inhibitors, CHAP31 and FR901228 (FK228), on adenovirus-mediated transgene expression.

PubMed

Taura, Kojiro; Yamamoto, Yuzo; Nakajima, Akio; Hata, Koichiro; Uchinami, Hiroshi; Yonezawa, Kei; Hatano, Etsuro; Nishino, Norikazu; Yamaoka, Yoshio

2004-05-01

Histone deacetylase inhibitors (HDIs) are known to enhance adenovirus (Ad)-mediated transgene expression. Recently, novel HDIs, including cyclic hydroxamic-acid-containing peptide 31 (CHAP31) and FR901228 (FK228), have been developed. The effects of these two novel HDIs on Ad-transduced or endogenous gene expression were investigated. Acetylation of core histones and the expression of the coxsackie and adenovirus receptor (CAR) in HDI-treated cells were examined using Western blot and a quantitative reverse transcription polymerase chain reaction (TaqMan RT-PCR), respectively. Their in vivo effect on adenoviral gene expression was investigated in BALB/c mice. Both compounds enhanced and prolonged Ad-mediated beta-galactosidase expression more effectively than did trichostatin A, a classic HDI. The same effect was observed in Ad-transduced heat shock protein 72 (HSP72), but not in hyperthermia-induced endogenous expression of HSP72, suggesting that the effect is specific for transduced gene expression. Hyperacetylation of core histones induced by HDIs was considered responsible for the augmentative effects of gene expression. Intravenous administration of either CHAP31 or FR901228 enhanced beta-galactosidase expression in mice infected with AdLacZ. CHAP31 and FR901228 amplified Ad-mediated transgene expression. The enhancement of transgene expression by HDIs may result in fewer vector doses for necessary gene expression, helping to alleviate disadvantages caused by Ad vectors. This could be a useful tool in overcoming current limitations of gene therapy using adenovirus vectors. Copyright 2004 John Wiley & Sons, Ltd.

The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter*

PubMed Central

Geisler, Christoph; Kotu, Varshika; Sharrow, Mary; Rendić, Dubravko; Pöltl, Gerald; Tiemeyer, Michael; Wilson, Iain B. H.; Jarvis, Donald L.

2012-01-01

Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac1 flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mistargeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac1 mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac1 Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac1 mutant. These results validate the Drosophila nac1 mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency. PMID:22745127

Engineering hepatitis B virus core particles for targeting HER2 receptors in vitro and in vivo.

PubMed

Mohamed Suffian, Izzat Fahimuddin Bin; Wang, Julie Tzu-Wen; Hodgins, Naomi O; Klippstein, Rebecca; Garcia-Maya, Mitla; Brown, Paul; Nishimura, Yuya; Heidari, Hamed; Bals, Sara; Sosabowski, Jane K; Ogino, Chiaki; Kondo, Akihiko; Al-Jamal, Khuloud T

2017-03-01

Hepatitis B Virus core (HBc) particles have been studied for their potential as drug delivery vehicles for cancer therapy. HBc particles are hollow nano-particles of 30-34 nm diameter and 7 nm thick envelopes, consisting of 180-240 units of 21 kDa core monomers. They have the capacity to assemble/dis-assemble in a controlled manner allowing encapsulation of various drugs and other biomolecules. Moreover, other functional motifs, i.e. receptors, receptor binding sequences, peptides and proteins can be expressed. This study focuses on the development of genetically modified HBc particles to specifically recognise and target human epidermal growth factor receptor-2 (HER2)-expressing cancer cells, in vitro and in vivo, for future cancer therapy. The non-specific binding capacity of wild type HBc particles was reduced by genetic deletion of the sequence encoding arginine-rich domains. A specific HER2-targeting was achieved by expressing the Z HER2 affibodies on the HBc particles surface. In vitro studies showed specific uptake of Z HER2 -ΔHBc particles in HER2 expressing cancer cells. In vivo studies confirmed positive uptake of Z HER2 -ΔHBc particles in HER2-expressing tumours, compared to non-targeted ΔHBc particles in intraperitoneal tumour-bearing mice models. The present results highlight the potential of these nanocarriers in targeting HER2-positive metastatic abdominal cancer following intra-peritoneal administration. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly

PubMed Central

Counihan, Natalie A.; Rawlinson, Stephen M.; Lindenbach, Brett D.

2011-01-01

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly. PMID:22028650

Paraspeckles: nuclear bodies built on long noncoding RNA

PubMed Central

Bond, Charles S.

2009-01-01

Paraspeckles are ribonucleoprotein bodies found in the interchromatin space of mammalian cell nuclei. These structures play a role in regulating the expression of certain genes in differentiated cells by nuclear retention of RNA. The core paraspeckle proteins (PSF/SFPQ, P54NRB/NONO, and PSPC1 [paraspeckle protein 1]) are members of the DBHS (Drosophila melanogaster behavior, human splicing) family. These proteins, together with the long nonprotein-coding RNA NEAT1 (MEN-ϵ/β), associate to form paraspeckles and maintain their integrity. Given the large numbers of long noncoding transcripts currently being discovered through whole transcriptome analysis, paraspeckles may be a paradigm for a class of subnuclear bodies formed around long noncoding RNA. PMID:19720872

Heat shock instructs hESCs to exit from the self-renewal program through negative regulation of OCT4 by SAPK/JNK and HSF1 pathway.

PubMed

Byun, Kyunghee; Kim, Taek-Kyun; Oh, Jeehyun; Bayarsaikhan, Enkhjargal; Kim, Daesik; Lee, Min Young; Pack, Chan-Gi; Hwang, Daehee; Lee, Bonghee

2013-11-01

Environmental factors affect self-renewal of stem cells by modulating the components of self-renewal networks. Heat shock, an environmental factor, induces heat shock factors (HSFs), which up-regulate stress response-related genes. However, the link of heat shock to self-renewal of stem cells has not been elucidated yet. Here, we present the direct link of heat shock to a core stem cell regulator, OCT4, in the self-renewal network through SAPK/JNK and HSF1 pathway. We first showed that heat shock initiated differentiation of human embryonic stem cells (hESCs). Gene expression analysis revealed that heat shock increased the expression of many genes involved in cellular processes related to differentiation of stem cells. We then examined the effects of HSFs induced by heat shock on core self-renewal factors. Among HSFs, heat shock induced mainly HSF1 in hESCs. The HSF1 repressed the expression of OCT4, leading to the differentiation of hESCs and the above differentiation-related gene expression change. We further examined the effects of the upstream MAP (mitogen-activated protein) kinases of HSF1 on the repression of OCT4 expression by HSF1. Among the MAP kinases, SAPK/JNK controlled predominantly the repression of the OCT4 expression by HSF1. The direct link of heat shock to the core self-renewal regulator through SAPK/JNK and HSF1 provides a fundamental basis for understanding the effect of heat and other stresses involving activation of HSF1 on the self-renewal program and further controlling differentiation of hESCs in a broad spectrum of stem cell applications using these stresses. © 2013.

Cholecystokinin (CCK)-expressing neurons in the suprachiasmatic nucleus: innervation, light responsiveness and entrainment in CCK-deficient mice.

PubMed

Hannibal, Jens; Hundahl, Christian; Fahrenkrug, Jan; Rehfeld, Jens F; Friis-Hansen, Lennart

2010-09-01

The suprachiasmatic nucleus (SCN) is the principal pacemaker driving circadian rhythms of physiology and behaviour. Neurons within the SCN express both classical and neuropeptide transmitters which regulate clock functions. Cholecyctokinin (CCK) is a potent neurotransmitter expressed in neurons of the mammalian SCN, but its role in circadian timing is not known. In the present study, CCK was demonstrated in a distinct population of neurons located in the shell region of the SCN and in a few cells in the core region. The CCK neurons did not express vasopressin or vasoactive intestinal peptide. However, CCK-containing processes make synaptic contacts with both groups of neurons and some CCK cell bodies were innervated by VIPergic neurons. The CCK neurons received no direct input from the three major pathways to the SCN, and the CCK neurons were not light-responsive as evaluated by induction of cFOS, and did not express the core clock protein PER1. Accordingly, CCK-deficient mice showed normal entrainment and had similar τ, light-induced phase shift and negative masking behaviour as wild-type animals. In conclusion, CCK signalling seems not to be involved directly in light-induced resetting of the clock or in regulating core clock function. The expression of CCK in a subpopulation of neurons, which do not belonging to either the VIP or AVP cells but which have synaptic contacts to both cell types and reverse innervation of CCK neurons from VIP neurons, suggests that the CCK neurons may act in non-photic regulation within the clock and/or, via CCK projections, mediate clock information to hypothalamic nuclei. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Core Histones and HIRIP3, a Novel Histone-Binding Protein, Directly Interact with WD Repeat Protein HIRA

PubMed Central

Lorain, Stéphanie; Quivy, Jean-Pierre; Monier-Gavelle, Frédérique; Scamps, Christine; Lécluse, Yann; Almouzni, Geneviève; Lipinski, Marc

1998-01-01

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second α helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development. PMID:9710638

Evaluation of the collaborative network of highly correlating skin proteins and its change following treatment with glucocorticoids

PubMed Central

2010-01-01

Background Glucocorticoids (GC) represent the core treatment modality for many inflammatory diseases. Its mode of action is difficult to grasp, not least because it includes direct modulation of many components of the extracellular matrix as well as complex anti-inflammatory effects. Protein expression profile of skin proteins is being changed with topical application of GC, however, the knowledge about singular markers in this regard is only patchy and collaboration is ill defined. Material/Methods Scar formation was observed under different doses of GC, which were locally applied on the back skin of mice (1 to 3 weeks). After euthanasia we analyzed protein expression of collagen I and III (picrosirius) in scar tissue together with 16 additional protein markers, which are involved in wound healing, with immunhistochemistry. For assessing GC's effect on co-expression we compared our results with a model of random figures to estimate how many significant correlations should be expected by chance. Results GC altered collagen and protein expression with distinct results in different areas of investigation. Most often we observed a reduced expression after application of low dose GC. In the scar infiltrate a multivariate analysis confirmed the significant impact of both GC concentrations. Calculation of Spearman's correlation coefficient similarly resulted in a significant impact of GC, and furthermore, offered the possibility to grasp the entire interactive profile in between all variables studied. The biological markers, which were connected by significant correlations could be arranged in a highly cross-linked network that involved most of the markers measured. A marker highly cross-linked with more than 3 significant correlations was indicated by a higher variation of all its correlations to the other variables, resulting in a standard deviation of > 0.2. Conclusion In addition to immunohistochemical analysis of single protein markers multivariate analysis of co-expressions by use of correlation coefficients reveals the complexity of biological relationships and identifies complex biological effects of GC on skin scarring. Depiction of collaborative clusters will help to understand functional pathways. The functional importance of highly cross-linked proteins will have to be proven in subsequent studies. PMID:20509951

Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

PubMed

Anand, Sanjeev K; Gaba, Amit; Singh, Jaswant; Tikoo, Suresh K

2014-02-01

Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

Exploring the key genes and pathways in enchondromas using a gene expression microarray.

PubMed

Shi, Zhongju; Zhou, Hengxing; Pan, Bin; Lu, Lu; Kang, Yi; Liu, Lu; Wei, Zhijian; Feng, Shiqing

2017-07-04

Enchondromas are the most common primary benign osseous neoplasms that occur in the medullary bone; they can undergo malignant transformation into chondrosarcoma. However, enchondromas are always undetected in patients, and the molecular mechanism is unclear. To identify key genes and pathways associated with the occurrence and development of enchondromas, we downloaded the gene expression dataset GSE22855 and obtained the differentially expressed genes (DEGs) by analyzing high-throughput gene expression in enchondromas. In total, 635 genes were identified as DEGs. Of these, 225 genes (35.43%) were up-regulated, and the remaining 410 genes (64.57%) were down-regulated. We identified the predominant gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were significantly over-represented in the enchondromas samples compared with the control samples. Subsequently the top 10 core genes were identified from the protein-protein interaction (PPI) network. The enrichment analyses of the genes mainly involved in two significant modules showed that the DEGs were principally related to ribosomes, protein digestion and absorption, ECM-receptor interaction, focal adhesion, amoebiasis and the PI3K-Akt signaling pathway.Together, these data elucidate the molecular mechanisms underlying the occurrence and development of enchondromas and provide promising candidates for therapeutic intervention and prognostic evaluation. However, further experimental studies are needed to confirm these results.

Prmt7 is dispensable in tissue culture models for adipogenic differentiation.

PubMed

Hu, Yu-Jie; Sif, Saïd; Imbalzano, Anthony N

2013-01-01

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models.

Prmt7 is dispensable in tissue culture models for adipogenic differentiation

PubMed Central

Imbalzano, Anthony N.

2013-01-01

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models. PMID:24715966

Quantifying the Effect of DNA Packaging on Gene Expression Level

NASA Astrophysics Data System (ADS)

Kim, Harold

2010-10-01

Gene expression, the process by which the genetic code comes alive in the form of proteins, is one of the most important biological processes in living cells, and begins when transcription factors bind to specific DNA sequences in the promoter region upstream of a gene. The relationship between gene expression output and transcription factor input which is termed the gene regulation function is specific to each promoter, and predicting this gene regulation function from the locations of transcription factor binding sites is one of the challenges in biology. In eukaryotic organisms (for example, animals, plants, fungi etc), DNA is highly compacted into nucleosomes, 147-bp segments of DNA tightly wrapped around histone protein core, and therefore, the accessibility of transcription factor binding sites depends on their locations with respect to nucleosomes - sites inside nucleosomes are less accessible than those outside nucleosomes. To understand how transcription factor binding sites contribute to gene expression in a quantitative manner, we obtain gene regulation functions of promoters with various configurations of transcription factor binding sites by using fluorescent protein reporters to measure transcription factor input and gene expression output in single yeast cells. In this talk, I will show that the affinity of a transcription factor binding site inside and outside the nucleosome controls different aspects of the gene regulation function, and explain this finding based on a mass-action kinetic model that includes competition between nucleosomes and transcription factors.

Identification and expression analysis of four 14-3-3 genes during fruit ripening in banana (Musa acuminata L. AAA group, cv. Brazilian).

PubMed

Li, Mei-Ying; Xu, Bi-Yu; Liu, Ju-Hua; Yang, Xiao-Liang; Zhang, Jian-Bin; Jia, Cai-Hong; Ren, Li-Cheng; Jin, Zhi-Qiang

2012-02-01

To investigate the regulation of 14-3-3 proteins in banana (Musa acuminata L. AAA group, cv. Brazilian) fruit postharvest ripening, four cDNAs encoding 14-3-3 proteins were isolated from banana and designated as Ma-14-3-3a, Ma-14-3-3c, Ma-14-3-3e, and Ma-14-3-3i, respectively. Amino acid sequence alignment showed that the four 14-3-3 proteins shared a highly conserved core structure and variable C-terminal as well as N-terminal regions with 14-3-3 proteins from other plant species. Phylogenetic analysis revealed that the four 14-3-3 genes belong to the non-ε groups. They were differentially and specifically expressed in various tissues. Real-time RT-PCR analysis indicated that these four genes function differentially during banana fruit postharvest ripening. Three genes, Ma-14-3-3a, Ma-14-3-3c, and Ma-14-3-3e, were significantly induced by exogenous ethylene treatment. However, gene function differed in naturally ripened fruits. Ethylene could induce Ma-14-3-3c expression during postharvest ripening, but expression patterns of Ma-14-3-3a and Ma-14-3-3e suggest that these two genes appear to be involved in regulating ethylene biosynthesis during fruit ripening. No obvious relationship emerged between Ma-14-3-3i expression in naturally ripened and 1-MCP (1-methylcyclopropene)-treated fruit groups during fruit ripening. These results indicate that the 14-3-3 proteins might be involved in various regulatory processes of banana fruit ripening. Further studies will mainly focus on revealing the detailed biological mechanisms of these four 14-3-3 genes in regulating banana fruit postharvest ripening.

A Method for Predicting Protein Complexes from Dynamic Weighted Protein-Protein Interaction Networks.

PubMed

Liu, Lizhen; Sun, Xiaowu; Song, Wei; Du, Chao

2018-06-01

Predicting protein complexes from protein-protein interaction (PPI) network is of great significance to recognize the structure and function of cells. A protein may interact with different proteins under different time or conditions. Existing approaches only utilize static PPI network data that may lose much temporal biological information. First, this article proposed a novel method that combines gene expression data at different time points with traditional static PPI network to construct different dynamic subnetworks. Second, to further filter out the data noise, the semantic similarity based on gene ontology is regarded as the network weight together with the principal component analysis, which is introduced to deal with the weight computing by three traditional methods. Third, after building a dynamic PPI network, a predicting protein complexes algorithm based on "core-attachment" structural feature is applied to detect complexes from each dynamic subnetworks. Finally, it is revealed from the experimental results that our method proposed in this article performs well on detecting protein complexes from dynamic weighted PPI networks.

Abiotic stress signaling and responses in plants

PubMed Central

Zhu, Jian-Kang

2016-01-01

Summary As sessile organisms, plants must cope with abiotic stress such as soil salinity, drought, and extreme temperatures. Core stress signaling pathways involve protein kinases related to the yeast SNF1 and mammalian AMPK, suggesting that stress signaling in plants evolved from energy sensing. Stress signaling regulates proteins critical for ion and water transport and for metabolic and gene-expression reprogramming to bring about ionic and water homeostasis and cellular stability under stress conditions. Understanding stress signaling and responses will increase our ability to improve stress resistance in crops to achieve agricultural sustainability and food security for a growing world population. PMID:27716505

RIC-7 Promotes Neuropeptide Secretion

PubMed Central

Hao, Yingsong; Hu, Zhitao; Sieburth, Derek; Kaplan, Joshua M.

2012-01-01

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV–mediated secretion. PMID:22275875

[The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016].

PubMed

Pumpens, P; Grens, E

2016-01-01

This review article is a continuation of the paper "Hepatitis B core particles as a universal display model: a structure-function basis for development" written by Pumpens P. and Grens E., ordered by Professor Lev Kisselev and published in FEBS Letters, 1999, 442, 1-6. The past 17 years have strengthened the paper's finding that the human hepatitis B virus core protein, along with other Hepadnaviridae family member core proteins, is a mysterious, multifunctional protein. The core gene of the Hepadnaviridae genome encodes five partially collinear proteins. The most important of these is the HBV core protein p21, or HBc. It can self-assemble by forming viral HBc particles, but also plays a crucial role in the regulation of viral replication. Since 1986, the HBc protein has been one of the first and the most successful tools of the virus-like particle (VLP) technology. Later, the woodchuck hepatitis virus core protein (WHc) was also used as a VLP carrier. The Hepadnaviridae core proteins remain favourite VLP candidates for the knowledge-based design of future vaccines, gene therapy vectors, specifically targeted nanocontainers, and other modern nanotechnological tools for prospective medical use.

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae.

PubMed

Ivanyi-Nagy, Roland; Lavergne, Jean-Pierre; Gabus, Caroline; Ficheux, Damien; Darlix, Jean-Luc

2008-02-01

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning-possibly mediated by intrinsically disordered protein segments-is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication.

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae

PubMed Central

Ivanyi-Nagy, Roland; Lavergne, Jean-Pierre; Gabus, Caroline; Ficheux, Damien; Darlix, Jean-Luc

2008-01-01

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication. PMID:18033802

Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1

PubMed Central

Mao, Yang; Resende, Mafalda; Daugaard, Mads; Riis Kristensen, Anders; Damm, Peter; G. Theander, Thor; R. Hansson, Stefan; Salanti, Ali

2016-01-01

During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells. PMID:27556547

Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

PubMed Central

Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J.; Molina, María

2013-01-01

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smialowska, Agata, E-mail: smialowskaa@gmail.com; School of Life Sciences, Södertörn Högskola, Huddinge 141-89; Djupedal, Ingela

Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its rolemore » in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.« less

Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli.

PubMed

Pesavento, Christina; Becker, Gisela; Sommerfeldt, Nicole; Possling, Alexandra; Tschowri, Natalia; Mehlis, Anika; Hengge, Regine

2008-09-01

During the transition from post-exponential to stationary phase, Escherichia coli changes from the motile-planktonic to the adhesive-sedentary "lifestyle." We demonstrate this transition to be controlled by mutual inhibition of the FlhDC/motility and sigma(S)/adhesion control cascades at two distinct hierarchical levels. At the top level, motility gene expression and the general stress response are inversely coordinated by sigma(70)/sigma(FliA)/sigma(S) competition for core RNA polymerase and the FlhDC-controlled FliZ protein acting as a sigma(S) inhibitor. At a lower level, the signaling molecule bis-(3'-5')-cyclic-diguanosine monophosphate (c-di-GMP) reduces flagellar activity and stimulates transcription of csgD, which encodes an essential activator of adhesive curli fimbriae expression. This c-di-GMP is antagonistically controlled by sigma(S)-regulated GGDEF proteins (mainly YegE) and YhjH, an EAL protein and c-di-GMP phosphodiesterase under FlhDC/FliA control. The switch from motility-based foraging to the general stress response and curli expression requires sigma(S)-modulated down-regulation of expression of the flagellar regulatory cascade as well as proteolysis of the flagellar master regulator FlhDC. Control of YhjH by FlhDC and of YegE by sigma(S) produces a fine-tuned checkpoint system that "unlocks" curli expression only after down-regulation of flagellar gene expression. In summary, these data reveal the logic and sequence of molecular events underlying the motile-to-adhesive "lifestyle" switch in E. coli.

Two Novel Rab2 Interactors Regulate Dense-core Vesicle Maturation

PubMed Central

Ailion, Michael; Hannemann, Mandy; Dalton, Susan; Pappas, Andrea; Watanabe, Shigeki; Hegermann, Jan; Liu, Qiang; Han, Hsiao-Fen; Gu, Mingyu; Goulding, Morgan Q.; Sasidharan, Nikhil; Schuske, Kim; Hullett, Patrick; Eimer, Stefan; Jorgensen, Erik M.

2014-01-01

Summary Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi, and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1 and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a new pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

Packing in protein cores

NASA Astrophysics Data System (ADS)

Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

2017-07-01

Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

Virus-producing cells determine the host protein profiles of HIV-1 virion cores

PubMed Central

2012-01-01

Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells. Conclusions Profiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN. PMID:22889230

The transcriptomic fingerprint of glucoamylase over-expression in Aspergillus niger

PubMed Central

2012-01-01

Background Filamentous fungi such as Aspergillus niger are well known for their exceptionally high capacity for secretion of proteins, organic acids, and secondary metabolites and they are therefore used in biotechnology as versatile microbial production platforms. However, system-wide insights into their metabolic and secretory capacities are sparse and rational strain improvement approaches are therefore limited. In order to gain a genome-wide view on the transcriptional regulation of the protein secretory pathway of A. niger, we investigated the transcriptome of A. niger when it was forced to overexpression the glaA gene (encoding glucoamylase, GlaA) and secrete GlaA to high level. Results An A. niger wild-type strain and a GlaA over-expressing strain, containing multiple copies of the glaA gene, were cultivated under maltose-limited chemostat conditions (specific growth rate 0.1 h-1). Elevated glaA mRNA and extracellular GlaA levels in the over-expressing strain were accompanied by elevated transcript levels from 772 genes and lowered transcript levels from 815 genes when compared to the wild-type strain. Using GO term enrichment analysis, four higher-order categories were identified in the up-regulated gene set: i) endoplasmic reticulum (ER) membrane translocation, ii) protein glycosylation, iii) vesicle transport, and iv) ion homeostasis. Among these, about 130 genes had predicted functions for the passage of proteins through the ER and those genes included target genes of the HacA transcription factor that mediates the unfolded protein response (UPR), e.g. bipA, clxA, prpA, tigA and pdiA. In order to identify those genes that are important for high-level secretion of proteins by A. niger, we compared the transcriptome of the GlaA overexpression strain of A. niger with six other relevant transcriptomes of A. niger. Overall, 40 genes were found to have either elevated (from 36 genes) or lowered (from 4 genes) transcript levels under all conditions that were examined, thus defining the core set of genes important for ensuring high protein traffic through the secretory pathway. Conclusion We have defined the A. niger genes that respond to elevated secretion of GlaA and, furthermore, we have defined a core set of genes that appear to be involved more generally in the intensified traffic of proteins through the secretory pathway of A. niger. The consistent up-regulation of a gene encoding the acetyl-coenzyme A transporter suggests a possible role for transient acetylation to ensure correct folding of secreted proteins. PMID:23237452

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

PubMed Central

Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

2012-01-01

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29(+/−)/MxCre(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine. PMID:23284733

Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

PubMed

Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

2012-01-01

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

L-type calcium channel blockers and substance P induce angiogenesis of cortical vessels associated with beta-amyloid plaques in an Alzheimer mouse model.

