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The expressions and roles of protein inhibitor of activated STAT (PIAS) proteins, a group of proteins with STAT inhibition and SUMOylation E3 ligase activity, are rarely revealed in endometrial cancer (EC). In this study, we analyzed the expressions of PIASs and their relationships with clinical features by mining online data through web servers, including UALCAN and Gene Expression Profiling Interactive Analysis (GEPIA) in EC. The expressions of PIASs in EC tissues were further validated by immunohistochemistry (IHC). The online analyses revealed only PIAS1 was consistently downregulated both at mRNA and protein level in EC, which was validated by the IHC. Subsequently, the mechanism of PIAS1 downregulation was explored with online tools like UALCAN, cBioPortal, LinkedOmics, and the Encyclopedia of RNA Interactomes (ENCORI). The results indicated that the mutation rate of PIAS1 was extremely low and not associated with PIAS1 expression. The promoter methylation level of PIAS1 was comparable between normal and EC tissues. miR-182-5p and miR-96-5p with negative association with PIAS1 in EC were predicted to target PIAS1. Dual luciferase reporter assay confirmed miR-182-5p and miR-96-5p could target PIAS1 in EC. MiR-182-5p and miR-96-5p inhibitors could upregulate PIAS1 in EC cells. Moreover, ectopic PIAS1 expression and STAT3 inhibitor treatment significantly inhibited STAT3's activity and the levels of miR-182-5p and miR-96-5p in EC cells. Collectively, our findings revealed PIAS1 was downregulated in EC, which was caused by upregulation of miR-182-5p and miR-96-5p, and PIAS1 downregulation further activated STAT3 and increased the expression of miR-182-5p and miR-96-5p, confirming miR-182-5p and miR-96-5p mediated the negative feedback regulatory loop between PIAS1 and STAT3 in EC.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease specific to motor neurons. Pathogenic mutations in an ALS-associated gene encoding superoxide dismutase 1 (SOD1) have been identified in familial ALS (fALS) cases. SOD1 with fALS-linked mutations is prone to form cytotoxic aggregates that cause cellular dysfunction. We previously demonstrated that the modification of SOD1 by small ubiquitin-like modifier (SUMO) 3 enhances the aggregation of fALS-linked SOD1 mutants. SUMOylation is a reversible post-translational modification targeting lysine residues. SUMO conjugation is mediated by the enzymes E1, E2, and E3, and deconjugation is catalyzed by deSUMOylation enzymes. To understand the process of SOD1 aggregation, we examined the involvement of protein inhibitor of activated STAT (PIAS) family and sentrin-specific protease (SENP) family proteins in the SUMOylation of SOD1 mutants. We found that all four types of PIAS family proteins, E3 ligase of SUMOylation, increased SUMOylation of SOD1 mutants. Among three SENP family proteins tested, deSUMOylation enzymes, SENP1, exhibited the most efficient deconjugation effect. In co-expression experiments, PIASy and SENP1 increased and decreased the number of cells exhibiting SOD1-mutant aggregation, respectively, confirming the effect of these enzymes on SOD1 aggregation. These findings suggest that regulation of SUMOylation affects the pathogenesis of ALS.

The SUMOylation machinery is a regulator of neuronal activity and synaptic plasticity. It is composed of SUMO isoforms and specialized enzymes named E1, E2 and E3 SUMO ligases. Recent studies have highlighted how SUMO isoforms and E2 enzymes localize with synaptic markers to support previous functional studies but less information is available on E3 ligases. PIAS proteins - belonging to the protein inhibitor of activated STAT (PIAS) SUMO E3-ligase family - are the best-characterized SUMO E3-ligases and have been linked to the formation of spatial memory in rodents. Whether however they exert their function co-localizing with synaptic markers is still unclear. In this study, we applied for the first time structured illumination microscopy (SIM) to PIAS ligases to investigate the co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal and cortical murine neurons. The results indicate partial co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal neurons and much rarer occurrence in cortical neurons. This is in line with previous super-resolution reports describing the co-localization with synaptic markers of other components of the SUMOylation machinery.

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits.To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 dpf and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits. To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 days post fertilization (dpf) and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.

Oxidative stress serves important roles in cardiac injury during the process of ischemia/reperfusion (I/R). Y‑box protein 1 (YB1), a member of the highly conserved Y‑box protein family, is closely associated with inflammation and stress responses by regulating gene transcription, RNA splicing and mRNA translation. However, the roles of YB1 in oxidative stress‑induced myocardial‑I/R (M‑I/R) injury are unknown. The aim of the present study was to examine the effects of YB1 on H2O2‑induced cardiomyocyte injury and its underlying mechanism. The results demonstrated that YB1 expression was upregulated during H2O2‑induced myocardial injury. YB1 knockdown through transfection of small interfering RNA significantly aggravated cardiac cell apoptosis. Furthermore, YB1 knockdown significantly reversed the H2O2‑mediated increase in phosphorylated signal transducer and activator of transcription (STAT)3, but did not affect the phosphorylation of P38, extracellular signal‑regulated kinases 1/2, c‑Jun N‑terminal kinases, P65, Janus kinase 1 and 2 or STAT1. Moreover, protein co‑immunoprecipitation and RNA‑binding protein immunoprecipitation assays revealed that YB1 interacted with protein inhibitor of activated STAT 3 (PIAS3) mRNA but not its translated protein. YB1 overexpression may have promoted PIAS3 mRNA decay, decreasing PIAS3 protein levels, and therefore increased the levels of phosphorylated STAT3. Finally, YB1 knockdown, mediated by a lentivirus carrying YB1 targeted short hairpin RNA, significantly decreased left ventricle percentage fractional shortening and ejection fraction values, while increasing the infarct sizes in a rat model of M‑I/R injury. These results demonstrated for the first time (to the best of our knowledge) that YB1 may protect cardiac myocytes against H2O2 or M‑I/R‑induced injury by binding to PIAS3 mRNA and resulting in the phosphorylation of STAT3.

