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Sample records for protein 3 regulatory

  1. Regulation of Airway Inflammation by G-protein Regulatory Motif Peptides of AGS3 protein

    PubMed Central

    Choi, IL-Whan; Ahn, Do Whan; Choi, Jang-Kyu; Cha, Hee-Jae; Ock, Mee Sun; You, EunAe; Rhee, SangMyung; Kim, Kwang Chul; Choi, Yung Hyun; Song, Kyoung Seob

    2016-01-01

    Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFβ but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment. PMID:27270970

  2. Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins

    NASA Technical Reports Server (NTRS)

    Toroser, D.; Athwal, G. S.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    We report an Mg2+-dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.

  3. Lung Angiogenesis Requires CD4+Forkhead Homeobox Protein-3+ Regulatory T Cells

    PubMed Central

    D’Alessio, Franco R.; Zhong, Qiong; Jenkins, John; Moldobaeva, Aigul

    2015-01-01

    Angiogenesis in ischemic organs is modulated by immune cells. Systemic neovascularization of the ischemic lung requires macrophages, with chemokines playing a central role in new vessel growth. Because regulatory T (Treg) cells modulate tumor-induced neovascularization, we questioned whether this CD4+ lymphocyte subset impacts blood vessel growth during ischemia. In a model of left lung ischemia, an increase in CD4+ CD25+ forkhead homeobox protein-3 (Foxp3)+ cells was observed 3–5 days after the onset of ischemia in wild-type C57Bl/6 mice. Using transgenic mice where Foxp3+ Treg cells can be depleted with diphtheria toxin (DT; Foxp3DTR), we unexpectedly found that Foxp3+ Treg depletion led to markedly reduced lung angiogenesis (90% reduction from Foxp3gfp controls). Adoptive transfer studies using CD4+ CD25+ splenocytes from congenic CD45.1 mice into Foxp3+ Treg–depleted mice showed an almost complete recovery of the angiogenic phenotype (80% of Foxp3gfp controls). A survey of lung gene expression of angiogenic (lipopolysaccharide-induced CXC chemokine [LIX], IL-6, IL-17) and angiostatic (IFN-γ, transforming growth factor-β, IL-10) cytokines showed Treg-dependent differences only in LIX (CXCL5) and IL-6. Protein confirmation demonstrated a significant reduction in LIX in Treg-deficient mice compared with controls 5 days after the onset of ischemia. Phenotyping other inflammatory cells in the lung by multicolor flow cytometry demonstrated a significantly reduced number of macrophages (major histocombatibility complex class II [MHCII]int, CD11C+) in Treg-deficient lungs compared with Treg-sufficient lungs. Treg cells are essential for maximal systemic angiogenesis after pulmonary ischemia. One likely mechanism responsible for the decrease in angiogenesis in Treg-depleted mice was the decline in the essential CXC chemokine, LIX. PMID:25275926

  4. Computational identification of new structured cis-regulatory elements in the 3'-untranslated region of human protein coding genes.

    PubMed

    Chen, Xiaowei Sylvia; Brown, Chris M

    2012-10-01

    Messenger ribonucleic acids (RNAs) contain a large number of cis-regulatory RNA elements that function in many types of post-transcriptional regulation. These cis-regulatory elements are often characterized by conserved structures and/or sequences. Although some classes are well known, given the wide range of RNA-interacting proteins in eukaryotes, it is likely that many new classes of cis-regulatory elements are yet to be discovered. An approach to this is to use computational methods that have the advantage of analysing genomic data, particularly comparative data on a large scale. In this study, a set of structural discovery algorithms was applied followed by support vector machine (SVM) classification. We trained a new classification model (CisRNA-SVM) on a set of known structured cis-regulatory elements from 3'-untranslated regions (UTRs) and successfully distinguished these and groups of cis-regulatory elements not been strained on from control genomic and shuffled sequences. The new method outperformed previous methods in classification of cis-regulatory RNA elements. This model was then used to predict new elements from cross-species conserved regions of human 3'-UTRs. Clustering of these elements identified new classes of potential cis-regulatory elements. The model, training and testing sets and novel human predictions are available at: http://mRNA.otago.ac.nz/CisRNA-SVM.

  5. Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3(+) regulatory T cells.

    PubMed

    Tanaka, H; Zhang, W; Yang, G-X; Ando, Y; Tomiyama, T; Tsuneyama, K; Leung, P; Coppel, R L; Ansari, A A; Lian, Z X; Ridgway, W M; Joh, T; Gershwin, M E

    2014-11-01

    Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (Treg ) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8(+) T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF-βRII) mice to recombination-activating gene (Rag)1(-/-) recipients. We then used this robust established adoptive transfer system and co-transferred CD8(+) T cells from dnTGF-βRII mice with either C57BL/6 or dnTGF-βRII forkhead box protein 3 (FoxP3(+) ) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of Treg from C57BL/6 but not dnTGF-βRII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down-regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 Treg versus dnTGF-βRII Treg on the ability to down-regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)-10 in Treg from C57BL/6 compared to dnTGF-βRII mice. Our data reflect the therapeutic potential of wild-type CD4(+) FoxP3(+) Treg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC.

  6. Pim-2 Kinase Influences Regulatory T Cell Function and Stability by Mediating Foxp3 Protein N-terminal Phosphorylation*

    PubMed Central

    Deng, Guoping; Nagai, Yasuhiro; Xiao, Yan; Li, Zhiyuan; Dai, Shujia; Ohtani, Takuya; Banham, Alison; Li, Bin; Wu, Shiaw-Lin; Hancock, Wayne; Samanta, Arabinda; Zhang, Hongtao; Greene, Mark I.

    2015-01-01

    Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4+CD25+Foxp3+ regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses. PMID:25987564

  7. Pim-2 Kinase Influences Regulatory T Cell Function and Stability by Mediating Foxp3 Protein N-terminal Phosphorylation.

    PubMed

    Deng, Guoping; Nagai, Yasuhiro; Xiao, Yan; Li, Zhiyuan; Dai, Shujia; Ohtani, Takuya; Banham, Alison; Li, Bin; Wu, Shiaw-Lin; Hancock, Wayne; Samanta, Arabinda; Zhang, Hongtao; Greene, Mark I

    2015-08-14

    Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses.

  8. Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB

    PubMed Central

    Coelho, Miguel B; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher WJ

    2015-01-01

    Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3. PMID:25599992

  9. Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

    PubMed

    Liu, Xia; Ji, Baoju; Sun, Mengyi; Wu, Weijiang; Huang, Lili; Sun, Aihua; Zong, Yangyong; Xia, Sheng; Shi, Liyun; Qian, Hui; Xu, Wenrong; Shao, Qixiang

    2015-07-01

    Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy.

  10. Glucokinase Regulatory Protein Genetic Variant Interacts with Omega-3 PUFA to Influence Insulin Resistance and Inflammation in Metabolic Syndrome

    PubMed Central

    Garcia-Rios, Antonio; Mc Monagle, Jolene; Gulseth, Hanne L.; Ordovas, Jose M.; Shaw, Danielle I.; Karlström, Brita; Kiec-Wilk, Beata; Blaak, Ellen E.; Helal, Olfa; Malczewska-Malec, Małgorzata; Defoort, Catherine; Risérus, Ulf; Saris, Wim H. M.; Lovegrove, Julie A.; Drevon, Christian A.; Roche, Helen M.; Lopez-Miranda, Jose

    2011-01-01

    Glucokinase Regulatory Protein (GCKR) plays a central role regulating both hepatic triglyceride and glucose metabolism. Fatty acids are key metabolic regulators, which interact with genetic factors and influence glucose metabolism and other metabolic traits. Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been of considerable interest, due to their potential to reduce metabolic syndrome (MetS) risk. Objective To examine whether genetic variability at the GCKR gene locus was associated with the degree of insulin resistance, plasma concentrations of C-reactive protein (CRP) and n-3 PUFA in MetS subjects. Design Homeostasis model assessment of insulin resistance (HOMA-IR), HOMA-B, plasma concentrations of C-peptide, CRP, fatty acid composition and the GCKR rs1260326-P446L polymorphism, were determined in a cross-sectional analysis of 379 subjects with MetS participating in the LIPGENE dietary cohort. Results Among subjects with n-3 PUFA levels below the population median, carriers of the common C/C genotype had higher plasma concentrations of fasting insulin (P = 0.019), C-peptide (P = 0.004), HOMA-IR (P = 0.008) and CRP (P = 0.032) as compared with subjects carrying the minor T-allele (Leu446). In contrast, homozygous C/C carriers with n-3 PUFA levels above the median showed lower plasma concentrations of fasting insulin, peptide C, HOMA-IR and CRP, as compared with individuals with the T-allele. Conclusions We have demonstrated a significant interaction between the GCKR rs1260326-P446L polymorphism and plasma n-3 PUFA levels modulating insulin resistance and inflammatory markers in MetS subjects. Further studies are needed to confirm this gene-diet interaction in the general population and whether targeted dietary recommendations can prevent MetS in genetically susceptible individuals. Trial Registration ClinicalTrials.gov NCT00429195 PMID:21674002

  11. 3 CFR - Regulatory Compliance

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... protecting the air we breathe and the water we drink. Consistent regulatory enforcement also levels the... can lead the Government to hold itself more accountable, encouraging agencies to identify and...

  12. 3 CFR - Regulatory Review

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... in general—should be revisited. I therefore direct the Director of OMB, in consultation with... delay; clarify the role of the behavioral sciences in formulating regulatory policy; and identify...

  13. The protein tyrosine phosphatase PTPN22 controls forkhead box protein 3 T regulatory cell induction but is dispensable for T helper type 1 cell polarization

    PubMed Central

    Fousteri, G; Jofra, T; Debernardis, I; Stanford, S M; Laurenzi, A; Bottini, N; Battaglia, M

    2014-01-01

    Protein tyrosine phosphatases (PTPs) regulate T cell receptor (TCR) signalling and thus have a role in T cell differentiation. Here we tested whether the autoimmune predisposing gene PTPN22 encoding for a PTP that inhibits TCR signalling affects the generation of forkhead box protein 3 (FoxP3)+ T regulatory (Treg) cells and T helper type 1 (Th1) cells. Murine CD4+ T cells isolated from Ptpn22 knock-out (Ptpn22KO) mice cultured in Treg cell polarizing conditions showed increased sensitivity to TCR activation compared to wild-type (WT) cells, and subsequently reduced FoxP3 expression at optimal-to-high levels of activation. However, at lower levels of TCR activation, Ptpn22KO CD4+ T cells showed enhanced expression of FoxP3. Similar experiments in humans revealed that at optimal levels of TCR activation PTPN22 knock-down by specific oligonucleotides compromises the differentiation of naive CD4+ T cells into Treg cells. Notably, in vivo Treg cell conversion experiments in mice showed delayed kinetic but overall increased frequency and number of Treg cells in the absence of Ptpn22. In contrast, the in vitro and in vivo generation of Th1 cells was comparable between WT and Ptpn22KO mice, thus suggesting PTPN22 as a FoxP3-specific regulating factor. Together, these results propose PTPN22 as a key factor in setting the proper threshold for FoxP3+ Treg cell differentiation. PMID:24905474

  14. Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis[C][W][OA

    PubMed Central

    Liu, Xiaomin; Qin, Tao; Ma, Qianqian; Sun, Jingbo; Liu, Ziqiang; Yuan, Ming; Mao, Tonglin

    2013-01-01

    Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation. PMID:23653471

  15. Effects of retinoic acid and hydrogen peroxide on sterol regulatory element-binding protein-1a activation during adipogenic differentiation of 3T3-L1 cells.

    PubMed

    Abd Eldaim, Mabrouk A; Okamatsu-Ogura, Yuko; Terao, Akira; Kimura, Kazuhiro

    2010-11-01

    Both retinoic acid (RA) and oxidative stress (H2O2) increased transcription and cleavage of membrane-bound sterol regulatory element-binding protein (SREBP)-1, leading to enhanced transcription of fatty acid synthase (FAS) in hepatoma cells. On the other hand, RA and H2O2 decreased and increased lipogenesis in adipocytes, respectively, although roles of SREBP-1 activation in these effects remain to be elucidated. To elucidate its involvement, we examined the activation of SREBP-la, expression of FAS genes and lipid accumulation in 3T3-L1 cells in the presence of RA and/or H2O2. RA (1 microM) treatment suppressed expression of SREBP-1a and FAS genes and lipid accumulation. H2O2 (2 microM) treatment induced increased cleavage of SREBP-1a, without affecting amounts of SREBP-1a mRNA and precursor protein, and enhanced expression of FAS gene and lipid accumulation. Increased cleavage of SREBP-1a by H2O2 was also observed even in the presence of RA. These results suggest that H2O2, enhances a cleavage of SREBP-1a precursor protein, which independently occurs with the RA suppression of SREBP-1a gene expression, and that RA itself has no role in the SREBP-1a activation in adipocytes.

  16. α -Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.

    PubMed

    Calla-Choque, Jaeson Santos; Figueroa-Angulo, Elisa Elvira; Ávila-González, Leticia; Arroyo, Rossana

    2014-01-01

    Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

  17. Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

    PubMed

    Stewart, Emerson V; Lloyd, S Julie-Ann; Burg, John S; Nwosu, Christine C; Lintner, Robert E; Daza, Riza; Russ, Carsten; Ponchner, Karen; Nusbaum, Chad; Espenshade, Peter J

    2012-01-02

    Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.

  18. Species and tissue distribution of the regulatory protein of glucokinase.

    PubMed

    Vandercammen, A; Van Schaftingen, E

    1993-09-01

    Rat liver is known to contain a regulatory protein that inhibits glucokinase (hexokinase IV or D) competitively versus glucose. This inhibition is greatly reinforced by the presence of fructose 6-phosphate and antagonized by fructose 1-phosphate and by KCl. This protein was now measured in various rat tissues and in the livers of various species by the inhibition it exerts on rat liver glucokinase. Rat, mouse, rabbit, guinea-pig and pig liver, all of which contain glucokinase, also contained between 60 and 200 units/g of tissue of a regulatory protein displaying the properties mentioned above. By contrast, this protein could not be detected in cat, goat, chicken or trout liver, or in rat brain, heart, skeletal muscle, kidney and spleen, all tissues from which glucokinase is missing. Fructose 1-phosphate stimulated glucokinase in extracts of human liver, indicating the presence of regulatory protein. In addition, antibodies raised against rat regulatory protein allowed the detection of an approximately 60 kDa polypeptide in rat, guinea pig, rabbit and human liver. The livers of the toad Bufo marinus, of Xenopus laevis and of the turtle Pseudemys scripta elegans contained a regulatory protein similar to that of the rat, with, however, the major difference that it was not sensitive to fructose 6-phosphate or fructose 1-phosphate. In rat liver, the regulatory protein was detectable 4 days before birth. Its concentration increased afterwards to reach the adult level at day 30 of extrauterine life, whereas glucokinase only appeared after day 15. In the liver of the adult rat, starvation and streptozotocin-diabetes caused a 50-60% decrease in the concentration of regulatory protein after 7 days, whereas glucokinase activity fell to about 20% of its initial level. When 4-day-starved rats were refed, or when diabetic rats were treated with insulin, the concentration of regulatory protein slowly increased to reach about 85% of the control level after 3 days, whereas the

  19. Disparate Regulatory Mechanisms Control Fat3 and P75NTR Protein Transport through a Conserved Kif5-Interaction Domain

    PubMed Central

    Birkness, Jacqueline E.; Trinidad, Jonathan C.

    2016-01-01

    Directed transport delivers proteins to specific cellular locations and is one mechanism by which cells establish and maintain polarized cellular architectures. The atypical cadherin Fat3 directs the polarized extension of dendrites in retinal amacrine cells by influencing the distribution of cytoskeletal regulators during retinal development, however the mechanisms regulating the distribution of Fat3 remain unclear. We report a novel Kinesin/Kif5 Interaction domain (Kif5-ID) in Fat3 that facilitates Kif5B binding, and determines the distribution of Fat3 cytosolic domain constructs in neurons and MDCK cells. The Kif5-ID sequence is conserved in the neurotrophin receptor P75NTR, which also binds Kif5B, and Kif5-ID mutations similarly result in P75NTR mislocalization. Despite these similarities, Kif5B-mediated protein transport is differentially regulated by these two cargos. For Fat3, the Kif5-ID is regulated by alternative splicing, and the timecourse of splicing suggests that the distribution of Fat3 may switch between early and later stages of retinal development. In contrast, P75NTR binding to Kif5B is enhanced by tyrosine phosphorylation and thus has the potential to be dynamically regulated on a more rapid time scale. PMID:27788242

  20. Functional Characterization of Rpn3 Uncovers a Distinct 19S Proteasomal Subunit Requirement for Ubiquitin-Dependent Proteolysis of Cell Cycle Regulatory Proteins in Budding Yeast

    PubMed Central

    Bailly, Eric; Reed, Steven I.

    1999-01-01

    By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G1/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G1-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G1 arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3

  1. Maturation and Activity of Sterol Regulatory Element Binding Protein 1 Is Inhibited by Acyl-CoA Binding Domain Containing 3

    PubMed Central

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F.

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis. PMID:23166793

  2. Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharomyces cerevisiae.

    PubMed

    Daugherty, J R; Rai, R; el Berry, H M; Cooper, T G

    1993-01-01

    We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.

  3. Swiss Cheese, a protein involved in progressive neurodegeneration acts as a non-canonical regulatory subunit for PKA-C3

    PubMed Central

    da Cruz, Alexandre Bettencourt; Wentzell, Jill; Kretzschmar, Doris

    2008-01-01

    The Drosophila Swiss Cheese (SWS) protein and its vertebrate orthologue Neuropathy Target Esterase (NTE) are required for neuronal survival and glial integrity. In humans, NTE is the target of organophosphorous compounds which cause a paralyzing axonal degeneration and recently mutations in NTE have been shown to cause a Hereditary Spastic Paraplegia called NTE-related Motor-Neuron Disorder. SWS and NTE are concentrated in the endoplasmic reticulum and both have been shown to have an esterase function against an artificial substrate. However, the functional mechanisms and the pathways in which SWS/NTE are involved in are still widely unknown. Here we show that SWS interacts specifically with the C3 catalytic subunit of cAMP activated protein kinase (PKA-C3) which together with orthologues in mouse (Pkare) and human (PrKX) forms a novel class of catalytic subunits of unknown function. This interaction requires a domain of SWS which shows homology to regulatory subunits of PKA and, like conventional regulatory subunits, the binding of SWS to the PKA-C3 inhibits is function. Consistent with this result, expression of additional PKA-C3 induces degeneration and enhances the neurodegenerative phenotype in sws mutants. We also show that the complex formation with the membrane-bound SWS tethers PKA-C3 to membranes. We therefore propose a model in which SWS acts as a non-canonical subunit for PKA-C3, whereby the complex formation regulates the localization and kinase activity of PKA-C3, and that disruption of this regulation can induce neurodegeneration. PMID:18945896

  4. The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism*

    PubMed Central

    McRae, Steven; Iqbal, Jawed; Sarkar-Dutta, Mehuli; Lane, Samantha; Nagaraj, Abhiram; Ali, Naushad; Waris, Gulam

    2016-01-01

    Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with

  5. Characterisation of the regulatory function of the H-NS protein in the benthic bacterium Shewanella piezotolerans WP3 under cold conditions.

    PubMed

    Jian, Huahua; Xu, Guanpeng; Wang, Fengping; Xiao, Xiang

    2017-02-01

    The histone-like nucleoid structuring (H-NS) protein is conserved in Gram-negative bacteria and plays important roles as a multifunctional bacterial modulator. However, its roles in deep-sea microorganisms warrant investigation. In this study, the relationship between temperature and the regulatory function of H-NS was characterised in the benthic bacterium Shewanella piezotolerans WP3. Comparative microarray analysis of the hns knockout mutant and wild-type strain identified a total of 264 differentially expressed genes at 4°C, which is significantly less than that at 20°C. Notably, H-NS was proved to be involved in the cold-mediated induction of lateral flagellar gene transcription. We further showed that the binding affinity of H-NS is higher at 4°C than 20°C, thus explaining the significant difference in the expression pattern at different temperatures.

  6. Subunit architecture of the Golgi Dsc E3 ligase required for sterol regulatory element-binding protein (SREBP) cleavage in fission yeast.

    PubMed

    Lloyd, S Julie-Ann; Raychaudhuri, Sumana; Espenshade, Peter J

    2013-07-19

    The membrane-bound sterol regulatory element-binding protein (SREBP) transcription factors regulate lipogenesis in mammalian cells and are activated through sequential cleavage by the Golgi-localized Site-1 and Site-2 proteases. The mechanism of fission yeast SREBP cleavage is less well defined and, in contrast, requires the Golgi-localized Dsc E3 ligase complex. The Dsc E3 ligase consists of five integral membrane subunits, Dsc1 through Dsc5, and resembles membrane E3 ligases that function in endoplasmic reticulum-associated degradation. Using immunoprecipitation assays and blue native electrophoresis, we determined the subunit architecture for the complex of Dsc1 through Dsc5, showing that the Dsc proteins form subcomplexes and display defined connectivity. Dsc2 is a rhomboid pseudoprotease family member homologous to mammalian UBAC2 and a central component of the Dsc E3 ligase. We identified conservation in the architecture of the Dsc E3 ligase and the multisubunit E3 ligase gp78 in mammals. Specifically, Dsc1-Dsc2-Dsc5 forms a complex resembling gp78-UBAC2-UBXD8. Further characterization of Dsc2 revealed that its C-terminal UBA domain can bind to ubiquitin chains but that the Dsc2 UBA domain is not essential for yeast SREBP cleavage. Based on the ability of rhomboid superfamily members to bind transmembrane proteins, we speculate that Dsc2 functions in SREBP recognition and binding. Homologs of Dsc1 through Dsc4 are required for SREBP cleavage and virulence in the human opportunistic pathogen Aspergillus fumigatus. Thus, these studies advance our organizational understanding of multisubunit E3 ligases involved in endoplasmic reticulum-associated degradation and fungal pathogenesis.

  7. Characterization and functional studies of forkhead box protein 3− lymphocyte activation gene 3+ CD4+ regulatory T cells induced by mucosal B cells

    PubMed Central

    Chu, K-H; Chiang, B-L

    2015-01-01

    The induction of mucosal tolerance has been demonstrated to be an effective therapeutic approach for the treatment of allergic diseases. Our previous study demonstrated that Peyer's patch B cells could convert naive T cells into regulatory T cells (so-called Treg-of-B(P) cells); however, it is important to characterize this particular subset of Treg-of-B cells for future applications. This study aimed to investigate the role of lymphocyte activating gene 3 (LAG3) in mediating the regulatory function of Treg-of-B(P) cells induced by mucosal follicular B (FOB) cells. Microarray analysis and real-time polymerase chain reaction (PCR) were used to assess the gene expression pattern of Treg-of-B(P) cells. To evaluate the role of LAG3, the in-vitro suppressive function and the alleviation of airway inflammation in a murine model of asthma was assessed. Our data indicated that FOB cells isolated from Peyer's patches had the ability to generate more suppressive Treg-of-B cells with LAG3 expression, compared with CD23loCD21lo B cells. LAG3 is not only a marker for Treg-of-B(P) cells, but also participate in the suppressive ability. Moreover, CCR4 and CCR6 could be detected on the LAG3+, not LAG3−, Treg-of-B(P) cells and would help cells homing to allergic lung. In the murine model of asthma, the adoptive transfer of LAG3+ Treg-of-B(P) cells was able to sufficiently suppress T helper type 2 (Th2) cytokine production, eosinophil infiltration and alleviate asthmatic symptoms. LAG3 was expressed in Treg-of-B(P) cells and was also involved in the function of Treg-of-B(P) cells. In the future, this particular subset of Treg-of-B cells might be used to alleviate allergic symptoms. PMID:25581421

  8. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

    SciTech Connect

    Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun; Xiao, Shaobo

    2010-08-13

    Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

  9. Synovial Regulatory T Cells Occupy a Discrete TCR Niche in Human Arthritis and Require Local Signals To Stabilize FOXP3 Protein Expression

    PubMed Central

    Giannakopoulou, Eirini; Lom, Hannah; Wedderburn, Lucy R.

    2015-01-01

    Although there is great interest in harnessing the immunosuppressive potential of FOXP3+ regulatory T cells (Tregs) for treating autoimmunity, a sizeable knowledge gap exists regarding Treg fate in human disease. In juvenile idiopathic arthritis (JIA) patients, we have previously reported that atypical CD25+FOXP3− Treg-like cells uniquely populate the inflamed site. Intriguingly, their proportions relative to CD25+FOXP3+ Tregs associate with arthritis course, suggesting a role in disease. The ontogeny of these FOXP3− Treg-like cells is, however, unknown. In this study, we interrogated clonal relationships between CD4+ T cell subsets in JIA, using high-throughput TCR repertoire analysis. We reveal that FOXP3+ Tregs possess highly exclusive TCRβ usage from conventional T cells, in blood, and also at the inflamed site, where they are clonally expanded. Intriguingly, the repertoires of FOXP3+ Tregs in synovial fluid are highly overlapping with CD25+FOXP3− Treg-like cells, indicating fluctuations in FOXP3 expression in the inflamed joint. Furthermore, cultured synovial Tregs rapidly downregulated FOXP3 protein (but not mRNA), and this process was prevented by addition of synovial fluid from JIA patients, through an IL-6–independent mechanism. Our findings suggest that most Tregs arise from a separate lineage from conventional T cells, and that this repertoire divergence is largely maintained under chronic inflammatory conditions. We propose that subsequent Treg expansions at the inflamed site creates an environment that leads to competition for limited resources within the synovium, resulting in the destabilization of FOXP3 expression in some Tregs. PMID:26561546

  10. Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

    PubMed Central

    Dong, Hui; Dumenil, Jack; Lu, Fu-Hao; Na, Li; Vanhaeren, Hannes; Naumann, Christin; Klecker, Maria; Prior, Rachel; Smith, Caroline; McKenzie, Neil; Saalbach, Gerhard; Chen, Liangliang; Xia, Tian; Gonzalez, Nathalie; Seguela, Mathilde; Inzé, Dirk; Dissmeyer, Nico; Li, Yunhai; Bevan, Michael W.

    2017-01-01

    The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins. PMID:28167503

  11. Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

    PubMed Central

    Xu, Yi; Wu, Jianxiang; Fu, Shuai; Li, Chenyang; Zhu, Zeng-Rong

    2015-01-01

    ABSTRACT The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants, but their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome is present in the small brown planthopper (SBPH) (Laodelphgax striatellus) and has components similar to those in plants and mammals. There was an increase in the accumulation of Rice stripe virus (RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome plays a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome, named RPN3, could interact with RSV NS3. Transient overexpression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH rpn3 to complement an rpn3 mutation in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3. In vivo, we found an accumulation of ubiquitinated protein in SBPH tissues where the RSV titer was high, and silencing of rpn3 resulted in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH via a direct interaction with the RPN3 subunit to attenuate the host defense response. IMPORTANCE We show, for the first time, that the 26S proteasome components are present in the small brown planthopper and play a role in defense against its vectored plant virus (RSV). In turn, RSV encodes a protein that subverts the SBPH 26S proteasome

  12. [The intracellular localization of the regulatory proteins of the densovirus of German cockroach, Blattella germanica].

    PubMed

    Martynova, E U; Kapelinskaia, T V; Schal, C; Mukha, D V

    2014-01-01

    The intracellular localization of the regulatory proteins encoded by the genome of the densovirus of German cockroach was analyzed using western-blotting of nuclear and cytoplasmic extracts of the densovirus-infected passaging cells tissue culture BGE-2. Two of the three regulatory proteins, NS1 and NS3, were shown to possess mainly nuclear localization, while NS2 protein was distributed between the nucleus and cytoplasm. Data obtained provide new information necessary for prediction of the functions of densovirus regulatory proteins. Intracellular localization of NS3 protein was described for the densoviruses for the first time.

  13. Sterol regulatory element binding protein-mediated effect of fluvastatin on cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase transcription.

    PubMed

    Mascaró, C; Ortiz, J A; Ramos, M M; Haro, D; Hegardt, F G

    2000-02-15

    The effects of acute treatment with fluvastatin, a hypocholesteremic drug, on the mRNA levels of several regulatory enzymes of cholesterogenesis and of the LDL receptor were determined in rat liver. Fluvastatin increased the hepatic mRNA levels for HMG-CoA reductase up to 12-fold in 5 weeks of treatment at a daily dose of 6. 3 mg/kg. The effect was less marked in cytosolic HMG-CoA synthase, farnesyl-PP synthase, squalene synthetase, and LDL receptor. SREBP-2 mRNA levels were also increased, but SREBP-1 were not. De novo synthesis of cholesterol in several cultured cells was reduced by increasing concentrations of fluvastatin, and the IC(50) values of fluvastatin in HepG2, CV-1, and CHO cells were respectively 0.01, 0. 05, and 0.1 microM. When CHO cells stably transfected with a chimeric gene composed of the promoter of cytosolic HMG-CoA synthase and the CAT gene as a reporter were incubated with fluvastatin, the CAT gene was overexpressed, an effect which was similar to the cotransfection with the processed form of SREBP-1a. Both ALLN and fluvastatin increased the transcriptional activity of cytosolic HMG-CoA synthase. Mutation in either SRE or NF-Y boxes abolished the increase in transcriptional rate caused by fluvastatin in the promoter of cytosolic HMG-CoA synthase. These results indicate that the increase in transcriptional activity in the HMG-CoA synthase gene attributable to fluvastatin is a consequence of the activation of the proteolytic cleavage of SREBPs by reduced levels of intracellular cholesterol.

  14. The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3' Immunoglobulin Heavy Chain Regulatory Region.

    PubMed

    Salisbury, Richard L; Sulentic, Courtney E W

    2015-12-01

    Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3'Igh regulatory region (3'IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3'IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3'IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3'IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3'IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3'IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3'IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels.

  15. The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3′ Immunoglobulin Heavy Chain Regulatory Region

    PubMed Central

    Salisbury, Richard L.; Sulentic, Courtney E. W.

    2015-01-01

    Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3′Igh regulatory region (3′IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3′IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3′IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3′IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3′IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3′IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3′IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels. PMID:26377645

  16. A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation.

    PubMed

    Peng, Di; Wang, Zining; Huang, Anfei; Zhao, Yong; Qin, F Xiao-Feng

    2017-01-15

    The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A.

  17. A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation

    PubMed Central

    Peng, Di; Wang, Zining; Huang, Anfei

    2017-01-01

    The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A. PMID:27956528

  18. Discovery of the First α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist Dependent upon Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8.

    PubMed

    Gardinier, Kevin M; Gernert, Douglas L; Porter, Warren J; Reel, Jon K; Ornstein, Paul L; Spinazze, Patrick; Stevens, F Craig; Hahn, Patric; Hollinshead, Sean P; Mayhugh, Daniel; Schkeryantz, Jeff; Khilevich, Albert; De Frutos, Oscar; Gleason, Scott D; Kato, Akihiko S; Luffer-Atlas, Debra; Desai, Prashant V; Swanson, Steven; Burris, Kevin D; Ding, Chunjin; Heinz, Beverly A; Need, Anne B; Barth, Vanessa N; Stephenson, Gregory A; Diseroad, Benjamin A; Woods, Tim A; Yu, Hong; Bredt, David; Witkin, Jeffrey M

    2016-05-26

    Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients.

  19. Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain.

    PubMed Central

    Minehart, P L; Magasanik, B

    1991-01-01

    The GLN3 gene of Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen. We cloned the GLN3 gene and constructed null alleles by gene disruption. GLN3 is not essential for growth, but increased copies of GLN3 lead to a drastic decrease in growth rate. The complete nucleotide sequence of the GLN3 gene was determined, revealing one open reading frame encoding a polypeptide of 730 amino acids, with a molecular weight of approximately 80,000. The GLN3 protein contains a single putative Cys2/Cys2 zinc finger which has homology to the Neurospora crassa NIT2 protein, the Aspergillus nidulans AREA protein, and the erythroid-specific transcription factor GATA-1. Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLN1, the gene encoding glutamine synthetase. Neither control of transcription nor control of initiation of translation of GLN3 is important for regulation in response to glutamine availability. Images PMID:1682800

  20. Candida albicans Kinesin Kar3 Depends on a Cik1-Like Regulatory Partner Protein for Its Roles in Mating, Cell Morphogenesis, and Bipolar Spindle Formation

    PubMed Central

    Frazer, Corey; Joshi, Monika; Delorme, Caroline; Davis, Darlene; Bennett, Richard J.

    2015-01-01

    Candida albicans is a major fungal pathogen whose virulence is associated with its ability to transition from a budding yeast form to invasive hyphal filaments. The kinesin-14 family member CaKar3 is required for transition between these morphological states, as well as for mitotic progression and karyogamy. While kinesin-14 proteins are ubiquitous, CaKar3 homologs in hemiascomycete fungi are unique because they form heterodimers with noncatalytic kinesin-like proteins. Thus, CaKar3-based motors may represent a novel antifungal drug target. We have identified and examined the roles of a kinesin-like regulator of CaKar3. We show that orf19.306 (dubbed CaCIK1) encodes a protein that forms a heterodimer with CaKar3, localizes CaKar3 to spindle pole bodies, and can bind microtubules and influence CaKar3 mechanochemistry despite lacking an ATPase activity of its own. Similar to CaKar3 depletion, loss of CaCik1 results in cell cycle arrest, filamentation defects, and an inability to undergo karyogamy. Furthermore, an examination of the spindle structure in cells lacking either of these proteins shows that a large proportion have a monopolar spindle or two dissociated half-spindles, a phenotype unique to the C. albicans kinesin-14 homolog. These findings provide new insights into mitotic spindle structure and kinesin motor function in C. albicans and identify a potentially vulnerable target for antifungal drug development. PMID:26024903

  1. Promoters, transcripts, and regulatory proteins of Mungbean yellow mosaic geminivirus.

    PubMed

    Shivaprasad, P V; Akbergenov, Rashid; Trinks, Daniela; Rajeswaran, R; Veluthambi, K; Hohn, Thomas; Pooggin, Mikhail M

    2005-07-01

    Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.

  2. Promoters, Transcripts, and Regulatory Proteins of Mungbean Yellow Mosaic Geminivirus†

    PubMed Central

    Shivaprasad, P. V.; Akbergenov, Rashid; Trinks, Daniela; Rajeswaran, R.; Veluthambi, K.; Hohn, Thomas; Pooggin, Mikhail M.

    2005-01-01

    Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing. PMID:15956560

  3. Regulatory elements of the Staphylococcus aureus protein A (Spa) promoter.

    PubMed

    Gao, Jinxin; Stewart, George C

    2004-06-01

    Staphylococcal protein A (Spa) is an important virulence factor of Staphylococcus aureus. Transcription of the spa determinant occurs during the exponential growth phase and is repressed when the cells enter the postexponential growth phase. Regulation of spa expression has been found to be complicated, with regulation involving multiple factors, including Agr, SarA, SarS, SarT, Rot, and MgrA. Our understanding of how these factors work on the spa promoter to regulate spa expression is incomplete. To identify regulatory sites within the spa promoter, analysis of deletion derivatives of the promoter in host strains deficient in one or more of the regulatory factors was undertaken, and several critical features of spa regulation were revealed. The transcriptional start sites of spa were determined by primer extension. The spa promoter sequences were subcloned in front of a promoterless chloramphenicol acetyltransferase reporter gene. Various lengths of spa truncations with the same 3' end were constructed, and the resultant plasmids were transduced into strains with different regulatory genetic backgrounds. Our results identified upstream promoter sequences necessary for Agr system regulation of spa expression. The cis elements for SarS activity, an activator of spa expression, and for SarA activity, a repressor of spa expression, were identified. The well-characterized SarA consensus sequence on the spa promoter was found to be insufficient for SarA repression of the spa promoter. Full repression required the presence of a second consensus site adjacent to the SarS binding site. Sequences directly upstream of the core promoter sequence were found to stimulate transcription.

  4. Functional Classification of Immune Regulatory Proteins

    SciTech Connect

    Rubinstein, Rotem; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.; Fiser, Andras

    2013-05-01

    Members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. We were guided by the Brotherhood approach and present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

  5. Sumoylation: a regulatory protein modification in health and disease.

    PubMed

    Flotho, Annette; Melchior, Frauke

    2013-01-01

    Posttranslational modification with small ubiquitin-related modifier (SUMO) proteins is now established as one of the key regulatory protein modifications in eukaryotic cells. Hundreds of proteins involved in processes such as chromatin organization, transcription, DNA repair, macromolecular assembly, protein homeostasis, trafficking, and signal transduction are subject to reversible sumoylation. Hence, it is not surprising that disease links are beginning to emerge and that interference with sumoylation is being considered for intervention. Here, we summarize basic mechanisms and highlight recent developments in the physiology of sumoylation.

  6. The Yeast Iron Regulatory Proteins Grx3/4 and Fra2 Form Heterodimeric Complexes Containing a [2Fe-2S] Cluster with Cysteinyl and Histidyl Ligation†

    PubMed Central

    Li, Haoran; Mapolelo, Daphne T.; Dingra, Nin N.; Naik, Sunil G.; Lees, Nicolas S.; Hoffman, Brian M.; Riggs-Gelasco, Pamela J.; Huynh, Boi Hanh; Johnson, Michael K.; Outten, Caryn E.

    2009-01-01

    The transcription of iron uptake and storage genes in S. cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear. To reconstitute and characterize components of this signaling pathway in vitro, we have overexpressed yeast Fra2 and Grx3/4 in E. coli. We have shown that co-expression of recombinant Fra2 with Grx3 or Grx4 allows purification of a stable [2Fe-2S]2+ cluster-containing Fra2-Grx3 or Fra2-Grx4 heterodimeric complex. Reconstitution of a [2Fe-2S] cluster on Grx3 or Grx4 without Fra2 produces a [2Fe-2S]-bridged homodimer. UV-visible absorption and CD, resonance Raman, EPR, ENDOR, Mössbauer, and EXAFS studies of [2Fe-2S] Grx3/4 homodimers and the [2Fe-2S] Fra2-Grx3/4 heterodimers indicate that inclusion of Fra2 in the Grx3/4 Fe-S complex causes a change in the cluster stability and coordination environment. Taken together, our analytical, spectroscopic, and mutagenesis data indicate that Grx3/4 and Fra2 form a Fe-S-bridged heterodimeric complex with Fe ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. Overall, these results suggest that the ability of the Fra2-Grx3/4 complex to assemble a [2Fe-2S] cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast. PMID:19715344

  7. Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

    PubMed Central

    Hovingh, Elise S.; van den Broek, Bryan; Jongerius, Ilse

    2016-01-01

    The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed. PMID:28066340

  8. CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS

    EPA Science Inventory

    IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic...

  9. Lyoniresinol 3α-O-β-D-glucopyranoside-mediated hypoglycaemia and its influence on apoptosis-regulatory protein expression in the injured kidneys of streptozotocin-induced mice.

    PubMed

    Wen, Qingwei; Liang, Tao; Qin, Feizhang; Wei, Jinbin; He, Qiaoling; Luo, Xiu; Chen, Xiaoyu; Zheng, Ni; Huang, Renbin

    2013-01-01

    Averrhoa carambola L. (Oxalidaceae) root (ACLR) has a long history of use in traditional Chinese medicine for treating diabetes and diabetic nephropathy (DN). (±)-Lyoniresinol 3α-O-β-D-glucopyranoside (LGP1, LGP2) were two chiral lignan glucosides that were isolated from the ACLR. The purpose of this study was to investigate the effect of LGP1 and LGP2-mediated hypoglycaemia on renal injury in streptozotocin (STZ)-induced diabetic mice. STZ-induced diabetic mice were administrated LGP1 and LGP2 orally (20, 40, 80 mg/kg body weight/d) for 14 days. Hyperglycaemia and the expression of related proteins such as nuclear factor-κB (NF-κB), caspase-3, -8, -9, and Bcl-associated X protein (Bax) were markedly decreased by LGP1 treatment. However, LGP2 treatment had no hypoglycaemic activity. Diabetes-dependent alterations in the kidney such as glomerular hypertrophy, excessive extracellular matrix amassing, and glomerular and tubular basement membrane thickening were improved after 14 days of LGP1 treatment. B cell lymphoma Leukaemia-2 (Bcl-2) expression was reduced in the STZ-induced diabetic mouse kidneys but was enhanced by LGP1 treatment. These findings suggest that LGP1 treatment may inhibit diabetic nephropathy progression and may regulate several pharmacological targets for treating or preventing diabetic nephropathy.

  10. Lyoniresinol 3α-O-β-D-Glucopyranoside-Mediated Hypoglycaemia and Its Influence on Apoptosis-Regulatory Protein Expression in the Injured Kidneys of Streptozotocin-Induced Mice

    PubMed Central

    Wen, Qingwei; Liang, Tao; Qin, Feizhang; Wei, Jinbin; He, Qiaoling; Luo, Xiu; Chen, Xiaoyu; Zheng, Ni; Huang, Renbin

    2013-01-01

    Averrhoa carambola L. (Oxalidaceae) root (ACLR) has a long history of use in traditional Chinese medicine for treating diabetes and diabetic nephropathy (DN). (±)-Lyoniresinol 3α-O-β-D-glucopyranoside (LGP1, LGP2) were two chiral lignan glucosides that were isolated from the ACLR. The purpose of this study was to investigate the effect of LGP1 and LGP2-mediated hypoglycaemia on renal injury in streptozotocin (STZ)-induced diabetic mice. STZ-induced diabetic mice were administrated LGP1 and LGP2 orally (20, 40, 80 mg/kg body weight/d) for 14 days. Hyperglycaemia and the expression of related proteins such as nuclear factor-κB (NF-κB), caspase-3, -8, -9, and Bcl-associated X protein (Bax) were markedly decreased by LGP1 treatment. However, LGP2 treatment had no hypoglycaemic activity. Diabetes-dependent alterations in the kidney such as glomerular hypertrophy, excessive extracellular matrix amassing, and glomerular and tubular basement membrane thickening were improved after 14 days of LGP1 treatment. B cell lymphoma Leukaemia-2 (Bcl-2) expression was reduced in the STZ-induced diabetic mouse kidneys but was enhanced by LGP1 treatment. These findings suggest that LGP1 treatment may inhibit diabetic nephropathy progression and may regulate several pharmacological targets for treating or preventing diabetic nephropathy. PMID:24312585

  11. The Arabidopsis pyruvate,orthophosphate dikinase regulatory proteins encode a novel, unprecedented Ser/Thr protein kinase primary structure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pyruvate,orthophosphate dikinase (PPDK) is a ubiquitous, low abundance metabolic enzyme of undetermined function in C3 plants. Its activity in C3 chloroplasts is light-regulated via reversible phosphorylation of an active-site Thr residue by the PPDK regulatory protein (RP), a most unusual, bifuncti...

  12. CUL3 and protein kinases

    PubMed Central

    Metzger, Thibaud; Kleiss, Charlotte; Sumara, Izabela

    2013-01-01

    Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22.1 In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases. PMID:24067371

  13. A tomato alternative oxidase protein with altered regulatory properties.

    PubMed

    Holtzapffel, Ruth C; Castelli, Joanne; Finnegan, Patrick M; Millar, A Harvey; Whelan, Jim; Day, David A

    2003-09-30

    We have investigated the expression and regulatory properties of the two alternative oxidase (Aox) proteins that are expressed in tomato (Lycopersicon esculentum L. Mill cv. Sweetie) after storage of green fruit at 4 degrees C. Four Aox genes were identified in the tomato genome, of which two (LeAox1a and LeAox1b) were demonstrated to be expressed in cold-treated fruit. The activity and regulatory properties of LeAox1a and LeAox1b were assayed after expression of each protein in yeast cells (Saccharomyces cerevisiae), proving that each is an active Aox protein. The LeAox1b protein was shown to have altered regulatory properties due to the substitution of a Ser for the highly conserved Cys(I) residue. LeAox1b could not form inactive disulfide-linked dimers and was activated by succinate instead of pyruvate. This is the first example of a dicot species expressing a natural Cys(I)/Ser isoform. The implications of the existence and expression of such Aox isoforms is discussed in the light of the hypothesised role for Aox in plant metabolism.

  14. Foxp3(+) regulatory T cells in tuberculosis.

    PubMed

    Larson, Ryan P; Shafiani, Shahin; Urdahl, Kevin B

    2013-01-01

    The immune response to Mycobacterium tuberculosis (Mtb) must be tightly regulated to mount a sufficient response to limit bacterial growth and dissemination while avoiding excessive inflammation that could damage host tissues. A wide variety of cell types, cell surface molecules, and cytokines are likely to contribute to this regulation, but recent studies have revealed that a subset of CD4 T cells expressing the transcription factor Foxp3, called regulatory T (reg) cells, play a critical role [1-3]. Although the first reports of T reg cells in tuberculosis (TB) occurred only recently (i.e., 2006) [4, 5], we have already gained many insights into their activity during TB. While it is likely that T reg cells do play some beneficial roles by preventing inflammation-mediated damage to host tissues during TB, this aspect of their function has not been well studied to date. What is clear, however, is that during the initial T cell response to Mtb infection, Mtb induces the expansions of T reg cells that delay the onset of adaptive immunity, suggesting that Mtb has hijacked T reg cell-mediated immune suppression to allow it to replicate unabated in the lung until T cells finally arrive [6]. In this chapter, we will first provide an overview of the delayed T cell response to Mtb and a brief introduction to regulatory T cells. We will then review what is known about T reg cells from observations in human populations, discuss mechanistic insights revealed in the mouse model, and speculate about the relevance of this understanding for future efforts to prevent and treat TB.

  15. Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.

    PubMed

    Lippens, S; Lefebvre, S; Gilbert, B; Sze, M; Devos, M; Verhelst, K; Vereecke, L; Mc Guire, C; Guérin, C; Vandenabeele, P; Pasparakis, M; Mikkola, M L; Beyaert, R; Declercq, W; van Loo, G

    2011-12-01

    The ubiquitin-editing enzyme A20 (tumor necrosis factor-α-induced protein 3) serves as a critical brake on nuclear factor κB (NF-κB) signaling. In humans, polymorphisms in or near the A20 gene are associated with several inflammatory disorders, including psoriasis. We show here that epidermis-specific A20-knockout mice (A20(EKO)) develop keratinocyte hyperproliferation, but no signs of skin inflammation, such as immune cell infiltration. However, A20(EKO) mice clearly developed ectodermal organ abnormalities, including disheveled hair, longer nails and sebocyte hyperplasia. This phenotype resembles that of mice overexpressing ectodysplasin-A1 (EDA-A1) or the ectodysplasin receptor (EDAR), suggesting that A20 negatively controls EDAR signaling. We found that A20 inhibited EDAR-induced NF-κB signaling independent from its de-ubiquitinating activity. In addition, A20 expression was induced by EDA-A1 in embryonic skin explants, in which its expression was confined to the hair placodes, known to be the site of EDAR expression. In summary, our data indicate that EDAR-induced NF-κB levels are controlled by A20, which functions as a negative feedback regulator, to assure proper skin homeostasis and epidermal appendage development.

  16. [Modulators of the regulatory protein activity acting at microdoses].

    PubMed

    Iamskova, V P; Krasnov, M S; Skripnikova, V S; Moliavka, A A; Il'ina, A P; Margasiuk, D V; Borisenko, A V; Berezin, B B; Iamskov, I A

    2009-01-01

    New, previously not studied bioregulators active in the ultra low doses corresponding of 10(-8) - 10(-17) mg/ml have been isolated from vitreoretinal tissue of eye. It has been shown that these bioregulators comprise some regulatory peptides-modulators represented by proteins with molecular weights 15-70 KDa one of which is bovine serum albumin. Correlation between the nanosize of bioregulators and their ability to show activity in ultra low doses is established.

  17. Herpes simplex virus 1-encoded tegument protein VP16 abrogates the production of beta interferon (IFN) by inhibiting NF-κB activation and blocking IFN regulatory factor 3 to recruit its coactivator CBP.

    PubMed

    Xing, Junji; Ni, Liwen; Wang, Shuai; Wang, Kezhen; Lin, Rongtuan; Zheng, Chunfu

    2013-09-01

    Host cells activate innate immune signaling pathways to defend against invading pathogens. To survive within an infected host, viruses have evolved intricate strategies to counteract host immune responses. Herpesviruses, including herpes simplex virus type 1 (HSV-1), have large genomes and therefore have the capacity to encode numerous proteins that modulate host innate immune responses. Here we define the contribution of HSV-1 tegument protein VP16 in the inhibition of beta interferon (IFN-β) production. VP16 was demonstrated to significantly inhibit Sendai virus (SeV)-induced IFN-β production, and its transcriptional activation domain was not responsible for this inhibition activity. Additionally, VP16 blocked the activation of the NF-κB promoter induced by SeV or tumor necrosis factor alpha treatment and expression of NF-κB-dependent genes through interaction with p65. Coexpression analysis revealed that VP16 selectively blocked IFN regulatory factor 3 (IRF-3)-mediated but not IRF-7-mediated transactivation. VP16 was able to bind to IRF-3 but not IRF-7 in vivo, based on coimmunoprecipitation analysis, but it did not affect IRF-3 dimerization, nuclear translocation, or DNA binding activity. Rather, VP16 interacted with the CREB binding protein (CBP) coactivator and efficiently inhibited the formation of the transcriptional complexes IRF-3-CBP in the context of HSV-1 infection. These results illustrate that VP16 is able to block the production of IFN-β by inhibiting NF-κB activation and interfering with IRF-3 to recruit its coactivator CBP, which may be important to the early events leading to HSV-1 infection.

  18. The RGK family: a regulatory tail of small GTP-binding proteins.

    PubMed

    Kelly, Kathleen

    2005-12-01

    RGK proteins are small Ras-related GTP-binding proteins that function as potent inhibitors of voltage-dependent calcium channels, and two members of the family, Gem and Rad, modulate Rho-dependent remodeling of the cytoskeleton. Within the Ras superfamily, RGK proteins have distinct structural and regulatory characteristics. It is an open question as to whether RGK proteins catalyze GTP hydrolysis in vivo. Binding of calmodulin and the 14-3-3 protein to RGK proteins controls downstream pathways. Here, we discuss the structural and functional properties of RGK proteins and highlight recent work by Beguin and colleagues addressing the mechanism of Gem regulation by calmodulin and 14-3-3.

  19. The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

    PubMed

    Gurzu, Simona; Jung, I; Prantner, I; Ember, I; Pávai, Z; Mezei, T

    2008-01-01

    The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

  20. The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins

    PubMed Central

    Rising, Lisa; Mak, Raymond; Webb, Kristofor; Kaiser, Stephen E.; Zuzow, Nathan; Riviere, Paul; Yang, Bing; Fenech, Emma; Tang, Xin; Lindsay, Scott A.; Christianson, John C.; Hampton, Randolph Y.; Wasserman, Steven A.; Bennett, Eric J.

    2015-01-01

    Summary Insults to endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as a previously uncharacterized and important facet of eukaryotic translational control. PMID:26051182

  1. The Evolution of the Secreted Regulatory Protein Progranulin

    PubMed Central

    Palfree, Roger G. E.; Bennett, Hugh P. J.; Bateman, Andrew

    2015-01-01

    Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

  2. Allosteric properties of PH domains in Arf regulatory proteins.

    PubMed

    Roy, Neeladri Sekhar; Yohe, Marielle E; Randazzo, Paul A; Gruschus, James M

    2016-01-01

    Pleckstrin Homology (PH) domains bind phospholipids and proteins. They are critical regulatory elements of a number enzymes including guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for Ras-superfamily guanine nucleotide binding proteins such as ADP-ribosylation factors (Arfs). Recent studies have indicated that many PH domains may bind more than one ligand cooperatively. Here we discuss the molecular basis of PH domain-dependent allosteric behavior of 2 ADP-ribosylation factor exchange factors, Grp1 and Brag2, cooperative binding of ligands to the PH domains of Grp1 and the Arf GTPase-activating protein, ASAP1, and the consequences for activity of the associated catalytic domains.

  3. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    PubMed Central

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  4. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks.

    PubMed

    Nuñez de Villavicencio-Diaz, Teresa; Rabalski, Adam J; Litchfield, David W

    2017-03-05

    Protein kinase CK2 is a small family of protein kinases that has been implicated in an expanding array of biological processes. While it is widely accepted that CK2 is a regulatory participant in a multitude of fundamental cellular processes, CK2 is often considered to be a constitutively active enzyme which raises questions about how it can be a regulatory participant in intricately controlled cellular processes. To resolve this apparent paradox, we have performed a systematic analysis of the published literature using text mining as well as mining of proteomic databases together with computational assembly of networks that involve CK2. These analyses reinforce the notion that CK2 is involved in a broad variety of biological processes and also reveal an extensive interplay between CK2 phosphorylation and other post-translational modifications. The interplay between CK2 and other post-translational modifications suggests that CK2 does have intricate roles in orchestrating cellular events. In this respect, phosphorylation of specific substrates by CK2 could be regulated by other post-translational modifications and CK2 could also have roles in modulating other post-translational modifications. Collectively, these observations suggest that the actions of CK2 are precisely coordinated with other constituents of regulatory cellular networks.

  5. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks

    PubMed Central

    Nuñez de Villavicencio-Diaz, Teresa; Rabalski, Adam J.; Litchfield, David W.

    2017-01-01

    Protein kinase CK2 is a small family of protein kinases that has been implicated in an expanding array of biological processes. While it is widely accepted that CK2 is a regulatory participant in a multitude of fundamental cellular processes, CK2 is often considered to be a constitutively active enzyme which raises questions about how it can be a regulatory participant in intricately controlled cellular processes. To resolve this apparent paradox, we have performed a systematic analysis of the published literature using text mining as well as mining of proteomic databases together with computational assembly of networks that involve CK2. These analyses reinforce the notion that CK2 is involved in a broad variety of biological processes and also reveal an extensive interplay between CK2 phosphorylation and other post-translational modifications. The interplay between CK2 and other post-translational modifications suggests that CK2 does have intricate roles in orchestrating cellular events. In this respect, phosphorylation of specific substrates by CK2 could be regulated by other post-translational modifications and CK2 could also have roles in modulating other post-translational modifications. Collectively, these observations suggest that the actions of CK2 are precisely coordinated with other constituents of regulatory cellular networks. PMID:28273877

  6. Structural Instability Tuning as a Regulatory Mechanism in Protein-Protein Interactions

    PubMed Central

    Chen, Li; Balabanidou, Vassilia; Remeta, David P.; Minetti, Conceição A.S.A.; Portaliou, Athina G.; Economou, Anastassios; Kalodimos, Charalampos G.

    2011-01-01

    SUMMARY Protein-protein interactions mediate a vast number of cellular processes. Here we present a regulatory mechanism in protein-protein interactions mediated by finely-tuned structural instability coupled with molecular mimicry. We show that a set of type III secretion (TTS) autoinhibited homodimeric chaperones adopt a molten-globule-like state that transiently exposes the substrate binding site as a means to become rapidly poised for binding to their cognate protein substrates. Packing defects at the homodimeric interface stimulate binding whereas correction of these defects results in less labile chaperones that give rise to non-functional biological systems. The protein substrates use structural mimicry to offset the “weak spots” in the chaperones and to counteract their autoinhibitory conformation. This regulatory mechanism of protein activity is evolutionary conserved among several TSS systems and presents a lucid example of functional advantage conferred upon a biological system by finely-tuned structural instability. PMID:22152477

  7. Pleiotropy constrains the evolution of protein but not regulatory sequences in a transcription regulatory network influencing complex social behaviors

    PubMed Central

    Molodtsova, Daria; Harpur, Brock A.; Kent, Clement F.; Seevananthan, Kajendra; Zayed, Amro

    2014-01-01

    It is increasingly apparent that genes and networks that influence complex behavior are evolutionary conserved, which is paradoxical considering that behavior is labile over evolutionary timescales. How does adaptive change in behavior arise if behavior is controlled by conserved, pleiotropic, and likely evolutionary constrained genes? Pleiotropy and connectedness are known to constrain the general rate of protein evolution, prompting some to suggest that the evolution of complex traits, including behavior, is fuelled by regulatory sequence evolution. However, we seldom have data on the strength of selection on mutations in coding and regulatory sequences, and this hinders our ability to study how pleiotropy influences coding and regulatory sequence evolution. Here we use population genomics to estimate the strength of selection on coding and regulatory mutations for a transcriptional regulatory network that influences complex behavior of honey bees. We found that replacement mutations in highly connected transcription factors and target genes experience significantly stronger negative selection relative to weakly connected transcription factors and targets. Adaptively evolving proteins were significantly more likely to reside at the periphery of the regulatory network, while proteins with signs of negative selection were near the core of the network. Interestingly, connectedness and network structure had minimal influence on the strength of selection on putative regulatory sequences for both transcription factors and their targets. Our study indicates that adaptive evolution of complex behavior can arise because of positive selection on protein-coding mutations in peripheral genes, and on regulatory sequence mutations in both transcription factors and their targets throughout the network. PMID:25566318

  8. Caerulomycin A Enhances Transforming Growth Factor-β (TGF-β)-Smad3 Protein Signaling by Suppressing Interferon-γ (IFN-γ)-Signal Transducer and Activator of Transcription 1 (STAT1) Protein Signaling to Expand Regulatory T Cells (Tregs)*

    PubMed Central

    Gurram, Rama Krishna; Kujur, Weshely; Maurya, Sudeep K.; Agrewala, Javed N.

    2014-01-01

    Cytokines play a very important role in the regulation of immune homeostasis. Regulatory T cells (Tregs) responsible for the generation of peripheral tolerance are under the tight regulation of the cytokine milieu. In this study, we report a novel role of a bipyridyl compound, Caerulomycin A (CaeA), in inducing the generation of Tregs. It was observed that CaeA substantially up-regulated the pool of Tregs, as evidenced by an increased frequency of CD4+ Foxp3+ cells. In addition, CaeA significantly suppressed the number of Th1 and Th17 cells, as supported by a decreased percentage of CD4+/IFN-γ+ and CD4+/IL-17+ cells, respectively. Furthermore, we established the mechanism and observed that CaeA interfered with IFN-γ-induced STAT1 signaling by augmenting SOCS1 expression. An increase in the TGF-β-mediated Smad3 activity was also noted. Furthermore, CaeA rescued Tregs from IFN-γ-induced inhibition. These results were corroborated by blocking Smad3 activity, which abolished the CaeA-facilitated generation of Tregs. In essence, our results indicate a novel role of CaeA in inducing the generation of Tregs. This finding suggests that CaeA has enough potential to be considered as a potent future drug for the treatment of autoimmunity. PMID:24811173

  9. Treatment with alpha-melanocyte stimulating hormone preserves calcium regulatory proteins in rat heart allografts.

    PubMed

    Colombo, Gualtiero; Sordi, Andrea; Lonati, Caterina; Carlin, Andrea; Turcatti, Flavia; Leonardi, Patrizia; Gatti, Stefano; Catania, Anna

    2008-08-01

    Prevention of graft dysfunction is a major objective in transplantation medicine. Previous research on experimental heart transplantation indicated that treatment with the immunomodulatory peptide alpha-melanocyte stimulating hormone (alpha-MSH) improves histopathology, prolongs allograft survival, and reduces expression of the main tissue injury mediators. Because calcium-handling is critical in heart graft function, we determined the effects of transplantation injury and influences of alpha-MSH treatment on representative calcium regulatory proteins in rat heart allografts. Hearts from Brown Norway rats were transplanted heterotopically into MHC incompatible Lewis rats. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), protein kinase C epsilon (PKC epsilon), sarcoplasmic/endoplasmic reticulum calcium-ATPase 2 (SERCA2a), arrestin-beta1 (Arrb1), cholinergic receptor M2 (Chrm2), and inositol 1,4,5-triphosphate receptor 1 (InsP(3)R1) were examined in: (1) non-transplanted donor hearts; (2) allografts from saline-treated rats; and (3) allografts from rats treated with the synthetic alpha-MSH analog Nle4-DPhe7-alpha-MSH (NDP-alpha-MSH) (100 microg i.p. every 12h). Transplantation injury was associated with severe reduction in calcium regulatory protein transcription and expression level. NDP-alpha-MSH administration partly reversed inhibition of protein transcription and almost completely prevented protein loss. Finally, because certain effects of cyclic 3'-5'-adenosine monophosphate (cAMP) signaling on calcium handling in cardiac myocytes depend on activation of exchange protein directly activated by cAMP 1 (Epac1), we determined Epac1 mRNA and protein expression in heart allografts. Transplantation injury markedly reduced Epac1. NDP-alpha-MSH treatment significantly preserved both Epac1 protein and mRNA in the allografts. Administration of alpha-MSH or related melanocortins could reduce transplantation-induced dysfunction through protection of heart calcium

  10. Dysregulation of sterol regulatory element binding protein-1c in livers of morbidly obese women is associated with altered suppressor of cytokine signaling-3 and signal transducer and activator of transcription-1 signaling.

    PubMed

    Elam, Marshall B; Yellaturu, Chandrahasa; Howell, George E; Deng, Xiong; Cowan, George S; Kumar, Poonam; Park, Edwards A; Hiler, M Lloyd; Wilcox, Henry G; Hughes, Thomas A; Cook, George A; Raghow, Rajendra

    2010-04-01

    We compared hepatic expression of genes that regulate lipid biosynthesis and metabolic signaling in liver biopsy specimens from women who were undergoing gastric bypass surgery (GBP) for morbid obesity with that in women undergoing ventral hernia repair who had experienced massive weight loss (MWL) after prior GBP. Comprehensive metabolic profiles of morbidly obese (MO) (22 subjects) and MWL (9 subjects) were also compared. Analyses of gene expression in liver biopsies from MO and MWL were accomplished by Affymetrix microarray, real-time polymerase chain reaction, and Western blotting techniques. After GBP, MWL subjects had lost on average 102 lb as compared with MO subjects. This was accompanied by effective reversal of the dyslipidemia and insulin resistance that were present in MO. As compared with MWL, livers of MO subjects exhibited increased expression of sterol regulatory element binding protein (SREBP)-1c and its downstream lipogenic targets, fatty acid synthase and acetyl-coenzyme A-carboxylase-1. Livers of MO subjects also exhibited enhanced expression of suppressor of cytokine signaling-3 protein and attenuated Janus kinase signal transducer and activator of transcription (JAK/STAT) signaling. Consistent with these findings, we found that the human SREBP-1c promoter was positively regulated by insulin and negatively regulated by STAT3. These data support the hypothesis that suppressor of cytokine signaling-3-mediated attenuation of the STAT signaling pathway and resulting enhanced expression of SREBP-1c, a key regulator of de novo lipid biosynthesis, are mechanistically related to the development of hepatic insulin resistance and dyslipidemia in MO women.

  11. Exploitation of complement regulatory proteins by Borrelia and Francisella.

    PubMed

    Madar, Marian; Bencurova, Elena; Mlynarcik, Patrik; Almeida, André M; Soares, Renata; Bhide, Katarina; Pulzova, Lucia; Kovac, Andrej; Coelho, Ana V; Bhide, Mangesh

    2015-06-01

    Pathogens have developed sophisticated mechanisms of complement evasion such as binding to the host complement regulatory proteins (CRPs) on their surface or expression of CRP mimicking molecules. The ability of pathogens to evade the complement system has been correlated with pathogenesis and host selectivity. Hitherto, little work has been undertaken to determine whether Borrelia and Francisella exploit various CRPs to block complement attack. Seventeen Borrelia (twelve species) and six Francisella (three subspecies) strains were used to assess their ability to bind human, sheep and cattle CRPs or mimic membrane associated complement regulators. A series of experiments including affinity ligand binding experiments, pull-down assays and mass spectrometry based protein identification, revealed an array of CRP binding proteins of Borrelia and Francisella. Unlike Francisella, Borrelia strains were able to bind multiple human CRPs. Three strains of Borrelia (SKT-4, SKT-2 and HO14) showed the presence of a human CD46-homologous motif, indicating their ability to possess putative human CD46 mimicking molecules. Similarly, five strains of Borrelia and two strains of Francisella may have surface proteins with human CD59-homologous motifs. Among ovine and bovine CRPs, the only CRP bound by Francisella (LVS, Tul4 strain) was vitronectin, while ovine C4BP, ovine factor H and bovine factor H were bound to Borrelia strains SKT-2, DN127 and Co53. This study presents an array of proteins of Borrelia and Francisella that bind CRPs or may mimic membrane-CRPs, thus enabling multiphasic complement evasion strategies of these pathogens.

  12. 14-3-3 proteins are promising LRRK2 interactors.

    PubMed

    Rudenko, Iakov N; Cookson, Mark R

    2010-09-15

    Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common cause of familial PD (Parkinson's disease). Mutations that cause PD are found in either the GTPase or kinase domains of LRRK2 or an intervening sequence called the COR [C-terminus of ROC (Ras of complex proteins)] domain. As well as the two catalytic domains, LRRK2 possesses several protein-protein interaction domains, but their function and the proteins with which they interact are poorly understood. In this issue of the Biochemical Journal, Nichols et al. study the interaction of the N-terminal region of LRRK2 with 14-3-3 proteins, regulatory proteins that often bind to phosphorylated regions of components of cell signalling pathways. Using a combination of techniques, Nichols et al. have identified two residues (Ser910 and Ser935) that are critically responsible for 14-3-3 binding. The interaction of LRRK2 with 14-3-3 proteins can prevent dephosphorylation of Ser910/Ser935 and stabilize LRRK2 structure, perhaps by influencing the dimerization of LRRK2. The ability to interact with 14-3-3 correlates with the pattern of intracellular LRRK2 distribution. Collectively, these new results identify a potentially important regulatory mechanism of this complex protein and might provide ways to think about therapeutic opportunities for PD.

  13. Iron Regulatory Proteins Mediate Host Resistance to Salmonella Infection.

    PubMed

    Nairz, Manfred; Ferring-Appel, Dunja; Casarrubea, Daniela; Sonnweber, Thomas; Viatte, Lydie; Schroll, Andrea; Haschka, David; Fang, Ferric C; Hentze, Matthias W; Weiss, Guenter; Galy, Bruno

    2015-08-12

    Macrophages are essential for systemic iron recycling, and also control iron availability to pathogens. Iron metabolism in mammalian cells is orchestrated posttranscriptionally by iron-regulatory proteins (IRP)-1 and -2. Here, we generated mice with selective and combined ablation of both IRPs in macrophages to investigate the role of IRPs in controlling iron availability. These animals are hyperferritinemic but otherwise display normal clinical iron parameters. However, mutant mice rapidly succumb to systemic infection with Salmonella Typhimurium, a pathogenic bacterium that multiplies within macrophages, with increased bacterial burdens in liver and spleen. Ex vivo infection experiments indicate that IRP function restricts bacterial access to iron via the EntC and Feo bacterial iron-acquisition systems. Further, IRPs contain Salmonella by promoting the induction of lipocalin 2, a host antimicrobial factor that inhibits bacterial uptake of iron-laden siderophores, and by suppressing the ferritin iron pool. This work reveals the importance of the IRPs in innate immunity.

  14. Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling.

    PubMed

    Londino, James D; Gulick, Dexter; Isenberg, Jeffrey S; Mallampalli, Rama K

    2015-12-25

    Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling.

  15. Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling*

    PubMed Central

    Londino, James D.; Gulick, Dexter; Isenberg, Jeffrey S.; Mallampalli, Rama K.

    2015-01-01

    Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling. PMID:26534964

  16. Iron regulatory proteins and their role in controlling iron metabolism.

    PubMed

    Kühn, Lukas C

    2015-02-01

    Cellular iron homeostasis is regulated by post-transcriptional feedback mechanisms, which control the expression of proteins involved in iron uptake, release and storage. Two cytoplasmic proteins with mRNA-binding properties, iron regulatory proteins 1 and 2 (IRP1 and IRP2) play a central role in this regulation. Foremost, IRPs regulate ferritin H and ferritin L translation and thus iron storage, as well as transferrin receptor 1 (TfR1) mRNA stability, thereby adjusting receptor expression and iron uptake via receptor-mediated endocytosis of iron-loaded transferrin. In addition splice variants of iron transporters for import and export at the plasma-membrane, divalent metal transporter 1 (DMT1) and ferroportin are regulated by IRPs. These mechanisms have probably evolved to maintain the cytoplasmic labile iron pool (LIP) at an appropriate level. In certain tissues, the regulation exerted by IRPs influences iron homeostasis and utilization of the entire organism. In intestine, the control of ferritin expression limits intestinal iron absorption and, thus, whole body iron levels. In bone marrow, erythroid heme biosynthesis is coordinated with iron availability through IRP-mediated translational control of erythroid 5-aminolevulinate synthase mRNA. Moreover, the translational control of HIF2α mRNA in kidney by IRP1 coordinates erythropoietin synthesis with iron and oxygen supply. Besides IRPs, body iron absorption is negatively regulated by hepcidin. This peptide hormone, synthesized and secreted by the liver in response to high serum iron, downregulates ferroportin at the protein level and thereby limits iron absorption from the diet. Hepcidin will not be discussed in further detail here.

  17. Protein modularity, cooperative binding, and hybrid regulatory states underlie transcriptional network diversification.

    PubMed

    Baker, Christopher R; Booth, Lauren N; Sorrells, Trevor R; Johnson, Alexander D

    2012-09-28

    We examine how different transcriptional network structures can evolve from an ancestral network. By characterizing how the ancestral mode of gene regulation for genes specific to a-type cells in yeast species evolved from an activating paradigm to a repressing one, we show that regulatory protein modularity, conversion of one cis-regulatory sequence to another, distribution of binding energy among protein-protein and protein-DNA interactions, and exploitation of ancestral network features all contribute to the evolution of a novel regulatory mode. The formation of this derived mode of regulation did not disrupt the ancestral mode and thereby created a hybrid regulatory state where both means of transcription regulation (ancestral and derived) contribute to the conserved expression pattern of the network. Finally, we show how this hybrid regulatory state has resolved in different ways in different lineages to generate the diversity of regulatory network structures observed in modern species.

  18. AraC regulatory protein mutants with altered effector specificity.

    PubMed

    Tang, Shuang-Yan; Fazelinia, Hossein; Cirino, Patrick C

    2008-04-16

    The AraC regulatory protein of the Escherichia coli ara operon has been engineered to activate transcription in response to D-arabinose and not in response to its native effector L-arabinose. Two different AraC mutant libraries, each with four randomized binding pocket residues, were subjected to FACS-mediated dual screening using a GFP reporter. Both libraries yielded mutants with the desired switch in effector specificity, and one mutant we describe maintains tight repression in the absence of effector. The presence of 100 mM L-arabinose does not influence the response of the reported mutants to D-arabinose, and the mutants are not induced by other sugars tested (D-xylose, D-fucose, D-lyxose). Co-expression of the FucP transporter in E. coli enabled induction by D-arabinose in the 0.1 mM range. Our results demonstrate the power of dual screening for altering AraC inducer specificity and represent steps toward the design of customized in vivo molecular reporters and gene switches for metabolic engineering.

  19. Iron regulatory protein-1 protects against mitoferrin-1-deficient porphyria.

    PubMed

    Chung, Jacky; Anderson, Sheila A; Gwynn, Babette; Deck, Kathryn M; Chen, Michael J; Langer, Nathaniel B; Shaw, George C; Huston, Nicholas C; Boyer, Leah F; Datta, Sumon; Paradkar, Prasad N; Li, Liangtao; Wei, Zong; Lambert, Amy J; Sahr, Kenneth; Wittig, Johannes G; Chen, Wen; Lu, Wange; Galy, Bruno; Schlaeger, Thorsten M; Hentze, Matthias W; Ward, Diane M; Kaplan, Jerry; Eisenstein, Richard S; Peters, Luanne L; Paw, Barry H

    2014-03-14

    Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.

  20. 14-3-3 proteins: regulators of numerous eukaryotic proteins.

    PubMed

    van Heusden, G Paul H

    2005-09-01

    14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins.

  1. Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A

    PubMed Central

    Kang, Yoon-Suk; Brame, Keenan; Jetter, Jonathan; Bothner, Brian B.; Wang, Gejiao

    2016-01-01

    ABSTRACT ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCE Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR. This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate

  2. Yeast 14-3-3 proteins.

    PubMed

    van Heusden, G Paul H; Steensma, H Yde

    2006-02-01

    14-3-3 proteins form a family of highly conserved proteins which are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. They interact with more than 200 different, mostly phosphorylated proteins. The molecular consequences of 14-3-3 binding are diverse: this binding may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization, to the interaction with other proteins or to shielding of binding sites. The binding partners, and hence the 14-3-3 proteins, are involved in almost every cellular process and 14-3-3 proteins have been linked to several diseases, such as cancer, Alzheimer's disease, the neurological Miller-Dieker and spinocerebellar ataxia type 1 diseases and bovine spongiform encephalopathy (BSE). The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both have two genes encoding 14-3-3 proteins, BMH1 and BMH2 and rad24 and rad25, respectively. In these yeasts, 14-3-3 proteins are essential in most laboratory strains. As in higher eukaryotes, yeast 14-3-3 proteins bind to numerous proteins involved in a variety of cellular processes. Recent genome-wide studies on yeast strains with impaired 14-3-3 function support the participation of 14-3-3 proteins in numerous yeast cellular processes. Given the high evolutionary conservation of the 14-3-3 proteins, the experimental accessibility and relative simplicity of yeasts make them excellent model organisms for elucidating the function of the 14-3-3 protein family.

  3. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

    NASA Astrophysics Data System (ADS)

    Lloyd, David J.; St Jean, David J.; Kurzeja, Robert J. M.; Wahl, Robert C.; Michelsen, Klaus; Cupples, Rod; Chen, Michelle; Wu, John; Sivits, Glenn; Helmering, Joan; Komorowski, Renée; Ashton, Kate S.; Pennington, Lewis D.; Fotsch, Christopher; Vazir, Mukta; Chen, Kui; Chmait, Samer; Zhang, Jiandong; Liu, Longbin; Norman, Mark H.; Andrews, Kristin L.; Bartberger, Michael D.; van, Gwyneth; Galbreath, Elizabeth J.; Vonderfecht, Steven L.; Wang, Minghan; Jordan, Steven R.; Véniant, Murielle M.; Hale, Clarence

    2013-12-01

    Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

  4. Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP.

    PubMed

    Bader, G; Schiffmann, S; Herrmann, A; Fischer, M; Gütlich, M; Auerbach, G; Ploom, T; Bacher, A; Huber, R; Lemm, T

    2001-10-05

    Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory protein (GFRP). The 2.6 A resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the interaction of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins.

  5. Control of alternative splicing by signal-dependent degradation of splicing-regulatory proteins.

    PubMed

    Katzenberger, Rebeccah J; Marengo, Matthew S; Wassarman, David A

    2009-04-17

    Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to down-regulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins.

  6. 3 CFR - Regulatory Flexibility, Small Business, and Job Creation

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 3 The President 1 2012-01-01 2012-01-01 false Regulatory Flexibility, Small Business, and Job... Flexibility, Small Business, and Job Creation Memorandum for the Heads of Executive Departments and Agencies... 60 percent of all new jobs in the Nation. Although small businesses and new companies provide...

  7. Regulatory Elements in Vectors for Efficient Generation of Cell Lines Producing Target Proteins

    PubMed Central

    Maksimenko, O.; Gasanov, N. B.; Georgiev, P.

    2015-01-01

    To date, there has been an increasing number of drugs produced in mammalian cell cultures. In order to enhance the expression level and stability of target recombinant proteins in cell cultures, various regulatory elements with poorly studied mechanisms of action are used. In this review, we summarize and discuss the potential mechanisms of action of such regulatory elements. PMID:26483956

  8. Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80.

    PubMed Central

    Salmeron, J M; Langdon, S D; Johnston, S A

    1989-01-01

    In Saccharomyces cerevisiae, transcriptional activation mediated by the GAL4 regulatory protein is repressed in the absence of galactose by the binding of the GAL80 protein, an interaction that requires the carboxy-terminal 28 amino acids of GAL4. The homolog of GAL4 from Kluyveromyces lactis, LAC9, activates transcription in S. cerevisiae and is highly similar to GAL4 in its carboxyl terminus but is not repressed by wild-type levels of GAL80 protein. Here we show that GAL80 does repress LAC9-activated transcription in S. cerevisiae if overproduced. We sought to determine the molecular basis for the difference in the responses of the LAC9 and GAL4 proteins to GAL80. Our results indicate that this difference is due primarily to the fact that under wild-type conditions, the level of LAC9 protein in S. cerevisiae is much higher than that of GAL4, which suggests that LAC9 escapes GAL80-mediated repression by titration of GAL80 protein in vivo. The difference in response to GAL80 is not due to amino acid sequence differences between the LAC9 and GAL4 carboxyl termini. We discuss the implications of these results for the mechanism of galactose metabolism regulation in S. cerevisiae and K. lactis. Images PMID:2550790

  9. 75 FR 45172 - Withdrawal of Regulatory Guides 3.44 and 3.49

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    ...: Withdrawal of Regulatory Guide 3.44, ``Standard Format and Content for the Safety Analysis Report for an... withdrawing Regulatory Guide (RG) 3.44, ``Standard Format and Content for the Safety Analysis Report for an... Guide 3.44 provides guidance to applicants on the format and content of the safety analysis report...

  10. Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

    SciTech Connect

    Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

    1988-07-12

    The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity (/sup 3/H)fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity (/sup 3/H)fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.

  11. Exceptionally high heterologous protein levels in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences.

    PubMed

    De Jaeger, Geert; Angenon, Geert; Depicker, Ann

    2003-01-01

    Seeds are concentrated sources of protein and thus may be ideal 'bioreactors' for the production of heterologous proteins. For this application, strong seed-specific expression signals are required. A set of expression cassettes were designed using 5' and 3' regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) from Phaseolus vulgaris, and evaluated for the production of heterologous proteins in dicotyledonous plant species. A murine single-chain variable fragment (scFv) was chosen as model protein because of the current industrial interest to produce antibodies and derived fragments in crops. Because the highest scFv accumulation in seed had previously been achieved in the endoplasmic reticulum (ER), the scFv-encoding sequence was provided with signal sequences for accumulation in the ER. Transgenic Arabidopsis seed stocks, expressing the scFv under control of the 35S promoter, contained scFv accumulation levels in the range of 1% of total soluble protein (TSP). However, the seed storage promoter constructs boosted the scFv to exceptionally high levels. Maximum scFv levels were obtained in homozygous seed stocks, being 12.5% of TSP under control of the arc5-I regulatory sequences and even up to 36.5% of TSP upon replacing the arc5-I promoter by the beta-phaseolin promoter of Phaseolus vulgaris. Even at such very high levels, the scFv proteins retain their full antigen-binding activity. Moreover, the presence of very high scFv levels has only minory effects on seed germination and no effect on seed production. These results demonstrate that the expression levels of arcelin 5-I and beta-phaseolin seed storage protein genes can be transferred to heterologous proteins, giving exceptionally high levels of heterologous proteins, which can be of great value for the molecular farming industry by raising production yield and lowering bio-mass production and purification costs. Finally, the feasibility of heterologous protein production using the

  12. Functional Characterization of a Novel Pro-Apoptotic Transcription Regulatory Protein in Ovarian Cancer

    DTIC Science & Technology

    2006-12-01

    of establishing stable cell lines in ovarian cancer as stated above, this project awaits the establishment of tetracycline -inducible ovarian cancer ...W81XWH-04-1-0085 TITLE: Functional Characterization of a Novel Pro-Apoptotic Transcription Regulatory Protein in Ovarian Cancer ...Transcription Regulatory Protein in Ovarian Cancer 5b. GRANT NUMBER W81XWH-04-1-0085 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER

  13. Development of coagulation regulatory proteins in the fetal and neonatal lamb.

    PubMed

    Manco-Johnson, Marilyn J; Jacobson, Linda J; Hacker, Michele R; Townsend, Susan F; Murphy, James; Hay, William

    2002-10-01

    To investigate the development of coagulation regulatory proteins-protein C (PC), protein S (PS), and antithrombin (AT)-in relationship to the procoagulant protein factor X (FX), a chronically catheterized fetal ovine model was used. Infusion and sampling catheters were placed into pregnant ewes and their fetuses and maintained from mid-gestation. From a total of 110 fetuses, 17 lambs, and 63 ewes that were studied on one to 15 occasions, 212 fetal, 88 neonatal, and 157 maternal samples were obtained. Liver tissue was obtained from 31 fetuses and 15 ewes. Plasma levels of all proteins studied were higher in the ewe than in the fetus (p < 0.0001). Plasma levels of FX, PC, and PS achieved neonatal levels by mid-gestation with mild but significant decreases during mid- and late gestation. Fetal and early neonatal plasma concentrations of these vitamin K-dependent proteins fit a model with both quadratic (p < 0.01) and linear (p < 0.01) components. The discrepant levels in mRNA relative to plasma concentration were consistent with regulatory control beyond the level of transcription. In contrast, a simple linear increase in plasma protein levels was determined for the vitamin K-independent coagulation regulatory protein, AT (p for quadratic component > 0.05). This study suggests that fetal regulation of coagulation proteins follows characteristic patterns relative to the vitamin K dependence of the protein rather than its role as a procoagulant versus regulatory protein.

  14. Regulatory roles of Oct proteins in the mammary gland.

    PubMed

    Qian, Xi; Zhao, Feng-Qi

    2016-06-01

    The expression of Oct-1 and -2 and their binding to the octamer motif in the mammary gland are developmentally and hormonally regulated, consistent with the expression of milk proteins. Both of these transcription factors constitutively bind to the proximal promoter of the milk protein gene β-casein and might be involved in the inhibition or activation of promoter activity via interactions with other transcription factors or cofactors at different developmental stages. In particular, the lactogenic hormone prolactin and glucocorticoids induce Oct-1 and Oct-2 binding and interaction with both the signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid receptor on the β-casein promoter to activate β-casein expression. In addition, increasing evidence has shown the involvement of another Oct factor, Oct-3/4, in mammary tumorigenesis, making Oct-3/4 an emerging prognostic marker of breast cancer and a molecular target for the gene-directed therapeutic intervention, prevention and treatment of breast cancer. This article is part of a Special Issue entitled: The Oct Transcription Factor Family, edited by Dr. Dean Tantin.

  15. Foxp3-transduced polyclonal regulatory T cells protect against chronic renal injury from adriamycin.

    PubMed

    Wang, Yuan Min; Zhang, Geoff Yu; Wang, Yiping; Hu, Min; Wu, Huiling; Watson, Debbie; Hori, Shohei; Alexander, Ian E; Harris, David C H; Alexander, Stephen I

    2006-03-01

    Chronic proteinuric renal injury is a major cause of ESRD. Adriamycin nephropathy is a murine model of chronic proteinuric renal disease whereby chemical injury is followed by immune and structural changes that mimic human disease. Foxp3 is a gene that induces a regulatory T cell (Treg) phenotype. It was hypothesized that Foxp3-transduced Treg could protect against renal injury in Adriamycin nephropathy. CD4+ T cells were transduced with either a Foxp3-containing retrovirus or a control retrovirus. Foxp3-transduced T cells had a regulatory phenotype by functional and phenotypic assays. Adoptive transfer of Foxp3-transduced T cells protected against renal injury. Urinary protein excretion and serum creatinine were reduced (P<0.05), and there was significantly less glomerulosclerosis, tubular damage, and interstitial infiltrates (P<0.01). It is concluded that Foxp3-transduced Treg cells may have a therapeutic role in protecting against immune injury and disease progression in chronic proteinuric renal disease.

  16. Proliferation of transformed somatotroph cells related to low or absent expression of protein kinase a regulatory subunit 1A protein.

    PubMed

    Lania, Andrea G; Mantovani, Giovanna; Ferrero, Stefano; Pellegrini, Caterina; Bondioni, Sara; Peverelli, Erika; Braidotti, Paola; Locatelli, Marco; Zavanone, Mario L; Ferrante, Emanuela; Bosari, Silvano; Beck-Peccoz, Paolo; Spada, Anna

    2004-12-15

    The two regulatory subunits (R1 and R2) of protein kinase A (PKA) are differentially expressed in cancer cell lines and exert diverse roles in growth control. Recently, mutations of the PKA regulatory subunit 1A gene (PRKAR1A) have been identified in patients with Carney complex. The aim of this study was to evaluate the expression of the PKA regulatory subunits R1A, R2A, and R2B in a series of 30 pituitary adenomas and the effects of subunit activation on cell proliferation. In these tumors, neither mutation of PRKAR1A nor loss of heterozygosity was identified. By real-time PCR, mRNA of the three subunits was detected in all of the tumors, R1A being the most represented in the majority of samples. By contrast, immunohistochemistry documented low or absent R1A levels in all tumors, whereas R2A and R2B were highly expressed, thus resulting in an unbalanced R1/R2 ratio. The low levels of R1A were, at least in part, due to proteasome-mediated degradation. The effect of the R1/R2 ratio on proliferation was assessed in GH3 cells, which showed a similar unbalanced pattern of R subunits expression, and in growth hormone-secreting adenomas. The R2-selective cAMP analog 8-Cl cAMP and R1A RNA silencing, stimulated cell proliferation and increased Cyclin D1 expression, respectively, in human and rat adenomatous somatotrophs. These data show that a low R1/R2 ratio promoted proliferation of transformed somatotrophs and are consistent with the Carney complex model in which R1A inactivating mutations further unbalance this ratio in favor of R2 subunits. These results suggest that low expression of R1A protein may favor cAMP-dependent proliferation of transformed somatotrophs.

  17. [The contractile, regulatory and structural proteins of myocardium].

    PubMed

    Adamcová, Michaela; Pelouch, Václav

    2003-01-01

    The myocardium consists of three basic categories of proteins. The myofibrillar proteins trasform the chemical energy of ATP to the mechanical work of the heart. The metabolic proteins located both in the cytosol and in the mitochondrial compartments provide energy for the cardiac contraction. The interstitial space between myocytes is occupied by the extracellular proteins (collagens, glycoproteins, glycosaminoglycans, elastins). By far the greater percentage of myofibrillar proteins (about 80%) is that concerned with contraction (actin and myosin), with about 10% concerned with its regulation (troponin, tropomyosin and tropomodulin) and another 10% concerned with maintenance of the structure of myofibril (C, M-, H-proteins, myomesin, nebulette, alpha-actinin, titin, CapZ protein). Most collagenous and non-collagenous proteins exist in many isoforms that originate from the same genom but are the product of alternative splicing of a primary RNA transcript.

  18. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed

    Fujita, H; Ishiwata, S

    1998-09-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.

  19. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed Central

    Fujita, H; Ishiwata, S

    1998-01-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself. PMID:9726945

  20. Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

    PubMed

    Wang, Honglin; Sun, Yue; Chang, Jianhong; Zheng, Fangfang; Pei, Haixia; Yi, Yanjun; Chang, Caren; Dong, Chun-Hai

    2016-07-01

    Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling.

  1. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    SciTech Connect

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M.

    2011-11-02

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  2. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    SciTech Connect

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. ); Pandey, S. )

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  3. Protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for thrombus formation

    PubMed Central

    Bowley, Sheryl R.; Fang, Chao; Merrill-Skoloff, Glenn; Furie, Barbara C.; Furie, Bruce

    2017-01-01

    Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells on vascular injury, is required for thrombus formation. Using PDI variants that form mixed disulfide complexes with their substrates, we identify by kinetic trapping multiple substrate proteins, including vitronectin. Plasma vitronectin does not bind to αvβ3 or αIIbβ3 integrins on endothelial cells and platelets. The released PDI reduces disulfide bonds on plasma vitronectin, enabling vitronectin to bind to αVβ3 and αIIbβ3. In vivo studies of thrombus generation in mice demonstrate that vitronectin rapidly accumulates on the endothelium and the platelet thrombus following injury. This process requires PDI activity and promotes platelet accumulation and fibrin generation. We hypothesize that under physiologic conditions in the absence of secreted PDI, thrombus formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury may serve as a regulatory switch that allows activation of proteins, among them vitronectin, critical for thrombus formation. PMID:28218242

  4. The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability

    PubMed Central

    Moncini, Silvia; Bevilacqua, Annamaria; Venturin, Marco; Fallini, Claudia; Ratti, Antonia; Nicolin, Angelo; Riva, Paola

    2007-01-01

    Background CDK5R1 plays a central role in neuronal migration and differentiation during central nervous system development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of CDK5R1 expression. Results The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of CDK5R1 3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins. Conclusion Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3'-UTR and support the hypothesis that CDK5R1 gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of CDK5R1 expression. The fine tuning of CDK5R1 expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders. PMID:18053171

  5. A feedback regulatory loop between methyltransferase PRMT1 and orphan receptor TR3

    PubMed Central

    Lei, Na-zi; Zhang, Xiao-yan; Chen, Hang-zi; Wang, Yuan; Zhan, Yan-yan; Zheng, Zhong-hui; Shen, Yue-mao; Wu, Qiao

    2009-01-01

    PRMT1, an arginine methyltransferase, plays an important role in numerous cellular processes. In this study, we demonstrate a feedback regulatory loop between PRMT1 and the orphan receptor TR3. Unlike another orphan receptor HNF4, TR3 is not methylated by PRMT1 although they physically interact with each other. By delaying the TR3 protein degradation, PRMT1 binding leads to the elevation of TR3 cellular protein level, thereby enhances the DNA binding and transactivation activity of TR3 in a non-methyltransferase manner. Another coactivator SRC-2 acts synergistically with PRMT1 to regulate TR3 functions. In turn, TR3 binding to the catalytic domain of PRMT1 causes an inhibition of the PRMT1 methyltransferase activity. This repression results in the functional changes in some of PRMT1 substrates, including STAT3 and Sam68. The negative regulation of PRMT1 by TR3 was further confirmed in both TR3-knockdown cells and TR3-knockout mice with the use of an agonist for TR3. Taken together, our study not only identifies a regulatory role of PRMT1, independent on methyltransferase activity, in TR3 transactivation, but also characterizes a novel function of TR3 in the repression of PRMT1 methyltransferase activity. PMID:19095693

  6. 14-3-3 Proteins: insights from genome-wide studies in yeast.

    PubMed

    van Heusden, G Paul H

    2009-11-01

    14-3-3 proteins form a family of highly conserved, acidic, dimeric proteins. These proteins have been identified in all eukaryotic species investigated, often in multiple isoforms, up to 13 in the plant Arabidopsis thaliana. Hundreds of proteins, from diverse eukaryotic organisms, implicated in numerous cellular processes, have been identified as binding partners of 14-3-3 proteins. Therefore, the major activity of 14-3-3 proteins seems to be its ability to bind other intracellular proteins. Binding to 14-3-3 proteins may result in a conformational change of the protein required for its full activity or for inhibition of its activity, in interaction between two binding partners or in a different subcellular localization. Most of these interactions take place after phosphorylation of the binding partners. These observations suggest a major role of 14-3-3 proteins in regulatory networks. Here, the information on 14-3-3 proteins gathered from several genome- and proteome-wide studies in the yeast Saccharomyces cerevisiae is reviewed. In particular, the protein kinases responsible for the phosphorylation of 14-3-3 binding partners, phosphorylation of 14-3-3 proteins themselves, the transcriptional regulation of the 14-3-3 genes, and the role of 14-3-3 proteins in transcription are addressed. These large scale studies may help understand the function of 14-3-3 proteins at a cellular level rather than at the level of a single process.

  7. Interferon regulatory factor 3 in adaptive immune responses.

    PubMed

    Ysebrant de Lendonck, Laure; Martinet, Valerie; Goriely, Stanislas

    2014-10-01

    Interferon regulatory factor (IRF) 3 plays a key role in innate responses against viruses. Indeed, activation of this transcription factor triggers the expression of type I interferons and downstream interferon-stimulated genes in infected cells. Recent evidences indicate that this pathway also modulates adaptive immune responses. This review focuses on the different mechanisms that are implicated in this process. We discuss the role of IRF3 within antigen-presenting cells and T lymphocytes in the polarization of the cellular immune response and its implication in the pathogenesis of immune disorders.

  8. DTDP-rhamnosyl transferase RfbF, is a newfound receptor-related regulatory protein for phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3.

    PubMed

    Liang, Junrong; Li, Xu; Zha, Tao; Chen, Yuhuang; Hao, Huijing; Liu, Chang; Duan, Ran; Xiao, Yuchun; Su, Mingming; Wang, Xin; Jing, Huaiqi

    2016-03-11

    Bacteriophages and their hosts are continuously engaged in evolutionary competition. Here we isolated a lytic phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3. We firstly described the phage receptor was regulated by DTDP-rhamnosyl transferase RfbF, encoded within the rfb cluster that was responsible for the biosynthesis of the O antigens. The deletion of DTDP-rhamnosyl transferase RfbF of wild type O:3 strain caused failure in phiYe-F10 adsorption; however, the mutation strain retained agglutination with O:3 antiserum; and complementation of its mutant converted its sensitivity to phiYe-F10. Therefore, DTDP-rhamnosyl transferase RfbF was responsible for the phage infection but did not affect recognition of Y. enterocolitica O:3 antiserum. Further, the deletions in the putative O-antigen biosynthesis protein precursor and outer membrane protein had no effect on sensitivity to phiYe-F10 infection. However, adsorption of phages onto mutant HNF10-ΔO-antigen took longer time than onto the WT, suggesting that deletion of the putative O-antigen biosynthesis protein precursor reduced the infection efficiency.

  9. Catecholamine Stress Hormones Regulate Cellular Iron Homeostasis by a Posttranscriptional Mechanism Mediated by Iron Regulatory Protein

    PubMed Central

    Tapryal, Nisha; Vivek G, Vishnu; Mukhopadhyay, Chinmay K.

    2015-01-01

    Adequate availability of iron is important for cellular energy metabolism. Catecholamines such as epinephrine and norepinephrine promote energy expenditure to adapt to conditions that arose due to stress. To restore the energy balance, epinephrine/norepinephrine-exposed cells may face higher iron demand. So far, no direct role of epinephrine/norepinephrine in cellular iron homeostasis has been reported. Here we show that epinephrine/norepinephrine regulates iron homeostasis components such as transferrin receptor-1 and ferritin-H in hepatic and skeletal muscle cells by promoting the binding of iron regulatory proteins to iron-responsive elements present in the UTRs of transferrin receptor-1 and ferritin-H transcripts. Increased transferrin receptor-1, decreased ferritin-H, and increased iron-responsive element-iron regulatory protein interaction are also observed in liver and muscle tissues of epinephrine/norepinephrine-injected mice. We demonstrate the role of epinephrine/norepinephrine-induced generation of reactive oxygen species in converting cytosolic aconitase (ACO1) into iron regulatory protein-1 to bind iron-responsive elements present in UTRs of transferrin receptor-1 and ferritin-H. Our study further reveals that mitochondrial iron content and mitochondrial aconitase (ACO2) activity are elevated by epinephrine/norepinephrine that are blocked by the antioxidant N-acetyl cysteine and iron regulatory protein-1 siRNA, suggesting involvement of reactive oxygen species and iron regulatory protein-1 in this mechanism. This study reveals epinephrine and norepinephrine as novel regulators of cellular iron homeostasis. PMID:25572399

  10. Complement regulatory protein genes in channel catfish and their involvement in disease defense response.

    PubMed

    Jiang, Chen; Zhang, Jiaren; Yao, Jun; Liu, Shikai; Li, Yun; Song, Lin; Li, Chao; Wang, Xiaozhu; Liu, Zhanjiang

    2015-11-01

    Complement system is one of the most important defense systems of innate immunity, which plays a crucial role in disease defense responses in channel catfish. However, inappropriate and excessive complement activation could lead to potential damage to the host cells. Therefore the complement system is controlled by a set of complement regulatory proteins to allow normal defensive functions, but prevent hazardous complement activation to host tissues. In this study, we identified nine complement regulatory protein genes from the channel catfish genome. Phylogenetic and syntenic analyses were conducted to determine their orthology relationships, supporting their correct annotation and potential functional inferences. The expression profiles of the complement regulatory protein genes were determined in channel catfish healthy tissues and after infection with the two main bacterial pathogens, Edwardsiella ictaluri and Flavobacterium columnare. The vast majority of complement regulatory protein genes were significantly regulated after bacterial infections, but interestingly were generally up-regulated after E. ictaluri infection while mostly down-regulated after F. columnare infection, suggesting a pathogen-specific pattern of regulation. Collectively, these findings suggested that complement regulatory protein genes may play complex roles in the host immune responses to bacterial pathogens in channel catfish.

  11. A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway.

    PubMed

    Bhattacharya, Bonhi S; Sweby, Peter K; Minihane, Anne-Marie; Jackson, Kim G; Tindall, Marcus J

    2014-05-21

    Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in a hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main regulator of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homeostasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

  12. A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

    PubMed Central

    Bhattacharya, Bonhi S.; Sweby, Peter K.; Minihane, Anne-Marie; Jackson, Kim G.; Tindall, Marcus J.

    2014-01-01

    Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in a hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main regulator of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homeostasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature. PMID:24444765

  13. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

    SciTech Connect

    Mao, Grace; Brody, James P.

    2007-11-09

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s{sup -1}. We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.

  14. Cleavage of Interferon Regulatory Factor 7 by Enterovirus 71 3C Suppresses Cellular Responses

    PubMed Central

    Lei, Xiaobo; Xiao, Xia; Xue, Qinghua; Jin, Qi

    2013-01-01

    Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses. PMID:23175366

  15. Regulatory Oversight Program, July 1, 1993--March 3, 1997. Volume 3

    SciTech Connect

    1997-12-31

    On July, 1993, a Regulatory Oversight (RO) organization was established within the US DOE, Oak Ridge Operations (ORO) to provide regulatory oversight of the DOE uranium enrichment facilities leased to the United States Enrichment Corporation (USEC). The purpose of the OR program was to ensure continued plant safety, safeguards and security while the plants were transitioned to regulatory oversight by the Nuclear Regulatory Commission (NRC). Volume 3 contains copies of two reports that document the DOE/ORO regulatory oversight inspection and enforcement history for each gaseous diffusion plant site. Each report provides a formal mechanism by which DOE/ORO could communicate the inspection and enforcement history to NRC. The reports encompass the inspection activities that occurred during July 1, 1993 through March 2, 1997.

  16. Glucokinase regulatory proten genetic variant interacts with omega-3 PUFA to influence insulin resistance and inflammation in metabolic syndrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucokinase Regulatory Protein (GCKR) plays a central role regulating both hepatic triglyceride and glucose metabolism. Fatty acids are key metabolic regulators, which interact with genetic factors and influence glucose metabolism and other metabolic traits. Omega-3 polyunsaturated fatty acids (n-3...

  17. Calyculin A Reveals Serine/Threonine Phosphatase Protein Phosphatase 1 as a Regulatory Nodal Point in Canonical Signal Transducer and Activator of Transcription 3 Signaling of Human Microvascular Endothelial Cells

    PubMed Central

    Zgheib, Carlos; Zouein, Fouad A.; Chidiac, Rony; Kurdi, Mazen

    2012-01-01

    Vascular inflammation is initiated by stimuli acting on endothelial cells. A clinical feature of vascular inflammation is increased circulating interleukin 6 (IL-6) type cytokines such as leukemia inhibitory factor (LIF), but their role in vascular inflammation is not fully defined. IL-6 type cytokines activate transcription factor signal transducer and activator of transcription 3 (STAT3), which has a key role in inflammation and the innate immune response. Canonical STAT3 gene induction is due to phosphorylation of (1) Y705, leading to STAT3 dimerization and DNA binding and (2) S727, enhancing homodimerization and DNA binding by recruiting p300/CBP. We asked whether enhancing S727 STAT3 phosphorylation using the protein phosphatase 1 (PP1) inhibitor, calyculin A, would enhance LIF-induced gene expression in human microvascular endothelial cells (HMEC-1). Cotreatment with calyculin A and LIF markedly increased STAT3 S727 phosphorylation, without affecting the increase in the nuclear fraction of STAT3 phosphorylated on Y705. PP2A inhibitors, okadaic acid and fostriecin, did not enhance STAT3 S727 phosphorylation. Surprisingly, calyculin A eliminated LIF-induced gene expression: (1) calyculin A reduced binding of nuclear extracts to a STAT3 consensus site, thereby reducing the overall level of binding observed with LIF; and (2) calyculin A caused p300/CBP phosphorylation, thus resulting in reduced acetylation activity and degradation. Together, these findings reveal a pivotal role of a protein serine/threonine phosphatases that is likely PP1 in HMEC in controlling STAT3 transcriptional activity. PMID:22142222

  18. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

    PubMed

    Yu, Chunxiao; Lopez, Carlos A; Hu, Han; Xia, Yu; Freedman, David S; Reddington, Alexander P; Daaboul, George G; Unlü, M Selim; Genco, Caroline Attardo

    2014-01-01

    The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  19. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  20. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  1. Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts.

    PubMed

    Nuez, B; Rojo, F; Salas, M

    1992-12-01

    Transcription from the late promoter, PA3, of Bacillus subtilis phage phi 29 is activated by the viral regulatory protein p4. A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a closed complex without significantly affecting further steps of the initiation process. Electrophoretic band-shift assays performed with a DNA fragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA. Similarly, when a DNA fragment containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binding of RNA polymerase to the DNA. These results strongly suggest that p4 and RNA polymerase contact each other at the PA3 promoter. In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process.

  2. Modelling gene and protein regulatory networks with answer set programming.

    PubMed

    Fayruzov, Timur; Janssen, Jeroen; Vermeir, Dirk; Cornelis, Chris; De Cock, Martine

    2011-01-01

    Recently, many approaches to model regulatory networks have been proposed in the systems biology domain. However, the task is far from being solved. In this paper, we propose an Answer Set Programming (ASP)-based approach to model interaction networks. We build a general ASP framework that describes the network semantics and allows modelling specific networks with little effort. ASP provides a rich and flexible toolbox that allows expanding the framework with desired features. In this paper, we tune our framework to mimic Boolean network behaviour and apply it to model the Budding Yeast and Fission Yeast cell cycle networks. The obtained steady states of these networks correspond to those of the Boolean networks.

  3. Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

    SciTech Connect

    Shetty, Nishant D.; Reddy, Manchi C.M.; Palaninathan, Satheesh K.; Owen, Joshua L.; Sacchettini, James C.

    2010-10-11

    PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 {angstrom} resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T-loop of one subunit and the C-loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T-loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T-loop makes direct contact with the {gamma}-phosphate of the ATP molecule replacing the Mg{sup 2+} position seen in the Methanococcus jannaschii GlnK1 structure. The C-loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C-terminal 3{sub 10} helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.

  4. 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

    PubMed Central

    Bolton, Diane L; Barnitz, Robert A; Sakai, Keiko; Lenardo, Michael J

    2008-01-01

    Background Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G2,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade. Results We found that in human T cells, 14-3-3 plays an active role in mediating Vpr-induced cell cycle arrest and reveal a dramatic increase in the amount of Cdk1, Cdc25C, and CyclinB1 bound to 14-3-3 θ during Vprv-induced G2,M arrest. By contrast, a cell-cycle-arrest-dead Vpr mutant failed to augment 14-3-3 θ association with Cdk1 and CyclinB1. Moreover, G2,M arrest caused by HIV-1 infection strongly correlated with a disruption in 14-3-3 θ binding to centrosomal proteins, Plk1 and centrin. Finally, Vpr caused elevated levels of CyclinB1, Plk1, and Cdk1 in a complex with the nuclear transport and spindle assembly protein, importin β. Conclusion Thus, our data reveal a new facet of Vpr-induced cell cycle arrest involving previously unrecognized abnormal rearrangements of multiprotein assemblies containing key cell cycle regulatory proteins. Reviewers This article was reviewed by David Kaplan, Nathaniel R. Landau and Yan Zhou. PMID:18445273

  5. Import of Soluble Proteins into Chloroplasts and Potential Regulatory Mechanisms

    PubMed Central

    Sjuts, Inga; Soll, Jürgen; Bölter, Bettina

    2017-01-01

    Chloroplasts originated from an endosymbiotic event in which a free-living cyanobacterium was engulfed by an ancestral eukaryotic host. During evolution the majority of the chloroplast genetic information was transferred to the host cell nucleus. As a consequence, proteins formerly encoded by the chloroplast genome are now translated in the cytosol and must be subsequently imported into the chloroplast. This process involves three steps: (i) cytosolic sorting procedures, (ii) binding to the designated receptor-equipped target organelle and (iii) the consecutive translocation process. During import, proteins have to overcome the two barriers of the chloroplast envelope, namely the outer envelope membrane (OEM) and the inner envelope membrane (IEM). In the majority of cases, this is facilitated by two distinct multiprotein complexes, located in the OEM and IEM, respectively, designated TOC and TIC. Plants are constantly exposed to fluctuating environmental conditions such as temperature and light and must therefore regulate protein composition within the chloroplast to ensure optimal functioning of elementary processes such as photosynthesis. In this review we will discuss the recent models of each individual import stage with regard to short-term strategies that plants might use to potentially acclimate to changes in their environmental conditions and preserve the chloroplast protein homeostasis. PMID:28228773

  6. In Vitro Treatment of Human Monocytes/Macrophages with Myristoylated Recombinant Nef of Human Immunodeficiency Virus Type 1 Leads to the Activation of Mitogen-Activated Protein Kinases, IκB Kinases, and Interferon Regulatory Factor 3 and to the Release of Beta Interferon▿

    PubMed Central

    Mangino, Giorgio; Percario, Zulema A.; Fiorucci, Gianna; Vaccari, Gabriele; Manrique, Santiago; Romeo, Giovanna; Federico, Maurizio; Geyer, Matthias; Affabris, Elisabetta

    2007-01-01

    The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-κB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the α and β subunits of the IκB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation. PMID:17182689

  7. Pestivirus Npro Directly Interacts with Interferon Regulatory Factor 3 Monomer and Dimer

    PubMed Central

    Holthauzen, Luis Marcelo F.; Ruggli, Nicolas

    2016-01-01

    ABSTRACT Interferon regulatory factor 3 (IRF3) is a transcription factor involved in the activation of type I alpha/beta interferon (IFN-α/β) in response to viral infection. Upon viral infection, the IRF3 monomer is activated into a phosphorylated dimer, which induces the transcription of interferon genes in the nucleus. Viruses have evolved several ways to target IRF3 in order to subvert the innate immune response. Pestiviruses, such as classical swine fever virus (CSFV), target IRF3 for ubiquitination and subsequent proteasomal degradation. This is mediated by the viral protein Npro that interacts with IRF3, but the molecular details for this interaction are largely unknown. We used recombinant Npro and IRF3 proteins and show that Npro interacts with IRF3 directly without additional proteins and forms a soluble 1:1 complex. The full-length IRF3 but not merely either of the individual domains is required for this interaction. The interaction between Npro and IRF3 is not dependent on the activation state of IRF3, since Npro binds to a constitutively active form of IRF3 in the presence of its transcriptional coactivator, CREB-binding protein (CBP). The results indicate that the Npro-binding site on IRF3 encompasses a region that is unperturbed by the phosphorylation and subsequent activation of IRF3 and thus excludes the dimer interface and CBP-binding site. IMPORTANCE The pestivirus N-terminal protease, Npro, is essential for evading the host's immune system by facilitating the degradation of interferon regulatory factor 3 (IRF3). However, the nature of the Npro interaction with IRF3, including the IRF3 species (inactive monomer versus activated dimer) that Npro targets for degradation, is largely unknown. We show that classical swine fever virus Npro and porcine IRF3 directly interact in solution and that full-length IRF3 is required for interaction with Npro. Additionally, Npro interacts with a constitutively active form of IRF3 bound to its transcriptional

  8. The potential function of steroid sulphatase activity in steroid production and steroidogenic acute regulatory protein expression.

    PubMed Central

    Sugawara, Teruo; Fujimoto, Seiichiro

    2004-01-01

    The first step in the biosynthesis of steroid hormones is conversion of cholesterol into pregnenolone. StAR (steroidogenic acute regulatory) protein plays a crucial role in the intra-mitochondrial movement of cholesterol. STS (steroid sulphatase), which is present ubiquitously in mammalian tissues, including the placenta, adrenal gland, testis and ovary, desulphates a number of 3beta-hydroxysteroid sulphates, including cholesterol sulphate. The present study was designed to examine the effect of STS on StAR protein synthesis and steroidogenesis in cells. Steroidogenic activities of COS-1 cells that had been co-transfected with a vector for the cholesterol P450scc (cytochrome P450 side-chain-cleavage enzyme) system, named F2, a StAR expression vector (pStAR), and an STS expression vector (pSTS) were assayed. Whole-cell extracts were subjected to SDS/PAGE and then to Western blot analysis. pSTS co-expressed in COS-1 cells with F2 and pStAR increased pregnenolone synthesis 2-fold compared with that of co-expression with F2 and pStAR. Western blot analysis using COS-1 cells that had been co-transfected with pSTS, F2 and pStAR revealed that StAR protein levels increased, whereas STS and P450scc protein levels did not change. The amount of StAR protein translation products increased when pSTS was added to an in vitro transcription-translation reaction mixture. Pulse-chase experiments demonstrated that the 37 kDa StAR pre-protein disappeared significantly ( P <0.01) more slowly in COS-1 cells that had been transfected with pSTS than in COS-1 cells that had not been transfected with pSTS. The increase in StAR protein level is not a result of an increase in StAR gene expression, but is a result of both an increase in translation and a longer half-life of the 37 kDa pre-StAR protein. In conclusion, STS increases StAR protein expression level and stimulates steroid production. PMID:14969586

  9. Evolutionary adaptation of an AraC-like regulatory protein in Citrobacter rodentium and Escherichia species.

    PubMed

    Tan, Aimee; Petty, Nicola K; Hocking, Dianna; Bennett-Wood, Vicki; Wakefield, Matthew; Praszkier, Judyta; Tauschek, Marija; Yang, Ji; Robins-Browne, Roy

    2015-04-01

    The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogen Citrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and the grlRA operon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genus Escherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated that regA has been horizontally transferred to Escherichia spp. and C. rodentium. Comparative studies of two RegA homologues, namely, those from C. rodentium and E. coli SMS-3-5, a multiresistant environmental strain of E. coli, showed that the two regulators acted similarly in vitro but differed in terms of their abilities to activate the virulence of C. rodentium in vivo, which evidently was due to their differential activation of grlRA. Our data indicate that RegA from C. rodentium has strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence by C. rodentium and on the evolution of virulence-regulatory genes of bacterial pathogens in general.

  10. Evolutionary Adaptation of an AraC-Like Regulatory Protein in Citrobacter rodentium and Escherichia Species

    PubMed Central

    Tan, Aimee; Petty, Nicola K.; Hocking, Dianna; Bennett-Wood, Vicki; Wakefield, Matthew; Praszkier, Judyta; Tauschek, Marija; Yang, Ji

    2015-01-01

    The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogen Citrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and the grlRA operon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genus Escherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated that regA has been horizontally transferred to Escherichia spp. and C. rodentium. Comparative studies of two RegA homologues, namely, those from C. rodentium and E. coli SMS-3-5, a multiresistant environmental strain of E. coli, showed that the two regulators acted similarly in vitro but differed in terms of their abilities to activate the virulence of C. rodentium in vivo, which evidently was due to their differential activation of grlRA. Our data indicate that RegA from C. rodentium has strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence by C. rodentium and on the evolution of virulence-regulatory genes of bacterial pathogens in general. PMID:25624355

  11. H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex.

    PubMed

    Bowman, Sarah K; Deaton, Aimee M; Domingues, Heber; Wang, Peggy I; Sadreyev, Ruslan I; Kingston, Robert E; Bender, Welcome

    2014-07-31

    The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a 'stairstep' pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity.

  12. Iron regulatory protein-2 knockout increases perihematomal ferritin expression and cell viability after intracerebral hemorrhage.

    PubMed

    Chen, Mai; Awe, Olatilewa O; Chen-Roetling, Jing; Regan, Raymond F

    2010-06-14

    Iron is deposited in perihematomal tissue after an intracerebral hemorrhage (ICH), and may contribute to oxidative injury. Cell culture studies have demonstrated that enhancing ferritin expression by targeting iron regulatory protein (IRP) binding activity reduces cellular vulnerability to iron and hemoglobin. In order to assess the therapeutic potential of this approach after striatal ICH, the effect of IRP1 or IRP2 gene knockout on ferritin expression and injury was quantified. Striatal ferritin in IRP1 knockout mice was similar to that in wild-type controls 3 days after stereotactic injection of artificial CSF or autologous blood. Corresponding levels in IRP2 knockouts were increased by 11-fold and 8.4-fold, respectively, compared with wild-type. Protein carbonylation, a sensitive marker of hemoglobin neurotoxicity, was increased by 2.4-fold in blood-injected wild-type striata, was not altered by IRP1 knockout, but was reduced by approximately 60% by IRP2 knockout. Perihematomal cell viability in wild-type mice, assessed by MTT assay, was approximately half of that in contralateral striata at 3 days, and was significantly increased in IRP2 knockouts but not in IRP1 knockouts. Protection was also observed when hemorrhage was induced by collagenase injection. These results suggest that IRP2 binding activity reduces ferritin expression in the striatum after ICH, preventing an optimal response to elevated local iron concentrations. IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries.

  13. Structure of the 'Escherichia Coli' Leucine-Responsive Regulatory Protein Lrp Reveals a Novel Octameric Assembly

    SciTech Connect

    de los Rios, S.; Perona, J.J.; /UC, Santa Barbara

    2007-07-09

    The structure of Escherichia coli leucine-responsive regulatory protein (Lrp) cocrystallized with a short duplex oligodeoxynucleotide reveals a novel quaternary assembly in which the protein octamer forms an open, linear array of four dimers. In contrast, structures of the Lrp homologs LrpA, LrpC and AsnC crystallized in the absence of DNA show that these proteins instead form highly symmetrical octamers in which the four dimers form a closed ring. Although the DNA is disordered within the Lrp crystal, comparative analyses suggest that the observed differences in quaternary state may arise from DNA interactions during crystallization. Interconversion of these conformations, possibly in response to DNA or leucine binding, provides an underlying mechanism to alter the relative spatial orientation of the DNA-binding domains. Breaking of the closed octamer symmetry may be a common essential step in the formation of active DNA complexes by all members of the Lrp/AsnC family of transcriptional regulatory proteins.

  14. The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1

    PubMed Central

    Lai, Tai-Yu; Yang, Yu-San; Hong, Wei-Fu; Chiang, Chi-Wu

    2016-01-01

    The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role. PMID:26684356

  15. Differential effects of thin and thick filament disruption on zebrafish smooth muscle regulatory proteins

    PubMed Central

    Davuluri, G.; Seiler, C.; Abrams, J.; Soriano, A. J.; Pack, M.

    2013-01-01

    Background The smooth muscle actin binding proteins Caldesmon and Tropomyosin (Tm) promote thin filament assembly by stabilizing actin polymerization, however, whether filament assembly affects either the stability or activation of these and other smooth muscle regulatory proteins is not known. Methods Measurement of smooth muscle regulatory protein levels in wild type zebrafish larvae following antisense knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins, and in colourless mutants that lack enteric nerves. Comparison of intestinal peristalsis in wild type and colourless larvae. Key Results Knockdown of Acta2 led to reduced levels of phospho-Caldesmon and Tm. Total Caldesmon and phospho-myosin light chain (p-Mlc) levels were unaffected. Knockdown of Myh11 had no effect on the levels of either of these proteins. Phospho-Caldesmon and p-Mlc levels were markedly reduced in colourless mutants that have intestinal motility comparable with wild type larvae. Conclusions & Inferences These in vivo findings provide new information regarding the activation and stability of smooth muscle regulatory proteins in zebrafish larvae and their role in intestinal peristalsis in this model organism. PMID:20591105

  16. Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation.

    PubMed

    Gibson, Candace; Schanen, Brian; Chakrabarti, Debopam; Chakrabarti, Ratna

    2006-06-01

    To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3'5'-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation.

  17. 18 CFR 367.1823 - Account 182.3, Other regulatory assets.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... GAS ACT Balance Sheet Chart of Accounts Deferred Debits § 367.1823 Account 182.3, Other regulatory... regulatory assets. 367.1823 Section 367.1823 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY REGULATIONS UNDER THE PUBLIC UTILITY HOLDING COMPANY ACT OF...

  18. Transmembrane AMPA receptor regulatory protein (TARP) dysregulation in anterior cingulate cortex in schizophrenia.

    PubMed

    Drummond, Jana B; Tucholski, Janusz; Haroutunian, Vahram; Meador-Woodruff, James H

    2013-06-01

    The glutamate hypothesis of schizophrenia proposes that abnormal glutamatergic neurotransmission occurs in this illness, and a major contribution may involve dysregulation of the AMPA subtype of ionotropic glutamate receptor (AMPAR). Transmembrane AMPAR regulatory proteins (TARPs) form direct associations with AMPARs to modulate the trafficking and biophysical functions of these receptors, and their dysregulation may alter the localization and activity of AMPARs, thus having a potential role in the pathophysiology of schizophrenia. We performed comparative quantitative real-time PCR and Western blot analysis to measure transcript (schizophrenia, N=25; comparison subjects, N=25) and protein (schizophrenia, N=36; comparison subjects, N=33) expression of TARPs (γ subunits 1-8) in the anterior cingulate cortex (ACC) in schizophrenia and a comparison group. TARP expression was also measured in frontal cortex of rats chronically treated with haloperidol decanoate (28.5mg/kg every three weeks for nine months) to determine the effect of antipsychotic treatment on the expression of these molecules. We found decreased transcript expression of TARP γ-8 in schizophrenia. At the protein level, γ-3 and γ-5 were increased, while γ-4, γ-7 and γ-8 were decreased in schizophrenia. No changes in any of the molecules were noted in the frontal cortex of haloperidol-treated rats. TARPs are abnormally expressed at transcript and protein levels in ACC in schizophrenia, and these changes are likely due to the illness and not to the antipsychotic treatment. Alterations in the expression of TARPs may contribute to the pathophysiology of schizophrenia, and represent a potential mechanism of glutamatergic dysregulation in this illness.

  19. Transmembrane AMPA receptor regulatory protein (TARP) dysregulation in anterior cingulate cortex in schizophrenia

    PubMed Central

    Drummond, Jana B.; Tucholski, Janusz; Haroutunian, Vahram; Meador-Woodruff, James H.

    2013-01-01

    The glutamate hypothesis of schizophrenia proposes that abnormal glutamatergic neurotransmission occurs in this illness, and a major contribution may involve dysregulation of the AMPA subtype of ionotropic glutamate receptor (AMPAR). Transmembrane AMPAR regulatory proteins (TARPs) form direct associations with AMPARs to modulate the trafficking and biophysical functions of these receptors, and their dysregulation may alter the localization and activity of AMPARs, thus having a potential role in the pathophysiology of schizophrenia. We performed comparative quantitative real-time PCR and Western blot analysis to measure transcript (schizophrenia, N = 25; comparison subjects, N = 25) and protein (schizophrenia, N = 36; comparison subjects, N = 33) expression of TARPs (γ subunits 1-8) in the anterior cingulate cortex (ACC) in schizophrenia and a comparison group. TARP expression was also measured in frontal cortex of rats chronically treated with haloperidol decanoate (28.5 mg/kg every three weeks for nine months) to determine the effect of antipsychotic treatment on the expression of these molecules. We found decreased transcript expression of TARP γ-8 in schizophrenia. At the protein level, γ-3 and γ-5 were increased, while γ-4, γ-7 and γ-8 were decreased in schizophrenia. No changes in any of the molecules were noted in the frontal cortex of haloperidol-treated rats. TARPs are abnormally expressed at transcript and protein levels in ACC in schizophrenia, and these changes are likely due to the illness and not antipsychotic treatment. Alterations in the expression of TARPs may contribute to the pathophysiology of schizophrenia, and represent a potential mechanism of glutamatergic dysregulation in this illness. PMID:23566497

  20. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  1. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

    PubMed Central

    Garbers, C; DeLong, A; Deruére, J; Bernasconi, P; Söll, D

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis. Images PMID:8641277

  2. UbiNet: an online resource for exploring the functional associations and regulatory networks of protein ubiquitylation

    PubMed Central

    Nguyen, Van-Nui; Huang, Kai-Yao; Weng, Julia Tzu-Ya; Lai, K. Robert; Lee, Tzong-Yi

    2016-01-01

    Protein ubiquitylation catalyzed by E3 ubiquitin ligases are crucial in the regulation of many cellular processes. Owing to the high throughput of mass spectrometry-based proteomics, a number of methods have been developed for the experimental determination of ubiquitylation sites, leading to a large collection of ubiquitylation data. However, there exist no resources for the exploration of E3-ligase-associated regulatory networks of for ubiquitylated proteins in humans. Therefore, the UbiNet database was developed to provide a full investigation of protein ubiquitylation networks by incorporating experimentally verified E3 ligases, ubiquitylated substrates and protein–protein interactions (PPIs). To date, UbiNet has accumulated 43 948 experimentally verified ubiquitylation sites from 14 692 ubiquitylated proteins of humans. Additionally, we have manually curated 499 E3 ligases as well as two E1 activating and 46 E2 conjugating enzymes. To delineate the regulatory networks among E3 ligases and ubiquitylated proteins, a total of 430 530 PPIs were integrated into UbiNet for the exploration of ubiquitylation networks with an interactive network viewer. A case study demonstrated that UbiNet was able to decipher a scheme for the ubiquitylation of tumor proteins p63 and p73 that is consistent with their functions. Although the essential role of Mdm2 in p53 regulation is well studied, UbiNet revealed that Mdm2 and additional E3 ligases might be implicated in the regulation of other tumor proteins by protein ubiquitylation. Moreover, UbiNet could identify potential substrates for a specific E3 ligase based on PPIs and substrate motifs. With limited knowledge about the mechanisms through which ubiquitylated proteins are regulated by E3 ligases, UbiNet offers users an effective means for conducting preliminary analyses of protein ubiquitylation. The UbiNet database is now freely accessible via http://csb.cse.yzu.edu.tw/UbiNet/. The content is regularly updated with the

  3. Mining 3D genome structure populations identifies major factors governing the stability of regulatory communities

    PubMed Central

    Dai, Chao; Li, Wenyuan; Tjong, Harianto; Hao, Shengli; Zhou, Yonggang; Li, Qingjiao; Chen, Lin; Zhu, Bing; Alber, Frank; Jasmine Zhou, Xianghong

    2016-01-01

    Three-dimensional (3D) genome structures vary from cell to cell even in an isogenic sample. Unlike protein structures, genome structures are highly plastic, posing a significant challenge for structure-function mapping. Here we report an approach to comprehensively identify 3D chromatin clusters that each occurs frequently across a population of genome structures, either deconvoluted from ensemble-averaged Hi-C data or from a collection of single-cell Hi-C data. Applying our method to a population of genome structures (at the macrodomain resolution) of lymphoblastoid cells, we identify an atlas of stable inter-chromosomal chromatin clusters. A large number of these clusters are enriched in binding of specific regulatory factors and are therefore defined as ‘Regulatory Communities.' We reveal two major factors, centromere clustering and transcription factor binding, which significantly stabilize such communities. Finally, we show that the regulatory communities differ substantially from cell to cell, indicating that expression variability could be impacted by genome structures. PMID:27240697

  4. Iron regulatory proteins control a mucosal block to intestinal iron absorption.

    PubMed

    Galy, Bruno; Ferring-Appel, Dunja; Becker, Christiane; Gretz, Norbert; Gröne, Hermann-Josef; Schümann, Klaus; Hentze, Matthias W

    2013-03-28

    Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs) that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated "mucosal block." We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin "mucosal block" and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.

  5. Structural and biological features of FOXP3 dimerization relevant to regulatory T cell function

    PubMed Central

    Song, Xiaomin; Li, Bin; Xiao, Yan; Chen, Chunxia; Wang, Qiang; Liu, Yujie; Berezov, Alan; Xu, Chen; Gao, Yayi; Wu, Shiaw-Lin; Zhang, Hongtao; Karger, Barry L.; Hancock, Wayne W.; Wells, Andrew D.; Zhou, Zhaocai; Greene, Mark I.

    2012-01-01

    FOXP3 is a key transcription factor for regulatory T cell function. We report the crystal structure of the FOXP3 coiled coil domain, through which a loose or transient dimeric association is formed and modulated, accounting for the activity variations introduced by disease-causing mutations or posttranslational modifications. Structure-guided mutagenesis revealed that FOXP3 coiled coil mediated homo-dimerization is essential for Treg function in vitro and in vivo. In particular, we identified human FOXP3 K250 and K252 as key residues for the conformational change and stability of the FOXP3 dimer, which can be regulated by protein posttranslational modifications such as reversible lysine acetylation. These studies provide structural and mechanistic explanations for certain disease-causing mutations in the coiled coil domain of FOXP3 that are commonly found in IPEX syndrome. Overall the regulatory machinery involving homo-oligomerization, acetylation, and hetero-association has been dissected, defining atomic insights into the biological and pathological characteristics of the FOXP3 complex. PMID:22813742

  6. Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli.

    PubMed Central

    Ernsting, B R; Atkinson, M R; Ninfa, A J; Matthews, R G

    1992-01-01

    The leucine-responsive regulatory protein (Lrp) has been shown to regulate, either positively or negatively, the transcription of several Escherichia coli genes in response to leucine. We have used two-dimensional gel electrophoresis to analyze the patterns of polypeptide expression in isogenic lrp+ and lrp mutant strains in the presence or absence of leucine. The absence of a functional Lrp protein alters the expression of at least 30 polypeptides. The expression of the majority of these polypeptides is not affected by the presence or absence of 10 mM exogenous leucine. Outer membrane porins OmpC and OmpF, glutamine synthetase (GlnA), the small subunit of glutamate synthase (GltD), lysyl-tRNA synthetase form II (LysU), a high-affinity periplasmic binding protein specific for branched-chain amino acids (LivJ), W protein, and the enzymes of the pathway converting threonine to glycine, namely, threonine dehydrogenase (Tdh) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), were identified as members of the Lrp regulon by electrophoretic analysis. We have shown that Lrp is a positive regulator of glutamate synthase and glutamine synthetase and that exogenous leucine has little or no effect on the expression of these proteins. In strains carrying a glnL deletion and in strains carrying the glnL2302 allele, which directs the synthesis of a GlnL protein that is constitutively active, expression of glutamine synthetase is no longer regulated by Lrp, demonstrating that the effect of Lrp on glutamine synthetase levels is indirect and requires an intact glnL gene. lrp::Tn10 strains grow poorly when arginine or ornithine is present as the sole nitrogen source in the medium. On the bases of present studies and previous research, we propose that Lrp is involved in the adaptation of E. coli cells to major shifts in environment, such as those which occur when E. coli leaves the intestinal tract of its animal host. Several genes required for amino acid and peptide transport and

  7. Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element*

    PubMed Central

    Crawford, Rebecca R.; Prescott, Eugenia T.; Sylvester, Charity F.; Higdon, Ashlee N.; Shan, Jixiu; Kilberg, Michael S.; Mungrue, Imran N.

    2015-01-01

    Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the −267 ATF/cAMP response element (CRE) site and a novel −248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein β binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease. PMID:25931127

  8. ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase.

    PubMed Central

    Hellyer, N J; Cheng, K; Koland, J G

    1998-01-01

    ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3. PMID:9677338

  9. ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase.

    PubMed

    Hellyer, N J; Cheng, K; Koland, J G

    1998-08-01

    ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.

  10. Foxp3+ Regulatory T Cells Are Required for Recovery from Severe Sepsis

    PubMed Central

    Schmoeckel, Katrin; Cziupka, Katharina; Nguyen, Huu Hung; Hildebrandt, Petra; Hünig, Thomas; Sparwasser, Tim; Huehn, Jochen; Pötschke, Christian; Bröker, Barbara M.

    2013-01-01

    The role of regulatory T cells (Tregs) in bacterial sepsis remains controversial because antibody-mediated depletion experiments gave conflicting results. We employed DEREG mice (DEpletion of REGulatory T cells) and a caecal ligation and puncture model to elucidate the role of CD4+Foxp3+ Tregs in sepsis. In DEREG mice natural Tregs can be visualized easily and selectively depleted by diphtheria toxin because the animals express the diphtheria toxin receptor and enhanced green fluorescent protein as a fusion protein under the control of the foxp3 locus. We confirmed rapid Treg-activation and an increased ratio of Tregs to Teffs in sepsis. Nevertheless, 24 h after sepsis induction, Treg-depleted and control mice showed equally strong inflammation, immune cell immigration into the peritoneum and bacterial dissemination. During the first 36 h of disease survival was not influenced by Treg-depletion. Later, however, only Treg-competent animals recovered from the insult. We conclude that the suppressive capacity of Tregs is not sufficient to control overwhelming inflammation and early mortality, but is a prerequisite for the recovery from severe sepsis. PMID:23724126

  11. The transcription factor regulatory factor X1 increases the expression of neuronal glutamate transporter type 3.

    PubMed

    Ma, Kaiwen; Zheng, Shuqiu; Zuo, Zhiyi

    2006-07-28

    Glutamate transporters (excitatory amino acid transporters, EAAT) play an important role in maintaining extracellular glutamate homeostasis and regulating glutamate neurotransmission. However, very few studies have investigated the regulation of EAAT expression. A binding sequence for the regulatory factor X1 (RFX1) exists in the promoter region of the gene encoding for EAAT3, a neuronal EAAT, but not in the promoter regions of the genes encoding for EAAT1 and EAAT2, two glial EAATs. RFX proteins are transcription factors binding to X-boxes of DNA sequences. Although RFX proteins are necessary for the normal function of sensory neurons in Caenorhabditis elegans, their roles in the mammalian brain are not known. We showed that RFX1 increased EAAT3 expression and activity in C6 glioma cells. RFX1 binding complexes were found in the nuclear extracts of C6 cells. The activity of EAAT3 promoter as measured by luciferase reporter activity was increased by RFX1 in C6 cells and the neuron-like SH-SY5Y cells. However, RFX1 did not change the expression of EAAT2 proteins in the NRK52E cells. RFX1 proteins were expressed in the neurons of rat brain. A high expression level of RFX1 proteins was found in the neurons of cerebral cortex and Purkinje cells. Knockdown of the RFX1 expression by RFX1 antisense oligonucleotides decreased EAAT3 expression in rat cortical neurons in culture. These results suggest that RFX1 enhances the activity of EAAT3 promoter to increase the expression of EAAT3 proteins. This study provides initial evidence for the regulation of gene expression in the nervous cells by RFX1.

  12. Regulatory landscape and clinical implication of MBD3 in human malignant glioma

    PubMed Central

    Weng, Ling; Wirbisky, Sara E.; Freeman, Jennifer L.; Liu, Jingping; Liu, Qing; Yuan, Xianrui; Irudayaraj, Joseph

    2016-01-01

    In this article we inspect the roles and functions of the methyl-CpG-binding domain protein 3 (MBD3) in human malignant glioma, to assess its potential as an epigenetic biomarker for prognosis. The regulatory effects of MBD3 on glioma transcriptome were first profiled by high-throughput microarray. Our results indicate that MBD3 is involved in both transcriptional activation and repression. Furthermore, MBD3 fine-controls a spectrum of proteins critical for cellular metabolism and proliferation, thereby contributing to an exquisite anti-glioma network. Specifically, the expression of MHC class II molecules was found to positively correlate with MBD3, which provides new insight into the immune escape of gliomagenesis. In addition, MBD3 participates in constraining a number of oncogenic non-coding RNAs whose over-activation could drive cells into excessive growth and higher malignancy. Having followed up a pilot cohort, we noted that the survival of malignant glioma patients was proportional to the content of MBD3 and 5-hydroxymethylcytosine (5hmC) in their tumor cells. The progression-free survival (PFS) and overall survival (OS) were relatively poor for patients with lower amount of MBD3 and 5hmC in the tissue biopsies. Taken together, this work enriches our understanding of the mechanistic involvement of MBD3 in malignant glioma. PMID:27835581

  13. Cloning, expression, purification and crystallization of a transcriptional regulatory protein (Rv3291c) from Mycobacterium tuberculosis H37Rv.

    PubMed

    Shrivastava, Tripti; Kumar, Sandeep; Ramachandran, Ravishankar

    2004-10-01

    Rv3291c, the translational product of the Mycobacterium tuberculosis Rv3291c gene, is an 18 kDa protein. It is a putative transcriptional regulatory protein belonging to the leucine-responsive regulatory protein/asparagine synthase C (Lrp/AsnC) family, which are proteins that have been identified in archaea and bacteria. Rv3291c probably plays a significant role during the persistent/latent phase of M. tuberculosis, as supported by its up-regulation several-fold during this stage. Orthorhombic crystals of recombinant Rv3291c have been grown from trisodium citrate dihydrate-buffered solutions containing monoammonium dihydrogen phosphate. Diffraction data extending to 2.7 A have been collected from a single crystal with unit-cell parameters a = 99.6, b = 100.7, c = 100.6 A. Assuming an octamer in the asymmetric unit results in a Matthews coefficient (VM) of 1.75 A3 Da(-1), corresponding to a solvent content of about 30%.

  14. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

    PubMed Central

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-01-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma. PMID:27573585

  15. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    SciTech Connect

    Volkman, Brian Finley

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a "receiver domain" in the family of "two-component" regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  16. Effects of combined radiofrequency radiation exposure on the cell cycle and its regulatory proteins.

    PubMed

    Lee, Kwan-Yong; Kim, Bong Cho; Han, Na-Kyung; Lee, Yun-Sil; Kim, Taehong; Yun, Jae-Hoon; Kim, Nam; Pack, Jeong-Ki; Lee, Jae-Seon

    2011-04-01

    The aim of this study was to investigate whether single or combined radio frequency (RF) radiation exposure has effects on the cell cycle and its regulatory proteins. Exposure of MCF7 cells to either single (837 MHz) or combined (837 and 1950 MHz) RF radiation was conducted at specific absorption rate values of 4 W/kg for 1 h. During the exposure period, the chamber was made isothermal by circulating water through the cavity. After RF radiation exposure, DNA synthesis rate and cell cycle distribution were assessed. The levels of cell cycle regulatory proteins, p53, p21, cyclins, and cyclin-dependent kinases were also examined. The positive control group was exposed to 0.5 and 4 Gy doses of ionizing radiation (IR) and showed changes in DNA synthesis and cell cycle distribution. The levels of p53, p21, cyclin A, cyclin B1, and cyclin D1 were also affected by IR exposure. In contrast to the IR-exposed group, neither the single RF radiation- nor the combined RF radiation-exposed group elicited alterations in DNA synthesis, cell cycle distribution, and levels of cell cycle regulatory proteins. These results indicate that neither single nor combined RF radiation affect cell cycle progression.

  17. Generation of novel bacterial regulatory proteins that detect priority pollutant phenols

    SciTech Connect

    Wise, A.A.; Kuske, C.R.

    2000-01-01

    The genetic systems of bacteria that have the ability to use organic pollutants as carbon and energy sources can be adapted to create bacterial biosensors for the detection of industrial pollution. The creation of bacterial biosensors is hampered by a lack of information about the genetic systems that control production of bacterial enzymes that metabolize pollutants. The authors have attempted to overcome this problem through modification of DmpR, a regulatory protein for the phenol degradation pathway of Pseudomonas sp. strain CF600. The phenol detection capacity of DmpR was altered by using mutagenic PCR targeted to the DmpR sensor domain. DmpR mutants were identified that both increased sensitivity to the phenolic effectors of wild-type DmpR and increased the range of molecules detected. The phenol detection characteristics of seven DmpR mutants were demonstrated through their ability to activate transcription of a lacZ reporter gene. Effectors of the DmpR derivatives included phenol, 2-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4-dimethylphenol, 2-nitrophenol, and 4-nitrophenol.

  18. Glucokinase regulatory protein (GCKR) gene polymorphism affects postprandial lipemic response in a dietary intervention study

    PubMed Central

    Shen, Haiqing; Pollin, Toni I.; Damcott, Coleen M.; McLenithan, John C.; Mitchell, Braxton D.; Shuldiner, Alan R.

    2010-01-01

    Postprandial triglyceridemia is an emerging risk factor for cardiovascular disease. However, most of the genes that influence postprandial triglyceridemia are not known. We evaluated whether a common nonsynonymous SNP rs1260326/P446L in the glucokinase regulatory protein (GCKR) gene influenced variation in the postprandial lipid response after a high-fat challenge in seven hundred and seventy participants in the Amish HAPI Heart Study who underwent an oral high-fat challenge and had blood samples taken in the fasting state and during the postprandial phase at 1, 2, 3, 4, and 6 hours. We found that the minor T allele at rs1260326 was associated with significantly higher fasting TG levels after adjusting for age, sex, and family structure (Pa = 0.06 for additive model, and Pr=0.0003 for recessive model). During the fat challenge, the T allele was associated with significantly higher maximum TG level (Pa = 0.006), incremental maximum TG level (Pa = 0.006), TG area under the curve (Pa = 0.02) and incremental TG area under the curve (Pa = 0.03). Our data indicate that the rs1260326 T allele of GCKR is associated with both higher fasting levels of TG as well as the postprandial TG response, which may result in higher atherogenic risk. PMID:19526250

  19. Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP.

    PubMed

    Maita, Nobuo; Okada, Kengo; Hatakeyama, Kazuyuki; Hakoshima, Toshio

    2002-02-05

    In the presence of phenylalanine, GTP cyclohydrolase I feedback regulatory protein (GFRP) forms a stimulatory 360-kDa complex with GTP cyclohydrolase I (GTPCHI), which is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. The crystal structure of the stimulatory complex reveals that the GTPCHI decamer is sandwiched by two GFRP homopentamers. Each GFRP pentamer forms a symmetrical five-membered ring similar to beta-propeller. Five phenylalanine molecules are buried inside each interface between GFRP and GTPCHI, thus enhancing the binding of these proteins. The complex structure suggests that phenylalanine-induced GTPCHI x GFRP complex formation enhances GTPCHI activity by locking the enzyme in the active state.

  20. Systematic identification of transcriptional regulatory modules from protein–protein interaction networks

    PubMed Central

    Diez, Diego; Hutchins, Andrew Paul; Miranda-Saavedra, Diego

    2014-01-01

    Transcription factors (TFs) combine with co-factors to form transcriptional regulatory modules (TRMs) that regulate gene expression programs with spatiotemporal specificity. Here we present a novel and generic method (rTRM) for the reconstruction of TRMs that integrates genomic information from TF binding, cell type-specific gene expression and protein–protein interactions. rTRM was applied to reconstruct the TRMs specific for embryonic stem cells (ESC) and hematopoietic stem cells (HSC), neural progenitor cells, trophoblast stem cells and distinct types of terminally differentiated CD4+ T cells. The ESC and HSC TRM predictions were highly precise, yielding 77 and 96 proteins, of which ∼75% have been independently shown to be involved in the regulation of these cell types. Furthermore, rTRM successfully identified a large number of bridging proteins with known roles in ESCs and HSCs, which could not have been identified using genomic approaches alone, as they lack the ability to bind specific DNA sequences. This highlights the advantage of rTRM over other methods that ignore PPI information, as proteins need to interact with other proteins to form complexes and perform specific functions. The prediction and experimental validation of the co-factors that endow master regulatory TFs with the capacity to select specific genomic sites, modulate the local epigenetic profile and integrate multiple signals will provide important mechanistic insights not only into how such TFs operate, but also into abnormal transcriptional states leading to disease. PMID:24137002

  1. Promoting Adoption of the 3Rs through Regulatory Qualification.

    PubMed

    Walker, Elizabeth Gribble; Baker, Amanda F; Sauer, John-Michael

    2016-12-01

    One mechanism to advance the application of novel safety assessment methodologies in drug development, including in silico or in vitro approaches that reduce the use of animals in toxicology studies, is regulatory qualification. Regulatory qualification, a formal process defined at the the U. S. Food and Drug Administration and the European Medicines Agency, hinges on a central concept of stating an appropriate "context of use" for a novel drug development tool (DDT) that precisely defines how that DDT can be used to support decision making in a regulated drug development setting. When accumulating the data to support a particular "context-of-use," the concept of "fit-for-purpose" often guides assay validation, as well as the type and amount of data or evidence required to evaluate the tool. This paper will review pathways for regulatory acceptance of novel DDTs and discuss examples of safety projects considered for regulatory qualification. Key concepts to be considered when defining the evidence required to formally adopt and potentially replace animal-intensive traditional safety assessment methods using qualified DDTs are proposed. Presently, the use of qualified translational kidney safety biomarkers can refine and reduce the total numbers of animals used in drug development. We propose that the same conceptual regulatory framework will be appropriate to assess readiness of new technologies that may eventually replace whole animal models.

  2. Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease

    PubMed Central

    Shen, Meng-Ru; Chou, Cheng-Yang; Browning, Joseph A; Wilkins, Robert J; Ellory, J Clive

    2001-01-01

    This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl− channel. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl− channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells. PMID:11731569

  3. Phosphorylation of sterol regulatory element binding protein-1a by protein kinase A (PKA) regulates transcriptional activity.

    PubMed

    Dong, Qingming; Giorgianni, Francesco; Deng, Xiong; Beranova-Giorgianni, Sarka; Bridges, Dave; Park, Edwards A; Raghow, Rajendra; Elam, Marshall B

    2014-07-11

    The counter-regulatory hormone glucagon inhibits lipogenesis via downregulation of sterol regulatory element binding protein 1 (SREBP-1). The effect of glucagon is mediated via protein kinase A (PKA). To determine if SREBP-1 is a direct phosphorylation target of PKA, we conducted mass spectrometry analysis of recombinant n-terminal SREBP-1a following PKA treatment in vitro. This analysis identified serines 331/332 as bona-fide phosphorylation targets of PKA. To determine the functional consequences of phosphorylation at these sites, we constructed mammalian expression vector for both nSREBP-1a and 1c isoforms in which the candidate PKA phosphorylation sites were mutated to active phosphomimetic or non-phosphorylatable amino acids. The transcriptional activity of SREBP was reduced by the phosphomimetic mutation of S332 of nSREBP-1a and the corresponding serine (S308) of nSREBP-1c. This site is a strong candidate for mediating the negative regulatory effect of glucagon on SREBP-1 and lipogenesis.

  4. Physical and functional domains of the herpes simplex virus transcriptional regulatory protein ICP4.

    PubMed Central

    DeLuca, N A; Schaffer, P A

    1988-01-01

    A characteristic common to DNA animal viruses is the expression early in infection of viral proteins that act in trans to regulate subsequent RNA polymerase II-dependent transcription of the remainder of the viral genome. The predominant transcriptional regulatory protein specified by herpes simplex virus type 1 is the immediate-early protein ICP4. ICP4 is a complex multifunctional protein required for the activation of many herpes simplex virus type 1 transcriptional units and for repression of its own transcription. In the present study we have introduced nonsense and deletion mutations into both genome copies of the ICP4 gene such that the resulting mutants express only defined subsets of the primary ICP4 amino acid sequence. The partial peptides retain activities and physical properties of the intact ICP4 molecule, permitting one to attribute individual activities and properties to defined amino acid sequences. Images PMID:2828668

  5. The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis

    PubMed Central

    Jones, Danielle M.; Murray, Christian M.; Ketelaar, KassaDee J.; Thomas, Joseph J.; Villalobos, Jose A.; Wallace, Ian S.

    2016-01-01

    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites. PMID:27252710

  6. Enhanced suppressor function of TIM-3+ FoxP3+ regulatory T cells.

    PubMed

    Gautron, Anne-Sophie; Dominguez-Villar, Margarita; de Marcken, Marine; Hafler, David A

    2014-09-01

    T-cell immunoglobulin and mucin domain 3 (TIM-3) is an Ig-superfamily member expressed on IFN-γ-secreting Th1 and Tc1 cells and was identified as a negative regulator of immune tolerance. TIM-3 is expressed by a subset of activated CD4(+) T cells, and anti-CD3/anti-CD28 stimulation increases both the level of expression and the number of TIM-3(+) T cells. In mice, TIM-3 is constitutively expressed on natural regulatory T (Treg) cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4(+) T-cell differentiation. Here, we examined TIM-3 expression on human Treg cells to determine its role in T-cell suppression. In contrast to mice, TIM-3 is not expressed on Treg cells ex vivo but is upregulated after activation. While TIM-3(+) Treg cells with increased gene expression of LAG3, CTLA4, and FOXP3 are highly efficient suppressors of effector T (Teff) cells, TIM-3(-) Treg cells poorly suppressed Th17 cells as compared with their suppression of Th1 cells; this decreased suppression ability was associated with decreased STAT-3 expression and phosphorylation and reduced gene expression of IL10, EBI3, GZMB, PRF1, IL1Rα, and CCR6. Thus, our results suggest that TIM-3 expression on Treg cells identifies a population highly effective in inhibiting pathogenic Th1- and Th17-cell responses.

  7. Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection*

    PubMed Central

    Heard, William; Sklenář, Jan; Tomé, Daniel F. A.; Robatzek, Silke; Jones, Alexandra M. E.

    2015-01-01

    The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein

  8. Nuclear Regulatory Commission issuances, September 1995. Volume 42, Number 3

    SciTech Connect

    1995-09-01

    This book contains an issuance of the Atomic Safety and Licensing Board and a Director`s Decision, both of the US Nuclear Regulatory Commission. The issuance concerns the dismissal of a case by adopting a settlement reached by the Staff of the Nuclear Regulatory Commission and a Radiation Safety Officer of a hospital in which the safety officer pled guilty to deliberate misconduct. The Director`s Decision was to deny a petition to impose a fine on Tennessee Valley Authority concerning alleged harassment of the petitioner and to appoint an independent arbitration board to review all past complaints filed against TVA concerning the Watts Bar Nuclear Plant.

  9. NRF2-ome: an integrated web resource to discover protein interaction and regulatory networks of NRF2.

    PubMed

    Türei, Dénes; Papp, Diána; Fazekas, Dávid; Földvári-Nagy, László; Módos, Dezső; Lenti, Katalin; Csermely, Péter; Korcsmáros, Tamás

    2013-01-01

    NRF2 is the master transcriptional regulator of oxidative and xenobiotic stress responses. NRF2 has important roles in carcinogenesis, inflammation, and neurodegenerative diseases. We developed an online resource, NRF2-ome, to provide an integrated and systems-level database for NRF2. The database contains manually curated and predicted interactions of NRF2 as well as data from external interaction databases. We integrated NRF2 interactome with NRF2 target genes, NRF2 regulating TFs, and miRNAs. We connected NRF2-ome to signaling pathways to allow mapping upstream NRF2 regulatory components that could directly or indirectly influence NRF2 activity totaling 35,967 protein-protein and signaling interactions. The user-friendly website allows researchers without computational background to search, browse, and download the database. The database can be downloaded in SQL, CSV, BioPAX, SBML, PSI-MI, and in a Cytoscape CYS file formats. We illustrated the applicability of the website by suggesting a posttranscriptional negative feedback of NRF2 by MAFG protein and raised the possibility of a connection between NRF2 and the JAK/STAT pathway through STAT1 and STAT3. NRF2-ome can also be used as an evaluation tool to help researchers and drug developers to understand the hidden regulatory mechanisms in the complex network of NRF2.

  10. Construction of hormonally responsive intact cell hybrids by cell fusion: transfer of. beta. -adrenergic receptor and nucleotide regulatory protein(s) in normal and desensitized cells

    SciTech Connect

    Schulster, D.; Salmon, D.M.

    1985-01-01

    Fusion of normal, untreated human erythrocytes with desensitized turkey erythrocytes increases isoproterenol stimulation of cyclic (/sup 3/H)AMP accumulation over basal rates. Moreover, pretreatment of the human erythrocytes with cholera toxin before they are fused with desensitized turkey erthythrocytes leads to a large stimulation with isoproterenol. This is even greater and far more rapid than the response obtained if turkey erythrocytes are treated directly with cholera toxin. It is concluded that the stimulation in the fused system is due to the transfer of an ADP-ribosylated subunit of nucleotide regulatory protein.

  11. Renal mass reduction results in accumulation of lipids and dysregulation of lipid regulatory proteins in the remnant kidney.

    PubMed

    Kim, Hyun Ju; Moradi, Hamid; Yuan, Jun; Norris, Keith; Vaziri, Nosratola D

    2009-06-01

    A significant reduction of renal mass results in proteinuria, glomerulosclerosis, and tubulointerstitial injury, culminating in end-stage chronic renal failure (CRF). The accumulation of lipids in the kidney can cause renal disease. Uptake of oxidized lipoproteins via scavenger receptors, reabsorption of filtered protein-bound lipids via the megalin-cubilin complex, and increased glucose load per nephron can promote lipid accumulation in glomerular, tubular, and interstitial cells in CRF. Cellular lipid homeostasis is regulated by lipid influx, synthesis, catabolism, and efflux. We examined lipid-regulatory factors in the remnant kidney of rats 11 wk after nephrectomy (CRF) or sham operation. CRF resulted in azotemia, proteinuria, lipid accumulation in the kidney, upregulation of megalin, cubilin, mediators of lipid influx (scavenger receptor class A and lectin-like oxidized receptor-1), lipid efflux (liver X receptor alpha/beta and ATP-binding cassette transporter), and fatty acid biosynthesis (carbohydrate-response element binding protein, fatty acid synthase, and acetyl-CoA carboxylase). However, factors involved in cholesterol biosynthesis (sterol regulatory element binding protein, 3-hydroxy-3-methylglutaryl coenzyme A reductase, SCAP, Insig-1, and Insig-2) and fatty acid oxidation (peroxisome proliferator-activated receptor, acyl-CoA oxidase, and liver-type fatty acid binding protein) were reduced in the remnant kidney. Thus CRF results in heavy lipid accumulation in the remnant kidney, which is mediated by upregulation of pathways involved in tubular reabsorption of filtered protein-bound lipids, influx of oxidized lipoproteins and synthesis of fatty acids, and downregulation of pathways involved in fatty acid catabolism.

  12. RNA regulatory networks diversified through curvature of the PUF protein scaffold

    PubMed Central

    Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P.; Nevil, Markus; Campbell, Zachary T.; Tanaka Hall, Traci M.; Wickens, Marvin

    2015-01-01

    Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks. PMID:26364903

  13. RNA regulatory networks diversified through curvature of the PUF protein scaffold

    DOE PAGES

    Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P.; ...

    2015-09-14

    Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extendedmore » interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.« less

  14. RNA regulatory networks diversified through curvature of the PUF protein scaffold

    SciTech Connect

    Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P.; Nevil, Markus; Campbell, Zachary T.; Tanaka Hall, Traci M.; Wickens, Marvin

    2015-09-14

    Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.

  15. Protective role of interferon regulatory factor 3-mediated signaling against prion infection.

    PubMed

    Ishibashi, Daisuke; Atarashi, Ryuichiro; Fuse, Takayuki; Nakagaki, Takehiro; Yamaguchi, Naohiro; Satoh, Katsuya; Honda, Kenya; Nishida, Noriyuki

    2012-05-01

    Abnormal prion protein (PrP(Sc)) generated from the cellular isoform of PrP (PrP(C)) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the innate immune system in prion infection, we investigated the function of interferon regulatory factor 3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited significantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrP(Sc) was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrP(Sc) formation. Our findings provide new insight into the role of the host innate immune system in the pathogenesis of prion diseases.

  16. Protective Role of Interferon Regulatory Factor 3-Mediated Signaling against Prion Infection

    PubMed Central

    Atarashi, Ryuichiro; Fuse, Takayuki; Nakagaki, Takehiro; Yamaguchi, Naohiro; Satoh, Katsuya; Honda, Kenya; Nishida, Noriyuki

    2012-01-01

    Abnormal prion protein (PrPSc) generated from the cellular isoform of PrP (PrPC) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the innate immune system in prion infection, we investigated the function of interferon regulatory factor 3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited significantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrPSc was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrPSc formation. Our findings provide new insight into the role of the host innate immune system in the pathogenesis of prion diseases. PMID:22379081

  17. Behavioral and regulatory abnormalities in mice deficient in the NPAS1 and NPAS3 transcription factors.

    PubMed

    Erbel-Sieler, Claudia; Dudley, Carol; Zhou, Yudong; Wu, Xinle; Estill, Sandi Jo; Han, Tina; Diaz-Arrastia, Ramon; Brunskill, Eric W; Potter, S Steven; McKnight, Steven L

    2004-09-14

    Laboratory mice bearing inactivating mutations in the genes encoding the NPAS1 and NPAS3 transcription factors have been shown to exhibit a spectrum of behavioral and neurochemical abnormalities. Behavioral abnormalities included diminished startle response, as measured by prepulse inhibition, and impaired social recognition. NPAS1/NPAS3-deficient mice also exhibited stereotypic darting behavior at weaning and increased locomotor activity. Immunohistochemical staining assays showed that the NPAS1 and NPAS3 proteins are expressed in inhibitory interneurons and that the viability and anatomical distribution of these neurons are unaffected by the absence of either transcription factor. Adult brain tissues from NPAS3- and NPAS1/NPAS3-deficient mice exhibited a distinct reduction in reelin, a large, secreted protein whose expression has been reported to be attenuated in the postmortem brain tissue of patients with schizophrenia. These observations raise the possibility that a regulatory program controlled in inhibitory interneurons by the NPAS1 and NPAS3 transcription factors may be either substantively or tangentially relevant to psychosis.

  18. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    SciTech Connect

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

    2013-07-12

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  19. APG: an Active Protein-Gene network model to quantify regulatory signals in complex biological systems.

    PubMed

    Wang, Jiguang; Sun, Yidan; Zheng, Si; Zhang, Xiang-Sun; Zhou, Huarong; Chen, Luonan

    2013-01-01

    Synergistic interactions among transcription factors (TFs) and their cofactors collectively determine gene expression in complex biological systems. In this work, we develop a novel graphical model, called Active Protein-Gene (APG) network model, to quantify regulatory signals of transcription in complex biomolecular networks through integrating both TF upstream-regulation and downstream-regulation high-throughput data. Firstly, we theoretically and computationally demonstrate the effectiveness of APG by comparing with the traditional strategy based only on TF downstream-regulation information. We then apply this model to study spontaneous type 2 diabetic Goto-Kakizaki (GK) and Wistar control rats. Our biological experiments validate the theoretical results. In particular, SP1 is found to be a hidden TF with changed regulatory activity, and the loss of SP1 activity contributes to the increased glucose production during diabetes development. APG model provides theoretical basis to quantitatively elucidate transcriptional regulation by modelling TF combinatorial interactions and exploiting multilevel high-throughput information.

  20. Distribution of transmembrane AMPA receptor regulatory protein (TARP) isoforms in the rat spinal cord.

    PubMed

    Larsson, M; Agalave, N; Watanabe, M; Svensson, C I

    2013-09-17

    The transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor regulatory proteins (TARPs) are a family of auxiliary AMPA receptor subunits that differentially modulate trafficking and many functional properties of the receptor. To investigate which TARP isoforms may be involved in AMPA receptor-mediated spinal synaptic transmission, we have mapped the localization of five of the known TARP isoforms, namely γ-2 (also known as stargazin), γ-3, γ-4, γ-7 and γ-8, in the rat spinal cord. Immunoblotting showed expression of all isoforms in the spinal cord to varying degrees. At the light microscopic level, immunoperoxidase labeling of γ-4, γ-7 and γ-8 was found throughout spinal gray matter. In white matter, γ-4 and γ-7 immunolabeling was observed in astrocytic processes and in mature oligodendrocytes. In pepsin-treated spinal cord, γ-7 often colocalized with GluA2 immunopositive puncta in the deep dorsal horn as well as in the ventral horn, but not in the superficial dorsal horn. Postembedding immunogold labeling was further used to assess the synaptic localization of γ-2, γ-7 and γ-8 in the dorsal horn. Synaptic immunogold labeling of γ-2 was sparse throughout the dorsal horn, with some primary afferent synapses weakly labeled, whereas relatively strong γ-7 immunogold labeling was found at deep dorsal horn synapses, including at synapses formed by low-threshold mechanosensitive primary afferent terminals. Prominent immunogold labeling of γ-8 was frequently detected at synapses established by primary afferent fibers. The spinal localization patterns of TARP isoforms reported here suggest that AMPA receptors at spinal synaptic populations and in glial cells may exhibit different functional characteristics owing to differences in auxiliary subunit composition.

  1. A maize protein associated with the G-box binding complex has homology to brain regulatory proteins.

    PubMed Central

    de Vetten, N C; Lu, G; Feri, R J

    1992-01-01

    The G-box element is a moderately conserved component of the promoter of many inducible genes, including the alcohol dehydrogenase genes of Arabidopsis and maize. We used monoclonal antibodies generated against partially purified G-box binding factor (GBF) activity to characterize maize proteins that are part of the DNA binding complex. Antibodies interacted with partially purified maize GBF complexes to produce a slower migrating complex in the gel retardation assay. Immunoprecipitation experiments suggested that the protein recognized by the antibody is not a DNA binding protein in and of itself, but rather is associated with the DNA binding complex. These monoclonal antibodies were used to isolate cDNA clones encoding a protein that we have designated GF14. Maize GF14 contains a region resembling a leucine zipper and acidic carboxy and amino termini, of which the latter can form an amphipathic alpha-helix similar to known transcriptional activators such as VP16 and GAL4. Protein gel blot analysis of cell culture extract showed that a single, major protein of approximately 30 kD is recognized by anti-GF14; the protein is also present predominantly in the kernel and root. The deduced amino acid sequence of maize GF14 is more than 80% identical to Arabidopsis GF14 and Oenothera PHP-O, and is more than 60% identical to a class of mammalian brain proteins described as both protein kinase C inhibitors and activators of tyrosine and tryptophan hydroxylases. GF14 is found in a variety of monocotyledons and dicotyledons, gymnosperms, and yeast. This suggests a deep evolutionary conservation of a potential regulatory protein associated with a core sequence found in the promoter region of many genes. PMID:1446170

  2. Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA

    SciTech Connect

    Walden, William E.; Selezneva, Anna I.; Dupuy, Jérôme; Volbeda, Anne; Fontecilla-Camps, Juan C.; Theil, Elizabeth C.; Volz1, Karl

    2011-07-27

    Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 angstrom resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by {approx}30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator or enzyme.

  3. Junction-mediating and regulatory protein (JMY) is essential for early porcine embryonic development.

    PubMed

    Lin, Zi Li; Cui, Xiang-Shun; Namgoong, Suk; Kim, Nam-Hyung

    2015-01-01

    Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway.

  4. Junction-mediating and regulatory protein (JMY) is essential for early porcine embryonic development

    PubMed Central

    LIN, Zi Li; CUI, Xiang-Shun; NAMGOONG, Suk; KIM, Nam-Hyung

    2015-01-01

    Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway. PMID:26052154

  5. Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

    NASA Technical Reports Server (NTRS)

    Mednieks, M. I.; Popova, I.; Grindeland, R. E.

    1992-01-01

    Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

  6. Geminivirus C3 Protein: Replication Enhancement and Protein Interactions

    PubMed Central

    Settlage, Sharon B.; See, Renee G.; Hanley-Bowdoin, Linda

    2005-01-01

    Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants. PMID:16014949

  7. Tyrosine phosphorylation of the herpes simplex virus type 1 regulatory protein ICP22 and a cellular protein which shares antigenic determinants with ICP22.

    PubMed Central

    Blaho, J A; Zong, C S; Mortimer, K A

    1997-01-01

    At least eight herpes simplex virus type 1 (HSV-1) and five HSV-2 proteins were tyrosine phosphorylated in infected cells. The first viral tyrosine phosphoprotein identified was the HSV-1 regulatory protein ICP22. Also, two novel phosphotyrosine proteins were bound by anti-ICP22 antibodies. H(R22) is a cellular protein, while the F(R10) protein is observed only in HSV-1-infected cells. PMID:9371655

  8. 3 CFR 13579 - Executive Order 13579 of July 11, 2011. Regulation and Independent Regulatory Agencies

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 3 The President 1 2012-01-01 2012-01-01 false Executive Order 13579 of July 11, 2011. Regulation and Independent Regulatory Agencies 13579 Order 13579 Presidential Documents Executive Orders Executive Order 13579 of July 11, 2011 EO 13579 Regulation and Independent Regulatory Agencies By...

  9. 3 CFR 13563 - Executive Order 13563 of January 18, 2011. Improving Regulation and Regulatory Review

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 3 The President 1 2012-01-01 2012-01-01 false Executive Order 13563 of January 18, 2011. Improving Regulation and Regulatory Review 13563 Order 13563 Presidential Documents Executive Orders Executive Order... improve regulation and regulatory review, it is hereby ordered as follows: Section 1. General...

  10. Cytosolic Na+ Controls an Epithelial Na+ Channel Via the Go Guanine Nucleotide-Binding Regulatory Protein

    NASA Astrophysics Data System (ADS)

    Komwatana, P.; Dinudom, A.; Young, J. A.; Cook, D. I.