PubMed

Daschil, Nina; Kniewallner, Kathrin M; Obermair, Gerald J; Hutter-Paier, Birgit; Windisch, Manfred; Marksteiner, Josef; Humpel, Christian

2015-03-01

It is well established that L-type calcium channels (LTCCs) are expressed in astroglia. However, their functional role is still speculative, especially under pathologic conditions. We recently showed that the α1 subunit-like immunoreactivity of the CaV1.2 channel is strongly expressed in reactive astrocytes around beta-amyloid plaques in 11-month-old Alzheimer transgenic (tg) mice with the amyloid precursor protein London and Swedish mutations. The aim of the present study was to examine the cellular expression of all LTCC subunits around beta-amyloid plaques by in situ hybridization using (35)S-labeled oligonucleotides. Our data show that messenger RNAs (mRNAs) of the LTCC CaV1.2 α1 subunit as well as all auxiliary β and α2δ subunits, except α2δ-4, were expressed in the hippocampus of age-matched wild-type mice. It was unexpected to see, that cells directly located in the plaque core in the cortex expressed mRNAs for CaV1.2 α1, β2, β4, and α2δ-1, whereas no expression was detected in the halo. Furthermore, cells in the plaque core also expressed preprotachykinin-A mRNA, the precursor for substance P. By means of confocal microscopy, we demonstrated that collagen-IV-stained brain vessels in the cortex were associated with the plaque core and were immunoreactive for substance P. In cortical organotypic brain slices of adult Alzheimer mice, we could demonstrate that LTCC blockers increased angiogenesis, which was further potentiated by substance P. In conclusion, our data show that brain vessels associated with beta-amyloid plaques express substance P and an LTCC and may play a role in angiogenesis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Molecular Cloning, Expression Profile and 5′ Regulatory Region Analysis of Two Chemosensory Protein Genes from the Diamondback Moth, Plutella xylostella

PubMed Central

Gong, Liang; Zhong, Guo-Hua; Hu, Mei-Ying; Luo, Qian; Ren, Zhen-Zhen

2010-01-01

Chemosensory proteins play an important role in transporting chemical compounds to their receptors on dendrite membranes. In this study, two full-length cDNA codings for chemosensory proteins of Plutella xylostella (Lepidoptera: Plutellidae) were obtained by RACE-PCR. PxylCSP3 and Pxyl-CSP4, with GenBank accession numbers ABM92663 and ABM92664, respectively, were cloned and sequenced. The gene sequences both consisted of three exons and two introns. RT-PCR analysis showed that Pxyl-CSP3 and Pxyl-CSP4 had different expression patterns in the examined developmental stages, but were expressed in all larval stages. Phylogenetic analysis indicated that lepidopteran insects consist of three branches, and Pxyl-CSP3 and Pxyl-CSP4 belong to different branches. The 5′regulatory regions of Pxyl-CSP3 and Pxyl-CSP4 were isolated and analyzed, and the results consist of not only the core promoter sequences (TATA-box), but also several transcriptional elements (BR-C Z4, Hb, Dfd, CF2-II, etc.). This study provides clues to better understanding the various physiological functions of CSPs in P. xylostella and other insects. PMID:21073345

Global identification and expression analysis of stress-responsive genes of the Argonaute family in apple.

PubMed

Xu, Ruirui; Liu, Caiyun; Li, Ning; Zhang, Shizhong

2016-12-01

Argonaute (AGO) proteins, which are found in yeast, animals, and plants, are the core molecules of the RNA-induced silencing complex. These proteins play important roles in plant growth, development, and responses to biotic stresses. The complete analysis and classification of the AGO gene family have been recently reported in different plants. Nevertheless, systematic analysis and expression profiling of these genes have not been performed in apple (Malus domestica). Approximately 15 AGO genes were identified in the apple genome. The phylogenetic tree, chromosome location, conserved protein motifs, gene structure, and expression of the AGO gene family in apple were analyzed for gene prediction. All AGO genes were phylogenetically clustered into four groups (i.e., AGO1, AGO4, MEL1/AGO5, and ZIPPY/AGO7) with the AGO genes of Arabidopsis. These groups of the AGO gene family were statistically analyzed and compared among 31 plant species. The predicted apple AGO genes are distributed across nine chromosomes at different densities and include three segment duplications. Expression studies indicated that 15 AGO genes exhibit different expression patterns in at least one of the tissues tested. Additionally, analysis of gene expression levels indicated that the genes are mostly involved in responses to NaCl, PEG, heat, and low-temperature stresses. Hence, several candidate AGO genes are involved in different aspects of physiological and developmental processes and may play an important role in abiotic stress responses in apple. To the best of our knowledge, this study is the first to report a comprehensive analysis of the apple AGO gene family. Our results provide useful information to understand the classification and putative functions of these proteins, especially for gene members that may play important roles in abiotic stress responses in M. hupehensis.

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro

PubMed Central

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3′ untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication. PMID:15141033

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro.

PubMed

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection

PubMed Central

Suomalainen, Maarit; Zheng, Yueting; Boucke, Karin

2017-01-01

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein—protein VII. Two other Ad core proteins, V and X/μ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII−virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection. PMID:28628648

Genetic ablation of phosphatidylcholine transfer protein/StarD2 in ob/ob mice improves glucose tolerance without increasing energy expenditure.

PubMed

Krisko, Tibor I; LeClair, Katherine B; Cohen, David E

2017-03-01

Phosphatidylcholine transfer protein (PC-TP; synonym StarD2) is highly expressed in liver and oxidative tissues. PC-TP promotes hepatic glucose production during fasting and aggravates glucose intolerance in high fat fed mice. However, because PC-TP also suppresses thermogenesis in brown adipose tissue (BAT), its direct contribution to obesity-associated diabetes in mice remains unclear. Here we examined the effects of genetic PC-TP ablation on glucose homeostasis in leptin-deficient ob/ob mice, which exhibit both diabetes and altered thermoregulation. Mice lacking both PC-TP and leptin (Pctp -/- ;ob/ob) were prepared by crossing Pctp -/- with ob/+ mice. Glucose homeostasis was assessed by standard assays, and energy expenditure was determined by indirect calorimetry using a comprehensive laboratory animal monitoring system, which also recorded physical activity and food intake. Body composition was determined by NMR and hepatic lipids by enzymatic assays. Core body temperature was measured using a rectal thermocouple probe. Pctp -/- ;ob/ob mice demonstrated improved glucose homeostasis, as evidenced by markedly improved glucose and pyruvate tolerance tests, without changes in insulin tolerance. However, there were no differences in EE at any ambient temperature. There were also no effects of PC-TP expression on physical activity, food intake or core body temperature. Improved glucose tolerance in Pctp -/- ;ob/ob mice in the absence of increases in energy expenditure or core body temperature indicates a direct pathogenic role for PC-TP in diabetes in leptin deficient mice. Copyright © 2016 Elsevier Inc. All rights reserved.

Circadian locomotor output cycles kaput affects the proliferation and migration of breast cancer cells by regulating the expression of E-cadherin via IQ motif containing GTPase activating protein 1.

PubMed

Li, Xiaoxue; Wang, Siyang; Yang, Shuhong; Ying, Junjie; Yu, Hang; Yang, Chunlei; Liu, Yanyou; Wang, Yuhui; Cheng, Shuting; Xiao, Jing; Guo, Huiling; Jiang, Zhou; Wang, Zhengrong

2018-05-01

The circadian rhythm regulates numerous physiological activities, including sleep and wakefulness, behavior, immunity and metabolism. Previous studies have demonstrated that circadian rhythm disorder is associated with the occurrence of tumors. Responsible for regulating a number of functions, the Circadian locomotor output cycles kaput ( Clock ) gene is one of the core regulatory genes of circadian rhythm. The Clock gene has also been implicated in the occurrence and development of tumors in previously studies. The present study evaluated the role of the Clock gene in the proliferation and migration of mouse breast cancer 4T1 cells, and investigated its possible regulatory pathways and mechanisms. It was reported that downregulation of Clock facilitated the proliferation and migration of breast cancer cells. Further investigation revealed the involvement of IQ motif containing GTPase activating protein 1 (IQGAP1) protein expression in the Clock regulatory pathway, further influencing the expression of E-cadherin, a known proprietor of tumor cell migration and invasion. To the best of our knowledge, the present study is the first to report that Clock , acting through the regulation of the scaffolding protein IQGAP1, regulates the downstream expression of E-cadherin, thereby affecting tumor cell structure and motility. These results confirmed the role of Clock in breast cancer tumor etiology and provide insight regarding the molecular avenues of its regulatory nature, which may translate beyond breast cancer into other known functions of the gene.

A DNA replicon system for rapid high-level production of virus-like particles in plants.

PubMed

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles; Mason, Hugh

2009-07-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. (c) 2009 Wiley Periodicals, Inc.

A DNA replicon system for rapid high-level production of virus-like particles in plants

PubMed Central

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles

2009-01-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low level antigen accumulation and long time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this paper, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within five days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without p19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. PMID:19309755

Systems and Photosystems: Cellular Limits of Autotrophic Productivity in Cyanobacteria

PubMed Central

Burnap, Robert L.

2014-01-01

Recent advances in the modeling of microbial growth and metabolism have shown that growth rate critically depends upon the optimal allocation of finite proteomic resources among different cellular functions and that modeling growth rates becomes more realistic with the explicit accounting for the costs of macromolecular synthesis, most importantly, protein expression. The “proteomic constraint” is considered together with its application to understanding photosynthetic microbial growth. The central hypothesis is that physical limits of cellular space (and corresponding solvation capacity) in conjunction with cell surface-to-volume ratios represent the underlying constraints on the maximal rate of autotrophic microbial growth. The limitation of cellular space thus constrains the size the total complement of macromolecules, dissolved ions, and metabolites. To a first approximation, the upper limit in the cellular amount of the total proteome is bounded this space limit. This predicts that adaptation to osmotic stress will result in lower maximal growth rates due to decreased cellular concentrations of core metabolic proteins necessary for cell growth owing the accumulation of compatible osmolytes, as surmised previously. The finite capacity of membrane and cytoplasmic space also leads to the hypothesis that the species-specific differences in maximal growth rates likely reflect differences in the allocation of space to niche-specific proteins with the corresponding diminution of space devoted to other functions including proteins of core autotrophic metabolism, which drive cell reproduction. An optimization model for autotrophic microbial growth, the autotrophic replicator model, was developed based upon previous work investigating heterotrophic growth. The present model describes autotrophic growth in terms of the allocation protein resources among core functional groups including the photosynthetic electron transport chain, light-harvesting antennae, and the ribosome groups. PMID:25654078

Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope1[OPEN

PubMed Central

Tanabe, Noriaki; Clarke, Adrian K.

2016-01-01

The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone’s interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import. PMID:26586836

Structure of human Fe-S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP-ISD11 interactions.

PubMed

Cory, Seth A; Van Vranken, Jonathan G; Brignole, Edward J; Patra, Shachin; Winge, Dennis R; Drennan, Catherine L; Rutter, Jared; Barondeau, David P

2017-07-03

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.

The malaria PTEX component PTEX88 interacts most closely with HSP101 at the host-parasite interface.

PubMed

Chisholm, Scott A; Kalanon, Ming; Nebl, Thomas; Sanders, Paul R; Matthews, Kathryn M; Dickerman, Benjamin K; Gilson, Paul R; de Koning-Ward, Tania F

2018-04-10

The pathogenic nature of malaria infections is due in part to the export of hundreds of effector proteins that actively remodel the host erythrocyte. The Plasmodium translocon of exported proteins (PTEX) has been shown to facilitate the trafficking of proteins into the host cell, a process that is essential for the survival of the parasite. The role of the auxiliary PTEX component PTEX88 remains unclear, as previous attempts to elucidate its function through reverse genetic approaches showed that in contrast to the core components PTEX150 and HSP101, knockdown of PTEX88 did not give rise to an export phenotype. Here, we have used biochemical approaches to understand how PTEX88 assembles within the translocation machinery. Proteomic analysis of the PTEX88 interactome showed that PTEX88 interacts closely with HSP101 but has a weaker affinity with the other core constituents of PTEX. PTEX88 was also found to associate with other PV-resident proteins, including chaperones and members of the exported protein-interacting complex that interacts with the major virulence factor PfEMP1, the latter contributing to cytoadherence and parasite virulence. Despite being expressed for the duration of the blood-stage life cycle, PTEX88 was only discretely observed at the parasitophorous vacuole membrane during ring stages and could not always be detected in the major high molecular weight complex that contains the other core components of PTEX, suggesting that its interaction with the PTEX complex may be dynamic. Together, these data have enabled the generation of an updated model of PTEX that now includes how PTEX88 assembles within the complex. © 2018 Federation of European Biochemical Societies.

Analysis of the distribution and expression of claudin-1 tight junction protein in the oral cavity.

PubMed

Ouban, Abderrahman; Ahmed, Atif

2015-07-01

Claudins are the main sealing proteins of the intercellular tight junctions and play an important role in cancer cell progression and dissemination. The authors have previously shown that overexpression of claudin-1 is associated with angiolymphatic and perineural invasion, consistent with aggressive tumor behavior and with advanced stage disease in oral squamous cell carcinomas (OSCCs). Our goal in this study was to examine claudin-1 expression in a tissue microarray of OSCCs taken from multiple sites within the oral cavity. This study examined and compared the expression of claudin-1 by immunohistochemistry in 60 tissue samples (49 OSCCs and 10 cases of non-neoplastic tissue, single core per case) were analyzed for claudin-1 expression by immunohistochemistry. The tumors included SCCs from the tongue (n=28), the cheek (n=9), gingival (n=4), lip (n=3), and oral cavity (n=5). Nonmalignant normal oral mucosa from the tongue (unmatched cases, n=2). Cancer adjacent tissue samples were taken from the tongue (n=6), gingival (n=2), and palate (n=1). This study demonstrates the expression of claudin-1 protein across a sample of OSCCs originating from multiple locations in the oral cavity. The highest expression of claudin-1 was observed in well-differentiated OSCCs, whereas poorly differentiated OSCCs exhibited mostly negative staining for claudin-1. In addition, we hereby report differential pattern of expression among tumors of different sites within the oral cavity, and between benign and cancerous samples. Our understanding of the exact function and role of claudin-1 in tumorigenesis is expanding exponentially.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

The structure of the core NuRD repression complex provides insights into its interaction with chromatin

PubMed Central

Millard, Christopher J; Varma, Niranjan; Saleh, Almutasem; Morris, Kyle; Watson, Peter J; Bottrill, Andrew R; Fairall, Louise; Smith, Corinne J; Schwabe, John WR

2016-01-01

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin. DOI: http://dx.doi.org/10.7554/eLife.13941.001 PMID:27098840

Heat treatment of unclarified Escherichia coli homogenate improved the recovery efficiency of recombinant hepatitis B core antigen.

PubMed

Ng, Michelle Y T; Tan, Wen Siang; Abdullah, Norhafizah; Ling, Tau Chuan; Tey, Beng Ti

2006-10-01

Heat precipitation procedure has been regularly incorporated as a selective purification step in various thermostable proteins expressed in different hosts. This method is efficient in precipitation of most of the host proteins and also deactivates various host proteases that can be harmful to the desired gene products. In this study, introduction of heat treatment procedure in the purification of hepatitis B core antigen (HBcAg) produced in Escherichia coli has been investigated. Thermal treatment of the cell homogenate at 60 degrees C for 30 min prior to subsequent clarification steps has resulted in 1.4 times and 18% higher in purity and recovery yield, respectively, compared to the non-heat-treated cell homogenate. In direct capture of HBcAg by using anion-exchangers from unclarified feedstock, pre-conditioning the feedstock by heat treatment at 60 degrees C for 45 min has increased the recovery yield of HBcAg by 2.9-fold and 42% in purity compared to that treated for 10 min. Enzyme-linked immunosorbent assay (ELISA) analysis showed that the antigenicity of the core particles was not affected by the heat treatment process.

Genomic analysis reveals a tight link between transcription factor dynamics and regulatory network architecture.

PubMed

Jothi, Raja; Balaji, S; Wuster, Arthur; Grochow, Joshua A; Gsponer, Jörg; Przytycka, Teresa M; Aravind, L; Babu, M Madan

2009-01-01

Although several studies have provided important insights into the general principles of biological networks, the link between network organization and the genome-scale dynamics of the underlying entities (genes, mRNAs, and proteins) and its role in systems behavior remain unclear. Here we show that transcription factor (TF) dynamics and regulatory network organization are tightly linked. By classifying TFs in the yeast regulatory network into three hierarchical layers (top, core, and bottom) and integrating diverse genome-scale datasets, we find that the TFs have static and dynamic properties that are similar within a layer and different across layers. At the protein level, the top-layer TFs are relatively abundant, long-lived, and noisy compared with the core- and bottom-layer TFs. Although variability in expression of top-layer TFs might confer a selective advantage, as this permits at least some members in a clonal cell population to initiate a response to changing conditions, tight regulation of the core- and bottom-layer TFs may minimize noise propagation and ensure fidelity in regulation. We propose that the interplay between network organization and TF dynamics could permit differential utilization of the same underlying network by distinct members of a clonal cell population.

The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion

PubMed Central

Etienne, Loïc; Blanchard, Emmanuelle; Boyer, Audrey; Desvignes, Virginie; Gaillard, Julien; Meunier, Jean-Christophe; Roingeard, Philippe; Hourioux, Christophe

2015-01-01

Hepatitis C virus (HCV) assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD) surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER) membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis. PMID:26339783

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

PubMed Central

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

RSR-2, the Caenorhabditis elegans Ortholog of Human Spliceosomal Component SRm300/SRRM2, Regulates Development by Influencing the Transcriptional Machinery

PubMed Central

Fontrodona, Laura; Porta-de-la-Riva, Montserrat; Morán, Tomás; Niu, Wei; Díaz, Mònica; Aristizábal-Corrales, David; Villanueva, Alberto; Schwartz, Simó; Reinke, Valerie; Cerón, Julián

2013-01-01

Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the Caenorhabditis elegans ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway. Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences C. elegans development. PMID:23754964

Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett

Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specificmore » antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.« less

Lactobacillus casei Low-Temperature, Dairy-Associated Proteome Promotes Persistence in the Mammalian Digestive Tract.

PubMed

Lee, Bokyung; Tachon, Sybille; Eigenheer, Richard A; Phinney, Brett S; Marco, Maria L

2015-08-07

We found that incubation of probiotic Lactobacillus casei BL23 in milk at 4 °C prior to ingestion increased its survival in the mammalian digestive tract. To investigate the specific molecular adaptations of L. casei to milk, we used tandem mass spectrometry to compare proteins produced by L. casei BL23 at 4 °C in milk to those in exponential and stationary phase cells in laboratory culture medium at either 37 or 4 °C. These comparisons revealed a core of expressed L. casei proteins as well as proteins produced in either a growth-phase or temperature-specific manner. In total, 205 L. casei proteins were uniquely expressed or detected in higher abundance specifically as a result of incubation in milk and included an over-representation of proteins for cell surface modification, fatty acid metabolism, amino acid transport and metabolism, and inorganic ion transport. Genes for DltD (d-alanine transfer protein), FabH (3-oxoacyl-ACP synthase), RecA (recombinase A), and Sod (superoxide dismutase) were targeted for inactivation. The competitive fitness of the mutants was altered in the mouse intestine compared with wild-type cells. These results show that the food matrix can have a profound influence on dietary (probiotic) bacteria and their functional significance in the mammalian gut.

Revisiting the substrate specificity of mammalian α1,6-fucosyltransferase reveals that it catalyzes core fucosylation of N-glycans lacking α1,3-arm GlcNAc.

PubMed

Yang, Qiang; Zhang, Roushu; Cai, Hui; Wang, Lai-Xi

2017-09-08

The mammalian α1,6-fucosyltransferase (FUT8) catalyzes the core fucosylation of N -glycans in the biosynthesis of glycoproteins. Previously, intensive in vitro studies with crude extract or purified enzyme concluded that the attachment of a GlcNAc on the α1,3 mannose arm of N -glycan is essential for FUT8-catalyzed core fucosylation. In contrast, we have recently shown that expression of erythropoietin in a GnTI knock-out, FUT8-overexpressing cell line results in the production of fully core-fucosylated glycoforms of the oligomannose substrate Man 5 GlcNAc 2 , suggesting that FUT8 can catalyze core fucosylation of N -glycans lacking an α1,3-arm GlcNAc in cells. Here, we revisited the substrate specificity of FUT8 by examining its in vitro activity toward an array of selected N -glycans, glycopeptides, and glycoproteins. Consistent with previous studies, we found that free N -glycans lacking an unmasked α1,3-arm GlcNAc moiety are not FUT8 substrates. However, Man 5 GlcNAc 2 glycan could be efficiently core-fucosylated by FUT8 in an appropriate protein/peptide context, such as with the erythropoietin protein, a V3 polypeptide derived from HIV-1 gp120, or a simple 9-fluorenylmethyl chloroformate-protected Asn moiety. Interestingly, when placed in the V3 polypeptide context, a mature bi-antennary complex-type N -glycan also could be core-fucosylated by FUT8, albeit at much lower efficiency than the Man 5 GlcNAc 2 peptide. This study represents the first report of in vitro FUT8-catalyzed core fucosylation of N -glycans lacking the α1,3-arm GlcNAc moiety. Our results suggest that an appropriate polypeptide context or other adequate structural elements in the acceptor substrate could facilitate the core fucosylation by FUT8. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Border patrol: insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders.

PubMed

Farach-Carson, Mary C; Warren, Curtis R; Harrington, Daniel A; Carson, Daniel D

2014-02-01

The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550 M years) extracellular matrix molecules. In vertebrates, perlecan's five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously suggested, to that of a critical agent for establishing and patrolling tissue borders in complex tissues in metazoans. We also propose that understanding these unique functions of the individual portions of the perlecan molecule can provide new insights and tools for engineering of complex multi-layered tissues including providing the necessary cues for establishing neotissue borders. © 2013.

Border Patrol: Insights into the Unique Role of Perlecan/Heparan Sulfate Proteoglycan2 at Cell and Tissue Borders

PubMed Central

Farach-Carson, Mary C.; Warren, Curtis R.; Harrington, Daniel A.; Carson, Daniel D.

2013-01-01

The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550M years) extracellular matrix molecules. In vertebrates, perlecan’s five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously suggested, to that of a critical agent for establishing and patrolling tissue borders in complex tissues in metazoans. We also propose that understanding these unique functions of the individual portions of the perlecan molecule can provide new insights and tools for engineering of complex multi-layered tissues including providing the necessary cues for establishing neotissue borders. PMID:24001398

Adolescent social defeat increases adult amphetamine conditioned place preference and alters D2 dopamine receptor expression

PubMed Central

Burke, Andrew R.; Watt, Michael J.; Forster, Gina L.

2011-01-01

Components of the brain’s dopaminergic system, such as dopamine receptors, undergo final maturation in adolescence. Exposure to social stress during human adolescence contributes to substance abuse behaviors. We utilized a rat model of adolescent social stress to investigate the neural mechanisms underlying this correlation. Rats exposed to repeated social defeat in adolescence (P35–P39) exhibited increased conditioned place preference (CPP) for amphetamine (1 mg/kg) in adulthood (P70). In contrast, rats experiencing foot-shock during the same developmental period exhibited amphetamine CPP levels similar to non-stressed controls. Our previous experiments suggested adolescent defeat alters dopamine activity in the mesocorticolimbic system. Furthermore, dopamine receptors have been implicated in the expression of amphetamine CPP. Therefore, we hypothesized that alteration to dopamine receptor expression in the mesocorticolimbic system may be associated with to heightened amphetamine CPP of adult rats exposed to adolescence defeat. We measured D1 and D2 dopamine receptor protein content in the medial prefrontal cortex, nucleus accumbens (NAc) and dorsal striatum following either adolescent social defeat or foot-shock stress and then adult amphetamine CPP. In controls, amphetamine CPP training reduced D2 receptor protein content in the NAc core. However, this down-regulation of NAc core D2 receptors was blocked by exposure to social defeat but not foot-shock stress in adolescence. These results suggest social defeat stress in adolescence alters the manner in which later amphetamine exposure down-regulates D2 receptors. Furthermore, persistent alterations to adult D2 receptor expression and amphetamine responses may depend on the type of stress experienced in adolescence. PMID:21933700

Small RNA sorting: matchmaking for Argonautes

PubMed Central

Czech, Benjamin; Hannon, Gregory J.

2013-01-01

Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes — microRNAs (miRNAs) and small interfering RNAs (siRNAs) — in plants and animals. PMID:21116305

Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.

PubMed

Guan, Chengran; Cui, Wenjing; Cheng, Jintao; Zhou, Li; Guo, Junling; Hu, Xu; Xiao, Guoping; Zhou, Zhemin

2015-09-21

Bacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA). The promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the -10 and -35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level. The auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression system that has an expression pattern that is split between cell-growth and over-expression, leading to an increase in cell density and elevating the overall expression levels of heterologously expressed proteins. The broad applicability of this system and inducer-free expression property in B. subtilis facilitate the industrial scale-up and medical applications for the over-production of a variety of desired proteins.

Dysregulation of gene expression in the striatum of BACHD rats expressing full-length mutant huntingtin and associated abnormalities on molecular and protein levels.

PubMed

Yu-Taeger, Libo; Bonin, Michael; Stricker-Shaver, Janice; Riess, Olaf; Nguyen, Hoa Huu Phuc

2017-05-01

Huntington disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for the huntingtin protein (HTT). Mutant HTT (mHTT) has been proposed to cause neuronal dysfunction and neuronal loss through multiple mechanisms. Transcriptional changes may be a core pathogenic feature of HD. Utilizing the Affymetrix platform we performed a genome-wide RNA expression analysis in two BACHD transgenic rat lines (TG5 and TG9) at 12 months of age, both of which carry full-length human mHTT but with different expression levels. By defining the threshold of significance at p < 0.01, we found 1608 genes and 871 genes differentially expressed in both TG5 and TG9 rats when compared to the wild type littermates, respectively. We only chose the highly up-/down-regulated genes for further analysis by setting an additional threshold of 1.5 fold change. Comparing gene expression profiles of human HD brains and BACHD rats revealed a high concordance in both functional and IPA (Ingenuity Pathway Analysis) canonical pathways relevant to HD. In addition, we investigated the causes leading to gene expression changes at molecular and protein levels in BACHD rats including the involvement of polyQ-containing transcription factors TATA box-binding protein (TBP), Sp1 and CBP as well as the chromatin structure. We demonstrate that the BACHD rat model recapitulates the gene expression changes of the human disease supporting its role as a preclinical research animal model. We also show for the first time that TFIID complex formation is reduced, while soluble TBP is increased in an HD model. This finding suggests that mHTT is a competitor instead of a recruiter of polyQ-containing transcription factors in the transcription process in HD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Patterns of gene expression associated with recovery and injury in heat-stressed rats.