Classical swine fever (CSF) is a highly contagious viral disease causing severe economic losses to the swine industry. As viroporins of viruses modulate the cellular ion balance and then take over the cellular machinery, blocking the activity of viroporin or developing viroporin-defective attenuated vaccines offers new approaches to treat or prevent viral infection. Non-structural protein p7 of CSF virus (CSFV) is a viroporin, which was highly involved in CSFV virulence. Deciphering the interaction between p7 and host proteins will aid our understanding of the mechanism of p7-cellular protein interaction affecting CSFV replication. In the present study, seven host cellular proteins including microtubule-associated protein RP/EB family member 1 (MAPRE1), voltage-dependent anion channel 1 (VDAC1), proteasome maturation protein (POMP), protein inhibitor of activated STAT 1 (PIAS1), gametogenetin binding protein 2 (GGNBP2), COP9 signalosome subunit 2 (COPS2), and contactin 1 (CNTN1) were identified as the potential interactive cellular proteins of CSFV p7 by using yeast two-hybrid (Y2H) screening. Plus, the interaction of CSFV p7 with MAPRE1 and VDAC1 was further evaluated by co-immunoprecipitation and GST-pulldown assay. Besides, the p7-cellular protein interaction network was constructed based on these seven host cellular proteins and the STRING database. Enrichment analysis of GO and KEGG indicated that many host proteins in the p7-cellular protein interaction network were mainly related to the ubiquitin-proteasome system, cGMP-PKG signaling pathway, calcium signaling pathway, and JAK-STAT pathway. Overall, this study identified potential interactive cellular proteins of CSFV p7, constructed the p7-cellular protein interaction network, and predicted the potential pathways involved in the interaction between CSFV p7 and host cells.

Previously, we demonstrated that the homeoprotein Msx1 interaction with p53 inhibited tumor growth by inducing apoptosis. However, Msx1 can exert its tumor suppressive effect through the inhibition of angiogenesis since growth of the tumor relies on sufficient blood supply from the existing vessels to provide oxygen and nutrients for tumor growth. We hypothesized that the inhibition of tumor growth by Msx1 might be due to the inhibition of angiogenesis. Here, we explored the role of Msx1 in angiogenesis. Overexpression of Msx1 in HUVECs inhibited angiogenesis, and silencing of Msx1 by siRNA abrogated its anti-angiogenic effects. Furthermore, forced expression of Msx1 in mouse muscle tissue inhibited vessel sprouting, and application of an Ad-Msx1-transfected conditioned medium onto the chicken chorioallantoic membrane (CAM) led to a significant inhibition of new vessel formation. To explore the underlying mechanism of Msx1-mediated angiogenesis, yeast two-hybrid screening was performed, and we identified PIASy (protein inhibitor of activated STAT Y) as a novel Msx1-interacting protein. We mapped the homeodomain of Msx1 and the C-terminal domain of PIASy as respective interacting domains. Consistent with its anti-angiogenic function, overexpression of Msx1 suppressed the reporter activity of VEGF. Interestingly, PIASy stabilized Msx1 protein, whereas deletion of the Msx1-interacting domain in PIASy abrogated the inhibition of tube formation and the stabilization of Msx1 protein. Our findings suggest the functional importance of PIASy-Msx1 interaction in Msx1-mediated angiogenesis inhibition.

The effects of interferon-γ (IFN-γ) on cholesterol accumulation and the development of foam cells are still unclear. In the present study, we found that IFN-γ promoted liver X receptor (LXR)-α degradation through the ubiquitin-proteasome system in macrophages. The process was dependent on its interactions with phosphorylated signal transducer and activator of transcription 1 (p-STAT1) and protein inhibitor of activated STAT 1 (PIAS1) because both fludarabine and PIAS1 shRNA reversed the decrease in LXR-α protein expression induced by IFN-γ. Additionally, IFN-γ enhanced the interactions of ubiquitin-conjugating enzyme 9 (UBC9), small ubiquitin-like modifier (SUMO)-1 and SUMO-2/3 with LXR-α. Moreover, treatment with shRNA specific for them not only reduced LXR-α polyubiquitination but also reversed the IFN-γ-induced decrease in its expression. Two specific sumoylation sites in LXR-α, K22 and K326, were indispensable for its IFN-γ-induced polyubiquitination because the K22R and K326R mutations inhibited the polyubiquitination and degradation of LXR-α in IFN-γ-treated macrophages. In addition, K22R or K326R mutation almost completely restored ATP-binding cassette subfamily G member 1 (ABCG1)-mediated cholesterol efflux in IFN-γ-treated macrophages. