Protein and Signaling Networks in Vertebrate Photoreceptor Cells

PubMed Central

Koch, Karl-Wilhelm; Dell’Orco, Daniele

2015-01-01

Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cyclic guanosine monophosphate (cGMP) and Ca2+. The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase (GRK1) under control of Ca2+-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases (GCs) and is regulated by specific neuronal Ca2+-sensor proteins called guanylate cyclase-activating proteins (GCAPs). At least one GC (ROS-GC1) was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca2+-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated (CNG) channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments. PMID:26635520

Tat-APE1/ref-1 protein inhibits TNF-{alpha}-induced endothelial cell activation

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

Song, Yun Jeong; Lee, Ji Young; Joo, Hee Kyoung

2008-03-28

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-{alpha}-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1 h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-{alpha}-induced monocyte adhesion and vascular cell adhesion molecule-1 expressionmore » in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.« less

Endocytosis of the seven-transmembrane RGS1 protein activates G-protein-coupled signalling in Arabidopsis.

PubMed

Urano, Daisuke; Phan, Nguyen; Jones, Janice C; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J Philip; Jones, Alan M

2012-10-01

Signal transduction typically begins by ligand-dependent activation of a concomitant partner that is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (regulator of G-protein signalling 1), which is the prototype of a seven-transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase-accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required for both G-protein-mediated sugar signalling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis mechanisms.

Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.

PubMed

Liu, Songling; Premont, Richard T; Rockey, Don C

2014-06-27

Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitogen Activated Protein Kinase Phosphatase-1 (MKP-1) in Retinal Ischemic Preconditioning

PubMed Central

Dreixler, John C.; Bratton, Anthony; Du, Eugenie; Shaikh, Afzhal R.; Savoie, Brian; Michael, Alexander; Marcet, Marcus; Roth, Steven

2011-01-01

We previously described the phenomenon of retinal ischemic preconditioning (IPC) and we have shown the role of various signaling proteins in the protective pathways, including the mitogen-activated protein kinase p38. In this study we examined the role in IPC of mitogen-activated protein kinase phosphatase-1 (MKP-1), which inactivates p38. Ischemia was produced by elevation of intraocular pressure above systolic arterial blood pressure in adult Wistar rats. Preconditioning was produced by transient retinal ischemia for 5 min, 24 h prior to ischemia. Small interfering RNA (siRNA) to MKP-1 or a control non-silencing siRNA, was injected into the vitreous 6 h prior to IPC. Recovery was assessed by electroretinography (ERG) and histology. The a- and b-waves, and oscillatory potentials (OPs), measured before and 1 week after ischemia, were then normalized relative to pre-ischemic baseline, and corrected for diurnal variation in the normal non-ischemic eye. The P2, or post-photoreceptor component of the ERG (which reflects function of the rod bipolar cells in the inner retina), was derived using the Hood-Birch model. MKP-1 was localized in specific retinal cells using immunohistochemistry; levels of mitogen-activated protein kinases were measured using Western blotting. Injection of siRNA to MKP-1 significantly attenuated the protective effect of IPC as reflected by decreased recovery of the electroretinogram a- and b-waves and the P2 after ischemia. The injection of siRNA to MKP-1 reduced the number of cells in the retinal ganglion cell and outer nuclear layers after IPC and ischemia. Blockade of MKP-1 by siRNA also increased the activation of p38 at 24 h following IPC. MKP-1 siRNA did not alter the levels of phosphorylated jun N-terminal kinase (JNK) or extracellular signal-regulated kinase (ERK) after IPC. The results suggest the involvement of dual-specificity phosphatase MKP-1 in IPC and that MKP-1 is involved in IPC by regulating levels of activated MAPK p38. PMID

Role of activator protein-1 on the effect of arginine-glycine-aspartic acid containing peptides on transforming growth factor-beta1 promoter activity.

PubMed

Ruiz-Torres, M P; Perez-Rivero, G; Diez-Marques, M L; Griera, M; Ortega, R; Rodriguez-Puyol, M; Rodríguez-Puyol, D

2007-01-01

While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-beta1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-beta1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-beta1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-beta1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use.

Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis1

PubMed Central

Moshkov, Igor E.; Mur, Luis A.J.; Novikova, Galina V.; Smith, Aileen R.; Hall, Michael A.

2003-01-01

Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction. PMID:12692329

An unfolded protein-induced conformational switch activates mammalian IRE1

PubMed Central

Acosta-Alvear, Diego; Nguyen, Hieu T; Lee, Crystal P; Chu, Feixia

2017-01-01

The unfolded protein response (UPR) adjusts the cell’s protein folding capacity in the endoplasmic reticulum (ER) according to need. IRE1 is the most conserved UPR sensor in eukaryotic cells. It has remained controversial, however, whether mammalian and yeast IRE1 use a common mechanism for ER stress sensing. Here, we show that similar to yeast, human IRE1α’s ER-lumenal domain (hIRE1α LD) binds peptides with a characteristic amino acid bias. Peptides and unfolded proteins bind to hIRE1α LD’s MHC-like groove and induce allosteric changes that lead to its oligomerization. Mutation of a hydrophobic patch at the oligomerization interface decoupled peptide binding to hIRE1α LD from its oligomerization, yet retained peptide-induced allosteric coupling within the domain. Importantly, impairing oligomerization of hIRE1α LD abolished IRE1’s activity in living cells. Our results provide evidence for a unifying mechanism of IRE1 activation that relies on unfolded protein binding-induced oligomerization. PMID:28971800

The recombinant expression and activity detection of MAF-1 fusion protein.

PubMed

Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

2015-10-01

This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

PubMed Central

Ganong, B R; Loomis, C R; Hannun, Y A; Bell, R M

1986-01-01

The specificity of protein kinase C activation by sn-1,2-diacylglycerols and analogues was investigated by using a Triton X-100 mixed micellar assay [Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043]. Analogues containing acyl or alkyl chains eight carbons in length were synthesized because sn-1,2-dioctanoylglycerol is an effective cell-permeant activator of protein kinase C. These analogues were tested as activators and antagonists of rat brain protein kinase C to determine the exact structural features important for activity. The analogues established that activation of protein kinase C by diacylglycerols is highly specific. Several analogues established that both carbonyl moieties of the oxygen esters are required for maximal activity and that the 3-hydroxyl moiety is also required. None of the analogues were antagonists. These data, combined with previous investigations, permitted formulation of a model of protein kinase C activation. A three-point attachment of sn-1,2-diacylglycerol to the surface-bound protein kinase C-phosphatidylserine-Ca2+ complex is envisioned to cause activation. Direct ligation of diacylglycerol to Ca2+ is proposed to be an essential step in the mechanism of activation of protein kinase C. Images PMID:3456578

Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

PubMed

Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

2013-01-01

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation

Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress

PubMed Central

Topolska-Woś, Agnieszka M.; Shell, Steven M.; Kilańczyk, Ewa; Szczepanowski, Roman H.; Chazin, Walter J.; Filipek, Anna

2015-01-01

CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.—Topolska-Woś, A. M., Shell, S. M., Kilańczyk, E., Szczepanowski, R. H., Chazin, W. J., Filipek, A. Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress. PMID:25609429

Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors.

PubMed

Mills, Ian G; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E

2005-07-18

Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors

PubMed Central

Mills, Ian G.; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E.

2005-01-01

Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription. PMID:16027218

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

PubMed Central

Nishimura, Akiko; Yamamoto, Katsuyoshi; Oyama, Masaaki; Kozuka-Hata, Hiroko

2016-01-01

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 subbranch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domain. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1Δ cells, whereas robust Hog1 activation occurred in AHK1+ cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 subbranch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross talk. Thus, Ahk1 is a scaffold protein in the HKR1 subbranch and prevents incorrect signal flow from Hkr1 to Kss1. PMID:26787842

Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

PubMed

Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

2014-01-17

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPAR{gamma}

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

Kim, Kook Hwan; Hong, Sung Pyo; Kim, KyeongJin

2007-04-20

Hepatic steatosis is a common feature in patients with chronic hepatitis C virus (HCV) infection. HCV core protein plays an important role in the development of hepatic steatosis in HCV infection. Because SREBP1 (sterol regulatory element binding protein 1) and PPAR{gamma} (peroxisome proliferators-activated receptor {gamma}) are involved in the regulation of lipid metabolism of hepatocyte, we sought to determine whether HCV core protein may impair the expression and activity of SREBP1 and PPAR{gamma}. In this study, it was demonstrated that HCV core protein increases the gene expression of SREBP1 not only in Chang liver, Huh7, and HepG2 cells transiently transfectedmore » with HCV core protein expression plasmid, but also in Chang liver-core stable cells. Furthermore, HCV core protein enhanced the transcriptional activity of SREBP1. In addition, HCV core protein elevated PPAR{gamma} transcriptional activity. However, HCV core protein had no effect on PPAR{gamma} gene expression. Finally, we showed that HCV core protein stimulates the genes expression of lipogenic enzyme and fatty acid uptake associated protein. Therefore, our finding provides a new insight into the mechanism of hepatic steatosis by HCV infection.« less

Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1.

PubMed

Sangadala, Sreedhara; Yoshioka, Katsuhito; Enyo, Yoshio; Liu, Yunshan; Titus, Louisa; Boden, Scott D

2014-01-01

Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquitination and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding

The Role of the Pleckstrin Homology Domain-containing Protein CKIP-1 in Activation of p21-activated Kinase 1 (PAK1)*

PubMed Central

Kim, Yong-Bae; Shin, Yong Jae; Roy, Adhiraj; Kim, Jeong-Ho

2015-01-01

Upon growth factor stimulation, PAK1 is recruited to the plasma membrane and activated by a mechanism that requires its phosphorylation at Ser-223 by the protein kinase CK2. However, the upstream signaling molecules that regulate this phosphorylation event are not clearly defined. Here, we demonstrate a major role of the CK2α-interacting protein CKIP-1 in activation of PAK1. CK2α, CKIP-1, and PAK1 are translocated to membrane ruffles in response to the epidermal growth factor (EGF), where CKIP-1 mediates the interaction between CK2α and PAK1 in a PI3K-dependent manner. Consistently, PAK1 mediates phosphorylation and modulation of the activity of p41-Arc, one of its plasma membrane substrate, in a fashion that requires PI3K and CKIP-1. Moreover, CKIP-1 knockdown or PI3K inhibition suppresses PAK1-mediated cell migration and invasion, demonstrating the physiological significance of the PI3K-CKIP-1-CK2-PAK1 signaling pathway. Taken together, these findings identify a novel mechanism for the activation of PAK1 at the plasma membrane, which is critical for cell migration and invasion. PMID:26160174

The Homeodomain of PDX-1 Mediates Multiple Protein-Protein Interactions in the Formation of a Transcriptional Activation Complex on the Insulin Promoter

PubMed Central

Ohneda, Kinuko; Mirmira, Raghavendra G.; Wang, Juehu; Johnson, Jeffrey D.; German, Michael S.

2000-01-01

Activation of insulin gene transcription specifically in the pancreatic β cells depends on multiple nuclear proteins that interact with each other and with sequences on the insulin gene promoter to build a transcriptional activation complex. The homeodomain protein PDX-1 exemplifies such interactions by binding to the A3/4 region of the rat insulin I promoter and activating insulin gene transcription by cooperating with the basic-helix-loop-helix (bHLH) protein E47/Pan1, which binds to the adjacent E2 site. The present study provides evidence that the homeodomain of PDX-1 acts as a protein-protein interaction domain to recruit multiple proteins, including E47/Pan1, BETA2/NeuroD1, and high-mobility group protein I(Y), to an activation complex on the E2A3/4 minienhancer. The transcriptional activity of this complex results from the clustering of multiple activation domains capable of interacting with coactivators and the basal transcriptional machinery. These interactions are not common to all homeodomain proteins: the LIM homeodomain protein Lmx1.1 can also activate the E2A3/4 minienhancer in cooperation with E47/Pan1 but does so through different interactions. Cooperation between Lmx1.1 and E47/Pan1 results not only in the aggregation of multiple activation domains but also in the unmasking of a potent activation domain on E47/Pan1 that is normally silent in non-β cells. While more than one activation complex may be capable of activating insulin gene transcription through the E2A3/4 minienhancer, each is dependent on multiple specific interactions among a unique set of nuclear proteins. PMID:10629047

Ppm1E is an in cellulo AMP-activated protein kinase phosphatase.

PubMed

Voss, Martin; Paterson, James; Kelsall, Ian R; Martín-Granados, Cristina; Hastie, C James; Peggie, Mark W; Cohen, Patricia T W

2011-01-01

Activation of 5'-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5'-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase Mg/Mn(2+)-dependent [corrected] (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the Mg(2+)/Mn(2+)-dependent [corrected] protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest [corrected] that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target. Copyright © 2010 Elsevier Inc. All rights reserved.

Antisense protein tyrosine phosphatase 1B reverses activation of p38 mitogen-activated protein kinase in liver of ob/ob mice.

PubMed

Gum, Rebecca J; Gaede, Lori L; Heindel, Matthew A; Waring, Jeffrey F; Trevillyan, James M; Zinker, Bradley A; Stark, Margery E; Wilcox, Denise; Jirousek, Michael R; Rondinone, Cristina M; Ulrich, Roger G

2003-06-01

Phosphorylation of stress-activated kinase p38, a MAPK family member, was increased in liver of ob/ob diabetic mice relative to lean littermates. Treatment of ob/ob mice with protein tyrosine phosphatase 1B (PTP1B) antisense oligonucleotides (ASO) reduced phosphorylation of p38 in liver-to below lean littermate levels-and normalized plasma glucose while reducing plasma insulin. Phosphorylation of ERK, but not JNK, was also decreased in ASO-treated mice. PTP1B ASO decreased TNFalpha protein levels and phosphorylation of the transcription factor cAMP response element binding protein (CREB) in liver, both of which can occur through decreased phosphorylation of p38 and both of which have been implicated in insulin resistance or hyperglycemia. Decreased p38 phosphorylation was not directly due to decreased phosphorylation of the kinases that normally phosphorylate p38-MKK3 and MKK6. Additionally, p38 phosphorylation was not enhanced in liver upon insulin stimulation of ASO-treated ob/ob mice (despite increased activation of other signaling molecules) corroborating that p38 is not directly affected via the insulin receptor. Instead, decreased phosphorylation of p38 may be due to increased expression of MAPK phosphatases, particularly the p38/ERK phosphatase PAC1 (phosphatase of activated cells). This study demonstrates that reduction of PTP1B protein using ASO reduces activation of p38 and its substrates TNFalpha and CREB in liver of diabetic mice, which correlates with decreased hyperglycemia and hyperinsulinemia.

Corticosteroids inhibit sphingosine 1-phosphate-induced interleukin-6 secretion from human airway smooth muscle via mitogen-activated protein kinase phosphatase 1-mediated repression of mitogen and stress-activated protein kinase 1.

PubMed

Che, Wenchi; Parmentier, Johannes; Seidel, Petra; Manetsch, Melanie; Ramsay, Emma E; Alkhouri, Hatem; Ge, Qi; Armour, Carol L; Ammit, Alaina J

2014-02-01

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays an important proinflammatory role in asthmatic airways. Corticosteroids are first-line antiinflammatories in asthma; however, their repressive effects on S1P-induced cytokine secretion have not been investigated. To address this, our in vitro study reveals the molecular mechanisms by which corticosteroids inhibit S1P-induced IL-6 expression in the pivotal immunomodulatory cell type, airway smooth muscle (ASM). We first uncover the cellular signaling pathways responsible: S1P activates a cyclic adenosine monophosphate/cAMP response-element-binding protein (CREB)/CRE-dependent pathway to induce IL-6 transcription, concomitant with stimulation of the mitogen-activated protein kinase (MAPK) superfamily and downstream mitogen and stress-activated protein kinase 1 (MSK1) and histone H3 phosphorylation. In this way, S1P stimulates parallel signaling pathways to induce IL-6 secretion via CRE-driven transcription of the IL-6 gene promoter in a relaxed chromatin environment achieved through histone H3 phosphorylation. Second, we investigated how corticosteroids mediate their repressive effects. The corticosteroid dexamethasone inhibits S1P-induced IL-6 protein secretion and mRNA expression, but CREB/CRE transrepression, inhibition of IL-6 mRNA stability, or subcellular relocation of MSK1 were not responsible for the repressive effects of dexamethasone. Rather, we show that dexamethasone rapidly induces up-regulation of the MAPK deactivator MAPK phosphatase 1 (MKP-1) and that MKP-1 blocks the MAPK-driven activation of MSK1 and phosphorylation of histone H3. This was confirmed by treatment with triptolide, an inhibitor of MKP-1 up-regulation, where repressive effects of corticosteroids were reversed. Our study reveals the molecular mechanism underlying the antiinflammatory capacity of corticosteroids to repress proinflammatory functions induced by the potent bioactive sphingolipid S1P in the lung.

Antioxidant Activity of Oxygen Evolving Enhancer Protein 1 Purified from Capsosiphon fulvescens.

PubMed

Kim, Eun-Young; Choi, Youn Hee; Lee, Jung Im; Kim, In-Hye; Nam, Taek-Jeong

2015-06-01

This study was conducted to determine the antioxidant activity of a protein purified from Capsosiphon fulvescens. The purification steps included sodium acetate (pH 6) extraction and diethylaminoethyl-cellulose, reversed phase Shodex C4P-50 column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the molecular weight of the purified protein was 33 kDa. The N-terminus and partial peptide amino acid sequence of this protein was identical to the sequence of oxygen evolving enhancer (OEE) 1 protein. The antioxidant activity of the OEE 1 was determined in vitro using a scavenging test with 4 types of reactive oxygen species (ROS), including the 2,2-diphenyl-1-picrylhydrazyl radical, hydroxyl radical, superoxide anion, and hydrogen peroxide (H2 O2 ). OEE 1 had higher H2 O2 scavenging activity, which proved to be the result of enzymatic antioxidants rather than nonenzymatic antioxidants. In addition, OEE 1 showed less H2 O2 -mediated ROS formation in HepG2 cells. In conclusion, this study demonstrates that OEE 1 purified from C. fulvescens is an excellent antioxidant. © 2015 Institute of Food Technologists®

Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

PubMed

Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M

1999-03-01

Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

Chemical-controlled Activation of Antiviral Myxovirus Resistance Protein 1*

PubMed Central

Verhelst, Judith; Van Hoecke, Lien; Spitaels, Jan; De Vlieger, Dorien; Kolpe, Annasaheb

2017-01-01

The antiviral myxovirus resistance protein 1 (MX1) is an interferon-induced GTPase that plays an important role in the defense of mammalian cells against influenza A viruses. Mouse MX1 interacts with the influenza ribonucleoprotein complexes (vRNPs) and can prevent the interaction between polymerase basic 2 (PB2) and the nucleoprotein (NP) of influenza A viruses. However, it is unclear whether mouse MX1 disrupts the PB2-NP interaction in the context of pre-existing vRNPs or prevents the assembly of new vRNP components. Here, we describe a conditionally active mouse MX1 variant that only exerts antiviral activity in the presence of a small molecule drug. Once activated, this MX1 construct phenocopies the antiviral and NP binding activity of wild type MX1. The interaction between PB2 and NP is disrupted within minutes after the addition of the small molecule activator. These findings support a model in which mouse MX1 interacts with the incoming influenza A vRNPs and inhibits their activity by disrupting the PB2-NP interaction. PMID:28011636

Constitutive Gαi coupling activity of very large G protein-coupled receptor 1 (VLGR1) and its regulation by PDZD7 protein.

PubMed

Hu, Qiao-Xia; Dong, Jun-Hong; Du, Hai-Bo; Zhang, Dao-Lai; Ren, Hong-Ze; Ma, Ming-Liang; Cai, Yuan; Zhao, Tong-Chao; Yin, Xiao-Lei; Yu, Xiao; Xue, Tian; Xu, Zhi-Gang; Sun, Jin-Peng

2014-08-29

The very large G protein-coupled receptor 1 (VLGR1) is a core component in inner ear hair cell development. Mutations in the vlgr1 gene cause Usher syndrome, the symptoms of which include congenital hearing loss and progressive retinitis pigmentosa. However, the mechanism of VLGR1-regulated intracellular signaling and its role in Usher syndrome remain elusive. Here, we show that VLGR1 is processed into two fragments after autocleavage at the G protein-coupled receptor proteolytic site. The cleaved VLGR1 β-subunit constitutively inhibited adenylate cyclase (AC) activity through Gαi coupling. Co-expression of the Gαiq chimera with the VLGR1 β-subunit changed its activity to the phospholipase C/nuclear factor of activated T cells signaling pathway, which demonstrates the Gαi protein coupling specificity of this subunit. An R6002A mutation in intracellular loop 2 of VLGR1 abolished Gαi coupling, but the pathogenic VLGR1 Y6236fsx1 mutant showed increased AC inhibition. Furthermore, overexpression of another Usher syndrome protein, PDZD7, decreased the AC inhibition of the VLGR1 β-subunit but showed no effect on the VLGR1 Y6236fsx1 mutant. Taken together, we identified an independent Gαi signaling pathway of the VLGR1 β-subunit and its regulatory mechanisms that may have a role in the development of Usher syndrome. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Sss1p Is Required to Complete Protein Translocon Activation*

PubMed Central

Wilkinson, Barrie M.; Brownsword, Judith K.; Mousley, Carl J.; Stirling, Colin J.

2010-01-01

Protein translocation across the endoplasmic reticulum membrane occurs at the Sec61 translocon. This has two essential subunits, the channel-forming multispanning membrane protein Sec61p/Sec61α and the tail-anchored Sss1p/Sec61γ, which has been proposed to “clamp” the channel. We have analyzed the function of Sss1p using a series of domain mutants and found that both the cytosolic and transmembrane clamp domains of Sss1p are essential for protein translocation. Our data reveal that the cytosolic domain is required for Sec61p interaction but that the transmembrane clamp domain is required to complete activation of the translocon after precursor targeting to Sec61p. PMID:20709746

Detection of Expansin Proteins and Activity during Tomato Fruit Ontogeny1

PubMed Central

Rose, Jocelyn K.C.; Cosgrove, Daniel J.; Albersheim, Peter; Darvill, Alan G.; Bennett, Alan B.

2000-01-01

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955–5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities. PMID:10938374

Ethylene Rapidly Up-Regulates the Activities of Both Monomeric GTP-Binding Proteins and Protein Kinase(s) in Epicotyls of Pea1

PubMed Central

Moshkov, Igor E.; Novikova, Galina V.; Mur, Luis A.J.; Smith, Aileen R.; Hall, Michael A.

2003-01-01

It is demonstrated that, in etiolated pea (Pisum sativum) epicotyls, ethylene affects the activation of both monomeric GTP-binding proteins (monomeric G-proteins) and protein kinases. For monomeric G-proteins, the effect may be a rapid (2 min) and bimodal up-regulation, a transiently unimodal activation, or a transient down-regulation. Pretreatment with 1-methylcyclopropene abolishes the response to ethylene overall. Immunoprecipitation studies indicate that some of the monomeric G-proteins affected may be of the Rab class. Protein kinase activity is rapidly up-regulated by ethylene, the effect is inhibited by 1-methylcyclopropene, and the activation is bimodal. Immunoprecipitation indicates that the kinase(s) are of the MAP kinase ERK1 group. It is proposed that the data support the hypothesis that a transduction chain exists that is separate and antagonistic to that currently revealed by studies on Arabidopsis mutants. PMID:12692330

Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

PubMed

Disatnik, M H; Hernandez-Sotomayor, S M; Jones, G; Carpenter, G; Mochly-Rosen, D

1994-01-18

Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

ADP Regulates SNF1, the Saccharomyces cerevisiae Homolog of AMP-Activated Protein Kinase

PubMed Central

Mayer, Faith V.; Heath, Richard; Underwood, Elizabeth; Sanders, Matthew J.; Carmena, David; McCartney, Rhonda R.; Leiper, Fiona C.; Xiao, Bing; Jing, Chun; Walker, Philip A.; Haire, Lesley F.; Ogrodowicz, Roksana; Martin, Stephen R.; Schmidt, Martin C.; Gamblin, Steven J.; Carling, David

2011-01-01

Summary The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK. PMID:22019086

Mitogen-activated protein kinase phosphatase-1: a critical phosphatase manipulating mitogen-activated protein kinase signaling in cardiovascular disease (review).

PubMed

Li, Chang-Yi; Yang, Ling-Chao; Guo, Kai; Wang, Yue-Peng; Li, Yi-Gang

2015-04-01

Mitogen-activated protein kinase (MAPK) cascades are important players in the overall representation of cellular signal transduction pathways, and the deregulation of MAPKs is involved in a variety of diseases. The activation of MAPK signals occurs through phosphorylation by MAPK kinases at conserved threonine and tyrosine (Thr-Xaa-Tyr) residues. The mitogen-activated protein kinase phosphatases (MKPs) are a major part of the dual-specificity family of phosphatases and specifically inactivate MAPKs by dephosphorylating both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate. MAPKs binding to MKPs can enhance MKP stability and activity, providing an important negative-feedback control mechanism that limits the MAPK cascades. In recent years, accumulating and compelling evidence from studies mainly employing cultured cells and mouse models has suggested that the archetypal MKP family member, MKP-1, plays a pivotal role in cardiovascular disease as a major negative modulator of MAPK signaling pathways. In the present review, we summarize the current knowledge on the pathological properties and the regulation of MKP-1 in cardiovascular disease, which may provide valuable therapeutic options.

Chemical-controlled Activation of Antiviral Myxovirus Resistance Protein 1.

PubMed

Verhelst, Judith; Van Hoecke, Lien; Spitaels, Jan; De Vlieger, Dorien; Kolpe, Annasaheb; Saelens, Xavier

2017-02-10

The antiviral myxovirus resistance protein 1 (MX1) is an interferon-induced GTPase that plays an important role in the defense of mammalian cells against influenza A viruses. Mouse MX1 interacts with the influenza ribonucleoprotein complexes (vRNPs) and can prevent the interaction between polymerase basic 2 (PB2) and the nucleoprotein (NP) of influenza A viruses. However, it is unclear whether mouse MX1 disrupts the PB2-NP interaction in the context of pre-existing vRNPs or prevents the assembly of new vRNP components. Here, we describe a conditionally active mouse MX1 variant that only exerts antiviral activity in the presence of a small molecule drug. Once activated, this MX1 construct phenocopies the antiviral and NP binding activity of wild type MX1. The interaction between PB2 and NP is disrupted within minutes after the addition of the small molecule activator. These findings support a model in which mouse MX1 interacts with the incoming influenza A vRNPs and inhibits their activity by disrupting the PB2-NP interaction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

V-1 regulates capping protein activity in vivo.

PubMed

Jung, Goeh; Alexander, Christopher J; Wu, Xufeng S; Piszczek, Grzegorz; Chen, Bi-Chang; Betzig, Eric; Hammer, John A

2016-10-25

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

PubMed Central

Andersson, Helena M.; Arantes, Márcia J.; Crawley, James T. B.; Luken, Brenda M.; Tran, Sinh; Dahlbäck, Björn; Rezende, Suely M.

2010-01-01

Protein S has an established role in the protein C anticoagulant pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of activated protein C (APC). Despite its physiological role and clinical importance, the molecular basis of its action is not fully understood. To clarify the mechanism of the protein S interaction with APC, we have constructed and expressed a library of composite or point variants of human protein S, with residue substitutions introduced into the Gla, thrombin-sensitive region (TSR), epidermal growth factor 1 (EGF1), and EGF2 domains. Cofactor activity for APC was evaluated by calibrated automated thrombography (CAT) using protein S–deficient plasma. Of 27 variants tested initially, only one, protein S D95A (within the EGF1 domain), was largely devoid of functional APC cofactor activity. Protein S D95A was, however, γ-carboxylated and bound phospholipids with an apparent dissociation constant (Kdapp) similar to that of wild-type (WT) protein S. In a purified assay using FVa R506Q/R679Q, purified protein S D95A was shown to have greatly reduced ability to enhance APC-induced cleavage of FVa Arg306. It is concluded that residue Asp95 within EGF1 is critical for APC cofactor function of protein S and could define a principal functional interaction site for APC. PMID:20308596

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.

PubMed

Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, T M; Meiler, Jens; Hamm, Heidi E

2016-09-09

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

V-1 regulates capping protein activity in vivo

PubMed Central

Jung, Goeh; Wu, Xufeng S.; Piszczek, Grzegorz; Chen, Bi-Chang; Betzig, Eric; Hammer, John A.

2016-01-01

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1–null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1’s ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their “actin phenotype.” PMID:27791032

The roles of MCP-1 and protein kinase C delta activation in human eosinophilic leukemia EoL-1 cells.

PubMed

Lee, Ji-Sook; Yang, Eun Ju; Kim, In Sik

2009-12-01

Idiopathic hypereosinophilc syndrome is a disorder associated with clonally eosinophilic proliferation. The importance of FIP1-like-1-platelet-derived growth factor receptor-alpha (FIP1L1-PDGFRA) in the pathogenesis and classification of HES has been recently reported. In this study, we investigated the contribution of monocyte chemoattractant protein-1 (MCP-1)/CCL2 to chemotactic activity and protein kinase C delta (PKC delta in the human eosinophilic leukemia cell line EoL-1. These cells express CCR2 protein among the CC chemokine receptors (CCR1-5). MCP-1 induces strong migration of EoL-1 cells and the chemotaxis signal in response to MCP-1 involves a G(i)/G(o) protein, phospholipase C (PLC), PKC delta, p38 MAPK and NF-kappaB. MCP-1 activates p38 MAPK via G(i)/G(o) protein, PLC and PKC delta cascade. MCP-1 also induces NF-kappaB translocation and the activation is inhibited by PKC delta activation. The increase in the basal expression and activity of PKC delta in EoL-1 cells, compared to normal eosinophils, inhibits apoptosis in EoL-1 cells. Anti-apoptotic mechanism of PKC delta is related to inhibition of caspase 3 and caspase 9, but not to FIP1L1-PDGFRA. PKC delta functions as an anti-apoptotic molecule, and is involved in EoL-1 cell movement stimulated by MCP-1. This study contributes to an understanding of MCP-1 in eosinophil biology and pathogenic mechanism of eosinophilic disorders.

EHV-1 EICP22 protein sequences that mediate its physical interaction with the immediate-early protein are not sufficient to enhance the trans-activation activity of the IE protein.

PubMed

Derbigny, Wilbert A; Kim, Seong K; Jang, Hyung K; O'Callaghan, Dennis J

2002-03-20

The early 293 amino acid EICP22 protein (EICP22P) of equine herpesvirus 1 localizes within the nucleus and functions as an accessory regulatory protein (J. Virol. 68 (1994) 4329). Transient transfection assays indicated that although the EICP22P by itself only minimally trans-activates EHV-1 promoters, the EICP22P functions synergistically with the immediate-early protein (IEP) to enhance expression of EHV-1 early genes (J. Virol. 71 (1997) 1004). We previously showed that the EICP22 protein enhances the DNA-binding activity of the EHV-1 IEP and that it also physically interacts with the IEP (J. Virol. 74 (2000) 1425). In this communication, we employed transient trans-activation assays utilizing EICP22P deletion mutants to address whether the sequences required for EICP22P-IEP physical interactions are essential for EICP22P's ability to interact synergistically with the IEP. Assays employing various classes of the EHV-1 promoters fused to the chloramphenicol acetyl-transferase (CAT) reporter gene indicated that: (1) neither full length nor any of the EICP22P mutants tested was able to overcome repression of the IE promoter elicited by the IEP, (2) the full-length EICP22P interacted synergistically with the IEP to trans-activate the early and late promoters tested, and (3) all of the EICP22P mutants, including those that were able to physically interact with IEP and itself, failed to function synergistically with the IEP to trans-activate representative EHV-1 early and late promoters. The results suggest that EICP22P sequences required for its interaction with the IE protein are not sufficient to mediate its synergistic effect on the trans-activation function of the IEP. The possible explanations as to why sequences in addition to those that mediate EICP22P-IEP interaction and EICP22P self-interactions are essential for the synergistic function of EICP22P are discussed.

Mitogen-activated protein kinase phosphatase-1 expression in macrophages is controlled by lymphocytes during macrophage activation.

PubMed

Luo, Chong; Yang, Xiqiang; Yao, Lan; Jiang, Liping; Liu, Wei; Li, Xin; Wang, Lijia

2012-01-01

The viewpoints on the control of innate immune cells by the adaptive immune system during sepsis remain controversial. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential to the negative control of innate immunity and suppresses the activation of macrophages by inhibiting activated mitogen-activated protein kinase (MAPK). The purpose of the current study was to observe inflammatory response and macrophage activation in mice with severe combined immunodeficiency (SCID) with endotoxemia and to determine the role of MKP-1 in the control of macrophage activation by the adaptive immune system. Endotoxemia was induced in wild-type and SCID mice by an intraperitoneal injection of lipopolysaccharide (LPS), and all of the SCID mice died. SCID mice produced more inflammatory cytokines than BALB/c mice systemically and locally. TNF-α mRNA expression was higher and MKP-1 mRNA expression was lower in peritoneal macrophages (PMa) from SCID mice compared to PMa from wild-type mice after and even before LPS injection. Thioglycollate-stimulated PMa from wild-type mice were stimulated with LPS in vitro in the presence or absence of pan-T cells. The levels of TNF-α and IL-6 were higher in the supernatants from PMa cultured alone compared to PMa co-cultured with pan-T cells, and PMa MKP-1 mRNA and protein expression were higher when PMa were co-cultured with pan-T cells. Therefore, pan-T cells can up-regulate MKP-1 expression in macrophages and inhibit the secretion of inflammatory cytokines secretion by macrophages. In SCID mice, lymphocyte deficiency, especially T cell deficiency, causes insufficient MKP-1 expression in macrophages, which can be responsible for the severe inflammation and bad prognosis of septic SCID mice. MKP-1 plays an important role in the control of macrophage activation by the adaptive immune system.

AMP-activated protein kinase (AMPK)-induced preconditioning in primary cortical neurons involves activation of MCL-1.

PubMed

Anilkumar, Ujval; Weisová, Petronela; Düssmann, Heiko; Concannon, Caoimhín G; König, Hans-Georg; Prehn, Jochen H M

2013-03-01

Neuronal preconditioning is a phenomenon where a previous exposure to a sub-lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP-activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling-induced preconditioning in neurons, however, the molecular basis of AMPK-mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5-aminoimidazole-4-carboxamide riboside (AICAR) in a model of NMDA-mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA-induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) in AICAR-preconditioned neurons. Interestingly, over-expression of MCL-1 protected neurons against NMDA-induced excitotoxicity while MCL-1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca²⁺ levels during NMDA excitation revealed that MCL-1 over-expressing neurons exhibited improved bioenergetics and markedly reduced Ca²⁺ elevations, suggesting a potential mechanism through which MCL-1 confers neuroprotection. This study identifies MCL-1 as a key effector of AMPK-induced preconditioning in neurons. © 2012 International Society for Neurochemistry.

Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades

PubMed Central

Jiang, Cheng-shi; Liang, Lin-fu; Guo, Yue-wei

2012-01-01

This article provides an overview of approximately 300 secondary metabolites with inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), which were isolated from various natural sources or derived from synthetic process in the last decades. The structure-activity relationship and the selectivity of some compounds against other protein phosphatases were also discussed. Potential pharmaceutical applications of several PTP1B inhibitors were presented. PMID:22941286

Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

PubMed Central

Johnson, Amanda N.; Weil, P. Anthony

2017-01-01

Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae. These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1AS). We used Rap1AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways. PMID:28196871

WFIKKN1 and WFIKKN2: "Companion" proteins regulating TGFB activity.

PubMed

Monestier, Olivier; Blanquet, Véronique

2016-12-01

The WFIKKN (WAP, Follistatin/kazal, Immunoglobulin, Kunitz and Netrin domain-containing) protein family is composed of two multidomain proteins: WFIKKN1 and WFIKKN2. They were formed by domain shuffling and are likely present in deuterostoms. The WFIKKN (also called GASP) proteins are well known for their function in muscle and skeletal tissues, namely, inhibition of certain members of the transforming growth factor beta (TGFB) superfamily such as myostatin (MSTN) and growth and differentiation factor 11 (GDF11). However, the role of the WFIKKN proteins in other tissues is still poorly understood in spite of evidence suggesting possible action in the inner ear, brain and reproduction. Further, several recent studies based on next generation technologies revealed differential expression of WFIKKN1 and WFIKKN2 in various tissues suggesting that their function is not limited to MSTN and GDF11 inhibition in musculoskeletal tissue. In this review, we summarize current knowledge about the WFIKKN proteins and propose that they are "companion" proteins for various growth factors by providing localized and sustained presentation of TGFB proteins to their respective receptors, thus regulating the balance between the activation of Smad and non-Smad pathways by TGFB. Copyright © 2016 Elsevier Ltd. All rights reserved.

Plasma Membrane Intrinsic Proteins from Maize Cluster in Two Sequence Subgroups with Differential Aquaporin Activity1

PubMed Central

Chaumont, François; Barrieu, François; Jung, Rudolf; Chrispeels, Maarten J.

2000-01-01

The transport of water through membranes is regulated in part by aquaporins or water channel proteins. These proteins are members of the larger family of major intrinsic proteins (MIPs). Plant aquaporins are categorized as either tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs). Sequence analysis shows that PIPs form several subclasses. We report on the characterization of three maize (Zea mays) PIPs belonging to the PIP1 and PIP2 subfamilies (ZmPIP1a, ZmPIP1b, and ZmPIP2a). The ZmPIP2a clone has normal aquaporin activity in Xenopus laevis oocytes. ZmPIP1a and ZmPIP1b have no activity, and a review of the literature shows that most PIP1 proteins identified in other plants have no or very low activity in oocytes. Arabidopsis PIP1 proteins are the only exception. Control experiments show that this lack of activity of maize PIP1 proteins is not caused by their failure to arrive at the plasma membrane of the oocytes. ZmPIP1b also does not appear to facilitate the transport of any of the small solutes tried (glycerol, choline, ethanol, urea, and amino acids). These results are discussed in relationship to the function and regulation of the PIP family of aquaporins. PMID:10759498

Pofut1 point-mutations that disrupt O-fucosyltransferase activity destabilize the protein and abolish Notch1 signaling during mouse somitogenesis

PubMed Central

Suzuki, Emiko; Saga, Yumiko

2017-01-01

The segmental pattern of the vertebrate body is established via the periodic formation of somites from the presomitic mesoderm (PSM). This periodical process is controlled by the cyclic and synchronized activation of Notch signaling in the PSM. Protein O-fucosyltransferase1 (Pofut1), which transfers O-fucose to the EGF domains of the Notch1 receptor, is indispensable for Notch signaling activation. The Drosophila homologue Ofut1 was reported to control Notch localization via two different mechanisms, working as a chaperone for Notch or as a regulator of Notch endocytosis. However, these were found to be independent of O-fucosyltransferase activity because the phenotypes were rescued by Ofut1 mutants lacking O-fucosyltransferase activity. Pofut1 may also be involved in the Notch receptor localization in mice. However, the contribution of enzymatic activity of Pofut1 to the Notch receptor dynamics remains to be elucidated. In order to clarify the importance of the O-fucosyltransferase activity of Pofut1 for Notch signaling activation and the protein localization in the PSM, we established mice carrying point mutations at the 245th a.a. or 370-372th a.a., highly conserved amino-acid sequences whose mutations disrupt the O-fucosyltransferase activity of both Drosophila Ofut1 and mammalian Pofut1, with the CRISPR/Cas9 mediated genome-engineering technique. Both mutants displayed the same severely perturbed somite formation and Notch1 subcellular localization defects as the Pofut1 null mutants. In the mutants, Pofut1 protein, but not RNA, became undetectable by E9.5. Furthermore, both wild-type and mutant Pofut1 proteins were degraded through lysosome dependent machinery. Pofut1 protein loss in the point mutant embryos caused the same phenotypes as those observed in Pofut1 null embryos. PMID:29095923

Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

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

Reimers, Kerstin; Buchholz, Katja; Werchau, Hermann

2005-01-20

Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cellsmore » revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.« less

Dietary protein-induced hepatic IGF-1 secretion mediated by PPARγ activation.

PubMed

Wan, Xiaojuan; Wang, Songbo; Xu, Jingren; Zhuang, Lu; Xing, Kongping; Zhang, Mengyuan; Zhu, Xiaotong; Wang, Lina; Gao, Ping; Xi, Qianyun; Sun, Jiajie; Zhang, Yongliang; Li, Tiejun; Shu, Gang; Jiang, Qingyan

2017-01-01

Dietary protein or amino acid (AA) is a crucial nutritional factor to regulate hepatic insulin-like growth factor-1 (IGF-1) expression and secretion. However, the underlying intracellular mechanism by which dietary protein or AA induces IGF-1 expression remains unknown. We compared the IGF-1 gene expression and plasma IGF-1 level of pigs fed with normal crude protein (CP, 20%) and low-protein levels (LP, 14%). RNA sequencing (RNA-seq) was performed to detect transcript expression in the liver in response to dietary protein. The results showed that serum concentrations and mRNA levels of IGF-1 in the liver were higher in the CP group than in the LP group. RNA-seq analysis identified a total of 1319 differentially expressed transcripts (667 upregulated and 652 downregulated), among which the terms "oxidative phosphorylation", "ribosome", "gap junction", "PPAR signaling pathway", and "focal adhesion" were enriched. In addition, the porcine primary hepatocyte and HepG2 cell models also demonstrated that the mRNA and protein levels of IGF-1 and PPARγ increased with the increasing AA concentration in the culture. The PPARγ activator troglitazone increased IGF-1 gene expression and secretion in a dose dependent manner. Furthermore, inhibition of PPARγ effectively reversed the effects of the high AA concentration on the mRNA expression of IGF-1 and IGFBP-1 in HepG2 cells. Moreover, the protein levels of IGF-1 and PPARγ, as well as the phosphorylation of mTOR, significantly increased in HepG2 cells under high AA concentrations. mTOR phosphorylation can be decreased by the mTOR antagonist, rapamycin. The immunoprecipitation results also showed that high AA concentrations significantly increased the interaction of mTOR and PPARγ. In summary, PPARγ plays an important role in the regulation of IGF-1 secretion and gene expression in response to dietary protein.

Human protein Staufen-2 promotes HIV-1 proliferation by positively regulating RNA export activity of viral protein Rev.

PubMed

Banerjee, Atoshi; Benjamin, Ronald; Balakrishnan, Kannan; Ghosh, Payel; Banerjee, Sharmistha

2014-02-13

The export of intron containing viral RNAs from the nucleus to the cytoplasm is an essential step in the life cycle of Human Immunodeficiency Virus-1 (HIV-1). As the eukaryotic system does not permit the transport of intron containing RNA out of the nucleus, HIV-1 makes a regulatory protein, Rev, that mediates the transportation of unspliced and partially spliced viral mRNA from the nucleus to the cytoplasm, thereby playing a decisive role in the generation of new infectious virus particles. Therefore, the host factors modulating the RNA export activity of Rev can be major determinants of virus production in an infected cell. In this study, human Staufen-2 (hStau-2) was identified as a host factor interacting with HIV-1 Rev through affinity chromatography followed by MALDI analyses. Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis. We demonstrated that Rev-hStau-2 interactions positively regulated the RNA export activity of Rev and promoted progeny virus synthesis. The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction. The expression of this positive regulator was elevated upon HIV-1 infection in both human T-lymphocyte and astrocyte cell lines. With this study, we establish that human Staufen-2, a host factor which is up-regulated upon HIV-1 infection, interacts with HIV-1 Rev, thereby promoting its RNA export activity and progeny virus formation. Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively

Mitogen-Activated Protein Kinase Phosphatase 1 Disrupts Proinflammatory Protein Synthesis in Endotoxin-Adapted Monocytes

PubMed Central

Brudecki, Laura; Ferguson, Donald A.; McCall, Charles E.

2013-01-01

Autotoxic production of proinflammatory mediators during early sepsis induces excessive inflammation, and their later suppression may limit the immune response. We previously reported that sepsis differentially represses transcription and translation of tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) to reprogram sepsis inflammation. This switch is gene specific and plays a crucial role in the clinically relevant syndrome of endotoxin adaptation/tolerance, multiorgan failure, and poor sepsis outcome. To further define the mechanisms responsible for translation disruption that follows inflammation induction, we used THP-1 human promonocytes as a model of Toll-like receptor 4 (TLR4) responses found in sepsis. We showed that phosphorylation-dependent activation of p38 mitogen-activated protein kinase (MAPK) and translation disruption of TNF-α and IL-6 follow increased MAPK phosphatase 1 (MKP-1) expression and that MKP-1 knockdown rephosphorylates p38 and restores the capacity to translate TNF-α and IL-6 mRNAs. We also observed that the RNA-binding protein motif 4 (RBM4), a p38 MAPK target, accumulates in an unphosphorylated form in the cytosol in endotoxin-adapted cells, suggesting that dephosphorylated RBM4 may function as a translational repressor. Moreover, MKP-1 knockdown promotes RBM4 phosphorylation, blocks its transfer from the nucleus to the cytosol, and reverses translation repression. We also found that microRNA 146a (miR-146a) knockdown prevents and miR-146a transfection induces MKP-1 expression, which lead to increases or decreases in TNF-α and IL-6 translation, respectively. We conclude that a TLR4-, miR-146a-, p38 MAPK-, and MKP-1-dependent autoregulatory pathway regulates the translation of proinflammatory genes during the acute inflammatory response by spatially and temporally modifying the phosphorylation state of RBM4 translational repressor protein. PMID:23825193

Chios mastic treatment of patients with active Crohn’s disease

PubMed Central

Kaliora, Andriana C; Stathopoulou, Maria G; Triantafillidis, John K; Dedoussis, George VZ; Andrikopoulos, Nikolaos K

2007-01-01

AIM: To evaluate the effectiveness of mastic administration on the clinical course and plasma inflammatory mediators of patients with active Crohn’s disease (CD). METHODS: This pilot study was conducted in patients with established mild to moderately active CD, attending the outpatient clinics of the hospital, and in healthy controls. Ten patients and 8 controls were recruited for a 4-wk treatment with mastic caps (6 caps/d, 0.37 g/cap). All patients successfully completed the protocol. CD Activity Index (CDAI), Nutritional Risk Index (NRI), C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and total antioxidant potential (TAP) were evaluated in the plasma at baseline and at the end of the treatment period. Results were expressed as mean values ± SE and P < 0.05 was considered to indicate statistical significance. RESULTS: Patients exhibited significant reduction of CDAI (222.9 ± 18.7 vs 136.3 ± 12.3, P = 0.05) as compared to pretreament values. Plasma IL-6 was significantly decreased (21.2 ± 9.3 pg/mL vs 7.2 ± 2.8 pg/ mL, P = 0.027), and so did CRP (40.3 ± 13.1 mg/mL vs 19.7 ± 5.5, P = 0.028). TAP was significantly increased (0.15 ± 0.09 vs 0.57 ± 0.15 mmol/L uric acid, P = 0.036). No patient or control exhibited any kind of side effects. CONCLUSION: The results suggest that mastic significantly decreased the activity index and the plasma levels of IL-6 and CRP in patients with mildly to moderately active CD. Further double-blind, placebo-controlled studies in a larger number of patients are required to clarify the role of this natural product in the treatment of patients with CD. PMID:17278198

The sterol-binding activity of PATHOGENESIS-RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein.

PubMed

Gamir, Jordi; Darwiche, Rabih; Van't Hof, Pieter; Choudhary, Vineet; Stumpe, Michael; Schneiter, Roger; Mauch, Felix

2017-02-01

Pathogenesis-related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS-RELATED 1 (PR-1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR-1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR-1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol-auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR-1, whereas sterol-prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR-1 expression are particularly well protected against oomycete pathogens. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Crystal structure of TBC1D15 GTPase-activating protein (GAP) domain and its activity on Rab GTPases.

PubMed

Chen, Yan-Na; Gu, Xin; Zhou, X Edward; Wang, Weidong; Cheng, Dandan; Ge, Yinghua; Ye, Fei; Xu, H Eric; Lv, Zhengbing

2017-04-01

TBC1D15 belongs to the TBC (Tre-2/Bub2/Cdc16) domain family and functions as a GTPase-activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark-TBC1D15 and Sus-TBC1D15 belong to the same subfamily of TBC domain-containing proteins, and their GAP-domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost. © 2017 The Protein Society.

Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation

NASA Technical Reports Server (NTRS)

D'Alonzo, Richard C.; Selvamurugan, Nagarajan; Karsenty, Gerard; Partridge, Nicola C.

2002-01-01

Previously, we determined that the activator protein-1 (AP-1)-binding site and the runt domain (RD)-binding site and their binding proteins, c-Fos.c-Jun and Cbfa, regulate the collagenase-3 promoter in parathyroid hormone-treated and differentiating osteoblasts. Here we show that Cbfa1 and c-Fos.c-Jun appear to cooperatively bind the RD- and AP-1-binding sites and form ternary structures in vitro. Both in vitro and in vivo co-immunoprecipitation and yeast two-hybrid studies further demonstrate interaction between Cbfa1 with c-Fos and c-Jun in the absence of phosphorylation and without binding to DNA. Additionally, only the runt domain of Cbfa1 was required for interaction with c-Jun and c-Fos. In mammalian cells, overexpression of Cbfa1 enhanced c-Jun activation of AP-1-binding site promoter activity, demonstrating functional interaction. Finally, insertion of base pairs that disrupted the helical phasing between the AP-1- and RD-binding sites also inhibited collagenase-3 promoter activation. Thus, we provide direct evidence that Cbfa1 and c-Fos.c-Jun physically interact and cooperatively bind the AP-1- and RD-binding sites in the collagenase-3 promoter. Moreover, the AP-1- and RD-binding sites appear to be organized in a specific required helical arrangement that facilitates transcription factor interaction and enables promoter activation.

Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation.

PubMed

Suryawan, Agus; Jeyapalan, Asumthia S; Orellana, Renan A; Wilson, Fiona A; Nguyen, Hanh V; Davis, Teresa A

2008-10-01

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.

Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

PubMed Central

Suryawan, Agus; Jeyapalan, Asumthia S.; Orellana, Renan A.; Wilson, Fiona A.; Nguyen, Hanh V.; Davis, Teresa A.

2008-01-01

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E·eIF4G complex and increased eIF4E·4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein β-subunit-like protein (GβL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors. PMID:18682538

RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response.

PubMed

Lee, Yuan-Cho; Zhou, Qing; Chen, Junjie; Yuan, Jingsong

2016-12-19

ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control. Copyright © 2016 Elsevier Ltd. All rights reserved.

PPAR{gamma} activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

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

Mogilenko, Denis A., E-mail: denis@iem.sp.ru; Department of Embryology, St. Petersburg State University, 199034 St. Petersburg; Shavva, Vladimir S.

Research highlights: {yields} PPAR{gamma} activates ABCA1 gene expression but decreases ABCA1 protein content in human hepatoma cell line HepG2. {yields} Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1-LXR{beta} complex. {yields} Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex. {yields} Activation of PPAR{gamma} leads to increasing of the level of LXR{beta} associated with LXRE within ABCA1 gene promoter. -- Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPAR{gamma} is known as activator of ABCA1 expression, but details of PPAR{gamma}-mediatedmore » regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPAR{gamma} activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXR{beta} binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1/LXR{beta} complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24 h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex, but does not block PPAR{gamma}-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPAR{gamma} may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPAR{gamma}, LXR{beta} and MEK1/2 in regulation of ABCA1 mRNA and protein expression.« less

Structural basis of divergent cyclin-dependent kinase activation by Spy1/RINGO proteins

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

McGrath, Denise A.; Fifield, Bre‐Anne; Marceau, Aimee H.

Cyclin-dependent kinases (Cdks) are principal drivers of cell division and are an important therapeutic target to inhibit aberrant proliferation. Cdk enzymatic activity is tightly controlled through cyclin interactions, posttranslational modifications, and binding of inhibitors such as the p27 tumor suppressor protein. Spy1/RINGO (Spy1) proteins bind and activate Cdk but are resistant to canonical regulatory mechanisms that establish cell-cycle checkpoints. Cancer cells exploit Spy1 to stimulate proliferation through inappropriate activation of Cdks, yet the mechanism is unknown. We have determined crystal structures of the Cdk2-Spy1 and p27-Cdk2-Spy1 complexes that reveal how Spy1 activates Cdk. We find that Spy1 confers structural changesmore » to Cdk2 that obviate the requirement of Cdk activation loop phosphorylation. Spy1 lacks the cyclin-binding site that mediates p27 and substrate affinity, explaining why Cdk-Spy1 is poorly inhibited by p27 and lacks specificity for substrates with cyclin-docking sites. We identify mutations in Spy1 that ablate its ability to activate Cdk2 and to proliferate cells. Our structural description of Spy1 provides important mechanistic insights that may be utilized for targeting upregulated Spy1 in cancer.« less

Plasma protein hydroperoxides during aging in humans: correlation with paraoxonase 1 (PON1) arylesterase activity and plasma total thiols.

PubMed

Mehdi, Mohammad Murtaza; Rizvi, Syed Ibrahim

2013-02-01

Oxidative stress is thought to play a major role in the development of several age-dependent diseases. Proteins are major targets for oxidative attack. Protein hydroperoxides are formed by hydroxyl and singlet oxygen attack on protein, forming relatively stable hydroperoxides on histidine, tyrosine and tryptophan residues. This study investigated the levels of plasma protein hydroperoxides and antioxidant potential of plasma during aging in humans. We correlated the protein hydroperoxide formation with plasma antioxidant potential, paraoxonase 1 (PON1) arylesterase activity and plasma total thiols. The protein hydroperoxides and antioxidant potential were measured in plasma of human subjects aged between 20 and 81 years of both genders. Increase in plasma protein hydroperoxides and decrease in plasma antioxidant potential were observed as function of human age. This study provides strong correlation between plasma protein hydroperoxides formation and decrease in plasma antioxidant potential during aging. PON1 arylesterase activity and plasma total thiols levels were also found to show significant correlation with increasing levels of plasma protein hydroperoxides during aging. The plasma protein hydroperoxides provide a reliable marker of long-term redox balance and degree of oxidative stress during aging process. Copyright © 2013 IMSS. Published by Elsevier Inc. All rights reserved.

Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein*

PubMed Central

Peng, Wentao; Lin, Zhijie; Li, Weirong; Lu, Jing; Shen, Yuequan; Wang, Chunguang

2013-01-01

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function. PMID:23979136

Structural insights into the unusually strong ATPase activity of the AAA domain of the Caenorhabditis elegans fidgetin-like 1 (FIGL-1) protein.

PubMed

Peng, Wentao; Lin, Zhijie; Li, Weirong; Lu, Jing; Shen, Yuequan; Wang, Chunguang

2013-10-11

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.

The microtubule-associated protein EB1 maintains cell polarity through activation of protein kinase C.

PubMed

Schober, Joseph M; Kwon, Guim; Jayne, Debbie; Cain, Jeanine M

2012-01-06

The plus-ends of microtubules target the cell cortex to modulate actin protrusion dynamics and polarity, but little is known of the molecular mechanism that couples the interaction. EB1 protein associates with the plus-ends of microtubules, placing EB1 in an ideal spatial position to mediate microtubule-actin cross talk. The objective of the current study was to further understand intracellular signaling involved in EB1-dependent cell polarity and motility. B16F10 mouse melanoma cells were depleted of EB1 protein using short hair-pin RNA interference. Correlative live cell-immunofluorescence microscopy was performed to determine localization of WAVE2 and IQGAP1 to protruding versus retracting edges. EB1 knock down caused poor subcellular separation of WAVE2 and IQGAP1, and overall decreased localization. Activation of PKC corrected defects in WAVE2 and IQGAP1 localization, cell spreading and cell shape to levels observed in control cells, but did not correct defects in cell migration. Consistent with these findings, decreased PKC phosphorylation was observed in EB1 knock down cells. These findings support a model where EB1 protein links microtubules to actin protrusion and cell polarity through signaling pathways involving PKC. Copyright © 2011 Elsevier Inc. All rights reserved.

[Design and activity verification of human parathyroid hormone (1-34) mutant protein].

PubMed

Qiu, Shuang; Jiang, Yue-Shui; Li, Zhi-Qin; Lei, Jian-Yong; Chen, Yun; Jin, Jian

2012-07-01

Through protein-protein BLAST of homologous sequences in different species in NCBI database and preliminary simulating molecular docking and molecular dynamics by computer software discovery studio 3.1, three amino acids R25K26K27 of natural human parathyroid hormone (1-34) with Q25E26L27 were mutated and the biological activity of the mutant peptide was evaluated. Result showed that: root mean superposition deviation RMSD value between PTH (1-34)-(RKK-QEL) and PTH (1-34) peptide main chain was 2.509 3, indicating that the differences between the two main chain structural conformation was relatively small; the interaction energy between PTH (1-34)-(RKK-QEL) and its receptor protein PTH1R had been enhanced by 7.5% compared to nature PTH (1-34), from -554.083 kcal x mol(-1) to -599.253 kcal x mol(-1); the number of hydrogen bonds was increased from 32 to 38; PTH (1-34)-(RKK-QEL) can significantly stimulate the RANKL gene expression (P < 0.01) while inhibiting the OPG gene expression (P < 0.01) in UAMS-32P cells; in the co-culture system of UAMS-32P cells and mouse primary femur bone marrow cells, PTH (1-34)-(RKK-QEL) stimulated the formation of osteoclasts (P < 0.01) and had a higher biological activity than PTH (1-34) standard reagents.

Mdm2 mediates FMRP- and Gp1 mGluR-dependent protein translation and neural network activity.

PubMed

Liu, Dai-Chi; Seimetz, Joseph; Lee, Kwan Young; Kalsotra, Auinash; Chung, Hee Jung; Lu, Hua; Tsai, Nien-Pei

2017-10-15

Activating Group 1 (Gp1) metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, elicits translation-dependent neural plasticity mechanisms that are crucial to animal behavior and circuit development. Dysregulated Gp1 mGluR signaling has been observed in numerous neurological and psychiatric disorders. However, the molecular pathways underlying Gp1 mGluR-dependent plasticity mechanisms are complex and have been elusive. In this study, we identified a novel mechanism through which Gp1 mGluR mediates protein translation and neural plasticity. Using a multi-electrode array (MEA) recording system, we showed that activating Gp1 mGluR elevates neural network activity, as demonstrated by increased spontaneous spike frequency and burst activity. Importantly, we validated that elevating neural network activity requires protein translation and is dependent on fragile X mental retardation protein (FMRP), the protein that is deficient in the most common inherited form of mental retardation and autism, fragile X syndrome (FXS). In an effort to determine the mechanism by which FMRP mediates protein translation and neural network activity, we demonstrated that a ubiquitin E3 ligase, murine double minute-2 (Mdm2), is required for Gp1 mGluR-induced translation and neural network activity. Our data showed that Mdm2 acts as a translation suppressor, and FMRP is required for its ubiquitination and down-regulation upon Gp1 mGluR activation. These data revealed a novel mechanism by which Gp1 mGluR and FMRP mediate protein translation and neural network activity, potentially through de-repressing Mdm2. Our results also introduce an alternative way for understanding altered protein translation and brain circuit excitability associated with Gp1 mGluR in neurological diseases such as FXS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The GIRK1 subunit potentiates G protein activation of cardiac GIRK1/4 hetero-tetramers

PubMed Central

Touhara, Kouki K; Wang, Weiwei; MacKinnon, Roderick

2016-01-01

G protein gated inward rectifier potassium (GIRK) channels are gated by direct binding of G protein beta-gamma subunits (Gβγ), signaling lipids, and intracellular Na+. In cardiac pacemaker cells, hetero-tetramer GIRK1/4 channels and homo-tetramer GIRK4 channels play a central role in parasympathetic slowing of heart rate. It is known that the Na+ binding site of the GIRK1 subunit is defective, but the functional difference between GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers remains unclear. Here, using purified proteins and the lipid bilayer system, we characterize Gβγ and Na+ regulation of GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. We find in GIRK4 homo-tetramers that Na+ binding increases Gβγ affinity and thereby increases the GIRK4 responsiveness to G protein stimulation. GIRK1/4 hetero-tetramers are not activated by Na+, but rather are in a permanent state of high responsiveness to Gβγ, suggesting that the GIRK1 subunit functions like a GIRK4 subunit with Na+ permanently bound. DOI: http://dx.doi.org/10.7554/eLife.15750.001 PMID:27074664

Role of protein kinase C alpha and mitogen-activated protein kinases in endothelin-1-stimulation of cytosolic phospholipase A2 in iris sphincter smooth muscle.

PubMed

Abdel-Latif, A A; Husain, S; Yousufzai, S Y

2000-11-01

We have investigated the roles of protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) in the phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in endothelin-1- (ET-1) stimulated cat iris sphincter smooth muscle (CISM) cells. We found that in these cells both PKC and p38 MAP kinases play a critical role in ET-1-induced cPLA, phosphorylation and arachidonic acid (AA) release. Our findings indicate that stimulation of the endothelin-A- (ET(A)) receptor leads to: (1) activation of Gq protein which stimulates phospholipase C to hydrolyze the polyphosphoinositide PIP, into diacylglycerol (DAG) and inositol trisphosphate (IP3), the DAG may then activate PKC to phosphorylate and activate cPLA2; and (2) activation of Gi protein, which, through a series of kinases, leads to the stimulation of p38 MAPK and subsequently to phosphorylation and activation of cPLA2. The ability of the activated ET(A)-receptor, which is coupled to both Gq and Gi proteins, to recruit and activate this complex signal transduction mechanism remains to be clarified.

Salvianolic Acid B Protects Normal Human Dermal Fibroblasts Against Ultraviolet B Irradiation-Induced Photoaging Through Mitogen-Activated Protein Kinase and Activator Protein-1 Pathways.

PubMed

Sun, Zhengwang; Park, Sang-Yong; Hwang, Eunson; Zhang, Mengyang; Jin, Fengxie; Zhang, Baochun; Yi, Tae Hoo

2015-01-01

Exposure to ultraviolet (UV) light causes increased matrix metalloproteinase (MMP) activity and decreased collagen synthesis, leading to skin photoaging. Salvianolic acid B (SAB), a polyphenol, was extracted and purified from salvia miltiorrhiza. We assessed effects of SAB on UVB-induced photoaging and investigated its molecular mechanism of action in UVB-irradiated normal human dermal fibroblasts. Our results show that SAB significantly inhibited the UVB-induced expression of metalloproteinases-1 (MMP-1) and interleukin-6 (IL-6) while promoting the production of type I procollagen and transforming growth factor β1 (TGF-β1). Moreover, treatment with SAB in the range of 1-100 μg/mL significantly inhibited UVB-induced extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 phosphorylation, which resulted in decreasing UVB-induced phosphorylation of c-Fos and c-Jun. These results indicate that SAB downregulates UV-induced MMP-1 expression by inhibiting Mitogen-activated protein kinase (MAPK) signaling pathways and activator protein-1 (AP-1) activation. Our results suggest a potential use for SAB in skin photoprotection. © 2015 The American Society of Photobiology.

G protein-coupled estrogen receptor 1 (GPER1)/GPR30 increases ERK1/2 activity through PDZ motif-dependent and -independent mechanisms.

PubMed

Gonzalez de Valdivia, Ernesto; Broselid, Stefan; Kahn, Robin; Olde, Björn; Leeb-Lundberg, L M Fredrik

2017-06-16

G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of G i/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that G i/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two G i/o -mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

PubMed Central

Caljon, Guy; Ridder, Karin De; Stijlemans, Benoît; Coosemans, Marc; Magez, Stefan; De Baetselier, Patrick; Van Den Abbeele, Jan

2012-01-01

Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents. PMID:23110062

The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

PubMed

An, Ningfei; Blumer, Joe B; Bernard, Michael L; Lanier, Stephen M

2008-09-05

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

Secretion of the Streptomyces tyrosinase is mediated through its trans-activator protein, MelC1.

PubMed

Leu, W M; Chen, L Y; Liaw, L L; Lee, Y H

1992-10-05

The tyrosinase of Streptomyces antibioticus is encoded by the second open reading frame, melC2 of the melanin operon (melC). The upstream open reading frame melC1 specifies a 146-amino acid protein with a typical NH2-terminal signal-peptide characteristic of a secretory protein. The MelC1 protein is involved in the transfer of copper ion to apotyrosinase MelC2 via binary complex formation (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81; Chen, L.-Y., Leu, W.-M., Wang, K.-T., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 20100-20107). To investigate whether the export of tyrosinase is also dependent on MelC1, a mutational study of its signal-peptide sequence was performed. Four different mutants were obtained. Mutation at the positively charged region (mutant M-6LE, Arg6-Arg7----Leu6-Glu7) or the hydrophobic region (mutant M-16D, Val16----Asp16) led to Mel- phenotypes. These lesions caused a severe 7-10-fold reduction of the export of both the MelC1 and MelC2 proteins and a concomitant accumulation of the two proteins in the cytosolic fraction. The cell-associated tyrosinase activity in M-6LE but not in the M-16D mutant was dramatically reduced to 4% of the activity found in the wild type strain, suggesting that the basic NH2 terminus of MelC1 is also important for the trans-activation function of this protein. Nevertheless, the defects on the trans-activation and/or secretory functions of MelC1 in mutants M-6LE and M-16D are not due to the impairment of the formation of the MelC1.MelC2 complex. The translation of melanin operon genes in these two mutants also decreased. In contrast, the tyrosinase activity and the secretion of MelC2 were not affected if the mutations occurred at the putative cleavage site of the signal peptidase (e.g. mutant M-29SM, Arg29-Ala30----Ser29-Met30 or mutant 29-SMG, Arg29-Ala30-Asp31----Ser29-Med30-Gly31+ ++). Additionally, tyrosinase activity and its export were

Activation of SIRT1 Attenuates Klotho Deficiency-Induced Arterial Stiffness and Hypertension by Enhancing AMP-Activated Protein Kinase Activity.

PubMed

Gao, Diansa; Zuo, Zhong; Tian, Jing; Ali, Quaisar; Lin, Yi; Lei, Han; Sun, Zhongjie

2016-11-01

Arterial stiffness is an independent risk factor for stroke and myocardial infarction. This study was designed to investigate the role of SIRT1, an important deacetylase, and its relationship with Klotho, a kidney-derived aging-suppressor protein, in the pathogenesis of arterial stiffness and hypertension. We found that the serum level of Klotho was decreased by ≈45% in patients with arterial stiffness and hypertension. Interestingly, Klotho haplodeficiency caused arterial stiffening and hypertension, as evidenced by significant increases in pulse wave velocity and blood pressure in Klotho-haplodeficient (KL +/- ) mice. Notably, the expression and activity of SIRT1 were decreased significantly in aortic endothelial and smooth muscle cells in KL +/- mice, suggesting that Klotho deficiency downregulates SIRT1. Treatment with SRT1720 (15 mg/kg/d, IP), a specific SIRT1 activator, abolished Klotho deficiency-induced arterial stiffness and hypertension in KL +/- mice. Klotho deficiency was associated with significant decreases in activities of AMP-activated protein kinase α (AMPKα) and endothelial NO synthase (eNOS) in aortas, which were abolished by SRT1720. Furthermore, Klotho deficiency upregulated NADPH oxidase activity and superoxide production, increased collagen expression, and enhanced elastin fragmentation in the media of aortas. These Klotho deficiency-associated changes were blocked by SRT1720. In conclusion, this study provides the first evidence that Klotho deficiency downregulates SIRT1 activity in arterial endothelial and smooth muscle cells. Pharmacological activation of SIRT1 may be an effective therapeutic strategy for arterial stiffness and hypertension. © 2016 American Heart Association, Inc.

HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation.

PubMed

Yin, M J; Christerson, L B; Yamamoto, Y; Kwak, Y T; Xu, S; Mercurio, F; Barbosa, M; Cobb, M H; Gaynor, R B

1998-05-29

NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

PubMed

Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

2017-08-25

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

IQ-domain GTPase-activating protein 1 promotes the malignant phenotype of invasive ductal breast carcinoma via canonical Wnt pathway.

PubMed

Zhao, Huan-Yu; Han, Yang; Wang, Jian; Yang, Lian-He; Zheng, Xiao-Ying; Du, Jiang; Wu, Guang-Ping; Wang, En-Hua

2017-06-01

IQ-domain GTPase-activating protein 1 is a scaffolding protein with multidomain which plays a role in modulating dishevelled (Dvl) nuclear translocation in canonical Wnt pathway. However, the biological function and mechanism of IQ-domain GTPase-activating protein 1 in invasive ductal carcinoma (IDC) remain unknown. In this study, we found that IQ-domain GTPase-activating protein 1 expression was elevated in invasive ductal carcinoma, which was positively correlated with tumor grade, lymphatic metastasis, and poor prognosis. Coexpression of IQ-domain GTPase-activating protein 1 and Dvl in the nucleus and cytoplasm of invasive ductal carcinoma was significantly correlated but not in the membrane. Postoperative survival in the patients with their coexpression in the nucleus and cytoplasm was obviously lower than that without coexpression. The positive expression rates of c-myc and cyclin D1 were significantly higher in the patients with nuclear coexpression of Dvl and IQ-domain GTPase-activating protein 1 than that with cytoplasmic coexpression, correlating with poor prognosis. IQ-domain GTPase-activating protein 1 significantly enhanced cell proliferation and invasion in invasive ductal carcinoma cell lines by interacting with Dvl in cytoplasm to promote Dvl nuclear translocation so as to upregulate the expression of c-myc and cyclin D1. Collectively, our data suggest that IQ-domain GTPase-activating protein 1 may promote the malignant phenotype of invasive ductal carcinoma via canonical Wnt signaling, and it could be used as a potential prognostic biomarker for breast cancer patients.

Conformational dynamics underlie the activity of the auxin-binding protein, Nt-abp1.

PubMed

David, K; Carnero-Diaz, E; Leblanc, N; Monestiez, M; Grosclaude, J; Perrot-Rechenmann, C

2001-09-14

The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.

Crystal structure of TBC1D15 GTPase‐activating protein (GAP) domain and its activity on Rab GTPases

PubMed Central

Chen, Yan‐Na; Gu, Xin; Zhou, X. Edward; Wang, Weidong; Cheng, Dandan; Ge, Yinghua; Ye, Fei

2017-01-01

Abstract TBC1D15 belongs to the TBC (Tre‐2/Bub2/Cdc16) domain family and functions as a GTPase‐activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark‐TBC1D15 and Sus‐TBC1D15 belong to the same subfamily of TBC domain‐containing proteins, and their GAP‐domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost. PMID:28168758

Pharmacological activation of CB1 receptor modulates long term potentiation by interfering with protein synthesis.

PubMed

Navakkode, Sheeja; Korte, Martin

2014-04-01

Cognitive impairment is one of the most important side effects associated with cannabis drug abuse, as well as the serious issue concerning the therapeutic use of cannabinoids. Cognitive impairments and neuropsychiatric symptoms are caused by early synaptic dysfunctions, such as loss of synaptic connections in different brain structures including the hippocampus, a region that is believed to play an important role in certain forms of learning and memory. We report here that metaplastic priming of synapses with a cannabinoid type 1 receptor (CB1 receptor) agonist, WIN55,212-2 (WIN55), significantly impaired long-term potentiation in the apical dendrites of CA1 pyramidal neurons. Interestingly, the CB1 receptor exerts its effect by altering the balance of protein synthesis machinery towards higher protein production. Therefore the activation of CB1 receptor, prior to strong tetanization, increased the propensity to produce new proteins. In addition, WIN55 priming resulted in the expression of late-LTP in a synaptic input that would have normally expressed early-LTP, thus confirming that WIN55 priming of LTP induces new synthesis of plasticity-related proteins. Furthermore, in addition to the effects on protein translation, WIN55 also induced synaptic deficits due to the ability of CB1 receptors to inhibit the release of acetylcholine, mediated by both muscarinic and nicotinic acetylcholine receptors. Taken together this supports the notion that the modulation of cholinergic activity by CB1 receptor activation is one mechanism that regulates the synthesis of plasticity-related proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Nrf1 CNC-bZIP protein is regulated by the proteasome and activated by hypoxia.

PubMed

Chepelev, Nikolai L; Bennitz, Joshua D; Huang, Ting; McBride, Skye; Willmore, William G

2011-01-01

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status. © 2011 Chepelev et al.

Interplay between SIRT1 and hepatitis B virus X protein in the activation of viral transcription.

PubMed

Deng, Jian-Jun; Kong, Ka-Yiu Edwin; Gao, Wei-Wei; Tang, Hei-Man Vincent; Chaudhary, Vidyanath; Cheng, Yun; Zhou, Jie; Chan, Chi-Ping; Wong, Danny Ka-Ho; Yuen, Man-Fung; Jin, Dong-Yan

2017-04-01

Hepatitis B virus (HBV) genome is organized into a minichromosome known as covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. SIRT1 is an NAD + -dependent protein deacetylase which activates HBV transcription by promoting the activity of cellular transcription factors and coactivators. How SIRT1 and viral transactivator X protein (HBx) might affect each other remains to be clarified. In this study we show synergy and mutual dependence between SIRT1 and HBx in the activation of HBV transcription. All human sirtuins SIRT1 through SIRT7 activated HBV gene expression. The steady-state levels of SIRT1 protein were elevated in HBV-infected liver tissues and HBV-replicating hepatoma cells. SIRT1 interacted with HBx and potentiated HBx transcriptional activity on precore promoter and covalently closed circular DNA (cccDNA) likely through a deacetylase-independent mechanism, leading to more robust production of cccDNA, pregenomic RNA and surface antigen. SIRT1 and HBx proteins were more abundant when both were expressed. SIRT1 promoted the recruitment of HBx as well as cellular transcriptional factors and coactivators such as PGC-1α and FXRα to cccDNA. Depletion of SIRT1 suppressed HBx recruitment. On the other hand, SIRT1 recruitment to cccDNA was compromised when HBx was deficient. Whereas pharmaceutical agonists of SIRT1 such as resveratrol activated HBV transcription, small-molecule inhibitors of SIRT1 including sirtinol and Ex527 exhibited anti-HBV activity. Taken together, our findings revealed not only the interplay between SIRT1 and HBx in the activation of HBV transcription but also new strategies and compounds for developing antivirals against HBV. Copyright © 2017 Elsevier B.V. All rights reserved.

Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery.

PubMed

Choy, Meng S; Li, Yang; Machado, Luciana E S F; Kunze, Micha B A; Connors, Christopher R; Wei, Xingyu; Lindorff-Larsen, Kresten; Page, Rebecca; Peti, Wolfgang

2017-02-16

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of Lipoic Acid on Serum Paraoxonase-1 and Paraoxonase-3 Protein Levels and Activities in Diabetic Rats.

PubMed

Ozgun, E; Ozgun, G S; Gokmen, S S; Eskıocak, S; Sut, N; Akıncı, M; Goncu, E; Cakır, E

2016-02-05

The aim of the present study was to investigate the effect of streptozotocin-induced diabetes mellitus and lipoic acid treatment on serum paraoxonase-1 and paraoxonase-3 protein levels and paraoxonase, arylesterase and lactonase activities.36 rats were equally and randomly divided into 4 groups as control, lipoic acid, diabetes and diabetes+lipoic acid. To induce diabetes, a single dose of streptozotocin (40 mg/kg) was injected intraperitoneally to diabetes and diabetes+lipoic acid groups. Lipoic acid (10 mg/kg/day) was injected intraperitoneally for 14 days to lipoic acid and diabetes+lipoic acid groups. Serum PON1 and PON3 protein levels were measured by western blotting. Serum paraoxonase, arylesterase and lactonase activities were determined by the measuring initial rate of substrate (paraoxon, phenylacetate and dihydrocoumarin) hydrolysis.Streptozotocin-induced diabetes mellitus caused a significant decrease whereas lipoic acid treatment caused a significant increase in serum PON1 and PON3 protein levels and paraoxonase, arylesterase and lactonase activities. The increase percent of serum PON3 protein was higher than that of serum PON1 protein and the increase percent of serum lactonase activity was higher than that of serum paraoxonase and arylesterase activities in diabetes+lipoic acid group.We can report that, like PON1 protein, PON3 protein and actually its lactonase activity may also have a role as an antioxidant in diabetes mellitus and lipoic acid treatment may be useful for the prevention of the atherosclerotic complications of diabetes by increasing serum PON1 and PON3 protein levels and serum enzyme activities. © Georg Thieme Verlag KG Stuttgart · New York.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

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

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

2015-03-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interruptsmore » the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.« less

Activator Protein-1: redox switch controlling structure and DNA-binding.