PubMed

Stallings, Jonathan D; Ippolito, Danielle L; Rakesh, Vineet; Baer, Christine E; Dennis, William E; Helwig, Bryan G; Jackson, David A; Leon, Lisa R; Lewis, John A; Reifman, Jaques

2014-12-03

The in vivo gene response associated with hyperthermia is poorly understood. Here, we perform a global, multiorgan characterization of the gene response to heat stress using an in vivo conscious rat model. We heated rats until implanted thermal probes indicated a maximal core temperature of 41.8°C (Tc,Max). We then compared transcriptomic profiles of liver, lung, kidney, and heart tissues harvested from groups of experimental animals at Tc,Max, 24 hours, and 48 hours after heat stress to time-matched controls kept at an ambient temperature. Cardiac histopathology at 48 hours supported persistent cardiac injury in three out of six animals. Microarray analysis identified 78 differentially expressed genes common to all four organs at Tc,Max. Self-organizing maps identified gene-specific signatures corresponding to protein-folding disorders in heat-stressed rats with histopathological evidence of cardiac injury at 48 hours. Quantitative proteomics analysis by iTRAQ (isobaric tag for relative and absolute quantitation) demonstrated that differential protein expression most closely matched the transcriptomic profile in heat-injured animals at 48 hours. Calculation of protein supersaturation scores supported an increased propensity of proteins to aggregate for proteins that were found to be changing in abundance at 24 hours and in animals with cardiac injury at 48 hours, suggesting a mechanistic association between protein misfolding and the heat-stress response. Pathway analyses at both the transcript and protein levels supported catastrophic deficits in energetics and cellular metabolism and activation of the unfolded protein response in heat-stressed rats with histopathological evidence of persistent heat injury, providing the basis for a systems-level physiological model of heat illness and recovery.

Inducible colitis-associated glycome capable of stimulating the proliferation of memory CD4+ T cells.

PubMed

Nishida, Atsushi; Nagahama, Kiyotaka; Imaeda, Hirotsugu; Ogawa, Atsuhiro; Lau, Cindy W; Kobayashi, Taku; Hisamatsu, Tadakazu; Preffer, Frederic I; Mizoguchi, Emiko; Ikeuchi, Hiroki; Hibi, Toshifumi; Fukuda, Minoru; Andoh, Akira; Blumberg, Richard S; Mizoguchi, Atsushi

2012-12-17

Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on the cell surface, which is known as the glycome. In this study, we propose that a unique glycome that can be identified through the binding of galectin-4 is created on local, but not systemic, memory CD4+ T cells under diverse intestinal inflammatory conditions, but not in the healthy state. The colitis-associated glycome (CAG) represents an immature core 1-expressing O-glycan. Development of CAG may be mediated by down-regulation of the expression of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, a key enzyme responsible for the production of core-2 O-glycan branch through addition of N-acetylglucosamine (GlcNAc) to a core-1 O-glycan structure. Mechanistically, the CAG seems to contribute to super raft formation associated with the immunological synapse on colonic memory CD4+ T cells and to the consequent stabilization of protein kinase C θ activation, resulting in the stimulation of memory CD4+ T cell expansion in the inflamed intestine. Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell-mediated experimental intestinal inflammations. Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

Effect of hypoxia on metabolic rate, core body temperature, and c-fos expression in the naked mole rat.

PubMed

Nathaniel, Thomas I; Otukonyong, Effiong; Abdellatif, Ahmed; Soyinka, Julius O

2012-10-01

Recent investigations of hypoxia physiology in the naked mole rat have opened up an interesting line of research into the basic physiological and genomic alterations that accompany hypoxia survival. The extent to which such findings connect the effect of hypoxia to metabolic rate (O₂ consumption), core body temperature (Tb), and transcripts encoding the immediate early gene product (such as c-fos) under a constant ambient temperature (Ta) is not well known. We investigated this issue in the current study. Our first sets of experiments measured Tb and metabolic rates during exposure of naked mole rats to hypoxia over a constant Ta. Hypoxia significantly decreased metabolic rates in the naked mole rat. Although core Tb also decreased during hypoxia, the effect of hypoxia in suppressing core Tb was not significant. The second series of experiments revealed that c-fos protein and mRNA expression in the hippocampus neurons (CA1) increased in naked mole rats that were repeatedly exposed to 3% O₂ for 60 min per day for 5 days when compared to normoxia. Our findings provide evidence for the up-regulation of c-fos and suppression of metabolic rate in hypoxia tolerating naked mole rats under constant ambient temperature. Metabolic suppression and c-fos upregulation constitute part of the physiological complex associated with adaptation to hypoxia. Published by Elsevier Ltd.

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

PubMed

Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

2017-08-01

The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many aspects of cell biology. The I2 protein is conserved in all poxviruses that infect vertebrates, suggesting an important role. The present study revealed that this protein is essential for vaccinia virus morphogenesis and that its absence results in an accumulation of deformed virus particles retaining the scaffold protein and deficient in surface proteins needed for cell entry. Copyright © 2017 American Society for Microbiology.

Identification of Major Signaling Pathways in Prion Disease Progression Using Network Analysis

PubMed Central

Newaz, Khalique; Sriram, K.; Bera, Debajyoti

2015-01-01

Prion diseases are transmissible neurodegenerative diseases that arise due to conformational change of normal, cellular prion protein (PrPC) to protease-resistant isofrom (rPrPSc). Deposition of misfolded PrpSc proteins leads to an alteration of many signaling pathways that includes immunological and apoptotic pathways. As a result, this culminates in the dysfunction and death of neuronal cells. Earlier works on transcriptomic studies have revealed some affected pathways, but it is not clear which is (are) the prime network pathway(s) that change during the disease progression and how these pathways are involved in crosstalks with each other from the time of incubation to clinical death. We perform network analysis on large-scale transcriptomic data of differentially expressed genes obtained from whole brain in six different mouse strain-prion strain combination models to determine the pathways involved in prion diseases, and to understand the role of crosstalks in disease propagation. We employ a notion of differential network centrality measures on protein interaction networks to identify the potential biological pathways involved. We also propose a crosstalk ranking method based on dynamic protein interaction networks to identify the core network elements involved in crosstalk with different pathways. We identify 148 DEGs (differentially expressed genes) potentially related to the prion disease progression. Functional association of the identified genes implicates a strong involvement of immunological pathways. We extract a bow-tie structure that is potentially dysregulated in prion disease. We also propose an ODE model for the bow-tie network. Predictions related to diseased condition suggests the downregulation of the core signaling elements (PI3Ks and AKTs) of the bow-tie network. In this work, we show using transcriptomic data that the neuronal dysfunction in prion disease is strongly related to the immunological pathways. We conclude that these immunological pathways occupy influential positions in the PFNs (protein functional networks) that are related to prion disease. Importantly, this functional network involvement is prevalent in all the five different mouse strain-prion strain combinations that we studied. We also conclude that the dysregulation of the core elements of the bow-tie structure, which belongs to PI3K-Akt signaling pathway, leads to dysregulation of the downstream components corresponding to other biological pathways. PMID:26646948

Abscisic acid-dependent regulation of small rubber particle protein gene expression in Taraxacum brevicorniculatum is mediated by TbbZIP1.

PubMed

Fricke, Julia; Hillebrand, Andrea; Twyman, Richard M; Prüfer, Dirk; Schulze Gronover, Christian

2013-04-01

Natural rubber is a high-molecular-mass biopolymer found in the latex of >2,500 plant species, including Hevea brasiliensis, Parthenium argentatum and Taraxacum spp. The active sites of rubber biosynthesis are rubber particles, which comprise a hydrophobic rubber core surrounded by a phospholipid monolayer membrane containing species-dependent lipids and associated proteins. Small rubber particle proteins are the most abundant rubber particle-associated proteins in Taraxacum brevicorniculatum (TbSRPPs) and may promote rubber biosynthesis by stabilizing the rubber particle architecture. We investigated the transcriptional regulation of genes encoding SRPPs and identified a bZIP transcription factor (TbbZIP.1) similar to the Arabidopsis thaliana ABI5-ABF-AREB subfamily, which is thought to include downstream targets of ABA and/or abiotic stress-inducible protein kinases. The TbbZIP.1 gene was predominantly expressed in laticifers and regulates the expression of TbSRPP genes in an ABA-dependent manner. The individual TbSRPP genes showed distinct induction profiles, suggesting diverse roles in rubber biosynthesis and stress adaptation. The potential involvement of TbSRPPs in the adaptation of T. brevicorniculatum plants to environmental stress is discussed based on our current knowledge of the stress-response roles of SRPPs and their homologs, and the protective function of latex and rubber against pathogens. Our data suggest that TbSRPPs contribute to stress tolerance in T. brevicorniculatum and that their effects are mediated by TbbZIP.1.

Comparative transcriptome analysis to investigate the potential role of miRNAs in milk protein/fat quality.

PubMed

Wang, Xuehui; Zhang, Li; Jin, Jing; Xia, Anting; Wang, Chunmei; Cui, Yingjun; Qu, Bo; Li, Qingzhang; Sheng, Chunyan

2018-04-19

miRNAs play an important role in the processes of cell differentiation, biological development, and physiology. Here we investigated the molecular mechanisms regulating milk secretion and quality in dairy cows via transcriptome analyses of mammary gland tissues from dairy cows during the high-protein/high-fat, low-protein/low-fat or dry periods. To characterize the important roles of miRNAs and mRNAs in milk quality and to elucidate their regulatory networks in relation to milk secretion and quality, an integrated analysis was performed. A total of 25 core miRNAs were found to be differentially expressed (DE) during lactation compared to non-lactation, and these miRNAs were involved in epithelial cell terminal differentiation and mammary gland development. In addition, comprehensive analysis of mRNA and miRNA expression between high-protein/high-fat group and low-protein/low-fat groups indicated that, 38 miRNAs and 944 mRNAs were differentially expressed between them. Furthermore, 38 DE miRNAs putatively negatively regulated 253 DE mRNAs. The putative genes (253 DE mRNAs) were enriched in lipid biosynthetic process and amino acid transmembrane transporter activity. Moreover, putative DE genes were significantly enriched in fatty acid (FA) metabolism, biosynthesis of amino acids, synthesis and degradation of ketone bodies and biosynthesis of unsaturated FAs. Our results suggest that DE miRNAs might play roles as regulators of milk quality and milk secretion during mammary gland differentiation.

Regulation and Functional Expression of Cinnamate 4-Hydroxylase from Parsley

PubMed Central

Koopmann, Edda; Logemann, Elke; Hahlbrock, Klaus

1999-01-01

A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10). Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley. The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues. These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit. PMID:9880345

Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis

PubMed Central

2011-01-01

Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland. PMID:21324116

Construction of nanometer cisplatin core-ferritin (NCC-F) and proteomic analysis of gastric cancer cell apoptosis induced with cisplatin released from the NCC-F.

PubMed

Ji, Xue-Tao; Huang, Lin; Huang, He-Qing

2012-06-18

Both transmission electron microscopy (TEM) and fluorescence spectrometry were used to reveal the characteristics of both subunit disassociation and recombination in apo-pig pancreas ferritin (apoPPF) in an alkaline medium ranging reversibly from pH 7.0 to 13.0. The experimental results indicated that apoPPF could be completely disassociated into 24 free subunits at pH 13.0, and then these subunits could be quickly reassembled into the original apoPPF once the pH of the reactive medium was returned to pH7.0. This novel and simple method could be used to effectively construct a novel nanometer cisplatin core-PPF (NCC-PPF). The major characteristics of NCC-PPF were investigated using various analytical methods such as ultraviolet-spectrometry, circular dichroism spectrometry and TEM, which indicated that its molecular structure was still similar to that of the original apoPPF. Results from the inductively coupled plasma mass spectrometer (ICP-MS) method showed that 11.26 cisplatin (CDDP) molecules were successfully packaged within the NCC-PPF shell, indicating that each molecule of apoPPF had the ability to enwrap 11.26 CDDP molecules for constructing the NCC-PPF. Flow cytometry showed that NCC-PPF also had the ability to release CDDP for inducing the apoptosis of gastric cancer cells BGC823 (GCC), but this phenomenon could scarcely be observed using apoPPF. A differential proteomic technique using two-dimensional electrophoresis (2-DE) gels selected and identified the differential proteins from cell apoptosis in order to reveal the molecular pathway of GCC apoptosis by both NCC-PPF and free CDDP, giving 13 differential expression proteins. These differential proteins could be further classified into six groups, which were described as being involved in the regulation of apoptosis, RNA transcription, oxidative stress response, signal transduction, cell metabolism, and cytoskeleton changes. In addition, a real-time PCR method was used to prove the expression level of mRNA and to identify the reliability of the protein expression according to these differential proteins, which indicated that the mRNA level changes of six differential proteins corresponded to those of its differential protein expression in 2-DE gels. These studies played an important role in reasonably revealing the different pathways of GCC apoptosis induced with both the CDDP released by NCC-PPF and the free CDDP. We thus suggest that apoPPF has great potential for constructing a nanometer carrier filled with various drugs for application in clinical work. Copyright © 2012 Elsevier B.V. All rights reserved.

Interferon-alpha-induced changes in metallothionein expression in liver biopsies from patients with chronic hepatitis C.

PubMed

Nagamine, Takeaki; Suzuki, Keiji; Kondo, Toshihiko; Nakazato, Kyomi; Kakizaki, Satoru; Takagi, Hitoshi; Nakajima, Katuyuki

2005-08-01

An association between reactive oxygen species and liver damage has been postulated in the course of hepatitis C virus (HCV) infection. Metallothionein (MT), induced by HCV core protein and interferon (IFN), plays a role in scavenging free radicals. MT expression in liver biopsies obtained from 21 patients with chronic HCV infection before and after IFN-alpha therapy was investigated. Changes in Knodell histological activity index (HAI) scores, MT protein levels (immunohistochemistry), MT-I and MT-II messenger (m)RNA expression levels (in situ hybridization) and proliferating cell nuclear antigen (PCNA) labelling index were determined and compared in serial liver specimens. MT staining was clustered around the portal tracts with inflammatory cells and fibrosis. The pattern of MT protein before IFN-alpha therapy was similar in all patients, but was higher in IFN-sustained responders than in nonresponders after IFN-alpha therapy. HAI scores and PCNA labelling indexes were significantly reduced after IFN-alpha therapy. MT-II mRNA expression correlated positively with PCNA index before therapy and with HAI scores after therapy (P<0.05). No correlation was found between MT-I mRNA and HAI scores or PCNA index. The findings indicate that IFN-alpha-induced hepatic MT may participate in the therapeutic effects of IFN-alpha for HCV. In addition, MT-II mRNA expression may be involved in cell proliferation in the livers of patients with chronic HCV infection.

Oil body proteins sequentially accumulate throughout seed development in Brassica napus.

PubMed

Jolivet, Pascale; Boulard, Céline; Bellamy, Annick; Valot, Benoît; d'Andréa, Sabine; Zivy, Michel; Nesi, Nathalie; Chardot, Thierry

2011-11-15

Despite the importance of seed oil bodies (OBs) as enclosed compartments for oil storage, little is known about lipid and protein accumulation in OBs during seed formation. OBs from rapeseed (Brassica napus) consist of a triacylglycerol (TAG) core surrounded by a phospholipid monolayer embedded with integral proteins which confer high stability to OBs in the mature dry seed. In the present study, we investigated lipid and protein accumulation patterns throughout seed development (from 5 to 65 days after pollination [DAP]) both in the whole seed and in purified OBs. Deposition of the major proteins (oleosins, caleosins and steroleosins) into OBs was assessed through (i) gene expression pattern, (ii) proteomics analysis, and (iii) protein immunodetection. For the first time, a sequential deposition of integral OB proteins was established. Accumulation of oleosins and caleosins was observed starting from early stages of seed development (12-17 DAP), while steroleosins accumulated later (~25 DAP) onwards. Copyright © 2011 Elsevier GmbH. All rights reserved.

Proteome Analysis of Borrelia burgdorferi Response to Environmental Change

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angel, Thomas E.; Luft, Benjamin J.; Yang, Xiaohua

2010-11-02

We examined global changes in protein expression in the B31 strain of Borrelia burgdorferi, in response to two environmental cues (pH and temperature) chosen for their reported similarity to those encountered at different stages of the organism’s life cycle. Multidimensional nano-liquid chromatographic separations coupled with tandem mass spectrometry were used to examine the array of proteins (i.e., the proteome) of B. burgdorferi for different pH and temperature culture conditions. Changes in pH and temperature elicited in vitro adaptations of this spirochete known to cause Lyme disease and led to alterations in protein expression that are associated with increased microbial pathogenesis.more » We identified 1031 proteins that represent 59% of the annotated genome of B. burgdorferi and elucidated a core proteome of 414 proteins that were present in all environmental conditions investigated. Observed changes in protein abundances indicated varied replicon usage, as well as proteome functional distributions between the in vitro cell culture conditions. Surprisingly, the pH and temperature conditions that mimicked B. burgdorferi residing in the gut of a fed tick showed a marked reduction in protein diversity. Additionally, the results provide us with leading candidates for exploring how B. burgdorferi adapts to and is able to survive in a wide variety of environmental conditions and lay a foundation for planned in situ studies of B. burgdorferi isolated from the tick midgut and infected animals.« less

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression.

PubMed

Robertson, M; Chandler, P M

1994-11-01

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich (core sequence KIKEK-LPG). This antiserum detected a novel M(r) 40,000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequenced differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin. The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance. The M(r) 40,000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.

FBXO11 promotes ubiquitination of the Snail family of transcription factors in cancer progression and epidermal development.

PubMed

Jin, Yue; Shenoy, Anitha K; Doernberg, Samuel; Chen, Hao; Luo, Huacheng; Shen, Huangxuan; Lin, Tong; Tarrash, Miriam; Cai, Qingsong; Hu, Xin; Fiske, Ryan; Chen, Ting; Wu, Lizi; Mohammed, Kamal A; Rottiers, Veerle; Lee, Siu Sylvia; Lu, Jianrong

2015-06-28

The Snail family of transcription factors are core inducers of epithelial-to-mesenchymal transition (EMT). Here we show that the F-box protein FBXO11 recognizes and promotes ubiquitin-mediated degradation of multiple Snail family members including Scratch. The association between FBXO11 and Snai1 in vitro is independent of Snai1 phosphorylation. Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and cellular invasiveness. Conversely, depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion, as well as loss of estrogen receptor expression in breast cancer cells. Expression of FBXO11 is downregulated by EMT-inducing signals TGFβ and nickel. In human cancer, high FBXO11 levels correlate with expression of epithelial markers and favorable prognosis. The results suggest that FBXO11 sustains the epithelial state and inhibits cancer progression. Inactivation of FBXO11 in mice leads to neonatal lethality, epidermal thickening, and increased Snail protein levels in epidermis, validating that FBXO11 is a physiological ubiquitin ligase of Snail. Moreover, in C. elegans, the FBXO11 mutant phenotype is attributed to the Snail factors as it is suppressed by inactivation/depletion of Snail homologs. Collectively, these findings suggest that the FBXO11-Snail regulatory axis is evolutionarily conserved and critically governs carcinoma progression and mammalian epidermal development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

De novo design of the hydrophobic core of ubiquitin.

PubMed Central

Lazar, G. A.; Desjarlais, J. R.; Handel, T. M.

1997-01-01

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins. PMID:9194177

Promoter activity of polypyrimidine tract-binding protein genes of potato responds to environmental cues.

PubMed

Butler, Nathaniel M; Hannapel, David J

2012-12-01

Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that target specific RNAs for post-transcriptional processing by binding cytosine/uracil motifs. PTBs have established functions in a range of RNA processes including splicing, translation, stability and long-distance transport. Six PTB-like genes identified in potato have been grouped into two clades based on homology to other known plant PTBs. StPTB1 and StPTB6 are closely related to a PTB protein discovered in pumpkin, designated CmRBP50, and contain four canonical RNA-recognition motifs. CmRBP50 is expressed in phloem tissues and functions as the core protein of a phloem-mobile RNA/protein complex. Sequence from the potato genome database was used to clone the upstream sequence of these two PTB genes and analyzed to identify conserved cis-elements. The promoter of StPTB6 was enriched for regulatory elements for light and sucrose induction and defense. Upstream sequence of both PTB genes was fused to β-glucuronidase and monitored in transgenic potato lines. In whole plants, the StPTB1 promoter was most active in leaf veins and petioles, whereas StPTB6 was most active in leaf mesophyll. Both genes are active in new tubers and tuber sprouts. StPTB6 expression was induced in stems and stolon sections in response to sucrose and in leaves or petioles in response to light, heat, drought and mechanical wounding. These results show that CmRBP50-like genes of potato exhibit distinct expression patterns and respond to both developmental and environmental cues.

Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India.

PubMed

Kaur, Gagandeep; Singh, Amninder; Sharma, Rohit; Sharma, Vinay; Verma, Swati; Sharma, Pushpender K

2016-06-01

In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg -1 and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0-14.0 pH and 30-80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of β-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel β-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

PubMed

Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Frequent loss of claudin-4 expression in dedifferentiated and undifferentiated endometrial carcinomas.

PubMed

Tessier-Cloutier, Basile; Soslow, Robert A; Stewart, Colin J R; Köbel, Martin; Lee, Cheng-Han

2018-04-19

Dedifferentiated endometrial carcinomas (DDECs)/undifferentiated endometrial carcinomas (UECs) are aggressive endometrial cancers with frequent genomic inactivation of core components of switch/sucrose non-fermentable (SWI/SNF) complex proteins. Claudin-4, an epithelial intercellular tight junction protein, was recently found to be expressed in SWI/SNF-deficient undifferentiated carcinomas but not in SWI/SNF-deficient sarcomas. The aim of this study was to examine claudin-4 expression in UECs/DDECs and other high-grade uterine carcinomas. We examined claudin-4 expression by immunohistochemistry (clone 3E2C1) on tissue microarrays that contained 44 UECs/DDECs (24 SWI/SNF-deficient), 50 carcinosarcomas, 164 grade 3 endometrioid carcinomas, 57 serous carcinomas, and 20 clear cell carcinomas. Tumours with <5% claudin-4 expression were considered to be negative. Nearly all SWI/SNF-deficient, and most SWI/SNF-proficient, UECs/DDECs showed a complete absence of claudin-4 expression in the undifferentiated component, whereas the differentiated component in DDECs showed consistent and diffuse claudin-4 expression. Only one SWI/SNF-deficient DDEC showed focal expression of claudin-4 in the undifferentiated component, as compared with diffuse expression in the corresponding differentiated component. Claudin-4 expression was consistently absent in the sarcomatous component of carcinosarcoma, and it was absent in 24% of grade 3 endometrioid carcinomas and serous carcinomas. Claudin-4 expression can be absent or very focal in a subset of high-grade endometrial carcinomas, and is almost always absent in the undifferentiated components of SWI/SNF-deficient UECs/DDECs, despite the apparent epithelial origin in the case of DDECs. Therefore, claudin-4 expression cannot be used to infer mesenchymal or epithelial tumour origin in the endometrium. The consistent loss or down-regulation of claudin-4, a tight junction protein, in SWI/SNF-deficient UECs/DDECs further supports the undifferentiated nature of these tumours. © 2018 John Wiley & Sons Ltd.

Promoter DNA methylation regulates progranulin expression and is altered in FTLD

PubMed Central

2013-01-01

Background Frontotemporal lobar degeneration (FTLD) is a heterogeneous group of neurodegenerative diseases associated with personality changes and progressive dementia. Loss-of-function mutations in the growth factor progranulin (GRN) cause autosomal dominant FTLD, but so far the pathomechanism of sporadic FTLD is unclear. Results We analyzed whether DNA methylation in the GRN core promoter restricts GRN expression and, thus, might promote FTLD in the absence of GRN mutations. GRN expression in human lymphoblast cell lines is negatively correlated with methylation at several CpG units within the GRN promoter. Chronic treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) strongly induces GRN mRNA and protein levels. In a reporter assay, CpG methylation blocks transcriptional activity of the GRN core promoter. In brains of FTLD patients several CpG units in the GRN promoter are significantly hypermethylated compared to age-matched healthy controls, Alzheimer and Parkinson patients. These CpG motifs are critical for GRN promoter activity in reporter assays. Furthermore, DNA methyltransferase 3a (DNMT3a) is upregulated in FTLD patients and overexpression of DNMT3a reduces GRN promoter activity and expression. Conclusion These data suggest that altered DNA methylation is a novel pathomechanism for FTLD that is potentially amenable to targeted pharmacotherapy. PMID:24252647

A core functional region of the RFP1 promoter from Chinese wild grapevine is activated by powdery mildew pathogen and heat stress.

PubMed

Yu, Yihe; Xu, Weirong; Wang, Jie; Wang, Lei; Yao, Wenkong; Xu, Yan; Ding, Jiahua; Wang, Yuejin

2013-01-01

RING-finger proteins (RFP) function as ubiquitin ligases and play key roles in plant responses to biotic and abiotic stresses. However, little information is available on the regulation of RFP expression. Here, we isolate and characterize the RFP promoter sequence from the disease-resistant Chinese wild grape Vitis pseudoreticulata accession Baihe-35-1. Promoter-GUS fusion assays revealed that defense signaling molecules, powdery mildew infection, and heat stress induce VpRFP1 promoter activity. By contrast, the RFP1 promoter isolated from Vitis vinifera was only slightly induced by pathogen infection and heat treatment. By promoter deletion analysis, we found that the -148 bp region of the VpRFP1 promoter was the core functional promoter region. We also found that, in Arabidopsis, VpRFP1 expressed under its own promoter activated defense-related gene expression and improved disease resistance, but the same construct using the VvRFP1 promoter slightly improve disease resistance. Our results demonstrated that the -148 bp region of the VpRFP1 promoter plays a key role in response to pathogen and heat stress, and suggested that expression differences between VpRFP1 and VvRFP1 may be key for the differing disease resistance phenotypes of the two Vitis genotypes.

Effects of Folic Acid on Secretases Involved in Aβ Deposition in APP/PS1 Mice

PubMed Central

Tian, Tian; Bai, Dong; Li, Wen; Huang, Guo-Wei; Liu, Huan

2016-01-01

Alzheimer’s disease (AD) is the most common type of dementia. Amyloid-β protein (Aβ) is identified as the core protein of neuritic plaques. Aβ is generated by the sequential cleavage of the amyloid precursor protein (APP) via the APP cleaving enzyme (α-secretase, or β-secretase) and γ-secretase. Previous studies indicated that folate deficiency elevated Aβ deposition in APP/PS1 mice, and this rise was prevented by folic acid. In the present study, we aimed to investigate whether folic acid could influence the generation of Aβ by regulating α-, β-, and γ-secretase. Herein, we demonstrated that folic acid reduced the deposition of Aβ42 in APP/PS1 mice brain by decreasing the mRNA and protein expressions of β-secretase [beta-site APP-cleaving enzyme 1 (BACE1)] and γ-secretase complex catalytic component—presenilin 1 (PS1)—in APP/PS1 mice brain. Meanwhile, folic acid increased the levels of ADAM9 and ADAM10, which are important α-secretases in ADAM (a disintegrin and metalloprotease) family. However, folic acid has no impact on the protein expression of nicastrin (Nct), another component of γ-secretase complex. Moreover, folic acid regulated the expression of miR-126-3p and miR-339-5p, which target ADAM9 and BACE1, respectively. Taken together, the effect of folic acid on Aβ deposition may relate to making APP metabolism through non-amyloidogenic pathway by decreasing β-secretase and increasing α-secretase. MicroRNA (miRNA) may involve in the regulation mechanism of folic acid on secretase expression. PMID:27618097

Reduced expression of the NMDA receptor-interacting protein SynGAP causes behavioral abnormalities that model symptoms of Schizophrenia.