    1996-07-01

    In tight Na+-absorbing epithelial cells, the rate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-β -S, pertussis toxin, and antibodies against the α -subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

  11. Cytosolic Na+ controls and epithelial Na+ channel via the Go guanine nucleotide-binding regulatory protein.

    PubMed Central

    Komwatana, P; Dinudom, A; Young, J A; Cook, D I

    1996-01-01

    In tight Na+-absorbing epithelial cells, the fate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-beta-S, pertussis toxin, and antibodies against the alpha-subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-. Images Fig. 4 PMID:8755611

  12. A large family of anti-activators accompanying XylS/AraC family regulatory proteins.

    PubMed

    Santiago, Araceli E; Yan, Michael B; Tran, Minh; Wright, Nathan; Luzader, Deborah H; Kendall, Melissa M; Ruiz-Perez, Fernando; Nataro, James P

    2016-07-01

    AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.

  13. A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

    PubMed Central

    Yan, Michael B.; Tran, Minh; Wright, Nathan; Luzader, Deborah H.; Kendall, Melissa M.; Ruiz‐Perez, Fernando; Nataro, James P.

    2016-01-01

    Summary AraC Negative Regulators (ANR) suppress virulence genes by directly down‐regulating AraC/XylS members in Gram‐negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR‐activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC‐like member AggR. ANR‐AggR binding disrupted AggR dimerization and prevented AggR‐DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α‐helices. Site‐directed mutagenesis studies suggest that at least predicted α‐helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners. PMID:27038276

  14. Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls

    PubMed Central

    Jain, Varsha; Patel, Brijesh; Umar, Farhat Paul; Ajithakumar, H. M.; Gurjar, Suraj K.; Gupta, I. D.; Verma, Archana

    2017-01-01

    Aim: This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls. Materials and Methods: Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme. Results: The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study. Conclusion: A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible. PMID:28344410

  15. Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes.

    PubMed Central

    Muntz, K H; Sternweis, P C; Gilman, A G; Mumby, S M

    1992-01-01

    Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma. Images PMID:1550955

  16. Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding.

    PubMed

    Korkuć, Paula; Walther, Dirk

    2016-05-01

    Phosphorylation is an important post-translational modification that regulates protein function by the attachment of negatively charged phosphate groups to phosphorylatable amino acid residues. As a mode of action, an influence of phosphorylation on the binding of compounds to proteins has been discussed and described for a number of proteins in the literature. However, a systematic statistical survey probing for enriched phosphorylation sites close to compound binding sites in support of this notion and with properly chosen random reference distributions has not been presented yet. Using high-resolution protein structures from the Protein Data Bank including their co-crystallized non-covalently bound compounds and experimentally determined phosphorylation sites, we analyzed the pairwise distance distributions of phosphorylation and compound binding sites on protein surfaces. We found that phosphorylation sites are indeed located at significantly closer distances to compounds than expected by chance holding true specifically also for the subset of compound binding sites serving as catalytic sites of metabolic reactions. This tendency was particularly evident when treating phosphorylation sites as collective sets supporting the relevance of phosphorylation hotspots. Interestingly, phosphorylation sites were found to be closer to negatively charged than to positively charged compounds suggesting a stronger modulation of the binding of negatively charged compounds in dependence on phosphorylation status than on positively charged compounds. The enrichment of phosphorylation sites near compound binding sites confirms a regulatory role of phosphorylation in compound binding and provides a solid statistical basis for the literature-reported selected events.

  17. Regulatory-auxiliary subunits of CLC chloride channel-transport proteins.

    PubMed

    Barrallo-Gimeno, Alejandro; Gradogna, Antonella; Zanardi, Ilaria; Pusch, Michael; Estévez, Raúl

    2015-09-15

    The CLC family of chloride channels and transporters is composed by nine members, but only three of them, ClC-Ka/b, ClC-7 and ClC-2, have been found so far associated with auxiliary subunits. These CLC regulatory subunits are small proteins that present few common characteristics among them, both structurally and functionally, and their effects on the corresponding CLC protein are different. Barttin, a protein with two transmembrane domains, is essential for the membrane localization of ClC-K proteins and their activity in the kidney and inner ear. Ostm1 is a protein with a single transmembrane domain and a highly glycosylated N-terminus. Unlike the other two CLC auxiliary subunits, Ostm1 shows a reciprocal relationship with ClC-7 for their stability. The subcellular localization of Ostm1 depends on ClC-7 and not the other way around. ClC-2 is active on its own, but GlialCAM, a transmembrane cell adhesion molecule with two extracellular immunoglobulin (Ig)-like domains, regulates its subcellular localization and activity in glial cells. The common theme for these three proteins is their requirement for a proper homeostasis, since their malfunction leads to distinct diseases. We will review here their properties and their role in normal chloride physiology and the pathological consequences of their improper function.

  18. Tim-3 pathway controls regulatory and effector T cell balance during hepatitis C virus infection.

    PubMed

    Moorman, Jonathan P; Wang, Jia M; Zhang, Ying; Ji, Xiao J; Ma, Cheng J; Wu, Xiao Y; Jia, Zhan S; Wang, Ke S; Yao, Zhi Q

    2012-07-15

    Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Upregulation of inhibitory signaling pathways (such as T cell Ig and mucin domain protein-3 [Tim-3]) and accumulation of regulatory T cells (Tregs) play pivotal roles in suppressing antiviral effector T cell (Teff) responses that are essential for viral clearance. Although the Tim-3 pathway has been shown to negatively regulate Teffs, its role in regulating Foxp3(+) Tregs is poorly explored. In this study, we investigated whether and how the Tim-3 pathway alters Foxp3(+) Treg development and function in patients with chronic HCV infection. We found that Tim-3 was upregulated, not only on IL-2-producing CD4(+)CD25(+)Foxp3(-) Teffs, but also on CD4(+)CD25(+)Foxp3(+) Tregs, which accumulate in the peripheral blood of chronically HCV-infected individuals when compared with healthy subjects. Tim-3 expression on Foxp3(+) Tregs positively correlated with expression of the proliferation marker Ki67 on Tregs, but it was inversely associated with proliferation of IL-2-producing Teffs. Moreover, Foxp3(+) Tregs were found to be more resistant to, and Foxp3(-) Teffs more sensitive to, TCR activation-induced cell apoptosis, which was reversible by blocking Tim-3 signaling. Consistent with its role in T cell proliferation and apoptosis, blockade of Tim-3 on CD4(+)CD25(+) T cells promoted expansion of Teffs more substantially than Tregs through improving STAT-5 signaling, thus correcting the imbalance of Foxp3(+) Tregs/Foxp3(-) Teffs that was induced by HCV infection. Taken together, the Tim-3 pathway appears to control Treg and Teff balance through altering cell proliferation and apoptosis during HCV infection.

  19. Individual interferon regulatory factor-3 thiol residues are not critical for its activation following virus infection.

    PubMed

    Zucchini, Nicolas; Williams, Virginie; Grandvaux, Nathalie

    2012-09-01

    The interferon regulatory factor (IRF)-3 transcription factor plays a central role in the capacity of the host to mount an efficient innate antiviral immune defense, mainly through the regulation of type I Interferon genes. A tight regulation of IRF-3 is crucial for an adapted intensity and duration of the response. Redox-dependent processes are now well known to regulate signaling cascades. Recent reports have revealed that signaling molecules upstream of IRF-3, including the mitochondrial antiviral-signalling protein (MAVS) and the TNF receptor associated factors (TRAFs) adaptors, are sensitive to redox regulation. In the present study, we assessed whether redox regulation of thiol residues contained in IRF-3, which are priviledged redox sensors, play a role in its regulation following Sendai virus infection, using a combination of mutation of Cysteine (Cys) residues into Alanine and thiols alkylation using N-ethyl maleimide. Alkylation of IRF-3 on Cys289 appears to destabilize IRF-3 dimer in vitro. However, a detailed analysis of IRF-3 phosphorylation, dimerization, nuclear accumulation, and induction of target gene promoter in vivo led us to conclude that IRF-3 specific, individual Cys residues redox status does not play an essential role in its activation in vivo.

  20. Individual Interferon Regulatory Factor-3 Thiol Residues Are Not Critical for Its Activation Following Virus Infection

    PubMed Central

    Zucchini, Nicolas; Williams, Virginie

    2012-01-01

    The interferon regulatory factor (IRF)-3 transcription factor plays a central role in the capacity of the host to mount an efficient innate antiviral immune defense, mainly through the regulation of type I Interferon genes. A tight regulation of IRF-3 is crucial for an adapted intensity and duration of the response. Redox-dependent processes are now well known to regulate signaling cascades. Recent reports have revealed that signaling molecules upstream of IRF-3, including the mitochondrial antiviral-signalling protein (MAVS) and the TNF receptor associated factors (TRAFs) adaptors, are sensitive to redox regulation. In the present study, we assessed whether redox regulation of thiol residues contained in IRF-3, which are priviledged redox sensors, play a role in its regulation following Sendai virus infection, using a combination of mutation of Cysteine (Cys) residues into Alanine and thiols alkylation using N-ethyl maleimide. Alkylation of IRF-3 on Cys289 appears to destabilize IRF-3 dimer in vitro. However, a detailed analysis of IRF-3 phosphorylation, dimerization, nuclear accumulation, and induction of target gene promoter in vivo led us to conclude that IRF-3 specific, individual Cys residues redox status does not play an essential role in its activation in vivo. PMID:22817838

  1. The role of complement regulatory proteins (CD55 and CD59) in the pathogenesis of autoimmune hemocytopenias.

    PubMed

    Ruiz-Argüelles, Alejandro; Llorente, Luis

    2007-01-01

    Mammalian cells are provided with surface-bound complement regulatory proteins that protect them from uncontrolled complement-mediated lysis. Two of these regulators in humans, CD55 and CD59, are glycosylphosphatidylinositol-anchored, type I cell surface proteins, which inhibit formation of the C3 convertases and prevent the terminal polymerization of the membrane attack complex, respectively. Paroxysmal nocturnal hemoglobinuria is a genetic disorder due to the impaired conformation of the glycosylphosphatidylinositol-anchor, that results in the deficient expression of CD55 and CD50 which leads to excessive destruction of red cells and leukocytes. We have studied the expression of these two molecules in patients with autoimmune hemolytic anemia, autoimmune thrombocytopenia, and patients with systemic lupus erythematosus showing lymphopenia, and found that all three types of cytopenias are associated to deficient expression of CD55 and CD59 on the involved hematopoietic lineage. These are the first descriptions of acquired deficiencies of complement regulatory molecules in human disease, and it seems, from our results, that such deficiencies might play a pathogenic role in the mechanism of cell destruction. Although autoantibodies appeal as the best candidates to cause underexpression of CD55 and CD59, the search for an association to the presence and titers of most frequent self-reactive antibodies has proved non-existent.

  2. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures.

    PubMed

    Slinger, Betty L; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M

    2015-12-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition.

  3. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures

    PubMed Central

    Slinger, Betty L.; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M.

    2015-01-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition. PMID:26675164

  4. Nuclear Regulatory legislation: 103d Congress. Volume 2, No. 3

    SciTech Connect

    1995-08-01

    This document is a compilation of nuclear regulatory legislation and other relevant material through the 103d Congress, 2d Session. This compilation has been prepared for use as a resource document, which the NRC intends to update at the end of every Congress. The contents of NUREG-0980 include the Atomic Energy Act of 1954, as amended; Energy Reorganization Act of 1974, as amended, Uranium Mill Tailings Radiation Control Act of 1978; Low-Level Radioactive Waste Policy Act; Nuclear Waste Policy Act of 1982; and NRC Authorization and Appropriations Acts. Other materials included are statutes and treaties on export licensing, nuclear non-proliferation, and environmental protection.

  5. Nuclear Regulatory legislation: 103d Congress. Volume 1, No. 3

    SciTech Connect

    1995-08-01

    This document is a compilation of nuclear regulatory legislation and other relevant material through the 103d Congress, 2d Session. This compilation has been prepared for use as a resource document, which the NRC intends to update at the end of every Congress. The contents of NUREG-0980 include the Atomic Energy Act of 1954, as amended; Energy Reorganization Act of 1974, as amended, Uranium Mill Tailings Radiation Control Act of 1978; Low-Level Radioactive Waste Policy Act; Nuclear Waste Policy Act of 1982; and NRC Authorization and Appropriations Acts. Other materials included are statutes and treaties on export licensing, nuclear non-proliferation, and environmental protection.

  6. Development and function of Foxp3(+) regulatory T cells.

    PubMed

    Wang, Yuan Min; Ghali, Joanna; Zhang, Geoff Yu; Hu, Min; Wang, Ya; Sawyer, Andrew; Zhou, Jimmy Jianheng; Hapudeniya, Dhanushka A; Wang, Yiping; Cao, Qi; Zheng, Guoping; Harris, David C; Alexander, Stephen I

    2016-02-01

    Regulatory T cells (Tregs) have been recognized as having a major role in maintaining peripheral tolerance and preventing and limiting autoimmune and chronic inflammatory diseases. Tregs derive from the thymus and also develop peripherally. In this review, we discuss recent progress in our understanding of the basic mechanisms involved in Treg development and function in protecting against autoimmunity in the periphery, including thymic selection, peripheral induction and the many mechanisms of Treg suppression. Specifically in kidney disease, Tregs have been shown to play a role in limiting injury and may potentially have a therapeutic role.

  7. A 3D bioprinting exemplar of the consequences of the regulatory requirements on customized processes.

    PubMed

    Hourd, Paul; Medcalf, Nicholas; Segal, Joel; Williams, David J

    2015-01-01

    Computer-aided 3D printing approaches to the industrial production of customized 3D functional living constructs for restoration of tissue and organ function face significant regulatory challenges. Using the manufacture of a customized, 3D-bioprinted nasal implant as a well-informed but hypothetical exemplar, we examine how these products might be regulated. Existing EU and USA regulatory frameworks do not account for the differences between 3D printing and conventional manufacturing methods or the ability to create individual customized products using mechanized rather than craft approaches. Already subject to extensive regulatory control, issues related to control of the computer-aided design to manufacture process and the associated software system chain present additional scientific and regulatory challenges for manufacturers of these complex 3D-bioprinted advanced combination products.

  8. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    NASA Astrophysics Data System (ADS)

    Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

    2015-02-01

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  9. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    SciTech Connect

    Matsushita, Y. Murakawa, T. Shimamura, K. Oishi, M. Ohyama, T. Kurita, N.

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  10. Downregulation of key regulatory proteins in androgen dependent prostate tumor cells by oncolytic reovirus.

    PubMed

    Gupta-Saraf, Pooja; Meseke, Tyler; Miller, Cathy L

    2015-11-01

    As prostate tumor cell growth depends on hormones, androgen ablation is an effective therapy for prostate cancer (PCa). However, progression of PCa cells to androgen independent growth (castrate resistant prostate cancer, CRPC) results in relapse and mortality. Hypoxia, a microenvironment of low oxygen that modifies the activity of PCa regulatory proteins including the androgen receptor (AR), plays a critical role in progression to CRPC. Therapies targeting hypoxia and the AR may lengthen the time to CRPC progression thereby increasing survival time of PCa patients. Mammalian Orthoreovirus (MRV) has shown promise for the treatment of prostate tumors in vitro and in vivo. In this study, we found that MRV infection induces downregulation of proteins implicated in CRPC progression, interferes with hypoxia-induced AR activity, and induces apoptosis in androgen dependent cells. This suggests MRV possesses traits that could be exploited to create novel therapies for the inhibition of progression to CRPC.

  11. Elucidating residue roles in engineered variants of AraC regulatory protein

    PubMed Central

    Tang, Shuang-Yan; Cirino, Patrick C

    2010-01-01

    The AraC regulatory protein was previously engineered to control gene expression specifically in response to d-arabinose and not the native effector l-arabinose (Tang et al., J Am Chem Soc 2008;130:5267–5271). Mutations were targeted in the ligand-binding pocket and on the AraC N-terminal arm, which plays an important role in maintaining repressing or activating conformations in the absence or presence of effector, respectively. In this study, we analyze the contributions of individual mutations toward the overall mutant functions in an attempt to streamline future AraC design efforts. For a variety of point mutants, we quantify the induced expression response to d-arabinose (level of leaky expression, induction fold, half-maximal dose response, and effector specificity) and the binding affinity of the purified ligand-binding domain for d-arabinose. We find that mutations introduced in the N-terminal arm (design Position 8) strengthen the induction response, most likely by weakening interactions with the DNA-binding domain, but are not involved in ligand binding. Meanwhile, binding pocket mutations occurring further away from the arm (Positions 80 and 82) primarily contribute to maintaining repression in the absence of effector and do not show response to d-arabinose without the accompanying mutations. Combinations of mutations cooperatively couple molecular recognition to transcriptional activation, demonstrating the complexity of the AraC regulatory switch and the power of combinatorial protein design to alter effector specificity while maintaining regulatory function. PMID:20014443

  12. Protein turnover in 3T3 cells transformed with the oncogene c-H-ras1.

    PubMed Central

    Gunn, J M; James, G

    1992-01-01

    We have examined protein turnover, growth, DNA synthesis and proliferation in three independent clones of 3T3-NR6 cells transformed with the oncogene c-H-ras1. We find that, firstly, the half-maximum concentration of serum and insulin regulating protein turnover in ras-transformed cells is significantly reduced from 0.5 to 0.3% for serum and from 4 nM to 0.5 nM for insulin, and, secondly, ras-transformed cells consistently have lower rates of protein degradation. The catabolic effect of conditioned medium or serum withdrawal is attenuated in transformed lines by maintaining lower basal rates of protein breakdown and higher basal rates of DNA and protein synthesis. Serum stimulation of growth in transformed cells is achieved in the short term by lower rates of protein breakdown rather than higher rates of protein synthesis: rates of protein synthesis become significantly higher 24 h after serum stimulation. Therefore transformed cells have higher rates of proliferation and grow to higher densities, but display characteristics common to normal cells because rates of protein synthesis decrease and protein degradation increase as a function of cell density. We conclude that higher basal rates of protein synthesis and growth with retention of the normal proliferative response to serum result from the pleiotropic nature of ras transformation, whereas lower rates of protein degradation and increased sensitivity to serum and insulin imply a direct regulatory role for ras. PMID:1575687

  13. Mitochondrial fusion and ERK activity regulate steroidogenic acute regulatory protein localization in mitochondria.

    PubMed

    Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J; Poderoso, Cecilia

    2014-01-01

    The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR.

  14. Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

    PubMed Central

    Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

    2014-01-01

    The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

  15. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness

    PubMed Central

    Wu, Guiqin; Shi, Fengying; Liu, Aiqiao; Yang, Ning

    2016-01-01

    Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57) would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 (SLC25A5) and down-regulated translocator protein (TSPO) would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF). In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation. PMID:28006025

  16. Evolution of context dependent regulation by expansion of feast/famine regulatory proteins

    SciTech Connect

    Plaisier, Christopher L.; Lo, Fang -Yin; Ashworth, Justin; Brooks, Aaron N.; Beer, Karlyn D.; Kaur, Amardeep; Pan, Min; Reiss, David J.; Facciotti, Marc T.; Baliga, Nitin S.

    2014-11-14

    Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions that mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.

  17. Evolution of context dependent regulation by expansion of feast/famine regulatory proteins

    DOE PAGES

    Plaisier, Christopher L.; Lo, Fang -Yin; Ashworth, Justin; ...

    2014-11-14

    Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions thatmore » mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.« less

  18. Epstein-Barr Virus BGLF4 Kinase Suppresses the Interferon Regulatory Factor 3 Signaling Pathway▿ †

    PubMed Central

    Wang, Jiin-Tarng; Doong, Shin-Lian; Teng, Shu-Chun; Lee, Chung-Pei; Tsai, Ching-Hwa; Chen, Mei-Ru

    2009-01-01

    The BGLF4 protein kinase of Epstein-Barr virus (EBV) is a member of the conserved family of herpesvirus protein kinases which, to some extent, have a function similar to that of the cellular cyclin-dependent kinase in regulating multiple cellular and viral substrates. In a yeast two-hybrid screening assay, a splicing variant of interferon (IFN) regulatory factor 3 (IRF3) was found to interact with the BGLF4 protein. This interaction was defined further by coimmunoprecipitation in transfected cells and glutathione S-transferase (GST) pull-down in vitro. Using reporter assays, we show that BGLF4 effectively suppresses the activities of the poly(I:C)-stimulated IFN-β promoter and IRF3-responsive element. Moreover, BGLF4 represses the poly(I:C)-stimulated expression of endogenous IFN-β mRNA and the phosphorylation of STAT1 at Tyr701. In searching for a possible mechanism, BGLF4 was shown not to affect the dimerization, nuclear translocation, or CBP recruitment of IRF3 upon poly(I:C) treatment. Notably, BGLF4 reduces the amount of active IRF3 recruited to the IRF3-responsive element containing the IFN-β promoter region in a chromatin immunoprecipitation assay. BGLF4 phosphorylates GST-IRF3 in vitro, but Ser339-Pro340 phosphorylation-dependent, Pin1-mediated downregulation is not responsible for the repression. Most importantly, we found that three proline-dependent phosphorylation sites at Ser123, Ser173, and Thr180, which cluster in a region between the DNA binding and IRF association domains of IRF3, contribute additively to the BGLF4-mediated repression of IRF3(5D) transactivation activity. IRF3 signaling is activated in reactivated EBV-positive NA cells, and the knockdown of BGLF4 further stimulates IRF3-responsive reporter activity. The data presented here thus suggest a novel mechanism by which herpesviral protein kinases suppress host innate immune responses and facilitate virus replication. PMID:19052084

  19. Development of neurodevelopmental disorders: a regulatory mechanism involving bromodomain-containing proteins.

    PubMed

    Li, Junlin; Zhao, Guifang; Gao, Xiaocai

    2013-02-20

    Neurodevelopmental disorders are classified as diseases that cause abnormal functions of the brain or central nervous system. Children with neurodevelopmental disorders show impaired language and speech abilities, learning and memory damage, and poor motor skills. However, we still know very little about the molecular etiology of these disorders. Recent evidence implicates the bromodomain-containing proteins (BCPs) in the initiation and development of neurodevelopmental disorders. BCPs have a particular domain, the bromodomain (Brd), which was originally identified as specifically binding acetyl-lysine residues at the N-terminus of histone proteins in vitro and in vivo. Other domains of BCPs are responsible for binding partner proteins to form regulatory complexes. Once these complexes are assembled, BCPs alter chromosomal states and regulate gene expression. Some BCP complexes bind nucleosomes, are involved in basal transcription regulation, and influence the transcription of many genes. However, most BCPs are involved in targeting. For example, some BCPs function as a recruitment platform or scaffold through their Brds-binding targeting sites. Others are recruited to form a complex to bind the targeting sites of their partners. The regulation mediated by these proteins is especially critical during normal and abnormal development. Mutant BCPs or dysfunctional BCP-containing complexes are implicated in the initiation and development of neurodevelopmental disorders. However, the pathogenic molecular mechanisms are not fully understood. In this review, we focus on the roles of regulatory BCPs associated with neurodevelopmental disorders, including mental retardation, Fragile X syndrome (FRX), Williams syndrome (WS), Rett syndrome and Rubinstein-Taybi syndrome (RTS). A better understanding of the molecular pathogenesis, based upon the roles of BCPs, will lead to screening of targets for the treatment of neurodevelopmental disorders.

  20. Lineage stability and phenotypic plasticity of Foxp3regulatory T cells.

    PubMed

    Hori, Shohei

    2014-05-01

    Regulatory T (Treg) cells expressing the transcription factor forkhead box protein 3 (Foxp3) constitute a unique T-cell lineage committed to suppressive functions. While their differentiation state is remarkably stable in the face of various perturbations from the extracellular environment, they are able to adapt to diverse and fluctuating tissue environments by changing their phenotype. The lineage stability and phenotypic plasticity of Treg cells thus ensure the robustness of self-tolerance and tissue homeostasis. Recent studies have suggested, however, that Treg cells may retain lineage plasticity, the ability to switch their cell fate to various effector T-cell types under certain circumstances such as inflammation, a notion that remains highly contentious. While accumulating evidence indicates that some Treg cells can downregulate Foxp3 expression and/or acquire effector T-helper cell-like phenotypes, results from my laboratory have shown that Treg cells retain epigenetic memory of, and thus remain committed to, Foxp3 expression and suppressive functions despite such phenotypic plasticity. It has also become evident that Foxp3 can be promiscuously and transiently expressed in activated T cells. Here, I argue that the current controversy stems partly from the lack of the lineage specificity of Foxp3 expression and also from the confusion between phenotypic plasticity and lineage plasticity, and discuss implications of our findings in Treg cell fate determination and maintenance.

  1. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

    PubMed Central

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  2. Quercetin and vitamin E attenuate Bonny Light crude oil-induced alterations in testicular apoptosis, stress proteins and steroidogenic acute regulatory protein in Wistar rats.

    PubMed

    Ebokaiwe, Azubuike P; Mathur, Premendu P; Farombi, Ebenezer O

    2016-10-01

    Studies have shown the reproductive effects of Bonny Light crude oil (BLCO) via the mechanism of oxidative stress and testicular apoptosis. We investigated the protective role of quercetin and vitamin E on BLCO-induced testicular apoptosis. Experimental rats were divided into four groups of four each. Animals were orally administered 2 ml/kg corn oil (control: group 1), BLCO-800 mg/kg body weight + 10 mg/kg quercetin (group 2), BLCO-800 mg/kg body weight + 50 mg/kg vitamin E (group 3) and BLCO-800 mg/kg body weight only (group 4) for 7 d. Protein levels of caspase 3, FasL, NF-kB, steroidogenic acute regulatory protein and stress response proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunofluorescence staining was used to quantify the expression of caspase 3, FasL and NF-kB. Apoptosis was quantified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptosis-related proteins by 50% and above after 7 d following BLCO exposure and a concomitant increase in expression of caspase 3, FasL and NF-kB expression by immunofluorescence staining. Apoptosis showed a significant increase in TUNEL positive cells. Co-administration with quercetin or vitamin E reversed BLCO-induced apoptosis and levels of stress protein, relative to control. These findings suggest that quercetin and vitamin E may confer protection against BLCO-induced testicular oxidative stress-related apoptosis.

  3. Hormone-dependent expression of a steroidogenic acute regulatory protein natural antisense transcript in MA-10 mouse tumor Leydig cells.

    PubMed

    Castillo, Ana Fernanda; Fan, Jinjiang; Papadopoulos, Vassilios; Podestá, Ernesto J

    2011-01-01

    Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3'-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5' and 3' RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis.

  4. Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

    PubMed Central

    Castillo, Ana Fernanda; Fan, Jinjiang; Papadopoulos, Vassilios; Podestá, Ernesto J.

    2011-01-01

    Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis. PMID:21829656

  5. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    SciTech Connect

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  6. FOXP3+ regulatory T-cells in chronic kidney disease: molecular pathways and clinical implications.

    PubMed

    Meier, Pascal

    2009-01-01

    CD4+/FOXP3+ regulatory T-cells (Tregs) are essential for the maintenance of self-tolerance and Tregs deficiency results in spontaneous autoimmunity in both mice and humans. The forkhead box P3 (FOXP3) expression is required for both survival of Tregs precursors as well as their function. This suggests that Tregs may use multiple mechanisms to limit autoimmunity and may reflect functional heterogeneity among Tregs subsets that localize to distinct tissue environments. Both cell contact- and cytokine-based immunosuppressive mechanisms would require that Tregs be in close proximity to their targets. The fundamental regulatory activity that can be consistently demonstrated by Tregs in vivo and in vitro has stimulated great interest in developing novel strategies for treating ongoing inflammatory conditions. Patients with end-stage kidney disease (ESKD) are known to display a cellular immune dysfunction. Uremic solutes that accumulate during ESKD may be involved in these processes. In these patients, oxidative stress induced by oxLDL may increase Tregs sensitivity to Fas-mediated apoptosis in part as a consequence of 26S proteasome activation. The 26S proteasome, an ATP-dependent multisubunit protease complex found in the cytoplasm and in the nucleus of all eukaryotic cells, constitutes the central proteolytic machinery of the ubiquitin/proteasome system. Considering the effect of uremia and oxLDL, Tregs from patients with ESKD exhibit early cell-cycle arrest and become apoptotic. These phenomena are the consequence of the oxLDL inhibited proteasome proteolytic activity of p27(Kipl) and Bax proteins in Tregs. This may be one mechanistic explanation of the cellular immune dysfunction in patients with ESKD and may have important implications in clinics, since this response could contribute to the micro-inflammation and atherogenesis encountered in this population.

  7. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  8. Ikkepsilon regulates viral-induced interferon regulatory factor-3 activation via a redox-sensitive pathway

    SciTech Connect

    Indukuri, Hemalatha; Castro, Shawn M.; Liao, S.-M.; Feeney, Lee Ann; Dorsch, Marion; Coyle, Anthony J.; Garofalo, Roberto P.; Brasier, Allan R.; Casola, Antonella . E-mail: ancasola@utmb.edu

    2006-09-15

    Respiratory syncytial virus (RSV)-induced chemokine gene expression occurs through the activation of a subset of transcription factors, including Interferon Regulatory Factor (IRF)-3. In this study, we have investigated the signaling pathway leading to RSV-induced IRF-3 activation and whether it is mediated by intracellular reactive oxygen species (ROS) generation. Our results show that RSV infection induces expression and catalytic activity of IKK{epsilon}, a noncanonical IKK-like kinase. Expression of a kinase-inactive IKK{epsilon} blocks RSV-induced IRF-3 serine phosphorylation, nuclear translocation and DNA-binding, leading to inhibition of RANTES gene transcription, mRNA expression and protein synthesis. Treatment of alveolar epithelial cells with antioxidants or with NAD(P)H oxidase inhibitors abrogates RSV-induced chemokine secretion, IRF-3 phosphorylation and IKK{epsilon} induction, indicating that ROS generation plays a fundamental role in the signaling pathway leading to IRF-3 activation, therefore, identifying a novel molecular target for the development of strategies aimed to modify the inflammatory response associated with RSV infection of the lung.

  9. Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

    PubMed Central

    Kong, Lingbao; Fujimoto, Akira; Nakamura, Mariko; Aoyagi, Haruyo; Matsuda, Mami; Watashi, Koichi; Suzuki, Ryosuke; Arita, Minetaro; Yamagoe, Satoshi; Dohmae, Naoshi; Suzuki, Takehiro; Sakamaki, Yuriko; Ichinose, Shizuko; Suzuki, Tetsuro; Wakita, Takaji

    2016-01-01

    ABSTRACT It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication

  10. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins

    PubMed Central

    Wilson, Rashaun S.; Swatek, Kirby N.; Thelen, Jay J.

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants. PMID:27242818

  11. Regulatory mechanisms of exoribonuclease PNPase and regulatory small RNA on T3SS of dickeya dadantii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we ...

  12. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues.

    PubMed

    Suzuki, Takashi; Swift, Larry L

    2016-06-03

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5'-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity.

  13. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues

    PubMed Central

    Suzuki, Takashi; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5′-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5′-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  14. The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli.

    PubMed Central

    Calvo, J M; Matthews, R G

    1994-01-01

    The leucine-responsive regulatory protein (Lrp) regulates the expression of more than 40 genes and proteins in Escherichia coli. Among the operons that are positively regulated by Lrp are operons involved in amino acid biosynthesis (ilvIH, serA)), in the biosynthesis of pili (pap, fan, fim), and in the assimilation of ammonia (glnA, gltBD). Negatively regulated operons include operons involved in amino acid catabolism (sdaA, tdh) and peptide transport (opp) and the operon coding for Lrp itself (lrp). Detailed studies of a few members of the regulon have shown that Lrp can act directly to activate or repress transcription of target operons. A substantial fraction of operons regulated by Lrp are also regulated by leucine, and the effect of leucine on expression of these operons requires a functional Lrp protein. The patterns of regulation are surprising and interesting: in some cases activation or repression mediated by Lrp is antagonized by leucine, in other cases Lrp-mediated activation or repression is potentiated by leucine, and in still other cases leucine has no effect on Lrp-mediated regulation. Current research is just beginning to elucidate the detailed mechanisms by which Lrp can mediate such a broad spectrum of regulatory effects. Our view of the role of Lrp in metabolism may change as more members of the regulon are identified and their regulation characterized, but at this point Lrp seems to be important in regulating nitrogen metabolism and one-carbon metabolism, permitting adaptations to feast and to famine. PMID:7968922

  15. Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  16. An account of fungal 14-3-3 proteins.

    PubMed

    Kumar, Ravinder

    2017-02-24

    14-3-3s are a group of relatively low molecular weight, acidic, dimeric, protein(s) conserved from single-celled yeast to multicellular vertebrates including humans. Despite lacking catalytic activity, these proteins have been shown to be involved in multiple cellular processes. Apart from their role in normal cellular physiology, recently these proteins have been implicated in various medical consequences. In this present review, fungal 14-3-3 protein localization, interactions, transcription, regulation, their role in the diverse cellular process including DNA duplication, cell cycle, protein trafficking or secretion, apoptosis, autophagy, cell viability under stress, gene expression, spindle positioning, role in carbon metabolism have been discussed. In the end, I also highlighted various roles of yeasts 14-3-3 proteins in tabular form. Thus this review with primary emphasis on yeast will help in appreciating the significance of 14-3-3 proteins in cell physiology.

  17. Molecular mechanism underlying the regulatory specificity of a Drosophila homeodomain protein that specifies myoblast identity

    PubMed Central

    Busser, Brian W.; Shokri, Leila; Jaeger, Savina A.; Gisselbrecht, Stephen S.; Singhania, Aditi; Berger, Michael F.; Zhou, Bo; Bulyk, Martha L.; Michelson, Alan M.

    2012-01-01

    A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, we first applied genome-wide mRNA expression profiling to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh). Next, we used protein-binding microarrays to define the sequences that are bound by Slou, Msh and other HD TFs that have mesodermal expression. These studies revealed that a large class of HDs, including Slou and Msh, predominantly recognize TAAT core sequences but that each HD also binds to unique sites that deviate from this canonical motif. To understand better the regulatory specificity of an individual FC identity HD, we evaluated the functions of atypical binding sites that are preferentially bound by Slou relative to other HDs within muscle enhancers that are either activated or repressed by this TF. These studies showed that Slou regulates the activities of particular myoblast enhancers through Slou-preferred sequences, whereas swapping these sequences for sites that are capable of binding to multiple HD family members does not support the normal regulatory functions of Slou. Moreover, atypical Slou-binding sites are overrepresented in putative enhancers associated with additional Slou-responsive FC genes. Collectively, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and suggest that the diversity of HD binding preferences can confer regulatory specificity. PMID:22296846

  18. Distribution of regulatory subunits of protein kinase A and A kinase anchor proteins (AKAP 95, 150) in rat pinealocytes.

    PubMed

    Koch, M; Korf, H-W

    2002-12-01

    The rat pineal organ is an established model to study signal transduction cascades that are activated by norepinephrine (NE) and cause increases in cAMP levels and stimulation of protein kinase A (PKA). PKA type II catalyzes the phosphorylation of the transcription factor cAMP-response-element-binding protein (CREB) which is essential for the transcriptional induction of the arylalkylamine- N-acetyltransferase (AANAT), the rate limiting enzyme of melatonin biosynthesis. Moreover, PKA may control protein levels and enzyme activity via two PKA-dependent phosphorylation sites in the AANAT molecule. Despite the functional importance of PKA very little is known about the distribution of its isoenzymes and of A-kinase anchor proteins (AKAPs) that target the PKA to specific membrane areas and organelles by binding to the regulatory (R) subunits of PKA. We have addressed this problem by demonstrating the R subunits alpha and beta of PKA type I and II and two AKAPs (150 and 95) in NE-stimulated and untreated rat pinealocytes by immunoblot and immunocytochemistry. The immunoreactions (IR) of all four R subunits were confined to granules evenly distributed in the pinealocyte cytoplasm. Immunoreactions of RIIalpha and RIIbeta were stronger than those of RIalpha and RIbeta. AKAP 150-IR was concentrated at the cell periphery; AKAP 95-IR was restricted to the nucleus. Amount and subcellular distribution of the immunoreactions of all proteins investigated did not change upon NE stimulation. A substantial colocalization was observed between RII-subunits and AKAP 150-IR, suggesting that, in rat pinealocytes, AKAP 150 primarily anchors the R subunits of PKA II.

  19. E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma.

    PubMed

    Obeid, Jean-Pierre; Zeidan, Youssef H; Zafar, Nawal; El Hokayem, Jimmy

    2017-02-18

    E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

  20. cAMP-dependent posttranscriptional regulation of steroidogenic acute regulatory (STAR) protein by the zinc finger protein ZFP36L1/TIS11b.

    PubMed

    Duan, Haichuan; Cherradi, Nadia; Feige, Jean-Jacques; Jefcoate, Colin

    2009-04-01

    Star is expressed in steroidogenic cells as 3.5- and 1.6-kb transcripts that differ only in their 3'-untranslated regions (3'-UTR). In mouse MA10 testis and Y-1 adrenal lines, Br-cAMP preferentially stimulates 3.5-kb mRNA. ACTH is similarly selective in primary bovine adrenocortical cells. The 3.5-kb form harbors AU-rich elements (AURE) in the extended 3'-UTR, which enhance turnover. After peak stimulation of 3.5-kb mRNA, degradation is seen. Star mRNA turnover is enhanced by the zinc finger protein ZFP36L1/TIS11b, which binds to UAUUUAUU repeats in the extended 3'-UTR. TIS11b is rapidly stimulated in each cell type in parallel with Star mRNA. Cotransfection of TIS11b selectively decreases cytomegalovirus-promoted Star mRNA and luciferase-Star 3'-UTR reporters harboring the extended 3'-UTR. Direct complex formation was demonstrated between TIS11b and the extended 3'-UTR of the 3.5-kb Star. AURE mutations revealed that TIS11b-mediated destabilization required the first two UAUUUAUU motifs. HuR, which also binds AURE, did not affect Star expression. Targeted small interfering RNA knockdown of TIS11b specifically enhanced stimulation of 3.5-kb Star mRNA in bovine adrenocortical cells, MA-10, and Y-1 cells but did not affect the reversals seen after peak stimulation. Direct transfection of Star mRNA demonstrated that Br-cAMP stimulated a selective turnover of 3.5-kb mRNA independent of AURE, which may correspond to these reversal processes. Steroidogenic acute regulatory (STAR) protein induction was halved by TIS11b knockdown, concomitant with decreased cholesterol metabolism. TIS11b suppression of 3.5-kb mRNA is therefore surprisingly coupled to enhanced Star translation leading to increased cholesterol metabolism.

  1. Preparation and crystallization of the stimulatory and inhibitory complexes of GTP cyclohydrolase I and its feedback regulatory protein GFRP.

    PubMed

    Maita, N; Okada, K; Hirotsu, S; Hatakeyama, K; Hakoshima, T

    2001-08-01

    Mammalian GTP cyclohydrolase I is a decameric enzyme in the first and rate-limiting step in the biosynthesis of tetrahydrobiopterin, which is an essential cofactor for enzymes producing neurotransmitters such as catecholamines and for nitric oxide synthases. The enzyme is dually regulated by its feedback regulatory protein GFRP in the presence of its stimulatory effector phenylalanine and its inhibitory effector biopterin. Here, both the stimulatory and inhibitory complexes of rat GTP cyclohydrolase I bound to GFRP were crystallized by vapour diffusion. Diffraction data sets at resolutions of 3.0 and 2.64 A were collected for the stimulatory and inhibitory complexes, respectively. Each complex consists of two GTPCHI pentamer rings and two GFRP pentamer rings, with pseudo-52 point-group symmetry.

  2. Sterol Regulatory Element Binding Protein Is a Principal Regulator of Anaerobic Gene Expression in Fission Yeast†

    PubMed Central

    Todd, Bridget L.; Stewart, Emerson V.; Burg, John S.; Hughes, Adam L.; Espenshade, Peter J.

    2006-01-01

    Fission yeast sterol regulatory element binding protein (SREBP), called Sre1p, functions in an oxygen-sensing pathway to allow adaptation to fluctuating oxygen concentrations. The Sre1p-Scp1p complex responds to oxygen-dependent sterol synthesis as an indirect measure of oxygen availability. To examine the role of Sre1p in anaerobic gene expression in Schizosaccharomyces pombe, we performed transcriptional profiling experiments after a shift to anaerobic conditions for 1.5 h. Of the 4,940 genes analyzed, expression levels of 521 (10.5%) and 686 (13.9%) genes were significantly increased and decreased, respectively, under anaerobic conditions. Sre1p controlled 68% of genes induced ≥2-fold. Oxygen-requiring biosynthetic pathways for ergosterol, heme, sphingolipid, and ubiquinone were primary targets of Sre1p. Induction of glycolytic genes and repression of mitochondrial oxidative phosphorylation genes largely did not require Sre1p. Using chromatin immunoprecipitation, we demonstrated that Sre1p acts directly at target gene promoters and stimulates its own transcription under anaerobic conditions. sre1+ promoter analysis identified two DNA elements that are both necessary and sufficient for oxygen-dependent, Sre1p-dependent transcription. Interestingly, these elements are homologous to sterol regulatory elements bound by mammalian SREBP, highlighting the evolutionary conservation between Sre1p and SREBP. We conclude that Sre1p is a principal activator of anaerobic gene expression, upregulating genes required for nonrespiratory oxygen consumption. PMID:16537923

  3. Arthritis protective regulatory potential of self–heat shock protein cross-reactive T cells

    PubMed Central

    van Eden, Willem; Wendling, Uwe; Paul, Liesbeth; Prakken, Berent; van Kooten, Peter; van der Zee, Ruurd

    2000-01-01

    Immunization with heat shock proteins has protective effects in models of induced arthritis. Analysis has shown a reduced synovial inflammation in such protected animals. Adoptive transfer and immunization with selected T cell epitopes (synthetic peptides) have indicated the protection to be mediated by T cells directed to conserved hsp epitopes. This was shown first for mycobacterial hsp60 and later for mycobacterial hsp70. Fine specificity analysis showed that such T cells were cross-reactive with the homologous self hsp. Therefore protection by microbial hsp reactive T cells can be by cross-recognition of self hsp overexpressed in the inflamed tissue. Preimmunization with hsp leads to a relative expansion of such self hsp cross-responsive T cells. The regulatory nature of such T cells may originate from mucosal tolerance maintained by commensal flora derived hsp or from partial activation through recognition of self hsp as a partial agonist (Altered Peptide Ligand) or in the absence of proper costimulation. Recently, we reported the selective upregulation of B7.2 on microbial hsp60 specific T cells in response to self hsp60. Through a preferred interaction with CTLA-4 on proinflammatory T cells this may constitute an effector mechanism of regulation. Also, regulatory T cells produced IL10. PMID:11189451

  4. Regulatory Advances in 11 Sub-Saharan Countries in Year 3 of the African Health Profession Regulatory Collaborative for Nurses and Midwives (ARC).

    PubMed

    Dynes, Michelle; Tison, Laura; Johnson, Carla; Verani, Andre; Zuber, Alexandra; Riley, Patricia L

    2016-01-01

    Sub-Saharan Africa carries the greatest burden of the HIV pandemic. Enhancing the supply and use of human resources through policy and regulatory reform is a key action needed to improve the quality of HIV services in this region. In year 3 of the African Health Profession Regulatory Collaborative for Nurses and Midwives (ARC), a President's Emergency Plan for AIDS Relief initiative, 11 country teams of nursing and midwifery leaders ("Quads") received small grants to carry out regulatory improvement projects. Four countries advanced a full stage on the Regulatory Function Framework (RFF), a staged capability maturity model used to evaluate progress in key regulatory functions. While the remaining countries did not advance a full stage on the RFF, important gains were noted. The year-3 evaluation highlighted limitations of the ARC evaluation strategy to capture nuanced progress and provided insight into how the RFF might be adapted for future use.

  5. New polymorphisms for the BoLA-DRB3 upstream regulatory region.

    PubMed

    Ripoli, M V; Villegas-Castagnasso, E E; Peral-Garcia, P; Giovambattista, G

    2005-08-01

    Two new alleles, named BoLA-DRB3-P*06 and BoLA-DRB3-P*07, have been identified for the upstream regulatory region of the BoLA-DRB3 gene. The 228-bp nucleotide sequences of the promoter comprising the W, X, Y, CAAT and TATA regulatory boxes were analysed. The BoLA-DRB3-P*06 exhibits one insertion between the W and X boxes, and one transition between the X and Y boxes. On the other hand, the BoLA-DRB3-P*07 showed one insertion in the X box.

  6. Molecular Dynamics Simulations and Structural Analysis of Giardia duodenalis 14-3-3 Protein-Protein Interactions.

    PubMed

    Cau, Ylenia; Fiorillo, Annarita; Mori, Mattia; Ilari, Andrea; Botta, Maurizo; Lalle, Marco

    2015-12-28

    Giardiasis is a gastrointestinal diarrheal illness caused by the protozoan parasite Giardia duodenalis, which affects annually over 200 million people worldwide. The limited antigiardial drug arsenal and the emergence of clinical cases refractory to standard treatments dictate the need for new chemotherapeutics. The 14-3-3 family of regulatory proteins, extensively involved in protein-protein interactions (PPIs) with pSer/pThr clients, represents a highly promising target. Despite homology with human counterparts, the single 14-3-3 of G. duodenalis (g14-3-3) is characterized by a constitutive phosphorylation in a region critical for target binding, thus affecting the function and the conformation of g14-3-3/clients interaction. However, to approach the design of specific small molecule modulators of g14-3-3 PPIs, structural elucidations are required. Here, we present a detailed computational and crystallographic study exploring the implications of g14-3-3 phosphorylation on protein structure and target binding. Self-Guided Langevin Dynamics and classical molecular dynamics simulations show that phosphorylation affects locally and globally g14-3-3 conformation, inducing a structural rearrangement more suitable for target binding. Profitable features for g14-3-3/clients interaction were highlighted using a hydrophobicity-based descriptor to characterize g14-3-3 client peptides. Finally, the X-ray structure of g14-3-3 in complex with a mode-1 prototype phosphopeptide was solved and combined with structure-based simulations to identify molecular features relevant for clients binding to g14-3-3. The data presented herein provide a further and structural understanding of g14-3-3 features and set the basis for drug design studies.

  7. Interaction of protein-bound polysaccharide (PSK) with smooth muscle myosin regulatory light chain.

    PubMed

    Fujii, Toshihiro; Kunimatsu, Mitoshi

    2003-06-01

    The interaction of a protein-bound polysaccharide (PSK) isolated from Basidiomycetes with smooth muscle myosin components was evaluated by limited digestion, urea/glycerol gel electrophoresis, affinity chromatography and overlay assay using a peptide array. PSK was bound to the regulatory light chain (RLC) of myosin, but not to the essential light chain. The binding to PSK was definitely observed for unphosphorylated RLC, compared to phosphorylated one. From the amino acid sequence of the RLC, 490 peptides were synthesized on a cellulose membrane. Overlay assays showed that the PSK-binding on the molecule of RLC were localized in the N- and C-terminal basic regions and these sites were conserved in RLC from the human smooth muscle and nonmuscle cells.

  8. Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Richardson, Dale N.; Wiehe, Thomas

    Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

  9. Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway.

    PubMed

    Murthy, Karnam S; Zhou, Huiping; Grider, John R; Brautigan, David L; Eto, Masumi; Makhlouf, Gabriel M

    2003-08-15

    Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagammai3 with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44+/-5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28+/-3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities

  10. Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway.

    PubMed Central

    Murthy, Karnam S; Zhou, Huiping; Grider, John R; Brautigan, David L; Eto, Masumi; Makhlouf, Gabriel M

    2003-01-01

    Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagammai3 with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44+/-5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28+/-3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities

  11. Ethanol utilization regulatory protein: profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion.

    PubMed Central

    Nicholas, H. B.; Persson, B.; Jörnvall, H.; Hempel, J.

    1995-01-01

    The suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 146:145-158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same composition. This level of similarity fails to support the suggested gene fusion. PMID:8580855

  12. Construction of a Comprehensive Protein-Protein Interaction Map for Vitiligo Disease to Identify Key Regulatory Elements: A Systemic Approach.

    PubMed

    Malhotra, Anvita Gupta; Jha, Mohit; Singh, Sudha; Pandey, Khushhali M

    2017-03-13

    Vitiligo is an idiopathic disorder characterized by depigmented patches on the skin due to progressive loss of melanocytes. Several genetic, immunological, and pathophysiological investigations have established vitiligo as a polygenetic disorder with multifactorial etiology. However, no definite model explaining the interplay between these causative factors has been established hitherto. Therefore, we studied the disorder at the system level to identify the key proteins involved by exploring their molecular connectivity in terms of topological parameters. The existing research data helped us in collating 215 proteins involved in vitiligo onset or progression. Interaction study of these proteins leads to a comprehensive vitiligo map with 4845 protein nodes linked with 107,416 edges. Based on centrality measures, a backbone network with 500 nodes has been derived. This has presented a clear overview of the proteins and processes involved and the crosstalk between them. Clustering backbone proteins revealed densely connected regions inferring major molecular interaction modules essential for vitiligo. Finally, a list of top order proteins that play a key role in the disease pathomechanism has been formulated. This includes SUMO2, ESR1, COPS5, MYC, SMAD3, and Cullin proteins. While this list is in fair agreement with the available literature, it also introduces new candidate proteins that can be further explored. A subnetwork of 64 vitiligo core proteins was built by analyzing the backbone and seed protein networks. Our finding suggests that the topology, along with functional clustering, provides a deep insight into the behavior of proteins. This in turn aids in the illustration of disease condition and discovery of significant proteins involved in vitiligo.

  13. Heat shock proteins (HSPs) in the homeostasis of regulatory T cells (Tregs)

    PubMed Central

    Marek-Trzonkowska, Natalia; Trzonkowski, Piotr; Siebert, Janusz

    2016-01-01

    Heat shock proteins (HSPs) belong to the family of conservative polypeptides with a high homology of the primary structure. The uniqueness of this family lies in their ability to interact with a large number of different proteins and provide protection from cellular and environmental stress factors as molecular chaperones to keep protein homeostasis. While intracellular HSPs play a mainly protective role, extracellular or membrane-bound HSPs mediate immunological functions and immunomodulatory activity. In immune system are subsets of cells including regulatory T cells (Tregs) with suppressive functions. HSPs are implicated in the function of innate and adaptive immune systems, stimulate T lymphocyte proliferation and immunomodulatory functions, increase the effectiveness of cross-presentation of antigens, and induce the secretion of cytokines. HSPs are also important in the induction, proliferation, suppressive function, and cytokine production of Tregs, which are a subset of CD4+ T cells maintaining peripheral tolerance. Together HSPs and Tregs are potential tools for future clinical interventions in autoimmune disease. PMID:27833451

  14. Protein Phosphatase 2A in the Regulatory Network Underlying Biotic Stress Resistance in Plants

    PubMed Central

    Durian, Guido; Rahikainen, Moona; Alegre, Sara; Brosché, Mikael; Kangasjärvi, Saijaliisa

    2016-01-01

    Biotic stress factors pose a major threat to plant health and can significantly deteriorate plant productivity by impairing the physiological functions of the plant. To combat the wide range of pathogens and insect herbivores, plants deploy converging signaling pathways, where counteracting activities of protein kinases and phosphatases form a basic mechanism for determining appropriate defensive measures. Recent studies have identified Protein Phosphatase 2A (PP2A) as a crucial component that controls pathogenesis responses in various plant species. Genetic, proteomic and metabolomic approaches have underscored the versatile nature of PP2A, which contributes to the regulation of receptor signaling, organellar signaling, gene expression, metabolic pathways, and cell death, all of which essentially impact plant immunity. Associated with this, various PP2A subunits mediate post-translational regulation of metabolic enzymes and signaling components. Here we provide an overview of protein kinase/phosphatase functions in plant immunity signaling, and position the multifaceted functions of PP2A in the tightly inter-connected regulatory network that controls the perception, signaling and responding to biotic stress agents in plants. PMID:27375664

  15. Role of the steroidogenic acute regulatory protein in health and disease

    PubMed Central

    Manna, Pulak R.; Stetson, Cloyce L.; Slominski, Andrzej T.; Pruitt, Kevin

    2015-01-01

    Steroid hormones are an important class of regulatory molecules that are synthesized in steroidogenic cells of the adrenal, ovary, testis, placenta, brain and skin, and influence a spectrum of developmental and physiological processes. The steroidogenic acute regulatory protein (STAR) predominantly mediates the rate-limiting step in steroid biosynthesis, i.e., the transport of the substrate of all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane. At the inner membrane, cytochrome P450 cholesterol side chain cleavage enzyme cleaves the cholesterol side-chain to form the first steroid, pregnenolone, which is converted by a series of enzymes to various steroid hormones in specific tissues. Both basic and clinical evidence have demonstrated the crucial involvement of the STAR protein in the regulation of steroid biosynthesis. Multiple levels of regulation impinge on STAR action. Recent findings demonstrate that hormone-sensitive lipase, through its action on the hydrolysis of cholesteryl esters, plays an important role in regulating StAR expression and steroidogenesis which involve the liver X receptor pathway. Activation of the latter influences macrophage cholesterol efflux that is a key process in the prevention of atherosclerotic cardiovascular disease. Appropriate regulation of steroid hormones is vital for proper functioning of many important biological activities, which are also paramount for geriatric populations to live longer and healthier. This review summarizes the current level of understanding on tissue-specific and hormone-induced regulation of STAR expression and steroidogenesis, and provides insights into a number of cholesterol and/or steroid coupled physiological and pathophysiological consequences. PMID:26271515

  16. High concentrations of protein test substances may have non-toxic effects on Daphnia magna: implications for regulatory study designs and ecological risk assessments for GM crops.

    PubMed

    Raybould, Alan; Burns, Andrea; Hamer, Mick

    2014-01-01

    Laboratory testing for possible adverse effects of insecticidal proteins on non-target organisms (NTOs) is an important part of many ecological risk assessments for regulatory decision-making about the cultivation of insect-resistant genetically modified (IRGM) crops. To increase confidence in the risk assessments, regulatory guidelines for effects testing specify that representative surrogate species for NTOs are exposed to concentrations of insecticidal proteins that are in excess of worst-case predicted exposures in the field. High concentrations in effects tests are achieved by using protein test substances produced in microbes, such as Escherichia coli. In a study that exposed Daphnia magna to a single high concentration of a microbial test substance containing Vip3Aa20, the insecticidal protein in MIR162 maize, small reductions in growth were observed. These effects were surprising as many other studies strongly suggest that the activity of Vip3Aa20 is limited to Lepidoptera. A plausible explanation for the effect on growth is that high concentrations of test substance have a non-toxic effect on Daphnia, perhaps by reducing its feeding rate. A follow-up study tested that hypothesis by exposing D. magna to several concentrations of Vip3Aa20, and a high concentration of a non-toxic protein, bovine serum albumin (BSA). Vip3Aa20 and BSA had sporadic effects on the reproduction and growth of D. magna. The pattern of the effects suggests that they result from non-toxic effects of high concentrations of protein, and not from toxicity. The implications of these results for regulatory NTO effects testing and ERA of IRGM crops are discussed.

  17. High concentrations of protein test substances may have non-toxic effects on Daphnia magna: Implications for regulatory study designs and ecological risk assessments for GM crops

    PubMed Central

    Raybould, Alan; Burns, Andrea; Hamer, Mick

    2014-01-01

    Laboratory testing for possible adverse effects of insecticidal proteins on non-target organisms (NTOs) is an important part of many ecological risk assessments for regulatory decision-making about the cultivation of insect-resistant genetically modified (IRGM) crops. To increase confidence in the risk assessments, regulatory guidelines for effects testing specify that representative surrogate species for NTOs are exposed to concentrations of insecticidal proteins that are in excess of worst-case predicted exposures in the field. High concentrations in effects tests are achieved by using protein test substances produced in microbes, such as Escherichia coli. In a study that exposed Daphnia magna to a single high concentration of a microbial test substance containing Vip3Aa20, the insecticidal protein in MIR162 maize, small reductions in growth were observed. These effects were surprising as many other studies strongly suggest that the activity of Vip3Aa20 is limited to Lepidoptera. A plausible explanation for the effect on growth is that high concentrations of test substance have a non-toxic effect on Daphnia, perhaps by reducing its feeding rate. A follow-up study tested that hypothesis by exposing D. magna to several concentrations of Vip3Aa20, and a high concentration of a non-toxic protein, bovine serum albumin (BSA). Vip3Aa20 and BSA had sporadic effects on the reproduction and growth of D. magna. The pattern of the effects suggests that they result from non-toxic effects of high concentrations of protein, and not from toxicity. The implications of these results for regulatory NTO effects testing and ERA of IRGM crops are discussed. PMID:25523175

  18. Transmembrane protein 88: a Wnt regulatory protein that specifies cardiomyocyte development

    PubMed Central

    Palpant, Nathan J.; Pabon, Lil; Rabinowitz, Jeremy S.; Hadland, Brandon K.; Stoick-Cooper, Cristi L.; Paige, Sharon L.; Bernstein, Irwin D.; Moon, Randall T.; Murry, Charles E.

    2013-01-01

    Genetic regulation of the cell fate transition from lateral plate mesoderm to the specification of cardiomyocytes requires suppression of Wnt/β-catenin signaling, but the mechanism for this is not well understood. By analyzing gene expression and chromatin dynamics during directed differentiation of human embryonic stem cells (hESCs), we identified a suppressor of Wnt/β-catenin signaling, transmembrane protein 88 (TMEM88), as a potential regulator of cardiovascular progenitor cell (CVP) specification. During the transition from mesoderm to the CVP, TMEM88 has a chromatin signature of genes that mediate cell fate decisions, and its expression is highly upregulated in advance of key cardiac transcription factors in vitro and in vivo. In early zebrafish embryos, tmem88a is expressed broadly in the lateral plate mesoderm, including the bilateral heart fields. Short hairpin RNA targeting of TMEM88 during hESC cardiac differentiation increases Wnt/β-catenin signaling, confirming its role as a suppressor of this pathway. TMEM88 knockdown has no effect on NKX2.5 or GATA4 expression, but 80% of genes most highly induced during CVP development have reduced expression, suggesting adoption of a new cell fate. In support of this, analysis of later stage cell differentiation showed that TMEM88 knockdown inhibits cardiomyocyte differentiation and promotes endothelial differentiation. Taken together, TMEM88 is crucial for heart development and acts downstream of GATA factors in the pre-cardiac mesoderm to specify lineage commitment of cardiomyocyte development through inhibition of Wnt/β-catenin signaling. PMID:23924634

  19. High-yield soluble expression, purification and characterization of human steroidogenic acute regulatory protein (StAR) fused to a cleavable Maltose-Binding Protein (MBP).

    PubMed

    Sluchanko, Nikolai N; Tugaeva, Kristina V; Faletrov, Yaroslav V; Levitsky, Dmitrii I

    2016-03-01

    Steroidogenic acute regulatory protein (StAR) is responsible for the rapid delivery of cholesterol to mitochondria where the lipid serves as a source for steroid hormones biosynthesis in adrenals and gonads. Despite many successful investigations, current understanding of the mechanism of StAR action is far from being completely clear. StAR was mostly obtained using denaturation/renaturation or in minor quantities in a soluble form at decreased temperatures that, presumably, limited the possibilities for its consequent detailed exploration. In our hands, existing StAR expression constructs could be bacterially expressed almost exclusively as insoluble forms, even upon decreased expression temperatures and in specific strains of Escherichia coli, and isolated protein tended to aggregate and was difficult to handle. To maximize the yield of soluble protein, optimized StAR sequence encompassing functional domain STARD1 (residues 66-285) was fused to the C-terminus of His-tagged Maltose-Binding Protein (MBP) with the possibility to cleave off the whole tag by 3C protease. The developed protocol of expression and purification comprising of a combination of subtractive immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography allowed us to obtain up to 25 mg/1 L culture of completely soluble StAR protein, which was (i) homogenous according to SDS-PAGE, (ii) gave a single symmetrical peak on a gel-filtration, (iii) showed the characteristic CD spectrum and (iv) pH-dependent ability to bind a fluorescently-labeled cholesterol analogue. We conclude that our strategy provides fully soluble and native StAR protein which in future could be efficiently used for biotechnology and drug discovery aimed at modulation of steroids production.

  20. Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

    PubMed Central

    Breunig, K D; Kuger, P

    1987-01-01

    As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription. Images PMID:2830492

  1. DANGER, a novel regulatory protein of inositol 1,4,5-trisphosphate-receptor activity.

    PubMed

    van Rossum, Damian B; Patterson, Randen L; Cheung, King-Ho; Barrow, Roxanne K; Syrovatkina, Viktoriya; Gessell, Gregory S; Burkholder, Scott G; Watkins, D Neil; Foskett, J Kevin; Snyder, Solomon H

    2006-12-01

    We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP(3)R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP(3)R. DANGER interacts with IP(3)R in vitro and co-immunoprecipitates with IP(3)R from cellular preparations. DANGER robustly enhances Ca(2+)-mediated inhibition of IP(3) RCa(2+) release without affecting IP(3) binding in microsomal assays and inhibits gating in single-channel recordings of IP(3)R. DANGER appears to allosterically modulate the sensitivity of IP(3) RtoCa(2+) inhibition, which likely alters IP(3)R-mediated Ca(2+) dynamics in cells where DANGER and IP(3)R are co-expressed.

  2. Nucleotide-specific recognition of iron-responsive elements by iron regulatory protein 1.

    PubMed

    Selezneva, Anna I; Walden, William E; Volz, Karl W

    2013-09-23

    IRP1 [iron regulatory protein (IRP) 1] is a bifunctional protein with mutually exclusive end-states. In one mode of operation, IRP1 binds iron-responsive element (IRE) stem-loops in messenger RNAs encoding proteins of iron metabolism to control their rate of translation. In its other mode, IRP1 serves as cytoplasmic aconitase to correlate iron availability with the energy and oxidative stress status of the cell. IRP1/IRE binding occurs through two separate interfaces, which together contribute about two-dozen hydrogen bonds. Five amino acids make base-specific contacts and are expected to contribute significantly to binding affinity and specificity of this protein:RNA interaction. In this mutagenesis study, each of the five base-specific amino acids was changed to alter binding at each site. Analysis of IRE binding affinity and translational repression activity of the resulting IRP1 mutants showed that four of the five contact points contribute uniquely to the overall binding affinity of the IRP1:IRE interaction, while one site was found to be unimportant. The stronger-than-expected effect on binding affinity of mutations at Lys379 and Ser681, residues that make contact with the conserved nucleotides G16 and C8, respectively, identified them as particularly critical for providing specificity and stability to IRP1:IRE complex formation. We also show that even though the base-specific RNA-binding residues are not part of the aconitase active site, their substitutions can affect the aconitase activity of holo-IRP1, positively or negatively.

  3. The Multifaceted Activity of the VirF Regulatory Protein in the Shigella Lifestyle

    PubMed Central

    Di Martino, Maria Letizia; Falconi, Maurizio; Micheli, Gioacchino; Colonna, Bianca; Prosseda, Gianni

    2016-01-01

    Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV). The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non-invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis. PMID:27747215

  4. Steroidogenic acute regulatory protein (StAR) overexpression reduces inflammation and insulin resistance in obese mice.

    PubMed

    Qiu, Yanyan; Sui, Xianxian; Cao, Shengxuan; Li, Xiaobo; Ning, Yanxia; Wang, Songmei; Yin, Lianhua; Zhi, Xiuling

    2017-04-12

    Steroidogenic acute regulatory protein (StAR), a mitochondrial cholesterol delivery protein, plays a beneficial role in hyperlipidemia, NAFLD and endothelial inflammation. Elevated circulating fatty acids and low grade inflammation are known as key risk factors of insulin resistance and type 2 diabetes. In the present study, C57BL/6J mice were fed with a HFD and infected with recombinant adenovirus expressing StAR by tail-vein injection. Intraperitoneal glucose/insulin tolerance test was performed to assess the insulin sensitivity. Morphological analysis and intramuscular lipid determination were used to illustrate the adipose hypertrophy and ectopic fat accumulation in skeletal muscle. The levels of inflammatory factor and nitric oxide were determined by ELISA and classic Griess reagent methods respectively. The fatty acids composition was analysis using gas chromatography -mass spectrometry (GC-MS). The expression of genes associated with inflammation and insulin resistance were determined by Western blotting and qPCR to elucidate the underlying mechanism.We demonstrated that StAR overexpression ameliorated insulin resistance and systemic inflammatory response with the reduction of adipose hypertrophy and intramuscular lipid in HFD fed mice. In addition, StAR overexpression increased serum unsaturated fatty acids and PPARγ expression in muscle and adipose tissue of obese mice. In conclusion, StAR may activate PPARγ by increasing unsaturated fatty acids, which leads to a protective role in systemic inflammation and insulin resistance in obese mice. This article is protected by copyright. All rights reserved.

  5. Iron-independent phosphorylation of iron regulatory protein 2 regulates ferritin during the cell cycle.

    PubMed

    Wallander, Michelle L; Zumbrennen, Kimberly B; Rodansky, Eva S; Romney, S Joshua; Leibold, Elizabeth A

    2008-08-29

    Iron regulatory protein 2 (IRP2) is a key iron sensor that post-transcriptionally regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) in mRNAs that encode proteins involved in iron metabolism (e.g. ferritin and transferrin receptor 1). During iron deficiency, IRP2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency IRP2 is degraded by the proteasome. Here, we identify an iron-independent IRP2 phosphorylation site that is regulated by the cell cycle. IRP2 Ser-157 is phosphorylated by Cdk1/cyclin B1 during G(2)/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Ser-157 phosphorylation during G(2)/M reduces IRP2 RNA-binding activity and increases ferritin synthesis, whereas Ser-157 dephosphorylation during mitotic exit restores IRP2 RNA-binding activity and represses ferritin synthesis. These data show that reversible phosphorylation of IRP2 during G(2)/M has a role in modulating the iron-independent expression of ferritin and other IRE-containing mRNAs during the cell cycle.

  6. Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

    PubMed

    Werner, Ernst R; Bahrami, Soheyl; Heller, Regine; Werner-Felmayer, Gabriele

    2002-03-22

    GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine.

  7. GTP cyclohydrolase I feedback regulatory protein-dependent and -independent inhibitors of GTP cyclohydrolase I.

    PubMed

    Yoneyama, T; Wilson, L M; Hatakeyama, K

    2001-04-01

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates the feedback inhibition of GTP cyclohydrolase I activity by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) through protein complex formation. Since guanine and BH4 have a common pyrimidine ring structure, we examined the inhibitory effect of guanine and its analogs on the enzyme activity. Guanine, 8-hydroxyguanine, 8-methylguanine, and 8-bromoguanine inhibited the enzyme activity in a GFRP-dependent and pH-dependent manner and induced complex formation between GTP cyclohydrolase I and GFRP. The type of inhibition by this group is a mixed type. All these properties were shared with BH4. In striking contrast, inhibition by 8-azaguanine and 8-mercaptoguanine was GFRP-independent and pH-independent. The type of inhibition by 8-azaguanine and 8-mercaptoguanine was a competitive type. The two compounds did not induce complex formation between the enzyme and GFRP. These results demonstrate that guanine compounds of the first group bind to the BH4-binding site of the GTP cyclohydrolase I/GFRP complex, whereas 8-azaguanine and 8-mercaptoguanine bind to the active site of the enzyme. Finally, the possible implications in Lesch-Nyhan syndrome and Parkinson diseases of the inhibition of GTP cyclohydrolase I by guanine and 8-hydroxyguanine are discussed.

  8. Dynamic localization of glucokinase and its regulatory protein in hypothalamic tanycytes.

    PubMed

    Salgado, Magdiel; Tarifeño-Saldivia, Estefanía; Ordenes, Patricio; Millán, Carola; Yañez, María José; Llanos, Paula; Villagra, Marcos; Elizondo-Vega, Roberto; Martínez, Fernando; Nualart, Francisco; Uribe, Elena; de Los Angeles García-Robles, María

    2014-01-01

    Glucokinase (GK), the hexokinase involved in glucose sensing in pancreatic β cells, is also expressed in hypothalamic tanycytes, which cover the ventricular walls of the basal hypothalamus and are implicated in an indirect control of neuronal activity by glucose. Previously, we demonstrated that GK was preferentially localized in tanycyte nuclei in euglycemic rats, which has been reported in hepatocytes and is suggestive of the presence of the GK regulatory protein, GKRP. In the present study, GK intracellular localization in hypothalamic and hepatic tissues of the same rats under several glycemic conditions was compared using confocal microscopy and Western blot analysis. In the hypothalamus, increased GK nuclear localization was observed in hyperglycemic conditions; however, it was primarily localized in the cytoplasm in hepatic tissue under the same conditions. Both GK and GKRP were next cloned from primary cultures of tanycytes. Expression of GK by Escherichia coli revealed a functional cooperative protein with a S0.5 of 10 mM. GKRP, expressed in Saccharomyces cerevisiae, inhibited GK activity in vitro with a Ki 0.2 µM. We also demonstrated increased nuclear reactivity of both GK and GKRP in response to high glucose concentrations in tanycyte cultures. These data were confirmed using Western blot analysis of nuclear extracts. Results indicate that GK undergoes short-term regulation by nuclear compartmentalization. Thus, in tanycytes, GK can act as a molecular switch to arrest cellular responses to increased glucose.

  9. MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice

    PubMed Central

    Horie, Takahiro; Nishino, Tomohiro; Baba, Osamu; Kuwabara, Yasuhide; Nakao, Tetsushi; Nishiga, Masataka; Usami, Shunsuke; Izuhara, Masayasu; Sowa, Naoya; Yahagi, Naoya; Shimano, Hitoshi; Matsumura, Shigenobu; Inoue, Kazuo; Marusawa, Hiroyuki; Nakamura, Tomoyuki; Hasegawa, Koji; Kume, Noriaki; Yokode, Masayuki; Kita, Toru; Kimura, Takeshi; Ono, Koh

    2013-01-01

    MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33, which is located within the intron of sterol regulatory element-binding protein (SREBP) 2, controls cholesterol homoeostasis and may be a potential therapeutic target for the treatment of atherosclerosis. Here we show that deletion of miR-33 results in marked worsening of high-fat diet-induced obesity and liver steatosis. Using miR-33−/−Srebf1+/− mice, we demonstrate that SREBP-1 is a target of miR-33 and that the mechanisms leading to obesity and liver steatosis in miR-33−/− mice involve enhanced expression of SREBP-1. These results elucidate a novel interaction between SREBP-1 and SREBP-2 mediated by miR-33 in vivo. PMID:24300912

  10. Iron Regulatory Protein-2 Knockout Increases Perihematomal Ferritin Expression and Cell Viability after Intracerebral Hemorrhage

    PubMed Central

    Chen, Mai; Awe, Olatilewa O.; Chen-Roetling, Jing; Regan, Raymond F.

    2010-01-01

    Iron is deposited in perihematomal tissue after an intracerebral hemorrhage (ICH), and may contribute to oxidative injury. Cell culture studies have demonstrated that enhancing ferritin expression by targeting iron regulatory protein (IRP) binding activity reduces cellular vulnerability to iron and hemoglobin. In order to assess the therapeutic potential of this approach after striatal ICH, the effect of IRP1 or IRP2 gene knockout on ferritin expression and injury was quantified. Striatal ferritin in IRP1 knockout mice was similar to that in wild-type controls three days after stereotactic injection of artificial CSF or autologous blood. Corresponding levels in IRP2 knockouts were increased by 11-fold and 8.4-fold, respectively, compared with wild-type. Protein carbonylation, a sensitive marker of hemoglobin neurotoxicity, was increased by 2.4-fold in blood-injected wild-type striata, was not altered by IRP1 knockout, but was reduced by approximately 60% by IRP2 knockout. Perihematomal cell viability in wild-type mice, assessed by MTT assay, was approximately half of that in contralateral striata at three days, and was significantly increased in IRP2 knockouts but not in IRP1 knockouts. Protection was also observed when hemorrhage was induced by collagenase injection. These results suggest that IRP2 binding activity reduces ferritin expression in the striatum after ICH, preventing an optimal response to elevated local iron concentrations. IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries. PMID:20399759

  11. Dynamic Localization of Glucokinase and Its Regulatory Protein in Hypothalamic Tanycytes

    PubMed Central

    Ordenes, Patricio; Millán, Carola; Yañez, María José; Llanos, Paula; Villagra, Marcos; Elizondo-Vega, Roberto; Martínez, Fernando; Nualart, Francisco; Uribe, Elena; de los Angeles García-Robles, María

    2014-01-01

    Glucokinase (GK), the hexokinase involved in glucose sensing in pancreatic β cells, is also expressed in hypothalamic tanycytes, which cover the ventricular walls of the basal hypothalamus and are implicated in an indirect control of neuronal activity by glucose. Previously, we demonstrated that GK was preferentially localized in tanycyte nuclei in euglycemic rats, which has been reported in hepatocytes and is suggestive of the presence of the GK regulatory protein, GKRP. In the present study, GK intracellular localization in hypothalamic and hepatic tissues of the same rats under several glycemic conditions was compared using confocal microscopy and Western blot analysis. In the hypothalamus, increased GK nuclear localization was observed in hyperglycemic conditions; however, it was primarily localized in the cytoplasm in hepatic tissue under the same conditions. Both GK and GKRP were next cloned from primary cultures of tanycytes. Expression of GK by Escherichia coli revealed a functional cooperative protein with a S0.5 of 10 mM. GKRP, expressed in Saccharomyces cerevisiae, inhibited GK activity in vitro with a Ki 0.2 µM. We also demonstrated increased nuclear reactivity of both GK and GKRP in response to high glucose concentrations in tanycyte cultures. These data were confirmed using Western blot analysis of nuclear extracts. Results indicate that GK undergoes short-term regulation by nuclear compartmentalization. Thus, in tanycytes, GK can act as a molecular switch to arrest cellular responses to increased glucose. PMID:24739934

  12. Chromatin reader L(3)mbt requires the Myb–MuvB/DREAM transcriptional regulatory complex for chromosomal recruitment

    PubMed Central

    Blanchard, Daniel P.; Georlette, Daphne; Antoszewski, Lisa; Botchan, Michael R.

    2014-01-01

    Lethal malignant brain tumors (lmbt) result from the loss of the conserved transcriptional repressor l(3)mbt, in Drosophila melanogaster. Similar mutations in the human homolog L3MBTL1 correlate with some cancers. The protein’s C-terminal MBT repeats bind mono and dimethylated histones in vitro, which could influence recruitment of L3MBTL1 to its target sites. The L(3)mbt chromatin targeting mechanism, however, is controversial and several studies suggest insufficiency or a minor role for histone methylation in determining the site specificity for recruitment. We report that L(3)mbt colocalizes with core members of the Myb–MuvB/DREAM (MMB/DREAM) transcriptional regulatory complex genome-wide, and that L(3)mbt-mediated repression requires this complex in salivary glands and larval brains. Loss of l(3)mbt or of MMB components through mutation cause similar spurious expression of genes, including the transposon regulatory gene piwi, in terminally differentiated cells. The DNA-binding MMB core component Mip120 (Lin54) is required for L(3)mbt recruitment to chromosomes, whereas Mip130 (Lin9) (an MMB core protein) and E2f2 (an MMB transcriptional repressor) are not, but are essential for repression. Cytolocalization experiments suggest the presence of site-specific differential composition of MMB in polytene chromosomes where some loci were bound by a Myb-containing or alternatively, an E2f2 and L(3)mbt form of the complex. PMID:25249635

  13. Evidence for regulatory genes on mouse chromosome 7 that affect the quantitative expression of proteins in the fetal and newborn liver.

    PubMed Central

    Giometti, C S; Gemmell, M A; Taylor, J; Tollaksen, S L; Angeletti, R; Gluecksohn-Waelsch, S

    1992-01-01

    A series of deletions around the albino locus on mouse chromosome 7 is believed to include one or more regulatory genes that control the activities of a cluster of liver enzymes. To further characterize the functions of this region of the mouse genome, we have used quantitative two-dimensional electrophoresis to analyze the effects of two of these deletions, c3H and c14CoS, on the expression of liver proteins. More than 400 distinct protein gene products were quantitated in livers from fetal and newborn wild-type homozygous (cch/cch), heterozygous (cch/c3H or cch/c14CoS), and deletion homozygous (c3H/c3H or c14CoS/c14CoS) mice. Livers of fetal and newborn c3H heterozygotes and homozygous wild-type littermates produced qualitatively identical protein patterns after two-dimensional electrophoresis. In livers of c3H homozygous fetuses, however, abnormal amounts (either increased or decreased relative to homozygous wild-type and heterozygous littermates) of 29 proteins were found. Twenty-eight of these 29 protein anomalies were also found in livers of newborn c3H homozygotes. Livers of fetal and newborn mice homozygous for the c14CoS deletion, which overlaps the c3H deletion and produces a similar phenotype, expressed normal amounts of these proteins. One of the 29 proteins (MSN807) has an amino-terminal sequence similar to a 23-kDa translationally controlled protein abundant in mouse erythroleukemia and sarcoma-180 cells. These results suggest that normal chromosome 7 contains genes, located within the region of the c3H but not the c14CoS deletion, that regulate the abundance of specific proteins in the liver. These proteins cannot be related to the phenotypic alterations shared by the c3H and c14CoS deletions. Images PMID:1549608

  14. Influence of energy supply on expression of genes encoding for lipogenic enzymes and regulatory proteins in growing beef steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty crossbred beef steers were used to determine the effects metabolizable energy (ME) intake and of site and complexity of carbohydrate (CHO) infusion on expression of genes encoding lipogenic enzymes and regulatory proteins in subcutaneous (SC), mesenteric (MES) and omental (OM) adipose. Treatm...

  15. High affinity binding proteins for the regulatory subunit of cAMP-dependent protein kinase: Cloning, characterization and expression of P150 and P75

    SciTech Connect

    Bregman, D.B.

    1989-01-01

    cAMP-dependent protein kinase II-B appears to be adapted for function in the mammalian central nervous system (CNS) via the properties of its regulatory subunit (RII-B). RII-B is selectively expressed in brain, tightly associated with cerebral cortex membranes and avidly complexed by the bovine brain calmodulin-binding protein designated P75. Complexes of RII-B and P75 polypeptides can be purified to near-homogeneity from either membrane or cytosolic fractions of brain homogenates, suggested that the binding protein plays a role in determining the subcellular localization and/or other CNS-specific properties of protein kinase II-B. In general, a single high-affinity RII-B binding protein is expressed in the brains of mammals, but the size of the protein varies (e.g., cow: 75 kDa; rat: 150 kDa). To investigate these non-abundant and CNS-enriched RII-B binding proteins, cDNAs for bovine brain P75 and rat brain P150, the homologue of P75, have been cloned and characterized. The cDNAs were retrieved from bovine and rat lambda gt11 expression libraries using {sup 32}P-RII-B as a functional probe. cDNA inserts subcloned into pIN-IA2 or pET-3b expression plasmids directed the production of partial P75 and P150 polypeptides in E. coli that exhibited RII-B binding activity. P150 cDNAs hybridize with 3 rat mRNAs (7.3, 5.0, 4.2 kb) that are present in brain and lung, but not other tissues. These mRNAs are not detected in fetal brain, but are expressed during the period of post-natal synaptogenesis and in adult rats. The P75 cDNA hybridizes to a 6 kb bovine brain mRNA. Finally, 3'-deletion analysis demonstrated that the C-terminal 15-25 amino acids of P150 or P75 are essential for binding with RII-B.

  16. Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

    PubMed

    Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

    2016-03-01

    The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

  17. GTP cyclohydrolase I feedback regulatory protein is expressed in serotonin neurons and regulates tetrahydrobiopterin biosynthesis.

    PubMed

    Kapatos, G; Hirayama, K; Shimoji, M; Milstien, S

    1999-02-01

    Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalanine, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit, the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L-phenylalanine specifically stimulates tetrahydrobiopterin synthesis by displacing tetrahydrobiopterin from the GTPCH-GFRP complex. To explore the role of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double-label in situ hybridization. GFRP mRNA expression was abundant in serotonin neurons of the dorsal raphe nucleus but was undetectable in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrain. Two species of GFRP mRNA differing by approximately 20 nucleotides in length were detected in brainstem but not in other tissues, with the longer, more abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mRNA, although GTPCH mRNA was abundant in both. Primary neuronal cultures were used to examine the role of GFRP-mediated regulation of GTPCH on tetrahydrobiopterin synthesis within brainstem serotonin neurons and midbrain dopamine neurons. L-Phenylalanine increased tetrahydrobiopterin levels in serotonin neurons to a maximum of twofold in a concentration-dependent manner, whereas D-phenylalanine and L-tryptophan were without effect. In contrast, tetrahydrobiopterin levels within cultured dopamine neurons were not altered by L-phenylalanine. The time course of this effect was very rapid, with a maximal response observed within 60 min. Inhibitors of tetrahydrobiopterin biosynthesis prevented the L

  18. Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

    PubMed

    Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

    2015-02-01

    sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo.

  19. Identification of a regulatory subunit of protein phosphatase 1 which mediates blue light signaling for stomatal opening.

    PubMed

    Takemiya, Atsushi; Yamauchi, Shota; Yano, Takayuki; Ariyoshi, Chie; Shimazaki, Ken-ichiro

    2013-01-01

    Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase comprised of a catalytic subunit (PP1c) and a regulatory subunit that modulates catalytic activity, subcellular localization and substrate specificity. PP1c positively regulates stomatal opening through blue light signaling between phototropins and the plasma membrane H(+)-ATPase in guard cells. However, the regulatory subunit functioning in this process is unknown. We identified Arabidopsis PRSL1 (PP1 regulatory subunit2-like protein1) as a regulatory subunit of PP1c. Tautomycin, a selective inhibitor of PP1c, inhibited blue light responses of stomata in the single mutants phot1 and phot2, supporting the idea that signals from phot1 and phot2 converge on PP1c. We obtained PRSL1 based on the sequence similarity to Vicia faba PRS2, a PP1c-binding protein isolated by a yeast two-hybrid screen. PRSL1 bound to Arabidopsis PP1c through its RVxF motif, a consensus PP1c-binding sequence. Arabidopsis prsl1 mutants were impaired in blue light-dependent stomatal opening, H(+) pumping and phosphorylation of the H(+)-ATPase, but showed normal phototropin activities. PRSL1 complemented the prsl1 phenotype, but not if the protein carried a mutation in the RVxF motif, suggesting that PRSL1 functions through binding PP1c via the RVxF motif. PRSL1 did not affect the catalytic activity of Arabidopsis PP1c but it stimulated the localization of PP1c in the cytoplasm. We conclude that PRSL1 functions as a regulatory subunit of PP1 and regulates blue light signaling in stomata.

  20. Identification of novel regulatory cholesterol metabolite, 5-cholesten, 3β,25-diol, disulfate.

    PubMed

    Ren, Shunlin; Kim, Jin Koung; Kakiyama, Genta; Rodriguez-Agudo, Daniel; Pandak, William M; Min, Hae-Ki; Ning, Yanxia

    2014-01-01

    Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3β, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.

  1. Thogoto virus ML protein suppresses IRF3 function

    SciTech Connect

    Jennings, Stephanie . E-mail: stephanie.jennings@uniklinik-freiburg.de; Martinez-Sobrido, Luis . E-mail: Luis.Martinez@mssm.edu; Garcia-Sastre, Adolfo . E-mail: adolfo.garcia-sastre@mssm.edu; Weber, Friedemann . E-mail: friedemann.weber@uniklinik-freiburg.de; Kochs, Georg . E-mail: georg.kochs@uniklinik-freiburg.de

    2005-01-05

    The Thogoto virus (THOV) is a member of the family Orthomyxoviridae. It prevents induction of alpha/beta interferons (IFN) in cell culture and in vivo via the action of the viral ML protein. Phenotypically, the effect of THOV ML resembles that of the NS1 protein of influenza A virus (FLUAV) in that it blocks the expression of IFN genes. IFN expression depends on IFN regulatory factor 3 (IRF3). Upon activation, IRF3 forms homodimers and accumulates in the nucleus where it binds the transcriptional coactivator CREB-binding protein (CBP). Here, we show that expression of ML blocked the transcriptional activity of IRF3 after stimulation by virus infection. Further biochemical analysis revealed that ML acts by blocking IRF3 dimerization and association with CBP. Surprisingly, however, ML did not interfere with the nuclear transport of IRF3. Thus, the action of ML differs strikingly from that of FLUAV NS1 that prevents IFN induction by retaining IRF3 in the cytoplasm.

  2. A novel microRNA-1207-3p/FNDC1/FN1/AR regulatory pathway in prostate cancer

    PubMed Central

    Das, Dibash K.; Ogunwobi, Olorunseun O.

    2017-01-01

    Prostate cancer (PCa) is the second most common cause of cancer-specific deaths in the U.S. Unfortunately, the underlying molecular mechanisms for its development and progression remain unclear. Studies have established that microRNAs (miRNAs) are dysregulated in PCa. The intron-derived microRNA-1207-3p (miR-1207-3p) is encoded at the non-protein coding gene locus PVT1 on the 8q24 human chromosomal region, an established PCa susceptibility locus. However, miR-1207-3p in PCa had not previously been investigated. Therefore, we explored if miR-1207-3p plays any regulatory role in PCa. We discovered that miR-1207-3p is significantly underexpressed in PCa cell lines in comparison to normal prostate epithelial cells, and that increased expression of microRNA-1207-3p in PCa cells significantly inhibits proliferation, migration, and induces apoptosis via direct molecular targeting of fibronectin type III domain containing 1 (FNDC1). Our studies also revealed significant overexpression of FNDC1, fibronectin (FN1) and the androgen receptor (AR) in human PCa cell lines as well as tissues, and FNDC1, FN1, and AR positively correlate with aggressive PCa. These findings, recently published in Experimental Cell Research, are the first to describe a novel miR-1207-3p/FNDC1/FN1/AR novel regulatory pathway in PCa. PMID:28251177

  3. STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide.

    PubMed

    Starzynski, Rafal Radoslaw; Gonçalves, Ana Sofia; Muzeau, Françoise; Tyrolczyk, Zofia; Smuda, Ewa; Drapier, Jean-Claude; Beaumont, Carole; Lipinski, Pawel

    2006-12-01

    RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-gamma-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1-DNA and STAT-DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression.

  4. A Common Missense Variant in the Glucokinase Regulatory Protein Gene (GCKR) Is Associated with Increased Plasma Triglyceride and C-Reactive Protein but Lower Fasting Glucose Concentrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVE-Using the genome-wide-association approach, we recently identified the glucokinase regulatory protein gene (GCKR, rs780094) region as a novel quantitative trait locus for plasma triglyceride concentration in Europeans. Here, we sought to study the association of GCKR variants with metaboli...

  5. Absence of mutations in the regulatory domain of the gap junction protein connexin 43 in patients with visceroatrial heterotaxy.

    PubMed Central

    Penman Splitt, M.; Tsai, M. Y.; Burn, J.; Goodship, J. A.

    1997-01-01

    OBJECTIVE: To determine the frequency of mutations in the regulatory domain of the gap junction protein connexin 43 in patients with visceroatrial heterotaxy. DESIGN: Mutation screening of the terminal 200 base pairs of connexin43 gene coding sequence in a series of patients from tertiary care centres. PATIENTS: 48 patients with visceroatrial heterotaxy attending UK Regional Paediatric Cardiology Centres. RESULTS: No changes from the published connexin43 consensus sequence were found in any of the 48 patients studied. CONCLUSIONS: Germline mutations of the phosphorylation sites in teh regulatory domain of the connexin43 gene are rare in patients with visceroatrial heterotaxy. PMID:9155619

  6. Isolation and computer analysis of the 5'-regulatory region of the seed storage protein gene from buckwheat (Fagopyrum esculentum Moench).

    PubMed

    Milisavljević, Mira Dj; Konstantinović, Miroslav M; Brkljacić, Jelena M; Maksimović, Vesna R

    2005-03-23

    Using the modified rapid amplification of cDNA ends (5'-RACE) approach, a fragment containing the 955 bp long 5'-regulatory region of the buckwheat storage globulin gene (FeLEG1) has been amplified from the genomic DNA of buckwheat. The entire fragment was sequenced, and the sequence was analyzed by computer prediction of cis-regulatory elements possibly involved in tissue-specific and developmentally controlled seed storage protein gene expression. The promoter obtained might be interesting not only for fundamental research but also as a useful tool for biotechnological application.

  7. Protein-Protein Docking Benchmark Version 3.0

    PubMed Central

    Hwang, Howook; Pierce, Brian; Mintseris, Julian; Janin, Joël; Weng, Zhiping

    2009-01-01

    We present version 3.0 of our publicly available protein-protein docking benchmark. This update includes 40 new test cases, representing a 48% increase from Benchmark 2.0. For all of the new cases, the crystal structures of both binding partners are available. As with Benchmark 2.0, SCOP1 (Structural Classification of Proteins) was used to remove redundant test cases. The 124 unbound-unbound test cases in Benchmark 3.0 are classified into 88 rigid-body cases, 19 medium difficulty cases, and 17 difficult cases, based on the degree of conformational change at the interface upon complex formation. In addition to providing the community with more test cases for evaluating docking methods, the expansion of Benchmark 3.0 will facilitate the development of new algorithms that require a large number of training examples. Benchmark 3.0 is available to the public at http://zlab.bu.edu/benchmark. PMID:18491384

  8. Identification of Functional Regulatory Residues of the β -Lactam Inducible Penicillin Binding Protein in Methicillin-Resistant Staphylococcus aureus.

    PubMed

    Mbah, Andreas N; Isokpehi, Raphael D

    2013-01-01

    Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA) isolates acquired a new protein called β -lactam inducible penicillin binding protein (PBP-2'). The PBP-2' functions by substituting other penicillin binding proteins which have been inhibited by β -lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2' protein. We conducted a complete structural and functional regulatory analysis of PBP-2' protein. Our analysis revealed that the PBP-2' is very stable with more hydrophilic amino acids expressing antigenic sites. PBP-2' has three striking regulatory points constituted by first penicillin binding site at Ser25, second penicillin binding site at Ser405, and finally a single metallic ligand binding site at Glu657 which binds to Zn(2+) ions. This report highlights structural features of PBP-2' that can serve as targets for developing new chemotherapeutic agents and conducting site direct mutagenesis experiments.

  9. Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

    SciTech Connect

    Senogles, S.E.; Caron, M.G.

    1986-05-01

    The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..S binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.

  10. Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis.

    PubMed

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Sayadi, Ahmed; Crescenzi, Marco; Pozio, Edoardo

    2012-05-04

    14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.

  11. Time-course changes in immunoreactivities of glucokinase and glucokinase regulatory protein in the gerbil hippocampus following transient cerebral ischemia.

    PubMed

    Park, Joon Ha; Lee, Choong Hyun; Kim, In Hye; Ahn, Ji Hyeon; Cho, Jeong-Hwi; Yan, Bing Chun; Lee, Jae-Chul; Lee, Tae Hun; Seo, Jeong Yeol; Cho, Jun Hwi; Won, Moo-Ho; Kang, Il-Jun

    2013-12-01

    Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia-reperfusion (I-R). Five days after I-R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.

  12. Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP.

    PubMed

    Milstien, S; Jaffe, H; Kowlessur, D; Bonner, T I

    1996-08-16

    The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and -phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern

  13. Direct interaction of the Polycomb protein with Antennapedia regulatory sequences in polytene chromosomes of Drosophila melanogaster.

    PubMed Central

    Zink, B; Engström, Y; Gehring, W J; Paro, R

    1991-01-01

    The Polycomb (Pc) gene is responsible for the elaboration and maintenance of the expression pattern of the homeotic genes during development of Drosophila. In mutant Pc- embryos, homeotic transcripts are ectopically expressed, leading to abdominal transformations in all segments. From this it was suggested that PC+ acts as a repressor of homeotic gene transcription. We have mapped the cis-acting control sequences of the homeotic Antennapedia (Antp) gene regulated by Pc. Using Antp P1 and P2 promoter fragments linked to the E. coli lacZ reporter gene we show different expression patterns of beta-galactosidase (beta-gal) in transformed Pc+ and Pc- embryos. In addition we are able to visualize by immunocytochemical techniques on polytene chromosomes the direct binding of the Pc protein to the transposed cis-regulatory promoter fragments. However, short Antp P1 promoter constructs which are--due to position effects--ectopically activated in salivary glands, do not reveal a Pc binding signal. Images PMID:1671215

  14. Regulatory mechanism of protein metabolic pathway during the differentiation process of chicken male germ cell.

    PubMed

    Li, Dong; Zuo, Qisheng; Lian, Chao; Zhang, Lei; Shi, Qingqing; Zhang, Zhentao; Wang, Yingjie; Ahmed, Mahmoud F; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

    2015-08-01

    We explored the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro. We sequenced the transcriptome of embryonic stem cells, primordial germ cells, and spermatogonial stem cells with RNA sequencing (RNA-Seq), bioinformatics analysis methods, and detection of the key genes by quantitative reverse transcription PCR (qRT-PCR). Finally, we found 16 amino acid metabolic pathways enriched in the biological metabolism during the differentiation process of embryonic stem cells to primordial germ cells and 15 amino acid metabolic pathways enriched in the differentiation stage of primordial germ cells to spermatogonial stem cells. We found three pathways, arginine-proline metabolic pathway, tyrosine metabolic pathway, and tryptophan metabolic pathway, significantly enriched in the whole differentiation process of embryonic stem cells to spermatogonial stem cells. Moreover, for these three pathways, we screened key genes such as NOS2, ADC, FAH, and IDO. qRT-PCR results showed that the expression trend of these genes were the same to RNA-Seq. Our findings showed that the three pathways and these key genes play an important role in the differentiation process of embryonic stem cells to male germ cells. These results provide basic information for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro.

  15. Acetylation of glucokinase regulatory protein decreases glucose metabolism by suppressing glucokinase activity

    PubMed Central

    Park, Joo-Man; Kim, Tae-Hyun; Jo, Seong-Ho; Kim, Mi-Young; Ahn, Yong-Ho

    2015-01-01

    Glucokinase (GK), mainly expressed in the liver and pancreatic β-cells, is critical for maintaining glucose homeostasis. GK expression and kinase activity, respectively, are both modulated at the transcriptional and post-translational levels. Post-translationally, GK is regulated by binding the glucokinase regulatory protein (GKRP), resulting in GK retention in the nucleus and its inability to participate in cytosolic glycolysis. Although hepatic GKRP is known to be regulated by allosteric mechanisms, the precise details of modulation of GKRP activity, by post-translational modification, are not well known. Here, we demonstrate that GKRP is acetylated at Lys5 by the acetyltransferase p300. Acetylated GKRP is resistant to degradation by the ubiquitin-dependent proteasome pathway, suggesting that acetylation increases GKRP stability and binding to GK, further inhibiting GK nuclear export. Deacetylation of GKRP is effected by the NAD+-dependent, class III histone deacetylase SIRT2, which is inhibited by nicotinamide. Moreover, the livers of db/db obese, diabetic mice also show elevated GKRP acetylation, suggesting a broader, critical role in regulating blood glucose. Given that acetylated GKRP may affiliate with type-2 diabetes mellitus (T2DM), understanding the mechanism of GKRP acetylation in the liver could reveal novel targets within the GK-GKRP pathway, for treating T2DM and other metabolic pathologies. PMID:26620281

  16. Regulatory effect of porcine plasma protein hydrolysates on pasting and gelatinization action of corn starch.

    PubMed

    Kong, Baohua; Niu, Haili; Sun, Fangda; Han, Jianchun; Liu, Qian

    2016-01-01

    The objective of this study was to investigate the regulatory effect of porcine plasma protein hydrolysates (PPPH) on the physicochemical, pasting, and gelatinization properties of corn starch (CS). The results showed that the solubility of CS markedly increased, whereas swelling power and gel penetration force decreased with increased PPPH concentration (P<0.05). Compared with native CS, PPPH significantly lowered peak viscosity, minimum viscosity, final viscosity, and total setback, whereas it increased breakdown and pasting temperature in rapid visco analyzer (RVA) measurement (P<0.05) and obviously enhanced the gelatinization temperature as determined in differential scanning calorimetry (DSC) (P<0.05). Confocal laser scanning microscopy (CLSM) showed that PPPH surrounded the starch granules at room temperature (25°C) and then formed a network with swollen starch granules during gelatinization. Atomic force microscopy (AFM) images indicated that the blocklet sizes of gelatinized CS-PPPH mixtures were smaller and more uniform than native CS. The results proved that pasting and gelatinization abilities of CS can be effectively influenced by adding PPPH.

  17. Importance of fumarate and nitrate reduction regulatory protein for intestinal proliferation of Vibrio vulnificus.

    PubMed

    Kado, Takehiro; Kashimoto, Takashige; Yamazaki, Kohei; Ueno, Shunji

    2017-01-01

    The sepsis caused by Vibrio vulnificus is characterized by an average incubation period of 26 h and a high mortality rate exceeding 50%. The fast growth and dissemination of V. vulnificus in vivo lead to poor clinical outcomes in patients. Therefore, elucidation of the proliferation mechanisms of this organism in vivo may lead to the development of an effective therapeutic strategy. In this study, we focused on the low oxygen concentration in the intestinal milieu because of its drastic difference from that in air. Fumarate and nitrate reduction regulatory protein (FNR) is known to be a global transcriptional regulator for adaptation to anaerobic conditions in various bacteria. We generated a strain of V. vulnificus in which the fnr gene was replaced with an erythromycin resistance gene (fnr::erm mutant). When the fnr::erm mutant was tested in a growth competition assay against the wild-type (WT) in vivo, the competitive index of fnr::erm mutant to WT in the intestinal loop and liver was 0.378 ± 0.192 (mean ± SD) and 0.243 ± 0.123, respectively. These data suggested that FNR is important for the proliferation of V. vulnificus in the intestine to achieve a critical mass to be able to invade the systemic circulation.

  18. Loss of Regulatory Protein RfaH Attenuates Virulence of Uropathogenic Escherichia coli

    PubMed Central

    Nagy, Gábor; Dobrindt, Ulrich; Schneider, György; Khan, A. Salam; Hacker, Jörg; Emödy, Levente

    2002-01-01

    RfaH is a regulatory protein in Escherichia coli and Salmonella enterica serovar Typhimurium. Although it enhances expression of different factors that are proposed to play a role in bacterial virulence, a direct effect of RfaH on virulence has not been investigated so far. We report that inactivation of rfaH dramatically decreases the virulence of uropathogenic E. coli strain 536 in an ascending mouse model of urinary tract infection. The mortality rate caused by the wild-type strain in this assay is 100%, whereas that of its isogenic rfaH mutant does not exceed 18%. In the case of coinfection, the wild-type strain 536 shows higher potential to colonize the urinary tract even when it is outnumbered 100-fold by its rfaH mutant in the inoculum. In contrast to the wild-type strain, serum resistance of strain 536rfaH::cat is fully abolished. Furthermore, we give evidence that, besides a major decrease in the amount of hemin receptor ChuA (G. Nagy, U. Dobrindt, M. Kupfer, L. Emody, H. Karch, and J. Hacker, Infect. Immun. 69:1924-1928, 2001), loss of the RfaH protein results in an altered lipopolysaccharide phenotype as well as decreased expression of K15 capsule and alpha-hemolysin, whereas levels of other pathogenicity factors such as siderophores, flagella, Prf, and S fimbriae appear to be unaltered in strain 536rfaH::cat in comparison to the wild-type strain. trans complementation of the mutant strain with the rfaH gene restores wild-type levels of the affected virulence factors and consequently restitutes virulence in the mouse model of ascending urinary tract infection. PMID:12117951

  19. Heat Shock Protein 90 Modulates Lipid Homeostasis by Regulating the Stability and Function of Sterol Regulatory Element-binding Protein (SREBP) and SREBP Cleavage-activating Protein.

    PubMed

    Kuan, Yen-Chou; Hashidume, Tsutomu; Shibata, Takahiro; Uchida, Koji; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2017-02-17

    Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis.

  20. Structure of a Construct of a Human Poly(C)-binding Protein Containing the First and Second KH Domains Reveals Insights into Its Regulatory Mechanisms*

    PubMed Central

    Du, Zhihua; Fenn, Sebastian; Tjhen, Richard; James, Thomas L.

    2008-01-01

    Poly(C)-binding proteins (PCBPs) are important regulatory proteins that contain three KH (hnRNP K homology) domains. Binding poly(C) D/RNA sequences via KH domains is essential for multiple PCBP functions. To reveal the basis for PCBP-D/RNA interactions and function, we determined the structure of a construct containing the first two domains (KH1-KH2) of human PCBP2 by NMR. KH1 and KH2 form an intramolecular pseudodimer. The large hydrophobic dimerization surface of each KH domain is on the side opposite the D/RNA binding interface. Chemical shift mapping indicates both domains bind poly(C) DNA motifs without disrupting the KH1-KH2 interaction. Spectral comparison of KH1-KH2, KH3, and full-length PCBP2 constructs suggests that the KH1-KH2 pseudodimer forms, but KH3 does not interact with other parts of the protein. From NMR studies and modeling, we propose possible modes of cooperative binding tandem poly(C) motifs by the KH domains. D/RNA binding may induce pseudodimer dissociation or stabilize dissociated KH1 and KH2, making protein interaction surfaces available to PCBP-binding partners. This conformational change may represent a regulatory mechanism linking D/RNA binding to PCBP functions. PMID:18701464

  1. 3 CFR - Proposed Revised Habitat for the Spotted Owl: Minimizing Regulatory Burdens

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 3 The President 1 2013-01-01 2013-01-01 false Proposed Revised Habitat for the Spotted Owl..., 2012 Proposed Revised Habitat for the Spotted Owl: Minimizing Regulatory Burdens Memorandum for the...) proposed critical habitat for the northern spotted owl. The proposal is an initial step in...

  2. 3 CFR 13610 - Executive Order 13610 of May 10, 2012. Identifying and Reducing Regulatory Burdens

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 3 The President 1 2013-01-01 2013-01-01 false Executive Order 13610 of May 10, 2012. Identifying and Reducing Regulatory Burdens 13610 Order 13610 Presidential Documents Executive Orders Executive... in light of changed circumstances, including the rise of new technologies. Executive Order 13563...

  3. miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 by directly targeting E2F3 in gastric cancer cells

    PubMed Central

    Chang, Su'e; Gao, Ling; Yang, Yang; Tong, Dongdong; Guo, Bo; Liu, Liying; Li, Zongfang; Song, Tusheng; Huang, Chen

    2015-01-01

    VitaminD3 signaling is involved in inhibiting the development and progression of gastric cancer (GC), while the active vitamin D metabolite 1-alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)-mediated gene regulatory mechanisms in GC remain unclear. We found that miR-145 is induced by 1,25(OH)2D3 in a dose- and vitamin D receptor (VDR)-dependent manner in GC cells. Inhibition of miR-145 reverses the antiproliferative effect of 1,25(OH)2D3. Furthermore, miR-145 expression was lower in tumors compared with matched normal samples and correlated with increased the E2F3 transcription factor protein staining. Overexpression of miR-145 inhibited colony formation, cell viability and induced cell arrest in S-phase in GC cells by targeting E2F3 and CDK6. miR-145 inhibition consistently abrogates the 1,25(OH)2D3-mediated suppression of E2F3, CDK6, CDK2 and CCNA2 genes. Altogether, our results indicate that miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 in GC cells and might hold promise for prognosis and therapeutic strategies for GC treatment. PMID:25762621

  4. Quantitative assessment of regulatory proteins in blood as markers of radiation effects in the late period after occupational exposure.

    PubMed

    Kirillova, Evgenia N; Zakharova, Maria L; Muksinova, Klara N; Drugova, Elena D; Pavlova, Olga S; Sokolova, Svetlana N

    2012-07-01

    The objective of this research was quantitative assessment of serum and membrane regulatory proteins in blood from nuclear workers as markers of radiation-induced alterations in immune homeostasis in the late period after protracted exposure of nuclear workers with different doses. The effector and regulatory lymphocytes were measured using a flow cytofluorometer in workers from the main facilities of the Mayak PA (aged ∼60 y up to 80 y) in the late period after combined exposure to external gamma-rays and internal alpha-radiation from incorporated 239Pu. The control group included non-occupationally exposed members of the Ozyorsk population matched by gender and age to the group of Mayak workers. Thirty serum proteins involved in regulation of immune homeostasis, such as growth factors, multifunctional interleukins, pro- and anti-inflammatory cytokines, and their receptors, were measured using ELISA in blood serum specimens from the Radiobiology Human Tissue Repository. The dosimetry estimates were obtained using Doses-2005. The correlation analysis revealed a statistically significant direct relationship of T-killers and plutonium body burden and a decreasing level of T-helpers with accumulated external dose in exposed individuals. There were differences in expression of membrane markers in young regulatory cells (double null T-lymphocytes, NKT-lymphocytes, regulatory T-cells, and an increase of activated forms of T-lymphocytes), which indicated an active role of regulatory cells in maintaining immune homeostasis in terms of protracted exposure. The assessment of regulatory proteins in blood indicated that growth factors (EGF, TGF-β1, PDGF), multifunctional interleukins (IL-17A, IL-18), and pro-inflammatory cytokines (IL-1β and INF-γ) could be potential markers of radiation-induced alterations in protein status. An imbalance of pro- and antiinflammatory proteins in blood and variations of protein profiles at the lower exposure levels (gamma-ray dose <1 Gy

  5. Pancreatic Islets Engineered with SA-FasL Protein Establish Robust Localized Tolerance by Inducing T Regulatory Cells in Mice

    PubMed Central

    Yolcu, Esma S; Zhao, Hong; Bandura-Morgan, Laura; Lacelle, Chantale; Woodward, Kyle B; Askenasy, Nadir; Shirwan, Haval

    2011-01-01

    Allogeneic islet transplantation is an important therapeutic approach for the treatment of T1D. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic T effector cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating T effector cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of FasL protein chimeric with streptavidin (SA-FasL), and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for over a week in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% C57BL/6 recipients. Tolerance was initiated and maintained by CD4+CD25+FoxP3+ T regulatory (Treg) cells as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of T1D. PMID:22068235

  6. Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice.

    PubMed

    Yolcu, Esma S; Zhao, Hong; Bandura-Morgan, Laura; Lacelle, Chantale; Woodward, Kyle B; Askenasy, Nadir; Shirwan, Haval

    2011-12-01

    Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.

  7. Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

    SciTech Connect

    Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg . E-mail: jkobarg@lnls.br

    2006-03-17

    Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro.

  8. Structural basis for protein–protein interactions in the 14-3-3 protein family

    PubMed Central

    Yang, Xiaowen; Lee, Wen Hwa; Sobott, Frank; Papagrigoriou, Evangelos; Robinson, Carol V.; Grossmann, J. Günter; Sundström, Michael; Doyle, Declan A.; Elkins, Jonathan M.

    2006-01-01

    The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein–protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners. PMID:17085597

  9. A new regulatory function of the region proximal to the RGG box in the fragile X mental retardation protein.

    PubMed

    Blackwell, Ernest; Ceman, Stephanie

    2011-09-15

    Fragile X mental retardation protein (FMRP) is required for normal cognition. FMRP has two autosomal paralogs, which although similar to FMRP, cannot compensate for the loss of FMRP expression in brain. The arginine- and glycine-rich region of FMRP (the RGG box) is unique; it is the high-affinity RNA-binding motif in FMRP and is encoded by exon 15. Alternative splicing occurs in the 5' end of exon 15, which is predicted to affect the structure of the distally encoded RGG box. Here, we provide evidence that isoform 3, which removes 25 amino acids from the 5' end of exon 15, has an altered conformation that reduces binding of a specific antibody and renders the RGG box unable to efficiently associate with polyribosomes. Isoform 3 is also compromised in its ability to form granules and to associate with a key messenger ribonucleoprotein Yb1 (also known as p50, NSEP1 and YBX1). Significantly, these functions are similarly compromised when the RGG box is absent from FMRP, suggesting an important regulatory role of the N-terminal region encoded by exon 15.

  10. Regulatory and pro-inflammatory phenotypes of myelin basic protein-autoreactive T cells in multiple sclerosis

    PubMed Central

    Li, Haiyan; Chen, Meiyue; Zang, Ying C. Q.; Skinner, Sheri M.; Killian, James M.; Zhang, Jingwu Z.

    2009-01-01

    MBP-specific autoreactive T cells are considered pro-inflammatory T cells and thought to play an important role in the pathogenesis of multiple sclerosis (MS). Here, we report that MBP83–99-specific T cells generated from MS patients (n = 7) were comprised of pro-inflammatory and regulatory subsets of distinct phenotypes. The pro-inflammatory phenotype was characterized by high production of IFN-γ, IL-6, IL-21 and IL-17 and low expression of FOXP3, whereas the regulatory subset expressed high levels of FOXP3 and exhibited potent regulatory functions. The regulatory subset of MBP-specific T cells appeared to expand from the CD4+CD25− T-cell pool. Their FOXP3 expression was stable, independent of the activation state and it correlated with suppressive function and inversely with the production of IFN-γ, IL-6, IL-21 and IL-17. In contrast, the phenotype and function of FOXP3low MBP-specific T cells were adaptive and dependent on IL-6. The higher frequency of FOXP3high MBP-specific T cells was observed when IL-6 was neutralized in the culture of PBMC with MBP. The study provides new evidence that MBP-specific T cells are susceptible to pro-inflammatory cytokine milieu and act as either pro-inflammatory or regulatory T cells. PMID:19822525

  11. Conserved cell cycle regulatory properties within the amino terminal domain of the Epstein-Barr virus nuclear antigen 3C

    SciTech Connect

    Sharma, Nikhil; Knight, Jason S.; Robertson, Erle S. . E-mail: erle@mail.med.upenn.edu

    2006-03-15

    The gammaherpesviruses Rhesus lymphocryptovirus (LCV) and Epstein-Barr virus (EBV) are closely related phylogenetically. Rhesus LCV efficiently immortalizes Rhesus B cells in vitro. However, despite a high degree of conservation between the Rhesus LCV and EBV genomes, Rhesus LCV fails to immortalize human B cells in vitro. This species restriction may, at least in part, be linked to the EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), known to be essential for B cell transformation. We compared specific properties of EBNA3C, a well-characterized and essential EBV protein, with its Rhesus counterpart to determine whether EBNA3C phenotypes which contribute to cell cycle regulation are conserved in the Rhesus LCV. We show that both EBNA3C and Rhesus EBNA3C bind to a conserved region of mammalian cyclins, regulate pRb stability, and modulate SCF{sup Skp2}-dependent ubiquitination. These results suggest that Rhesus LCV restriction from human B cell immortalization is independent of the conserved cell cycle regulatory functions of the EBNA3C protein.

  12. 14-3-3 Proteins in Guard Cell Signaling

    PubMed Central

    Cotelle, Valérie; Leonhardt, Nathalie

    2016-01-01

    Guard cells are specialized cells located at the leaf surface delimiting pores which control gas exchanges between the plant and the atmosphere. To optimize the CO2 uptake necessary for photosynthesis while minimizing water loss, guard cells integrate environmental signals to adjust stomatal aperture. The size of the stomatal pore is regulated by movements of the guard cells driven by variations in their volume and turgor. As guard cells perceive and transduce a wide array of environmental cues, they provide an ideal system to elucidate early events of plant signaling. Reversible protein phosphorylation events are known to play a crucial role in the regulation of stomatal movements. However, in some cases, phosphorylation alone is not sufficient to achieve complete protein regulation, but is necessary to mediate the binding of interactors that modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multiple isoforms in eukaryotes and have been shown to be involved in the regulation of stomatal movements. In this review, we describe the current knowledge about 14-3-3 roles in the regulation of their binding partners in guard cells: receptors, ion pumps, channels, protein kinases, and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatal movements in response to abiotic or biotic stresses. PMID:26858725

  13. Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants.

    PubMed Central

    Varagona, M J; Schmidt, R J; Raikhel, N V

    1991-01-01

    Protein targeting to the nucleus has been studied extensively in animal and yeast systems; however, nothing is known about nuclear targeting in plants. The Opaque-2 (O2) gene produces a regulatory protein that is responsible for inducing transcription of the alpha-zein class of storage proteins in maize kernels. The cloned O2 gene encodes a protein that contains a leucine zipper DNA binding domain that can interact with zein gene promoters. We have used immunolocalization to show that the O2 protein is present in nuclei in the maize endosperm tissues known to produce alpha-zeins. In addition, neither embryo tissue from wild-type kernels nor endosperm from kernels harboring a null o2 allele contain the O2 protein. Analysis of a transposable, element-induced o2 allele, o2-m20, revealed that sectors of endosperm cells contained the nuclear-localized O2 protein, indicating excision of the transposable element. To study further the nuclear transport of the O2 protein, we have transformed this gene, under the control of a constitutive promoter, into tobacco. Plants were shown to have detectable levels of steady-state O2 mRNA and O2 protein. Immunolocalization of O2 protein in transformed tobacco plants indicated that the O2 protein was transported into tobacco nuclei. Therefore, we have developed a system to study nuclear targeting in plants and have established that the nuclear transport machinery is similar in monocots and dicots. PMID:1840902

  14. Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants.

    PubMed

    Varagona, M J; Schmidt, R J; Raikhel, N V

    1991-02-01

    Protein targeting to the nucleus has been studied extensively in animal and yeast systems; however, nothing is known about nuclear targeting in plants. The Opaque-2 (O2) gene produces a regulatory protein that is responsible for inducing transcription of the alpha-zein class of storage proteins in maize kernels. The cloned O2 gene encodes a protein that contains a leucine zipper DNA binding domain that can interact with zein gene promoters. We have used immunolocalization to show that the O2 protein is present in nuclei in the maize endosperm tissues known to produce alpha-zeins. In addition, neither embryo tissue from wild-type kernels nor endosperm from kernels harboring a null o2 allele contain the O2 protein. Analysis of a transposable, element-induced o2 allele, o2-m20, revealed that sectors of endosperm cells contained the nuclear-localized O2 protein, indicating excision of the transposable element. To study further the nuclear transport of the O2 protein, we have transformed this gene, under the control of a constitutive promoter, into tobacco. Plants were shown to have detectable levels of steady-state O2 mRNA and O2 protein. Immunolocalization of O2 protein in transformed tobacco plants indicated that the O2 protein was transported into tobacco nuclei. Therefore, we have developed a system to study nuclear targeting in plants and have established that the nuclear transport machinery is similar in monocots and dicots.

  15. A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

    PubMed Central

    Steer, Beatrix; Adler, Barbara; Jonjic, Stipan; Stewart, James P.; Adler, Heiko

    2010-01-01

    Background Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. Methodology/Principal Findings Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. Conclusions/Significance In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein. PMID:20657771

  16. Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma.

    PubMed

    Florea, Ana-Maria; Varghese, Elizabeth; McCallum, Jennifer E; Mahgoub, Safa; Helmy, Irfan; Varghese, Sharon; Gopinath, Neha; Sass, Steffen; Theis, Fabian J; Reifenberger, Guido; Büsselberg, Dietrich

    2017-02-11

    Neuroblastoma (NB) is a pediatric cancer treated with poly-chemotherapy including platinum complexes (e.g. cisplatin (CDDP), carboplatin), DNA alkylating agents, and topoisomerase I inhibitors (e.g. topotecan (TOPO)). Despite aggressive treatment, NB may become resistant to chemotherapy. We investigated whether CDDP and TOPO treatment of NB cells interacts with the expression and function of proteins involved in regulating calcium signaling. Human neuroblastoma cell lines SH-SY5Y, IMR-32 and NLF were used to investigate the effects of CDDP and TOPO on cell viability, apoptosis, calcium homeostasis, and expression of selected proteins regulating intracellular calcium concentration ([Ca2+]i). In addition, the impact of pharmacological inhibition of [Ca2+]i-regulating proteins on neuroblastoma cell survival was studied. Treatment of neuroblastoma cells with increasing concentrations of CDDP (0.1-10 μM) or TOPO (0.1 nM-1 μM) induced cytotoxicity and increased apoptosis in a concentration- and time-dependent manner. Both drugs increased [Ca2+]i over time. Treatment with CDDP or TOPO also modified mRNA expression of selected genes encoding [Ca2+]i-regulating proteins. Differentially regulated genes included S100A6, ITPR1, ITPR3, RYR1 and RYR3. With FACS and confocal laser scanning microscopy experiments we validated their differential expression at the protein level. Importantly, treatment of neuroblastoma cells with pharmacological modulators of [Ca2+]i-regulating proteins in combination with CDDP or TOPO increased cytotoxicity. Thus, our results confirm an important role of calcium signaling in the response of neuroblastoma cells to chemotherapy and suggest [Ca2+]i modulation as a promising strategy for adjunctive treatment.

  17. Identification and characterization of the minimal 5'-regulatory region of the human riboflavin transporter-3 (SLC52A3) in intestinal epithelial cells.

    PubMed

    Ghosal, Abhisek; Sabui, Subrata; Said, Hamid M

    2015-01-15

    The human riboflavin (RF) transporter-3 (product of the SLC52A3 gene) plays an important role in intestinal RF absorption. Our aims in this study were to identify the minimal 5'-regulatory region of the SLC52A3 gene and the regulatory element(s) involved in its activity in intestinal epithelial cells, as well as to confirm promoter activity and establish physiological relevance in vivo in transgenic mice. With the use of transiently transfected human intestinal epithelial HuTu 80 cells and 5'-deletion analysis, the minimal SLC52A3 promoter was found to be encoded between -199 and +8 bp (using the start of the transcription start site as position 1). Although several putative cis-regulatory elements were predicted in this region, only the stimulating protein-1 (Sp1) binding site (at position -74/-71 bp) was found to play a role in promoter activity, as indicated by mutational analysis. Binding of Sp1 to the minimal SLC52A3 promoter was demonstrated by means of EMSA and supershift assays and by chromatin immunoprecipitation analysis. Studies with Drosophila SL2 cells (which lack Sp activity) confirmed the importance of Sp1 in driving the activity of the SLC52A3 minimal promoter; they further showed that Sp3 can also do the activation. Finally, with the use of luciferase gene fusions, the activity of the cloned SLC52A3 promoter was confirmed in vivo in transgenic mice. These studies report, for the first time, on the identification and characterization of the SLC52A3 promoter and also demonstrate the importance of Sp1 in regulating its activity in intestinal epithelial cells.

  18. EGF Uptake and Degradation Assay to Determine the Effect of HTLV Regulatory Proteins on the ESCRT-Dependent MVB Pathway.

    PubMed

    Murphy, Colin; Sheehy, Noreen

    2017-01-01

    The endosomal sorting complex required for transport (ESCRT) pathway plays key roles in multivesicular bodies (MVBs) formation and lysosomal degradation of membrane receptors, viral budding, and midbody abscission during cytokinesis. The epidermal growth factor receptor (EGFR) is regarded as a prototypical cargo of the MVB/ESCRT pathway and following stimulation by epidermal growth factor (EGF) EGFR/EGF complexes are internalized, sorted into MVBs, and degraded by lysosomes or recycled back to the cell membrane. Here, we describe an assay to analyze the effect of human T-cell leukemia (HTLV) regulatory proteins on the functionality of ESCRT-dependent MVB/lysosomal trafficking of EGFR/EGF complexes. This is performed by direct visualization and quantification of the rate of EGF-Alexa595/EGFR internalization and degradation in HeLa cells expressing HTLV regulatory proteins by immunofluorescence and western blot.

  19. PDZD8 is a novel moesin-interacting cytoskeletal regulatory protein that suppresses infection by herpes simplex virus type 1.

    PubMed

    Henning, Matthew S; Stiedl, Patricia; Barry, Denis S; McMahon, Robert; Morham, Scott G; Walsh, Derek; Naghavi, Mojgan H

    2011-07-05

    The host cytoskeleton plays a central role in the life cycle of many viruses yet our knowledge of cytoskeletal regulators and their role in viral infection remains limited. Recently, moesin and ezrin, two members of the ERM (Ezrin/Radixin/Moesin) family of proteins that regulate actin and plasma membrane cross-linking and microtubule (MT) stability, have been shown to inhibit retroviral infection. To further understand how ERM proteins function and whether they also influence infection by other viruses, we identified PDZD8 as a novel moesin-interacting protein. PDZD8 is a poorly understood protein whose function is unknown. Exogenous expression of either moesin or PDZD8 reduced the levels of stable MTs, suggesting that these proteins functioned as part of a cytoskeletal regulatory complex. Additionally, exogenous expression or siRNA-mediated knockdown of either factor affected Herpes Simplex Virus type 1 (HSV-1) infection, identifying a cellular function for PDZD8 and novel antiviral properties for these two cytoskeletal regulatory proteins.

  20. Hormonal regulation of the steroidogenic acute regulatory protein (StAR) in gonadal tissues of the Atlantic croaker (Micropogonias undulatus).

    PubMed

    Nunez, B Scott; Evans, Andrew N

    2007-02-01

    The steroidogenic acute regulatory protein (StAR), a member of the StAR-related lipid transfer domain (START) family, is critical to regulated steroidogenesis in vertebrates. We have isolated a cDNA encoding StAR from a well-studied model of teleost physiology, the Atlantic croaker Micropogonias undulatus. This cDNA (1204 nucleotides total length) contains an open reading frame of 858 nucleotides encoding a protein of 286 amino acids. Molecular phylogenetic analysis indicates the putative Atlantic croaker StAR protein is more closely related to StAR proteins (62-85% identity) than to the related START protein MLN-64 (28-31% identity). Green monkey kidney cells (COS-1) cotransfected with Atlantic croaker StAR and human cholesterol side chain cleavage (SCC) expression constructs are able to produce significantly more pregnenolone than cells transfected with SCC alone. StAR mRNA is detected in the Atlantic croaker head kidney by reverse transcriptase-polymerase chain reaction (RT-PCR) and in the kidney and hypothalamus in some individuals. Gonadal StAR gene expression is below the level of detection by RT-PCR in most individuals, but can be detected using fluorescent probes in quantitative RT-PCR. StAR mRNA is not detected in the Atlantic croaker brain. Six hour in vitro treatment of Atlantic croaker ovarian follicles with human chorionic gonadotropin (hCG) is insufficient to significantly alter StAR mRNA levels; however, 24 h hCG treatment induces StAR mRNA levels 17-fold over untreated controls. Neither 6 nor 24 h treatment with hCG significantly alters StAR mRNA levels in Atlantic croaker testicular minces. Likewise, 6h in vitro treatment with estradiol, testosterone or the maturation-inducing steroid 17,20beta,21-trihydroxy-4-pregnen-3-one is without effect on gonadal StAR mRNA levels.

  1. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    Adaptive responses associated with environmental stressors are critical to cell survival. These involve the modulation of central signaling protein functions through site-specific and enzymatically reversible oxidative modifications of methionines to coordinate cellular metabolism, energy utilization, and calcium signaling. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. Thus, depending on either the ecological niche of the organism or the cellular milieu of different organ systems, cellular metabolism can be fine-tuned to maintain optimal function in the face of variable amounts of collateral oxidative damage. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met{sup 144} or Met{sup 145} results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylationdependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in

  2. Expression of regulatory proteins in choroid plexus changes in early stages of Alzheimer disease.

    PubMed

    Krzyzanowska, Agnieszka; García-Consuegra, Inés; Pascual, Consuelo; Antequera, Desiree; Ferrer, Isidro; Carro, Eva

    2015-04-01

    Recent studies indicate that the choroid plexus has important physiologic and pathologic roles in Alzheimer disease (AD). To obtain additional insight on choroid plexus function, we performed a proteomic analysis of choroid plexus samples from patients with AD stages I to II (n = 16), III to IV (n = 16), and V to VI (n = 11) and 7 age-matched control subjects. We used 2-dimensional differential gel electrophoresis coupled with mass spectrometry to generate a complete picture of changes in choroid plexus protein expression occurring in AD patients. We identified 6 proteins: 14-3-3 β/α, 14-3-3 ε, moesin, proteasome activator complex subunit 1, annexin V, and aldehyde dehydrogenase, which were significantly regulated in AD patient samples (p < 0.05, >1.5-fold variation in expression vs control samples). These proteins are implicated in major physiologic functions including mitochondrial dysfunction and apoptosis regulation. These findings contribute additional significance to the emerging importance of molecular and functional changes of choroid plexus function in the pathophysiology of AD.

  3. Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA.

    PubMed

    Lease, Richard A; Woodson, Sarah A

    2004-12-10

    Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.

  4. Emotional regulatory function of Receptor Interacting Protein 140 revealed in the ventromedial hypothalamus

    PubMed Central

    Flaisher-Grinberg, S; Tsai, HC; Feng, X; Wei, LN

    2014-01-01

    Receptor-interacting protein (RIP140) is a transcription co-regulator highly expressed in macrophages to regulate inflammatory and metabolic processes. However, its implication in neurological, cognitive and emotional conditions, and the cellular systems relevant to its biological activity within the central nervous system are currently less clear. A transgenic mouse line with macrophage-specific knockdown of RIP140 was generated (MΦRIPKD mice) and brain-region specific RIP140 knockdown efficiency evaluated. Mice were subjected to a battery of tests, designed to evaluate multiple behavioral domains at naïve or following site-specific RIP140 re-expression. Gene expression analysis assessed TNF-α, IL-1β, TGF-1β, IL1-RA and Neuropeptide Y (NPY) expression, and in-vitro studies examined the effects of macrophage’s RIP140 on astrocytes’ NPY production. We found RIP140 expression was dramatically reduced in macrophages within the ventromedial hypothalamus (VMH) and the cingulate cortex of MΦRIPKD mice. These animals exhibited increased anxiety- and depressive-like behaviors. VMH-targeted RIP140 re-expression in MΦRIPKD mice reversed its depressive- but not its anxiety-like phenotype. Analysis of specific neurochemical changes revealed reduced astrocytic-NPY expression within the hypothalamus of MΦRIPKD mice, and in-vitro analysis confirmed that conditioned medium of RIP140-silnenced macrophage culture could no longer stimulate NPY production from astrocytes. The current study revealed an emotional regulatory function of macrophage-derived RIP140 in the VMH, and secondary dysregulation of NPY within hypothalamic astrocyte population, which might be associated with the observed behavioral phenotype of MΦRIPKD mice. This study highlights RIP140 as a novel target for the development of potential therapeutic and intervention strategies for emotional regulation disorders. PMID:24726835

  5. Regulatory Considerations in the Design and Manufacturing of Implantable 3D-Printed Medical Devices

    PubMed Central

    Morrison, Robert J.; Kashlan, Khaled N.; Flanangan, Colleen L.; Wright, Jeanne K.; Green, Glenn E.; Hollister, Scott J.; Weatherwax, Kevin J.

    2015-01-01

    Three-dimensional (3D) printing, or additive manufacturing, technology has rapidly penetrated the medical device industry over the past several years, and innovative groups have harnessed it to create devices with unique composition, structure, and customizability. These distinctive capabilities afforded by 3D printing have introduced new regulatory challenges. The customizability of 3D-printed devices introduces new complexities when drafting a design control model for FDA consideration of market approval. The customizability and unique build processes of 3D-printed medical devices pose unique challenges in meeting regulatory standards related to the manufacturing quality assurance. Consistent material powder properties and optimal printing parameters such as build orientation and laser power must be addressed and communicated to the FDA to ensure a quality build. Post-printing considerations unique to 3D-printed devices, such as cleaning, finishing and sterilization are also discussed. In this manuscript we illustrate how such regulatory hurdles can be navigated by discussing our experience with our group’s 3D-printed bioresorbable implantable device. PMID:26243449

  6. Regulatory Considerations in the Design and Manufacturing of Implantable 3D-Printed Medical Devices.

    PubMed

    Morrison, Robert J; Kashlan, Khaled N; Flanangan, Colleen L; Wright, Jeanne K; Green, Glenn E; Hollister, Scott J; Weatherwax, Kevin J

    2015-10-01

    Three-dimensional (3D) printing, or additive manufacturing, technology has rapidly penetrated the medical device industry over the past several years, and innovative groups have harnessed it to create devices with unique composition, structure, and customizability. These distinctive capabilities afforded by 3D printing have introduced new regulatory challenges. The customizability of 3D-printed devices introduces new complexities when drafting a design control model for FDA consideration of market approval. The customizability and unique build processes of 3D-printed medical devices pose unique challenges in meeting regulatory standards related to the manufacturing quality assurance. Consistent material powder properties and optimal printing parameters such as build orientation and laser power must be addressed and communicated to the FDA to ensure a quality build. Postprinting considerations unique to 3D-printed devices, such as cleaning, finishing and sterilization are also discussed. In this manuscript we illustrate how such regulatory hurdles can be navigated by discussing our experience with our group's 3D-printed bioresorbable implantable device.

  7. The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

    PubMed

    Satoh, Jun-ichi; Onoue, Hiroyuki; Arima, Kunimasa; Yamamura, Takashi

    2005-10-01

    The 14-3-3 protein family consists of acidic 30-kDa proteins composed of 7 isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 protein identified in the cerebrospinal fluid provides a surrogate marker for premortem diagnosis of Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of prion diseases remains unknown. By protein overlay and mass spectrometric analysis of protein extract of NTera2-derived differentiated neurons, we identified heat shock protein Hsp60 as a 14-3-3-interacting protein. The 14-3-3zeta and gamma isoforms interacted with Hsp60, suggesting that the interaction is not isoform-specific. Furthermore, the interaction was identified in SK-N-SH neuroblastoma, U-373MG astrocytoma, and HeLa cervical carcinoma cells. The cellular prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain protein extract. By protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning amino acid residues 27-287 and the NTF of PrPC spanning amino acid residues 23-137. By immunostaining, the 14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the 14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of prion diseases.

  8. Relationship between COX-2 and cell cycle-regulatory proteins in patients with esophageal squamous cell carcinoma

    PubMed Central

    Huang, Jun-Xing; Xiao, Wei; Chen, Wei-Chang; Lin, Mao-Song; Song, Zheng-Xiang; Chen, Ping; Zhang, Yun-Lei; Li, Feng-Yue; Qian, Rong-Yu; Salminen, Eeva

    2010-01-01

    AIM: To investigate the correlation between cyclooxygenase-2 (COX-2) and cell cycle-regulatory proteins in patients with esophageal squamous cell carcinoma (ESCC). METHODS: One hundred and two surgically obtained specimens of ESCC were randomly collected. All specimens were obtained from patients who had not received chemo- or radiotherapy prior to surgical resection. Twenty-eight specimens of normal squamous epithelium served as controls. The expression of COX-2, Ki-67, cyclin A and p27 was examined by immunohistochemistry. The Pearson test was used to analyze the relationship between groups. RESULTS: The protein level of COX-2, Ki-67 and cyclin A was significantly higher in ESCC than in normal squamous epithelium (74.7 ± 61.2 vs 30.2 ± 43.4, 64.0 ± 51.6 vs 11.6 ± 2.3, 44.2 ± 32.2 vs 11.7 ± 5.0, respectively, all P < 0.01). In contrast, the protein level of p27 was significantly lower in ESCC than in normal squamous epithelium (182.0 ± 69.0 vs 266.4 ± 28.0, P < 0.01). In ESCC, COX-2 expression was correlated with T stage, the score of T1-T2 stage was lower than that of T3-T4 stage (55.0 ± 42.3 vs 83.0 ± 66.5, P < 0.05), and Ki-67, cyclin A and p27 expressions were correlated with the tumor differentiation (43.8 ± 31.7 vs 98.4 ± 84.8, 32.0 ± 19.0 vs 54.1 ± 53.7, 206.2 ± 61.5 vs 123.5 ± 68.3, respectively, all P < 0.01). COX-2 expression was positively correlated to Ki-67, cyclin A and negatively correlated to p27 expression in ESCC (r = 0.270, 0.233 and -0.311, respectively, all P < 0.05). CONCLUSION: The expression of COX-2 is correlated with tumor cell invasion and is closely related to the cell proliferation in patients with ESCC. PMID:21157974

  9. Contribution of Mesenteric Lymph Nodes and GALT to the Intestinal Foxp3+ Regulatory T-Cell Compartment.

    PubMed

    Geem, Duke; Ngo, Vu; Harusato, Akihito; Chassaing, Benoit; Gewirtz, Andrew T; Newberry, Rodney D; Denning, Timothy L

    2016-05-01

    This study showed that the absence of CCR7 or mesenteric lymph nodes/gut-associated lymphoid tissue did not appreciably impact total intestinal Foxp3+ regulatory T cell representation in the steady-state. However, mesenteric lymph nodes/GALT are required for normal peripherally induced Foxp3+ regulatory T cell differentiation in the small intestine, but not in the large intestine.

  10. Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide

    PubMed Central

    Roy, Sophie; Bayly, Christopher I.; Gareau, Yves; Houtzager, Vicky M.; Kargman, Stacia; Keen, Sabina L. C.; Rowland, Kathleen; Seiden, Isolde M.; Thornberry, Nancy A.; Nicholson, Donald W.

    2001-01-01

    Caspase-3 is synthesized as a dormant proenzyme and is maintained in an inactive conformation by an Asp-Asp-Asp “safety-catch” regulatory tripeptide contained within a flexible loop near the large-subunit/small-subunit junction. Removal of this “safety catch” results in substantially enhanced autocatalytic maturation as well as increased vulnerability to proteolytic activation by upstream proteases in the apoptotic pathway such as caspase-9 and granzyme B. The safety catch functions through multiple ionic interactions that are disrupted by acidification, which occurs in the cytosol of cells during the early stages of apoptosis. We propose that the caspase-3 safety catch is a key regulatory checkpoint in the apoptotic cascade that regulates terminal events in the caspase cascade by modulating the triggering of caspase-3 activation. PMID:11353841

  11. HCF-1 self-association via an interdigitated Fn3 structure facilitates transcriptional regulatory complex formation.

    PubMed

    Park, Jihye; Lammers, Fabienne; Herr, Winship; Song, Ji-Joon

    2012-10-23

    Host-cell factor 1 (HCF-1) is an unusual transcriptional regulator that undergoes a process of proteolytic maturation to generate N- (HCF-1(N)) and C- (HCF-1(C)) terminal subunits noncovalently associated via self-association sequence elements. Here, we present the crystal structure of the self-association sequence 1 (SAS1) including the adjacent C-terminal HCF-1 nuclear localization signal (NLS). SAS1 elements from each of the HCF-1(N) and HCF-1(C) subunits form an interdigitated fibronectin type 3 (Fn3) tandem repeat structure. We show that the C-terminal NLS recruited by the interdigitated SAS1 structure is required for effective formation of a transcriptional regulatory complex: the herpes simplex virus VP16-induced complex. Thus, HCF-1(N)-HCF-1(C) association via an integrated Fn3 structure permits an NLS to facilitate formation of a transcriptional regulatory complex.

  12. Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein.

    PubMed

    Maita, Nobuo; Hatakeyama, Kazuyuki; Okada, Kengo; Hakoshima, Toshio

    2004-12-03

    GTP cyclohydrolase I (GTPCHI) is the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin, a key cofactor necessary for nitric oxide synthase and for the hydroxylases that are involved in the production of catecholamines and serotonin. In animals, the GTPCHI feedback regulatory protein (GFRP) binds GTPCHI to mediate feed-forward activation of GTPCHI activity in the presence of phenylalanine, whereas it induces feedback inhibition of enzyme activity in the presence of biopterin. Here, we have reported the crystal structure of the biopterin-induced inhibitory complex of GTPCHI and GFRP and compared it with the previously reported phenylalanine-induced stimulatory complex. The structure reveals five biopterin molecules located at each interface between GTPCHI and GFRP. Induced fitting structural changes by the biopterin binding expand large conformational changes in GTPCHI peptide segments forming the active site, resulting in inhibition of the activity. By locating 3,4-dihydroxy-phenylalanine-responsive dystonia mutations in the complex structure, we found mutations that may possibly disturb the GFRP-mediated regulation of GTPCHI.

  13. Forkhead transcription factor 1 inhibits endometrial cancer cell proliferation via sterol regulatory element-binding protein 1

    PubMed Central

    Zhang, Yifang; Zhang, Lili; Sun, Hengzi; Lv, Qingtao; Qiu, Chunping; Che, Xiaoxia; Liu, Zhiming; Jiang, Jie

    2017-01-01

    The morbidity and mortality associated with endometrial cancer (EC) has increased in recent years. Regarded as a tumor suppressor, forkhead transcription factor 1 (FOXO1) has various biological activities and participates in cell cycle progression, apoptosis and differentiation. Notably, FOXO1 also functions in the regulation of lipogenesis and energy metabolism. Lipogenesis is a feature of cancer and is upregulated in EC. Sterol regulatory element-binding protein 1 (SREBP1) is a transcription factor that is also able to regulate lipogenesis. Increased expression of SREBP1 is directly correlated with malignant transformation of tumors. A previous study demonstrated that SREBP1 was highly expressed in EC and directly resulted in tumorigenesis. However, the association between FOXO1 and SREBP1 in EC is not clear. In the present study, lentiviruses overexpressing FOXO1 were used in cell transfection and transduction. Cell viability assays demonstrated that the overexpression of FOXO1 was able to suppress cell proliferation significantly in Ishikawa and AN3 CA cell lines. In addition, FOXO1 overexpression significantly inhibited cell migration and invasion ability in vitro. In xenograft models, overexpression of FOXO1 suppressed cell tumorigenesis, and western blot analysis demonstrated that SREBP1 expression was markedly reduced in the FOXO1-overexpressing cells. It may therefore be concluded that FOXO1 is able to inhibit the proliferative capacity of cells in vitro and in vivo, in addition to the migratory and invasive capacities in vitro by directly targeting SREBP1. PMID:28356952

  14. Distinct Regulatory Mechanisms Act to Establish and Maintain Pax3 Expression in the Developing Neural Tube

    PubMed Central

    Moore, Steven; Ribes, Vanessa; Terriente, Javier; Wilkinson, David; Relaix, Frédéric; Briscoe, James

    2013-01-01

    Pattern formation in developing tissues is driven by the interaction of extrinsic signals with intrinsic transcriptional networks that together establish spatially and temporally restricted profiles of gene expression. How this process is orchestrated at the molecular level by genomic cis-regulatory modules is one of the central questions in developmental biology. Here we have addressed this by analysing the regulation of Pax3 expression in the context of the developing spinal cord. Pax3 is induced early during neural development in progenitors of the dorsal spinal cord and is maintained as pattern is subsequently elaborated, resulting in the segregation of the tissue into dorsal and ventral subdivisions. We used a combination of comparative genomics and transgenic assays to define and dissect several functional cis-regulatory modules associated with the Pax3 locus. We provide evidence that the coordinated activity of two modules establishes and refines Pax3 expression during neural tube development. Mutational analyses of the initiating element revealed that in addition to Wnt signaling, Nkx family homeodomain repressors restrict Pax3 transcription to the presumptive dorsal neural tube. Subsequently, a second module mediates direct positive autoregulation and feedback to maintain Pax3 expression. Together, these data indicate a mechanism by which transient external signals are converted into a sustained expression domain by the activities of distinct regulatory elements. This transcriptional logic differs from the cross-repression that is responsible for the spatiotemporal patterns of gene expression in the ventral neural tube, suggesting that a variety of circuits are deployed within the neural tube regulatory network to establish and elaborate pattern formation. PMID:24098141

  15. Evolving New Skeletal Traits by cis-Regulatory Changes in Bone Morphogenetic Proteins

    PubMed Central

    Indjeian, Vahan B.; Kingman, Garrett A.; Jones, Felicity C.; Guenther, Catherine A.; Grimwood, Jane; Schmutz, Jeremy; Myers, Richard M.; Kingsley, David M.

    2016-01-01

    SUMMARY Changes in bone size and shape are defining features of many vertebrates. Here we use genetic crosses and comparative genomics to identify specific regulatory DNA alterations controlling skeletal evolution. Armor bone size differences in sticklebacks maps to a major effect locus overlapping BMP family member GDF6. Freshwater fish express more GDF6 due in part to a transposon insertion, and transgenic overexpression of GDF6 phenocopies evolutionary changes in armor plate size. The human GDF6 locus also has undergone distinctive regulatory evolution, including complete loss of an enhancer that is otherwise highly conserved between chimps and other mammals. Functional tests show that the ancestral enhancer drives expression in hindlimbs but not forelimbs, in locations that have been specifically modified during the human transition to bipedalism. Both gain and loss of regulatory elements can localize BMP changes to specific anatomical locations, providing a flexible regulatory basis for evolving species-specific changes in skeletal form. PMID:26774823

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

    SciTech Connect

    Shi, T.; Niepel, M.; McDermott, J. E.; Gao, Y.; Nicora, C. D.; Chrisler, W. B.; Markillie, L. M.; Petyuk, V. A.; Smith, R. D.; Rodland, K. D.; Sorger, P. K.; Qian, W. -J.; Wiley, H. S.

    2016-07-12

    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 abundance 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.

  17. Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

    PubMed Central

    Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

    2012-01-01

    The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

  18. Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

    PubMed Central

    Singh, K K; Samson, L

    1995-01-01

    Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7761422

  19. Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: Involvement of the effector proteins in the regulatory mechanism

    SciTech Connect

    Saxena, Sunil K. . E-mail: ssaxena@stevens.edu; Kaur, Simarna

    2006-07-21

    Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.

  20. FoxP3+ regulatory T cells are not important for rotavirus clearance or the early antibody response to rotavirus.

    PubMed

    Miller, Amber D; Blutt, Sarah E; Conner, Margaret E

    2014-01-01

    Regulatory T cells produce TGF-β that contributes to IgA induction by intestinal commensal bacteria but their importance in IgA responses to pathogens has not been determined. Immunity against the enteropathogen, rotavirus, is dependent on intestinal IgA, but whether FoxP3(+) regulatory T cells contribute to this IgA is unknown. Infection with rotavirus increased the numbers of intestinal FoxP3(+) regulatory T cells. Depletion of FoxP3(+) regulatory T cells altered leukocyte activation but did not significantly alter rotavirus clearance or specific antibody levels. These data suggest FoxP3(+) regulatory T cells are not critical for the early antibody response to rotavirus infection.

  1. Integrin associated proteins differentially regulate neutrophil polarity and directed migration in 2D and 3D.

    PubMed

    Yamahashi, Yukie; Cavnar, Peter J; Hind, Laurel E; Berthier, Erwin; Bennin, David A; Beebe, David; Huttenlocher, Anna

    2015-10-01

    Directed neutrophil migration in blood vessels and tissues is critical for proper immune function; however, the mechanisms that regulate three-dimensional neutrophil chemotaxis remain unclear. It has been shown that integrins are dispensable for interstitial three-dimensional (3D) leukocyte migration; however, the role of integrin regulatory proteins during directed neutrophil migration is not known. Using a novel microfluidic gradient generator amenable to 2D and 3D analysis, we found that the integrin regulatory proteins Kindlin-3, RIAM, and talin-1 differentially regulate neutrophil polarization and directed migration to gradients of chemoattractant in 2D versus 3D. Both talin-1-deficient and RIAM-deficient neutrophil-like cells had impaired adhesion, polarization, and migration on 2D surfaces whereas in 3D the cells polarized but had impaired 3D chemotactic velocity. Kindlin-3 deficient cells were able to polarize and migrate on 2D surfaces but had impaired directionality. In a 3D environment, Kindlin-3 deficient cells displayed efficient chemotaxis. These findings demonstrate that the role of integrin regulatory proteins in cell polarity and directed migration can be different in 2D and 3D.

  2. Eimeria tenella: 14-3-3 protein interacts with telomerase.

    PubMed

    Zhao, Na; Gong, Pengtao; Cheng, Baiqi; Li, Jianhua; Yang, Zhengtao; Li, He; Yang, Ju; Zhang, Guocai; Zhang, Xichen

    2014-10-01

    Telomerase, consisting of telomerase RNA and telomerase reverse transcriptase (TERT), is responsible for the maintenance of the end of linear chromosomes. TERT, as the catalytic subunit of telomerase, plays a critical role in telomerase activity. Researches indicate TERT-associated proteins participate in the regulation of telomerase assembly, posttranslational modification, localization, and enzymatic function. Here, the telomerase RNA-binding domain of Eimeria tenella TERT (EtTRBD) was cloned into pGBKT7 and performed as the bait. α-Galactosidase assay showed that the bait plasmid did not activate Gal4 reporter gene. Further, we isolated an EtTRBD-associated protein, 14-3-3, by yeast two-hybrid screening using the constructed bait plasmid. To confirm the interaction, EtTRBD and 14-3-3 were expressed by prokaryotic and eukaryotic expression systems. Pull-down assays by purified proteins demonstrated a direct bind between EtTRBD and 14-3-3. Co-immunoprecipitation techniques successfully validated that 14-3-3 interacted with EtTRBD in 293T cells. The protein-protein interaction provides a starting point for more in-depth studies on telomerase and telomere regulation in E. tenella.

  3. Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases

    PubMed Central

    Swatek, Kirby N.; Wilson, Rashaun S.; Ahsan, Nagib; Tritz, Rebecca L.; Thelen, Jay J.

    2014-01-01

    Plant 14-3-3 proteins are phosphorylated at multiple sites in vivo; however, the protein kinase(s) responsible are unknown. Of the 34 CPK (calcium-dependent protein kinase) paralogues in Arabidopsis thaliana, three (CPK1, CPK24 and CPK28) contain a canonical 14-3-3-binding motif. These three, in addition to CPK3, CPK6 and CPK8, were tested for activity against recombinant 14-3-3 proteins χ and ε. Using an MS-based quantitative assay we demonstrate phosphorylation of 14-3-3 χ and ε at a total of seven sites, one of which is an in vivo site discovered in Arabidopsis. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analysed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF-hand domain of CPK28. Of all CPKs assayed, CPK28, which contained an autophosphorylation site (Ser43) within a canonical 14-3-3-binding motif, showed the highest activity against 14-3-3 proteins. Phosphomimetic mutagenesis of Ser72 to aspartate on 14-3-3χ, which is adjacent to the 14-3-3-binding cleft and conserved among all 14-3-3 isoforms, prevented 14-3-3-mediated inhibition of phosphorylated nitrate reductase. PMID:24438037

  4. X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase.

    PubMed

    Sazinsky, Matthew H; Dunten, Pete W; McCormick, Michael S; DiDonato, Alberto; Lippard, Stephen J

    2006-12-26

    Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket

  5. Role of calpain in eccentric contraction-induced proteolysis of Ca(2+)-regulatory proteins and force depression in rat fast-twitch skeletal muscle.

    PubMed

    Kanzaki, Keita; Watanabe, Daiki; Kuratani, Mai; Yamada, Takashi; Matsunaga, Satoshi; Wada, Masanobu

    2017-02-01

    The aim of this study was to examine the in vivo effects of eccentric contraction (ECC) on calpain-dependent proteolysis of Ca(2+)-regulatory proteins and force production in fast-twitch skeletal muscles. Rat extensor digitorum longus muscles were exposed to 200 repeated ECC in situ and excised immediately [recovery 0 (REC0)] or 3 days [recovery 3 (REC3)] after cessation of ECC. Calpain inhibitor (CI)-treated rats were intraperitoneally injected with MDL-28170 before ECC and during REC3. Tetanic force was markedly reduced at REC0 and remained reduced at REC3. CI treatment ameliorated the ECC-induced force decline but only at REC3. No evidence was found for proteolysis of dihydropyridine receptor (DHPR), junctophilin (JP)1, JP2, ryanodine receptor (RyR), sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)1a, or junctional face protein-45 at REC0. At REC3, ECC resulted in decreases in DHPR, JP1, JP2, RyR, and SERCA1a. CI treatment prevented the decreases in DHPR, JP1, and JP2, whereas it had little effect on RyR and SERCA1a. These findings suggest that DHPR, JP1, and JP2, but not RyR and SERCA1a, undergo calpain-dependent proteolysis in in vivo muscles subjected to ECC and that impaired function of DHPR and/or JP might cause prolonged force deficits with ECC.NEW & NOTEWORTHY Calpain-dependent proteolysis is one of the contributing factors to muscle damage that occurs with eccentric contraction (ECC). It is unclear, however, whether calpains account for proteolysis of Ca(2+)-regulatory proteins in in vivo muscles subjected to ECC. Here, we provide evidence that dihydropyridine receptor and junctophilin, but not ryanodine receptor and sarcoplasmic reticulum Ca(2+)-ATPase, undergo calpain-dependent proteolysis.

  6. Variations at regulatory regions of the milk protein genes are associated with milk traits and coagulation properties in the Sarda sheep.

    PubMed

    Noce, A; Pazzola, M; Dettori, M L; Amills, M; Castelló, A; Cecchinato, A; Bittante, G; Vacca, G M

    2016-12-01

    Regulatory variation at the ovine casein genes could have important effects on the composition and coagulation properties of milk. Herewith, we have partially resequenced the promoters and the 3'-UTR of the four casein genes in 25 Sarda sheep. Alignment of these sequences allowed us to identify a total of 29 SNPs. This level of polymorphism (one SNP every 250 bp) is remarkably high if compared with SNP densities estimated in human genic regions (approximately one SNP per bp). The 29 SNPs identified in our resequencing experiment, plus three previously reported SNPs mapping to the lactalbumin, alpha (LALBA) and β-lactoglobulin (BLG, also known as progestagen-associated endometrial protein, PAEP) genes, were genotyped with a multiplex TaqMan Open Array Real-Time PCR assay in 760 Sarda sheep with records for milk composition and coagulation properties. Association analysis revealed the existence of significant associations of CSN1S2 and CSN3 genotypes with milk protein and casein contents. Moreover, genotypes at CSN1S1 were significantly associated with rennet coagulation time, curd firming time and curd firmness, whereas CSN2 was associated with curd firming time. These results suggest that SNPs mapping to the promoters and 3'-UTRs of ovine casein genes may exert regulatory effects on gene expression and that they could be used for improving sheep milk quality and technological traits at the population level through marker assisted selection.

  7. Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

    PubMed

    Mitchison, A

    1997-01-01

    In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.

  8. Construction of proteins with molecular recognition capabilities using α3β3 de novo protein scaffolds.

    PubMed

    Okura, Hiromichi; Mihara, Hisakazu; Takahashi, Tsuyoshi

    2013-10-01

    The molecular recognition ability of proteins is essential in biological systems, and therefore a considerable amount of effort has been devoted to constructing desired target-binding proteins using a variety of naturally occurring proteins as scaffolds. However, since generating a binding site in a native protein can often affect its structural properties, highly stable de novo protein scaffolds may be more amenable than the native proteins. We previously reported the generation of de novo proteins comprising three α-helices and three β-strands (α3β3) from a genetic library coding simplified amino acid sets. Two α3β3 de novo proteins, vTAJ13 and vTAJ36, fold into a native-like stable and molten globule-like structures, respectively, even though the proteins have similar amino acid compositions. Here, we attempted to create binding sites for the vTAJ13 and vTAJ36 proteins to prove the utility of de novo designed artificial proteins as a molecular recognition tool. Randomization of six amino acids at two linker sites of vTAJ13 and vTAJ36 followed by biopanning generated binding proteins that recognize the target molecules, fluorescein and green fluorescent protein, with affinities of 10(-7)-10(-8) M. Of note, the selected proteins from the vTAJ13-based library tended to recognize the target molecules with high specificity, probably due to the native-like stable structure of vTAJ13. Our studies provide an example of the potential of de novo protein scaffolds, which are composed of a simplified amino acid set, to recognize a variety of target compounds.

  9. A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs.

    PubMed Central

    Korthout, H A; de Boer, A H

    1994-01-01

    The fusicoccin binding protein (FCBP) is a highly conserved plasma membrane protein present in all higher plants tested thus far. It exhibits high- and low-affinity binding for the fungal toxin fusicoccin (FC). We purified the active FCBP from a fraction highly enriched in plasma membrane by selective precipitation and anion exchange chromatography. After SDS-PAGE, the two FCBP subunits of 30 and 31 kD were detected as major bands. Amino acid sequence analysis of the 31-kD polypeptide displayed a high degree of identity with so-called 14-3-3 proteins, a class of mammalian brain proteins initially described as regulators of neurotransmitter synthesis and protein kinase C inhibitors. Thereafter, we affinity purified the 30- and 31-kD FCBP subunits, using biotinylated FC in combination with a monomeric avidin column. Immunodecoration of these 30- and 31-kD FCBP subunits with polyclonal antibodies raised against a 14-3-3 homolog from yeast confirmed the identity of the FCBP as a 14-3-3 homolog. Similar to all 14-3-3 protein homologs, the FCBP seems to exist as a dimer in native form. Thus far, the FCBP is the only 14-3-3 homolog with a receptor-like function. The conserved structure of the 14-3-3 protein family is a further indication that the FCBP plays an important role in the physiology of higher plants. PMID:7827499

  10. Protein kinase CK2 phosphorylation regulates the interaction of Kaposi's sarcoma-associated herpesvirus regulatory protein ORF57 with its multifunctional partner hnRNP K

    PubMed Central

    Malik, Poonam; Clements, J. Barklie

    2004-01-01

    ORF57 protein of Kaposi's sarcoma-associated herpesvirus has a counterpart in all herpesvirus of mammals and birds and regulates gene expression at transcriptional and post-transcriptional levels. ORF57 was capable of self-interaction and bound a rapidly migrating form of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional cellular protein involved in gene expression. In virus infected cell extracts, ORF57 was present in a complex with hnRNP K that had protein kinase CK2 activity, and was phosphorylated by CK2. Different regions of ORF57 bound both catalytic α/α′ and regulatory β subunits of CK2. CK2 modification enhanced the ORF57–hnRNP K interaction, and may regulate the presence and activities of components in the complex. We suggest that ORF57 and hnRNP K interaction may modulate ORF57-mediated regulation of viral gene expression. Herpesviral ORF57 (Rhadinovirus) and ICP27 (Simplexvirus) proteins both interact with hnRNP K and CK2 implying that adaptation of the ancestral hnRNP K and CK2 to associate with viral regulatory ancestor protein likely pre-dates divergence of these Herpesviridae genera that occurred 200 million years ago. PMID:15486205

  11. Class-Switch Recombination in the Absence of the IgH 3' Regulatory Region.

    PubMed

    Kim, Ahrom; Han, Li; Santiago, Gabriel E; Verdun, Ramiro E; Yu, Kefei

    2016-10-01

    The ∼28-kb 3' regulatory region (3'RR), which is located at the most distal 3' region of the Ig H chain locus, has multiple regulatory functions that control IgH expression, class-switch recombination (CSR), and somatic hypermutation. In this article, we report that deletion of the entire 3'RR in a mouse B cell line that is capable of robust cytokine-dependent CSR to IgA results in reduced, but not abolished, CSR. These data suggest that 3'RR is not absolutely required for CSR and, thus, is not essential for targeting activation-induced cytidine deaminase to S regions, as was suggested. Moreover, replacing 3'RR with a DNA fragment including only its four DNase I hypersensitive sites (lacking the large spacer regions) restores CSR to a level equivalent to or even higher than in wild-type cells, suggesting that the four hypersensitive sites contain most of the CSR-promoting functions of 3'RR. Stimulated cells express abundant germline transcripts, with the presence or absence of 3'RR, providing evidence that 3'RR has a role in promoting CSR that is unique from enhancing S region transcription.

  12. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    SciTech Connect

    Lee, Andrew Loyd

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a βαβ-βαβ pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition 15N T1, T2, and 15N/1H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone 1H, 13C, and 15N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and 15N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

  13. Egr2 and Egr3 in regulatory T cells cooperatively control systemic autoimmunity through Ltbp3-mediated TGF-β3 production.

    PubMed

    Morita, Kaoru; Okamura, Tomohisa; Inoue, Mariko; Komai, Toshihiko; Teruya, Shuzo; Iwasaki, Yukiko; Sumitomo, Shuji; Shoda, Hirofumi; Yamamoto, Kazuhiko; Fujio, Keishi

    2016-12-13

    Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by autoantibodies. Early growth response gene 2 (Egr2), a transcription factor essential for T-cell anergy induction, controls systemic autoimmunity in mice and humans. We have previously identified a subpopulation of CD4(+) regulatory T cells, CD4(+)CD25(-)LAG3(+) cells, that characteristically express both Egr2 and LAG3 and control mice model of lupus via TGF-β3 production. However, due to the mild phenotype of lymphocyte-specific Egr2-deficient mice, the presence of an additional regulator has been speculated. Here, we show that Egr2 and Egr3 expressed in T cells cooperatively prevent humoral immune responses by supporting TGF-β3 secretion. T cell-specific Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously developed an early onset lupus-like disease that was more severe than in T cell-specific Egr2-deficient mice. In accordance with the observation that CD4(+)CD25(-)LAG3(+) cells from Egr2/3DKO mice completely lost the capacity to produce TGF-β3, the excessive germinal center reaction in Egr2/3DKO mice was suppressed by the adoptive transfer of WT CD4(+)CD25(-)LAG3(+) cells or treatment with a TGF-β3-expressing vector. Intriguingly, latent TGF-β binding protein (Ltbp)3 expression maintained by Egr2 and Egr3 was required for TGF-β3 production from CD4(+)CD25(-)LAG3(+) cells. Because Egr2 and Egr3 did not demonstrate cell intrinsic suppression of the development of follicular helper T cells, Egr2- and Egr3-dependent TGF-β3 production by CD4(+)CD25(-)LAG3(+) cells is critical for controlling excessive B-cell responses. The unique attributes of Egr2/Egr3 in T cells may provide an opportunity for developing novel therapeutics for autoantibody-mediated diseases including SLE.

  14. Egr2 and Egr3 in regulatory T cells cooperatively control systemic autoimmunity through Ltbp3-mediated TGF-β3 production

    PubMed Central

    Morita, Kaoru; Okamura, Tomohisa; Inoue, Mariko; Komai, Toshihiko; Teruya, Shuzo; Iwasaki, Yukiko; Sumitomo, Shuji; Shoda, Hirofumi; Yamamoto, Kazuhiko; Fujio, Keishi

    2016-01-01

    Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by autoantibodies. Early growth response gene 2 (Egr2), a transcription factor essential for T-cell anergy induction, controls systemic autoimmunity in mice and humans. We have previously identified a subpopulation of CD4+ regulatory T cells, CD4+CD25−LAG3+ cells, that characteristically express both Egr2 and LAG3 and control mice model of lupus via TGF-β3 production. However, due to the mild phenotype of lymphocyte-specific Egr2-deficient mice, the presence of an additional regulator has been speculated. Here, we show that Egr2 and Egr3 expressed in T cells cooperatively prevent humoral immune responses by supporting TGF-β3 secretion. T cell-specific Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously developed an early onset lupus-like disease that was more severe than in T cell-specific Egr2-deficient mice. In accordance with the observation that CD4+CD25−LAG3+ cells from Egr2/3DKO mice completely lost the capacity to produce TGF-β3, the excessive germinal center reaction in Egr2/3DKO mice was suppressed by the adoptive transfer of WT CD4+CD25−LAG3+ cells or treatment with a TGF-β3–expressing vector. Intriguingly, latent TGF-β binding protein (Ltbp)3 expression maintained by Egr2 and Egr3 was required for TGF-β3 production from CD4+CD25−LAG3+ cells. Because Egr2 and Egr3 did not demonstrate cell intrinsic suppression of the development of follicular helper T cells, Egr2- and Egr3-dependent TGF-β3 production by CD4+CD25−LAG3+ cells is critical for controlling excessive B-cell responses. The unique attributes of Egr2/Egr3 in T cells may provide an opportunity for developing novel therapeutics for autoantibody-mediated diseases including SLE. PMID:27911796

  15. The Golgi protein ACBD3 facilitates Enterovirus 71 replication by interacting with 3A

    PubMed Central

    Lei, Xiaobo; Xiao, Xia; Zhang, Zhenzhen; Ma, Yijie; Qi, Jianli; Wu, Chao; Xiao, Yan; Zhou, Zhuo; He, Bin; Wang, Jianwei

    2017-01-01

    Enterovirus 71 (EV71) is a human pathogen that causes hand, foot, mouth disease and neurological complications. Although EV71, as well as other enteroviruses, initiates a remodeling of intracellular membrane for genomic replication, the regulatory mechanism remains elusive. By screening human cDNA library, we uncover that the Golgi resident protein acyl-coenzyme A binding domain-containing 3 (ACBD3) serves as a target of the 3A protein of EV71. This interaction occurs in cells expressing 3A or infected with EV71. Genetic inhibition or deletion of ACBD3 drastically impairs viral RNA replication and plaque formation. Such defects are corrected upon restoration of ACBD3. In infected cells, EV71 3A redirects ACBD3, to the replication sites. I44A or H54Y substitution in 3A interrupts the binding to ACBD3. As such, viral replication is impeded. These results reveal a mechanism of EV71 replication that involves host ACBD3 for viral replication. PMID:28303920

  16. Structure and Function of the SWIRM Domain, a Conserved Protein Module Found in Chromatin Regulatory Complexes

    SciTech Connect

    Da,G.; Lenkart, J.; Zhao, K.; Shiekhattar, R.; Cairns, B.; Marmorstein, R.

    2006-01-01

    The SWIRM domain is a module found in the Swi3 and Rsc8 subunits of SWI/SNF-family chromatin remodeling complexes, and the Ada2 and BHC110/LSD1 subunits of chromatin modification complexes. Here we report the high-resolution crystal structure of the SWIRM domain from Swi3 and characterize the in vitro and in vivo function of the SWIRM domains from Saccharomyces cerevisiae Swi3 and Rsc8. The Swi3 SWIRM forms a four-helix bundle containing a pseudo 2-fold axis and a helix-turn-helix motif commonly found in DNA-binding proteins. We show that the Swi3 SWIRM binds free DNA and mononucleosomes with high and comparable affinity and that a subset of Swi3 substitution mutants that display growth defects in vivo also show impaired DNA-binding activity in vitro, consistent with a nucleosome targeting function of this domain. Genetic and biochemical studies also reveal that the Rsc8 and Swi3 SWIRM domains are essential for the proper assembly and in vivo functions of their respective complexes. Together, these studies identify the SWIRM domain as an essential multifunctional module for the regulation of gene expression.

  17. Structure and function of the SWIRM domain, a conserved protein module found in chromatin regulatory complexes.

    PubMed

    Da, Guoping; Lenkart, Jeffrey; Zhao, Kehao; Shiekhattar, Ramin; Cairns, Bradley R; Marmorstein, Ronen

    2006-02-14

    The SWIRM domain is a module found in the Swi3 and Rsc8 subunits of SWI/SNF-family chromatin remodeling complexes, and the Ada2 and BHC110/LSD1 subunits of chromatin modification complexes. Here we report the high-resolution crystal structure of the SWIRM domain from Swi3 and characterize the in vitro and in vivo function of the SWIRM domains from Saccharomyces cerevisiae Swi3 and Rsc8. The Swi3 SWIRM forms a four-helix bundle containing a pseudo 2-fold axis and a helix-turn-helix motif commonly found in DNA-binding proteins. We show that the Swi3 SWIRM binds free DNA and mononucleosomes with high and comparable affinity and that a subset of Swi3 substitution mutants that display growth defects in vivo also show impaired DNA-binding activity in vitro, consistent with a nucleosome targeting function of this domain. Genetic and biochemical studies also reveal that the Rsc8 and Swi3 SWIRM domains are essential for the proper assembly and in vivo functions of their respective complexes. Together, these studies identify the SWIRM domain as an essential multifunctional module for the regulation of gene expression.

  18. Fourteen Ways to Reroute Cooperative Communication in the Lactose Repressor: Engineering Regulatory Proteins with Alternate Repressive Functions.

    PubMed

    Richards, David H; Meyer, Sarai; Wilson, Corey J

    2017-01-20

    The lactose repressor (LacI) is a classic genetic switch that has been used as a fundamental component in a host of synthetic genetic networks. To expand the function of LacI for use in the development of novel networks and other biotechnological applications, we engineered alternate communication in the LacI scaffold via laboratory evolution. Here we produced 14 new regulatory elements based on the LacI topology that are responsive to isopropyl β-d-1-thiogalactopyranoside (IPTG) with variation in repression strengths and ligand sensitivities-on solid media. The new variants exhibit repressive as well as antilac (i.e., inverse-repression + IPTG) functions and variations in the control of gene output upon exposure to different concentrations of IPTG. In addition, examination of this collection of variants in solution results in the controlled output of a canonical florescent reporter, demonstrating the utility of this collection of new regulatory proteins under standard conditions.

  19. The Golgi Protein ACBD3, an Interactor for Poliovirus Protein 3A, Modulates Poliovirus Replication

    PubMed Central

    Téoulé, François; Brisac, Cynthia; Pelletier, Isabelle; Vidalain, Pierre-Olivier; Jégouic, Sophie; Mirabelli, Carmen; Bessaud, Maël; Combelas, Nicolas; Autret, Arnaud; Tangy, Frédéric; Delpeyroux, Francis

    2013-01-01

    We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence. PMID:23926333

  20. Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3β protein.

    PubMed

    Ge, Qiang; Huang, Nian; Wynn, R Max; Li, Yang; Du, Xinlin; Miller, Bonnie; Zhang, Hong; Uyeda, Kosaku

    2012-12-07

    Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β·ChREBP α2 complex.

  1. LGP2 downregulates interferon production during infection with seasonal human influenza A viruses that activate interferon regulatory factor 3.

    PubMed

    Malur, Meghana; Gale, Michael; Krug, Robert M

    2012-10-01

    LGP2, a member of the RIG-I-like receptor family, lacks the amino-terminal caspase activation recruitment domains (CARDs) required for initiating the activation of interferon regulatory factor 3 (IRF3) and interferon (IFN) transcription. The role of LGP2 in virus infection is controversial, and the only LGP2 experiments previously carried out with mammalian influenza A viruses employed an attenuated, mouse-adapted H1N1 A/PR/8/34 (PR8) virus that does not encode the NS1 protein. Here we determine whether LGP2 has a role during infection with wild-type, nonattenuated influenza A viruses that have circulated in the human population, specifically two types of seasonal influenza A viruses: (i) H3N2 and H1N1 viruses that activate IRF3 and IFN transcription and (ii) recent H1N1 viruses that block these two activations. In human cells infected with an H3N2 virus that activates IRF3, overexpression of LGP2 or its repressor domain decreased STAT1 activation and IFN-β transcription approximately 10-fold. Overexpression of LGP2 also caused a 10-fold decrease of STAT1 activation during infection with other seasonal influenza A viruses that activate IRF3. Using LGP2(+/+) and LGP2(-/-) mouse cells, we show that endogenous LGP2 decreased IFN production during H3N2 virus infection 3- to 4-fold. In contrast, in both mouse and human cells infected with H1N1 viruses that do not activate IRF3, LGP2 had no detectable role. These results demonstrate that LGP2 downregulates IFN production during infection by seasonal influenza A viruses that activate IRF3 and IFN transcription. It is intriguing that LGP2, a host protein induced during influenza A virus infection, downregulates the host antiviral IFN response.

  2. The 15N and 46R Residues of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Enhance Regulatory T Lymphocytes Proliferation

    PubMed Central

    Bai, Juan; Li, Yufeng; Zhang, Qiaoya; Jiang, Ping

    2015-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) negatively modulates host immune responses, resulting in persistent infection and immunosuppression. PRRSV infection increases the number of PRRSV-specific regulatory T lymphocytes (Tregs) in infected pigs. However, the target antigens for Tregs proliferation in PRRSV infection have not been fully understood. In this study, we demonstrated that the highly pathogenic PRRSV (HP-PRRSV) induced more CD4+CD25+Foxp3+ Tregs than classical PRRSV (C-PRRSV) strain. Of the recombinant GP5, M and N proteins of HP-PRRSV expressed in baculovirus expression systems, only N protein induced Tregs proliferation. The Tregs assays showed that three amino-acid regions, 15–21, 42–48 and 88–94, in N protein played an important role in induction of Tregs proliferation with synthetic peptides covering the whole length of N protein. By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation. The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine. These data should be useful for understanding the mechanism of immunity to PRRSV and development of infection control strategies in the future. PMID:26397116

  3. Magnolol causes alterations in the cell cycle in androgen insensitive human prostate cancer cells in vitro by affecting expression of key cell cycle regulatory proteins.

    PubMed

    McKeown, Brendan T; McDougall, Luke; Catalli, Adriana; Hurta, Robert A R

    2014-01-01

    Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

  4. Conformation-selective ATP-competitive inhibitors control regulatory interactions and noncatalytic functions of mitogen-activated protein kinases.

    PubMed

    Hari, Sanjay B; Merritt, Ethan A; Maly, Dustin J

    2014-05-22

    Most potent protein kinase inhibitors act by competing with ATP to block the phosphotransferase activity of their targets. However, emerging evidence demonstrates that ATP-competitive inhibitors can affect kinase interactions and functions in ways beyond blocking catalytic activity. Here, we show that stabilizing alternative ATP-binding site conformations of the mitogen-activated protein kinases (MAPKs) p38α and Erk2 with ATP-competitive inhibitors differentially, and in some cases divergently, modulates the abilities of these kinases to interact with upstream activators and deactivating phosphatases. Conformation-selective ligands are also able to modulate Erk2's ability to allosterically activate the MAPK phosphatase DUSP6, highlighting how ATP-competitive ligands can control noncatalytic kinase functions. Overall, these studies underscore the relationship between the ATP-binding and regulatory sites of MAPKs and provide insight into how ATP-competitive ligands can be designed to confer graded control over protein kinase function.

  5. Preservation of Gene Duplication Increases the Regulatory Spectrum of Ribosomal Protein Genes and Enhances Growth under Stress.

    PubMed

    Parenteau, Julie; Lavoie, Mathieu; Catala, Mathieu; Malik-Ghulam, Mustafa; Gagnon, Jules; Abou Elela, Sherif

    2015-12-22

    In baker's yeast, the majority of ribosomal protein genes (RPGs) are duplicated, and it was recently proposed that such duplications are preserved via the functional specialization of the duplicated genes. However, the origin and nature of duplicated RPGs' (dRPGs) functional specificity remain unclear. In this study, we show that differences in dRPG functions are generated by variations in the modality of gene expression and, to a lesser extent, by protein sequence. Analysis of the sequence and expression patterns of non-intron-containing RPGs indicates that each dRPG is controlled by specific regulatory sequences modulating its expression levels in response to changing growth conditions. Homogenization of dRPG sequences reduces cell tolerance to growth under stress without changing the number of expressed genes. Together, the data reveal a model where duplicated genes provide a means for modulating the expression of ribosomal proteins in response to stress.

  6. Regulatory role of the 90-kDa-heat-shock protein (Hsp90) and associated factors on gene expression.

    PubMed

    Erlejman, Alejandra G; Lagadari, Mariana; Toneatto, Judith; Piwien-Pilipuk, Graciela; Galigniana, Mario D

    2014-02-01

    The term molecular chaperone was first used to describe the ability of nucleoplasmin to prevent the aggregation of histones with DNA during the assembly of nucleosomes. Subsequently, the name was extended to proteins that mediate the post-translational assembly of oligomeric complexes protecting them from denaturation and/or aggregation. Hsp90 is a 90-kDa molecular chaperone that represents the major soluble protein of the cell. In contrast to most conventional chaperones, Hsp90 functions as a refined sensor of protein function and its principal role in the cell is to facilitate biological activity to properly folded client proteins that already have a preserved tertiary structure. Consequently, Hsp90 is related to basic cell functions such as cytoplasmic transport of soluble proteins, translocation of client proteins to organelles, and regulation of the biological activity of key signaling factors such as protein kinases, ubiquitin ligases, steroid receptors, cell cycle regulators, and transcription factors. A growing amount of evidence links the protective action of this molecular chaperone to mechanisms related to posttranslational modifications of soluble nuclear factors as well as histones. In this article, we discuss some aspects of the regulatory action of Hsp90 on transcriptional regulation and how this effect could have impacted genetic assimilation mechanism in some organisms.

  7. Control of tissue size and development by a regulatory element in the yorkie 3’UTR

    PubMed Central

    Umegawachi, Takanari; Yoshida, Hideki; Koshida, Hiromu; Yamada, Momoko; Ohkawa, Yasuyuki; Sato, Tetsuya; Suyama, Mikita; Krause, Henry M; Yamaguchi, Masamitsu

    2017-01-01

    Regulation of the Hippo pathway via phosphorylation of Yorkie (Yki), the Drosophila homolog of human Yes-associated protein 1, is conserved from Drosophila to humans. Overexpression of a non-phosphorylatable form of Yki induces severe overgrowth in adult fly eyes. Here, we show that yki mRNA associates with microsomal fractions and forms foci that partially colocalize to processing bodies in the vicinity of endoplasmic reticulum. This localization is dependent on a stem-loop (SL) structure in the 3’ untranslated region of yki. Surprisingly, expression of SL deleted yki in eye imaginal discs also results in severe overgrowth phenotypes. When the structure of the SL is disrupted, Yki protein levels increase without a significant effect on RNA levels. When the SL is completely removed, protein levels drastically increase, but in this case, due to increased RNA stability. In the latter case, we show that the increased RNA accumulation is due to removal of a putative miR-8 seed sequence in the SL. These data demonstrate the function of two novel regulatory mechanisms, both controlled by the yki SL element, that are essential for proper Hippo pathway mediated growth regulation.

  8. Print Me an Organ? Ethical and Regulatory Issues Emerging from 3D Bioprinting in Medicine.

    PubMed

    Gilbert, Frederic; O'Connell, Cathal D; Mladenovska, Tajanka; Dodds, Susan

    2017-02-09

    Recent developments of three-dimensional printing of biomaterials (3D bioprinting) in medicine have been portrayed as demonstrating the potential to transform some medical treatments, including providing new responses to organ damage or organ failure. However, beyond the hype and before 3D bioprinted organs are ready to be transplanted into humans, several important ethical concerns and regulatory questions need to be addressed. This article starts by raising general ethical concerns associated with the use of bioprinting in medicine, then it focuses on more particular ethical issues related to experimental testing on humans, and the lack of current international regulatory directives to guide these experiments. Accordingly, this article (1) considers whether there is a limit as to what should be bioprinted in medicine; (2) examines key risks of significant harm associated with testing 3D bioprinting for humans; (3) investigates the clinical trial paradigm used to test 3D bioprinting; (4) analyses ethical questions of irreversibility, loss of treatment opportunity and replicability; (5) explores the current lack of a specific framework for the regulation and testing of 3D bioprinting treatments.

  9. Superior Cervical Ganglia Neurons Induce Foxp3+ Regulatory T Cells via Calcitonin Gene-Related Peptide.

    PubMed

    Szklany, Kirsten; Ruiter, Evelyn; Mian, Firoz; Kunze, Wolfgang; Bienenstock, John; Forsythe, Paul; Karimi, Khalil

    2016-01-01

    The nervous and immune systems communicate bidirectionally, utilizing diverse molecular signals including cytokines and neurotransmitters to provide an integrated response to changes in the body's internal and external environment. Although, neuro-immune interactions are becoming better understood under inflammatory circumstances and it has been evidenced that interaction between neurons and T cells results in the conversion of encephalitogenic T cells to T regulatory cells, relatively little is known about the communication between neurons and naïve T cells. Here, we demonstrate that following co-culture of naïve CD4+ T cells with superior cervical ganglion neurons, the percentage of Foxp3 expressing CD4+CD25+ cells significantly increased. This was mediated in part by immune-regulatory cytokines TGF-β and IL-10, as well as the neuropeptide calcitonin gene-related peptide while vasoactive intestinal peptide was shown to play no role in generation of T regulatory cells. Additionally, T cells co-cultured with neurons showed a decrease in the levels of pro-inflammatory cytokine IFN-γ released upon in vitro stimulation. These findings suggest that the generation of Tregs may be promoted by naïve CD4+ T cell: neuron interaction through the release of neuropeptide CGRP.

  10. Drug-drug interactions related to altered absorption and plasma protein binding: theoretical and regulatory considerations, and an industry perspective.

    PubMed

    Hochman, Jerome; Tang, Cuyue; Prueksaritanont, Thomayant

    2015-03-01

    Drug-drug interactions (DDIs) related to altered drug absorption and plasma protein binding have received much less attention from regulatory agencies relative to DDIs mediated via drug metabolizing enzymes and transporters. In this review, a number of theoretical bases and regulatory framework are presented for these DDI aspects. Also presented is an industry perspective on how to approach these issues in support of drug development. Overall, with the exception of highly permeable and highly soluble (BCS 1) drugs, DDIs related to drug-induced changes in gastrointestinal (GI) physiology can be substantial, thus warranting more attentions. For a better understanding of absorption-associated DDI potential in a clinical setting, mechanistic studies should be conducted based on holistic integration of the pharmaceutical profiles (e.g., pH-dependent solubility) and pharmacological properties (e.g., GI physiology and therapeutic margin) of drug candidates. Although majority of DDI events related to altered plasma protein binding are not expected to be of clinical significance, exceptions exist for a subset of compounds with certain pharmacokinetic and pharmacological properties. Knowledge of the identity of binding proteins and the binding extent in various clinical setting (including disease states) can be valuable in aiding clinical DDI data interpretations, and ensuring safe and effective use of new drugs.

  11. Rethinking gene regulatory networks in light of alternative splicing, intrinsically disordered protein domains, and post-translational modifications

    PubMed Central

    Niklas, Karl J.; Bondos, Sarah E.; Dunker, A. Keith; Newman, Stuart A.

    2015-01-01

    Models for genetic regulation and cell fate specification characteristically assume that gene regulatory networks (GRNs) are essentially deterministic and exhibit multiple stable states specifying alternative, but pre-figured cell fates. Mounting evidence shows, however, that most eukaryotic precursor RNAs undergo alternative splicing (AS) and that the majority of transcription factors contain intrinsically disordered protein (IDP) domains whose functionalities are context dependent as well as subject to post-translational modification (PTM). Consequently, many transcription factors do not have fixed cis-acting regulatory targets, and developmental determination by GRNs alone is untenable. Modeling these phenomena requires a multi-scale approach to explain how GRNs operationally interact with the intra- and intercellular environments. Evidence shows that AS, IDP, and PTM complicate gene expression and act synergistically to facilitate and promote time- and cell-specific protein modifications involved in cell signaling and cell fate specification and thereby disrupt a strict deterministic GRN-phenotype mapping. The combined effects of AS, IDP, and PTM give proteomes physiological plasticity, adaptive responsiveness, and developmental versatility without inefficiently expanding genome size. They also help us understand how protein functionalities can undergo major evolutionary changes by buffering mutational consequences. PMID:25767796

  12. Stress-induced Start Codon Fidelity Regulates Arsenite-inducible Regulatory Particle-associated Protein (AIRAP) Translation*

    PubMed Central

    Zach, Lolita; Braunstein, Ilana; Stanhill, Ariel

    2014-01-01

    Initial steps in protein synthesis are highly regulated processes as they define the reading frame of the translation machinery. Eukaryotic translation initiation is a process facilitated by numerous factors (eIFs), aimed to form a “scanning” mechanism toward the initiation codon. Translation initiation of the main open reading frame (ORF) in an mRNA transcript has been reported to be regulated by upstream open reading frames (uORFs) in a manner of re-initiation. This mode of regulation is governed by the phosphorylation status of eIF2α and controlled by cellular stresses. Another mode of translational initiation regulation is leaky scanning, and this regulatory process has not been extensively studied. We have identified arsenite-inducible regulatory particle-associated protein (AIRAP) transcript to be translationally induced during arsenite stress conditions. AIRAP transcript contains a single uORF in a poor-kozak context. AIRAP translation induction is governed by means of leaky scanning and not re-initiation. This induction of AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is phosphorylated under specific conditions that induce protein misfolding and have biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning like re-initiation is responsive to stress conditions and that leaky scanning can induce ORF translation by bypassing poor kozak context of a single uORF transcript. PMID:24898249

  13. An Ancient P-Loop GTPase in Rice Is Regulated by a Higher Plant-specific Regulatory Protein*

    PubMed Central

    Cheung, Ming-Yan; Xue, Yan; Zhou, Liang; Li, Man-Wah; Sun, Samuel Sai-Ming; Lam, Hon-Ming

    2010-01-01

    YchF is a subfamily of the Obg family in the TRAFAC class of P-loop GTPases. The wide distribution of YchF homologues in both eukarya and bacteria suggests that they are descendents of an ancient protein, yet their physiological roles remain unclear. Using the OsYchF1-OsGAP1 pair from rice as the prototype, we provide evidence for the regulation of GTPase/ATPase activities and RNA binding capacity of a plant YchF (OsYchF1) by its regulatory protein (OsGAP1). The effects of OsGAP1 on the subcellular localization/cycling and physiological functions of OsYchF1 are also discussed. The finding that OsYchF1 and OsGAP1 are involved in plant defense response might shed light on the functional roles of YchF homologues in plants. This work suggests that during evolution, an ancestral P-loop GTPase/ATPase may acquire new regulation and function(s) by the evolution of a lineage-specific regulatory protein. PMID:20876569

  14. Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.

    PubMed Central

    Germino, J; Gray, J G; Charbonneau, H; Vanaman, T; Bastia, D

    1983-01-01

    The initiation of DNA replication of plasmid R6K is triggered by a 35-kilodalton initiator protein. The initiator protein had been elusive because of its lability and the lack of a convenient assay procedure to aid its purification. Using recombinant DNA techniques, we have fused the cistron of the initiator near its COOH-terminal end, in the correct reading frame, to the lacZ cistron of Escherichia coli at the ninth codon from the NH2 terminus. The fused cistron yielded a protein that was not only stable in vivo but also had dual activities: initiation of DNA replication in vivo and in vitro and hydrolysis of beta-galactoside. Using an affinity column that is specific for beta-galactosidase, we have demonstrated the rapid purification of the hybrid protein to near homogeneity. Exploiting the polymeric structure of the initiator, we have also isolated the nonfused form of the initiator protein, associated through subunit interaction with the beta-galactosidase-fused protein, which permits its purification by affinity chromatography. NH2-terminal amino acid sequence analysis of the heteropolymer has not only shown that the fused and nonfused initiators have the same sequence but also confirmed the protein sequence of the initiator as predicted from its nucleotide sequence. The techniques described here should be generally useful for the isolation of other proteins that are difficult to purify by conventional procedures. Images PMID:6316329

  15. Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

    PubMed Central

    Biondi, R M; Baehler, P J; Reymond, C D; Véron, M

    1998-01-01

    The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit. PMID:9776758

  16. Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

    PubMed

    Biondi, R M; Baehler, P J; Reymond, C D; Véron, M

    1998-11-01

    The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.

  17. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent Lipogenic Pathway*

    PubMed Central

    Zhou, Jie; Zhai, Yonggong; Mu, Ying; Gong, Haibiao; Uppal, Hirdesh; Toma, David; Ren, Songrong; Evans, Ronald M.; Xie, Wen

    2014-01-01

    The pregnane X receptor (PXR) was isolated as a xenosensor regulating xenobiotic responses. In this study, we show that PXR plays an endobiotic role by impacting lipid homeostasis. Expression of an activated PXR in the livers of transgenic mice resulted in an increased hepatic deposit of triglycerides. This PXR-mediated lipid accumulation was independent of the activation of the lipogenic transcriptional factor SREBP-1c (sterol regulatory element-binding protein 1c) and its primary lipogenic target enzymes, including fatty-acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC-1). Instead, the lipid accumulation in transgenic mice was associated with an increased expression of the free fatty acid transporter CD36 and several accessory lipogenic enzymes, such as stearoyl-CoA desaturase-1 (SCD-1) and long chain free fatty acid elongase. Studies using transgenic and knock-out mice showed that PXR is both necessary and sufficient for Cd36 activation. Promoter analyses revealed a DR-3-type of PXR-response element in the mouse Cd36 promoter, establishing Cd36 as a direct transcriptional target of PXR. The hepatic lipid accumulation and Cd36 induction were also seen in the hPXR “humanized” mice treated with the hPXR agonist rifampicin. The activation of PXR was also associated with an inhibition of pro-β-oxidative genes, such as peroxisome proliferator-activated receptor α (PPARα) and thiolase, and an up-regulation of PPARγ, a positive regulator of CD36. The cross-regulation of CD36 by PXR and PPARγ suggests that this fatty acid transporter may function as a common target of orphan nuclear receptors in their regulation of lipid homeostasis. PMID:16556603

  18. Complement regulatory proteins in early human fetal life: CD59, membrane co-factor protein (MCP) and decay-accelerating factor (DAF) are differentially expressed in the developing liver.

    PubMed Central

    Simpson, K L; Houlihan, J M; Holmes, C H

    1993-01-01

    The human fetus appears to be capable of protecting itself from maternal complement (C) from an early stage in development by expressing the C regulatory proteins decay-accelerating factor (DAF), membrane co-factor protein (MCP) and CD59 on fetally derived trophoblast at the feto-maternal interface. In this study we have examined the ontogeny of these proteins within the fetus itself and have focused on the liver which represents a major site of haemopoiesis during development. Immunostaining revealed that DAF, MCP and CD59 are all expressed from at least 6 weeks of gestation in the liver but that these proteins display distinct distribution patterns. CD59 was broadly distributed both within the epithelial and haemopoietic compartments, but expression of C3 convertase regulators was more restricted. DAF expression was limited to isolated cells within haemopoietic nests and the epithelium was DAF-negative. Although MCP expression on haemopoietic cells was also limited, by contrast with DAF the developing hepatic epithelium was strongly MCP-positive. Typical CD59 and MCP components were observed in fetal liver extracts by immunoblotting, although liver MCP components consistently migrated 4000-5000 MW ahead of those observed on placental trophoblast. Differences in the distribution of these proteins were also observed between the fetal and adult liver. In particular, by comparison with fetal hepatic epithelium, there was an apparent loss of MCP expression from adult hepatocytes. Thus, MCP appears to be developmentally regulated in the human liver and is expressed in the absence of DAF on the early hepatic epithelium. Overall, this study suggests that C regulatory proteins, and in particular CD59 and MCP, are required from the very early stages of gestation within the fetus itself. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7505254

  19. Essential role of mitochondrial energy metabolism in Foxp3+ T-regulatory cell function and allograft survival

    PubMed Central

    Beier, Ulf H.; Angelin, Alessia; Akimova, Tatiana; Wang, Liqing; Liu, Yujie; Xiao, Haiyan; Koike, Maya A.; Hancock, Saege A.; Bhatti, Tricia R.; Han, Rongxiang; Jiao, Jing; Veasey, Sigrid C.; Sims, Carrie A.; Baur, Joseph A.; Wallace, Douglas C.; Hancock, Wayne W.

    2015-01-01

    Conventional T (Tcon) cells and Foxp3+ T-regulatory (Treg) cells are thought to have differing metabolic requirements, but little is known of mitochondrial functions within these cell populations in vivo. In murine studies, we found that activation of both Tcon and Treg cells led to myocyte enhancer factor 2 (Mef2)-induced expression of genes important to oxidative phosphorylation (OXPHOS). Inhibition of OXPHOS impaired both Tcon and Treg cell function compared to wild-type cells but disproportionally affected Treg cells. Deletion of Pgc1α or Sirt3, which are key regulators of OXPHOS, abrogated Treg-dependent suppressive function and impaired allograft survival. Mef2 is inhibited by histone/protein deacetylase-9 (Hdac9), and Hdac9 deletion increased Treg suppressive function. Hdac9−/− Treg showed increased expression of Pgc1α and Sirt3, and improved mitochondrial respiration, compared to wild-type Treg cells. Our data show that key OXPHOS regulators are required for optimal Treg function and Treg-dependent allograft acceptance. These findings provide a novel approach to increase Treg function and give insights into the fundamental mechanisms by which mitochondrial energy metabolism regulates immune cell functions in vivo.—Beier, U. H., Angelin, A., Akimova, T., Wang, L., Liu, Y., Xiao, H., Koike, M. A., Hancock, S. A., Bhatti, T. R., Han, R., Jiao, J., Veasey, S. C., Sims, C. A., Baur, J. A., Wallace, D. C., Hancock, W. W. Essential role of mitochondrial energy metabolism in Foxp3+ T-regulatory cell function and allograft survival. PMID:25681462

  20. Protein kinase A type II-α regulatory subunit regulates the response of prostate cancer cells to taxane treatment

    PubMed Central

    Zynda, Evan R; Matveev, Vitaliy; Makhanov, Michael; Chenchik, Alexander; Kandel, Eugene S

    2014-01-01

    In the last decade taxane-based therapy has emerged as a standard of care for hormone-refractory prostate cancer. Nevertheless, a significant fraction of tumors show no appreciable response to the treatment, while the others develop resistance and recur. Despite years of intense research, the mechanisms of taxane resistance in prostate cancer and other malignancies are poorly understood and remain a topic of intense investigation. We have used improved mutagenesis via random insertion of a strong promoter to search for events, which enable survival of prostate cancer cells after Taxol exposure. High-throughput mapping of the integration sites pointed to the PRKAR2A gene, which codes for a type II-α regulatory subunit of protein kinase A, as a candidate modulator of drug response. Both full-length and N-terminally truncated forms of the PRKAR2A gene product markedly increased survival of prostate cancer cells lines treated with Taxol and Taxotere. Suppression of protein kinase A enzymatic activity is the likely mechanism of action of the overexpressed proteins. Accordingly, protein kinase A inhibitor PKI (6–22) amide reduced toxicity of Taxol to prostate cancer cells. Our findings support the role of protein kinase A and its constituent proteins in cell response to chemotherapy. PMID:25485509

  1. Basolateral Na+/HCO3– cotransport activity is regulated by the dissociable Na+/H+ exchanger regulatory factor

    PubMed Central

    Bernardo, Angelito A.; Kear, Felicidad T.; Santos, Anna V.P.; Ma, Jianfei; Steplock, Debra; Robey, R. Brooks; Weinman, Edward J.

    1999-01-01

    In the renal proximal tubule, the activities of the basolateral Na+/HCO3– cotransporter (NBC) and the apical Na+/H+ exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif–containing protein, the Na+/H+ exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10–8M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-RF–expressing B-SC-1 cells (B-SC-RF) exhibited markedly lower basal levels of NBC activity than did wild-type controls. Inhibition of NBC activity in B-SC-RF cells was enhanced after 10 μM of forskolin treatment, consistent with a postulated role for NHE-RF in mediating the inhibition of NBC activity by PKA. These findings not only suggest NHE-RF involvement in PKA-regulated NBC activity, but also provide a unique molecular mechanism whereby

  2. Arf-like Protein 3 (ARL3) Regulates Protein Trafficking and Ciliogenesis in Mouse Photoreceptors.

    PubMed

    Hanke-Gogokhia, Christin; Wu, Zhijian; Gerstner, Cecilia D; Frederick, Jeanne M; Zhang, Houbin; Baehr, Wolfgang

    2016-03-25

    Arf-like protein 3 (ARL3) is a ubiquitous small GTPase expressed in ciliated cells of plants and animals. Germline deletion ofArl3in mice causes multiorgan ciliopathy reminiscent of Bardet-Biedl or Joubert syndromes. As photoreceptors are elegantly compartmentalized and have cilia, we probed the function of ARL3 (ADP-ribosylation factor (Arf)-like 3 protein) by generating rod photoreceptor-specific (prefix(rod)) and retina-specific (prefix(ret))Arl3deletions. In predegenerate(rod)Arl3(-/-)mice, lipidated phototransduction proteins showed trafficking deficiencies, consistent with the role of ARL3 as a cargo displacement factor for lipid-binding proteins. By contrast,(ret)Arl3(-/-)rods and cones expressing Cre recombinase during embryonic development formed neither connecting cilia nor outer segments and degenerated rapidly. Absence of cilia infers participation of ARL3 in ciliogenesis and axoneme formation. Ciliogenesis was rescued, and degeneration was reversed in part by subretinal injection of adeno-associated virus particles expressing ARL3-EGFP. The conditional knock-out phenotypes permitted identification of two ARL3 functions, both in the GTP-bound form as follows: one as a regulator of intraflagellar transport participating in photoreceptor ciliogenesis and the other as a cargo displacement factor transporting lipidated protein to the outer segment. Surprisingly, a farnesylated inositol polyphosphate phosphatase only trafficked from the endoplasmic reticulum to the Golgi, thereby excluding it from a role in photoreceptor cilia physiology.

  3. [Interaction of 8-substituted derivatives and adenosine-3',5'-cyclophosphate esters with protein kinase from pig brain].

    PubMed

    Guliaev, N N; Tunitskaia, V L; Nesterova, M V; Mazurova, L A; Murtuzaev, I M

    1977-11-01

    A synthesis of previously unknown 8-substituted derivatives and alkyl esters of cyclic adenosine-3',5'-monophosphate, containing reactive groups, was carried out. The interaction of the compounds obtained with a homogeneous preparation of protein kinase from pig brain was studied. It was found that all compounds, with the exception of neutral esters of 3',5'-AMP, activate the enzyme and competitively inhibit 3H-labelled 3',5'-cAMP binding by the regulatory subunit of protein kinase. The activating effect and affinity of 8-(beta-aminoethylamino)-3',5'-cAMP for protein kinase was 10 times lower than that for 3',5'-cAMP and other 8-substituted derivatives of the cyclic nucleotide. It was found that 8-(N-chloroacetylaminoethylamino)-3',5'-cAMP interaction with the enzyme is of irreversible type, which suggest covalent blocking of the nucleophilic group of the 3',5'-cAMP binding site of protein kinase. The data obtained indicate that the 3',5'-cAMP molecule is bound to the regulatory site of protein kinase in the syn-conformation. The previously made assumption on the crucial importance of the negative charge in the 3',5'-cyclophosphate system for the interaction of cyclic AMP with the regulatory subunit of protein kinase has been thus confirmed.

  4. PARP-2 and PARP-3 are selectively activated by 5′ phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1

    PubMed Central

    Langelier, Marie-France; Riccio, Amanda A.; Pascal, John M.

    2014-01-01

    PARP-1, PARP-2 and PARP-3 are DNA-dependent PARPs that localize to DNA damage, synthesize poly(ADP-ribose) (PAR) covalently attached to target proteins including themselves, and thereby recruit repair factors to DNA breaks to increase repair efficiency. PARP-1, PARP-2 and PARP-3 have in common two C-terminal domains—Trp-Gly-Arg (WGR) and catalytic (CAT). In contrast, the N-terminal region (NTR) of PARP-1 is over 500 residues and includes four regulatory domains, whereas PARP-2 and PARP-3 have smaller NTRs (70 and 40 residues, respectively) of unknown structural composition and function. Here, we show that PARP-2 and PARP-3 are preferentially activated by DNA breaks harboring a 5′ phosphate (5′P), suggesting selective activation in response to specific DNA repair intermediates, in particular structures that are competent for DNA ligation. In contrast to PARP-1, the NTRs of PARP-2 and PARP-3 are not strictly required for DNA binding or for DNA-dependent activation. Rather, the WGR domain is the central regulatory domain of PARP-2 and PARP-3. Finally, PARP-1, PARP-2 and PARP-3 share an allosteric regulatory mechanism of DNA-dependent catalytic activation through a local destabilization of the CAT. Collectively, our study provides new insights into the specialization of the DNA-dependent PARPs and their specific roles in DNA repair pathways. PMID:24928857

  5. PARP-2 and PARP-3 are selectively activated by 5' phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1.

    PubMed

    Langelier, Marie-France; Riccio, Amanda A; Pascal, John M

    2014-07-01

    PARP-1, PARP-2 and PARP-3 are DNA-dependent PARPs that localize to DNA damage, synthesize poly(ADP-ribose) (PAR) covalently attached to target proteins including themselves, and thereby recruit repair factors to DNA breaks to increase repair efficiency. PARP-1, PARP-2 and PARP-3 have in common two C-terminal domains-Trp-Gly-Arg (WGR) and catalytic (CAT). In contrast, the N-terminal region (NTR) of PARP-1 is over 500 residues and includes four regulatory domains, whereas PARP-2 and PARP-3 have smaller NTRs (70 and 40 residues, respectively) of unknown structural composition and function. Here, we show that PARP-2 and PARP-3 are preferentially activated by DNA breaks harboring a 5' phosphate (5'P), suggesting selective activation in response to specific DNA repair intermediates, in particular structures that are competent for DNA ligation. In contrast to PARP-1, the NTRs of PARP-2 and PARP-3 are not strictly required for DNA binding or for DNA-dependent activation. Rather, the WGR domain is the central regulatory domain of PARP-2 and PARP-3. Finally, PARP-1, PARP-2 and PARP-3 share an allosteric regulatory mechanism of DNA-dependent catalytic activation through a local destabilization of the CAT. Collectively, our study provides new insights into the specialization of the DNA-dependent PARPs and their specific roles in DNA repair pathways.

  6. Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates

    PubMed Central

    Tinti, Michele; Johnson, Catherine; Toth, Rachel; Ferrier, David E. K.; MacKintosh, Carol

    2012-01-01

    14-3-3 proteins regulate cellular responses to stimuli by docking onto pairs of phosphorylated residues on target proteins. The present study shows that the human 14-3-3-binding phosphoproteome is highly enriched in 2R-ohnologues, which are proteins in families of two to four members that were generated by two rounds of whole genome duplication at the origin of the vertebrates. We identify 2R-ohnologue families whose members share a ‘lynchpin’, defined as a 14-3-3-binding phosphosite that is conserved across members of a given family, and aligns with a Ser/Thr residue in pro-orthologues from the invertebrate chordates. For example, the human receptor expression enhancing protein (REEP) 1–4 family has the commonest type of lynchpin motif in current datasets, with a phosphorylatable serine in the –2 position relative to the 14-3-3-binding phosphosite. In contrast, the second 14-3-3-binding sites of REEPs 1–4 differ and are phosphorylated by different kinases, and hence the REEPs display different affinities for 14-3-3 dimers. We suggest a conceptual model for intracellular regulation involving protein families whose evolution into signal multiplexing systems was facilitated by 14-3-3 dimer binding to lynchpins, which gave freedom for other regulatory sites to evolve. While increased signalling complexity was needed for vertebrate life, these systems also generate vulnerability to genetic disorders. PMID:22870394

  7. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    SciTech Connect

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L.

    2010-05-25

    Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  8. p38 MAPK down-regulates fibulin 3 expression through methylation of gene regulatory sequences: role in migration and invasion.

    PubMed

    Arechederra, María; Priego, Neibla; Vázquez-Carballo, Ana; Sequera, Celia; Gutiérrez-Uzquiza, Álvaro; Cerezo-Guisado, María Isabel; Ortiz-Rivero, Sara; Roncero, Cesáreo; Cuenda, Ana; Guerrero, Carmen; Porras, Almudena

    2015-02-13

    p38 MAPKs regulate migration and invasion. However, the mechanisms involved are only partially known. We had previously identified fibulin 3, which plays a role in migration, invasion, and tumorigenesis, as a gene regulated by p38α. We have characterized in detail how p38 MAPK regulates fibulin 3 expression and its role. We describe here for the first time that p38α, p38γ, and p38δ down-regulate fibulin 3 expression. p38α has a stronger effect, and it does so through hypermethylation of CpG sites in the regulatory sequences of the gene. This would be mediated by the DNA methylase, DNMT3A, which is down-regulated in cells lacking p38α, but once re-introduced represses Fibulin 3 expression. p38α through HuR stabilizes dnmt3a mRNA leading to an increase in DNMT3A protein levels. Moreover, by knocking-down fibulin 3, we have found that Fibulin 3 inhibits migration and invasion in MEFs by mechanisms involving p38α/β inhibition. Hence, p38α pro-migratory/invasive effect might be, at least in part, mediated by fibulin 3 down-regulation in MEFs. In contrast, in HCT116 cells, Fibulin 3 promotes migration and invasion through a mechanism dependent on p38α and/or p38β activation. Furthermore, Fibulin 3 promotes in vitro and in vivo tumor growth of HCT116 cells through a mechanism dependent on p38α, which surprisingly acts as a potent inducer of tumor growth. At the same time, p38α limits fibulin 3 expression, which might represent a negative feed-back loop.

  9. Age-dependent increase of brain copper levels and expressions of copper regulatory proteins in the subventricular zone and choroid plexus

    PubMed Central

    Fu, Sherleen; Jiang, Wendy; Zheng, Wei

    2015-01-01

    Our recent data suggest a high accumulation of copper (Cu) in the subventricular zone (SVZ) along the wall of brain ventricles. Anatomically, SVZ is in direct contact with cerebrospinal fluid (CSF), which is secreted by a neighboring tissue choroid plexus (CP). Changes in Cu regulatory gene expressions in the SVZ and CP as the function of aging may determine Cu levels in the CSF and SVZ. This study was designed to investigate the associations between age, Cu levels, and Cu regulatory genes in SVZ and plexus. The SVZ and CP were dissected from brains of 3-week, 10-week, or 9-month old male rats. Analyses by atomic absorption spectroscopy revealed that the SVZ of adult and old animals contained the highest Cu level compared with other tested brain regions. Significantly positive correlations between age and Cu levels in SVZ and plexus were observed; the SVZ Cu level of old animals was 7.5- and 5.8-fold higher than those of young and adult rats (p < 0.01), respectively. Quantitation by qPCR of the transcriptional expressions of Cu regulatory proteins showed that the SVZ expressed the highest level of Cu storage protein metallothioneins (MTs), while the CP expressed the high level of Cu transporter protein Ctr1. Noticeably, Cu levels in the SVZ were positively associated with type B slow proliferating cell marker Gfap (p < 0.05), but inversely associated with type A proliferating neuroblast marker Dcx (p < 0.05) and type C transit amplifying progenitor marker Nestin (p < 0.01). Dmt1 had significant positive correlations with age and Cu levels in the plexus (p < 0.01). These findings suggest that Cu levels in all tested brain regions are increased as the function of age. The SVZ shows a different expression pattern of Cu-regulatory genes from the CP. The age-related increase of MTs and decrease of Ctr1 may contribute to the high Cu level in this neurogenesis active brain region. PMID:26106293

  10. A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators.

    PubMed Central

    Ueda, K; Miyake, K; Horinouchi, S; Beppu, T

    1993-01-01

    Mutants of Streptomyces griseus deficient in A-factor production are sporulation negative, since A-factor is an essential hormonal regulator for the induction of morphological and physiological differentiation in this bacterium. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this mutant strain. Subcloning experiments and nucleotide sequencing showed that two open reading frames, ORF1 with 656 amino acids and ORF2 with 201 amino acids, were required in order to induce sporulation. The amino acid sequence of ORF1 significantly resembled that of the Escherichia coli HlyB protein, a member of a family of bacterial membrane proteins engaged in ATP-dependent secretion mechanisms. Conserved features of this surface translocator family, such as the transmembrane structure predicted by their hydropathy profiles and the amino acid sequence forming an ATP-binding fold, were also conserved in ORF1. The ORF1 gene appeared to constitute a transcriptional unit with an additional upstream gene encoding ORF3, which was greatly similar to ORF1 in size and amino acid sequence. The other protein, ORF2, showed significant end-to-end homology with the E. coli uhpA product, a regulatory protein for the uptake of sugar phosphates. Like UhpA as a response regulator of a bacterial two-component regulatory system, ORF2 contained a helix-turn-helix DNA-binding domain at its COOH-terminal portion and an Asp residue (Asp-54) probably to be phosphorylated at its NH2-terminal portion. An amino acid replacement from Asp-54 to Asn resulted in the loss of the ability of ORF2 to induce sporulation in strain HH1. Images PMID:8458843

  11. Increased expression of the maize immunoglobulin binding protein homolog b-70 in three zein regulatory mutants.

    PubMed Central

    Boston, R S; Fontes, E B; Shank, B B; Wrobel, R L

    1991-01-01

    Plants carrying floury-2, Defective endosperm-B30, or Mucronate mutations overproduce b-70, a maize homolog of the mammalian immunoglobulin binding protein. During endosperm development in these mutants, levels of both b-70 protein and RNA increase dramatically between 14 days and 20 days after pollination. At later stages, b-70 RNA levels decline while protein levels remain high. The increase in b-70 RNA levels is endosperm specific and dependent on gene dosage in the floury-2 mutant. In all three mutants, the increases in b-70 RNA and protein levels are inversely proportional to changes in zein synthesis. Although b-70 polypeptides can be extracted from purified protein bodies, they carry a carboxy-terminal endoplasmic reticulum retention signal, HDEL. We propose that induction of b-70 in these mutants is a cellular response to abnormally folded or improperly assembled storage proteins and probably reflects its role as a polypeptide chain binding protein. PMID:1840924

  12. Protein inhibitor of activated STAT3 (PIAS3) protein promotes SUMOylation and nuclear sequestration of the intracellular domain of ErbB4 protein.

    PubMed

    Sundvall, Maria; Korhonen, Anna; Vaparanta, Katri; Anckar, Julius; Halkilahti, Kalle; Salah, Zaidoun; Aqeilan, Rami I; Palvimo, Jorma J; Sistonen, Lea; Elenius, Klaus

    2012-06-29

    ErbB4 is a receptor tyrosine kinase implicated in the development and homeostasis of the heart, central nervous system, and mammary gland. Cleavable isoforms of ErbB4 release a soluble intracellular domain (ICD) that can translocate to the nucleus and function as a transcriptional coregulator. In search of regulatory mechanisms of ErbB4 ICD function, we identified PIAS3 as a novel interaction partner of ErbB4 ICD. In keeping with the small ubiquitin-like modifier (SUMO) E3 ligase function of protein inhibitor of activated STAT (PIAS) proteins, we showed that the ErbB4 ICD is modified by SUMO, and that PIAS3 stimulates the SUMOylation. Upon overexpression of PIAS3, the ErbB4 ICD generated from the full-length receptor accumulated into the nucleus in a manner that was dependent on the functional nuclear localization signal of ErbB4. In the nucleus, ErbB4 colocalized with PIAS3 and SUMO-1 in promyelocytic leukemia nuclear bodies, nuclear domains involved in regulation of transcription. Accordingly, PIAS3 overexpression had an effect on the transcriptional coregulatory activity of ErbB4, repressing its ability to coactivate transcription with Yes-associated protein. Finally, knockdown of PIAS3 with siRNA partially rescued the inhibitory effect of the ErbB4 ICD on differentiation of MDA-MB-468 breast cancer and HC11 mammary epithelial cells. Our findings illustrate that PIAS3 is a novel regulator of ErbB4 receptor tyrosine kinase, controlling its nuclear sequestration and function.

  13. Protein Inhibitor of Activated STAT3 (PIAS3) Protein Promotes SUMOylation and Nuclear Sequestration of the Intracellular Domain of ErbB4 Protein*

    PubMed Central

    Sundvall, Maria; Korhonen, Anna; Vaparanta, Katri; Anckar, Julius; Halkilahti, Kalle; Salah, Zaidoun; Aqeilan, Rami I.; Palvimo, Jorma J.; Sistonen, Lea; Elenius, Klaus

    2012-01-01

    ErbB4 is a receptor tyrosine kinase implicated in the development and homeostasis of the heart, central nervous system, and mammary gland. Cleavable isoforms of ErbB4 release a soluble intracellular domain (ICD) that can translocate to the nucleus and function as a transcriptional coregulator. In search of regulatory mechanisms of ErbB4 ICD function, we identified PIAS3 as a novel interaction partner of ErbB4 ICD. In keeping with the small ubiquitin-like modifier (SUMO) E3 ligase function of protein inhibitor of activated STAT (PIAS) proteins, we showed that the ErbB4 ICD is modified by SUMO, and that PIAS3 stimulates the SUMOylation. Upon overexpression of PIAS3, the ErbB4 ICD generated from the full-length receptor accumulated into the nucleus in a manner that was dependent on the functional nuclear localization signal of ErbB4. In the nucleus, ErbB4 colocalized with PIAS3 and SUMO-1 in promyelocytic leukemia nuclear bodies, nuclear domains involved in regulation of transcription. Accordingly, PIAS3 overexpression had an effect on the transcriptional coregulatory activity of ErbB4, repressing its ability to coactivate transcription with Yes-associated protein. Finally, knockdown of PIAS3 with siRNA partially rescued the inhibitory effect of the ErbB4 ICD on differentiation of MDA-MB-468 breast cancer and HC11 mammary epithelial cells. Our findings illustrate that PIAS3 is a novel regulator of ErbB4 receptor tyrosine kinase, controlling its nuclear sequestration and function. PMID:22584572

  14. Origin and functions of pro-inflammatory cytokine producing Foxp3+ regulatory T cells.

    PubMed

    Pandiyan, Pushpa; Zhu, Jinfang

    2015-11-01

    CD4(+)CD25(+)Foxp3(+) regulatory cells (Tregs) are a special lineage of cells central in the maintenance of immune homeostasis, and are targeted for human immunotherapy. They are conventionally associated with the production of classical anti-inflammatory cytokines such as IL-10, TGF-β and IL-35, consistent to their anti-inflammatory functions. However, emerging evidence show that they also express effector cytokines such as IFN-γ and IL-17A under inflammatory conditions. While some studies reveal that these pro-inflammatory cytokine producing Foxp3(+) regulatory cells retain their suppressive ability, others believe that these cells are dys-regulated and are associated with perpetuation of immunopathology. Therefore the development of these cells may challenge the efficacy of human Treg therapy. Mechanistically, toll-like receptor (TLR) ligands and the pro-inflammatory cytokine milieu have been shown to play important roles in the induction of effector cytokines in Tregs. Here we review the mechanisms of development and the possible functions of pro-inflammatory cytokine producing Foxp3+ Tregs.

  15. MAR binding protein SMAR1 favors IL-10 mediated regulatory T cell function in acute colitis

    SciTech Connect

    Mirlekar, Bhalchandra; Patil, Sachin; Bopanna, Ramanamurthy; Chattopadhyay, Samit

    2015-08-21

    T{sub reg} cells are not only crucial for controlling immune responses to autoantigens but also prevent those directed towards commensal pathogens. Control of effector immune responses by T{sub reg} cells depend on their capacity to accumulate at inflammatory site and accordingly accommodate to inflammatory environment. Till date, the factors associated with maintaining these aspects of T{sub reg} phenotype is not understood properly. Here we have shown that a known nuclear matrix binding protein SMAR1 is selectively expressed more in colonic T{sub reg} cells and is required for their ability to accumulate at inflammatory site and to sustain high levels of Foxp3 and IL-10 expression during acute colitis. Elimination of anti-inflammatory subsets revealed a protective role for IL-10 producing T{sub reg} cells in SMAR1{sup −/−} mice. Moreover, a combined action of Foxp3 and SMAR1 restricts effector cytokine production and enhance the production of IL-10 by colonic T{sub reg} cells that controls acute colitis. This data highlights a critical role of SMAR1 in maintaining T{sub reg} physiology during inflammatory disorders. - Highlights: • SMAR1 is essential to sustain high level of Foxp3 and IL-10 in T{sub reg} cells. • SMAR1{sup −/−} T{sub reg} cells produce pro-inflammatory cytokine IL-17 leads to inflammation. • IL-10 administration can control the inflammation in SMAR1{sup −/−} mice. • Both Foxp3 and SMAR1 maintain T{sub reg} phenotype that controls colitis.

  16. APOBEC3 Proteins in Viral Immunity

    PubMed Central

    Stavrou, Spyridon; Ross, Susan R.

    2015-01-01

    Apolipoprotein B Editing Complex (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to infection by retroviruses and have also been implicated in the control of other viruses such as parvoviruses, herpesviruses, papillomaviruses, hepatitis B virus and retrotransposons. While their direct effect on modification of viral DNA has been clearly demonstrated, whether they play additional roles in innate and adaptive immunity to viruses is less clear. Here we review the data regarding the various steps in the innate and adaptive immune response to virus infection in which APOBEC3 proteins have been implicated. PMID:26546688

  17. NMR structure of the (1-51) N-terminal domain of the HIV-1 regulatory protein Vpr.

    PubMed

    Wecker, K; Roques, B P

    1999-12-01

    The human immunodeficiency virus type 1 (HIV-1) genome encodes a highly conserved 16 kDa regulatory gene product, Vpr (viral protein of regulation, 96 amino acid residues), which is incorporated into virions, in quantities equivalent to those of the viral Gag proteins. In the infected cells, Vpr is believed to function in the early phase of HIV-1 replication, including nuclear migration of preintegration complex, transcription of the provirus genome and viral multiplication by blocking cells in the G2 phase. Vpr has a critical role in long-term AIDS disease by inducing infection in nondividing cells such as monocytes and macrophages. Mutations have suggested that the N-terminal domain of Vpr encompassing the first 40 residues could be required for nuclear localization, packaging into virions and binding of transcription factor (TFIIB, Sp1), viral proteins (p6) and cellular proteins (RIP1, UNG, karyopherins). To gain insight into the structure-function relationship of Vpr, (1-51)Vpr was synthesized and its structure analyzed by circular dichroism and two-dimensional 1H NMR in aqueous trifluoroethanol (30%) solution and refined by restrained molecular dynamics. The structure is characterized by three turns around the first three prolines, Pro5, Pro10, Pro14, followed by a long amphipathic alpha helix-turn-alpha helix (Asp17-Ile46) motif ended by a turn extending from Tyr47 to Thr49. The alpha helix-turn-alpha helix motif and the amphipathic helix are well known for being implicated in protein-protein or protein-nucleic acid interaction. Therefore structural characteristics of the (1-51) N-terminal fragment of Vpr could explain why this region of Vpr plays a role in several biological functions of this protein.

  18. Regulatory protein phosphorylation in Mycoplasma pneumoniae. A PP2C-type phosphatase serves to dephosphorylate HPr(Ser-P).

    PubMed

    Halbedel, Sven; Busse, Julia; Schmidl, Sebastian R; Stülke, Jörg

    2006-09-08

    Among the few regulatory events in the minimal bacterium Mycoplasma pneumoniae is the phosphorylation of the HPr phosphocarrier protein of the phosphotransferase system. In the presence of glycerol, HPr is phosphorylated in an ATP-dependent manner by the HPr kinase/phosphorylase. The role of the latter enzyme was studied by constructing a M. pneumoniae hprK mutant defective in HPr kinase/phosphorylase. This mutant strain no longer exhibited HPr kinase activity but, surprisingly, still had phosphatase activity toward serine-phosphorylated HPr (HPr(Ser-P)). An inspection of the genome sequence revealed the presence of a gene (prpC) encoding a presumptive protein serine/threonine phosphatase of the PP2C family. The phosphatase PrpC was purified and its biochemical activity in HPr(Ser-P) dephosphorylation demonstrated. Moreover, a prpC mutant strain was isolated and found to be impaired in HPr(Ser-P) dephosphorylation. Homologues of PrpC are present in many bacteria possessing HPr(Ser-P), suggesting that PrpC may play an important role in adjusting the cellular HPr phosphorylation state and thus controlling the diverse regulatory functions exerted by the different forms of HPr.