PubMed

Yin, Zhou; Machius, Mischa; Nestler, Eric J; Rudenko, Gabby

2017-11-02

The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a 'redox switch' centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the 'OFF' state, and show that the mid-point redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Activator Protein-1: redox switch controlling structure and DNA-binding

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

Yin, Zhou; Machius, Mischa; Nestler, Eric J.

The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a ‘redox switch’ centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the ‘OFF’ state, and show that the mid-pointmore » redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins.« less

The heat-shock protein Apg-2 binds to the tight junction protein ZO-1 and regulates transcriptional activity of ZONAB.

PubMed

Tsapara, Anna; Matter, Karl; Balda, Maria S

2006-03-01

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1-ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G(1)/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1-ZONAB signaling in epithelial cells in response to cellular stress.

The Heat-Shock Protein Apg-2 Binds to the Tight Junction Protein ZO-1 and Regulates Transcriptional Activity of ZONAB

PubMed Central

Tsapara, Anna; Matter, Karl; Balda, Maria S.

2006-01-01

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1–ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G1/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1–ZONAB signaling in epithelial cells in response to cellular stress. PMID:16407410

Mitogen activated protein kinase (MAPK) pathway regulates heme oxygenase-1 gene expression by hypoxia in vascular cells.

PubMed

Ryter, Stefan W; Xi, Sichuan; Hartsfield, Cynthia L; Choi, Augustine M K

2002-08-01

Hypoxia induces the stress protein heme oxygenase-1 (HO-1), which participates in cellular adaptation. The molecular pathways that regulate ho-1 gene expression under hypoxia may involve mitogen activated protein kinase (MAPK) signaling and reactive oxygen. Hypoxia (8 h) increased HO-1 mRNA in rat pulmonary aortic endothelial cells (PAEC), and also activated both extracellular signal-regulated kinase 1 (ERK1)/ERK2 and p38 MAPK pathways. The role of these kinases in hypoxia-induced ho-1 gene expression was examined using chemical inhibitors of these pathways. Surprisingly, SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK1), strongly enhanced hypoxia-induced HO-1 mRNA expression in PAEC. UO126, a MEK1/2 inhibitor, enhanced HO-1 expression in PAEC under normoxia, but not hypoxia. Diphenylene iodonium, an inhibitor of NADPH oxidase, also induced the expression of HO-1 in PAEC under both normoxia and hypoxia. Similar results were observed in aortic vascular smooth muscle cells. Furthermore, hypoxia induced activator protein (AP-1) DNA-binding activity in PAEC. Pretreatment with SB203580 and PD98059 enhanced AP-1 binding activity under hypoxia in PAEC; UO126 stimulated AP-1 binding under normoxia, whereas diphenylene iodonium stimulated AP-1 binding under normoxia and hypoxia. These results suggest a relationship between MAPK and hypoxic regulation of ho-1 in vascular cells, involving AP-1.

Mutant human tumor suppressor p53 modulates the activation of mitogen-activated protein kinase and nuclear factor-kappaB, but not c-Jun N-terminal kinase and activated protein-1.

PubMed

Gulati, Anthony P; Yang, Yang-Ming; Harter, David; Mukhopadhyay, Asok; Aggarwal, Bharat B; Aggarwal, Bharat A; Benzil, Deborah L; Whysner, John; Albino, Anthony P; Murali, Raj; Jhanwar-Uniyal, Meena

2006-01-01

The roles of the mitogen-activated kinase protein (MAPK) pathway, nuclear factor-kappa B (NF-kappaB), and activator protein-1 (AP-1) in cellular responses to growth factors and mitogen are well established. However, the manner by which these proliferative pathways are affected by the tumor suppressor protein p53 is not fully understood. We report here the results of an investigation of the status of p53 on two human melanoma cell lines with wild-type p53 (SK-Mel-186) or mutant p53 (SK-Mel-110). The basal levels of the activated extracellular-signal regulated kinases 1 and 2 (ERK1/2) were high in cells with wild-type p53, but low in cells with mutant p53. The 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of ERK1/2 through the phosphorylation of threonine and tyrosine at 202 and 204, respectively, was demonstrated in both cell lines, however, in a discrete manner. TPA-induced activation of ERK1/2 was sustained in wild-type p53 cells, while only a transient activation was seen in mutant p53 cells. Inhibition of MAPK kinase (MEK), an upstream kinase, by U0126, blocked TPA-induced activation of ERK1/2 in wild-type p53 cells and in mutant p53 cells. Treatment of wild-type p53 (SK-Mel 186) cells with small interfering RNA (siRNA) of p53 displayed a transient induction of activation of ERK1/2 following TPA treatment, indicating that p53 has a role in the regulation of the activation of ERK1/2. NF-kappaB activity decreased significantly in cells with wild-type p53, while enhanced NF-kappaB activity was evident in cells with mutant p53. The expression of either wild-type or mutant p53 had a similar effect on TPA-induced Jun N-terminal kinase (JNK) activation, indicating specificity for the ERK pathway. Similarly, AP-1 binding activity showed a transient variation in both cell lines after TPA treatment but with different kinetics. These observations suggest that both wild-type and mutant p53 can modulate the activation pathways for ERK1/2, and NF

Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

PubMed

Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

2017-01-01

Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

Timely Degradation of Wip1 Phosphatase by APC/C Activator Protein Cdh1 is Necessary for Normal Mitotic Progression.

PubMed

Jeong, Ho-Chang; Gil, Na-Yeon; Lee, Ho-Soo; Cho, Seung-Ju; Kim, Kyungtae; Chun, Kwang-Hoon; Cho, Hyeseong; Cha, Hyuk-Jin

2015-08-01

Wip1 belongs to the protein phosphatase C (PP2C) family, of which expression is up-regulated by a number of external stresses, and serves as a stress modulator in normal physiological conditions. When overexpressed, premature dephosphorylation of stress-mediators by Wip1 results in abrogation of tumor surveillance, thus Wip1 acts as an oncogene. Previously, the functional regulation of Wip1 in cell-cycle progression by counteracting cellular G1 and G2/M checkpoint activity in response to DNA damage was reported. However, other than in stress conditions, the function and regulatory mechanism of Wip1 has not been fully determined. Herein, we demonstrated that protein regulation of Wip1 occurs in a cell cycle-dependent manner, which is directly governed by APC/C(Cdh1) at the end of mitosis. In particular, we also showed evidence that Wip1 phosphatase activity is closely associated with its own protein stability, suggesting that reduced phosphatase activity of Wip1 during mitosis could trigger its degradation. Furthermore, to verify the physiological role of its phosphatase activity during mitosis, we established doxycycline-inducible cell models, including a Wip1 wild type (WT) and phosphatase dead mutant (Wip1 DA). When ectopically expressing Wip1 WT, we observed a delay in the transition from metaphase to anaphase. In conclusion, these studies show that mitotic degradation of Wip1 by APC/C(Cdh1) is important for normal mitotic progression. © 2015 Wiley Periodicals, Inc.

Role of Bioavailable Iron in Coal Dust-Induced Activation of Activator Protein-1 and Nuclear Factor of Activated T Cells

PubMed Central

Huang, Chuanshu; Li, Jingxia; Zhang, Qi; Huang, Xi

2010-01-01

Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers’ pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH2-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions. PMID:12397016

Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells.

PubMed

Tsai, Chung-Che; Kuo, Ting-Yu; Hong, Zhi-Wei; Yeh, Ying-Chieh; Shih, Kuo-Shun; Du, Shin-Yi; Fu, Hua-Wen

2015-01-01

Helicobacter pylori neutrophil-activating protein (HP-NAP) activates several innate leukocytes including neutrophils, monocytes, and mast cells. It has been reported that HP-NAP induces degranulation and interleukin-6 (IL-6) secretion of rat peritoneal mast cells. However, the molecular mechanism is not very clear. Here, we show that HP-NAP activates human mast cell line-1 (HMC-1) cells to secrete histamine and IL-6. The secretion depends on pertussis toxin (PTX)-sensitive heterotrimeric G proteins but not on Toll-like receptor 2. Moreover, HP-NAP induces PTX-sensitive G protein-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38-mitogen-activated protein kinase (p38 MAPK), and Akt in HMC-1 cells. Inhibition of ERK1/2, p38 MAPK, or phosphatidylinositol 3-kinase (PI3K) suppresses HP-NAP-induced release of histamine and IL-6 from HMC-1 cells. Thus, the activation of HMC-1 cells by HP-NAP is through Gi-linked G protein-coupled receptor-mediated MAPKs and PI3K/Akt pathways.

Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2

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

Nakatani, Miyuki; Ito, Jumpei; Japan Society for the Promotion of Science, Tokyo, 102-0083

Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the firstmore » step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. -- Highlights: •Enigma Homolog 1 (ENH1) is a scaffold protein. •ENH1 binds to inhibitor of DNA binding 2 (Id2) in myoblasts. •ENH1 overexpression overcomes the Id2's repression of myogenesis. •The Id2-ENH1 complex play an important role in the activation of myogenesis.« less

Thyroid hormone activates rat liver adenosine 5,-monophosphate-activated protein kinase: relation to CaMKKb, TAK1 and LKB1 expression and energy status.

PubMed

Vargas, R; Ortega, Y; Bozo, V; Andrade, M; Minuzzi, G; Cornejo, P; Fernandez, V; Videla, L A

2013-01-01

AMP-activated protein kinase (AMPK) is a sensor of energy status supporting cellular energy homeostasis that may represent the metabolic basis for 3,3,,5-triiodo-L-thyronine (T3) liver preconditioning. Functionally transient hyperthyroid state induced by T3 (single dose of 0.1 mg/kg) in fed rats led to upregulation of mRNA expression (RT-PCR) and protein phosphorylation (Western blot) of hepatic AMPK at 8 to 36 h after treatment. AMPK Thr 172 phosphorylation induced by T3 is associated with enhanced mRNA expression of the upstream kinases Ca2+ -calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) and transforming growth-factor-beta-activated kinase-1 (TAK1), with increased protein levels of CaMKKbeta and higher TAK1 phosphorylation, without changes in those of the liver kinase B1 (LKB1) signaling pathway. Liver contents of AMP and ADP were augmented by 291 percent and 44 percent by T3 compared to control values (p less than 0.05), respectively, whereas those of ATP decreased by 64% (p less than 0.05), with no significant changes in the total content of adenine nucleotides (AMP + ADP + ATP) at 24 h after T3 administration. Consequently, hepatic ATP/ADP content ratios exhibited 64 percent diminution (p less than 0.05) and those of AMP/ATP increased by 425 percent (p less than 0.05) in T3-treated rats over controls. It is concluded that in vivoT3 administration triggers liver AMPK upregulation in association with significant enhancements in AMPK mRNA expression, AMPK phosphorylation coupled to CaMKKbeta and TAK1 activation, and in AMP/ATP ratios, which may promote enhanced AMPK activity to support T3-induced energy consuming processes such as those of liver preconditioning.

The influence of acute resistance exercise on cyclooxygenase-1 and -2 activity and protein levels in human skeletal muscle.

PubMed

Carroll, Chad C; O'Connor, Devin T; Steinmeyer, Robert; Del Mundo, Jonathon D; McMullan, David R; Whitt, Jamie A; Ramos, Jahir E; Gonzales, Rayna J

2013-07-01

This study evaluated the activity and content of cyclooxygenase (COX)-1 and -2 in response to acute resistance exercise (RE) in human skeletal muscle. Previous work suggests that COX-1, but not COX-2, is the primary COX isoform elevated with resistance exercise in human skeletal muscle. COX activity, however, has not been assessed after resistance exercise in humans. It was hypothesized that RE would increase COX-1 but not COX-2 activity. Muscle biopsies were taken from the vastus lateralis of nine young men (25 ± 1 yr) at baseline (preexercise), 4, and 24 h after a single bout of knee extensor RE (three sets of 10 repetitions at 70% of maximum). Tissue lysate was assayed for COX-1 and COX-2 activity. COX-1 and COX-2 protein levels were measured via Western blot analysis. COX-1 activity increased at 4 h (P < 0.05) compared with preexercise, but returned to baseline at 24 h (PRE: 60 ± 10, 4 h: 106 ± 22, 24 h: 72 ± 8 nmol PGH2·g total protein(-1)·min(-1)). COX-2 activity was elevated at 4 and 24 h after RE (P < 0.05, PRE: 51 ± 7, 4 h: 100 ± 19, 24 h: 98 ± 14 nmol PGH2·g total protein(-1)·min(-1)). The protein level of COX-1 was not altered (P > 0.05) with acute RE. In contrast, COX-2 protein levels were nearly 3-fold greater (P > 0.05) at 4 h and 5-fold greater (P = 0.06) at 24 h, compared with preexercise. In conclusion, COX-1 activity increases transiently with exercise independent of COX-1 protein levels. In contrast, both COX-2 activity and protein levels were elevated with exercise, and this elevation persisted to at least 24 h after RE.

Platelet-activating factor mediates monocyte chemoattractant protein-1 expression in glomerular immune injury.

PubMed

Jocks, T; Freudenberg, J; Zahner, G; Stahl, R A

1998-01-01

These studies were designed to determine the possible role of platelet-activating factor (PAF) in the production of monocyte chemoattractant protein-1 (MCP-1) in glomerular immune injury. The glomerular lesion was induced in isolated perfused rat kidneys by a rabbit anti-rat-thymocyte serum (ATS) and rat serum (RS) as a complement source. Perfusion of kidneys with ATS and RS results in the selective binding of the antiserum to the glomerular mesangium with consecutive intraglomerular activation of complement. Antibody binding and complement activation induced a significant increase in glomerular MCP-1 mRNA levels when assessed by Northern blotting or RT-PCR. Decomplemented RS or non antibody rabbit IgG had only moderate effects on glomerular MCP-1 mRNA levels. The PAF receptor antagonist WEB 2170 almost completely blocked the ATS and RS induced MCP-1 mRNA levels. Perfusion of control kidneys with PAF increased MCP-1 mRNA expression, an effect which was blocked by WEB 2170. Glomerular MCP-1 protein formation, assessed by Western blotting, was stimulated following ATS and RS and PAF, respectively, was blocked by WEB 2170. These data show that PAF, derived from glomerular resident cells following antibody and complement induced injury, stimulates MCP-1 expression. In addition to the direct effects on leukocyte adhesion and activation PAF may mediate inflammatory cell influx in glomerular injuries due to the release of MCP-1.

Nucleic Acid Chaperone Activity of the ORF1 Protein from the Mouse LINE-1 Retrotransposon

PubMed Central

Martin, Sandra L.; Bushman, Frederic D.

2001-01-01

Non-LTR retrotransposons such as L1 elements are major components of the mammalian genome, but their mechanism of replication is incompletely understood. Like retroviruses and LTR-containing retrotransposons, non-LTR retrotransposons replicate by reverse transcription of an RNA intermediate. The details of cDNA priming and integration, however, differ between these two classes. In retroviruses, the nucleocapsid (NC) protein has been shown to assist reverse transcription by acting as a “nucleic acid chaperone,” promoting the formation of the most stable duplexes between nucleic acid molecules. A protein-coding region with an NC-like sequence is present in most non-LTR retrotransposons, but no such sequence is evident in mammalian L1 elements or other members of its class. Here we investigated the ORF1 protein from mouse L1 and found that it does in fact display nucleic acid chaperone activities in vitro. L1 ORF1p (i) promoted annealing of complementary DNA strands, (ii) facilitated strand exchange to form the most stable hybrids in competitive displacement assays, and (iii) facilitated melting of an imperfect duplex but stabilized perfect duplexes. These findings suggest a role for L1 ORF1p in mediating nucleic acid strand transfer steps during L1 reverse transcription. PMID:11134335

Chaperone activity of human small heat shock protein-GST fusion proteins.

PubMed

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

Canine parvovirus NS1 protein exhibits anti-tumor activity in a mouse mammary tumor model.

PubMed

Gupta, Shishir Kumar; Yadav, Pavan Kumar; Gandham, Ravi Kumar; Sahoo, A P; Harish, D R; Singh, Arvind Kumar; Tiwari, A K

2016-02-02

Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity. Copyright © 2015 Elsevier B.V. All rights reserved.

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed

Husain, S; Abdel-Latif, A A

1999-08-15

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed Central

Husain, S; Abdel-Latif, A A

1999-01-01

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals.

PubMed

Prochazka, Radek; Blaha, Milan

2015-01-01

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.

Clarinet (CLA-1), a novel active zone protein required for synaptic vesicle clustering and release

PubMed Central

Nelson, Jessica; Richmond, Janet E; Colón-Ramos, Daniel A; Shen, Kang

2017-01-01

Active zone proteins cluster synaptic vesicles at presynaptic terminals and coordinate their release. In forward genetic screens, we isolated a novel Caenorhabditis elegans active zone gene, clarinet (cla-1). cla-1 mutants exhibit defects in synaptic vesicle clustering, active zone structure and synapse number. As a result, they have reduced spontaneous vesicle release and increased synaptic depression. cla-1 mutants show defects in vesicle distribution near the presynaptic dense projection, with fewer undocked vesicles contacting the dense projection and more docked vesicles at the plasma membrane. cla-1 encodes three isoforms containing common C-terminal PDZ and C2 domains with homology to vertebrate active zone proteins Piccolo and RIM. The C-termini of all isoforms localize to the active zone. Specific loss of the ~9000 amino acid long isoform results in vesicle clustering defects and increased synaptic depression. Our data indicate that specific isoforms of clarinet serve distinct functions, regulating synapse development, vesicle clustering and release. PMID:29160205

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

PubMed Central

Reedijk, M; Liu, X Q; Pawson, T

1990-01-01

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages. Images PMID:2172781

BAR Proteins PSTPIP1/2 Regulate Podosome Dynamics and the Resorption Activity of Osteoclasts

PubMed Central

Sztacho, Martin; Segeletz, Sandra; Sanchez-Fernandez, Maria Arantzazu; Czupalla, Cornelia; Niehage, Christian; Hoflack, Bernard

2016-01-01

Bone resorption in vertebrates relies on the ability of osteoclasts to assemble F-actin-rich podosomes that condense into podosomal belts, forming sealing zones. Sealing zones segregate bone-facing ruffled membranes from other membrane domains, and disassemble when osteoclasts migrate to new areas. How podosome/sealing zone dynamics is regulated remains unknown. We illustrate the essential role of the membrane scaffolding F-BAR-Proline-Serine-Threonine Phosphatase Interacting Proteins (PSTPIP) 1 and 2 in this process. Whereas PSTPIP2 regulates podosome assembly, PSTPIP1 regulates their disassembly. PSTPIP1 recruits, through its F-BAR domain, the protein tyrosine phosphatase non-receptor type 6 (PTPN6) that de-phosphophorylates the phosphatidylinositol 5-phosphatases SHIP1/2 bound to the SH3 domain of PSTPIP1. Depletion of any component of this complex prevents sealing zone disassembly and increases osteoclast activity. Thus, our results illustrate the importance of BAR domain proteins in podosome structure and dynamics, and identify a new PSTPIP1/PTPN6/SHIP1/2-dependent negative feedback mechanism that counterbalances Src and PI(3,4,5)P3 signalling to control osteoclast cell polarity and activity during bone resorption. PMID:27760174

Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

PubMed

Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T; Scheaffer, Suzanne M; Roswit, William T; Alevy, Yael G; Patel, Anand C; Heier, Richard F; Romero, Arthur G; Nichols, Colin G; Holtzman, Michael J; Brett, Tom J

2012-12-07

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

Role of hypoxia-inducible factor-{alpha} in hepatitis-B-virus X protein-mediated MDR1 activation

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

Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa

2007-06-01

The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and induced the nuclear translocation of C/EBP{beta}. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1{alpha} siRNAmore » but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1{alpha} activation, and suggest HIF-1{alpha} for the therapeutic target of HBV-mediated chemoresistance.« less

Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

PubMed Central

Echevarria-Lima, Juliana; Kyle-Cezar, Fernanda; Leite, Daniela F P; Capella, Luiz; Capella, Márcia A M; Rumjanek, Vivian M

2005-01-01

Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2′-7′-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation. PMID:15804283

In silico study of protein to protein interaction analysis of AMP-activated protein kinase and mitochondrial activity in three different farm animal species

NASA Astrophysics Data System (ADS)

Prastowo, S.; Widyas, N.

2018-03-01

AMP-activated protein kinase (AMPK) is cellular energy censor which works based on ATP and AMP concentration. This protein interacts with mitochondria in determine its activity to generate energy for cell metabolism purposes. For that, this paper aims to compare the protein to protein interaction of AMPK and mitochondrial activity genes in the metabolism of known animal farm (domesticated) that are cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In silico study was done using STRING V.10 as prominent protein interaction database, followed with biological function comparison in KEGG PATHWAY database. Set of genes (12 in total) were used as input analysis that are PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PPARGC1, ACC, CPT1B, NRF2 and SOD. The first 7 genes belong to gene in AMPK family, while the last 5 belong to mitochondrial activity genes. The protein interaction result shows 11, 8 and 5 metabolism pathways in Bos taurus, Sus scrofa and Gallus gallus, respectively. The top pathway in Bos taurus is AMPK signaling pathway (10 genes), Sus scrofa is Adipocytokine signaling pathway (8 genes) and Gallus gallus is FoxO signaling pathway (5 genes). Moreover, the common pathways found in those 3 species are Adipocytokine signaling pathway, Insulin signaling pathway and FoxO signaling pathway. Genes clustered in Adipocytokine and Insulin signaling pathway are PRKAA2, PPARGC1A, PRKAB1 and PRKAG2. While, in FoxO signaling pathway are PRKAA2, PRKAB1, PRKAG2. According to that, we found PRKAA2, PRKAB1 and PRKAG2 are the common genes. Based on the bioinformatics analysis, we can demonstrate that protein to protein interaction shows distinct different of metabolism in different species. However, further validation is needed to give a clear explanation.

Protein tyrosine phosphatase 1B inhibitory activity of lavandulyl flavonoids from roots of Sophora flavescens.

PubMed

Sasaki, Tatsunori; Li, Wei; Higai, Koji; Quang, Tran Hong; Kim, Young Ho; Koike, Kazuo

2014-05-01

Protein tyrosine phosphatase 1B is a non-transmembrane protein tyrosine phosphatase and major negative regulator in insulin signaling cascades, and much attention has been paid to protein tyrosine phosphatase 1B inhibitors as potential therapies for diabetes. The screening of a natural compound library led to the discovery of five lavandulyl flavonoids, which were isolated from the roots of Sophora flavescens, as novel PTP1B inhibitors: kuraridin (1), norkurarinone (2), kurarinone (3), 2'-methoxykurarinone (4), and kushenol T (5). The three most potent compounds, 1, 2, and 4 (IC50 < 30 µM), were demonstrated to be noncompetitive inhibitors of protein tyrosine phosphatase 1B based on a kinetic analysis, and they exhibited different inhibitory selectivities against four homologous protein tyrosine phosphatases (T cell protein tyrosine phosphatase, vaccinia H1-related phosphatase, and Src homology domain 2-containing protein tyrosine phosphatases 1 and 2). Compounds 1, 2, and 4 also exhibited cellular activity in the insulin signaling pathway by increasing the insulin-stimulated Akt phosphorylation level in human hepatocellular liver carcinoma HepG2 cells, suggesting their potential for new anti-insulin-resistant drug developments. Georg Thieme Verlag KG Stuttgart · New York.

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

NASA Technical Reports Server (NTRS)

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

2000-01-01

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

An Arabidopsis Ran-binding protein, AtRanBP1c, is a co-activator of Ran GTPase-activating protein and requires the C-terminus for its cytoplasmic localization

NASA Technical Reports Server (NTRS)

Kim, Soo-Hwan; Roux, Stanley J.

2003-01-01

Ran-binding proteins (RanBPs) are a group of proteins that bind to Ran (Ras-related nuclear small GTP-binding protein), and thus either control the GTP/GDP-bound states of Ran or help couple the Ran GTPase cycle to a cellular process. AtRanBP1c is a Ran-binding protein from Arabidopsis thaliana (L.) Heynh. that was recently shown to be critically involved in the regulation of auxin-induced mitotic progression [S.-H. Kim et al. (2001) Plant Cell 13:2619-2630]. Here we report that AtRanBP1c inhibits the EDTA-induced release of GTP from Ran and serves as a co-activator of Ran-GTPase-activating protein (RanGAP) in vitro. Transient expression of AtRanBP1c fused to a beta-glucuronidase (GUS) reporter reveals that the protein localizes primarily to the cytosol. Neither the N- nor C-terminus of AtRanBP1c, which flank the Ran-binding domain (RanBD), is necessary for the binding of PsRan1-GTP to the protein, but both are needed for the cytosolic localization of GUS-fused AtRanBP1c. These findings, together with a previous report that AtRanBP1c is critically involved in root growth and development, imply that the promotion of GTP hydrolysis by the Ran/RanGAP/AtRanBP1c complex in the cytoplasm, and the resulting concentration gradient of Ran-GDP to Ran-GTP across the nuclear membrane could be important in the regulation of auxin-induced mitotic progression in root tips of A. thaliana.

GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes

PubMed Central

Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

2014-01-01

Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12–ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex. PMID:25046113

Role of Accessory Proteins of HTLV-1 in Viral Replication, T Cell Activation, and Cellular Gene Expression

PubMed Central

Michael, Bindhu; Nair, Amithraj; Lairmore, Michael D.

2010-01-01

Human T-cell lymphotropic virus type 1 (HTLV-1), causes adult T cell leukemia/lymphoma (ATLL), and initiates a variety of immune mediated disorders. The viral genome encodes common structural and enzymatic proteins characteristic of all retroviruses and utilizes alternative splicing and alternate codon usage to make several regulatory and accessory proteins encoded in the pX region (pX ORF I to IV). Recent studies indicate that the accessory proteins p12I, p27I, p13II, and p30II, encoded by pX ORF I and II, contribute to viral replication and the ability of the virus to maintain typical in vivo expression levels. Proviral clones that are mutated in either pX ORF I or II, while fully competent in cell culture, are severely limited in their replicative capacity in a rabbit model. These HTLV-1 accessory proteins are critical for establishment of viral infectivity, enhance T- lymphocyte activation and potentially alter gene transcription and mitochondrial function. HTLV-1 pX ORF I expression is critical to the viral infectivity in resting primary lymphocytes suggesting a role for the calcineurin-binding protein p12I in lymphocyte activation. The endoplasmic reticulum and cis-Golgi localizing p12I activates NFAT, a key T cell transcription factor, through calcium-mediated signaling pathways and may lower the threshold of lymphocyte activation via the JAK/STAT pathway. In contrast p30II localizes to the nucleus and represses viral promoter activity, but may regulate cellular gene expression through p300/CBP or related co-activators of transcription. The mitochondrial localizing p13II induces morphologic changes in the organelle and may influence energy metabolism infected cells. Future studies of the molecular details HTLV-1 “accessory” proteins interactions will provide important new directions for investigations of HTLV-1 and related viruses associated with lymphoproliferative diseases. Thus, the accessory proteins of HTLV-1, once thought to be dispensable for

Hox proteins activate the IGFBP-1 promoter and suppress the function of hPR in human endometrial cells.

PubMed

Gao, Jiaguo; Mazella, James; Tseng, Linda

2002-11-01

Previous studies have shown that progestin activates the transcription of IGFBP-1 (insulin-like growth factor binding protein-1). Four regions in the IGFBP-1 promotor have been identified to enhance the transcription. Two of the regions, located at -73 to -65 bp and -319 to -311 bp formed identical DNA-protein complexes with the nuclear extracts of endometrial stromal/decidual cells. To identify the binding protein(s) in endometrial cells that interact with these two regions, we have used the TGTCAATTA repeats (-319 to -11 bp of the IGFBP-1 promoter) to screen the human decidual cDNA library by yeast one-hybrid system. We found that Hox A10, HoxA11, HoxB2, HoxB4, and HoxD11 interacted with the TGTCAATTA repeats in yeast cells. Among these hox genes, the full-length coding region of HoxA10, HoxA11, and HoxB4 were used for functional analysis in three types of endometrial cells, undifferentiated endometrial stromal cells, decidual cells (differentiated stromal cells) and endometrial adenocarcinoma cell line (HEC1-B). All these endometrial cells produce IGFBP-1. Transient transfection assay showed that HoxA10 expression vector increased the promoter activity (the IGFBP-1 proximal promoter containing TGC/TCAATTA and two functional PRE sites) in endometrial stromal cells and in HEC-1B cells, but not in decidual cells. HoxB4 enhanced the promoter activity only in decidual cells, while HoxA11 had no apparent effect in all three types of cells. To evaluate whether Hox proteins would interact with progesterone receptor (hPR), cells were transfected with the promoter construct, Hox and hPR expression vectors. hPR alone activated the IGFBP-1 promoter activity, but expression of Hox gene suppressed the activation. Hox proteins also suppressed the hPR enhanced promoter activities of MMTV (containing consensus-PRE sites) and glycodelin (GdA, containing Sp1 site which mediates the hPR function). These data showed that Hox genes selectively activate the transcription of the IGFBP

Salt Stress in Arabidopsis: Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

PubMed Central

Pitzschke, Andrea

2014-01-01

A plant’s capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase (MAPK) cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein (LTP)-related hybrid proline-rich protein (HyPRP), as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azi1 are hypersensitive to salt stress, while AZI1-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZI1-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT–PCR data point to a role of MPK3 as positive regulator of AZI1 abundance. PMID:24214892

The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain.

PubMed

Ma, Kai-Ge; Lv, Jia; Hu, Xiao-Dan; Shi, Li-Li; Chang, Ke-Wei; Chen, Xin-Lin; Qian, Yi-Hua; Yang, Wei-Na; Qu, Qiu-Min

2016-07-01

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transcriptional activation by MEIS1A in response to protein kinase A signaling requires the transducers of regulated CREB family of CREB co-activators.

PubMed

Goh, Siew-Lee; Looi, Yvonne; Shen, Hui; Fang, Jun; Bodner, Caroline; Houle, Martin; Ng, Andy Cheuk-Him; Screaton, Robert A; Featherstone, Mark

2009-07-10

The transcription factor encoded by the murine ecotropic integration site 1 gene (MEIS1) is a partner of HOX and PBX proteins. It has been implicated in embryonic patterning and leukemia, and causally linked to restless legs syndrome. The MEIS1A C terminus harbors a transcriptional activation domain that is stimulated by protein kinase A (PKA) in a manner dependent on the co-activator of cAMP response element-binding protein (CREB), CREB-binding protein (CBP). We explored the involvement of another mediator of PKA-inducible transcription, namely the CREB co-activators transducers of regulated CREB activity (TORCs). Overexpression of TORC1 or TORC2 bypassed PKA for activation by MEIS1A. Co-immunoprecipitation experiments demonstrated a physical interaction between MEIS1 and TORC2 that is dependent on the MEIS1A C terminus, whereas chromatin immunoprecipitation revealed PKA-inducible recruitment of MEIS1, PBX1, and TORC2 on the MEIS1 target genes Hoxb2 and Meis1. The MEIS1 interaction domain on TORC1 was mapped to the N-terminal coiled-coil region, and TORC1 mutants lacking this domain attenuated the response to PKA on a natural MEIS1A target enhancer. Thus, TORCs physically cooperate with MEIS1 to achieve PKA-inducible transactivation through the MEIS1A C terminus, suggesting a concerted action in developmental and oncogenic processes.

Transcriptional Activation by MEIS1A in Response to Protein Kinase A Signaling Requires the Transducers of Regulated CREB Family of CREB Co-activators*

PubMed Central

Goh, Siew-Lee; Looi, Yvonne; Shen, Hui; Fang, Jun; Bodner, Caroline; Houle, Martin; Ng, Andy Cheuk-Him; Screaton, Robert A.; Featherstone, Mark

2009-01-01

The transcription factor encoded by the murine ecotropic integration site 1 gene (MEIS1) is a partner of HOX and PBX proteins. It has been implicated in embryonic patterning and leukemia, and causally linked to restless legs syndrome. The MEIS1A C terminus harbors a transcriptional activation domain that is stimulated by protein kinase A (PKA) in a manner dependent on the co-activator of cAMP response element-binding protein (CREB), CREB-binding protein (CBP). We explored the involvement of another mediator of PKA-inducible transcription, namely the CREB co-activators transducers of regulated CREB activity (TORCs). Overexpression of TORC1 or TORC2 bypassed PKA for activation by MEIS1A. Co-immunoprecipitation experiments demonstrated a physical interaction between MEIS1 and TORC2 that is dependent on the MEIS1A C terminus, whereas chromatin immunoprecipitation revealed PKA-inducible recruitment of MEIS1, PBX1, and TORC2 on the MEIS1 target genes Hoxb2 and Meis1. The MEIS1 interaction domain on TORC1 was mapped to the N-terminal coiled-coil region, and TORC1 mutants lacking this domain attenuated the response to PKA on a natural MEIS1A target enhancer. Thus, TORCs physically cooperate with MEIS1 to achieve PKA-inducible transactivation through the MEIS1A C terminus, suggesting a concerted action in developmental and oncogenic processes. PMID:19473990

Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein*

PubMed Central

Law, Jennifer; Salla, Mohamed; Zare, Alaa; Wong, Yoke; Luong, Le; Volodko, Natalia; Svystun, Orysya; Flood, Kayla; Lim, Jonathan; Sung, Miranda; Dyck, Jason R. B.; Tan, Chong Teik; Su, Yu-Chin; Yu, Victor C.; Mackey, John; Baksh, Shairaz

2015-01-01

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis. PMID:26269600

Preparation and characterization of human recombinant protein 1/Clara cell M(r) 10,000 protein.

PubMed

Okutani, R; Itoh, Y; Yamada, T; Yamaguchi, T; Singh, G; Yagisawa, H; Kawai, T

1996-09-01

Protein 1, which is identical to human Clara cell M(r) 10(4) protein, is a homodimeric, low molecular mass protein (M(r) 14,000) and an effective inhibitor of phospholipase A2 activity. We have expressed this protein in E. coli and characterized its physiochemical and biological properties. Using a pET expression system, about 1.7 mg of purified recombinant protein 1 was obtained from 250 ml of E. coli culture. The amino-terminal sequence of recombinant protein 1 up to the 20th residue was identical to that of native protein 1 except for an extra methionine at the amino-terminus. On reversed-phase HPLC, recombinant protein 1 eluted at the same retention time as native protein 1. The dose-response curves of recombinant protein 1 and native protein 1 in an enzyme-linked immunosorbent assay for protein 1 were identical. Recombinant protein 1 inhibited both porcine pancreas and cobra venom phospholipase A2 activities. These results indicated that recombinant protein 1 is structurally and biologically identical to native protein 1. We found that recombinant protein 1 also inhibits phosphatidylinositol-specific phospholipase C activity.

The biological activity of ABA-1-like protein from Ascaris lumbricoides.

PubMed

Muto, R; Imai, S; Tezuka, H; Furuhashi, Y; Fujita, K

2001-09-01

The elevation of non-specific IgE (total IgE) in Ascaris infection can be seen one week after infection, and reaches a peak after approximately two weeks. It has been reported that ABA-1 protein is the main constituent in the pseudocoelomic fluid of Ascaris suum. To investigate the effect of the ABA-1-like protein from Ascaris lumbricoides (ALB), the cDNA was cloned by reverse transcriptase polymerase chain reaction, using original primers based on the consensus sequences of ABA-1 and TBA-1, that is an ABA-1-like protein from Toxocara canis. The clone was sequenced, we constructed the recombinant polyprotein of ALB (rALB14 and rALB7) based on the ALB sequence, and rALB was administrated to BALB/c mice. Fourteen days after inoculation with rALB14 which is the full length of ALB, the elevation of total IgE which we supposed to contain non-specific IgE was observed, and the results were as we expected. Furthermore, in an in-vitro experiment, we confirmed that the spleen cells proliferated when stimulated by rALB14 and concanavalin A. Therefore, the whole conformation of ALB is considered to be involved in the elevation of non-specific IgE, and is involved in the activation of T cells.

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

PubMed

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Network-Based Methods for Identifying Key Active Proteins in the Extracellular Electron Transfer Process in Shewanella oneidensis MR-1.

PubMed

Ding, Dewu; Sun, Xiao

2018-01-16

Shewanella oneidensis MR-1 can transfer electrons from the intracellular environment to the extracellular space of the cells to reduce the extracellular insoluble electron acceptors (Extracellular Electron Transfer, EET). Benefiting from this EET capability, Shewanella has been widely used in different areas, such as energy production, wastewater treatment, and bioremediation. Genome-wide proteomics data was used to determine the active proteins involved in activating the EET process. We identified 1012 proteins with decreased expression and 811 proteins with increased expression when the EET process changed from inactivation to activation. We then networked these proteins to construct the active protein networks, and identified the top 20 key active proteins by network centralization analysis, including metabolism- and energy-related proteins, signal and transcriptional regulatory proteins, translation-related proteins, and the EET-related proteins. We also constructed the integrated protein interaction and transcriptional regulatory networks for the active proteins, then found three exclusive active network motifs involved in activating the EET process-Bi-feedforward Loop, Regulatory Cascade with a Feedback, and Feedback with a Protein-Protein Interaction (PPI)-and identified the active proteins involved in these motifs. Both enrichment analysis and comparative analysis to the whole-genome data implicated the multiheme c -type cytochromes and multiple signal processing proteins involved in the process. Furthermore, the interactions of these motif-guided active proteins and the involved functional modules were discussed. Collectively, by using network-based methods, this work reported a proteome-wide search for the key active proteins that potentially activate the EET process.

The Scaffold Protein TANK/I-TRAF Inhibits NF-κB Activation by Recruiting Polo-like Kinase 1

PubMed Central

Zhang, Wanqiao; Zhang, Ying; Yuan, Yanzhi; Guan, Wei; Jin, Chaozhi; Chen, Hui; Wang, Xiaohui

2010-01-01

TANK/I-TRAF is a TRAF-binding protein that negatively regulates NF-κB activation. The underlying mechanism of this activity remains unclear. Here we show that TANK directly interacts with PLK1, a conserved cell cycle–regulated kinase. PLK1 inhibits NF-κB transcriptional activation induced by TNF-α, IL-1β, or several activators, but not by nuclear transcription factor p65. PLK1 expression reduces the DNA-binding activity of NF-κB induced by TNF-α. Moreover, endogenous activation of PLK1 reduces the TNF-induced phosphorylation of endogenous IκBα. PLK1 is bound to NEMO (IKKγ) through TANK to form a ternary complex in vivo. We describe a new regulatory mechanism for PLK1: PLK1 negatively regulates TNF-induced IKK activation by inhibiting the ubiquitination of NEMO. These findings reveal that the scaffold protein TANK recruits PLK1 to negatively regulate NF-κB activation and provide direct evidence that PLK1 is required for the repression function of TANK. PMID:20484576

FGF-1-induced matrix metalloproteinase-9 expression in breast cancer cells is mediated by increased activities of NF-kappaB and activating protein-1.

PubMed

Lungu, Gina; Covaleda, Lina; Mendes, Odete; Martini-Stoica, Heidi; Stoica, George

2008-06-01

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK. (c) 2007 Wiley-Liss, Inc.

Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes.

PubMed

Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer; Jensen, Thomas Elbenhardt; Sakamoto, Kei; Göransson, Olga

2011-05-01

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes remains elusive. Previous studies have identified LKB1 as a major AMPK kinase in muscle, liver, and other tissues. In certain cell types, Ca(2+) /calmodulin-dependent protein kinase kinase β (CaMKKβ) has been shown to activate AMPK in response to increases of intracellular Ca(2+) levels. Our aim was to investigate if LKB1 and/or CaMKK function as AMPK kinases in adipocytes. We used adipose tissue and isolated adipocytes from mice in which the expression of LKB1 was reduced to 10-20% of that of wild-type (LKB1 hypomorphic mice). We show that adipocytes from LKB1 hypomorphic mice display a 40% decrease in basal AMPK activity and a decrease of AMPK activity in the presence of the AMPK activator phenformin. We also demonstrate that stimulation of 3T3L1 adipocytes with intracellular [Ca(2+) ]-raising agents results in an activation of the AMPK pathway. The inhibition of CaMKK isoforms, particularly CaMKKβ, by the inhibitor STO-609 or by siRNAs, blocked Ca(2+) -, but not phenformin-, AICAR-, or forskolin-induced activation of AMPK, indicating that CaMKK activated AMPK in response to Ca(2+) . Collectively, we show that LKB1 is required to maintain normal AMPK-signaling in non-stimulated adipocytes and in the presence of phenformin. In addition, we demonstrate the existence of a Ca(2+) /CaMKK signaling pathway that can also regulate the activity of AMPK in adipocytes. Copyright © 2011 Wiley-Liss, Inc.

Phenformin Activates the Unfolded Protein Response in an AMP-activated Protein Kinase (AMPK)-dependent Manner*

PubMed Central

Yang, Liu; Sha, Haibo; Davisson, Robin L.; Qi, Ling

2013-01-01

Activation of the unfolded protein response (UPR) is associated with the disruption of endoplasmic reticulum (ER) homeostasis and has been implicated in the pathogenesis of many human metabolic diseases, including obesity and type 2 diabetes. However, the nature of the signals activating UPR under these conditions remains largely unknown. Using a method that we recently optimized to directly measure UPR sensor activation, we screened the effect of various metabolic drugs on UPR activation and show that the anti-diabetic drug phenformin activates UPR sensors IRE1α and pancreatic endoplasmic reticulum kinase (PERK) in both an ER-dependent and ER-independent manner. Mechanistically, AMP-activated protein kinase (AMPK) activation is required but not sufficient to initiate phenformin-mediated IRE1α and PERK activation, suggesting the involvement of additional factor(s). Interestingly, activation of the IRE1α (but not PERK) pathway is partially responsible for the cytotoxic effect of phenformin. Together, our data show the existence of a non-canonical UPR whose activation requires the cytosolic kinase AMPK, adding another layer of complexity to UPR activation upon metabolic stress. PMID:23548904

Chemical synthesis of biologically active tat trans-activating protein of human immunodeficiency virus type 1.

PubMed Central

Chun, R; Glabe, C G; Fan, H

1990-01-01

Full-length (86-residue) polypeptide corresponding to the human immunodeficiency virus type 1 tat trans-activating protein was chemically synthesized on a semiautomated apparatus, using an Fmoc amino acid continuous-flow strategy. The bulk material was relatively homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, and it showed trans-activating activity when scrape loaded into cells containing a human immunodeficiency virus long terminal repeat-chloramphenicol acetyl-transferase reporter plasmid. Reverse-phase high-pressure liquid chromatography yielded a rather broad elution profile, and assays across the column for biological activity indicated a sharper peak. Thus, high-pressure liquid chromatography provided for enrichment of biological activity. Fast atom bombardment-mass spectrometry of tryptic digests of synthetic tat identified several of the predicted tryptic peptides, consistent with accurate chemical synthesis. Images PMID:2186178

Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein.

PubMed

Gamba, Elia; Mori, Mattia; Kovalenko, Lesia; Giannini, Alessia; Sosic, Alice; Saladini, Francesco; Fabris, Dan; Mély, Yves; Gatto, Barbara; Botta, Maurizio

2018-02-10

In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation*♦

PubMed Central

Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T.; Scheaffer, Suzanne M.; Roswit, William T.; Alevy, Yael G.; Patel, Anand C.; Heier, Richard F.; Romero, Arthur G.; Nichols, Colin G.; Holtzman, Michael J.; Brett, Tom J.

2012-01-01

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface. PMID:23112050

Walleye dermal sarcoma virus Orf B functions through receptor for activated C kinase (RACK1) and protein kinase C

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

Daniels, Candelaria C.; Rovnak, Joel; Quackenbush, Sandra L.

2008-06-05

Walleye dermal sarcoma virus is a complex retrovirus that is associated with walleye dermal sarcomas that are seasonal in nature. Fall developing tumors contain low levels of spliced accessory gene transcripts A and B, suggesting a role for the encoded proteins, Orf A and Orf B, in oncogenesis. In explanted tumor cells the 35 kDa Orf B accessory protein is localized to the cell periphery in structures similar to focal adhesions and along actin stress fibers. Similar localization was observed in mammalian cells. The cellular protein, receptor for activated C kinase 1 (RACK1), bound Orf B in yeast two-hybrid assaysmore » and in cell culture. Sequence analysis of walleye RACK1 demonstrated high conservation to other known RACK1 sequences. RACK1 binds to activated protein kinase C (PKC). Orf B associates with PKC{alpha}, which is constitutively activated and localized at the membrane. Activated PKC promoted cell survival, proliferation, and increased cell viability in Orf B-expressing cells.« less

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

PubMed Central

Lindin, Inger; Wuxiuer, Yimingjiang; Ravna, Aina Westrheim; Moens, Ugo; Sylte, Ingebrigt

2014-01-01

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding. PMID:24651460

Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1.

PubMed Central

Bischoff, F R; Krebber, H; Smirnova, E; Dong, W; Ponstingl, H

1995-01-01

RCC1 (the regulator of chromosome condensation) stimulates guanine nucleotide dissociation on the Ras-related nuclear protein Ran. Both polypeptides are components of a regulatory pathway that has been implicated in regulating DNA replication, onset of and exit from mitosis, mRNA processing and transport, and import of proteins into the nucleus. In a search for further members of the RCC1-Ran signal pathway, we have identified proteins of 23, 45 and 300 kDa which tightly bind to Ran-GTP but not Ran-GDP. The purified soluble 23 kDa Ran binding protein RanBP1 does not activate RanGTPase, but increases GTP hydrolysis induced by the RanGTPase-activating protein RanGAP1 by an order of magnitude. In the absence of RanGAP, it strongly inhibits RCC1-induced exchange of Ran-bound GTP. In addition, it forms a stable complex with nucleotide-free RCC1-Ran. With these properties, it differs markedly from guanine diphosphate dissociation inhibitors which preferentially prevent the exchange of protein-bound GDP and in some cases were shown to inhibit GAP-induced GTP hydrolysis. RanBP1 is the first member of a new class of proteins regulating the binding and hydrolysis of GTP by Ras-related proteins. Images PMID:7882974

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

PubMed

Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J

2014-07-03

The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may

Regulation of RE1 Protein Silencing Transcription Factor (REST) Expression by HIP1 Protein Interactor (HIPPI)*

PubMed Central

Datta, Moumita; Bhattacharyya, Nitai P.

2011-01-01

Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease. PMID:21832040

Regulation of RE1 protein silencing transcription factor (REST) expression by HIP1 protein interactor (HIPPI).

PubMed

Datta, Moumita; Bhattacharyya, Nitai P

2011-09-30

Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease.

Phenformin activates the unfolded protein response in an AMP-activated protein kinase (AMPK)-dependent manner.

PubMed

Yang, Liu; Sha, Haibo; Davisson, Robin L; Qi, Ling

2013-05-10

The cross-talk between UPR activation and metabolic stress remains largely unclear. Phenformin treatment activates the IRE1α and PERK pathways in an AMPK-dependent manner. AMPK is required for phenformin-mediated IRE1α and PERK activation. Our findings demonstrate the cross-talk between UPR and metabolic signals. Activation of the unfolded protein response (UPR) is associated with the disruption of endoplasmic reticulum (ER) homeostasis and has been implicated in the pathogenesis of many human metabolic diseases, including obesity and type 2 diabetes. However, the nature of the signals activating UPR under these conditions remains largely unknown. Using a method that we recently optimized to directly measure UPR sensor activation, we screened the effect of various metabolic drugs on UPR activation and show that the anti-diabetic drug phenformin activates UPR sensors IRE1α and pancreatic endoplasmic reticulum kinase (PERK) in both an ER-dependent and ER-independent manner. Mechanistically, AMP-activated protein kinase (AMPK) activation is required but not sufficient to initiate phenformin-mediated IRE1α and PERK activation, suggesting the involvement of additional factor(s). Interestingly, activation of the IRE1α (but not PERK) pathway is partially responsible for the cytotoxic effect of phenformin. Together, our data show the existence of a non-canonical UPR whose activation requires the cytosolic kinase AMPK, adding another layer of complexity to UPR activation upon metabolic stress.

Active inhibitor-1 maintains protein hyper-phosphorylation in aging hearts and halts remodeling in failing hearts.

PubMed

Pritchard, Tracy J; Kawase, Yoshiaki; Haghighi, Kobra; Anjak, Ahmad; Cai, Wenfeng; Jiang, Min; Nicolaou, Persoulla; Pylar, George; Karakikes, Ioannis; Rapti, Kleopatra; Rubinstein, Jack; Hajjar, Roger J; Kranias, Evangelia G

2013-01-01

Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term.

NHERF1, a novel GPER associated protein, increases stability and activation of GPER in ER-positive breast cancer

PubMed Central

Xiong, Ying; Wang, Yan; Zheng, Junfang; Zhao, Yuan; Tao, Tao; Wang, Qiqi; Liu, Hua; Wang, Songlin; Jiang, Wen G.; He, Junqi

2016-01-01

G protein-coupled estrogen receptor (GPER) plays an important role in mediating the effects of estradiol. High levels of GPER have been implicated to associate with the malignant progress of invasive breast cancer (IBC). However, the mechanisms by which GPER protein levels were regulated remain unclear. In this study, PDZ protein Na+/H+ exchanger regulatory factor (NHERF1) was found to interact with GPER in breast cancer cells. This interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal PDZ binding motif of GPER. NHERF1 was demonstrated to facilitate GPER expression at post-transcriptional level and improve GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in a GPER/NHERF1 interaction-dependent manner. In addition, GPER protein levels are positively associated with NHERF1 protein levels in a panel of estrogen receptor (ER)-positive breast cancer cells. Furthermore, analysis of clinical IBC data from The Cancer Genome Atlas (TCGA) showed no significant difference in GPER mRNA levels between ER-positive IBC and normal breast tissues. However, gene set enrichment analysis (GSEA) showed that GPER signaling is ultra-activated in ER-positive IBC when compared with normal and its activation is positively associated with NHERF1 mRNA levels. Taken together, our findings identify NHERF1 as a new binding partner for GPER and its overexpression promotes protein stability and activation of GPER in ER-positive IBC. Our data indicate that regulation of GPER stability by NHERF1 may contribute to GPER-mediated carcinogenesis in ER-positive IBC. PMID:27448983

NHERF1, a novel GPER associated protein, increases stability and activation of GPER in ER-positive breast cancer.

PubMed

Meng, Ran; Qin, Qiong; Xiong, Ying; Wang, Yan; Zheng, Junfang; Zhao, Yuan; Tao, Tao; Wang, Qiqi; Liu, Hua; Wang, Songlin; Jiang, Wen G; He, Junqi

2016-08-23

G protein-coupled estrogen receptor (GPER) plays an important role in mediating the effects of estradiol. High levels of GPER have been implicated to associate with the malignant progress of invasive breast cancer (IBC). However, the mechanisms by which GPER protein levels were regulated remain unclear. In this study, PDZ protein Na+/H+ exchanger regulatory factor (NHERF1) was found to interact with GPER in breast cancer cells. This interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal PDZ binding motif of GPER. NHERF1 was demonstrated to facilitate GPER expression at post-transcriptional level and improve GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in a GPER/NHERF1 interaction-dependent manner. In addition, GPER protein levels are positively associated with NHERF1 protein levels in a panel of estrogen receptor (ER)-positive breast cancer cells. Furthermore, analysis of clinical IBC data from The Cancer Genome Atlas (TCGA) showed no significant difference in GPER mRNA levels between ER-positive IBC and normal breast tissues. However, gene set enrichment analysis (GSEA) showed that GPER signaling is ultra-activated in ER-positive IBC when compared with normal and its activation is positively associated with NHERF1 mRNA levels. Taken together, our findings identify NHERF1 as a new binding partner for GPER and its overexpression promotes protein stability and activation of GPER in ER-positive IBC. Our data indicate that regulation of GPER stability by NHERF1 may contribute to GPER-mediated carcinogenesis in ER-positive IBC.

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

PubMed

Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

2017-08-11

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

Regulation of Src homology 2-containing tyrosine phosphatase 1 during activation of human neutrophils. Role of protein kinase C.

PubMed

Brumell, J H; Chan, C K; Butler, J; Borregaard, N; Siminovitch, K A; Grinstein, S; Downey, G P

1997-01-10

The tyrosine phosphorylation of several proteins induced in neutrophils by soluble and particulate stimuli is thought to be crucial for initiating antimicrobial responses. Although activation of tyrosine kinases is thought to mediate this event, the role of tyrosine phosphatases in the initiation and modulation of neutrophil responses remains largely undefined. We investigated the role of Src homology 2-containing tyrosine phosphatase 1 (SHP-1; also known as protein tyrosine phosphatase 1C (PTP1C), hematopoetic cell phosphatase, PTP-N6, and SHPTP-1), a phosphatase expressed primarily in hemopoietic cells, in the activation of human neutrophils. SHP-1 mRNA and protein were detected in these cells, and the enzyme was found to be predominantly localized to the cytosol in unstimulated cells. Following stimulation with neutrophil agonists such as phorbol ester, chemotactic peptide, or opsonized zymosan, a fraction of the phosphatase redistributed to the cytoskeleton. Agonist treatment also induced significant decreases (30-60%) in SHP-1 activity, which correlated temporally with increases in the cellular phosphotyrosine content. Phosphorylation of SHP-1 on serine residues was associated with the inhibition of its enzymatic activity, suggesting a causal relationship. Accordingly, both the agonist-evoked phosphorylation of SHP-1 and the inhibition of its catalytic activity were blocked by treatment with bisindolylmaleimide I, a potent and specific inhibitor of protein kinase C (PKC) activity. Immunoprecipitated SHP-1 was found to be phosphorylated efficiently by purified PKC in vitro. Such phosphorylation also caused a decrease in the phosphatase activity of SHP-1. Together, these data suggest that inhibition of SHP-1 by PKC-mediated serine phosphorylation plays a role in facilitating the accumulation of tyrosine-phosphorylated proteins following neutrophil stimulation. These findings provide a new link between the PKC and tyrosine phosphorylation branches of the

Repeated immobilization stress increases uncoupling protein 1 expression and activity in Wistar rats.

PubMed

Gao, Bihu; Kikuchi-Utsumi, Kazue; Ohinata, Hiroshi; Hashimoto, Masaaki; Kuroshima, Akihiro

2003-06-01

Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

PubMed Central

Lvov, Anatoli; Gage, Steven D.; Berrios, Virla M.

2010-01-01

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K+ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K+ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening. PMID:20479109

The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis.

PubMed

Moreau, Fanny; Thévenon, Emmanuel; Blanvillain, Robert; Lopez-Vidriero, Irene; Franco-Zorrilla, Jose Manuel; Dumas, Renaud; Parcy, François; Morel, Patrice; Trehin, Christophe; Carles, Cristel C

2016-04-01

Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression. © 2016. Published by The Company of Biologists Ltd.

The PDZ protein tax-interacting protein-1 inhibits beta-catenin transcriptional activity and growth of colorectal cancer cells.

PubMed

Kanamori, Mutsumi; Sandy, Peter; Marzinotto, Stefania; Benetti, Roberta; Kai, Chikatoshi; Hayashizaki, Yoshihide; Schneider, Claudio; Suzuki, Harukazu

2003-10-03

Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is beta-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of beta-catenin that may control its transcriptional activity, we performed a mammalian two-hybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between beta-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of beta-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of beta-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous beta-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorage-independent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/beta-catenin signaling pathway.

GC-GAP, a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2.

PubMed

Zhao, Chunmei; Ma, Hong; Bossy-Wetzel, Ella; Lipton, Stuart A; Zhang, Zhuohua; Feng, Gen-Sheng

2003-09-05

Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.

Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

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

Guo, Shiguang; Mao, Li; Ji, Feng, E-mail: huaiaifengjidr@163.com

Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) exerts cytotoxic effect to cultured osteoblasts. Here, we investigated the potential activity of Compound 13 (C13), a novel α1 selective AMP-activated protein kinase (AMPK) activator, against the process. Our data revealed that C13 pretreatment significantly attenuated Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. AMPK activation mediated C13′ cytoprotective effect in osteoblasts. The AMPK inhibitor Compound C, shRNA-mediated knockdown of AMPKα1, or dominant negative mutation of AMPKα1 (T172A) almost abolished C13-induced AMPK activation and its pro-survival effect in osteoblasts. On the othermore » hand, forced AMPK activation by adding AMPK activator A-769662 or exogenous expression a constitutively-active (ca) AMPKα1 (T172D) mimicked C13's actions and inhibited Dex-induced osteoblast cell death. Meanwhile, A-769662 or ca-AMPKα1 almost nullified C13's activity in osteoblast. Further studies showed that C13 activated AMPK-dependent nicotinamide adenine dinucleotide phosphate (NADPH) pathway to inhibit Dex-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary murine osteoblasts. Such effects by C13 were almost reversed by Compound C or AMPKα1 depletion/mutation. Together, these results suggest that C13 alleviates Dex-induced osteoblast cell death via activating AMPK signaling pathway. - Highlights: • Compound 13 (C13) attenuates dexamethasone (Dex)-induced osteoblast cell death. • C13-induced cytoprotective effect against Dex in osteoblasts requires AMPK activation. • Forced AMPK activation protects osteoblasts from Dex, nullifying C13's activities. • C13 increases NADPH activity and inhibits Dex-induced oxidative stress in osteoblasts.« less

Novel prosurvival function of Yip1A in human cervical cancer cells: constitutive activation of the IRE1 and PERK pathways of the unfolded protein response.

PubMed

Taguchi, Yuki; Horiuchi, Yuta; Kano, Fumi; Murata, Masayuki

2017-03-30

Cancer cells are under chronic endoplasmic reticulum (ER) stress due to hypoxia, low levels of nutrients, and a high metabolic demand for proliferation. To survive, they constitutively activate the unfolded protein response (UPR). The inositol-requiring protein 1 (IRE1) and protein kinase RNA-like ER kinase (PERK) signaling branches of the UPR have been shown to have cytoprotective roles in cancer cells. UPR-induced autophagy is another prosurvival strategy of cancer cells, possibly to remove misfolded proteins and supply nutrients. However, the mechanisms by which cancer cells exploit the UPR and autophagy machinery to promote survival and the molecules that are essential for these processes remain to be elucidated. Recently, a multipass membrane protein, Yip1A, was shown to function in the activation of IRE1 and in UPR-induced autophagy. In the present study, we explored the possible role of Yip1A in activation of the UPR by cancer cells for their survival, and found that depletion of Yip1A by RNA interference (RNAi) induced apoptotic cell death in HeLa and CaSki cervical cancer cells. Intriguingly, Yip1A was found to activate the IRE1 and PERK pathways of the UPR constitutively in HeLa and CaSki cells. Yip1A mediated the phosphorylation of IRE1 and also engaged in the transcription of PERK. The activation of these signaling pathways upregulated the expression of anti-apoptotic proteins and autophagy-related proteins. These events might enhance resistance to apoptosis and promote cytoprotective autophagy in HeLa and CaSki cells. The present study is the first to uncover a key prosurvival modulator, Yip1A, which coordinates IRE1 signaling with PERK signaling to support the survival of HeLa and CaSki cervical cancer cells.

Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMP-activated protein kinase in DU145 human prostate cancer cells.

PubMed

Jung, Seung-Nam; Yang, Woo Kyeom; Kim, Joungmok; Kim, Hak Su; Kim, Eun Ju; Yun, Hee; Park, Hyunsung; Kim, Sung Soo; Choe, Wonchae; Kang, Insug; Ha, Joohun

2008-04-01

Hypoxia-inducible factor (HIF-1) plays a central role in the cellular adaptive response to hypoxic conditions, which are closely related to pathophysiological conditions, such as cancer. Although reactive oxygen species (ROS) have been implicated in the regulation of hypoxic and non-hypoxic induction of HIF-1 under various conditions, the role of ROS is quite controversial, and the mechanism underlying the HIF-1 regulation by ROS is not completely understood yet. Here, we investigated the biochemical mechanism for the ROS-induced HIF-1 by revealing a novel role of adenosine monophosphate-activated protein kinase (AMPK) and the upstream signal components. AMPK plays an essential role as energy-sensor under adenosine triphosphate-deprived conditions. Here we report that ROS induced by a direct application of H(2)O(2) and menadione to DU145 human prostate carcinoma resulted in accumulation of HIF-1alpha protein by attenuation of its degradation and activation of its transcriptional activity in an AMPK-dependent manner. By way of contrast, AMPK was required only for the transcriptional activity of HIF-1 under hypoxic condition, revealing a differential role of AMPK in these two stimuli. Furthermore, our data show that inhibition of AMPK enhances HIF-1alpha ubiquitination under ROS condition. Finally, we show that the regulation of HIF-1 by AMPK in response to ROS is under the control of c-Jun N-terminal kinase and Janus kinase 2 pathways. Collectively, our findings identify AMPK as a key determinant of HIF-1 functions in response to ROS and its possible role in the sophisticated HIF-1 regulatory mechanisms.

TRAF2-binding BIR1 domain of c-IAP2/MALT1 fusion protein is essential for activation of NF-kappaB.

PubMed

Garrison, J B; Samuel, T; Reed, J C

2009-04-02

Marginal zone mucosa-associated lymphoid tissue (MALT) B-cell lymphoma is the most common extranodal non-Hodgkin lymphoma. The t(11;18)(q21;q21) translocation occurs frequently in MALT lymphomas and creates a chimeric NF-kappaB-activating protein containing the baculoviral IAP repeat (BIR) domains of c-IAP2 (inhibitor of apoptosis protein 2) fused with portions of the MALT1 protein. The BIR1 domain of c-IAP2 interacts directly with TRAF2 (TNFalpha-receptor-associated factor-2), but its role in NF-kappaB activation is still unclear. Here, we investigated the role of TRAF2 in c-IAP2/MALT1-induced NF-kappaB activation. We show the BIR1 domain of c-IAP2 is essential for NF-kappaB activation, whereas BIR2 and BIR3 domains are not. Studies of c-IAP2/MALT1 BIR1 mutant (E47A/R48A) that fails to activate NF-kappaB showed loss of TRAF2 binding, but retention of TRAF6 binding, suggesting that interaction of c-IAP2/MALT1 with TRAF6 is insufficient for NF-kappaB induction. In addition, a dominant-negative TRAF2 mutant or downregulation of TRAF2 achieved by small interfering RNA inhibited NF-kappaB activation by c-IAP2/MALT1 showing that TRAF2 is indispensable. Comparisons of the bioactivity of intact c-IAP2/MALT1 oncoprotein and BIR1 E47A/R48A c-IAP2/MALT1 mutant that cannot bind TRAF2 in a lymphoid cell line provided evidence that TRAF2 interaction is critical for c-IAP2/MALT1-mediated increases in the NF-kappaB activity, increased expression of endogenous NF-kappaB target genes (c-FLIP, TRAF1), and resistance to apoptosis.

Differential activation of G-proteins by mu-opioid receptor agonists.

PubMed

Saidak, Zuzana; Blake-Palmer, Katherine; Hay, Debbie L; Northup, John K; Glass, Michelle

2006-03-01

We investigated the ability of the activated mu-opioid receptor (MOR) to differentiate between myristoylated G(alphai1) and G(alphaoA) type G(alpha) proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each G(alpha) protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The G(alpha) subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified G(alpha) protein by CB(1) cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[(35)S]GTP(gamma)S exchange was then compared for G(alphai1) and G(alphaoA). Activation of MOR by DAMGO produced a high-affinity saturable interaction for G(alphaoA) (K(m)=20+/-1 nM) but a low-affinity interaction with G(alphai1) (K(m)=116+/-12 nM). DAMGO, met-enkephalin and leucine-enkephalin displayed maximal G(alpha) activation among the agonists evaluated. Endomorphins 1 and 2, methadone and beta-endorphin activated both G(alpha) to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between G(alphai1) and G(alphaoA). Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two G(alpha). Differences in maximal activity and potency, for G(alphai1) versus G(alphaoA), are both indicative of agonist selective activation of G-proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects.

Differential activation of G-proteins by μ-opioid receptor agonists

PubMed Central

Saidak, Zuzana; Blake-Palmer, Katherine; Hay, Debbie L; Northup, John K; Glass, Michelle

2006-01-01

We investigated the ability of the activated μ-opioid receptor (MOR) to differentiate between myristoylated Gαi1 and GαoA type Gα proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each Gα protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The Gα subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified Gα protein by CB1 cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[35S]GTPγS exchange was then compared for Gαi1 and GαoA. Activation of MOR by DAMGO produced a high-affinity saturable interaction for GαoA (Km=20±1 nM) but a low-affinity interaction with Gαi1 (Km=116±12 nM). DAMGO, met-enkephalin and leucine-enkephalin displayed maximal Gα activation among the agonists evaluated. Endomorphins 1 and 2, methadone and β-endorphin activated both Gα to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between Gαi1 and GαoA. Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two Gα. Differences in maximal activity and potency, for Gαi1 versus GαoA, are both indicative of agonist selective activation of G-proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects. PMID:16415903

Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

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

Mochimaru, Yuta; Azuma, Morio; Oshima, Natsuki

2015-02-20

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1more » overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.« less

PDK1-dependent activation of atypical PKC leads to degradation of the p21 tumour modifier protein

PubMed Central

Scott, Mary T.; Ingram, Angela; Ball, Kathryn L.

2002-01-01

p21WAF1/CIP1 contributes to positive and negative growth control on multiple levels. We previously mapped phosphorylation sites within the C-terminal domain of p21 that regulate proliferating cell nucear antigen binding. In the current study, a kinase has been fractionated from mammalian cells that stoichiometrically phosphorylates p21 at the Ser146 site, and the enzyme has been identified as an insulin-responsive atypical protein kinase C (aPKC). Expression of PKCζ or activation of the endogenous kinase by 3-phosphoinositide dependent protein kinase-1 (PDK1) decreased the half-life of p21. Conversely, dnPKCζ or dnPDK1 increased p21 protein half-life, and a PDK1-dependent increase in the rate of p21 degradation was mediated by aPKC. Insulin stimulation gave a biphasic response with a rapid transient decrease in p21 protein levels during the initial signalling phase that was dependent on phosphatidylinositol 3- kinase, PKC and proteasome activity. Thus, aPKC provides a physiological signal for the degradation of p21. The rapid degradation of p21 protein during the signalling phase of insulin stimulation identifies a novel link between energy metabolism and a key modulator of cell cycle progression. PMID:12485998

The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

PubMed

Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

2002-09-01

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Broncho Vaxom (OM-85) modulates rhinovirus docking proteins on human airway epithelial cells via Erk1/2 mitogen activated protein kinase and cAMP

PubMed Central

Pasquali, Christian; Stolz, Daiana; Tamm, Michael

2017-01-01

Background Bronchial epithelial cells (BEC) are primary target for Rhinovirus infection through attaching to cell membrane proteins. OM-85, a bacterial extract, improves recovery of asthma and COPD patients after viral infections, but only part of the mechanism was addressed, by focusing on defined immune cells. Objective We therefore determined the effect of OM-85 on isolated primary human BEC of controls (n = 8), asthma patients (n = 10) and COPD patients (n = 9). Methods BEC were treated with OM-85 alone (24 hours) or infected with Rhinovirus. BEC survival was monitored by manual cell counting and Rhinovirus replication by lytic activity. Immuno-blotting and ELISA were used to determine the expression of Rhinovirus interacting proteins: intracellular adhesion molecule (ICAM), major histocompatibility complex class II (MHC-2), complement component C1q receptor (C1q-R), inducible T-Cell co-stimulator (ICOS), its ligand ICOSL, and myeloid differentiation primary response gene 88 (Myd88); as well as for signal transducers Erk1/2, p38, JNK mitogen activated protein kinases MAPK), and cAMP. Results OM-85 significantly reduced Rhinovirus-induced BEC death and virus replication. OM-85 significantly increased the expression of virus interacting proteins C1q-R and β-defensin in all 3 probes and groups, which was prevented by either Erk1/2 MAPK or cAMP inhibition. In addition, OM-85 significantly reduced Rhinovirus induced expression of ICAM1 involving p38 MAPK. In BEC OM-85 had no significant effect on the expression of ICOS, ICOSL and MHC-2 membrane proteins nor on the adaptor protein MyD88. Conclusion The OM-85-induced increased of C1q-R and β-defensin, both important for antigen presentation and phagocytosis, supports its activity in host cell’s defence against Rhinovirus infection. PMID:29182620

Broncho Vaxom (OM-85) modulates rhinovirus docking proteins on human airway epithelial cells via Erk1/2 mitogen activated protein kinase and cAMP.

PubMed

Roth, Michael; Pasquali, Christian; Stolz, Daiana; Tamm, Michael

2017-01-01

Bronchial epithelial cells (BEC) are primary target for Rhinovirus infection through attaching to cell membrane proteins. OM-85, a bacterial extract, improves recovery of asthma and COPD patients after viral infections, but only part of the mechanism was addressed, by focusing on defined immune cells. We therefore determined the effect of OM-85 on isolated primary human BEC of controls (n = 8), asthma patients (n = 10) and COPD patients (n = 9). BEC were treated with OM-85 alone (24 hours) or infected with Rhinovirus. BEC survival was monitored by manual cell counting and Rhinovirus replication by lytic activity. Immuno-blotting and ELISA were used to determine the expression of Rhinovirus interacting proteins: intracellular adhesion molecule (ICAM), major histocompatibility complex class II (MHC-2), complement component C1q receptor (C1q-R), inducible T-Cell co-stimulator (ICOS), its ligand ICOSL, and myeloid differentiation primary response gene 88 (Myd88); as well as for signal transducers Erk1/2, p38, JNK mitogen activated protein kinases MAPK), and cAMP. OM-85 significantly reduced Rhinovirus-induced BEC death and virus replication. OM-85 significantly increased the expression of virus interacting proteins C1q-R and β-defensin in all 3 probes and groups, which was prevented by either Erk1/2 MAPK or cAMP inhibition. In addition, OM-85 significantly reduced Rhinovirus induced expression of ICAM1 involving p38 MAPK. In BEC OM-85 had no significant effect on the expression of ICOS, ICOSL and MHC-2 membrane proteins nor on the adaptor protein MyD88. The OM-85-induced increased of C1q-R and β-defensin, both important for antigen presentation and phagocytosis, supports its activity in host cell's defence against Rhinovirus infection.

Nedd4 Family Interacting Protein 1 (Ndfip1) Is Required for Ubiquitination and Nuclear Trafficking of BRCA1-associated ATM Activator 1 (BRAT1) during the DNA Damage Response*

PubMed Central

Low, Ley-Hian; Chow, Yuh-Lit; Li, Yijia; Goh, Choo-Peng; Putz, Ulrich; Silke, John; Ouchi, Toru; Howitt, Jason; Tan, Seong-Seng

2015-01-01

During injury, cells are vulnerable to apoptosis from a variety of stress conditions including DNA damage causing double-stranded breaks. Without repair, these breaks lead to aberrations in DNA replication and transcription, leading to apoptosis. A major response to DNA damage is provided by the protein kinase ATM (ataxia telangiectasia mutated) that is capable of commanding a plethora of signaling networks for DNA repair, cell cycle arrest, and even apoptosis. A key element in the DNA damage response is the mobilization of activating proteins into the cell nucleus to repair damaged DNA. BRAT1 is one of these proteins, and it functions as an activator of ATM by maintaining its phosphorylated status while also keeping other phosphatases at bay. However, it is unknown how BRAT1 is trafficked into the cell nucleus to maintain ATM phosphorylation. Here we demonstrate that Ndfip1-mediated ubiquitination of BRAT1 leads to BRAT1 trafficking into the cell nucleus. Without Ndfip1, BRAT1 failed to translocate to the nucleus. Under genotoxic stress, cells showed increased expression of both Ndfip1 and phosphorylated ATM. Following brain injury, neurons show increased expression of Ndfip1 and nuclear translocation of BRAT1. These results point to Ndfip1 as a sensor protein during cell injury and Ndfip1 up-regulation as a cue for BRAT1 ubiquitination by Nedd4 E3 ligases, followed by nuclear translocation of BRAT1. PMID:25631046

Fatty Acid-binding Proteins 1 and 2 Differentially Modulate the Activation of Peroxisome Proliferator-activated Receptor α in a Ligand-selective Manner*

PubMed Central

Hughes, Maria L. R.; Liu, Bonan; Halls, Michelle L.; Wagstaff, Kylie M.; Patil, Rahul; Velkov, Tony; Jans, David A.; Bunnett, Nigel W.; Scanlon, Martin J.; Porter, Christopher J. H.

2015-01-01

Nuclear hormone receptors (NHRs) regulate the expression of proteins that control aspects of reproduction, development and metabolism, and are major therapeutic targets. However, NHRs are ubiquitous and participate in multiple physiological processes. Drugs that act at NHRs are therefore commonly restricted by toxicity, often at nontarget organs. For endogenous NHR ligands, intracellular lipid-binding proteins, including the fatty acid-binding proteins (FABPs), can chaperone ligands to the nucleus and promote NHR activation. Drugs also bind FABPs, raising the possibility that FABPs similarly regulate drug activity at the NHRs. Here, we investigate the ability of FABP1 and FABP2 (intracellular lipid-binding proteins that are highly expressed in tissues involved in lipid metabolism, including the liver and intestine) to influence drug-mediated activation of the lipid regulator peroxisome proliferator-activated receptor (PPAR) α. We show by quantitative fluorescence imaging and gene reporter assays that drug binding to FABP1 and FABP2 promotes nuclear localization and PPARα activation in a drug- and FABP-dependent manner. We further show that nuclear accumulation of FABP1 and FABP2 is dependent on the presence of PPARα. Nuclear accumulation of FABP on drug binding is driven largely by reduced nuclear egress rather than an increased rate of nuclear entry. Importin binding assays indicate that nuclear access occurs via an importin-independent mechanism. Together, the data suggest that specific drug-FABP complexes can interact with PPARα to effect nuclear accumulation of FABP and NHR activation. Because FABPs are expressed in a regionally selective manner, this may provide a means to tailor the patterns of NHR drug activation in a tissue-specific manner. PMID:25847235

Carboxy-terminal truncation activates glp-1 protein to specify vulval fates in Caenorhabditis elegans.

PubMed

Mango, S E; Maine, E M; Kimble, J

1991-08-29

The glp-1 and lin-12 genes encode homologous transmembrane proteins that may act as receptors for cell interactions during development. The glp-1 product is required for induction of germ-line proliferation and for embryogenesis. By contrast, lin-12 mediates somatic cell interactions, including those between the precursor cells that form the vulval hypodermis (VPCs). Here we analyse an unusual allele of glp-1, glp-1(q35), which displays a semidominant multivulva phenotype (Muv), as well as the typical recessive, loss-of-function Glp phenotypes (sterility and embryonic lethality). We find that the effects of glp-1(q35) on VPC development mimic those of dominant lin-12 mutations, even in the absence of lin-12 activity. The glp-1(q35) gene bears a nonsense mutation predicted to eliminate the 122 C-terminal amino acids, including a ProGluSerThr (PEST) sequence thought to destabilize proteins. We suggest that the carboxy terminus bears a negative regulatory domain which normally inactivates glp-1 in the VPCs. We propose that inappropriate glp-1(q35) activity can substitute for lin-12 to determine vulval fate, perhaps by driving the VPCs to proliferate.

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

PubMed

Tong, X; Kono, T; Evans-Molina, C

2015-06-18

The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF1

PubMed Central

Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

2008-01-01

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized β-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

Activation of HIV Transcription by the Viral Tat Protein Requires a Demethylation Step Mediated by Lysine-specific Demethylase 1 (LSD1/KDM1)

PubMed Central

Sakane, Naoki; Kwon, Hye-Sook; Pagans, Sara; Kaehlcke, Katrin; Mizusawa, Yasuhiro; Kamada, Masafumi; Lassen, Kara G.; Chan, Jonathan; Greene, Warner C.; Schnoelzer, Martina; Ott, Melanie

2011-01-01

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear. We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells. Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation

The radical induced cell death protein 1 (RCD1) supports transcriptional activation of genes for chloroplast antioxidant enzymes

PubMed Central

Hiltscher, Heiko; Rudnik, Radoslaw; Shaikhali, Jehad; Heiber, Isabelle; Mellenthin, Marina; Meirelles Duarte, Iuri; Schuster, Günter; Kahmann, Uwe; Baier, Margarete

2014-01-01

The rimb1 (redox imbalanced 1) mutation was mapped to the RCD1 locus (radical-induced cell death 1; At1g32230) demonstrating that a major factor involved in redox-regulation genes for chloroplast antioxidant enzymes and protection against photooxidative stress, RIMB1, is identical to the regulator of disease response reactions and cell death, RCD1. Discovering this link let to our investigation of its regulatory mechanism. We show in yeast that RCD1 can physically interact with the transcription factor Rap2.4a which provides redox-sensitivity to nuclear expression of genes for chloroplast antioxidant enzymes. In the rimb1 (rcd1-6) mutant, a single nucleotide exchange results in a truncated RCD1 protein lacking the transcription factor binding site. Protein-protein interaction between full-length RCD1 and Rap2.4a is supported by H2O2, but not sensitive to the antioxidants dithiotreitol and ascorbate. In combination with transcript abundance analysis in Arabidopsis, it is concluded that RCD1 stabilizes the Rap2.4-dependent redox-regulation of the genes encoding chloroplast antioxidant enzymes in a widely redox-independent manner. Over the years, rcd1-mutant alleles have been described to develop symptoms like chlorosis, lesions along the leaf rims and in the mesophyll and (secondary) induction of extra- and intra-plastidic antioxidant defense mechanisms. All these rcd1 mutant characteristics were observed in rcd1-6 to succeed low activation of the chloroplast antioxidant system and glutathione biosynthesis. We conclude that RCD1 protects plant cells from running into reactive oxygen species (ROS)-triggered programs, such as cell death and activation of pathogen-responsive genes (PR genes) and extra-plastidic antioxidant enzymes, by supporting the induction of the chloroplast antioxidant system. PMID:25295044

Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

PubMed

Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

2014-12-01

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p<0.01) compared to rhBMP-2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Activation of AMP-activated Protein Kinase by Metformin Induces Protein Acetylation in Prostate and Ovarian Cancer Cells*

PubMed Central

Galdieri, Luciano; Gatla, Himavanth; Vancurova, Ivana; Vancura, Ales

2016-01-01

AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects. PMID:27733682

Defective interplay between mTORC1 activity and endoplasmic reticulum stress-unfolded protein response in uremic vascular calcification.

PubMed

Panda, Dibyendu K; Bai, Xiuying; Sabbagh, Yves; Zhang, Yan; Zaun, Hans-Christian; Karellis, Angeliki; Koromilas, Antonis E; Lipman, Mark L; Karaplis, Andrew C

2018-06-01

Vascular calcification increases the risk of cardiovascular disease and death in patients with chronic kidney disease (CKD). Increased activity of mammalian target of rapamycin complex 1 (mTORC1) and endoplasmic reticulum (ER) stress-unfolded protein response (UPR) are independently reported to partake in the pathogenesis of vascular calcification in CKD. However, the association between mTORC1 activity and ER stress-UPR remains unknown. We report here that components of the uremic state [activation of the receptor for advanced glycation end products (RAGE) and hyperphosphatemia] potentiate vascular smooth muscle cell (VSMC) calcification by inducing persistent and exaggerated activity of mTORC1. This gives rise to prolonged and excessive ER stress-UPR as well as attenuated levels of sestrin 1 ( Sesn1) and Sesn3 feeding back to inhibit mTORC1 activity. Activating transcription factor 4 arising from the UPR mediates cell death via expression of CCAAT/enhancer-binding protein (c/EBP) homologous protein (CHOP), impairs the generation of pyrophosphate, a potent inhibitor of mineralization, and potentiates VSMC transdifferentiation to the osteochondrocytic phenotype. Short-term treatment of CKD mice with rapamycin, an inhibitor of mTORC1, or tauroursodeoxycholic acid, a bile acid that restores ER homeostasis, normalized mTORC1 activity, molecular markers of UPR, and calcium content of aortas. Collectively, these data highlight that increased and/or protracted mTORC1 activity arising from the uremic state leads to dysregulated ER stress-UPR and VSMC calcification. Manipulation of the mTORC1-ER stress-UPR pathway opens up new therapeutic strategies for the prevention and treatment of vascular calcification in CKD.

A peptide fragment of ependymin neurotrophic factor uses protein kinase C and the mitogen-activated protein kinase pathway to activate c-Jun N-terminal kinase and a functional AP-1 containing c-Jun and c-Fos proteins in mouse NB2a cells.

PubMed

Adams, David S; Hasson, Brendan; Boyer-Boiteau, Anne; El-Khishin, Adam; Shashoua, Victor E

2003-05-01

Ependymin (EPN) is a goldfish brain neurotrophic factor previously shown to function in a variety of cellular events related to long-term memory formation and neuronal regeneration. CMX-8933, an 8-amino-acid synthetic peptide fragment of EPN, was designed for aiding an investigation of the biological properties of this glycoprotein. We reported from previous studies that treatment of mouse neuroblastoma (NB2a) cultures with CMX-8933 promotes activation of transcription factor AP-1, a characteristic previously associated with the following full-length neurotrophic factors: nerve growth factor, neurotropin-3, and brain-derived neurotrophic factor. The CMX-8933-activated AP-1 specifically bound an AP-1 consensus probe and appeared to contain c-Jun and c-Fos protein components in antibody supershift experiments. Because AP-1 influences a variety of positive and negative cellular processes, determined in part by its exact protein composition and mechanism of activation, we extended these initial AP-1 observations in the current study to confirm the identity of the CMX-8933-activated c-Jun and c-Fos components. CMX-8933 increases the enzymatic activity of c-Jun N-terminal kinase (JNK), increases the phosphorylation of JNK and c-Jun proteins, and increases the cellular titers of c-Jun and c-Fos mRNAs. Furthermore, the AP-1 activated by CMX-8933 is functional, insofar as it transactivates both synthetic and natural AP-1-dependent reporter plasmids. Inhibition studies indicate that activation of the 8933-induced AP-1 occurs via the mitogen-activated protein kinase pathway. These data are in agreement with the recently proposed model for the conversion of short- to long-term synaptic plasticity and memory, in which a JNK-activated transcription factor AP-1, containing c-Jun and c-Fos components, functions at the top of a hierarchy of transcription factors known to regulate long-term neural plasticity. Copyright 2003 Wiley-Liss, Inc.

Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

PubMed Central

Bandara, G; Mueller, G M; Galea-Lauri, J; Tindal, M H; Georgescu, H I; Suchanek, M K; Hung, G L; Glorioso, J C; Robbins, P D; Evans, C H

1993-01-01

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy. Images Fig. 1 Fig. 2 PMID:8248169

AMP-activated protein kinase protects against necroptosis via regulation of Keap1-PGAM5 complex.

PubMed

Wang, Yi-Shu; Yu, Peng; Wang, Yong; Zhang, Jing; Hang, Wei; Yin, Zhi-Xian; Liu, Gang; Chen, Jianfeng; Werle, Kaitlin D; Quan, Cheng-Shi; Gao, Hang; Zeng, Qinghua; Cui, Rutao; Liang, Jiyong; Ding, Qiang; Li, Yu-Lin; Xu, Zhi-Xiang

2018-05-15

The AMP-activated protein kinase (AMPK) plays critical roles in growth regulation and metabolism reprogramming. AMPK activation protects cells against apoptosis from injury in different cell and animal models. However, its function in necroptosis remains largely unclear. In the current study, we demonstrated that AMPK was activated upon necroptosis induction and protected mouse embryonic fibroblasts (MEFs) and cardiomyocytes from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and reactive oxygen species (ROS) induced necroptosis. Activation of AMPK with chemicals A-769662, 2-deoxyglucose (2-DG), and metformin or constitutively active (CA) AMPK markedly decreased necroptosis and cytotoxicity induced by MNNG. In contrast, AMPK inhibitor compound C, dominant negative (DN) AMPK, as well as AMPK shRNAs increased necroptosis and cytotoxicity induced by MNNG. We further showed that AMPK physically associated with a protein complex containing PGAM5 and Keap1 whereby facilitating Keap1-mediated PGAM5 ubiquitination upon necroptosis induction. The AMPK agonist metformin ameliorated myocardial ischemia and reperfusion (IR) injury and reduced necroptosis through down-regulating the expression of PGAM5 in the Langendorff-perfused rat hearts. Activation of AMPK protects against necroptosis via promoting Keap1-mediated PGAM5 degradation. Metformin may act as a valuable agent for the protection of myocardial ischemia and reperfusion injury by activating AMPK and reducing necroptosis. Copyright © 2018. Published by Elsevier B.V.

Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

PubMed

Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

2009-02-01

Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein.

PubMed Central

van Ooij, C; Snyder, R C; Paeper, B W; Duester, G

1992-01-01

The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver. Images PMID:1620113

Spirulina non-protein components induce BDNF gene transcription via HO-1 activity in C6 glioma cells.

PubMed

Morita, Kyoji; Itoh, Mari; Nishibori, Naoyoshi; Her, Song; Lee, Mi-Sook

2015-01-01

Blue-green algae are known to contain biologically active proteins and non-protein substances and considered as useful materials for manufacturing the nutritional supplements. Particularly, Spirulina has been reported to contain a variety of antioxidants, such as flavonoids, carotenoids, and vitamin C, thereby exerting their protective effects against the oxidative damage to the cells. In addition to their antioxidant actions, polyphenolic compounds have been speculated to cause the protection of neuronal cells and the recovery of neurologic function in the brain through the production of brain-derived neurotrophic factor (BDNF) in glial cells. Then, the protein-deprived extract was prepared by removing the most part of protein components from aqueous extract of Spirulina platensis, and the effect of this extract on BDNF gene transcription was examined in C6 glioma cells. Consequently, the protein-deprived extract was shown to cause the elevation of BDNF mRNA levels following the expression of heme oxygenase-1 (HO-1) in the glioma cells. Therefore, the non-protein components of S. platensis are considered to stimulate BDNF gene transcription through the HO-1 induction in glial cells, thus proposing a potential ability of the algae to indirectly modulate the brain function through the glial cell activity.

Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis.

PubMed

Serce, Nuran Bektas; Boesl, Andreas; Klaman, Irina; von Serényi, Sonja; Noetzel, Erik; Press, Michael F; Dimmler, Arno; Hartmann, Arndt; Sehouli, Jalid; Knuechel, Ruth; Beckmann, Matthias W; Fasching, Peter A; Dahl, Edgar

2012-12-13

Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the

Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis

PubMed Central

2012-01-01

Background Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Methods Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. Results SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. Conclusions The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour

Fatty Acid-binding Proteins 1 and 2 Differentially Modulate the Activation of Peroxisome Proliferator-activated Receptor α in a Ligand-selective Manner.

PubMed

Hughes, Maria L R; Liu, Bonan; Halls, Michelle L; Wagstaff, Kylie M; Patil, Rahul; Velkov, Tony; Jans, David A; Bunnett, Nigel W; Scanlon, Martin J; Porter, Christopher J H

2015-05-29

Nuclear hormone receptors (NHRs) regulate the expression of proteins that control aspects of reproduction, development and metabolism, and are major therapeutic targets. However, NHRs are ubiquitous and participate in multiple physiological processes. Drugs that act at NHRs are therefore commonly restricted by toxicity, often at nontarget organs. For endogenous NHR ligands, intracellular lipid-binding proteins, including the fatty acid-binding proteins (FABPs), can chaperone ligands to the nucleus and promote NHR activation. Drugs also bind FABPs, raising the possibility that FABPs similarly regulate drug activity at the NHRs. Here, we investigate the ability of FABP1 and FABP2 (intracellular lipid-binding proteins that are highly expressed in tissues involved in lipid metabolism, including the liver and intestine) to influence drug-mediated activation of the lipid regulator peroxisome proliferator-activated receptor (PPAR) α. We show by quantitative fluorescence imaging and gene reporter assays that drug binding to FABP1 and FABP2 promotes nuclear localization and PPARα activation in a drug- and FABP-dependent manner. We further show that nuclear accumulation of FABP1 and FABP2 is dependent on the presence of PPARα. Nuclear accumulation of FABP on drug binding is driven largely by reduced nuclear egress rather than an increased rate of nuclear entry. Importin binding assays indicate that nuclear access occurs via an importin-independent mechanism. Together, the data suggest that specific drug-FABP complexes can interact with PPARα to effect nuclear accumulation of FABP and NHR activation. Because FABPs are expressed in a regionally selective manner, this may provide a means to tailor the patterns of NHR drug activation in a tissue-specific manner. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Assessment of the potential pathogenicity of missense mutations identified in the GTPase-activating protein (GAP)-related domain of the neurofibromatosis type-1 (NF1) gene.

PubMed

Thomas, Laura; Richards, Mark; Mort, Matthew; Dunlop, Elaine; Cooper, David N; Upadhyaya, Meena

2012-12-01

Neurofibromatosis type-1 (NF1) is caused by constitutional mutations of the NF1 tumor-suppressor gene. Although ∼85% of inherited NF1 microlesions constitute truncating mutations, the remaining ∼15% are missense mutations whose pathological relevance is often unclear. The GTPase-activating protein-related domain (GRD) of the NF1-encoded protein, neurofibromin, serves to define its major function as a negative regulator of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. We have established a functional assay to assess the potential pathogenicity of 15 constitutional nonsynonymous NF1 missense mutations (11 novel and 4 previously reported but not functionally characterized) identified in the NF1-GRD (p.R1204G, p.R1204W, p.R1276Q, p.L1301R, p.I1307V, p.T1324N, p.E1327G, p.Q1336R, p.E1356G, p.R1391G, p.V1398D, p.K1409E, p.P1412R, p.K1436Q, p.S1463F). Individual mutations were introduced into an NF1-GRD expression vector and activated Ras was assayed by an enzyme-linked immunosorbent assay (ELISA). Ten NF1-GRD variants were deemed to be potentially pathogenic by virtue of significantly elevated levels of activated GTP-bound Ras in comparison to wild-type NF1 protein. The remaining five NF1-GRD variants were deemed less likely to be of pathological significance as they exhibited similar levels of activated Ras to the wild-type protein. These conclusions received broad support from both bioinformatic analysis and molecular modeling and serve to improve our understanding of NF1-GRD structure and function. © 2012 Wiley Periodicals, Inc.

Dimethyl Sulfoxide Promotes the Multiple Functions of the Tumor Suppressor HLJ1 through Activator Protein-1 Activation in NSCLC Cells

PubMed Central

Wang, Chi-Chung; Lin, Sheng-Yi; Lai, Yi-Hua; Liu, Ya-Jung; Hsu, Yuan-Lin; Chen, Jeremy J. W.

2012-01-01

Background Dimethyl sulfoxide (DMSO) is an amphipathic molecule that displays a diversity of antitumor activities. Previous studies have demonstrated that DMSO can modulate AP-1 activity and lead to cell cycle arrest at the G1 phase. HLJ1 is a newly identified tumor and invasion suppressor that inhibits tumorigenesis and cancer metastasis. Its transcriptional activity is regulated by the transcription factor AP-1. However, the effects of DMSO on HLJ1 are still unknown. In the present study, we investigate the antitumor effects of DMSO through HLJ1 induction and demonstrate the mechanisms involved. Methods and Findings Low-HLJ1-expressing highly invasive CL1–5 lung adenocarcinoma cells were treated with various concentrations of DMSO. We found that DMSO can significantly inhibit cancer cell invasion, migration, proliferation, and colony formation capabilities through upregulation of HLJ1 in a concentration-dependent manner, whereas ethanol has no effect. In addition, the HLJ1 promoter and enhancer reporter assay revealed that DMSO transcriptionally upregulates HLJ1 expression through an AP-1 site within the HLJ1 enhancer. The AP-1 subfamily members JunD and JunB were significantly upregulated by DMSO in a concentration-dependent manner. Furthermore, pretreatment with DMSO led to a significant increase in the percentage of UV-induced apoptotic cells. Conclusions Our results suggest that DMSO may be an important stimulator of the tumor suppressor protein HLJ1 through AP-1 activation in highly invasive lung adenocarcinoma cells. Targeted induction of HLJ1 represents a promising approach for cancer therapy, which also implied that DMSO may serve as a potential lead compound or coordinated ligand for the development of novel anticancer drugs. PMID:22529897

Pattern Recognition Protein Binds to Lipopolysaccharide and β-1,3-Glucan and Activates Shrimp Prophenoloxidase System*

PubMed Central

Amparyup, Piti; Sutthangkul, Jantiwan; Charoensapsri, Walaiporn; Tassanakajon, Anchalee

2012-01-01

The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP). However, shrimp LGBPs that are involved in the proPO system have yet to be clarified. Here, we focus on characterizing the role of a Penaeus monodon LGBP (PmLGBP) in the proPO system. We found that PmLGBP transcripts are expressed primarily in the hemocytes and are increased at 24 h after pathogenic bacterium Vibrio harveyi challenge. The binding studies carried out using ELISA indicated that recombinant (r)PmLGBP binds to β-1,3-glucan and LPS with a dissociation constant of 6.86 × 10−7 m and 3.55 × 10−7 m, respectively. Furthermore, we found that rPmLGBP could enhance the phenoloxidase (PO) activity of hemocyte suspensions in the presence of LPS or β-1,3-glucan. Using dsRNA interference-mediated gene silencing assay, we further demonstrated that knockdown of PmLGBP in shrimp in vivo significantly decreased the PmLGBP transcript level but had no effect on the expression of the other immune genes tested, including shrimp antimicrobial peptides (AMPs). However, suppression of proPO expression down-regulated PmLGBP, proPO-activating enzyme (PmPPAE2), and AMPs (penaeidin and crustin). Such PmLGBP down-regulated shrimp showed significantly decreased total PO activity. We conclude that PmLGBP functions as a pattern recognition protein for LPS and β-1,3-glucan in the shrimp proPO activating system. PMID:22235126

Dieckol, a phlorotannin isolated from a brown seaweed, Ecklonia cava, inhibits adipogenesis through AMP-activated protein kinase (AMPK) activation in 3T3-L1 preadipocytes.

PubMed

Ko, Seok-Chun; Lee, Myoungsook; Lee, Ji-Hyeok; Lee, Seung-Hong; Lim, Yunsook; Jeon, You-Jin

2013-11-01

In this study, we assessed the potential inhibitory effect of 5 species of brown seaweeds on adipogenesis the differentiation of 3T3-L1 preadipocytes into mature adipocytes by measuring Oil-Red O staining. The Ecklonia cava extract tested herein evidenced profound adipogenesis inhibitory effect, compared to that exhibited by the other four brown seaweed extracts. Thus, E. cava was selected for isolation of active compounds and finally the three polyphenol compounds of phlorotannins were obtained and their inhibitory effect on adipogenesis was observed. Among the phlorotannins, dieckol exhibited greatest potential adipogenesis inhibition and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding proteins (C/EBPα), sterol regulatory element-binding protein 1 (SREBP1) and fatty acid binding protein 4 (FABP4) in a dose-dependent manner. The specific mechanism mediating the effects of dieckol was confirmed by AMP-activated protein kinase (AMPK) activation. These results demonstrate inhibitory effect of dieckol compound on adipogenesis through the activation of the AMPK signal pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

Mitogen-activated protein kinase phosphatase (MKP)-1 in immunology, physiology, and disease.

PubMed

Wancket, Lyn M; Frazier, W Joshua; Liu, Yusen

2012-02-13

Mitogen-activated protein kinases (MAPKs) are key regulators of cellular physiology and immune responses, and abnormalities in MAPKs are implicated in many diseases. MAPKs are activated by MAPK kinases through phosphorylation of the threonine and tyrosine residues in the conserved Thr-Xaa-Tyr domain, where Xaa represents amino acid residues characteristic of distinct MAPK subfamilies. Since MAPKs play a crucial role in a variety of cellular processes, a delicate regulatory network has evolved to control their activities. Over the past two decades, a group of dual specificity MAPK phosphatases (MKPs) has been identified that deactivates MAPKs. Since MAPKs can enhance MKP activities, MKPs are considered as an important feedback control mechanism that limits the MAPK cascades. This review outlines the role of MKP-1, a prototypical MKP family member, in physiology and disease. We will first discuss the basic biochemistry and regulation of MKP-1. Next, we will present the current consensus on the immunological and physiological functions of MKP-1 in infectious, inflammatory, metabolic, and nervous system diseases as revealed by studies using animal models. We will also discuss the emerging evidence implicating MKP-1 in human disorders. Finally, we will conclude with a discussion of the potential for pharmacomodulation of MKP-1 expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1

PubMed Central

Beaulieu, Lea M.; Church, Frank C.

2014-01-01

Activated protein C (APC) is a serine protease that regulates thrombin (IIa) production through inactivation of blood coagulation factors Va and VIIIa. APC also has non-hemostatic functions related to inflammation, proliferation, and apoptosis through various mechanisms. Using two breast cancer cell lines, MDA-MB-231 and MDA-MB-435, we investigated the role of APC in cell chemotaxis and invasion. Treatment of cells with increasing APC concentrations (1–50 μg/ml) increased invasion and chemotaxis in a concentration-dependent manner. Only the active form of APC increased invasion and chemotaxis of the MDA-MB-231 cells when compared to 3 inactive APC derivatives. Using a modified “checkerboard” analysis, APC was shown to only affect migration when plated with the cells; therefore, APC is not a chemoattractant. Blocking antibodies to endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) attenuated the effects of APC on chemotaxis in the MDA-MB-231 cells. Finally, treatment of the MDA-MB-231 cells with the proliferation inhibitor, Na butyrate, showed that APC did not increase migration by increasing cell number. Therefore, APC increases invasion and chemotaxis of cells by binding to the cell surface and activating specific signaling pathways through EPCR and PAR-1. PMID:17254565

1-Aminocyclopropane-1-carboxylic acid oxidase reaction mechanism and putative post-translational activities of the ACCO protein

PubMed Central

Dilley, David R.; Wang, Zhenyong; Kadirjan-Kalbach, Deena K.; Ververidis, Fillipos; Beaudry, Randolph; Padmanabhan, Kallaithe

2013-01-01

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyses the final step in ethylene biosynthesis converting ACC to ethylene, cyanide, CO2, dehydroascorbate and water with inputs of Fe(II), ascorbate, bicarbonate (as activators) and oxygen. Cyanide activates ACCO. A ‘nest’ comprising several positively charged amino acid residues from the C-terminal α-helix 11 along with Lys158 and Arg299 are proposed as binding sites for ascorbate and bicarbonate to coordinately activate the ACCO reaction. The binding sites for ACC, bicarbonate and ascorbic acid for Malus domestica ACCO1 include Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. Glutamate 297, Phe300 and Glu301 in α-helix 11 are also important for the ACCO reaction. Our proposed reaction pathway incorporates cyanide as an ACCO/Fe(II) ligand after reaction turnover. The cyanide ligand is likely displaced upon binding of ACC and ascorbate to provide a binding site for oxygen. We propose that ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction. ACC oxidase has significant homology with Lycopersicon esculentum cysteine protease LeCp, which functions as a protease and as a regulator of 1-aminocyclopropane-1-carboxylic acid synthase (Acs2) gene expression. ACC oxidase may play a similar role in signal transduction after post-translational processing. ACC oxidase becomes inactivated by fragmentation and apparently has intrinsic protease and transpeptidase activity. ACC oxidase contains several amino acid sequence motifs for putative protein–protein interactions, phosphokinases and cysteine protease. ACC oxidase is subject to autophosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner. PMID:24244837

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil

USGS Publications Warehouse

Alexander, Courtney R.; Hooper, Michael J.; Cacela, Dave; Smelker, Kim D.; Calvin, Caleshia S.; Dean, Karen M.; Bursian, Steve J.; Cunningham, Fred L.; Hanson-Dorr, Katie C.; Horak, Katherine E.; Isanhart, John P.; Link, Jane E.; Shriner, Susan A.; Godard-Codding, Céline A.J.

2017-01-01

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20 ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5 ml/kg bw oil or 10 ml/kg bw oil, 21 day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10 ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to

Inhibitory effect of ginsenoside Rg1 on extracellular matrix production via extracellular signal-regulated protein kinase/activator protein 1 pathway in nasal polyp-derived fibroblasts.

PubMed

Cho, Jung-Sun; Moon, You-Mi; Um, Ji-Young; Moon, Jun-Hyeok; Park, Il-Ho; Lee, Heung-Man

2012-06-01

Nasal polyps are associated with chronic inflammation of the sinonasal mucosa and are involved in myofibroblast differentiation and extracellular matrix (ECM) accumulation. Ginsenoside Rg1, a compound derived from Panax ginseng, shows antifibrotic and anticancer effects. However, the molecular effects of Rg1 on myofibroblast differentiation and ECM production remain unknown. The aims of this study were to investigate the effect of Rg1 on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and ECM production and to determine the molecular mechanism of Rg1 in nasal polyp-derived fibroblasts (NPDFs). NPDFs were isolated from nasal polyps of seven patients who had chronic rhinosinusitis with nasal polyp. NPDFs were exposed to TGF-β1 with or without Rg1. Expression levels of α-smooth muscle actin (SMA), fibronectin and collagen type Iα1 were determined by reverse transcription polymerase chain reaction, Western blot and immunofluorescent staining. TGF-β1 signaling molecules, including Smad2/3, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were analyzed by Western blotting. Transcription factors involved with TGF-β1 signaling, nuclear factor (NF)-κB and activator protein 1 (AP-1) were also assessed by Western blot. The cytotoxic effect of Rg1 was measured by an established viability assay. The mRNA and protein expression levels of α-SMA, fibronectin and collagen type Iα1 were increased in TGF-β1-induced NPDFs. Rg1 inhibited these effects. The inhibitory molecular mechanism of Rg1 was involved in the ERK pathway. Rg1 inhibited the transcription factor activation of AP-1. Rg1 itself was not cytotoxic. The ginsenoside Rg1 has inhibitory effects on myofibroblast differentiation and ECM production. The inhibitory mechanism of Rg1 is involved with the ERK and AP-1 signaling pathways. Rg1 may be useful as an inhibitor of ECM deposition, and has potential to be used as a novel treatment option for nasal

SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1

PubMed Central

Brown, James R.; Conn, Kristen L.; Wasson, Peter; Charman, Matthew; Tong, Lily; Grant, Kyle; McFarlane, Steven

2016-01-01

ABSTRACT Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. IMPORTANCE Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against

SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1.

PubMed

Brown, James R; Conn, Kristen L; Wasson, Peter; Charman, Matthew; Tong, Lily; Grant, Kyle; McFarlane, Steven; Boutell, Chris

2016-07-01

Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against infection. PML

Exercise training decreases activation of the mitochondrial fission protein dynamin-related protein-1 in insulin-resistant human skeletal muscle.

PubMed

Fealy, Ciaran E; Mulya, Anny; Lai, Nicola; Kirwan, John P

2014-08-01

Defects in mitochondrial dynamics, the processes of fission, fusion, and mitochondrial autophagy, may contribute to metabolic disease including type 2 diabetes. Dynamin-related protein-1 (Drp1) is a GTPase protein that plays a central role in mitochondrial fission. We hypothesized that aerobic exercise training would decrease Drp1 Ser(616) phosphorylation and increase fat oxidation and insulin sensitivity in obese (body mass index: 34.6 ± 0.8 kg/m(2)) insulin-resistant adults. Seventeen subjects performed supervised exercise for 60 min/day, 5 days/wk at 80-85% of maximal heart rate for 12 wk. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp, and fat oxidation was determined by indirect calorimetry. Skeletal muscle biopsies were obtained from the vastus lateralis muscle before and after the 12-wk program. The exercise intervention increased insulin sensitivity 2.1 ± 0.2-fold (P < 0.01) and fat oxidation 1.3 ± 0.3-fold (P < 0.01). Phosphorylation of Drp1 at Ser(616) was decreased (pre vs. post: 0.81 ± 0.15 vs. 0.58 ± 0.14 arbitrary units; P < 0.05) following the intervention. Furthermore, reductions in Drp1 Ser(616) phosphorylation were negatively correlated with increases in fat oxidation (r = -0.58; P < 0.05) and insulin sensitivity (rho = -0.52; P < 0.05). We also examined expression of genes related to mitochondrial dynamics. Dynamin1-like protein (DNM1L; P < 0.01), the gene that codes for Drp1, and Optic atrophy 1 (OPA1; P = 0.05) were significantly upregulated following the intervention, while there was a trend towards an increase in expression of both mitofusin protein MFN1 (P = 0.08) and MFN2 (P = 0.07). These are the first data to suggest that lifestyle-mediated improvements in substrate metabolism and insulin sensitivity in obese insulin-resistant adults may be regulated through decreased activation of the mitochondrial fission protein Drp1. Copyright © 2014 the American Physiological Society.

Structure of the human protein kinase MPSK1 reveals an atypical activation loop architecture.

PubMed

Eswaran, Jeyanthy; Bernad, Antonio; Ligos, Jose M; Guinea, Barbara; Debreczeni, Judit E; Sobott, Frank; Parker, Sirlester A; Najmanovich, Rafael; Turk, Benjamin E; Knapp, Stefan

2008-01-01

The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a beta sheet and a large alpha-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-phi-H/Y-T*-N/G-X-X-X (phi is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.

The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila

PubMed Central

2013-01-01

Background The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription. Results and discussion Here we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters. Conclusions Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes. PMID:23442797

CDK5 Regulatory Subunit-Associated Protein 1-like 1 Negatively Regulates Adipocyte Differentiation through Activation of Wnt Signaling Pathway.

PubMed

Take, Kazumi; Waki, Hironori; Sun, Wei; Wada, Takahito; Yu, Jing; Nakamura, Masahiro; Aoyama, Tomohisa; Yamauchi, Toshimasa; Kadowaki, Takashi

2017-08-04

CDK5 Regulatory Subunit-Associated Protein 1-like 1 (CDKAL1) was identified as a susceptibility gene for type 2 diabetes and body mass index in genome-wide association studies. Although it was reported that CDKAL1 is a methylthiotransferase essential for tRNA Lys (UUU) and faithful translation of proinsulin generated in pancreatic β cells, the role of CDKAL1 in adipocytes has not been understood well. In this study, we found that CDKAL1 is expressed in adipose tissue and its expression is increased during differentiation. Stable overexpression of CDKAL1, however, inhibited adipocyte differentiation of 3T3-L1 cells, whereas knockdown of CDKAL1 promoted differentiation. CDKAL1 increased protein levels of β-catenin and its active unphosphorylated form in the nucleus, thereby promoting Wnt target gene expression, suggesting that CDKAL1 activated the Wnt/β-catenin pathway-a well-characterized inhibitory regulator of adipocyte differentiation. Mutant experiments show that conserved cysteine residues of Fe-S clusters of CDKAL1 are essential for its anti-adipogenic action. Our results identify CDKAL1 as novel negative regulator of adipocyte differentiation and provide insights into the link between CDKAL1 and metabolic diseases such as type 2 diabetes and obesity.

Targeting the MEF2-Like Transcription Factor Smp1 by the Stress-Activated Hog1 Mitogen-Activated Protein Kinase

PubMed Central

Nadal, Eulàlia de; Casadomé, Laura; Posas, Francesc

2003-01-01

Exposure of Saccharomyces cerevisiae to increases in extracellular osmolarity activates the stress-activated Hog1 mitogen-activated protein kinase (MAPK), which is essential for cell survival upon osmotic stress. Yeast cells respond to osmotic stress by inducing the expression of a very large number of genes, and the Hog1 MAPK plays a critical role in gene transcription upon stress. To understand how Hog1 controls gene expression, we designed a genetic screen to isolate new transcription factors under the control of the MAPK and identified the MEF2-like transcription factor, Smp1, as a target for Hog1. Overexpression of SMP1 induced Hog1-dependent expression of osmoresponsive genes such as STL1, whereas smp1Δ cells were defective in their expression. Consistently, smp1Δ cells displayed reduced viability upon osmotic shock. In vivo coprecipitation and phosphorylation studies showed that Smp1 and Hog1 interact and that Smp1 is phosphorylated upon osmotic stress in a Hog1-dependent manner. Hog1 phosphorylated Smp1 in vitro at the C-terminal region. Phosphorylation of Smp1 by the MAPK is essential for its function, since a mutant allele unable to be phosphorylated by the MAPK displays impaired stress responses. Thus, our data indicate that Smp1 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK. Moreover, Smp1 concentrates in the nucleus during the stationary phase, and the lack of SMP1 results in cells that lose viability in the stationary phase. Localization of Smp1 depends on HOG1, and consistently, hog1Δ cells also lose viability during this growth phase. These data suggest that Smp1 could be mediating a role for the Hog1 MAPK during the stationary phase. PMID:12482976

The AMP-activated protein kinase beta 1 subunit modulates erythrocyte integrity.

PubMed

Cambridge, Emma L; McIntyre, Zoe; Clare, Simon; Arends, Mark J; Goulding, David; Isherwood, Christopher; Caetano, Susana S; Reviriego, Carmen Ballesteros; Swiatkowska, Agnieszka; Kane, Leanne; Harcourt, Katherine; Adams, David J; White, Jacqueline K; Speak, Anneliese O

2017-01-01

Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia. Half-life is affected by numerous factors, including energy balance, electrolyte gradients, reactive oxygen species, and membrane plasticity. The heterotrimeric AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that acts as a critical regulator of cellular energy balance. Previous roles for the alpha 1 and gamma 1 subunits in the control of erythrocyte survival have been reported. In the work described here, we studied the role of the beta 1 subunit in erythrocytes and observed microcytic anemia with compensatory extramedullary hematopoiesis together with splenomegaly and increased osmotic resistance. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

PubMed Central

Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

2011-01-01

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

Association of the macrophage activating factor (MAF) precursor activity with polymorphism in vitamin D-binding protein.

PubMed

Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Kubo, Shinichi; Hori, Hitoshi

2004-01-01

Serum vitamin D-binding protein (Gc protein or DBP) is a highly expressed polymorphic protein, which is a precursor of the inflammation-primed macrophage activating factor, GcMAF, by a cascade of carbohydrate processing reactions. In order to elucidate the relationship between Gc polymorphism and GcMAF precursor activity, we estimated the phagocytic ability of three homotypes of Gc protein, Gc1F-1F, Gc1S-1S and Gc2-2, through processing of their carbohydrate moiety. We performed Gc typing of human serum samples by isoelectric focusing (IEF). Gc protein from human serum was purified by affinity chromatography with 25-hydroxyvitamin D3-sepharose. A phagocytosis assay of Gc proteins, modified using beta-glycosidase and sialidase, was carried out. The Gc1F-1F phenotype was revealed to possess Galbeta1-4GalNAc linkage by the analysis of GcMAF precursor activity using beta1-4 linkage-specific galactosidase from jack bean. The GcMAF precursor activity of the Gc1F-1F phenotype was highest among three Gc homotypes. The Gc polymorphism and carbohydrate diversity of Gc protein are significant for its pleiotropic effects.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

PubMed

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Dobesilate diminishes activation of the mitogen - activated protein kinase ERK1/2 in glioma cells

PubMed Central

Cuevas, P; Diaz-González, Diana; Garcia-Martin-Córdova, C; Sánchez, I; Lozano, Rosa Maria; Giménez-Gallego, G; Dujovny, M

2006-01-01

Fibroblast growth factors (FGFs) and their receptors, regularly expressed at high levels in gliomas, are further upregulated during the transition of the tumor from low- to high-grade malignancy, and are essential for glioma progression. FGFs induce upregulation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured glioma cells, which suggests that MAPK pathway participates in the FGF-dependent glioma development. Recently, it has been shown that dobesilate, an inhibitor of FGF mitogenic activity, shows antiproliferative and proapoptotic activities in glioma cell cultures. Accordingly, it should be expected this new synthetic FGF inhibitor to affect the activation levels of MAPK. Here we report that immunocytochemical and Western blot data unequivocally show that treatment of cell cultures with dobesilate causes a significant decrease of the intracellular levels of ERK1/2 activation, one of the components of the MAPK signalling cascade. This finding supports an important role for dobesilate in glioma growth, suggesting that dobesilate should be a treatment to be born in mind for glioma management. PMID:16563234

Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris.

PubMed

Huang, Mengmeng; Gao, Yanyun; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2017-03-01

Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.

Direct activation of RIP3/MLKL-dependent necrosis by herpes simplex virus 1 (HSV-1) protein ICP6 triggers host antiviral defense

PubMed Central

Wang, Xing; Li, Yun; Liu, Shan; Yu, Xiaoliang; Li, Lin; Shi, Cuilin; He, Wenhui; Li, Jun; Xu, Lei; Hu, Zhilin; Yu, Lu; Yang, Zhongxu; Chen, Qin; Ge, Lin; Zhang, Zili; Zhou, Biqi; Jiang, Xuejun; Chen, She; He, Sudan

2014-01-01

The receptor-interacting kinase-3 (RIP3) and its downstream substrate mixed lineage kinase domain-like protein (MLKL) have emerged as the key cellular components in programmed necrotic cell death. Receptors for the cytokines of tumor necrosis factor (TNF) family and Toll-like receptors (TLR) 3 and 4 are able to activate RIP3 through receptor-interacting kinase-1 and Toll/IL-1 receptor domain-containing adapter inducing IFN-β, respectively. This form of cell death has been implicated in the host-defense system. However, the molecular mechanisms that drive the activation of RIP3 by a variety of pathogens, other than the above-mentioned receptors, are largely unknown. Here, we report that human herpes simplex virus 1 (HSV-1) infection triggers RIP3-dependent necrosis. This process requires MLKL but is independent of TNF receptor, TLR3, cylindromatosis, and host RIP homotypic interaction motif-containing protein DNA-dependent activator of IFN regulatory factor. After HSV-1 infection, the viral ribonucleotide reductase large subunit (ICP6) interacts with RIP3. The formation of the ICP6–RIP3 complex requires the RHIM domains of both proteins. An HSV-1 ICP6 deletion mutant failed to cause effective necrosis of HSV-1–infected cells. Furthermore, ectopic expression of ICP6, but not RHIM mutant ICP6, directly activated RIP3/MLKL-mediated necrosis. Mice lacking RIP3 exhibited severely impaired control of HSV-1 replication and pathogenesis. Therefore, this study reveals a previously uncharacterized host antipathogen mechanism. PMID:25316792

A novel serine protease, Sep1, from Bacillus firmus DS-1 has nematicidal activity and degrades multiple intestinal-associated nematode proteins.

PubMed

Geng, Ce; Nie, Xiangtao; Tang, Zhichao; Zhang, Yuyang; Lin, Jian; Sun, Ming; Peng, Donghai

2016-04-27

Plant-parasitic nematodes (PPNs) cause serious harm to agricultural production. Bacillus firmus shows excellent control of PPNs and has been produced as a commercial nematicide. However, its nematicidal factors and mechanisms are still unknown. In this study, we showed that B. firmus strain DS-1 has high toxicity against Meloidogyne incognita and soybean cyst nematode. We sequenced the whole genome of DS-1 and identified multiple potential virulence factors. We then focused on a peptidase S8 superfamily protein called Sep1 and demonstrated that it had toxicity against the nematodes Caenorhabditis elegans and M. incognita. The Sep1 protein exhibited serine protease activity and degraded the intestinal tissues of nematodes. Thus, the Sep1 protease of B. firmus is a novel biocontrol factor with activity against a root-knot nematode. We then used C. elegans as a model to elucidate the nematicidal mechanism of Sep1, and the results showed that Sep1 could degrade multiple intestinal and cuticle-associated proteins and destroyed host physical barriers. The knowledge gained in our study will lead to a better understanding of the mechanisms of B. firmus against PPNs and will aid in the development of novel bio-agents with increased efficacy for controlling PPNs.

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

PubMed

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

2016-02-01

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

Mouse macrophages primed with alendronate down-regulate monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) production in response to Toll-like receptor (TLR) 2 and TLR4 agonist via Smad3 activation.

PubMed

Masuda, Takahiro; Deng, Xue; Tamai, Riyoko

2009-08-01

Alendronate is one of the nitrogen-containing bisphosphonates (NBPs) used as anti-bone resorptive drugs. However, NBPs have inflammatory side effects including osteomyelitis and osteonecrosis of the jaw. In the present study, we examined the effects of alendronate on chemokine production by the macrophage-like cell line, J774.1, when incubated with Pam(3)CSK(4) (a Toll-like receptor (TLR) 2 agonist) and Lipid A (a TLR4 agonist). Pretreatment of J774.1 cells with alendronate decreased the production of TLR ligand-induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) but did not influence nuclear factor-kappaB (NF-kappaB) activation. While this agent induced caspase-8 activation, a caspase-8 inhibitor did not affect the decrease in MCP-1 production by alendronate and TLR ligands. Thus, the alendronate-mediated decrease in chemokine production was independent of NF-kappaB and caspase-8 activation. Although transforming growth factor-beta1 (TGF-beta1) is known to inhibit chemokine production by various cell types via Smad3 activation, pretreatment with alendronate did not increase TGF-beta1 production by J774.1 cells incubated in the presence or absence of TLR ligands. However, alendronate directly activated Smad3. These results suggest that by down-regulating MCP-1 and MIP-1alpha production via Smad3, long-term use of alendronate might inhibit normal activation and migration of osteoclasts and cause osteonecrosis.

Genistein promotes insulin action through adenosine monophosphate-activated protein kinase activation and p70 ribosomal protein S6 kinase 1 inhibition in the skeletal muscle of mice fed a high energy diet.

PubMed

Arunkumar, Elumalai; Anuradha, Carani Venkatraman

2012-08-01

Genistein (GEN), a soy isoflavone, exerts insulin-sensitizing actions in animals; however, the underlying mechanisms have not been determined. Because GEN is a known activator of adenosine monophosphate-activated protein kinase (AMPK), we hypothesize that GEN activates insulin signaling through AMPK activation. To test this hypothesis, a high fat-high fructose diet (HFFD)-fed mice model of insulin resistance was administered GEN, and the insulin signaling pathway proteins in the skeletal muscle were examined. Hyperglycemia and hyperinsulinemia observed in HFFD-fed mice were significantly lowered by GEN. GEN increased insulin-stimulated tyrosine phosphorylation of insulin receptor-β and insulin receptor substrate (IRS) 1 but down-regulated IRS-1 serine phosphorylation in the skeletal muscle of HFFD-fed mice. Furthermore, GEN treatment improved muscle IRS-1-associated phospatidylinositol-3 kinase expression, phosphorylation of Akt at Ser(473), and translocation of glucose transporter subtype 4. Phosphorylation of AMPK at Thr(172) and acetyl coenzyme A carboxylase (ACC) at Ser(79) was augmented, whereas phosphorylation of p70 ribosomal protein S6 kinase 1 at Thr(389) was significantly decreased after GEN treatment in the skeletal muscle of HFFD-fed mice. These results suggest that GEN might improve insulin action in the skeletal muscle by targeting AMPK. Copyright © 2012 Elsevier Inc. All rights reserved.

mTORC1-Independent Reduction of Retinal Protein Synthesis in Type 1 Diabetes

PubMed Central

Losiewicz, Mandy K.; Pennathur, Subramaniam; Jefferson, Leonard S.; Kimball, Scot R.; Abcouwer, Steven F.; Gardner, Thomas W.

2014-01-01

Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2Akita diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy. PMID:24740573

Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis.

PubMed

Kumar, Naveen; Gupta, Sonal; Dabral, Surbhi; Singh, Shailja; Sehrawat, Seema

2017-06-01

Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death. Herein, we performed the analysis of the Cancer Genome Atlas (TCGA) dataset to evaluate the essential role of cAMP molecular network in breast cancer. We report that EPAC1, PKA, and AKAP9 along with other molecular partners are amplified in breast cancer patients, indicating the importance of this signaling network. To evaluate the functional role of few of these proteins, we used pharmacological modulators and analyzed their effect on cell migration and cell death in breast cancer cells. Hence, we report that inhibition of EPAC1 activity using pharmacological modulators leads to inhibition of cell migration and induces cell death. Additionally, we also observed that the inhibition of EPAC1 resulted in disruption of its association with the microtubule cytoskeleton and delocalization of AKAP9 from the centrosome as analyzed by in vitro imaging. Finally, this study suggests for the first time the mechanistic insights of mode of action of a primary cAMP-dependent sensor, Exchange protein activated by cAMP 1 (EPAC1), via its interaction with A-kinase anchoring protein 9 (AKAP9). This study provides a new cell signaling cAMP-EPAC1-AKAP9 direction to the development of additional biotherapeutics for breast cancer.

Apical Na(+)-D-glucose cotransporter 1 (SGLT1) activity and protein abundance are expressed along the jejunal crypt-villus axis in the neonatal pig

USDA-ARS?s Scientific Manuscript database

Gut apical Na(+)-glucose cotransporter 1 (SGLT1) activity is high at the birth and during suckling, thus contributing substantially to neonatal glucose homeostasis. We hypothesize that neonates possess high SGLT1 maximal activity by expressing apical SGLT1 protein along the intestinal crypt-villus a...

Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity.

PubMed

Kathum, Omer Abid; Schräder, Tobias; Anhlan, Darisuren; Nordhoff, Carolin; Liedmann, Swantje; Pande, Amit; Mellmann, Alexander; Ehrhardt, Christina; Wixler, Viktor; Ludwig, Stephan

2016-06-01

Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed. © 2016 The Authors Cellular Microbiology published by John Wiley & Sons Ltd.

Stretch and interleukin 1 beta: pro-labour factors with similar mitogen-activated protein kinase effects but differential patterns of transcription factor activation and gene expression.

PubMed

Sooranna, S R; Engineer, N; Liang, Z; Bennett, P R; Johnson, M R

2007-07-01

IL-1beta and stretch increase uterine smooth muscle cell (USMC) prostaglandin H synthase 2 (PGHS-2) and interleukin (IL)-8 mRNA expression in a mitogen-activated protein kinase (MAPK) dependent mechanism. We have tested our hypothesis that stretch and IL-1beta activate different components of the MAPK cascade in USMC and investigated the effects of specific MAPK inhibitors on these components. Further, we have used a Jun N-terminal kinase (JNK) and p38 activator, anisomycin, to compare the effect of differential MAPK activation on the expression of PGHS-2, IL-8 and oxytocin receptor (OTR) mRNA with that seen in response to stretch and IL-1beta. Stretch, IL-1beta and anisomycin activated similar components of the MAPK cascade and specific inhibitors of MAPK altered phosphorylation of MAPK and downstream cascade components as expected. Expression of OTR mRNA was increased by stretch and anisomycin in a MAPK-independent manner. All three stimuli increased PGHS-2 and IL-8 mRNA expression in a MAPK-dependent manner, but while the MAPK inhibitors reduced the IL-1beta-induced activation of activating transcription factor (ATF)-2, liver activating protein (LAP) and c-jun, the stretch-induced increase in LAP was unaffected by MAPK-inhibition and only JNK inhibition appeared to reduce c-jun activation. These observations show that stretch, IL-1beta and anisomycin activate the same components of the MAPK cascade, but differentially activate LAP and liver inhibitory protein (LIP) perhaps accounting for the increase in OTR by stretch and anisomycin but not IL-1beta observed in this study.

TRUSS, a Novel Tumor Necrosis Factor Receptor 1 Scaffolding Protein That Mediates Activation of the Transcription Factor NF-κB

PubMed Central

Soond, Surinder M.; Terry, Jennifer L.; Colbert, Jeff D.; Riches, David W. H.

2003-01-01

We describe the cloning and characterization of tumor necrosis factor receptor (TNF-R)-associated ubiquitous scaffolding and signaling protein (TRUSS), a novel TNF-R1-interacting protein of 90.7 kDa. TRUSS mRNA was ubiquitously expressed in mouse tissues but was enriched in heart, liver, and testis. Coimmunoprecipitation experiments showed that TRUSS was constitutively associated with unligated TNF-R1 and that the complex was relatively insensitive to stimulation with TNF-α. Deletion mutagenesis of TNF-R1 indicated that TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. In addition, the N-terminal region of TRUSS (residues 1 to 440) contains sequences that permit association with the cytoplasmic domain of TNF-R1. Transient overexpression of TRUSS activated NF-κB and increased NF-κB activation in response to ligation of TNF-R1. In contrast, a COOH-terminal-deletion mutant of TRUSS (TRUSS1-723) was found to inhibit NF-κB activation by TNF-α. Coprecipitation and coimmunoprecipitation assays revealed that TRUSS can interact with TRADD, TRAF2, and components of the IKK complex. These findings suggest that TRUSS may serve as a scaffolding protein that interacts with TNF-R1 signaling proteins and may link TNF-R1 to the activation of IKK. PMID:14585990

Mucin1 mediates autocrine transforming growth factor beta signaling through activating the c-Jun N-terminal kinase/activator protein 1 pathway in human hepatocellular carcinoma cells.

PubMed

Li, Qiongshu; Liu, Guomu; Shao, Dan; Wang, Juan; Yuan, Hongyan; Chen, Tanxiu; Zhai, Ruiping; Ni, Weihua; Tai, Guixiang

2015-02-01

In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-β) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-β signaling. To investigate the effect of MUC1 on TGF-β signaling, we determined TGF-β secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-β1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-β promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-β receptor (TβR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-β signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-β secretion and exogenous TGF-β1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TβR inhibitor or small interfering RNA silencing of TGF-β1 gene in HCC cells. The high correlation between MUC1 and TGF-β1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-β signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of the active protein in rice bran protein having an inhibitory activity of cholesterol micellar solubility.

PubMed

Wang, Jilite; Shimada, Masaya; Nagaoka, Satoshi

2017-06-01

In our previous study, rice bran protein (RBP) inhibited cholesterol micellar solubility in vitro and decreased serum cholesterol level in rats. In the present study, RBP was separated and purified by size-exclusion chromatography and reversed-phase chromatography. The active protein of RBP related to cholesterol micellar solubility was identified as lectin and non-specific lipid-transfer protein 1 using MALDI-TOF mass spectrometry analysis.

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

PubMed Central

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

2016-01-01

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

Polymerase Acidic Protein-Basic Protein 1 (PA-PB1) Protein-Protein Interaction as a Target for Next-Generation Anti-influenza Therapeutics.

PubMed

Massari, Serena; Goracci, Laura; Desantis, Jenny; Tabarrini, Oriana

2016-09-08

The limited therapeutic options against the influenza virus (flu) and increasing challenges in drug resistance make the search for next-generation agents imperative. In this context, heterotrimeric viral PA/PB1/PB2 RNA-dependent RNA polymerase is an attractive target for a challenging but strategic protein-protein interaction (PPI) inhibition approach. Since 2012, the inhibition of the polymerase PA-PB1 subunit interface has become an active field of research following the publication of PA-PB1 crystal structures. In this Perspective, we briefly discuss the validity of flu polymerase as a drug target and its inhibition through a PPI inhibition strategy, including a comprehensive analysis of available PA-PB1 structures. An overview of all of the reported PA-PB1 complex formation inhibitors is provided, and approaches used for identification of the inhibitors, the hit-to-lead studies, and the emerged structure-activity relationship are described. In addition to highlighting the strengths and weaknesses of all of the PA-PB1 heterodimerization inhibitors, we analyze their hypothesized binding modes and alignment with a pharmacophore model that we have developed.

G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

PubMed

Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

2016-12-30

G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser 318 , Ser 346 , Ser 612 , and Ser 789 , and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.