PubMed

Guo, Xiaochuan; Hamilton, Peter J; Reish, Nicholas J; Sweatt, J David; Miller, Courtney A; Rumbaugh, Gavin

2009-06-01

Abnormal function of NMDA receptors is believed to be a contributing factor to the pathophysiology of schizophrenia. NMDAR subunits and postsynaptic-interacting proteins of these channels are abnormally expressed in some patients with this illness. In mice, reduced NMDAR expression leads to behaviors analogous to symptoms of schizophrenia, but reports of animals with mutations in core postsynaptic density proteins having similar a phenotype have yet to be reported. Here we show that reduced expression of the neuronal RasGAP and NMDAR-associated protein, SynGAP, results in abnormal behaviors strikingly similar to that reported in mice with reduced NMDAR function. SynGAP mutant mice exhibited nonhabituating and persistent hyperactivity that was ameliorated by the antipsychotic clozapine. An NMDAR antagonist, MK-801, induced hyperactivity in normal mice but SynGAP mutants were less responsive, suggesting that NMDAR hypofunction contributes to this behavioral abnormality. SynGAP mutants exhibited enhanced startle reactivity and impaired sensory-motor gating. These mice also displayed a complete lack of social memory and a propensity toward social isolation. Finally, SynGAP mutants had deficits in cued fear conditioning and working memory, indicating abnormal function of circuits that control emotion and choice. Our results demonstrate that SynGAP mutant mice have gross neurological deficits similar to other mouse models of schizophrenia. Because SynGAP interacts with NMDARs, and the signaling activity of this protein is regulated by these channels, our data in dicate that SynGAP lies downstream of NMDARs and is a required intermediate for normal neural circuit function and behavior. Taken together, these data support the idea that schizophrenia may arise from abnormal signaling pathways that are mediated by NMDA receptors.

A single-point mutation in the extreme heat- and pressure-resistant sso7d protein from sulfolobus solfataricus leads to a major rearrangement of the hydrophobic core.

PubMed

Consonni, R; Santomo, L; Fusi, P; Tortora, P; Zetta, L

1999-09-28

Sso7d is a basic 7-kDa DNA-binding protein from Sulfolobus solfataricus, also endowed with ribonuclease activity. The protein consists of a double-stranded antiparallel beta-sheet, onto which an orthogonal triple-stranded antiparallel beta-sheet is packed, and of a small helical stretch at the C-terminus. Furthermore, the two beta-sheets enclose an aromatic cluster displaying a fishbone geometry. We previously cloned the Sso7d-encoding gene, expressed it in Escherichia coli, and produced several single-point mutants, either of residues located in the hydrophobic core or of Trp23, which is exposed to the solvent and plays a major role in DNA binding. The mutation F31A was dramatically destabilizing, with a loss in thermo- and piezostabilities by at least 27 K and 10 kbar, respectively. Here, we report the solution structure of the F31A mutant, which was determined by NMR spectroscopy using 744 distance constraints obtained from analysis of multidimensional spectra in conjunction with simulated annealing protocols. The most remarkable finding is the change in orientation of the Trp23 side chain, which in the wild type is completely exposed to the solvent, whereas in the mutant is largely buried in the aromatic cluster. This prevents the formation of a cavity in the hydrophobic core of the mutant, which would arise in the absence of structural rearrangements. We found additional changes produced by the mutation, notably a strong distortion in the beta-sheets with loss in several hydrogen bonds, increased flexibility of some stretches of the backbone, and some local strains. On one hand, these features may justify the dramatic destabilization provoked by the mutation; on the other hand, they highlight the crucial role of the hydrophobic core in protein stability. To the best of our knowledge, no similar rearrangement has been so far described as a result of a single-point mutation.

Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.

PubMed Central

Bäckström, Malin; Link, Thomas; Olson, Fredrik J; Karlsson, Hasse; Graham, Rosalind; Picco, Gianfranco; Burchell, Joy; Taylor-Papadimitriou, Joyce; Noll, Thomas; Hansson, Gunnar C

2003-01-01

We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer. PMID:12950230

Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Zhengliang L., E-mail: Leon.wu@bio-techne.com; Zhou, Hui; Ethen, Cheryl M.

The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6more » fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and {sup 3}H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. - Graphical abstract: Proposed mechanism for how core-fucose prohibits the tetramerization of the 1918 pandemic viral neuraminidase. Only the cross section of the stalk region with two N-linked glycans are depicted for clarity. (A) Carbohydrate–carbohydrate interaction on non-fucosylated monomer allows tetramerization. (B) Core-fucosylation disrupts the interaction and prevents the tetramerization. - Highlights: • Expressed 1918 pandemic influenza viral neuraminidase has inactive and active forms. • The inactive form contains high level of core-6 fucose, while the active form lacks such modification. • Core fucose could interfere the oligomerization of the neuraminidase and thus its activation. • This discovery may explain why 1918 pandemic influenza caused higher death rate among young population.« less

Protection of the Photosynthetic Apparatus from Extreme Dehydration and Oxidative Stress in Seedlings of Transgenic Tobacco

PubMed Central

Personat, José-María; Tejedor-Cano, Javier; Lindahl, Marika; Diaz-Espejo, Antonio; Jordano, Juan

2012-01-01

A genetic program that in sunflower seeds is activated by Heat Shock transcription Factor A9 (HaHSFA9) has been analyzed in transgenic tobacco seedlings. The ectopic overexpression of the HSFA9 program protected photosynthetic membranes, which resisted extreme dehydration and oxidative stress conditions. In contrast, heat acclimation of seedlings induced thermotolerance but not resistance to the harsh stress conditions employed. The HSFA9 program was found to include the expression of plastidial small Heat Shock Proteins that accumulate only at lower abundance in heat-stressed vegetative organs. Photosystem II (PSII) maximum quantum yield was higher for transgenic seedlings than for non-transgenic seedlings, after either stress treatment. Furthermore, protection of both PSII and Photosystem I (PSI) membrane protein complexes was observed in the transgenic seedlings, leading to their survival after the stress treatments. It was also shown that the plastidial D1 protein, a labile component of the PSII reaction center, and the PSI core protein PsaB were shielded from oxidative damage and degradation. We infer that natural expression of the HSFA9 program during embryogenesis may protect seed pro-plastids from developmental desiccation. PMID:23227265

Upregulation of miR-181c contributes to chemoresistance in pancreatic cancer by inactivating the Hippo signaling pathway.

PubMed

Chen, Meiyuan; Wang, Min; Xu, Simiao; Guo, Xingjun; Jiang, Jianxin

2015-12-29

The Hippo signaling pathway plays a crucial role in regulating tissue homeostasis, organ size, tumorigenesis and cancer chemoresistance when deregulated. Physiologically, the Hippo core kinase cassette that consists of mamma-lian STE20-like protein kinase 1/2 (MST1/2), and large tumour suppressor 1/2 (LATS1/2), together with the adaptor proteins Salvador homologue 1 (SAV1) and MOB kinase activator 1 (MOB1), tightly restricts the activities of homologous oncoproteins Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) to low levels. However, how the Hippo kinase cassette core components are simultaneously inhibited, to exhibit constitutively inactivated Hippo signaling and activated YAP/TAZ in cancer remains puzzling. Herein, we reported that miR-181c directly repressed MST1, LATS2, MOB1 and SAV1 expression in human pancreatic cancer cells. Overexpression of miR-181c induced hyperactivation of the YAP/TAZ and enhanced expression of the Hippo signaling downstream genes CTGF, BIRC5 and BLC2L1, leading to pancreatic cancer cell survival and chemoresistance in vitro and in vivo. Importantly, high miR-181c levels were significantly correlated with Hippo signaling inactivation in pancreatic cancer samples, and predicted a poor patient overall survival. These findings provide a novel mechanism for Hippo signaling inactivation in cancer, indicating not only a potentially pivotal role for miR-181c in the progression of pancreatic cancer, but also may represent a new therapeutic target and prognostic marker.

Quantitative assessment of effect of preanalytic cold ischemic time on protein expression in breast cancer tissues.

PubMed

Neumeister, Veronique M; Anagnostou, Valsamo; Siddiqui, Summar; England, Allison Michal; Zarrella, Elizabeth R; Vassilakopoulou, Maria; Parisi, Fabio; Kluger, Yuval; Hicks, David G; Rimm, David L

2012-12-05

Companion diagnostic tests can depend on accurate measurement of protein expression in tissues. Preanalytic variables, especially cold ischemic time (time from tissue removal to fixation in formalin) can affect the measurement and may cause false-negative results. We examined 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues. A series of 93 breast cancer specimens with known time-to-fixation represented in a tissue microarray and a second series of 25 matched pairs of core needle biopsies and breast cancer resections were used to evaluate changes in antigenicity as a function of cold ischemic time. Estrogen receptor (ER), progesterone receptor (PgR), HER2 or Ki67, and 19 other antigens were tested. Each antigen was measured using the AQUA method of quantitative immunofluorescence on at least one series. All statistical tests were two-sided. We found no evidence for loss of antigenicity with time-to-fixation for ER, PgR, HER2, or Ki67 in a 4-hour time window. However, with a bootstrapping analysis, we observed a trend toward loss for ER and PgR, a statistically significant loss of antigenicity for phosphorylated tyrosine (P = .0048), and trends toward loss for other proteins. There was evidence of increased antigenicity in acetylated lysine, AKAP13 (P = .009), and HIF1A (P = .046), which are proteins known to be expressed in conditions of hypoxia. The loss of antigenicity for phosphorylated tyrosine and increase in expression of AKAP13, and HIF1A were confirmed in the biopsy/resection series. Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions. Future studies that extend the time range and normalize for heterogeneity will provide more comprehensive information on preanalytic variation due to cold ischemic time.

Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.

PubMed

Hollingsworth, M A; Strawhecker, J M; Caffrey, T C; Mack, D R

1994-04-15

We examined the steady-state expression levels of mRNA for the MUC1, MUC2, MUC3 and MUC4 gene products in 12 pancreatic tumor cell lines, 6 colon tumor cell lines, and one ileocecal tumor cell line. The results showed that 10 of 12 pancreatic tumor cell lines expressed MUC1 mRNA and that 7 of these 12 lines also expressed relatively high levels of MUC4 mRNA. In contrast, MUC2 mRNA was expressed at only low levels and MUC3 was not detected in the pancreatic tumor cell lines. All 7 intestinal tumor cell lines examined expressed MUC2, and 5 of 7 expressed MUC3; however only one expressed significant levels of MUC1 and 2 expressed low levels of MUC4 mRNA. This report of high levels of MUC4 mRNA expression by pancreatic tumor cells raises the possibility that mucin carbohydrate epitopes defined by antibodies such as DuPan 2 may be expressed on a second mucin core protein produced by pancreatic tumor cells.

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

PubMed

Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

2016-03-10

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

Mammalian Peptidylglycine α-Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

PubMed Central

Brenet, Fabienne; Dussault, Nadège; Borch, Jonas; Ferracci, Géraldine; Delfino, Christine; Roepstorff, Peter; Miquelis, Raymond; Ouafik, L'Houcine

2005-01-01

Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3′ untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3′ UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3′ UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3′ UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules. PMID:16107699

Disturbed Laminar Blood Flow Vastly Augments Lipoprotein Retention in the Artery Wall: A Key Mechanism Distinguishing Susceptible From Resistant Sites.

PubMed

Steffensen, Lasse Bach; Mortensen, Martin Bødtker; Kjolby, Mads; Hagensen, Mette Kallestrup; Oxvig, Claus; Bentzon, Jacob Fog

2015-09-01

Atherosclerosis develops initially at branch points and in areas of high vessel curvature. Moreover, experiments in hypercholesterolemic mice have shown that the introduction of disturbed flow in straight, atherosclerosis-resistant arterial segments turns them highly atherosclerosis susceptible. Several biomechanical mechanisms have been proposed, but none has been demonstrated. In the present study, we examined whether a causal link exists between disturbed laminar flow and the ability of the arterial wall to retain lipoproteins. Lipoprotein retention was detected at natural predilection sites of the murine thoracic aorta 18 hours after infusion of fluorescently labeled low-density lipoprotein. To test for causality between blood flow and the ability of these areas to retain lipoproteins, we manipulated blood flow in the straight segment of the common carotid artery using a constrictive collar. Disturbed laminar flow did not affect low-density lipoprotein influx, but increased the ability of the artery wall to bind low-density lipoprotein. Concordantly, disturbed laminar flow led to differential expression of genes associated with phenotypic modulation of vascular smooth muscle cells, increased expression of proteoglycan core proteins associated with lipoprotein retention, and of enzymes responsible for chondroitin sulfate glycosaminoglycan synthesis and sulfation. Blood flow regulates genes associated with vascular smooth muscle cell phenotypic modulation, as well as the expression and post-translational modification of lipoprotein-binding proteoglycan core proteins, and the introduction of disturbed laminar flow vastly augments the ability of a previously resistant, straight arterial segment to retain lipoproteins. © 2015 American Heart Association, Inc.

Mouse genotypes drive the liver and adrenal gland clocks

NASA Astrophysics Data System (ADS)

Košir, Rok; Prosenc Zmrzljak, Uršula; Korenčič, Anja; Juvan, Peter; Ačimovič, Jure; Rozman, Damjana

2016-08-01

Circadian rhythms regulate a plethora of physiological processes. Perturbations of the rhythm can result in pathologies which are frequently studied in inbred mouse strains. We show that the genotype of mouse lines defines the circadian gene expression patterns. Expression of majority of core clock and output metabolic genes are phase delayed in the C56BL/6J line compared to 129S2 in the adrenal glands and the liver. Circadian amplitudes are generally higher in the 129S2 line. Experiments in dark - dark (DD) and light - dark conditions (LD), exome sequencing and data mining proposed that mouse lines differ in single nucleotide variants in the binding regions of clock related transcription factors in open chromatin regions. A possible mechanisms of differential circadian expression could be the entrainment and transmission of the light signal to peripheral organs. This is supported by the genotype effect in adrenal glands that is largest under LD, and by the high number of single nucleotide variants in the Receptor, Kinase and G-protein coupled receptor Panther molecular function categories. Different phenotypes of the two mouse lines and changed amino acid sequence of the Period 2 protein possibly contribute further to the observed differences in circadian gene expression.

Analysis of Autophagy Genes in Microalgae: Chlorella as a Potential Model to Study Mechanism of Autophagy

PubMed Central

Jiang, Qiao; Zhao, Li; Dai, Junbiao; Wu, Qingyu

2012-01-01

Background Microalgae, with the ability to mitigate CO2 emission and produce carbohydrates and lipids, are considered one of the most promising resources for producing bioenergy. Recently, we discovered that autophagy plays a critical role in the metabolism of photosynthetic system and lipids production. So far, more than 30-autophagy related (ATG) genes in all subtypes of autophagy have been identified. However, compared with yeast and mammals, in silico and experimental research of autophagy pathways in microalgae remained limited and fragmentary. Principal Findings In this article, we performed a genome-wide analysis of ATG genes in 7 microalgae species and explored their distributions, domain structures and evolution. Eighteen “core autophagy machinery” proteins, four mammalian-specific ATG proteins and more than 30 additional proteins (including “receptor-adaptor” complexes) in all subtypes of autophagy were analyzed. Data revealed that receptor proteins in cytoplasm-to-vacuole targeting and mitophagy seem to be absent in microalgae. However, most of the “core autophagy machinery” and mammalian-specific proteins are conserved among microalgae, except for the ATG9-cycling system in Chlamydomonas reinhardtii and the second ubiquitin-like protein conjugation complex in several algal species. The catalytic and binding residues in ATG3, ATG5, ATG7, ATG8, ATG10 and ATG12 are also conserved and the phylogenetic tree of ATG8 coincides well with the phylogenies. Chlorella contains the entire set of the core autophagy machinery. In addition, RT-PCR analysis verified that all crucial ATG genes tested are expressed during autophagy in both Chlorella and Chlamydomonas reinhardtii. Finally, we discovered that addition of 3-Methyladenine (a PI3K specific inhibitor) could suppress the formation of autophagic vacuoles in Chlorella. Conclusions Taken together, Chlorella may represent a potential model organism to investigate autophagy pathways in photosynthetic eukaryotes. The study will not only promote understanding of the general features of autophagic pathways, but also benefit the production of Chlorella-derived biofuel with future commercial applications. PMID:22848622

In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice.

PubMed

de la Rosa, Xavier; Santalucía, Tomàs; Fortin, Pierre-Yves; Purroy, Jesús; Calvo, Maria; Salas-Perdomo, Angélica; Justicia, Carles; Couillaud, Franck; Planas, Anna M

2013-02-01

Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core.

Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli.

PubMed

Soundrarajan, Nagasundarapandian; Cho, Hye-Sun; Ahn, Byeongyong; Choi, Minkyung; Thong, Le Minh; Choi, Hojun; Cha, Se-Yeoun; Kim, Jin-Hoi; Park, Choi-Kyu; Seo, Kunho; Park, Chankyu

2016-02-11

The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches.

Semi-synthesis of murine prion protein by native chemical ligation and chemical activation for preparation of polypeptide-α-thioester.

PubMed

Shi, Lei; Chen, Huai; Zhang, Si-Yu; Chu, Ting-Ting; Zhao, Yu-Fen; Chen, Yong-Xiang; Li, Yan-Mei

2017-06-01

Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three-dimensional structure domain was constructed from three segments murine PrP (mPrP)(90-177), mPrP(178-212), and mPrP(213-230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C-terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids.

PubMed

Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

2016-05-25

Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only.

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

PubMed

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli

PubMed Central

Soundrarajan, Nagasundarapandian; Cho, Hye-sun; Ahn, Byeongyong; Choi, Minkyung; Thong, Le Minh; Choi, Hojun; Cha, Se-Yeoun; Kim, Jin-Hoi; Park, Choi-Kyu; Seo, Kunho; Park, Chankyu

2016-01-01

The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches. PMID:26864123

APOBEC3B edits HBV DNA and inhibits HBV replication during reverse transcription.

PubMed

Chen, Yanmeng; Hu, Jie; Cai, Xuefei; Huang, Yao; Zhou, Xing; Tu, Zeng; Hu, Jieli; Tavis, John E; Tang, Ni; Huang, Ailong; Hu, Yuan

2018-01-01

Hepatitis B virus is a partially double-stranded DNA virus that replicates by reverse transcription, which occurs within viral core particles in the cytoplasm. The cytidine deaminase APOBEC3B is a cellular restriction factor for HBV. Recently, it was reported that APOBEC3B can edit HBV cccDNA in the nucleus, causing its degradation. However, whether and how it can edit HBV core-associated DNAs during reverse transcription is unclear. Our studies to address this question revealed the following: First, silencing endogenous APOBEC3B in an HBV infection system lead to upregulation of HBV replication. Second, APOBEC3B can inhibit replication of HBV isolates from genotypes (gt) A, B, C, and D as determined by employing transfection of plasmids expressing isolates from four different HBV genotypes. For HBV inhibition, APOBEC3B-mediated inhibition of replication primarily depends on the C-terminal active site of APOBEC3B. In addition, employing the HBV RNaseH-deficient D702A mutant and a polymerase-deficient YMHA mutant, we demonstrated that APOBEC3B can edit both the HBV minus- and plus-strand DNAs, but not the pregenomic RNA in core particles. Furthermore, we found by co-immunoprecipitation assays that APOBEC3B can interact with HBV core protein in an RNA-dependent manner. Our results provide evidence that APOBEC3B can interact with HBV core protein and edit HBV DNAs during reverse transcription. These data suggest that APOBEC3B exerts multifaceted antiviral effects against HBV. Copyright © 2017 Elsevier B.V. All rights reserved.

Meta-analysis of gene expression patterns in animal models of prenatal alcohol exposure suggests role for protein synthesis inhibition and chromatin remodeling

PubMed Central

Rogic, Sanja; Wong, Albertina; Pavlidis, Paul

2017-01-01

Background Prenatal alcohol exposure (PAE) can result in an array of morphological, behavioural and neurobiological deficits that can range in their severity. Despite extensive research in the field and a significant progress made, especially in understanding the range of possible malformations and neurobehavioral abnormalities, the molecular mechanisms of alcohol responses in development are still not well understood. There have been multiple transcriptomic studies looking at the changes in gene expression after PAE in animal models, however there is a limited apparent consensus among the reported findings. In an effort to address this issue, we performed a comprehensive re-analysis and meta-analysis of all suitable, publically available expression data sets. Methods We assembled ten microarray data sets of gene expression after PAE in mouse and rat models consisting of samples from a total of 63 ethanol-exposed and 80 control animals. We re-analyzed each data set for differential expression and then used the results to perform meta-analyses considering all data sets together or grouping them by time or duration of exposure (pre- and post-natal, acute and chronic, respectively). We performed network and Gene Ontology enrichment analysis to further characterize the identified signatures. Results For each sub-analysis we identified signatures of differential expressed genes that show support from multiple studies. Overall, the changes in gene expression were more extensive after acute ethanol treatment during prenatal development than in other models. Considering the analysis of all the data together, we identified a robust core signature of 104 genes down-regulated after PAE, with no up-regulated genes. Functional analysis reveals over-representation of genes involved in protein synthesis, mRNA splicing and chromatin organization. Conclusions Our meta-analysis shows that existing studies, despite superficial dissimilarity in findings, share features that allow us to identify a common core signature set of transcriptome changes in PAE. This is an important step to identifying the biological processes that underlie the etiology of FASD. PMID:26996386

A focused microarray approach to functional glycomics: transcriptional regulation of the glycome.

PubMed

Comelli, Elena M; Head, Steven R; Gilmartin, Tim; Whisenant, Thomas; Haslam, Stuart M; North, Simon J; Wong, Nyet-Kui; Kudo, Takashi; Narimatsu, Hisashi; Esko, Jeffrey D; Drickamer, Kurt; Dell, Anne; Paulson, James C

2006-02-01

Glycosylation is the most common posttranslational modification of proteins, yet genes relevant to the synthesis of glycan structures and function are incompletely represented and poorly annotated on the commercially available arrays. To fill the need for expression analysis of such genes, we employed the Affymetrix technology to develop a focused and highly annotated glycogene-chip representing human and murine glycogenes, including glycosyltransferases, nucleotide sugar transporters, glycosidases, proteoglycans, and glycan-binding proteins. In this report, the array has been used to generate glycogene-expression profiles of nine murine tissues. Global analysis with a hierarchical clustering algorithm reveals that expression profiles in immune tissues (thymus [THY], spleen [SPL], lymph node, and bone marrow [BM]) are more closely related, relative to those of nonimmune tissues (kidney [KID], liver [LIV], brain [BRN], and testes [TES]). Of the biosynthetic enzymes, those responsible for synthesis of the core regions of N- and O-linked oligosaccharides are ubiquitously expressed, whereas glycosyltransferases that elaborate terminal structures are expressed in a highly tissue-specific manner, accounting for tissue and ultimately cell-type-specific glycosylation. Comparison of gene expression profiles with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) profiling of N-linked oligosaccharides suggested that the alpha1-3 fucosyltransferase 9, Fut9, is the enzyme responsible for terminal fucosylation in KID and BRN, a finding validated by analysis of Fut9 knockout mice. Two families of glycan-binding proteins, C-type lectins and Siglecs, are predominately expressed in the immune tissues, consistent with their emerging functions in both innate and acquired immunity. The glycogene chip reported in this study is available to the scientific community through the Consortium for Functional Glycomics (CFG) (http://www.functionalglycomics.org).

Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block.

PubMed

Kawakami, Toru; Yoshikawa, Ryo; Fujiyoshi, Yuki; Mishima, Yuichi; Hojo, Hironobu; Tajima, Shoji; Suetake, Isao

2015-11-01

The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected. To this end, a phenacyl group was successfully utilized to protect endogenous Cys residue(s), and the recombinant peptide was ligated with a peptide containing a Cys-Pro ester (CPE) sequence as a thioester precursor. Using this approach it was possible to prepare all of the core histones H2A, H2B, H3 and H4 with any modifications. The resulting proteins could then be used to prepare a core histone library of proteins that have been post-translationally modified. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Overall Architecture of the Intraflagellar Transport (IFT)-B Complex Containing Cluap1/IFT38 as an Essential Component of the IFT-B Peripheral Subcomplex.

PubMed

Katoh, Yohei; Terada, Masaya; Nishijima, Yuya; Takei, Ryota; Nozaki, Shohei; Hamada, Hiroshi; Nakayama, Kazuhisa

2016-05-20

Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wild-type and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Overall Architecture of the Intraflagellar Transport (IFT)-B Complex Containing Cluap1/IFT38 as an Essential Component of the IFT-B Peripheral Subcomplex*

PubMed Central

Katoh, Yohei; Terada, Masaya; Nishijima, Yuya; Takei, Ryota; Nozaki, Shohei; Hamada, Hiroshi; Nakayama, Kazuhisa

2016-01-01

Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wild-type and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex. PMID:26980730

Glycomic characterization of basal tears and changes with diabetes and diabetic retinopathy.

PubMed

Nguyen-Khuong, Terry; Everest-Dass, Arun V; Kautto, Liisa; Zhao, Zhenjun; Willcox, Mark D P; Packer, Nicolle H

2015-03-01

As a secreted fluid, the state of tear glycosylation is particularly important in the role of immunity of the ocular surface. Tears are a valuable source of non-invasive biomarkers for disease and there are continued efforts to characterize their components thoroughly. In this study, a small volume of basal tears (5 μL) was collected from healthy controls, patients with diabetes without retinopathy and patients with diabetes and retinopathy. The detailed N- and O-linked tear protein glycome was characterized and the relative abundance of each structure determined. Of the 50 N-linked glycans found, 89% were complex with 50% containing a bisecting N-acetylglucosamine, 65% containing a core fucose whilst 33% were sialylated. Of the 8 O-linked glycans detected, 3 were of cores 1 and 5 of core 2 type, with a majority of them being sialylated (90%). Additionally, these glycan structures were profiled across the three diabetic disease groups. Whilst the higher abundant structures did not alter across the three groups, only five low abundance N-linked glycans and 1 O-linked glycan did alter with the onset of diabetes mellitus and diabetic retinopathy (DR). These results suggest the conservation of glycan types on basal tear proteins between individuals and point to only small changes in glycan expression on the proteins in tears with the development of diabetes and DR. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Ankrd6 is a mammalian functional homolog of Drosophila planar cell polarity gene diego and regulates coordinated cellular orientation in the mouse inner ear.

PubMed

Jones, Chonnettia; Qian, Dong; Kim, Sun Myoung; Li, Shuangding; Ren, Dongdong; Knapp, Lindsey; Sprinzak, David; Avraham, Karen B; Matsuzaki, Fumio; Chi, Fanglu; Chen, Ping

2014-11-01

The coordinated polarization of neighboring cells within the plane of the tissue, known as planar cell polarity (PCP), is a recurring theme in biology. It is required for numerous developmental processes for the form and function of many tissues and organs across species. The genetic pathway regulating PCP was first discovered in Drosophila, and an analogous but distinct pathway is emerging in vertebrates. It consists of membrane protein complexes known as core PCP proteins that are conserved across species. Here we report that the over-expression of the murine Ankrd6 (mAnkrd6) gene that shares homology with Drosophila core PCP gene diego causes a typical PCP phenotype in Drosophila, and mAnkrd6 can rescue the loss of function of diego in Drosophila. In mice, mAnkrd6 protein is asymmetrically localized in cells of the inner ear sensory organs, characteristic of components of conserved core PCP complexes. The loss of mAnkrd6 causes PCP defects in the inner ear sensory organs. Moreover, canonical Wnt signaling is significantly increased in mouse embryonic fibroblasts from mAnkrd6 knockout mice in comparison to wild type controls. Together, these results indicated that mAnkrd6 is a functional homolog of the Drosophila diego gene for mammalian PCP regulation and act to suppress canonical Wnt signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

The insulator protein BEAF-32 is required for Hippo pathway activity in the terminal differentiation of neuronal subtypes.

PubMed

Jukam, David; Viets, Kayla; Anderson, Caitlin; Zhou, Cyrus; DeFord, Peter; Yan, Jenny; Cao, Jinshuai; Johnston, Robert J

2016-07-01

The Hippo pathway is crucial for not only normal growth and apoptosis but also cell fate specification during development. What controls Hippo pathway activity during cell fate specification is incompletely understood. In this article, we identify the insulator protein BEAF-32 as a regulator of Hippo pathway activity in Drosophila photoreceptor differentiation. Though morphologically uniform, the fly eye is composed of two subtypes of R8 photoreceptor neurons defined by expression of light-detecting Rhodopsin proteins. In one R8 subtype, active Hippo signaling induces Rhodopsin 6 (Rh6) and represses Rhodopsin 5 (Rh5), whereas in the other subtype, inactive Hippo signaling induces Rh5 and represses Rh6. The activity state of the Hippo pathway in R8 cells is determined by the expression of warts, a core pathway kinase, which interacts with the growth regulator melted in a double-negative feedback loop. We show that BEAF-32 is required for expression of warts and repression of melted Furthermore, BEAF-32 plays a second role downstream of Warts to induce Rh6 and prevent Rh5 fate. BEAF-32 is dispensable for Warts feedback, indicating that BEAF-32 differentially regulates warts and Rhodopsins. Loss of BEAF-32 does not noticeably impair the functions of the Hippo pathway in eye growth regulation. Our study identifies a context-specific regulator of Hippo pathway activity in post-mitotic neuronal fate, and reveals a developmentally specific role for a broadly expressed insulator protein. © 2016. Published by The Company of Biologists Ltd.

Four regulatory elements in the human c-fos promoter mediate transactivation by HTLV-1 Tax protein.

PubMed

Alexandre, C; Verrier, B

1991-04-01

Expression of the human c-fos proto-oncogene is activated in trans by the Tax protein encoded by human T-cell leukemia virus type-1 (HTLV-1). Indeed, we show here that a HeLa clone stably transfected by Tax expresses Fos at a high level. We also show that multiple elements of the human c-fos promoter, i.e. the v-sis conditioned medium inducible element (SIE), the dyad symmetry element (DSE) necessary for growth factor induction, the octanucleotide direct repeat element (DR), and the cyclic AMP response element (CRE) centred at -60, can all mediate Tax transactivation. In the DSE, the 10bp central core that binds the serum response factor (SRF) is, by itself, sufficient to mediate Tax transactivation. Moreover, a CRE-binding protein is involved in Tax activation through the CRE-60 element. Since Fos is a transregulator of cellular genes, our results suggest that the oncoprotein plays a crucial role in T-cell transformation by HTLV-1 in conjunction with other Tax-inducible genes.

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.

PubMed

Glozman, Rina; Okiyoneda, Tsukasa; Mulvihill, Cory M; Rini, James M; Barriere, Herve; Lukacs, Gergely L

2009-03-23

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

High Mobility Group N Proteins Modulate the Fidelity of the Cellular Transcriptional Profile in a Tissue- and Variant-specific Manner*

PubMed Central

Kugler, Jamie E.; Horsch, Marion; Huang, Di; Furusawa, Takashi; Rochman, Mark; Garrett, Lillian; Becker, Lore; Bohla, Alexander; Hölter, Sabine M.; Prehn, Cornelia; Rathkolb, Birgit; Racz, Ildikó; Aguilar-Pimentel, Juan Antonio; Adler, Thure; Adamski, Jerzy; Beckers, Johannes; Busch, Dirk H.; Eickelberg, Oliver; Klopstock, Thomas; Ollert, Markus; Stöger, Tobias; Wolf, Eckhard; Wurst, Wolfgang; Yildirim, Ali Önder; Zimmer, Andreas; Gailus-Durner, Valérie; Fuchs, Helmut; Hrabě de Angelis, Martin; Garfinkel, Benny; Orly, Joseph; Ovcharenko, Ivan; Bustin, Michael

2013-01-01

The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family, which bind specifically to nucleosome core particles and affect chromatin structure and function, including transcription. Here, we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1tm1/tm1, Hmgn3tm1/tm1, and Hmgn5tm1/tm1 mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgntm1/tm1 lines reveals very little overlap between genes affected by specific variants in different tissues. Pathway analysis reveals that loss of an HMGN variant subtly affects expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner. PMID:23620591

Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-β Protein Precursor Transgenic Mice.

PubMed

Liu, Peng; Reichl, John H; Rao, Eshaan R; McNellis, Brittany M; Huang, Eric S; Hemmy, Laura S; Forster, Colleen L; Kuskowski, Michael A; Borchelt, David R; Vassar, Robert; Ashe, Karen H; Zahs, Kathleen R

2017-01-01

There exist several dozen lines of transgenic mice that express human amyloid-β protein precursor (AβPP) with Alzheimer's disease (AD)-linked mutations. AβPP transgenic mouse lines differ in the types and amounts of Aβ that they generate and in their spatiotemporal patterns of expression of Aβ assemblies, providing a toolkit to study Aβ amyloidosis and the influence of Aβ aggregation on brain function. More complete quantitative descriptions of the types of Aβ assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aβ toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AβPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ∼4.5 times that of 21-month-old Tg2576 mice and ∼15 times that of 21-24-month-old rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort.

Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-β Precursor Protein Transgenic Mice

PubMed Central

Liu, Peng; Reichl, John H.; Rao, Eshaan R.; McNellis, Brittany M.; Huang, Eric S.; Hemmy, Laura S.; Forster, Colleen L.; Kuskowski, Michael A.; Borchelt, David R.; Vassar, Robert; Ashe, Karen H.; Zahs, Kathleen R.

2016-01-01

There exist several dozen lines of transgenic mice that express human amyloid-β precursor protein (AβPP) with Alzheimer’s disease (AD)-linked mutations. AβPP transgenic mouse lines differ in the types and amounts of Aβ that they generate and in their spatiotemporal patterns of expression of Aβ assemblies, providing a toolkit to study Aβ amyloidosis and the influence of Aβ aggregation on brain function. More complete quantitative descriptions of the types of Aβ assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aβ toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AβPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ~4.5 times that of 21-month Tg2576 mice and ~15 times that of 21–24-month rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort. PMID:28059792

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong

2010-02-19

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less

Temperature-dependent resetting of the molecular circadian oscillator in Drosophila

PubMed Central

Goda, Tadahiro; Sharp, Brandi; Wijnen, Herman

2014-01-01

Circadian clocks responsible for daily time keeping in a wide range of organisms synchronize to daily temperature cycles via pathways that remain poorly understood. To address this problem from the perspective of the molecular oscillator, we monitored temperature-dependent resetting of four of its core components in the fruitfly Drosophila melanogaster: the transcripts and proteins for the clock genes period (per) and timeless (tim). The molecular circadian cycle in adult heads exhibited parallel responses to temperature-mediated resetting at the levels of per transcript, tim transcript and TIM protein. Early phase adjustment specific to per transcript rhythms was explained by clock-independent temperature-driven transcription of per. The cold-induced expression of Drosophila per contrasts with the previously reported heat-induced regulation of mammalian Period 2. An altered and more readily re-entrainable temperature-synchronized circadian oscillator that featured temperature-driven per transcript rhythms and phase-shifted TIM and PER protein rhythms was found for flies of the ‘Tim 4’ genotype, which lacked daily tim transcript oscillations but maintained post-transcriptional temperature entrainment of tim expression. The accelerated molecular and behavioural temperature entrainment observed for Tim 4 flies indicates that clock-controlled tim expression constrains the rate of temperature cycle-mediated circadian resetting. PMID:25165772

Circadian systems biology in Metazoa.

PubMed

Lin, Li-Ling; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2015-11-01

Systems biology, which can be defined as integrative biology, comprises multistage processes that can be used to understand components of complex biological systems of living organisms and provides hierarchical information to decoding life. Using systems biology approaches such as genomics, transcriptomics and proteomics, it is now possible to delineate more complicated interactions between circadian control systems and diseases. The circadian rhythm is a multiscale phenomenon existing within the body that influences numerous physiological activities such as changes in gene expression, protein turnover, metabolism and human behavior. In this review, we describe the relationships between the circadian control system and its related genes or proteins, and circadian rhythm disorders in systems biology studies. To maintain and modulate circadian oscillation, cells possess elaborative feedback loops composed of circadian core proteins that regulate the expression of other genes through their transcriptional activities. The disruption of these rhythms has been reported to be associated with diseases such as arrhythmia, obesity, insulin resistance, carcinogenesis and disruptions in natural oscillations in the control of cell growth. This review demonstrates that lifestyle is considered as a fundamental factor that modifies circadian rhythm, and the development of dysfunctions and diseases could be regulated by an underlying expression network with multiple circadian-associated signals. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Co-expression network with protein-protein interaction and transcription regulation in malaria parasite Plasmodium falciparum.

PubMed

Yu, Fu-Dong; Yang, Shao-You; Li, Yuan-Yuan; Hu, Wei

2013-04-10

Malaria continues to be one of the most severe global infectious diseases, as a major threat to human health and economic development. Network-based biological analysis is a promising approach to uncover key genes and biological processes from a network viewpoint, which could not be recognized from individual gene-based signatures. We integrated gene co-expression profile with protein-protein interaction and transcriptional regulation information to construct a comprehensive gene co-expression network of Plasmodium falciparum. Based on this network, we identified 10 core modules by using ICE (Iterative Clique Enumeration) algorithm, which were essential for malaria parasite development in intraerythrocytic developmental cycle (IDC) stages. In each module, all genes were highly correlated probably due to co-regulation or formation of a protein complex. Some of these genes were recognized to be differentially coexpressed among three close-by IDC stages. The gene of prpf8 (PFD0265w) encoding pre-mRNA processing splicing factor 8 product was identified as DCGs (differentially co-expressed genes) among IDC stages, although this gene function was seldom reported in previous researches. Integrating the species-specific gene prediction and differential co-expression gene detection, we found some modules could perform species-specific functions according to some of genes in these modules were species-specific genes, like the module 10. Furthermore, in order to reveal the underlying mechanisms of the erythrocyte invasion by P. falciparum, Steiner Tree algorithm was employed to identify the invasion subnetwork from our gene co-expression network. The subnetwork-based analysis indicated that some important Plasmodium parasite specific genes could corporate with each other and be co-regulated during the parasite invasion process, which including a head-to-head gene pair of PfRH2a (PF13_0198) and PfRH2b (MAL13P1.176). This study based on gene co-expression network could shed new insights on the mechanisms of pathogenesis, even virulence and P. falciparum development. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Hey bHLH transcription factors.

PubMed

Weber, David; Wiese, Cornelia; Gessler, Manfred

2014-01-01

Hey bHLH transcription factors are direct targets of canonical Notch signaling. The three mammalian Hey proteins are closely related to Hes proteins and they primarily repress target genes by either directly binding to core promoters or by inhibiting other transcriptional activators. Individual candidate gene approaches and systematic screens identified a number of Hey target genes, which often encode other transcription factors involved in various developmental processes. Here, we review data on interaction partners and target genes and conclude with a model for Hey target gene regulation. Furthermore, we discuss how expression of Hey proteins affects processes like cell fate decisions and differentiation, e.g., in cardiovascular, skeletal, and neural development or oncogenesis and how this relates to the observed developmental defects and phenotypes observed in various knockout mice. © 2014 Elsevier Inc. All rights reserved.

Production and pathogenicity of hepatitis C virus core gene products

PubMed Central

Li, Hui-Chun; Ma, Hsin-Chieh; Yang, Chee-Hing; Lo, Shih-Yen

2014-01-01

Hepatitis C virus (HCV) is a major cause of chronic liver diseases, including steatosis, cirrhosis and hepatocellular carcinoma, and its infection is also associated with insulin resistance and type 2 diabetes mellitus. HCV, belonging to the Flaviviridae family, is a small enveloped virus whose positive-stranded RNA genome encoding a polyprotein. The HCV core protein is cleaved first at residue 191 by the host signal peptidase and further cleaved by the host signal peptide peptidase at about residue 177 to generate the mature core protein (a.a. 1-177) and the cleaved peptide (a.a. 178-191). Core protein could induce insulin resistance, steatosis and even hepatocellular carcinoma through various mechanisms. The peptide (a.a. 178-191) may play a role in the immune response. The polymorphism of this peptide is associated with the cellular lipid drop accumulation, contributing to steatosis development. In addition to the conventional open reading frame (ORF), in the +1 frame, an ORF overlaps with the core protein-coding sequence and encodes the alternative reading frame proteins (ARFP or core+1). ARFP/core+1/F protein could enhance hepatocyte growth and may regulate iron metabolism. In this review, we briefly summarized the current knowledge regarding the production of different core gene products and their roles in viral pathogenesis. PMID:24966583

Simultaneous spatiotemporal mapping of in situ pH and bacterial activity within an intact 3D microcolony structure

NASA Astrophysics Data System (ADS)

Hwang, Geelsu; Liu, Yuan; Kim, Dongyeop; Sun, Victor; Aviles-Reyes, Alejandro; Kajfasz, Jessica K.; Lemos, Jose A.; Koo, Hyun

2016-09-01

Biofilms are comprised of bacterial-clusters (microcolonies) enmeshed in an extracellular matrix. Streptococcus mutans can produce exopolysaccharides (EPS)-matrix and assemble microcolonies with acidic microenvironments that can cause tooth-decay despite the surrounding neutral-pH found in oral cavity. How the matrix influences the pH and bacterial activity locally remains unclear. Here, we simultaneously analyzed in situ pH and gene expression within intact biofilms and measured the impact of damage to the surrounding EPS-matrix. The spatiotemporal changes of these properties were characterized at a single-microcolony level following incubation in neutral-pH buffer. The middle and bottom-regions as well as inner-section within the microcolony 3D structure were resistant to neutralization (vs. upper and peripheral-region), forming an acidic core. Concomitantly, we used a green fluorescent protein (GFP) reporter to monitor expression of the pH-responsive atpB (PatpB::gfp) by S. mutans within microcolonies. The atpB expression was induced in the acidic core, but sharply decreased at peripheral/upper microcolony regions, congruent with local pH microenvironment. Enzymatic digestion of the surrounding matrix resulted in nearly complete neutralization of microcolony interior and down-regulation of atpB. Altogether, our data reveal that biofilm matrix facilitates formation of an acidic core within microcolonies which in turn activates S. mutans acid-stress response, mediating both the local environment and bacterial activity in situ.

A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins

PubMed Central

Strutt, Helen; Searle, Elizabeth; Thomas-MacArthur, Victoria; Brookfield, Rosalind; Strutt, David

2013-01-01

The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry. PMID:23487316

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly.

PubMed

Wu, Shuo; Zhao, Qiong; Zhang, Pinghu; Kulp, John; Hu, Lydia; Hwang, Nicky; Zhang, Jiming; Block, Timothy M; Xu, Xiaodong; Du, Yanming; Chang, Jinhong; Guo, Ju-Tao

2017-08-15

Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the heteroaryldihydropyrimidine (HAP) pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated more slowly in native agarose gel electrophoresis from A36V mutant than from the wild-type core protein. Moreover, we showed that the assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence, HBV core protein is a dominant antiviral target that may suppress the selection of drug-resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV. IMPORTANCE HBV core protein plays essential roles in many steps of the viral replication cycle. In addition to packaging viral pregenomic RNA (pgRNA) and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several different quaternary structures in infected hepatocytes, participate in and regulate HBV virion assembly, capsid uncoating, and covalently closed circular DNA (cccDNA) formation. It is anticipated that small molecular core protein assembly modulators may disrupt one or multiple steps of HBV replication, depending on their interaction with the distinct quaternary structures of core protein. The discovery of novel core protein-targeting antivirals, such as benzamide derivatives reported here, and investigation of their antiviral mechanism may lead to the identification of antiviral therapeutics for the cure of chronic hepatitis B. Copyright © 2017 American Society for Microbiology.

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

PubMed

Huang, Yuehua; Tai, Andrew W; Tong, Shuping; Lok, Anna S F

2013-06-01

Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2. We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs. CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB. HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

ZmDof3, a maize endosperm-specific Dof protein gene, regulates starch accumulation and aleurone development in maize endosperm.

PubMed

Qi, Xin; Li, Shixue; Zhu, Yaxi; Zhao, Qian; Zhu, Dengyun; Yu, Jingjuan

2017-01-01

To explore the function of Dof transcription factors during kernel development in maize, we first identified Dof genes in the maize genome. We found that ZmDof3 was exclusively expressed in the endosperm of maize kernel and had the features of a Dof transcription factor. Suppression of ZmDof3 resulted in a defective kernel phenotype with reduced starch content and a partially patchy aleurone layer. The expression levels of starch synthesis-related genes and aleurone differentiation-associated genes were down-regulated in ZmDof3 knockdown kernels, indicating that ZmDof3 plays an important role in maize endosperm development. The maize endosperm, occupying a large proportion of the kernel, plays an important role in seed development and germination. Current knowledge regarding the regulation of endosperm development is limited. Dof proteins, a family of plant-specific transcription factors, play critical roles in diverse biological processes. In this study, an endosperm-specific Dof protein gene, ZmDof3, was identified in maize through genome-wide screening. Suppression of ZmDof3 resulted in a defective kernel phenotype. The endosperm of ZmDof3 knockdown kernels was loosely packed with irregular starch granules observed by electronic microscope. Through genome-wide expression profiling, we found that down-regulated genes were enriched in GO terms related to carbohydrate metabolism. Moreover, ZmDof3 could bind to the Dof core element in the promoter of starch biosynthesis genes Du1 and Su2 in vitro and in vivo. In addition, the aleurone at local position in mature ZmDof3 knockdown kernels varied from one to three layers, which consisted of smaller and irregular cells. Further analyses showed that knockdown of ZmDof3 reduced the expression of Nkd1, which is involved in aleurone cell differentiation, and that ZmDof3 could bind to the Dof core element in the Nkd1 promoter. Our study reveals that ZmDof3 functions in maize endosperm development as a positive regulator in the signaling system controlling starch accumulation and aleurone development.

Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042

PubMed Central

2018-01-01

ABSTRACT Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae, cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli, out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue (hns2). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae. IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool. PMID:29577085

Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042.

PubMed

Prieto, A; Bernabeu, M; Aznar, S; Ruiz-Cruz, S; Bravo, A; Queiroz, M H; Juárez, A

2018-01-01

Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae , cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli , out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue ( hns2 ). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae . IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool.

Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study.

PubMed

Hayashi, K; Hayashi, M; Jalkanen, M; Firestone, J H; Trelstad, R L; Bernfield, M

1987-10-01

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.

Alternative Splicing of Barley Clock Genes in Response to Low Temperature

PubMed Central

Calixto, Cristiane P. G.; Simpson, Craig G.; Waugh, Robbie; Brown, John W. S.

2016-01-01

Alternative splicing (AS) is a regulated mechanism that generates multiple transcripts from individual genes. It is widespread in eukaryotic genomes and provides an effective way to control gene expression. At low temperatures, AS regulates Arabidopsis clock genes through dynamic changes in the levels of productive mRNAs. We examined AS in barley clock genes to assess whether temperature-dependent AS responses also occur in a monocotyledonous crop species. We identify changes in AS of various barley core clock genes including the barley orthologues of Arabidopsis AtLHY and AtPRR7 which showed the most pronounced AS changes in response to low temperature. The AS events modulate the levels of functional and translatable mRNAs, and potentially protein levels, upon transition to cold. There is some conservation of AS events and/or splicing behaviour of clock genes between Arabidopsis and barley. In addition, novel temperature-dependent AS of the core clock gene HvPPD-H1 (a major determinant of photoperiod response and AtPRR7 orthologue) is conserved in monocots. HvPPD-H1 showed a rapid, temperature-sensitive isoform switch which resulted in changes in abundance of AS variants encoding different protein isoforms. This novel layer of low temperature control of clock gene expression, observed in two very different species, will help our understanding of plant adaptation to different environments and ultimately offer a new range of targets for plant improvement. PMID:27959947

Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex

PubMed Central

Andrejka, Laura; Wen, Hong; Ashton, Jonathan; Grant, Megan; Iori, Kevin; Wang, Amy; Manak, J. Robert; Lipsick, Joseph S.

2011-01-01

Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics. PMID:21969598

The WD40 Domain Protein MSI1 Functions in a Histone Deacetylase Complex to Fine-Tune Abscisic Acid Signaling.

PubMed

Mehdi, Saher; Derkacheva, Maria; Ramström, Margareta; Kralemann, Lejon; Bergquist, Jonas; Hennig, Lars

2016-01-01

MSI1 belongs to a family of histone binding WD40-repeat proteins. Arabidopsis thaliana contains five genes encoding MSI1-like proteins, but their functions in diverse chromatin-associated complexes are poorly understood. Here, we show that MSI1 is part of a histone deacetylase complex. We copurified HISTONE DEACETYLASE19 (HDA19) with MSI1 and transcriptional regulatory SIN3-like proteins and provide evidence that MSI1 and HDA19 associate into the same complex in vivo. These data suggest that MSI1, HDA19, and HISTONE DEACETYLATION COMPLEX1 protein form a core complex that can integrate various SIN3-like proteins. We found that reduction of MSI1 or HDA19 causes upregulation of abscisic acid (ABA) receptor genes and hypersensitivity of ABA-responsive genes. The MSI1-HDA19 complex fine-tunes ABA signaling by binding to the chromatin of ABA receptor genes and by maintaining low levels of acetylation of histone H3 at lysine 9, thereby affecting the expression levels of ABA receptor genes. Reduced MSI1 or HDA19 levels led to increased tolerance to salt stress corresponding to the increased ABA sensitivity of gene expression. Together, our results reveal the presence of an MSI1-HDA19 complex that fine-tunes ABA signaling in Arabidopsis. © 2016 American Society of Plant Biologists. All rights reserved.

Genome-wide expression profiling analysis to identify key genes in the anti-HIV mechanism of CD4+ and CD8+ T cells.

PubMed

Gao, Lijie; Wang, Yunqi; Li, Yi; Dong, Ya; Yang, Aimin; Zhang, Jie; Li, Fengying; Zhang, Rongqiang

2018-07-01

Comprehensive bioinformatics analyses were performed to explore the key biomarkers in response to HIV infection of CD4 + and CD8 + T cells. The numbers of CD4 + and CD8 + T cells of HIV infected individuals were analyzed and the GEO database (GSE6740) was screened for differentially expressed genes (DEGs) in HIV infected CD4 + and CD8 + T cells. Gene Ontology enrichment, KEGG pathway analyses, and protein-protein interaction (PPI) network were performed to identify the key pathway and core proteins in anti-HIV virus process of CD4 + and CD8 + T cells. Finally, we analyzed the expressions of key proteins in HIV-infected T cells (GSE6740 dataset) and peripheral blood mononuclear cells(PBMCs) (GSE511 dataset). 1) CD4 + T cells counts and ratio of CD4 + /CD8 + T cells decreased while CD8 + T cells counts increased in HIV positive individuals; 2) 517 DEGs were found in HIV infected CD4 + and CD8 + T cells at acute and chronic stage with the criterial of P-value <0.05 and fold change (FC) ≥2; 3) In acute HIV infection, type 1 interferon (IFN-1) pathway might played a critical role in response to HIV infection of T cells. The main biological processes of the DEGs were response to virus and defense response to virus. At chronic stage, ISG15 protein, in conjunction with IFN-1 pathway might play key roles in anti-HIV responses of CD4 + T cells; and 4) The expression of ISG15 increased in both T cells and PBMCs after HIV infection. Gene expression profile of CD4 + and CD8 + T cells changed significantly in HIV infection, in which ISG15 gene may play a central role in activating the natural antiviral process of immune cells. © 2018 Wiley Periodicals, Inc.

Systems analysis of apoptosis protein expression allows the case-specific prediction of cell death responsiveness of melanoma cells

PubMed Central

Passante, E; Würstle, M L; Hellwig, C T; Leverkus, M; Rehm, M

2013-01-01

Many cancer entities and their associated cell line models are highly heterogeneous in their responsiveness to apoptosis inducers and, despite a detailed understanding of the underlying signaling networks, cell death susceptibility currently cannot be predicted reliably from protein expression profiles. Here, we demonstrate that an integration of quantitative apoptosis protein expression data with pathway knowledge can predict the cell death responsiveness of melanoma cell lines. By a total of 612 measurements, we determined the absolute expression (nM) of 17 core apoptosis regulators in a panel of 11 melanoma cell lines, and enriched these data with systems-level information on apoptosis pathway topology. By applying multivariate statistical analysis and multi-dimensional pattern recognition algorithms, the responsiveness of individual cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or dacarbazine (DTIC) could be predicted with very high accuracy (91 and 82% correct predictions), and the most effective treatment option for individual cell lines could be pre-determined in silico. In contrast, cell death responsiveness was poorly predicted when not taking knowledge on protein–protein interactions into account (55 and 36% correct predictions). We also generated mathematical predictions on whether anti-apoptotic Bcl-2 family members or x-linked inhibitor of apoptosis protein (XIAP) can be targeted to enhance TRAIL responsiveness in individual cell lines. Subsequent experiments, making use of pharmacological Bcl-2/Bcl-xL inhibition or siRNA-based XIAP depletion, confirmed the accuracy of these predictions. We therefore demonstrate that cell death responsiveness to TRAIL or DTIC can be predicted reliably in a large number of melanoma cell lines when investigating expression patterns of apoptosis regulators in the context of their network-level interplay. The capacity to predict responsiveness at the cellular level may contribute to personalizing anti-cancer treatments in the future. PMID:23933815

Evidence for a Role of Chloroplastic m-Type Thioredoxins in the Biogenesis of Photosystem II in Arabidopsis1[C][W][OPEN

PubMed Central

Wang, Peng; Liu, Jun; Liu, Bing; Feng, Dongru; Da, Qingen; Wang, Peng; Shu, Shengying; Su, Jianbin; Zhang, Yang; Wang, Jinfa; Wang, Hong-Bin

2013-01-01

Chloroplastic m-type thioredoxins (TRX m) are essential redox regulators in the light regulation of photosynthetic metabolism. However, recent genetic studies have revealed novel functions for TRX m in meristem development, chloroplast morphology, cyclic electron flow, and tetrapyrrole synthesis. The focus of this study is on the putative role of TRX m1, TRX m2, and TRX m4 in the biogenesis of the photosynthetic apparatus in Arabidopsis (Arabidopsis thaliana). To that end, we investigated the impact of single, double, and triple TRX m deficiency on chloroplast development and the accumulation of thylakoid protein complexes. Intriguingly, only inactivation of three TRX m genes led to pale-green leaves and specifically reduced stability of the photosystem II (PSII) complex, implying functional redundancy between three TRX m isoforms. In addition, plants silenced for three TRX m genes displayed elevated levels of reactive oxygen species, which in turn interrupted the transcription of photosynthesis-related nuclear genes but not the expression of chloroplast-encoded PSII core proteins. To dissect the function of TRX m in PSII biogenesis, we showed that TRX m1, TRX m2, and TRX m4 interact physically with minor PSII assembly intermediates as well as with PSII core subunits D1, D2, and CP47. Furthermore, silencing three TRX m genes disrupted the redox status of intermolecular disulfide bonds in PSII core proteins, most notably resulting in elevated accumulation of oxidized CP47 oligomers. Taken together, our results suggest an important role for TRX m1, TRX m2, and TRX m4 proteins in the biogenesis of PSII, and they appear to assist the assembly of CP47 into PSII. PMID:24151299

Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor l-arginine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garnett, James A.; Baumberg, Simon; Stockley, Peter G.

2007-11-01

The crystal structure of the C-terminal domain hexameric core of AhrC, with bound corepressor (l-arginine), has been solved at 1.95 Å resolution. Binding of l-arginine results in a rotation between the two trimers of the hexamer, leading to the activation of the DNA-binding state. The arginine repressor/activator protein (AhrC) from Bacillus subtilis belongs to a large family of multifunctional transcription factors that are involved in the regulation of bacterial arginine metabolism. AhrC interacts with operator sites in the promoters of arginine biosynthetic and catabolic operons, acting as a transcriptional repressor at biosynthetic sites and an activator of transcription at catabolicmore » sites. AhrC is a hexamer of identical subunits, each having two domains. The C-terminal domains form the core of the protein and are involved in oligomerization and l-arginine binding. The N-terminal domains lie on the outside of the compact core and play a role in binding to 18 bp DNA operators called ARG boxes. The C-terminal domain of AhrC has been expressed, purified and characterized, and also crystallized as a hexamer with the bound corepressor l-arginine. Here, the crystal structure refined to 1.95 Å is presented.« less

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

DOE PAGES

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany; ...

2017-01-31

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

Comparative Genomics of Herpesviridae Family to Look for Potential Signatures of Human Infecting Strains

PubMed Central

2016-01-01

Herpesviridae family is one of the significant viral families which comprises major pathogens of a wide range of hosts. This family includes at least eight species of viruses which are known to infect humans. This family has evolved 180–220 million years ago and the present study highlights that it is still evolving and more genes can be added to the repertoire of this family. In addition, its core-genome includes important viral proteins including glycoprotein B and helicase. Most of the infections caused by human herpesviruses have no definitive cure; thus, search for new therapeutic strategies is necessary. The present study finds core-genome of human herpesviruses that differs from that of Herpesviridae family and nonhuman herpes strains of this family and might be a putative target for vaccine development. The phylogenetic reconstruction based upon the protein sequences of core gene set of Herpesviridae family reveals the sharp splits of its different subfamilies and supports the hypothesis of coevolution of viruses with their hosts. In addition, data mining for cis-elements in the genomes of human herpesviruses results in the prediction of numerous regulatory elements which can be used for regulating the expression of viral based vectors implicated in gene therapies. PMID:27314006

Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors.

PubMed

Schröder, R; Maassen, A; Lippoldt, A; Börner, T; von Baehr, R; Dobrowolski, P

1991-08-01

Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids.

What is shared, what is different? Core relational themes and expressive displays of eight positive emotions.

PubMed

Campos, Belinda; Shiota, Michelle N; Keltner, Dacher; Gonzaga, Gian C; Goetz, Jennifer L

2013-01-01

Understanding positive emotions' shared and differentiating features can yield valuable insight into the structure of positive emotion space and identify emotion states, or aspects of emotion states, that are most relevant for particular psychological processes and outcomes. We report two studies that examined core relational themes (Study 1) and expressive displays (Study 2) for eight positive emotion constructs--amusement, awe, contentment, gratitude, interest, joy, love, and pride. Across studies, all eight emotions shared one quality: high positive valence. Distinctive core relational theme and expressive display patterns were found for four emotions--amusement, awe, interest, and pride. Gratitude was associated with a distinct core relational theme but not an expressive display. Joy and love were each associated with a distinct expressive display but their core relational themes also characterised pride and gratitude, respectively. Contentment was associated with a distinct expressive display but not a core relational theme. The implications of this work for the study of positive emotion are discussed.

Quantitative Proteomic Analysis of Optimal Cutting Temperature (OCT) Embedded Core-Needle Biopsy of Lung Cancer

NASA Astrophysics Data System (ADS)

Zhao, Xiaozheng; Huffman, Kenneth E.; Fujimoto, Junya; Canales, Jamie Rodriguez; Girard, Luc; Nie, Guangjun; Heymach, John V.; Wistuba, Igacio I.; Minna, John D.; Yu, Yonghao

2017-10-01

With recent advances in understanding the genomic underpinnings and oncogenic drivers of pathogenesis in different subtypes, it is increasingly clear that proper pretreatment diagnostics are essential for the choice of appropriate treatment options for non-small cell lung cancer (NSCLC). Tumor tissue preservation in optimal cutting temperature (OCT) compound is commonly used in the surgical suite. However, proteins recovered from OCT-embedded specimens pose a challenge for LC-MS/MS experiments, due to the large amounts of polymers present in OCT. Here we present a simple workflow for whole proteome analysis of OCT-embedded NSCLC tissue samples, which involves a simple trichloroacetic acid precipitation step. Comparisons of protein recovery between frozen versus OCT-embedded tissue showed excellent consistency with more than 9200 proteins identified. Using an isobaric labeling strategy, we quantified more than 5400 proteins in tumor versus normal OCT-embedded core needle biopsy samples. Gene ontology analysis indicated that a number of proliferative as well as squamous cell carcinoma (SqCC) marker proteins were overexpressed in the tumor, consistent with the patient's pathology based diagnosis of "poorly differentiated SqCC". Among the most downregulated proteins in the tumor sample, we noted a number of proteins with potential immunomodulatory functions. Finally, interrogation of the aberrantly expressed proteins using a candidate approach and cross-referencing with publicly available databases led to the identification of potential druggable targets in DNA replication and DNA damage repair pathways. We conclude that our approach allows LC-MS/MS proteomic analyses on OCT-embedded lung cancer specimens, opening the way to bring powerful proteomics into the clinic. [Figure not available: see fulltext.

A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA-DNA Complex Structure and Binding Specificities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Xun; Guanga, Gerald P; Wan, Cheng

2012-11-13

MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G –5C –4 andmore » central C 0/G 0 bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.« less

Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations.

PubMed

Elizalde, María Mercedes; Sevic, Ina; González López Ledesma, María Mora; Campos, Rodolfo Héctor; Barbini, Luciana; Flichman, Diego Martin

2018-01-01

In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM.

PubMed

Ma, Junfeng; Sanda, Miloslav; Wei, Renhuizi; Zhang, Lihua; Goldman, Radoslav

2018-02-07

Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and selective LC-MS-MRM method. Despite an overall increase in core fucosylation of many of the glycoproteins that we examined, core fucosylation changed in a protein- and site-specific manner. Moreover, increased and decreased fucosylation was observed on different N-glycopeptides of the same protein. Altered core fucosylation of N-glycopeptides might be used as an alternative serologic assay for the evaluation of fibrotic liver disease. Copyright © 2018. Published by Elsevier B.V.

Comparison of cytotoxicity and expression of metal regulatory genes in zebrafish (Danio rerio) liver cells exposed to cadmium sulfate, zinc sulfate and quantum dots.

PubMed

Tang, Song; Allagadda, Vinay; Chibli, Hicham; Nadeau, Jay L; Mayer, Gregory D

2013-10-01

Recent advances in the ability to manufacture and manipulate materials at the nanometer scale have led to increased production and use of many types of nanoparticles. Quantum dots (QDs) are small, fluorescent nanoparticles composed of a core of semiconductor material (e.g. cadmium selenide, zinc sulfide) and shells or dopants of other elements. Particle core composition, size, shell, and surface chemistry have all been found to influence toxicity in cells. The aim of this study was to compare the toxicities of ionic cadmium (Cd) and zinc (Zn) and Cd- and Zn-containing QDs in zebrafish liver cells (ZFL). As expected, Cd(2+) was more toxic than Zn(2+), and the general trend of IC50-24 h values of QDs was determined to be CdTe < CdSe/ZnS or InP/ZnS, suggesting that ZnS-shelled CdSe/ZnS QDs were more cytocompatible than bare core CdTe crystals. Smaller QDs showed greater toxicity than larger QDs. Isolated mRNA from these exposures was used to measure the expression of metal response genes including metallothionein (MT), metal response element-binding transcription factor (MTF-1), divalent metal transporter (DMT-1), zrt and irt like protein (ZIP-1) and the zinc transporter, ZnT-1. CdTe exposure induced expression of these genes in a dose dependent manner similar to that of CdSO4 exposure. However, CdSe/ZnS and InP/ZnS altered gene expression of metal homeostasis genes in a manner different from that of the corresponding Cd or Zn salts. This implies that ZnS shells reduce QD toxicity attributed to the release of Cd(2+), but do not eliminate toxic effects caused by the nanoparticles themselves.

Formation of Orthopoxvirus Cytoplasmic A-Type Inclusion Bodies and Embedding of Virions Are Dynamic Processes Requiring Microtubules

PubMed Central

Howard, Amanda R.

2012-01-01

In cells infected with some orthopoxviruses, numerous mature virions (MVs) become embedded within large, cytoplasmic A-type inclusions (ATIs) that can protect infectivity after cell lysis. ATIs are composed of an abundant viral protein called ATIp, which is truncated in orthopoxviruses such as vaccinia virus (VACV) that do not form ATIs. To study ATI formation and occlusion of MVs within ATIs, we used recombinant VACVs that express the cowpox full-length ATIp or we transfected plasmids encoding ATIp into cells infected with VACV, enabling ATI formation. ATI enlargement and MV embedment required continued protein synthesis and an intact microtubular network. For live imaging of ATIs and MVs, plasmids expressing mCherry fluorescent protein fused to ATIp were transfected into cells infected with VACV expressing the viral core protein A4 fused to yellow fluorescent protein. ATIs appeared as dynamic, mobile bodies that enlarged by multiple coalescence events, which could be prevented by disrupting microtubules. Coalescence of ATIs was confirmed in cells infected with cowpox virus. MVs were predominantly at the periphery of ATIs early in infection. We determined that coalescence contributed to the distribution of MVs within ATIs and that microtubule-disrupting drugs abrogated coalescence-mediated MV embedment. In addition, MVs were shown to move from viral factories at speeds consistent with microtubular transport to the peripheries of ATIs, whereas disruption of microtubules prevented such trafficking. The data indicate an important role for microtubules in the coalescence of ATIs into larger structures, transport of MVs to ATIs, and embedment of MVs within the ATI matrix. PMID:22438543

In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica) *

PubMed Central

Fang, Yu; Feng, Mao; Han, Bin; Lu, Xiaoshan; Ramadan, Haitham; Li, Jianke

2014-01-01

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24–48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48–72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects. PMID:24895377

In-depth proteomics characterization of embryogenesis of the honey bee worker (Apis mellifera ligustica).

PubMed

Fang, Yu; Feng, Mao; Han, Bin; Lu, Xiaoshan; Ramadan, Haitham; Li, Jianke

2014-09-01

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24-48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48-72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Inositol trisphosphate receptor-mediated Ca2+ signalling stimulates mitochondrial function and gene expression in core myopathy patients.

PubMed

Suman, Matteo; Sharpe, Jenny A; Bentham, Robert B; Kotiadis, Vassilios N; Menegollo, Michela; Pignataro, Viviana; Molgó, Jordi; Muntoni, Francesco; Duchen, Michael R; Pegoraro, Elena; Szabadkai, Gyorgy

2018-07-01

Core myopathies are a group of childhood muscle disorders caused by mutations of the ryanodine receptor (RyR1), the Ca2+ release channel of the sarcoplasmic reticulum. These mutations have previously been associated with elevated inositol trisphosphate receptor (IP3R) levels in skeletal muscle myotubes derived from patients. However, the functional relevance and the relationship of IP3R mediated Ca2+ signalling with the pathophysiology of the disease is unclear. It has also been suggested that mitochondrial dysfunction underlies the development of central and diffuse multi-mini-cores, devoid of mitochondrial activity, which is a key pathological consequence of RyR1 mutations. Here we used muscle biopsies of central core and multi-minicore disease patients with RyR1 mutations, as well as cellular and in vivo mouse models of the disease to characterize global cellular and mitochondrial Ca2+ signalling, mitochondrial function and gene expression associated with the disease. We show that RyR1 mutations that lead to the depletion of the channel are associated with increased IP3-mediated nuclear and mitochondrial Ca2+ signals and increased mitochondrial activity. Moreover, western blot and microarray analysis indicated enhanced mitochondrial biogenesis at the transcriptional and protein levels and was reflected in increased mitochondrial DNA content. The phenotype was recapitulated by RYR1 silencing in mouse cellular myotube models. Altogether, these data indicate that remodelling of skeletal muscle Ca2+ signalling following loss of functional RyR1 mediates bioenergetic adaptation.

The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

PubMed Central

Vincent, Maxence S.; Durand, Eric; Cascales, Eric

2016-01-01

The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

Convergent roles of de novo mutations and common variants in schizophrenia in tissue-specific and spatiotemporal co-expression network.

PubMed

Jia, Peilin; Chen, Xiangning; Fanous, Ayman H; Zhao, Zhongming

2018-05-24

Genetic components susceptible to complex disease such as schizophrenia include a wide spectrum of variants, including common variants (CVs) and de novo mutations (DNMs). Although CVs and DNMs differ by origin, it remains elusive whether and how they interact at the gene, pathway, and network levels that leads to the disease. In this work, we characterized the genes harboring schizophrenia-associated CVs (CVgenes) and the genes harboring DNMs (DNMgenes) using measures from network, tissue-specific expression profile, and spatiotemporal brain expression profile. We developed an algorithm to link the DNMgenes and CVgenes in spatiotemporal brain co-expression networks. DNMgenes tended to have central roles in the human protein-protein interaction (PPI) network, evidenced in their high degree and high betweenness values. DNMgenes and CVgenes connected with each other significantly more often than with other genes in the networks. However, only CVgenes remained significantly connected after adjusting for their degree. In our gene co-expression PPI network, we found DNMgenes and CVgenes connected in a tissue-specific fashion, and such a pattern was similar to that in GTEx brain but not in other GTEx tissues. Importantly, DNMgene-CVgene subnetworks were enriched with pathways of chromatin remodeling, MHC protein complex binding, and neurotransmitter activities. In summary, our results unveiled that both DNMgenes and CVgenes contributed to a core set of biologically important pathways and networks, and their interactions may attribute to the risk for schizophrenia. Our results also suggested a stronger biological effect of DNMgenes than CVgenes in schizophrenia.

Enhancement of myofibrillar proteolysis following infusion of amino acid mixture correlates positively with elevation of core body temperature in rats.

PubMed

Yamaoka, Ippei; Mikura, Mayumi; Nishimura, Masuhiro; Doi, Masako; Kawano, Yuichi; Nakayama, Mitsuo

2008-12-01

Administration of an amino acid (AA) mixture stimulates muscle protein synthesis and elevates core body temperature (T(b)), as characteristically found under anesthetic conditions. We tested the hypothesis that not only AA given, but also AA produced by degradation of endogenous muscular protein are provided for muscle protein synthesis, which is further reflected in T(b) modifications. Rats were intravenously administered an AA mixture or saline in combination with the anesthetic propofol or lipid emulsion. We measured plasma 3-methylhistidine (MeHis) concentrations as an index of myofibrillar protein degradation, rectal temperature and mRNA expression of atrogin-1, MuRF-1 and ubiquitin in gastrocnemius and soleus muscles of rats following 3 h infusion of test solutions. T(b) did not differ significantly between conscious groups, but was higher in the AA group than in the saline group among anesthetized rats. Plasma MeHis concentrations were higher in the AA group than in the saline group under both conditions. Plasma MeHis levels correlated positively with T(b) of rats under both conditions. AA administration decreased mRNA levels of atrogin-1 and ubiquitin in gastrocnemius muscle and all mRNA levels in soleus muscle. These results suggest that AA administration enhances myofibrillar protein degradation and that the change is a determinant of T(b) modification by AA administration. However, the mechanisms underlying AA administration-associated enhancement of myofibrillar proteolysis remains yet to be determined.

Domain organization, genomic structure, evolution, and regulation of expression of the aggrecan gene family.

PubMed

Schwartz, N B; Pirok, E W; Mensch, J R; Domowicz, M S

1999-01-01

Proteoglycans are complex macromolecules, consisting of a polypeptide backbone to which are covalently attached one or more glycosaminoglycan chains. Molecular cloning has allowed identification of the genes encoding the core proteins of various proteoglycans, leading to a better understanding of the diversity of proteoglycan structure and function, as well as to the evolution of a classification of proteoglycans on the basis of emerging gene families that encode the different core proteins. One such family includes several proteoglycans that have been grouped with aggrecan, the large aggregating chondroitin sulfate proteoglycan of cartilage, based on a high number of sequence similarities within the N- and C-terminal domains. Thus far these proteoglycans include versican, neurocan, and brevican. It is now apparent that these proteins, as a group, are truly a gene family with shared structural motifs on the protein and nucleotide (mRNA) levels, and with nearly identical genomic organizations. Clearly a common ancestral origin is indicated for the members of the aggrecan family of proteoglycans. However, differing patterns of amplification and divergence have also occurred within certain exons across species and family members, leading to the class-characteristic protein motifs in the central carbohydrate-rich region exclusively. Thus the overall domain organization strongly suggests that sequence conservation in the terminal globular domains underlies common functions, whereas differences in the central portions of the genes account for functional specialization among the members of this gene family.

Large-Scale Gene Relocations following an Ancient Genome Triplication Associated with the Diversification of Core Eudicots.

PubMed

Wang, Yupeng; Ficklin, Stephen P; Wang, Xiyin; Feltus, F Alex; Paterson, Andrew H

2016-01-01

Different modes of gene duplication including whole-genome duplication (WGD), and tandem, proximal and dispersed duplications are widespread in angiosperm genomes. Small-scale, stochastic gene relocations and transposed gene duplications are widely accepted to be the primary mechanisms for the creation of dispersed duplicates. However, here we show that most surviving ancient dispersed duplicates in core eudicots originated from large-scale gene relocations within a narrow window of time following a genome triplication (γ) event that occurred in the stem lineage of core eudicots. We name these surviving ancient dispersed duplicates as relocated γ duplicates. In Arabidopsis thaliana, relocated γ, WGD and single-gene duplicates have distinct features with regard to gene functions, essentiality, and protein interactions. Relative to γ duplicates, relocated γ duplicates have higher non-synonymous substitution rates, but comparable levels of expression and regulation divergence. Thus, relocated γ duplicates should be distinguished from WGD and single-gene duplicates for evolutionary investigations. Our results suggest large-scale gene relocations following the γ event were associated with the diversification of core eudicots.

Large-Scale Gene Relocations following an Ancient Genome Triplication Associated with the Diversification of Core Eudicots

PubMed Central

Wang, Yupeng; Ficklin, Stephen P.; Wang, Xiyin; Feltus, F. Alex; Paterson, Andrew H.

2016-01-01

Different modes of gene duplication including whole-genome duplication (WGD), and tandem, proximal and dispersed duplications are widespread in angiosperm genomes. Small-scale, stochastic gene relocations and transposed gene duplications are widely accepted to be the primary mechanisms for the creation of dispersed duplicates. However, here we show that most surviving ancient dispersed duplicates in core eudicots originated from large-scale gene relocations within a narrow window of time following a genome triplication (γ) event that occurred in the stem lineage of core eudicots. We name these surviving ancient dispersed duplicates as relocated γ duplicates. In Arabidopsis thaliana, relocated γ, WGD and single-gene duplicates have distinct features with regard to gene functions, essentiality, and protein interactions. Relative to γ duplicates, relocated γ duplicates have higher non-synonymous substitution rates, but comparable levels of expression and regulation divergence. Thus, relocated γ duplicates should be distinguished from WGD and single-gene duplicates for evolutionary investigations. Our results suggest large-scale gene relocations following the γ event were associated with the diversification of core eudicots. PMID:27195960

Ecotypic variation in chloroplast small heat-shock proteins and related thermotolerance in Chenopodium album.

PubMed

Shakeel, Samina; Haq, Noor Ul; Heckathorn, Scott A; Hamilton, E William; Luthe, Dawn S

2011-08-01

Production of chloroplast-localized small heat-shock proteins (Cp-sHSP) is correlated with increased thermotolerance in plants. Ecotypic variation in function and expression of Cp-sHSPs was analyzed in two Chenopodium album ecotypes from cool vs. warm-temperate USA habitats [New York (NY) and Mississippi (MS) respectively]. P(et) was more heat tolerant in the MS than the NY ecotype, and MS ecotype derived proportionally greater protection of P(et) by Cp-sHSP during high temperatures. Four genes encoding Cp-sHSPs were isolated and characterized: CaHSP25.99n (NY-1) and CaHSP26.23n (NY-2) from NY ecotype, and CaHSP26.04m (MS-1) and CaHSP26.26m (MS-2) from MS ecotype. The genes were nearly identical in predicted amino-acid sequence and hydrophobicity. Gene expression analysis indicated that MS-1 and MS-2 transcripts were constitutively expressed at low levels at 25 °C, while no NY-1 and NY-2 transcripts were detected at this temperature. Maximum accumulation of NY-1 and NY-2 transcripts occurred at 33 °C and 40 °C for MS-1 and MS-2. Immunoblot analysis revealed that (1) protein expression was highest at 37 °C in both ecotypes, but was greater in MS than NY ecotype at 40 °C; and (2) import of Cp-sHSP into chloroplasts was more heat-labile in NY ecotype. The higher expression of one isoform in MS ecotype may contribute to its enhanced thermotolerance. Absence of correlation between protein and transcript levels, suggests the post-transcriptional regulation is occurring. Promoter analysis of these genes revealed significant variations in heat-shock elements (HSE), core motifs required for heat-shock-factor binding. We propose a correlation between unique promoter architecture, Cp-sHSP expression and thermotolerance in both ecotypes. Published by Elsevier Masson SAS.

Expression of the Long Intergenic Non-Protein Coding RNA 665 (LINC00665) Gene and the Cell Cycle in Hepatocellular Carcinoma Using The Cancer Genome Atlas, the Gene Expression Omnibus, and Quantitative Real-Time Polymerase Chain Reaction.

PubMed

Wen, Dong-Yue; Lin, Peng; Pang, Yu-Yan; Chen, Gang; He, Yun; Dang, Yi-Wu; Yang, Hong

2018-05-05

BACKGROUND Long non-coding RNAs (lncRNAs) have a role in physiological and pathological processes, including cancer. The aim of this study was to investigate the expression of the long intergenic non-protein coding RNA 665 (LINC00665) gene and the cell cycle in hepatocellular carcinoma (HCC) using database analysis including The Cancer Genome Atlas (TCGA), the Gene Expression Omnibus (GEO), and quantitative real-time polymerase chain reaction (qPCR). MATERIAL AND METHODS Expression levels of LINC00665 were compared between human tissue samples of HCC and adjacent normal liver, clinicopathological correlations were made using TCGA and the GEO, and qPCR was performed to validate the findings. Other public databases were searched for other genes associated with LINC00665 expression, including The Atlas of Noncoding RNAs in Cancer (TANRIC), the Multi Experiment Matrix (MEM), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) networks. RESULTS Overexpression of LINC00665 in patients with HCC was significantly associated with gender, tumor grade, stage, and tumor cell type. Overexpression of LINC00665 in patients with HCC was significantly associated with overall survival (OS) (HR=1.47795%; CI: 1.046-2.086). Bioinformatics analysis identified 469 related genes and further analysis supported a hypothesis that LINC00665 regulates pathways in the cell cycle to facilitate the development and progression of HCC through ten identified core genes: CDK1, BUB1B, BUB1, PLK1, CCNB2, CCNB1, CDC20, ESPL1, MAD2L1, and CCNA2. CONCLUSIONS Overexpression of the lncRNA, LINC00665 may be involved in the regulation of cell cycle pathways in HCC through ten identified hub genes.

Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes.

PubMed

Keller, Lani C; Romijn, Edwin P; Zamora, Ivan; Yates, John R; Marshall, Wallace F

2005-06-21

The centriole is one of the most enigmatic organelles in the cell. Centrioles are cylindrical, microtubule-based barrels found in the core of the centrosome. Centrioles also act as basal bodies during interphase to nucleate the assembly of cilia and flagella. There are currently only a handful of known centriole proteins. We used mass-spectrometry-based MudPIT (multidimensional protein identification technology) to identify the protein composition of basal bodies (centrioles) isolated from the green alga Chlamydomonas reinhardtii. This analysis detected the majority of known centriole proteins, including centrin, epsilon tubulin, and the cartwheel protein BLD10p. By combining proteomic data with information about gene expression and comparative genomics, we identified 45 cross-validated centriole candidate proteins in two classes. Members of the first class of proteins (BUG1-BUG27) are encoded by genes whose expression correlates with flagellar assembly and which therefore may play a role in ciliogenesis-related functions of basal bodies. Members of the second class (POC1-POC18) are implicated by comparative-genomics and -proteomics studies to be conserved components of the centriole. We confirmed centriolar localization for the human homologs of four candidate proteins. Three of the cross-validated centriole candidate proteins are encoded by orthologs of genes (OFD1, NPHP-4, and PACRG) implicated in mammalian ciliary function and disease, suggesting that oral-facial-digital syndrome and nephronophthisis may involve a dysfunction of centrioles and/or basal bodies. By analyzing isolated Chlamydomonas basal bodies, we have been able to obtain the first reported proteomic analysis of the centriole.

Characterization of SLCO5A1/OATP5A1, a Solute Carrier Transport Protein with Non-Classical Function

PubMed Central

Sebastian, Katrin; Detro-Dassen, Silvia; Rinis, Natalie; Fahrenkamp, Dirk; Müller-Newen, Gerhard; Merk, Hans F.; Schmalzing, Günther

2013-01-01

Organic anion transporting polypeptides (OATP/SLCO) have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (∼6.5-fold) and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of ∼105 kDa and ∼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of ∼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20) and genes implicated in developmental processes (e.g. TGM2). A single nucleotide polymorphism leading to the exchange of amino acid 33 (L→F) revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration. PMID:24376674

Network-based co-expression analysis for exploring the potential diagnostic biomarkers of metastatic melanoma.

PubMed

Wang, Li-Xin; Li, Yang; Chen, Guan-Zhi

2018-01-01

Metastatic melanoma is an aggressive skin cancer and is one of the global malignancies with high mortality and morbidity. It is essential to identify and verify diagnostic biomarkers of early metastatic melanoma. Previous studies have systematically assessed protein biomarkers and mRNA-based expression characteristics. However, molecular markers for the early diagnosis of metastatic melanoma have not been identified. To explore potential regulatory targets, we have analyzed the gene microarray expression profiles of malignant melanoma samples by co-expression analysis based on the network approach. The differentially expressed genes (DEGs) were screened by the EdgeR package of R software. A weighted gene co-expression network analysis (WGCNA) was used for the identification of DEGs in the special gene modules and hub genes. Subsequently, a protein-protein interaction network was constructed to extract hub genes associated with gene modules. Finally, twenty-four important hub genes (RASGRP2, IKZF1, CXCR5, LTB, BLK, LINGO3, CCR6, P2RY10, RHOH, JUP, KRT14, PLA2G3, SPRR1A, KRT78, SFN, CLDN4, IL1RN, PKP3, CBLC, KRT16, TMEM79, KLK8, LYPD3 and LYPD5) were treated as valuable factors involved in the immune response and tumor cell development in tumorigenesis. In addition, a transcriptional regulatory network was constructed for these specific modules or hub genes, and a few core transcriptional regulators were found to be mostly associated with our hub genes, including GATA1, STAT1, SP1, and PSG1. In summary, our findings enhance our understanding of the biological process of malignant melanoma metastasis, enabling us to identify specific genes to use for diagnostic and prognostic markers and possibly for targeted therapy.

Melatonin attenuates neuronal apoptosis through up-regulation of K(+) -Cl(-) cotransporter KCC2 expression following traumatic brain injury in rats.

PubMed

Wu, Haijian; Shao, Anwen; Zhao, Mingfei; Chen, Sheng; Yu, Jun; Zhou, Jingyi; Liang, Feng; Shi, Ligen; Dixon, Brandon J; Wang, Zhen; Ling, Chenhan; Hong, Yuan; Zhang, Jianmin

2016-09-01

Traumatic brain injury (TBI) initiates a complex cascade of neurochemical and signaling changes that leads to neuronal apoptosis, which contributes to poor outcomes for patients with TBI. The neuron-specific K(+) -Cl(-) cotransporter-2 (KCC2), the principal Cl(-) extruder in adult neurons, plays an important role in Cl(-) homeostasis and neuronal function. This present study was designed to investigate the expression pattern of KCC2 following TBI and to evaluate whether or not melatonin is able to prevent neuronal apoptosis by modulating KCC2 expression in a Sprague Dawley rat controlled cortical impact model of TBI. The time course study showed decreased mRNA and protein expression of KCC2 in the ipsilateral peri-core parietal cortex after TBI. Double immunofluorescence staining demonstrated that KCC2 is located in the plasma membrane of neurons. In addition, melatonin (10 mg/kg) was injected intraperitoneally at 5 minutes and repeated at 1, 2, 3, and 4 hours after brain trauma, and brain samples were extracted 24 hours after TBI. Compared to the vehicle group, melatonin treatment altered the down-regulation of KCC2 expression in both mRNA and protein levels after TBI. Also, melatonin treatment increased the protein levels of brain-derived neurotrophic factor (BDNF) and phosphorylated extracellular signal-regulated kinase (p-ERK). Simultaneously, melatonin administration ameliorated cortical neuronal apoptosis, reduced brain edema, and attenuated neurological deficits after TBI. In conclusion, our findings suggested that melatonin restores KCC2 expression, inhibits neuronal apoptosis and attenuates secondary brain injury after TBI, partially through activation of BDNF/ERK pathway. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genome-wide identification of translationally inhibited and degraded miR-155 targets using RNA-interacting protein-IP

PubMed Central

Meier, Jan; Hovestadt, Volker; Zapatka, Marc; Pscherer, Armin; Lichter, Peter; Seiffert, Martina

2013-01-01

MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, which fine-tune protein expression by degrading and/or translationally inhibiting mRNAs. Manipulation of miRNA expression in animal models frequently results in severe phenotypes indicating their relevance in controlling cellular functions, most likely by interacting with multiple targets. To better understand the effect of miRNA activities, genome-wide analysis of their targets are required. MicroRNA profiling as well as transcriptome analysis upon enforced miRNA expression were frequently used to investigate their relevance. However, these approaches often fail to identify relevant miRNAs targets. Therefore, we tested the precision of RNA-interacting protein immunoprecipitation (RIP) using AGO2-specific antibodies, a core component of the “RNA-induced silencing complex” (RISC), followed by RNA sequencing (Seq) in a defined cellular system, the HEK293T cells with stable, ectopic expression of miR-155. Thereby, we identified 100 AGO2-associated mRNAs in miR-155-expressing cells, of which 67 were in silico predicted miR-155 target genes. An integrated analysis of the corresponding expression profiles indicated that these targets were either regulated by mRNA decay or by translational repression. Of the identified miR-155 targets, 17 were related to cell cycle control, suggesting their involvement in the observed increase in cell proliferation of HEK293T cells upon miR-155 expression. Additional, secondary changes within the gene expression profile were detected and might contribute to this phenotype as well. Interestingly, by analyzing RIP-Seq data of HEK-293T cells and two B-cell lines we identified a recurrent disproportional enrichment of several miRNAs, including miR-155 and miRNAs of the miR-17-92 cluster, in the AGO2-associated precipitates, suggesting discrepancies in miRNA expression and activity. PMID:23673373

Spherical tensor analysis of polar liquid crystals with biaxial and chiral molecules

NASA Astrophysics Data System (ADS)

Iwamoto, Mitsumasa; Zhong-can, Ou-Yang

2012-11-01

With the help of spherical tensor expression, an irreducible calculus of a Lth-rank macroscopic susceptibility χ for a polar liquid crystal (PLC) of biaxial and chiral molecules written as the average of molecular hyperpolarizability tensor β associated with their spherical orientational order parameters (0⩽l⩽L) is presented. Comprehensive formulas of L=1,2 have been obtained and the latter explains the optical activity and spontaneous splay texture observed in bent-core PLC. The expression of L=3 specifies for the molecules with D2 symmetry which can be applied to analyze the nonlinear optical second harmonic generation (SHG) observed in proteins, peptides, and double-stranded DNA at interfaces.

CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline

PubMed Central

Campbell, Anne C.; Updike, Dustin L.

2015-01-01

Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription. PMID:25968310

Interaction between core protein of classical swine fever virus with cellular IQGAP1 proetin appears essential for virulence in swine

USDA-ARS?s Scientific Manuscript database

Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton affecting cell adhesion, polarization and migration, interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified a defined set of residues within CSFV Core prote...

Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kokusho, Ryuhei, E-mail: kokusho@ss.ab.a.u-tokyo.a

2016-11-15

Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulationmore » of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.« less

Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes.

PubMed

Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

2016-10-18

The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains-In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

The ESFRI Instruct Core Centre Frankfurt: automated high-throughput crystallization suited for membrane proteins and more.

PubMed

Thielmann, Yvonne; Koepke, Juergen; Michel, Hartmut

2012-06-01

Structure determination of membrane proteins and membrane protein complexes is still a very challenging field. To facilitate the work on membrane proteins the Core Centre follows a strategy that comprises four labs of protein analytics and crystal handling, covering mass spectrometry, calorimetry, crystallization and X-ray diffraction. This general workflow is presented and a capacity of 20% of the operating time of all systems is provided to the European structural biology community within the ESFRI Instruct program. A description of the crystallization service offered at the Core Centre is given with detailed information on screening strategy, screens used and changes to adapt high throughput for membrane proteins. Our aim is to constantly develop the Core Centre towards the usage of more efficient methods. This strategy might also include the ability to automate all steps from crystallization trials to crystal screening; here we look ahead how this aim might be realized at the Core Centre.

New Class of Cargo Protein in Tetrahymena thermophila Dense Core Secretory Granules

PubMed Central

Haddad, Alex; Bowman, Grant R.; Turkewitz, Aaron P.

2002-01-01

Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules. PMID:12456006

Expression of Proteins Involved in Epithelial-Mesenchymal Transition as Predictors of Metastasis and Survival in Breast Cancer Patients

DTIC Science & Technology

2015-01-01

were enrolled in the Data Bank and BioRepository at Roswell Park Cancer Institute. These results were presented at the 2011 AACR Annual Meeting and...participants with tumor tissue from the Pathology core facility at Roswell Park. We examined these samples for quality and quantity by several different...presented data at seminars and work-in-progress meetings at Roswell Park and the University at Buffalo. The trainee has also actively participated in

Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility.

PubMed

Etzkorn, Manuel; Raschle, Thomas; Hagn, Franz; Gelev, Vladimir; Rice, Amanda J; Walz, Thomas; Wagner, Gerhard

2013-03-05

Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Nonconventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here, we present a comparative study of the heptahelical membrane protein bacteriorhodopsin in detergent micelles, amphipols, and nanodiscs. Our results confirm that nonconventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are, in principle, accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguish regions of bacteriorhodopsin that mediate membrane/solvent contacts in the tested environments, whereas the protein's functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

E(y)2/Sus1 is required for blocking PRE silencing by the Wari insulator in Drosophila melanogaster.

PubMed

Erokhin, Maksim; Parshikov, Alexander; Georgiev, Pavel; Chetverina, Darya

2010-06-01

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers to the spread of repressive chromatin. Recently, we have found an insulator, named Wari, located on the 3' side of the white gene. Here, we show that the previously identified 368-bp core of this insulator is sufficient for blocking Polycomb response element-mediated silencing. Although Wari does not contain binding sites for known insulator proteins, the E(y)2 and CP190 proteins bind to Wari as well as to the Su(Hw)-containing insulators in vivo. It may well be that these proteins are recruited to the insulator by as yet unidentified DNA-binding protein. Partial inactivation of E(y)2 in a weak e(y)2 ( u1 ) mutation impairs only the anti-silencing but not the enhancer-blocking activity of the Wari insulator. Thus, the E(y)2 protein in different Drosophila insulators serves to protect gene expression from silencing.

Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part II. [4Fe-4S] and [3Fe-4S] iron-sulfur proteins.

PubMed

Zanello, Piero

2018-06-01

In the context of the plethora of proteins harboring iron-sulfur clusters we have already reviewed structure/electrochemistry of metalloproteins expressing single types of iron-sulfur clusters (namely: {Fe(Cys) 4 }, {[Fe 2 S 2 ](Cys) 4 }, {[Fe 2 S 2 ](Cys) 3 (X)} (X = Asp, Arg, His), {[Fe 2 S 2 ](Cys) 2 (His) 2 }, {[Fe 3 S 4 ](Cys) 3 }, {[Fe 4 S 4 ](Cys) 4 } and {[Fe 4 S 4 ](S γ Cys ) 3 (nonthiolate ligand)} cores) and their synthetic analogs. More recently we are focussing on structure/electrochemistry of metalloproteins harboring iron-sulfur centres of different nuclearities. Having started such a subject with proteins harboring [4Fe-4S] and [2Fe-2S] clusters, we now depict the state of art of proteins containing [4Fe-4S] and [3Fe-4S] clusters. Copyright © 2018 Elsevier Inc. All rights reserved.

VE-Cadherin–Mediated Epigenetic Regulation of Endothelial Gene Expression

PubMed Central

Morini, Marco F.; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I.; Conze, Lei L.; O’Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P.

2018-01-01

Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system. PMID:29233846

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

PubMed

Morini, Marco F; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I; Conze, Lei L; O'Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P; Dejana, Elisabetta; Taddei, Andrea

2018-01-19

The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5 , vascular endothelial-protein tyrosine phosphatase ( VE-PTP ), and von Willebrand factor ( vWf ). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5 , VE-PTP , and vWf . VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5 , VE-PTP , and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system. © 2017 The Authors.

Melatonin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress.

PubMed

Lin, Yu Wen; Chen, Tsung Ying; Hung, Chia Yang; Tai, Shih Huang; Huang, Sheng Yang; Chang, Che Chao; Hung, Hsin Yi; Lee, E Jian

2018-07-01

Endoplasmic reticulum (ER) stress plays a vital role in mediating ischemic reperfusion damage in brain. In this study, we evaluated whether melatonin inhibits ER stress in cultured neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to transient focal cerebral ischemia. Sprague-Dawley rats were treated with melatonin (5 mg/kg) or control at reperfusion onset after transient occlusion of the right middle cerebral artery (MCA) for 90 min. Brain infarction and hemorrhage within infarcts were measured. The expression of ER stress proteins of phosphorylation of PRKR‑like endoplasmic reticulum kinase (p-PERK), phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by western blotting and immunohistochemistry analysis. The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method, cleaved caspase-3 and cytochrome c were used to investigate cell apoptosis in OGD-induced cultured neurons. Our results demonstrated that animals treated with melatonin had significantly reduced infarction volumes and individual cortical lesion sizes as well as increased numbers of surviving neurons. Melatonin can significantly modulate protein levels by decreasing both p-PERK and p-eIF2α in the ischemic core and penumbra. Moreover, the expressions of ATF4 and CHOP were restrained in the ischemic core and penumbra, respectively. Furthermore, pretreatment with melatonin at 10-100 µM effectively reduced the levels of p-PERK and p-eIF2α in cultured neurons after OGD injury. Melatonin treatment also effectively decreased neuron apoptosis resulting from OGD-induced neuron injury. These results indicate that melatonin effectively attenuated post-ischemic ER stress after ischemic stroke.

Expression and Characterization of an Ice Binding Protein from a Bacterium Isolated at a Depth of 3,519 Meters in the Vostok Ice Core, Antarctica

NASA Astrophysics Data System (ADS)

Christner, B. C.; Achberger, A.; Brox, T. I.; Skidmore, M. L.

2011-12-01

The cryopreservation of microorganisms in ancient glacial ice is possible if lethal levels of macromolecular damage are not incurred and cellular integrity is not compromised via intracellular ice formation or recrystallization. There are numerous examples of cold-adapted species that prevent or limit ice crystal growth by producing ice-binding proteins (IBP). Previously, a bacterium (isolate 3519-10; Flavobacteriaceae family) recovered from a depth of 3,519 meters below the surface in the Vostok ice core was shown to produce and secrete an IBP that inhibits the recrystallization of ice. To explore the phenotypic advantage that IBPs confer to ice-entrapped cells, experiments were designed to examine the expression of 3519-10's IBP gene and protein at different temperatures, assess the effect of the IBP on bacterial viability in ice, and determine how the IBP influences the physical structure of the ice. Total RNA isolated from aerobic cultures grown at temperatures between 4C to 25C and analyzed by reverse transcription-PCR indicated constitutive expression of the IBP gene. Additionally, SDS-PAGE analysis of 3519-10's extracellular proteins revealed a polypeptide corresponding to the predicted size of the 54 kDa IBP at all temperatures tested. The total extracellular protein fraction was subsequently used in assays with Escherichia coli to examine the effect of the IBP on bacterial survival in warm ice (-5C) and after freeze-thaw cycling. In the presence of 100 μg mL-1 of extracellular protein from 3519-10, the survival of E. coli was increased by greater than 100-fold; however, the survival of E. coli suspensions containing the same concentration of bovine serum albumin was not significantly different than controls (p<0.05). Microscopic analysis of ice formed in the presence of the IBP indicated that in a mm^2 field of view, there were 5 times as many crystals as in ice formed in the presence of washed 3519-10 cells and non-IBP producing bacteria, and 10 times as many crystals as in particle-free deionized water. Presumably, the effect that the IBP has on bacterial viability and ice crystal structure is due to its activity as an inhibitor of ice recrystallization. Although a myriad of molecular adaptations are likely to play a role in bacterial persistence under frozen conditions, the ability of 3519-10's IBP to control ice crystal structure may provide one explanation for its successful survival deep within the Antarctic ice sheet for thousands of years.

Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

PubMed

Liu, Baoming; Panda, Debasis; Mendez-Rios, Jorge D; Ganesan, Sundar; Wyatt, Linda S; Moss, Bernard

2018-04-01

Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5. IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess. Copyright © 2018 American Society for Microbiology.

Core binding factor beta (Cbfβ) controls the balance of chondrocyte proliferation and differentiation by upregulating Indian hedgehog (Ihh) expression and inhibiting parathyroid hormone-related protein receptor (PPR) expression in postnatal cartilage and bone formation.

PubMed

Tian, Fei; Wu, Mengrui; Deng, Lianfu; Zhu, Guochun; Ma, Junqing; Gao, Bo; Wang, Lin; Li, Yi-Ping; Chen, Wei

2014-07-01

Core binding factor beta (Cbfβ) is essential for embryonic bone morphogenesis. Yet the mechanisms by which Cbfβ regulates chondrocyte proliferation and differentiation as well as postnatal cartilage and bone formation remain unclear. Hence, using paired-related homeobox transcription factor 1-Cre (Prx1-Cre) mice, mesenchymal stem cell-specific Cbfβ-deficient (Cbfβ(f/f) Prx1-Cre) mice were generated to study the role of Cbfβ in postnatal cartilage and bone development. These mutant mice survived to adulthood but exhibited severe sternum and limb malformations. Sternum ossification was largely delayed in the Cbfβ(f/f) Prx1-Cre mice and the xiphoid process was noncalcified and enlarged. In newborn and 7-day-old Cbfβ(f/f) Prx1-Cre mice, the resting zone was dramatically elongated, the proliferation zone and hypertrophic zone of the growth plates were drastically shortened and disorganized, and trabecular bone formation was reduced. Moreover, in 1-month-old Cbfβ(f/f) Prx1-Cre mice, the growth plates were severely deformed and trabecular bone was almost absent. In addition, Cbfβ deficiency impaired intramembranous bone formation both in vivo and in vitro. Interestingly, although the expression of Indian hedgehog (Ihh) was largely reduced, the expression of parathyroid hormone-related protein (PTHrP) receptor (PPR) was dramatically increased in the Cbfβ(f/f) Prx1-Cre growth plate, indicating that that Cbfβ deficiency disrupted the Ihh-PTHrP negative regulatory loop. Chromatin immunoprecipitation (ChIP) analysis and promoter luciferase assay demonstrated that the Runx/Cbfβ complex binds putative Runx-binding sites of the Ihh promoter regions, and also the Runx/Cbfβ complex directly upregulates Ihh expression at the transcriptional level. Consistently, the expressions of Ihh target genes, including CyclinD1, Ptc, and Pthlh, were downregulated in Cbfβ-deficient chondrocytes. Taken together, our study reveals not only that Cbfβ is essential for chondrocyte proliferation and differentiation for the growth and maintenance of the skeleton in postnatal mice, but also that it functions in upregulating Ihh expression to promoter chondrocyte proliferation and osteoblast differentiation, and inhibiting PPR expression to enhance chondrocyte differentiation. © 2014 American Society for Bone and Mineral Research.

PTEN induces apoptosis and cavitation via HIF-2-dependent Bnip3 upregulation during epithelial lumen formation.

PubMed

Qi, Y; Liu, J; Saadat, S; Tian, X; Han, Y; Fong, G-H; Pandolfi, P P; Lee, L Y; Li, S

2015-05-01

The tumor suppressor phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 and antagonizes the prosurvival PI3K-Akt pathway. Targeted deletion of PTEN in mice led to early embryonic lethality. To elucidate its role in embryonic epithelial morphogenesis and the underlying mechanisms, we used embryonic stem cell-derived embryoid body (EB), an epithelial cyst structurally similar to the periimplantation embryo. PTEN is upregulated during EB morphogenesis in parallel with apoptosis of core cells, which mediates EB cavitation. Genetic ablation of PTEN causes Akt overactivation, apoptosis resistance and cavitation blockade. However, rescue experiments using mutant PTEN and pharmacological inhibition of Akt suggest that the phosphatase activity of PTEN and Akt are not involved in apoptosis-mediated cavitation. Instead, hypoxia-induced upregulation of Bnip3, a proapoptotic BH3-only protein, mediates PTEN-dependent apoptosis and cavitation. PTEN inactivation inhibits hypoxia- and reactive oxygen species-induced Bnip3 elevation. Overexpression of Bnip3 in PTEN-null EBs rescues apoptosis of the core cells. Mechanistically, suppression of Bnip3 following PTEN loss is likely due to reduction of hypoxia-inducible factor-2α (HIF-2α) because forced expression of an oxygen-stable HIF-2α mutant rescues Bnip3 expression and apoptosis. Lastly, we show that HIF-2α is upregulated by PTEN at both transcriptional and posttranscriptional levels. Ablation of prolyl hydroxylase domain-containing protein 2 (PHD2) in normal EBs or inhibition of PHD activities in PTEN-null EBs stabilizes HIF-2α and induces Bnip3 and caspase-3 activation. Altogether, these results suggest that PTEN is required for apoptosis-mediated cavitation during epithelial morphogenesis by regulating the expression of HIF-2α and Bnip3.

The Role and Regulation of ABI5 (ABA-Insensitive 5) in Plant Development, Abiotic Stress Responses and Phytohormone Crosstalk

PubMed Central

Skubacz, Anna; Daszkowska-Golec, Agata; Szarejko, Iwona

2016-01-01

ABA Insensitive 5 (ABI5) is a basic leucine zipper transcription factor that plays a key role in the regulation of seed germination and early seedling growth in the presence of ABA and abiotic stresses. ABI5 functions in the core ABA signaling, which is composed of PYR/PYL/RCAR receptors, PP2C phosphatases and SnRK2 kinases, through the regulation of the expression of genes that contain the ABSCISIC ACID RESPONSE ELEMENT (ABRE) motif within their promoter region. The regulated targets include stress adaptation genes, e.g., LEA proteins. However, the expression and activation of ABI5 is not only dependent on the core ABA signaling. Many transcription factors such as ABI3, ABI4, MYB7 and WRKYs play either a positive or a negative role in the regulation of ABI5 expression. Additionally, the stability and activity of ABI5 are also regulated by other proteins through post-translational modifications such as phosphorylation, ubiquitination, sumoylation and S-nitrosylation. Moreover, ABI5 also acts as an ABA and other phytohormone signaling integrator. Components of auxin, cytokinin, gibberellic acid, jasmonate and brassinosteroid signaling and metabolism pathways were shown to take part in ABI5 regulation and/or to be regulated by ABI5. Monocot orthologs of AtABI5 have been identified. Although their roles in the molecular and physiological adaptations during abiotic stress have been elucidated, knowledge about their detailed action still remains elusive. Here, we describe the recent advances in understanding the action of ABI5 in early developmental processes and the adaptation of plants to unfavorable environmental conditions. We also focus on ABI5 relation to other phytohormones in the abiotic stress response of plants. PMID:28018412

Random close packing in protein cores

NASA Astrophysics Data System (ADS)

Gaines, Jennifer C.; Smith, W. Wendell; Regan, Lynne; O'Hern, Corey S.