  19. A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus

    PubMed Central

    Beccari, Leonardo; Yakushiji-Kaminatsui, Nayuta; Woltering, Joost M.; Necsulea, Anamaria; Lonfat, Nicolas; Rodríguez-Carballo, Eddie; Mascrez, Benedicte; Yamamoto, Shiori; Kuroiwa, Atsushi

    2016-01-01

    During vertebrate limb development, Hoxd genes are regulated following a bimodal strategy involving two topologically associating domains (TADs) located on either side of the gene cluster. These regulatory landscapes alternatively control different subsets of Hoxd targets, first into the arm and subsequently into the digits. We studied the transition between these two global regulations, a switch that correlates with the positioning of the wrist, which articulates these two main limb segments. We show that the HOX13 proteins themselves help switch off the telomeric TAD, likely through a global repressive mechanism. At the same time, they directly interact with distal enhancers to sustain the activity of the centromeric TAD, thus explaining both the sequential and exclusive operating processes of these two regulatory domains. We propose a model in which the activation of Hox13 gene expression in distal limb cells both interrupts the proximal Hox gene regulation and re-enforces the distal regulation. In the absence of HOX13 proteins, a proximal limb structure grows without any sign of wrist articulation, likely related to an ancestral fish-like condition. PMID:27198226

  20. Iron, lactoferrin and iron regulatory protein activity in the synovium; relative importance of iron loading and the inflammatory response

    PubMed Central

    Guillen, C; McInnes, I; Kruger, H; Brock, J

    1998-01-01

    OBJECTIVES—To determine the ability of lactoferrin in rheumatoid arthritis (RA) synovial fluid to bind "free" iron, and to study the regulatory mechanisms therein that control iron homeostasis.
METHODS—"Free" iron was determined by the bleomycin assay and lactoferrin concentrations by enzyme linked immunosorbent assay. The activities of iron regulatory protein (IRP) and NF-κB in synovial fluid cells were assayed by mobility shift assay.
RESULTS—30% of synovial fluids contained "free" iron and in these, lactoferrin concentrations were significantly lower than in those with no "free" iron (p<0.01). Addition of exogenous lactoferrin consistently reduced the amount of "free" iron in positive synovial fluids. IRP activity in synovial cells did not correlate with synovial fluid iron concentrations but did correlate with NF-κB activation and with serum C reactive protein.
CONCLUSION—Lactoferrin may prevent iron mediated tissue damage in RA by reducing "free" synovial iron concentration when inflammatory stimuli have disregulated IRP mediated iron homeostasis.

 Keywords: lactoferrin; rheumatoid arthritis; inflammation PMID:9741316

  1. Quantitative Phosphoproteomics Reveals Novel Phosphorylation Events in Insulin Signaling Regulated by Protein Phosphatase 1 Regulatory Subunit 12A

    PubMed Central

    Zhang, Xiangmin; Ma, Danjun; Caruso, Michael; Lewis, Monique; Qi, Yue; Yi, Zhengping

    2014-01-01

    Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes. PMID:24972320

  2. Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum

    PubMed Central

    DuBois, Juwen C.; Smulian, A. George

    2016-01-01

    The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence. PMID:27711233

  3. The mouse albumin enhancer contains a negative regulatory element that interacts with a novel DNA-binding protein.

    PubMed Central

    Herbst, R S; Boczko, E M; Darnell, J E; Babiss, L E

    1990-01-01

    The far-upstream mouse albumin enhancer (-10.5 to -8.43 kilobases) has both positive and negative regulatory domains which contribute to the rate and tissue specificity of albumin gene transcription. (R. S. Herbst, N. Friedman, J. E. Darnell, Jr., and L. E. Babiss, Proc. Natl. Acad. Sci. USA 86:1553-1557). In this work, the negative regulatory region has been functionally localized to sequences -8.7 to -8.43 kilobases upstream of the albumin gene cap site. In the absence of the albumin-modulating region (in which there are binding sites for the transcription factor C/EBP), the negative region can suppress a neighboring positive-acting element, thereby interfering with albumin enhancer function. The negative region is also capable of negating the positive action of the heterologous transthyretin enhancer in an orientation-independent fashion. Within this negative-acting region we can detect two DNA-binding sites, both of which are recognized by a protein present in all cell types tested. This DNA-binding activity is not competed for by any of a series of known DNA-binding sites, and hence this new protein is a candidate for a role in suppressing the albumin gene in nonhepatic cells. Images PMID:2370857

  4. Regulatory role of the second gelsolin-like domain of Caenorhabditis elegans gelsolin-like protein 1 (GSNL-1) in its calcium-dependent conformation and actin-regulatory activities

    PubMed Central

    Liu, Zhongmei; Ono, Shoichiro

    2013-01-01

    Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) is an unconventional member of the gelsolin family of actin-regulatory proteins. Unlike typical gelsolin-related proteins with three or six G domains, GSNL-1 has four gelsolin-like (G) domains (G1–G4) and exhibits calcium-dependent actin filament severing and capping activities. The first G domain (G1) of GSNL-1 is necessary for its actin-regulatory activities. However, how other domains in GSNL-1 participate in regulation of its functions is not understood. Here, we report biochemical evidence that the second G domain (G2) of GSNL-1 has a regulatory role in its calcium-dependent conformation and actin-regulatory activities. Comparison of the sequences of gelsolin-related proteins from various species indicates that sequences of G2 are highly conserved. Among the conserved residues in G2, we focused on D162 of GSNL-1, since equivalent residues in gelsolin and severin are part of the calcium-binding sites and is a pathogenic mutation site in human gelsolin causing familial amyloidosis, Finnish-type. The D162N mutation does not alter the inactive and fully calcium-activated states of GSNL-1 for actin filament severing (at 20 nM GSNL-1) and capping activities (at 50 nM GSNL-1). However, under these conditions, the mutant shows reduced calcium sensitivity for activation. By contrast, the D162N mutation strongly enhances susceptibility of GSNL-1 to chymotrypsin digestion only at high calcium concentrations but not at low calcium concentrations. The mutation also reduces affinity of GSNL-1 with actin monomers. These results suggest that G2 of GSNL-1 functions as a regulatory domain for its calcium-dependent actin-regulatory activities by mediating conformational changes of the GSNL-1 molecule. PMID:23475707

  5. [Seasonal changes in phosphorylation of myosin regulatory light chains and C-protein in myocardium of hibernating ground squirrel Citellus undulatus].

    PubMed

    Malyshev, S L; Osipova, D A; Vikhliantsev, I M; Podlubnaia, Z A

    2006-01-01

    A comparative study concerning the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of hibernating ground squirrel Citellus undulatus during the periods of hibernation and activity was carried out. During hibernation, regulatory light chains of ground squirrel were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averages 40-45% of their total amount. The extent of phosphorylation of the cardiac C-protein during hibernation is about two times higher than that in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to hibernation.

  6. The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

    PubMed

    Ogden, S; Haggerty, D; Stoner, C M; Kolodrubetz, D; Schleif, R

    1980-06-01

    The locations of DNA binding by the proteins involved with positive and negative regulation of transcription initiation of the L-arabinose operon in Escherichia coli have been determined by the DNase I protection method. Two cyclic AMP receptor protein sites were found, at positions -78 to -107 and -121 to -146, an araC protein--arabinose binding site was found at position -40 to -78, and an araC protein-fucose binding site was found at position -106 to -144. These locations, combined with in vivo data on induction of the two divergently oriented arabinose promoters, suggest the following regulatory mechanism: induction of the araBAD operon occurs when cyclic AMP receptor protein, araC protein, and RNA polymerase are all present and able to bind to DNA. Negative regulation is accomplished by the repressing form of araC protein binding to a site in the regulatory region such that it stimultaneously blocks access of cyclic AMP receptor protein to two sites on the DNA, one site of which serves each of the two promoters. Thus, from a single operator site, the negative regulator represses the two outwardly oriented ara promoters. This regulatory mechanism explains the known positive and negative regulatory properties of the ara promoters.

  7. Regulatory analysis of Regulatory Guide 1. 35 (Revision 3, Draft 2): In-service inspection of ungrouted tendons in prestressed concrete containments

    SciTech Connect

    Naus, D.J.

    1987-02-01

    The objectives of this study were to review all the changes in the latest version (Rev. 3, Draft 2) of Regulatory Guide 1.35 and to provide a regulatory analysis for all positions in the guide to determine if it is cost-effective to backfit the guide to the containments of existing plants. To meet these objectives, three tasks were undertaken. The first task outlined containment design criteria, traced the evolution of prestressed concrete containment configurations and prestressing systems, reviewed the history of the development of the guide, and summarized the applicability of the particular versions of the regulatory guide to the 43 nuclear plants that utilize a prestressed concrete containment. Under the second task a comparative regulatory analysis of Rev. 3 (Draft 2) of the guide was developed in which major decision factors affected by the proposed change were identified, and differential risk and cost factors were addressed in relation to the current version of the guide which is in effect (Rev. 2). Finally, under the third task a backfit analysis was conducted in accordance with the requirements of the ''Backfitting'' Rule, Section 50.109 for each of the revised or added positions contained in Rev. 3 (Draft 2). Application of the revision to operating plants will provide consistency in review and a uniform standard for assessing the in-service condition of the ungrouted tendons in prestressed concrete containments. It is concluded that the revisions will have a positive impact on safety and thus lower public risk. Backfitting of the revised guide is possible for most plants licensed since 1974.

  8. Signal Regulatory Protein alpha (SIRPalpha)+ Cells in the Adaptive Response to ESAT-6/CFP-10 Protein of Tuberculous Mycobacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10(CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the eff...

  9. Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration.

    PubMed

    Kaplan, Andrew; Morquette, Barbara; Kroner, Antje; Leong, SooYuen; Madwar, Carolin; Sanz, Ricardo; Banerjee, Sara L; Antel, Jack; Bisson, Nicolas; David, Samuel; Fournier, Alyson E

    2017-03-08

    Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.

  10. Effects of the combined blockade of EGFR and ErbB-2 on signal transduction and regulation of cell cycle regulatory proteins in breast cancer cells.

    PubMed

    D'Alessio, Amelia; De Luca, Antonella; Maiello, Monica R; Lamura, Luana; Rachiglio, Anna Maria; Napolitano, Maria; Gallo, Marianna; Normanno, Nicola

    2010-09-01

    Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27(kip1) and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27(kip1), with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27(kip1) mRNA and in the nuclear levels of the p27(kip1) transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27(kip1) mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27(kip1) mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27(kip1) expression.

  11. Non-small-cell lung cancer-induced immunosuppression by increased human regulatory T cells via Foxp3 promoter demethylation.

    PubMed

    Ke, Xing; Zhang, Shuping; Xu, Jian; Liu, Genyan; Zhang, Lixia; Xie, Erfu; Gao, Li; Li, Daqian; Sun, Ruihong; Wang, Fang; Pan, Shiyang

    2016-05-01

    Patients with non-small-cell lung cancer (NSCLC) have immune defects that are poorly understood. Forkhead box protein P3 (Foxp3) is crucial for immunosuppression by CD4(+) regulatory T cells (Tregs). It is not well known how NSCLC induces Foxp3 expression and causes immunosuppression in tumor-bearing patients. Our study found a higher percentage of CD4(+) Tregs in the peripheral blood of NSCLC compared with healthy donors. NSCLC patients showed demethylation of eight CpG sites within the Foxp3 promoter with methylation ratios negatively correlated with CD4(+)CD25(+)Foxp3(+) T levels. Foxp3 expression in CD4(+) Tregs was directly regulated by Foxp3 promoter demethylation and was involved in immunosuppression by NSCLC. To verify the effect of tumor cells on the phenotype and function of CD4(+) Tregs, we established a coculture system using NSCLC cell line and healthy CD4(+) T cells and showed that SPC-A1 induced IL-10 and TGF-β1 secretion by affecting the function of CD4(+) Tregs. The activity of DNA methyltransferases from CD4(+) T was decreased during this process. Furthermore, eight CpG sites within the Foxp3 promoter also appeared to have undergone demethylation. Foxp3 is highly expressed in CD4(+) T cells, and this may be caused by gene promoter demethylation. These induced Tregs are highly immunosuppressive and dramatically inhibit the proliferative activity of naïve CD4(+) T cells. Our study provides one possible mechanism describing Foxp3 promoter demethylation changes by which NSCLC down-regulates immune responses and contributes to tumor progression. Foxp3 represents an important target for NSCLC anti-tumor immunotherapy.

  12. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

    PubMed Central

    Matsui, Y; Kikuchi, A; Araki, S; Hata, Y; Kondo, J; Teranishi, Y; Takai, Y

    1990-01-01

    We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent. Images PMID:2115118

  13. TIM3+FOXP3+ regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer

    PubMed Central

    Sakuishi, Kaori; Ngiow, Shin Foong; Sullivan, Jenna M.; Teng, Michele W. L.; Kuchroo, Vijay K.; Smyth, Mark J.; Anderson, Ana C.

    2013-01-01

    T-cell immunoglobulin mucin 3 (TIM3) is an inhibitory molecule that has emerged as a key regulator of dysfunctional or exhausted CD8+ T cells arising in chronic diseases such as cancer. In addition to exhausted CD8+ T cells, highly suppressive regulatory T cells (Tregs) represent a significant barrier against the induction of antitumor immunity. We have found that the majority of intratumoral FOXP3+ Tregs express TIM3. TIM3+ Tregs co-express PD-1, are highly suppressive and comprise a specialized subset of tissue Tregs that are rarely observed in the peripheral tissues or blood of tumor-bearing mice. The co-blockade of the TIM3 and PD-1 signaling pathways in vivo results in the downregulation of molecules associated with TIM3+ Treg suppressor functions. This suggests that the potent clinical efficacy of co-blocking TIM3 and PD-1 signal transduction cascades likely stems from the reversal of T-cell exhaustion combined with the inhibition of regulatory T-cell function in tumor tissues. Interestingly, we find that TIM3+ Tregs accumulate in the tumor tissue prior to the appearance of exhausted CD8+ T cells, and that the depletion of Tregs at this stage interferes with the development of the exhausted phenotype by CD8+ T cells. Collectively, our data indicate that TIM3 marks highly suppressive tissue-resident Tregs that play an important role in shaping the antitumor immune response in situ, increasing the value of TIM3-targeting therapeutic strategies against cancer. PMID:23734331

  14. DPP4 truncated GM-CSF & IL-3 manifest distinct receptor binding & regulatory functions compared to their full length forms.

    PubMed

    O'Leary, H A; Capitano, M; Cooper, S; Mantel, C; Boswell, H S; Kapur, R; Ramdas, B; Chan, R; Deng, L; Qu, C-K; Broxmeyer, H E

    2017-03-27

    Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated factors [(T)-GM-CSF and- IL-3] on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and T-IL-3 had enhanced receptor binding, but decreased CSF activity, compared to their FL forms. Importantly, T-GM-CSF and T-IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and FL-IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster forming cells from patients with Acute Myeloid Leukemia (AML) regardless of cytogenetic or molecular alterations and in vivo utilizing animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared to their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.Leukemia accepted article preview online, 27 March 2017. doi:10.1038/leu.2017.98.

  15. Significance of 1,3-butadiene to the US air toxics regulatory effort.

    PubMed

    Morrow, N L

    2001-06-01

    Because of its prevalence, particularly as a combustion by-product, 1,3-butadiene is a particularly important air toxic. It plays a significant role in all air toxics regulatory efforts in the US. The various requirements of the Federal Clean Air Act (CAA) dealing with air toxics are reviewed and the significance of 1,3-butadiene in each area is discussed in light of what is known about its emissions and health effects. The impacts of the changes in the understanding of 1,3-butadiene cancer potency over the past 15 years demonstrates the possible impact of such benchmarks and the importance of using the best science in understanding public health risks.

  16. Regulatory effects on central carbon metabolism from poly-3-hydroxybutryate synthesis.

    PubMed

    Sekar, Karthik; Tyo, Keith E J

    2015-03-01

    Poly-3-hydroxybutyrate (PHB) synthesis in Escherichia coli elicits regulatory responses that affect product yield and productivity. We used controlled, steady-state cultures (chemostats) of a genetically stable strain to determine growth-independent metabolic flux regulation. We measured flux and steady-state intracellular metabolite concentrations across different dilution rates (0.05, 0.15, 0.3h(-1)), limitations (glucose, gluconate and nitrogen), and operon copy counts of the PHB pathway (0, 6, 17, and 29). As PHB flux increases, specific substrate consumption and lactate secretion increase while formate and acetate secretion decreases in N-limited, glucose-fed conditions. To understand the regulatory mechanisms that resulted in these macroscopic changes, we used a flux balance analysis model to analyze intracellular redox conditions. Our model shows that under N-limited conditions, synthesis of PHB creates excess reducing equivalents. Cells, under these conditions, secrete more reduced metabolites in order to recycle reducing equivalents. By switching to a more oxidized substrate (gluconate) that decreased excess reducing equivalents, PHB flux yield increased 1.6 fold compared to glucose-fed fermentations. High flux of PHB (~1.2 mmol/g DCWh) was maintained under these steady-state, oxidized conditions. These results imply redox imbalance is a driving force in industrial production of PHB, and substrates that are more oxidized than glucose can increase productivity.

  17. Evidence for the existence of an Ns-type regulatory protein in Trypanosoma cruzi membranes.

    PubMed Central

    Eisenschlos, C D; Paladini, A A; Molina y Vedia, L; Torres, H N; Flawiá, M M

    1986-01-01

    The existence of a GTP-binding protein of the Ns type in Trypanosoma cruzi was explored. Epimastigote membranes were labelled by cholera toxin in the presence of [adenine-14C]NAD+. After SDS/polyacrylamide-gel electrophoresis of extracted membrane proteins, a single labelled polypeptide band of apparent Mr approx. 45,000 was detected. Epimastigote cells were treated with N-ethylmaleimide and electrofused to lymphoma S49 cells lacking the Ns protein. Evidence indicates that in such electrofusion-generated cell hybrids a heterologous adenylate cyclase system was reconstituted with the Ns protein provided by T. cruzi epimastigotes. Images Fig. 2. PMID:3099761

  18. The complex between SOS3 and SOS2 regulatory domain from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis

    PubMed Central

    Sánchez-Barrena, María José; Moreno-Pérez, Sandra; Angulo, Iván; Martínez-Ripoll, Martín; Albert, Armando

    2007-01-01

    The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P212121, with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 Å. PMID:17620712

  19. Mutation analysis of TRPS1 gene including core promoter, 5'UTR, and 3'UTR regulatory sequences with insight into their organization.

    PubMed

    Solc, Roman; Klugerova, Michaela; Vcelak, Josef; Baxova, Alice; Kuklik, Miloslav; Vseticka, Jan; Beharka, Rastislav; Hirschfeldova, Katerina

    2017-01-01

    The TRPS1 protein is a potent regulator of proliferation, differentiation, and apoptosis. The TRPS1 gene aberrations are strongly associated with rare trichorhinophalangeal syndrome (TRPS) development. We have conducted MLPA analysis to capture deletion within the crucial 8q24.1 chromosomal region in combination with mutation analysis of TRPS1 gene including core promoter, 5'UTR, and 3'UTR sequences in nine TRPS patients. Low complexity or extent of untranslated regulatory sequences avoided them from analysis in previous studies. Amplicon based next generation sequencing used in our study bridge over these technical limitations. Finally, we have made extended in silico analysis of TRPS1 gene regulatory sequences organization. Single contiguous deletion and an intragenic deletion intervening several exons were detected. Mutation analysis revealed five TRPS1 gene aberrations (two structural rearrangements, two nonsense mutations, and one missense substitution) reaching the overall detection rate of 78%. Several polymorphic variants were detected within the analysed regulatory sequences but without proposed pathogenic effect. In silico analysis suggested alternative promoter usage and diverse expression effectivity for different TRPS1 transcripts. Haploinsufficiency of TRPS1 gene was responsible for most of the TRPS phenotype. Structure of TRPS1 gene regulatory sequences is indicative of generally low single allele expression and its tight control.

  20. Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes.

    PubMed

    Ledesma-García, Laura; Sánchez-Azqueta, Ana; Medina, Milagros; Reyes-Ramírez, Francisca; Santero, Eduardo

    2016-03-31

    Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H-ThnA4-ThnA3-ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction.

  1. Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes

    PubMed Central

    Ledesma-García, Laura; Sánchez-Azqueta, Ana; Medina, Milagros; Reyes-Ramírez, Francisca; Santero, Eduardo

    2016-01-01

    Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H–ThnA4–ThnA3–ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H–ThnA4–ThnA3–ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction. PMID:27030382

  2. The human T-cell leukemia virus type 1 Rex regulatory protein exhibits an impaired functionality in human lymphoblastoid Jurkat T cells.

    PubMed Central

    Hamaia, S; Cassé, H; Gazzolo, L; Duc Dodon, M

    1997-01-01

    The Rex protein of human T-cell leukemia virus type 1 (HTLV-1) intervenes in the posttranscriptional regulation of proviral gene expression. Its binding to the Rex response element (XRE) present in the 3' long terminal repeat ensures the coordinate cytoplasmic accumulation of spliced and unspliced forms of viral messengers. Consequently, synthesis of viral structural and enzymatic proteins is strictly dependent on the Rex posttranscriptional activity. Here we report that synthesis of HTLV-1 envelope glycoproteins by Jurkat T cells could be detected only when they were regulated in a Rex-independent manner. Indeed, Jurkat T cells transfected with a Rex-dependent env expression vector (encompassing both the env and pX open reading frames) do not produce significant levels of envelope glycoproteins despite the production of significant amounts of Rex protein. The analysis of levels and distribution patterns of the unspliced env and of the singly spliced tax/rex transcripts suggests that the failure in envelope glycoprotein synthesis may be ascribed to a deficiency of Rex in mediating the nucleocytoplasmic transport of unspliced env RNAs in these cells. Furthermore, despite the synthesis of regulatory proteins, HTLV-1 structural proteins were not detected in Jurkat T cells transfected with an HTLV-1 infectious provirus. Conversely, and as expected, structural proteins were produced by Jurkat cells transfected by a human immunodeficiency virus type 1 (HIV-1) infectious provirus. This phenotype appeared to be linked to a specific dysfunction of Rex, since the functionally equivalent Rev protein of HIV-1 was shown to be fully efficient in promoting the synthesis of HTLV-1 envelope glycoproteins in Jurkat cells. Therefore, it seems likely that the block to Rex function in these lymphoblastoid T cells is determined by inefficient Rex-XRE interactions. These observations suggest that the acquisition of this Rex-deficient phenotype by in vivo-infected HTLV-1 T cells may

  3. Drug Transporters and Na+/H+ Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

    PubMed Central

    Walsh, Dustin R.; Nolin, Thomas D.

    2015-01-01

    Drug transporters govern the absorption, distribution, and elimination of pharmacologically active compounds. Members of the solute carrier and ATP binding-cassette drug transporter family mediate cellular drug uptake and efflux processes, thereby coordinating the vectorial movement of drugs across epithelial barriers. To exert their physiologic and pharmacological function in polarized epithelia, drug transporters must be targeted and stabilized to appropriate regions of the cell membrane (i.e., apical versus basolateral). Despite the critical importance of drug transporter membrane targeting, the mechanisms that underlie these processes are largely unknown. Several clinically significant drug transporters possess a recognition sequence that binds to PSD-95/Drosophila discs large/ZO-1 (PDZ) proteins. PDZ proteins, such as the Na+/H+ exchanger regulatory factor (NHERF) family, act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is particularly high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia, tissues important to drug disposition. Several recent studies have highlighted NHERF proteins as determinants of drug transporter function secondary to their role in controlling membrane abundance and localization. Mounting evidence strongly suggests that NHERF proteins may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development. PMID:26092975

  4. DNA binding and transcriptional regulatory activity of mammalian achaete-scute homologous (MASH) proteins revealed by interaction with a muscle-specific enhancer.

    PubMed

    Johnson, J E; Birren, S J; Saito, T; Anderson, D J

    1992-04-15

    The MASH genes are vertebrate homologues of achaete-scute, genes required for neuronal determination in Drosophila. The sequence of MASH1 and MASH2 contains a basic helix-loop-helix (bHLH) motif that is present in other transcriptional regulators such as MyoD and E12. In the absence of an authentic target for the MASH proteins, we examined their DNA binding and transcriptional regulatory activity by using a binding site (the E box) from the muscle creatine kinase (MCK) gene, a target of MyoD. Like myogenic bHLH proteins, the MASH proteins form heterooligomers with E12 that bind the MCK E box with high affinity in vitro. Unexpectedly, however, MASH1 and MASH2 also activate transcription of both exogenous and endogenous MCK in transfected C3H/10T1/2 fibroblasts. However, they do not induce myogenesis. Myogenic activity is not exclusively a property of the MyoD basic region, as substitution of this domain fails to confer myogenic activity on MASH1. These data suggest that different bHLH proteins may activate overlapping but distinct sets of target genes in the same cell type.

  5. Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR.

    PubMed Central

    Benkirane, M; Neuveut, C; Chun, R F; Smith, S M; Samuel, C E; Gatignol, A; Jeang, K T

    1997-01-01

    TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double-stranded RNA-activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa. One member of this family, PKR, is a serine/threonine kinase which has anti-viral and anti-proliferative effects. In this study we show that TRBP is a cellular down-regulator of PKR function. Assaying expression from an infectious HIV-1 molecular clone, we found that PKR inhibited viral protein synthesis and that over-expression of TRBP effectively countered this inhibition. In intracellular and in cell-free assays we show that TRBP directly inhibits PKR autophosphorylation through an RNA binding-independent pathway. Biologically, TRBP serves a growth-promoting role; cells that overexpress TRBP exhibit transformed phenotypes. Our results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation. PMID:9034343

  6. Regulatory Oversight Program, July 1, 1993--March 3, 1997. Volume 2: Appendices

    SciTech Connect

    1997-12-31

    On July, 1993, a Regulatory Oversight (RO) organization was established within the US DOE, Oak Ridge Operations to provide regulatory oversight of the DOE uranium enrichment facilities leased to the United States Enrichment Corporation (USEC). The purpose of the OR program was to ensure continued plant safety, safeguards and security while the plants were transitioned to regulatory oversight by the Nuclear Regulatory Commission (NRC). Volume 2 contains copies of the documents which established the relationship between NRC, DOE, USEC, and DOL (Dept of Labor) required to facilitate regulatory oversight transition.

  7. OTX2 Activity at Distal Regulatory Elements Shapes the Chromatin Landscape of Group 3 Medulloblastoma.

    PubMed

    Boulay, Gaylor; Awad, Mary E; Riggi, Nicolo; Archer, Tenley C; Iyer, Sowmya; Boonseng, Wannaporn E; Rossetti, Nikki E; Naigles, Beverly; Rengarajan, Shruthi; Volorio, Angela; Kim, James C; Mesirov, Jill P; Tamayo, Pablo; Pomeroy, Scott L; Aryee, Martin J; Rivera, Miguel N

    2017-02-17

    Medulloblastoma is the most frequent malignant pediatric brain tumor and is divided into at least four subgroups known as WNT, SHH, Group 3, and Group 4. Here, we characterized gene regulation mechanisms in the most aggressive subtype, Group 3 tumors, through genome-wide chromatin and expression profiling. Our results show that most active distal sites in these tumors are occupied by the transcription factor OTX2. Highly active OTX2-bound enhancers are often arranged as clusters of adjacent peaks and are also bound by the transcription factor NEUROD1. These sites are responsive to OTX2 and NEUROD1 knockdown and could also be generated de novo upon ectopic OTX2 expression in primary cells, showing that OTX2 cooperates with NEUROD1 and plays a major role in maintaining and possibly establishing regulatory elements as a pioneer factor. Among OTX2 target genes, we identified the kinase NEK2, whose knockdown and pharmacologic inhibition decreased cell viability. Our studies thus show that OTX2 controls the regulatory landscape of Group 3 medulloblastoma through cooperative activity at enhancer elements and contributes to the expression of critical target genes.SIGNIFICANCE: The gene regulation mechanisms that drive medulloblastoma are not well understood. Using chromatin profiling, we find that the transcription factor OTX2 acts as a pioneer factor and, in cooperation with NEUROD1, controls the Group 3 medulloblastoma active enhancer landscape. OTX2 itself or its target genes, including the mitotic kinase NEK2, represent attractive targets for future therapies. Cancer Discov; 7(3); 1-14. ©2017 AACR.

  8. Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

    SciTech Connect

    Selezneva, Anna I.; Cavigiolio, Giorgio; Theil, Elizabeth C.; Walden, William E.; Volz, Karl

    2010-12-03

    Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe-4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe-4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe-S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P21, with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 {angstrom}, = 92.0{sup o}. Native data sets have been collected from several crystals with resolution extending to 2.8 {angstrom} and the structure has been solved by molecular replacement.

  9. In situ detection of a heat-shock regulatory element binding protein using a soluble synthetic enhancer sequence.

    PubMed Central

    Harel-Bellan, A; Brini, A T; Ferris, D K; Robin, P; Farrar, W L

    1989-01-01

    In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also it was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer. Images PMID:2740211

  10. CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN.

    PubMed

    Paust, Hans-Joachim; Riedel, Jan-Hendrik; Krebs, Christian F; Turner, Jan-Eric; Brix, Silke R; Krohn, Sonja; Velden, Joachim; Wiech, Thorsten; Kaffke, Anna; Peters, Anett; Bennstein, Sabrina B; Kapffer, Sonja; Meyer-Schwesinger, Catherine; Wegscheid, Claudia; Tiegs, Gisa; Thaiss, Friedrich; Mittrücker, Hans-Willi; Steinmetz, Oliver M; Stahl, Rolf A K; Panzer, Ulf

    2016-07-01

    Chemokines and chemokine receptors are implicated in regulatory T cell (Treg) trafficking to sites of inflammation and suppression of excessive immune responses in inflammatory and autoimmune diseases; however, the specific requirements for Treg migration into the inflamed organs and the positioning of these cells within the tissue are incompletely understood. Here, we report that Tregs expressing the TH1-associated chemokine receptor CXCR3 are enriched in the kidneys of patients with ANCA-associated crescentic GN and colocalize with CXCR3(+) effector T cells. To investigate the functional role of CXCR3(+) Tregs, we generated mice that lack CXCR3 in Tregs specifically (Foxp3(eGFP-Cre) × Cxcr3(fl/fl)) and induced experimental crescentic GN. Treg-specific deletion of CXCR3 resulted in reduced Treg recruitment to the kidney and an overwhelming TH1 immune response, with an aggravated course of the nephritis that was reversible on anti-IFNγ treatment. Together, these findings show that a subset of Tregs expresses CXCR3 and thereby, acquires trafficking properties of pathogenic CXCR3(+) TH1 cells, allowing Treg localization and control of excessive TH1 responses at sites of inflammation.

  11. Activation of sterol regulatory element-binding protein 1c and fatty acid synthase transcription by hepatitis C virus non-structural protein 2.

    PubMed

    Oem, Jae-Ku; Jackel-Cram, Candice; Li, Yi-Ping; Zhou, Yan; Zhong, Jin; Shimano, Hitoshi; Babiuk, Lorne A; Liu, Qiang

    2008-05-01

    Transcriptional factor sterol regulatory element-binding protein 1c (SREBP-1c) activates the transcription of lipogenic genes, including fatty acid synthase (FAS). Hepatitis C virus (HCV) infection is often associated with lipid accumulation within the liver, known as steatosis in the clinic. The molecular mechanisms of HCV-associated steatosis are not well characterized. Here, we showed that HCV non-structural protein 2 (NS2) activated SREBP-1c transcription in human hepatic Huh-7 cells as measured by using a human SREBP-1c promoter-luciferase reporter plasmid. We further showed that sterol regulatory element (SRE) and liver X receptor element (LXRE) in the SREBP-1c promoter were involved in SREBP-1c activation by HCV NS2. Furthermore, expression of HCV NS2 resulted in the upregulation of FAS transcription. We also showed that FAS upregulation by HCV NS2 was SREBP-1-dependent since deleting the SRE sequence in a FAS promoter and expressing a dominant-negative SREBP-1 abrogated FAS promoter upregulation by HCV NS2. Taken together, our results suggest that HCV NS2 can upregulate the transcription of SREBP-1c and FAS, and thus is probably a contributing factor for HCV-associated steatosis.

  12. Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP)*

    PubMed Central

    Shao, Wei; Espenshade, Peter J.

    2014-01-01

    Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipogenesis. Release of membrane-bound SREBP requires SREBP cleavage-activating protein (SCAP) to escort SREBP from the endoplasmic reticulum (ER) to the Golgi for cleavage by site-1 and site-2 proteases. SCAP then recycles to the ER for additional rounds of SREBP binding and transport. Mechanisms regulating ER-to-Golgi transport of SCAP-SREBP are understood in molecular detail, but little is known about SCAP recycling. Here, we have demonstrated that SCAP Golgi-to-ER transport requires cleavage of SREBP at site-1. Reductions in SREBP cleavage lead to SCAP degradation in lysosomes, providing additional negative feedback control to the SREBP pathway. Current models suggest that SREBP plays a passive role prior to cleavage. However, we show that SREBP actively prevents premature recycling of SCAP-SREBP until initiation of SREBP cleavage. SREBP regulates SCAP in human cells and yeast, indicating that this is an ancient regulatory mechanism. PMID:24478315

  13. Over-expression of GTP-cyclohydrolase 1 feedback regulatory protein attenuates LPS and cytokine-stimulated nitric oxide production.

    PubMed

    Nandi, Manasi; Kelly, Peter; Vallance, Patrick; Leiper, James

    2008-02-01

    GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate-limiting step for the de novo production of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS). The GTP-CH1-BH(4) pathway is emerging as an important regulator in a number of pathologies associated with over-production of nitric oxide (NO) and hence a more detailed understanding of this pathway may lead to novel therapeutic targets for the treatment of certain vascular diseases. GTP-CH1 activity can be inhibited by BH(4) through its protein-protein interactions with GTP-CH1 regulatory protein (GFRP), and transcriptional and post-translational modification of both GTP-CH1 and GFRP have been reported in response to proinflammatory stimuli. However, the functional significance of GFRP/GTP-CH1 interactions on NO pathways has not yet been demonstrated. We aimed to investigate whether over-expression of GFRP could affect NO production in living cells. Over-expression of N-terminally Myc-tagged recombinant human GFRP in the murine endothelial cell line sEnd 1 resulted in no significant effect on basal BH(4) nor NO levels but significantly attenuated the rise in BH(4) and NO observed following lipopolysaccharide and cytokine stimulation of cells. This study demonstrates that GFRP can play a direct regulatory role in iNOS-mediated NO synthesis and suggests that the allosteric regulation of GTP-CH1 activity by GFRP may be an important mechanism regulating BH(4) and NO levels in vivo.

  14. The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif

    PubMed Central

    Awad, Rida; Marion, Sévajol; Isabel, Ayala; Anne, Chouquet; Philippe, Frachet; Pierre, Gans; Jean-Baptiste, Reiser; Jean-Philippe, Kleman

    2015-01-01

    Eukaryotic EnguLfment and cell MOtility (ELMO) proteins form an evolutionary conserved family of regulators involved in small GTPase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the Downstream Of CrK (DOCK) family members. Gathered data strongly suggest that DOCK activation by ELMO and the subsequent signaling result from a subtle balance in the binding of partners to ELMO. Among its putative upward modulators, the Hematopoietic cell kinase (Hck), a member of the Src kinase superfamily, has been identified as a binding partner and a specific tyrosine kinase for ELMO1. Indeed, Hck is implicated in distinct molecular signaling pathways governing phagocytosis, cell adhesion, and migration of hematopoietic cells. Although ELMO1 has been shown to interact with the regulatory Src Homology 3 (SH3) domain of Hck, no direct evidence indicating the mode of interaction between Hck and ELMO1 have been provided in the literature. In the present study, we report convergent pieces of evidence that demonstrate the specific interaction between the SH3 domain of Hck and the polyproline motif of ELMO1. Our results also suggest that the tyrosine-phosphorylation state of ELMO1 tail might act as a putative modulator of Hck kinase activity towards ELMO1 that in turn participates in DOCK180 activation and further triggers subsequent signaling towards actin remodeling. PMID:25737835

  15. Inflammation-associated genes: risks and benefits to Foxp3+ regulatory T-cell function.

    PubMed

    O'Connor, Richard A; Anderton, Stephen M

    2015-10-01

    Foxp3(+) regulatory T (Treg) cells prevent the development of autoimmunity and immunopathology, as well as maintaining homeostasis and tolerance to commensal microorganisms. The suppressive activity of Treg cells is their defining characteristic, generating great interest in their therapeutic potential. However, suppressive and effector functions are not entirely exclusive. Considerable evidence points to the ability of supposedly anti-inflammatory Foxp3-expressing Treg cells to also express transcription factors that have been characterized as cardinal drivers of T effector cell function. We will consider the mounting evidence that Treg cells can function in non-suppressive capacities and review the impetus for this functional change, its relevance to developing immune and autoimmune responses and its significance to the development of Treg-based therapies.

  16. 14-3-3 and its binding partners are regulators of protein–protein interactions during spermatogenesis

    PubMed Central

    Sun, Shengyi; Wong, Elissa W P; Li, Michelle W M; Lee, Will M; Cheng, C Yan

    2009-01-01

    During spermatogenesis, spermiation takes place at the adluminal edge of the seminiferous epithelium at stage VIII of the epithelial cycle during which fully developed spermatids (i.e. spermatozoa) detach from the epithelium in adult rat testes. This event coincides with the migration of preleptotene/leptotene spermatocytes across the blood–testis barrier from the basal to the apical (or adluminal) compartment. At stage XIV of the epithelial cycle, Pachytene spermatocytes (diploid, 2n) differentiate into diplotene spermatocytes (tetraploid, 4n) in the apical compartment of the epithelium, which begin meiosis I to be followed by meiosis II to form spermatids (haploid, 1n) at stage XIVof the epithelial cycle. These spermatids, in turn, undergo extensive morphological changes and traverse the seminiferous epithelium until they differentiate into elongated spermatids. Thus, there are extensive changes at the Sertoli–Sertoli and Sertoli–germ cell interface via protein ‘coupling’ and ‘uncoupling’ between cell adhesion protein complexes, as well as changes in interactions between integral membrane proteins and their peripheral adaptors, regulatory protein kinases and phosphatases, and the cytoskeletal proteins. These precisely coordinated protein–protein interactions affect cell adhesion and cell movement. In this review, we focus on the 14-3-3 protein family, whose members have different binding partners in the seminiferous epithelium. Recent studies have illustrated that 14-3-3 affects protein–protein interactions in the seminiferous epithelium, and regulates cell adhesion possibly via its effects on intracellular protein trafficking and cell-polarity proteins. This review provides a summary on the latest findings regarding the role of 14-3-3 family of proteins and their potential implications on spermatogenesis. We also highlight research areas that deserve attentions by investigators. PMID:19366886

  17. TRAF3 regulates the effector function of regulatory T cells and humoral immune responses

    PubMed Central

    Chang, Jae-Hoon; Hu, Hongbo; Jin, Jin; Puebla-Osorio, Nahum; Xiao, Yichuan; Gilbert, Brian E.; Brink, Robert; Ullrich, Stephen E.

    2014-01-01

    Regulatory T cells (Treg cells) control different aspects of immune responses, but how the effector functions of Treg cells are regulated is incompletely understood. Here we identified TNF receptor–associated factor 3 (TRAF3) as a regulator of Treg cell function. Treg cell–specific ablation of TRAF3 impaired CD4 T cell homeostasis, characterized by an increase in the Th1 type of effector/memory T cells. Moreover, the ablation of TRAF3 in Treg cells resulted in increased antigen-stimulated activation of follicular T helper cells (TFH cells), coupled with heightened formation of germinal centers and production of high-affinity IgG antibodies. Although the loss of TRAF3 did not reduce the overall frequency of Treg cells, it attenuated the antigen-stimulated production of follicular Treg cells (TFR cells). TRAF3 signaling in Treg cells was required to maintain high level expression of inducible co-stimulator (ICOS), which in turn was required for TFR cell generation and inhibition of antibody responses. These findings establish TRAF3 as a mediator of Treg cell function in the regulation of antibody responses and suggest a role for TRAF3 in mediating ICOS expression in Treg cells. PMID:24378539

  18. TGF-beta-induced Foxp3+ regulatory T cells rescue scurfy mice.

    PubMed

    Huter, Eva N; Punkosdy, George A; Glass, Deborah D; Cheng, Lily I; Ward, Jerrold M; Shevach, Ethan M

    2008-07-01

    Scurfy mice have a deletion in the forkhead domain of the forkhead transcription factor p3 (Foxp3), fail to develop thymic-derived, naturally occurring Foxp3+ regulatory T cells (nTreg), and develop a fatal lymphoproliferative syndrome with multi-organ inflammation. Transfer of thymic-derived Foxp3+ nTreg into neonatal Scurfy mice prevents the development of disease. Stimulation of conventional CD4+Foxp3(-) via the TCR in the presence of TGF-beta and IL-2 induces the expression of Foxp3 and an anergic/suppressive phenotype. To determine whether the TGF-beta-induced Treg (iTreg) were capable of suppressing disease in the Scurfy mouse, we reconstituted newborn Scurfy mice with polyclonal iTreg. Scurfy mice treated with iTreg do not show any signs of disease and have drastically reduced cell numbers in peripheral lymph nodes and spleen in comparison to untreated Scurfy controls. The iTreg retained their expression of Foxp3 in vivo for 21 days, migrated into the skin, and prevented the development of inflammation in skin, liver and lung. Thus, TGF-beta-differentiated Foxp3+ Treg appear to possess all of the functional properties of thymic-derived nTreg and represent a potent population for the cellular immunotherapy of autoimmune and inflammatory diseases.

  19. Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes.

    PubMed

    Yoneyama, T; Hatakeyama, K

    2001-04-01

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively.

  20. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice

    PubMed Central

    Sheehan, Michael J.; Lee, Victoria; Corbett-Detig, Russell; Bi, Ke; Beynon, Robert J.; Hurst, Jane L.; Nachman, Michael W.

    2016-01-01

    Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation) and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup) gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP) scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones. PMID:26938775

  1. Temperature inducible β-sheet structure in the transactivation domains of retroviral regulatory proteins of the Rev family

    NASA Astrophysics Data System (ADS)

    Thumb, Werner; Graf, Christine; Parslow, Tristram; Schneider, Rainer; Auer, Manfred

    1999-11-01

    The interaction of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev with cellular cofactors is crucial for the viral life cycle. The HIV-1 Rev transactivation domain is functionally interchangeable with analog regions of Rev proteins of other retroviruses suggesting common folding patterns. In order to obtain experimental evidence for similar structural features mediating protein-protein contacts we investigated activation domain peptides from HIV-1, HIV-2, VISNA virus, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) by CD spectroscopy, secondary structure prediction and sequence analysis. Although different in polarity and hydrophobicity, all peptides showed a similar behavior with respect to solution conformation, concentration dependence and variations in ionic strength and pH. Temperature studies revealed an unusual induction of β-structure with rising temperatures in all activation domain peptides. The high stability of β-structure in this region was demonstrated in three different peptides of the activation domain of HIV-1 Rev in solutions containing 40% hexafluoropropanol, a reagent usually known to induce α-helix into amino acid sequences. Sequence alignments revealed similarities between the polar effector domains from FIV and EIAV and the leucine rich (hydrophobic) effector domains found in HIV-1, HIV-2 and VISNA. Studies on activation domain peptides of two dominant negative HIV-1 Rev mutants, M10 and M32, pointed towards different reasons for the biological behavior. Whereas the peptide containing the M10 mutation (L 78E 79→D 78L 79) showed wild-type structure, the M32 mutant peptide (L 78L 81L 83→A 78A 81A 83) revealed a different protein fold to be the reason for the disturbed binding to cellular cofactors. From our data, we conclude, that the activation domain of Rev proteins from different viral origins adopt a similar fold and that a β-structural element is involved in binding to a

  2. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice.

    PubMed

    Sheehan, Michael J; Lee, Victoria; Corbett-Detig, Russell; Bi, Ke; Beynon, Robert J; Hurst, Jane L; Nachman, Michael W

    2016-03-01

    Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation) and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup) gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP) scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones.

  3. Structural basis for the CsrA-dependent modulation of translation initiation by an ancient regulatory protein

    PubMed Central

    Altegoer, Florian; Rensing, Stefan A.; Bange, Gert

    2016-01-01

    Regulation of translation is critical for maintaining cellular protein levels, and thus protein homeostasis. The conserved RNA-binding protein CsrA (also called RsmA; for carbon storage regulator and regulator of secondary metabolism, respectively; hereafter called CsrA) represents a well-characterized example of regulation at the level of translation initiation in bacteria. Binding of a CsrA homodimer to the 5′UTR of an mRNA occludes the Shine–Dalgarno sequence, blocking ribosome access for translation. Small noncoding RNAs (sRNAs) can competitively antagonize CsrA activity by a well-understood mechanism. However, the regulation of CsrA by the protein FliW is just emerging. FliW antagonizes the CsrA-dependent repression of translation of the flagellar filament protein, flagellin. Crystal structures of the FliW monomer reveal a novel, minimal β-barrel-like fold. Structural analysis of the CsrA/FliW heterotetramer shows that FliW interacts with a C-terminal extension of CsrA. In contrast to the competitive regulation of CsrA by sRNAs, FliW allosterically antagonizes CsrA in a noncompetitive manner by excluding the 5′UTR from the CsrA–RNA binding site. Our phylogenetic analysis shows that the FliW-mediated regulation of CsrA regulation is the ancestral state in flagellated bacteria. We thus demonstrate fundamental mechanistic differences in the regulation of CsrA by sRNA in comparison with an ancient regulatory protein. PMID:27551070

  4. SIGNR3-dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis.

    PubMed

    Lightfoot, Yaíma L; Selle, Kurt; Yang, Tao; Goh, Yong Jun; Sahay, Bikash; Zadeh, Mojgan; Owen, Jennifer L; Colliou, Natacha; Li, Eric; Johannssen, Timo; Lepenies, Bernd; Klaenhammer, Todd R; Mohamadzadeh, Mansour

    2015-04-01

    Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3(-/-) mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.

  5. Immune regulatory functions of DOCK family proteins in health and disease.

    PubMed

    Nishikimi, Akihiko; Kukimoto-Niino, Mutsuko; Yokoyama, Shigeyuki; Fukui, Yoshinori

    2013-09-10

    DOCK proteins constitute a family of evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho family of GTPases. Although DOCK family proteins do not contain the Dbl homology domain typically found in GEFs, they mediate the GTP-GDP exchange reaction through DHR-2 domain. Accumulating evidence indicates that the DOCK proteins act as major GEFs in varied biological settings. For example, DOCK2, which is predominantly expressed in hematopoietic cells, regulates migration and activation of leukocytes through Rac activation. On the other hand, it was recently reported that mutations of DOCK8, another member of the DOCK family proteins, cause a combined immunodeficiency syndrome in humans. This article reviews the structure, functions and signaling of DOCK2 and DOCK8, especially focusing on their roles in immune responses.

  6. TGFβ-induced FoxP3+ Regulatory T Cells Rescue Scurfy Mice

    PubMed Central

    Huter, Eva N.; Punkosdy, George A.; Glass, Deborah D.; Cheng, Lily I.; Ward, Jerrold M.; Shevach, Ethan M.

    2008-01-01

    Scurfy mice have a deletion in the forkhead domain of Foxp3, fail to develop thymic-derived Foxp3+ regulatory T cells (nTreg), and develop a fatal lymphoproliferative syndrome with multi-organ inflammation. Transfer of thymic-derived Foxp3+ nTreg into neonatal Scurfy mice prevents the development of disease. Stimulation of conventional CD4+Foxp3− via the TCR in the presence of TGFβ and IL-2 induces the expression of Foxp3 and an anergic/suppressive phenotype. To determine whether the TGFβ-induced Treg (iTR) were capable of suppressing disease in the Scurfy mouse, we reconstituted newborn Scurfy mice with polyclonal iTR. iTR-treated Scurfy mice do not show any signs of disease and have drastically reduced cell numbers in peripheral lymph nodes and spleen in comparison to untreated Scurfy controls. The iTR retained their expression of FoxP3 in vivo for 21 days, migrated into the skin, and prevented the development of inflammation in skin, liver and lung. Thus, TGFβ-differentiated Foxp3+ Treg appear to possess all of the functional properties of thymic-derived nTreg and represent a potent population for the cellular immunotherapy of autoimmune and inflammatory diseases. PMID:18546144

  7. T Cell Receptor CDR3 Sequence but Not Recognition Characteristics Distinguish Autoreactive Effector and Foxp3+ Regulatory T Cells

    PubMed Central

    Liu, Xin; Nguyen, Phuong; Liu, Wei; Cheng, Cheng; Steeves, Meredith; Obenauer, John C.; Ma, Jing; Geiger, Terrence L.

    2010-01-01

    SUMMARY The source, specificity, and plasticity of the forkhead box transcription factor 3 (Foxp3)+ regulatory T (Treg) and conventional T (Tconv) cell populations active at sites of autoimmune pathology are not well characterized. To evaluate this, we combined global repertoire analyses and functional assessments of isolated T cell receptors (TCR) from TCRα retrogenic mice with autoimmune encephalomyelitis. Treg and Tconv cell TCR repertoires were distinct, and autoantigen-specific Treg and Tconv cells were enriched in diseased tissue. Autoantigen sensitivity and fine specificity of these cells intersected, implying that differences in responsiveness were not responsible for lineage specification. Notably, autoreactive Treg and Tconv cells could be fully distinguished by an acidic versus aliphatic variation at a single TCR CDR3 residue. Our results imply that ontogenically distinct Treg and Tconv cell repertoires with convergent specificities for autoantigen respond during autoimmunity and argue against more than limited plasticity between Treg and Tconv cells during autoimmune inflammation. PMID:20005134

  8. Interleukin-1 family cytokines and their regulatory proteins in normal pregnancy and pre-eclampsia.

    PubMed

    Southcombe, J H; Redman, C W G; Sargent, I L; Granne, I

    2015-09-01

    Maternal systemic inflammation is a feature of pre-eclampsia, a condition in pregnancy characterized by hypertension and proteinuria. Pre-eclampsia is caused by the placenta; many placental factors contribute to the syndrome's progression, and proinflammatory cytokines have been identified previously as one such mediator. The interleukin (IL)-1 family of cytokines are key regulators of the inflammatory network, and two naturally occurring regulatory molecules for IL-1 family cytokines, IL-1RA and sST2, have been found previously to be elevated in maternal blood from women with pre-eclampsia. Here we investigate more recently identified IL-1 family cytokines and regulatory molecules, IL-1RAcP, IL-37, IL-18BP, IL-36α/β/γ/Ra and IL-38 in pre-eclampsia. Pregnant women have more circulating IL-18BP and IL-36Ra than non-pregnant women, and sIL-1RAcP is elevated from women with pre-eclampsia compared to normal pregnancies. The placenta expresses all the molecules, and IL-37 and IL-18BP are up-regulated significantly in pre-eclampsia placentas compared to those from normal pregnancies. Together, these changes contribute to the required inhibition of maternal systemic cytotoxic immunity in normal pregnancy; however, in pre-eclampsia the same profile is not seen. Interestingly, the increased circulating levels of sIL-1RAcP and increased placental IL-18BP and IL-37, the latter of which we show to be induced by hypoxic damage to the placenta, are all factors which are anti-inflammatory. While the placenta is often held responsible for the damage and clinical symptoms of pre-eclampsia by the research community, here we show that the pre-eclampsia placenta is also trying to prevent inflammatory damage to the mother.

  9. Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance

    PubMed Central

    Soroosh, Pejman; Doherty, Taylor A.; Duan, Wei; Mehta, Amit Kumar; Choi, Heonsik; Adams, Yan Fei; Mikulski, Zbigniew; Khorram, Naseem; Rosenthal, Peter; Broide, David H.

    2013-01-01

    Airway tolerance is the usual outcome of inhalation of harmless antigens. Although T cell deletion and anergy are likely components of tolerogenic mechanisms in the lung, increasing evidence indicates that antigen-specific regulatory T cells (inducible Treg cells [iTreg cells]) that express Foxp3 are also critical. Several lung antigen-presenting cells have been suggested to contribute to tolerance, including alveolar macrophages (MØs), classical dendritic cells (DCs), and plasmacytoid DCs, but whether these possess the attributes required to directly promote the development of Foxp3+ iTreg cells is unclear. Here, we show that lung-resident tissue MØs coexpress TGF-β and retinal dehydrogenases (RALDH1 and RALDH 2) under steady-state conditions and that their sampling of harmless airborne antigen and presentation to antigen-specific CD4 T cells resulted in the generation of Foxp3+ Treg cells. Treg cell induction in this model depended on both TGF-β and retinoic acid. Transfer of the antigen-pulsed tissue MØs into the airways correspondingly prevented the development of asthmatic lung inflammation upon subsequent challenge with antigen. Moreover, exposure of lung tissue MØs to allergens suppressed their ability to generate iTreg cells coincident with blocking airway tolerance. Suppression of Treg cell generation required proteases and TLR-mediated signals. Therefore, lung-resident tissue MØs have regulatory functions, and strategies to target these cells might hold promise for prevention or treatment of allergic asthma. PMID:23547101