2016-03-01

Shortly after the determination of the first protein x-ray crystal structures, researchers analyzed their cores and reported packing fractions ϕ ≈0.75 , a value that is similar to close packing of equal-sized spheres. A limitation of these analyses was the use of extended atom models, rather than the more physically accurate explicit hydrogen model. The validity of the explicit hydrogen model was proved in our previous studies by its ability to predict the side chain dihedral angle distributions observed in proteins. In contrast, the extended atom model is not able to recapitulate the side chain dihedral angle distributions, and gives rise to large atomic clashes at side chain dihedral angle combinations that are highly probable in protein crystal structures. Here, we employ the explicit hydrogen model to calculate the packing fraction of the cores of over 200 high-resolution protein structures. We find that these protein cores have ϕ ≈0.56 , which is similar to results obtained from simulations of random packings of individual amino acids. This result provides a deeper understanding of the physical basis of protein structure that will enable predictions of the effects of amino acid mutations to protein cores and interfaces of known structure.

Random close packing in protein cores.

PubMed

Gaines, Jennifer C; Smith, W Wendell; Regan, Lynne; O'Hern, Corey S

2016-03-01

Shortly after the determination of the first protein x-ray crystal structures, researchers analyzed their cores and reported packing fractions ϕ ≈ 0.75, a value that is similar to close packing of equal-sized spheres. A limitation of these analyses was the use of extended atom models, rather than the more physically accurate explicit hydrogen model. The validity of the explicit hydrogen model was proved in our previous studies by its ability to predict the side chain dihedral angle distributions observed in proteins. In contrast, the extended atom model is not able to recapitulate the side chain dihedral angle distributions, and gives rise to large atomic clashes at side chain dihedral angle combinations that are highly probable in protein crystal structures. Here, we employ the explicit hydrogen model to calculate the packing fraction of the cores of over 200 high-resolution protein structures. We find that these protein cores have ϕ ≈ 0.56, which is similar to results obtained from simulations of random packings of individual amino acids. This result provides a deeper understanding of the physical basis of protein structure that will enable predictions of the effects of amino acid mutations to protein cores and interfaces of known structure.

Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses

PubMed Central

Szécsi, Judit; Boson, Bertrand; Johnsson, Per; Dupeyrot-Lacas, Pia; Matrosovich, Mikhail; Klenk, Hans-Dieter; Klatzmann, David; Volchkov, Viktor; Cosset, François-Loïc

2006-01-01

There is an urgent need to develop novel approaches to vaccination against the emerging, highly pathogenic avian influenza viruses. Here, we engineered influenza viral-like particles (Flu-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two highly pathogenic influenza viruses of either H7N1 or H5N1 antigenic subtype. We demonstrate that, upon recovery of viral RNAs from a field strain, one can easily generate expression vectors that encode the HA, NA and M2 surface proteins of either virus and prepare high-titre Flu-VLPs. We characterise these Flu-VLPs incorporating the HA, NA and M2 proteins and we show that they induce high-titre neutralising antibodies in mice. PMID:16948862

ReTrOS: a MATLAB toolbox for reconstructing transcriptional activity from gene and protein expression data.

PubMed

Minas, Giorgos; Momiji, Hiroshi; Jenkins, Dafyd J; Costa, Maria J; Rand, David A; Finkenstädt, Bärbel

2017-06-26

Given the development of high-throughput experimental techniques, an increasing number of whole genome transcription profiling time series data sets, with good temporal resolution, are becoming available to researchers. The ReTrOS toolbox (Reconstructing Transcription Open Software) provides MATLAB-based implementations of two related methods, namely ReTrOS-Smooth and ReTrOS-Switch, for reconstructing the temporal transcriptional activity profile of a gene from given mRNA expression time series or protein reporter time series. The methods are based on fitting a differential equation model incorporating the processes of transcription, translation and degradation. The toolbox provides a framework for model fitting along with statistical analyses of the model with a graphical interface and model visualisation. We highlight several applications of the toolbox, including the reconstruction of the temporal cascade of transcriptional activity inferred from mRNA expression data and protein reporter data in the core circadian clock in Arabidopsis thaliana, and how such reconstructed transcription profiles can be used to study the effects of different cell lines and conditions. The ReTrOS toolbox allows users to analyse gene and/or protein expression time series where, with appropriate formulation of prior information about a minimum of kinetic parameters, in particular rates of degradation, users are able to infer timings of changes in transcriptional activity. Data from any organism and obtained from a range of technologies can be used as input due to the flexible and generic nature of the model and implementation. The output from this software provides a useful analysis of time series data and can be incorporated into further modelling approaches or in hypothesis generation.

PDGFRα depletion attenuates glioblastoma stem cells features by modulation of STAT3, RB1 and multiple oncogenic signals.

PubMed

Cenciarelli, Carlo; Marei, Hany E; Felsani, Armando; Casalbore, Patrizia; Sica, Gigliola; Puglisi, Maria Ausiliatrice; Cameron, Angus J M; Olivi, Alessandro; Mangiola, Annunziato

2016-08-16

Platelet derived growth factor receptors (PDGFRs) play an important role in tumor pathogenesis, and they are frequently overexpressed in glioblastoma (GBM). Earlier we have shown a higher protein expression of PDGFR isoforms (α and β) in peritumoral-tissue derived cancer stem cells (p-CSC) than in tumor core (c-CSC) of several GBM affected patients. In the current study, in order to assess the activity of PDGFRα/PDGF-AA signaling axis, we performed time course experiments to monitor the effects of exogenous PDGF-AA on the expression of downstream target genes in c-CSC vs p-CSC. Interestingly, in p-CSC we detected the upregulation of Y705-phosphorylated Stat3, concurrent with a decrement of Rb1 protein in its active state, within minutes of PDGF-AA addition. This finding prompted us to elucidate the role of PDGFRα in self-renewal, invasion and differentiation in p-CSC by using short hairpin RNA depletion of PDGFRα expression. Notably, in PDGFRα-depleted cells, protein analysis revealed attenuation of stemness-related and glial markers expression, alongside early activation of the neuronal marker MAP2a/b that correlated with the induction of tumor suppressor Rb1. The in vitro reduction of the invasive capacity of PDGFRα-depleted CSC as compared to parental cells correlated with the downmodulation of markers of epithelial-mesenchymal transition phenotype and angiogenesis. Surprisingly, we observed the induction of anti-apoptotic proteins and compensatory oncogenic signals such as EDN1, EDNRB, PRKCB1, PDGF-C and PDGF-D. To conclude, we hypothesize that the newly discovered PDGFRα/Stat3/Rb1 regulatory axis might represent a potential therapeutic target for GBM treatment.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyn, Rodney K.; Department of Chemistry, University of Ottawa, Ottawa; Kennedy, David C.

Research highlights: {yields} Hepatitis C virus uses lipid droplets (LD) onto which HCV core proteins bind. {yields} HCV core proteins on LDs facilitate viral particle assembly. {yields} We used a novel combination of CARS, two-photon fluorescence, and DIC microscopies. {yields} Particle tracking experiments show that core slowly affects LD localization. {yields} Particle tracking measured the change in speed and directionality of LD movement. -- Abstract: The hepatitis C virus (HCV) is a global health problem, with limited treatment options and no vaccine available. HCV uses components of the host cell to proliferate, including lipid droplets (LD) onto which HCV coremore » proteins bind and facilitate viral particle assembly. We have measured the dynamics of HCV core protein-mediated changes in LDs and rates of LD movement on microtubules using a combination of coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), and differential interference contrast (DIC) microscopies. Results show that the HCV core protein induces rapid increases in LD size. Particle tracking experiments show that HCV core protein slowly affects LD localization by controlling the directionality of LD movement on microtubules. These dynamic processes ultimately aid HCV in propagating and the molecules and interactions involved represent novel targets for potential therapeutic intervention.« less

Structure of a BMI-1-Ring1B Polycomb Group Ubiquitin Ligase Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li,Z.; Cao, R.; Wang, M.

2006-01-01

Polycomb group (PcG) proteins Bmi-1 and Ring1B are core subunits of the PRC1 complex which plays important roles in the regulation of Hox gene expression, X-chromosome inactivation, tumorigenesis and stem cell self-renewal. The RING finger protein Ring1B is an E3 ligase that participates in the ubiquitination of lysine 119 of histone H2A, and the binding of Bmi-1 stimulates the E3 ligase activity. We have mapped the regions of Bmi-1 and Ring1B required for efficient ubiquitin transfer and determined a 2.5 Angstroms structure of the Bmi-1-Ring1B core domain complex. The structure reveals that Ring1B 'hugs' Bmi-1 through extensive RING domain contactsmore » and its N-terminal tail wraps around Bmi-1. The two regions of interaction have a synergistic effect on the E3 ligase activity. Our analyses suggest a model where the Bmi-1-Ring1B complex stabilizes the interaction between the E2 enzyme and the nucleosomal substrate to allow efficient ubiquitin transfer.« less

Molecular targets for diabetes mellitus-associated erectile dysfunction.

PubMed

Yohannes, Elizabeth; Chang, Jinsook; Tar, Moses T; Davies, Kelvin P; Chance, Mark R

2010-03-01

Protein expression profiles in rat corporal smooth muscle tissue were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls (AMCs) at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four AMC rat corpora tissues were prepared independently and analyzed together across multiple quantitative two-dimensional gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 170 spots were differential expressed among the four experimental groups. A subsequent mass spectrometry analysis of the 170 spots identified a total of 57 unique proteins. Network analysis of these proteins using MetaCore suggested altered activity of transcriptional factors that are of too low abundance to be detected by the two-dimensional gel method. The proteins that were down-regulated with diabetes include isoforms of collagen that are precursors to fibril-forming collagen type 1; Hsp47, which assists and mediates the proper folding of procollagen; and several proteins whose abundance is controlled by sex hormones (e.g. CRP1 and A2U). On the other hand, proteins seen or predicted to be up-regulated include proteins involved in cell apoptosis (e.g. p53, 14-3-3-gamma, Serpinf1, Cct4, Cct5, and Sepina3n), proteins that neutralize the biological activity of nerve growth factor (e.g. anti-NGF 30), and proteins involved in lipid metabolism (e.g. apoA-I and apoA-IV). Subsequent Western blot validation analysis of p53, 14-3-3-gamma, and Hsp47 confirmed increased p53 and 14-3-3-gamma and decreased Hsp47 levels in separate samples. According to the results from the Western blot analysis, Hsp47 protein showed a approximately 3-fold decrease at 1 week and was virtually undetectable at 2 months in diabetic versus control. Taken together, our results identify novel candidate proteins playing a role in erectile dysfunction in diabetes resulting from STZ treatment.

Molecular Targets for Diabetes Mellitus-associated Erectile Dysfunction*

PubMed Central

Yohannes, Elizabeth; Chang, Jinsook; Tar, Moses T.; Davies, Kelvin P.; Chance, Mark R.

2010-01-01

Protein expression profiles in rat corporal smooth muscle tissue were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls (AMCs) at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four AMC rat corpora tissues were prepared independently and analyzed together across multiple quantitative two-dimensional gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 170 spots were differential expressed among the four experimental groups. A subsequent mass spectrometry analysis of the 170 spots identified a total of 57 unique proteins. Network analysis of these proteins using MetaCoreTM suggested altered activity of transcriptional factors that are of too low abundance to be detected by the two-dimensional gel method. The proteins that were down-regulated with diabetes include isoforms of collagen that are precursors to fibril-forming collagen type 1; Hsp47, which assists and mediates the proper folding of procollagen; and several proteins whose abundance is controlled by sex hormones (e.g. CRP1 and A2U). On the other hand, proteins seen or predicted to be up-regulated include proteins involved in cell apoptosis (e.g. p53, 14-3-3-γ, Serpinf1, Cct4, Cct5, and Sepina3n), proteins that neutralize the biological activity of nerve growth factor (e.g. anti-NGF 30), and proteins involved in lipid metabolism (e.g. apoA-I and apoA-IV). Subsequent Western blot validation analysis of p53, 14-3-3-γ, and Hsp47 confirmed increased p53 and 14-3-3-γ and decreased Hsp47 levels in separate samples. According to the results from the Western blot analysis, Hsp47 protein showed a ∼3-fold decrease at 1 week and was virtually undetectable at 2 months in diabetic versus control. Taken together, our results identify novel candidate proteins playing a role in erectile dysfunction in diabetes resulting from STZ treatment. PMID:20007950

Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

PubMed Central

Schmidt, R J; Ketudat, M; Aukerman, M J; Hoschek, G

1992-01-01

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family. PMID:1392590

Regulation of PSMB5 Protein and β Subunits of Mammalian Proteasome by Constitutively Activated Signal Transducer and Activator of Transcription 3 (STAT3)

PubMed Central

Vangala, Janakiram Reddy; Dudem, Srikanth; Jain, Nishant; Kalivendi, Shasi V.

2014-01-01

The ubiquitin-proteasome system facilitates the degradation of ubiquitin-tagged proteins and performs a regulatory role in cells. Elevated proteasome activity and subunit expression are found in several cancers. However, the inherent molecular mechanisms responsible for increased proteasome function in cancers remain unclear despite the well investigated and defined role of the mammalian proteasome. This study was initiated to elucidate the mechanisms involved in the regulation of β subunits of the mammalian proteasome. Suppression of STAT3 tyrosine phosphorylation coordinately decreased the mRNA and protein levels of the β subunits of the 20 S core complex in DU145 cells. Notably, PSMB5, a molecular target of bortezomib, was shown to be a target of STAT3. Knockdown of STAT3 decreased PSMB5 protein. Inhibition of phospho-STAT3 substantially reduced PSMB5 protein levels in cells expressing constitutively active-STAT3. Accumulation of activated STAT3 resulted in the induction of PSMB5 promoter and protein levels. In addition, a direct correlation was observed between the endogenous levels of PSMB5 and constitutively active STAT3. PSMB5 and STAT3 protein levels remained unaltered following the inhibition of proteasome activity. The EGF-induced concerted increase of β subunits was blocked by inhibition of the EGF receptor or STAT3 but not by the PI3K/AKT or MEK/ERK pathways. Decreased proteasome activities were due to reduced protein levels of catalytic subunits of the proteasome in STAT3-inhibited cells. Combined treatments with bortezomib and inhibitor of STAT3 abrogated proteasome activity and enhanced cellular apoptosis. Overall, we demonstrate that aberrant activation of STAT3 regulates the expression of β subunits, in particular PSMB5, and the catalytic activity of the proteasome. PMID:24627483

Expression of the sodium potassium chloride cotransporter (NKCC1) and sodium chloride cotransporter (NCC) and their effects on rat lens transparency.

PubMed

Chee, K N; Vorontsova, I; Lim, J C; Kistler, J; Donaldson, P J

2010-05-04

To characterize the expression patterns of the Na+-K+-Cl(-) cotransporter (NKCC) 1 and NKCC2, and the Na+-Cl(-) cotransporter (NCC) in the rat lens and to determine if they play a role in regulating lens volume and transparency. RT-PCR was performed on RNA extracted from fiber cells to identify sodium dependent cotransporters expressed in the rat lens. Western blotting and immunohistochemistry, using NKCC1, NKCC2, and NCC antibodies, were used to verify expression at the protein level and to localize transporter expression. Organ cultured rat lenses were incubated in Artificial Aqueous Humor (AAH) of varying osmolarities or isotonic AAH that contained either the NKCC specific inhibitor bumetanide, or the NCC specific inhibitor thiazide for up to 18 h. Lens transparency was monitored with dark field microscopy, while tissue morphology and antibody labeling patterns were recorded using a confocal microscope. Molecular experiments showed that NKCC1 and NCC were expressed in the lens at both the transcript and protein levels, but NKCC2 was not. Immunohistochemistry showed that both NKCC1 and NCC were expressed in the lens cortex, but NCC expression was also found in the lens core. In the lens cortex the majority of labeling for both transporters was cytoplasmic in nature, while in the lens core, NCC labeling was associated with the membrane. Exposure of lenses to either hypotonic or hypertonic AAH had no noticeable effects on the predominantly cytoplasmic location of either transporter in the lens cortex. Incubation of lenses in isotonic AAH plus the NKCC inhibitor bumetanide for 18 h induced a cortical opacity that was initiated by a shrinkage of peripheral fiber cells and the dilation of the extracellular space between fiber cells in a deeper zone located some approximately 150 microm in from the capsule. In contrast, lenses incubated in isotonic AAH and the NCC inhibitor thiazide maintained both their transparency and their regular fiber cell morphology. We have confirmed the expression of NKCC1 in the rat lens and report for the first time the expression of NCC in lens fiber cells. The expression patterns of the two transporters and the differential effects of their specific inhibitors on fiber cell morphology indicate that these transporters play distinct roles in the lens. NKCC1 appears to mediate ion influx in the lens cortex while NCC may play a role in the lens nucleus.

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

PubMed

Pandey, Naresh; Kuypers, Brianna E; Nassif, Barbara; Thomas, Emily E; Alnahhas, Razan N; Segatori, Laura; Silberg, Jonathan J

2016-07-12

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.

Tolerance of a knotted near infrared fluorescent protein to random circular permutation

PubMed Central

Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

2016-01-01

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

Independent evolution of striated muscles in cnidarians and bilaterians.

PubMed

Steinmetz, Patrick R H; Kraus, Johanna E M; Larroux, Claire; Hammel, Jörg U; Amon-Hassenzahl, Annette; Houliston, Evelyn; Wörheide, Gert; Nickel, Michael; Degnan, Bernard M; Technau, Ulrich

2012-07-12

Striated muscles are present in bilaterian animals (for example, vertebrates, insects and annelids) and some non-bilaterian eumetazoans (that is, cnidarians and ctenophores). The considerable ultrastructural similarity of striated muscles between these animal groups is thought to reflect a common evolutionary origin. Here we show that a muscle protein core set, including a type II myosin heavy chain (MyHC) motor protein characteristic of striated muscles in vertebrates, was already present in unicellular organisms before the origin of multicellular animals. Furthermore, 'striated muscle' and 'non-muscle' myhc orthologues are expressed differentially in two sponges, compatible with a functional diversification before the origin of true muscles and the subsequent use of striated muscle MyHC in fast-contracting smooth and striated muscle. Cnidarians and ctenophores possess striated muscle myhc orthologues but lack crucial components of bilaterian striated muscles, such as genes that code for titin and the troponin complex, suggesting the convergent evolution of striated muscles. Consistently, jellyfish orthologues of a shared set of bilaterian Z-disc proteins are not associated with striated muscles, but are instead expressed elsewhere or ubiquitously. The independent evolution of eumetazoan striated muscles through the addition of new proteins to a pre-existing, ancestral contractile apparatus may serve as a model for the evolution of complex animal cell types.

Independent evolution of striated muscles in cnidarians and bilaterians

PubMed Central

Steinmetz, Patrick R.H.; Kraus, Johanna E.M.; Larroux, Claire; U. Hammel, Jörg; Amon-Hassenzahl, Annette; Houliston, Evelyn; Wörheide, Gert; Nickel, Michael; Degnan, Bernard M.; Technau, Ulrich

2012-01-01

Striated muscles are present in bilaterian animals (e.g. vertebrates, insects, annelids) and some non-bilaterian eumetazoans (i.e. cnidarians and ctenophores). The striking ultrastructural similarity of striated muscles between these animal groups is thought to reflect a common evolutionary origin1, 2. Here we show that a muscle protein core set, including a Myosin type II Heavy Chain motor protein characteristic of striated muscles in vertebrates (MyHC-st), was already present in unicellular organisms before the origin of multicellular animals. Furthermore, myhc-st and myhc-non-muscle (myhc-nm) orthologues are expressed differentially in two sponges, compatible with the functional diversification of myhc paralogues before the origin of true muscles and the subsequent deployment of MyHC-st in fast-contracting smooth and striated muscle. Cnidarians and ctenophores possess myhc-st orthologues but lack crucial components of bilaterian striated muscles, such as troponin complex and titin genes, suggesting the convergent evolution of striated muscles. Consistently, jellyfish orthologues of a shared set of bilaterian z-disc proteins are not associated with striated muscles, but are instead expressed elsewhere or ubiquitously. The independent evolution of eumetazoan striated muscles through the addition of novel proteins to a pre-existing, ancestral contractile apparatus may serve as a paradigm for the evolution of complex animal cell types. PMID:22763458

Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids

PubMed Central

Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

2016-01-01

Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only. PMID:27222167

COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein.

PubMed Central

Avram, Dorina; Fields, Andrew; Senawong, Thanaset; Topark-Ngarm, Acharawan; Leid, Mark

2002-01-01

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5'-GGCCGG-3' (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems. PMID:12196208

CIKS, a connection to IκB kinase and stress-activated protein kinase

PubMed Central

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

An Alternative Splice Product of IκB Kinase (IKKγ), IKKγ-Δ, Differentially Mediates Cytokine and Human T-Cell Leukemia Virus Type 1 Tax-Induced NF-κB Activation

PubMed Central

Hai, Tao; Yeung, Man-Lung; Wood, Thomas G.; Wei, Yuanfen; Yamaoka, Shoji; Gatalica, Zoran; Jeang, Kuan-Teh; Brasier, Allan R.

2006-01-01

NF-κB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IκB kinase (IKK) “signalsome”. The core IKK consists of two catalytic serine kinases, IKKα and IKKβ, and a noncatalytic subunit, IKKγ. IKKγ is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKγ mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKγ-Δ. Using a specific reverse transcription-PCR assay, we find that IKKγ-Δ is widely expressed in cultured human cells and normal human tissues. Because IKKγ-Δ protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-κB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKγ-Δ has strong homo- and heterotypic association with wild-type (WT) IKKγ and, like IKKγ WT, associates with the IKKβ kinase. Similarly, IKKγ-Δ mediates IKK kinase activity and downstream NF-κB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-κB-inducing kinase-IKKα signaling pathway. Surprisingly, however, in contrast to IKKγ WT, IKKγ-Δ is not able to mediate HTLV-1 Tax-induced NF-κB-dependent transcription, even though IKKγ-Δ binds and colocalizes with Tax. These observations suggest that IKKγ-Δ is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKγ-Δ expression, therefore, may affect signal transduction cascades coupling to IKK. PMID:16611882

Live imaging of dense-core vesicles in primary cultured hippocampal neurons.

PubMed

Kwinter, David M; Silverman, Michael A; Kwinter, David; Michael, Silverman

2009-05-29

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

HPV16 E7 Protein and hTERT Proteins Defective for Telomere Maintenance Cooperate to Immortalize Human Keratinocytes

PubMed Central

Miller, Jonathan; Dakic, Aleksandra; Chen, Renxiang; Palechor-Ceron, Nancy; Dai, Yuhai; Kallakury, Bhaskar; Schlegel, Richard; Liu, Xuefeng

2013-01-01

Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity. PMID:23592995

Circadian clock proteins and immunity.

PubMed

Curtis, Anne M; Bellet, Marina M; Sassone-Corsi, Paolo; O'Neill, Luke A J

2014-02-20

Immune parameters change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. A circadian-clock-controlled immune system might allow an organism to anticipate daily changes in activity and feeding and the associated risk of infection or tissue damage to the host. Responses to bacteria have been shown to vary depending on time of infection, with mice being more at risk of sepsis when challenged ahead of their activity phase. Studies highlight the extent to which the molecular clock, most notably the core clock proteins BMAL1, CLOCK, and REV-ERBα, control fundamental aspects of the immune response. Examples include the BMAL1:CLOCK heterodimer regulating toll-like receptor 9 (TLR9) expression and repressing expression of the inflammatory monocyte chemokine ligand (CCL2) as well as REV-ERBα suppressing the induction of interleukin-6. Understanding the daily rhythm of the immune system could have implications for vaccinations and how we manage infectious and inflammatory diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Proteoglycans and neuronal migration in the cerebral cortex during development and disease

PubMed Central

Maeda, Nobuaki

2015-01-01

Chondroitin sulfate proteoglycans and heparan sulfate proteoglycans are major constituents of the extracellular matrix and the cell surface in the brain. Proteoglycans bind with many proteins including growth factors, chemokines, axon guidance molecules, and cell adhesion molecules through both the glycosaminoglycan and the core protein portions. The functions of proteoglycans are flexibly regulated due to the structural variability of glycosaminoglycans, which are generated by multiple glycosaminoglycan synthesis and modifying enzymes. Neuronal cell surface proteoglycans such as PTPζ, neuroglycan C and syndecan-3 function as direct receptors for heparin-binding growth factors that induce neuronal migration. The lectican family, secreted chondroitin sulfate proteoglycans, forms large aggregates with hyaluronic acid and tenascins, in which many signaling molecules and enzymes including matrix proteases are preserved. In the developing cerebrum, secreted chondroitin sulfate proteoglycans such as neurocan, versican and phosphacan are richly expressed in the areas that are strategically important for neuronal migration such as the striatum, marginal zone, subplate and subventricular zone in the neocortex. These proteoglycans may anchor various attractive and/or repulsive cues, regulating the migration routes of inhibitory neurons. Recent studies demonstrated that the genes encoding proteoglycan core proteins and glycosaminoglycan synthesis and modifying enzymes are associated with various psychiatric and intellectual disorders, which may be related to the defects of neuronal migration. PMID:25852466

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Chun-Jun; Sun, Wei-Wen; Bruno, Kenneth S.

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes or non-ribosomal peptide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. More interestingly, further experiments revealedmore » that the aspulvinone E produced by two different genes accumulates in different fungal compartments. And this spatial control of aspulvinone E production is likely to be regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is inserted in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. The study also identified one trans-prenyltransferase AbpB which is capable of prenylating two different substrates aspulvinones and butyrolactones. In total, our study shows the first example in which the locally distribution of the same natural product could lead to its incorporation into different SM pathways.« less

Increasing Sequence Diversity with Flexible Backbone Protein Design: The Complete Redesign of a Protein Hydrophobic Core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Grant S.; Mills, Jeffrey L.; Miley, Michael J.

2015-10-15

Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures, most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helixmore » bundle protein. Only small perturbations to the backbone, 12 {angstrom}, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point >140C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 {angstrom}).« less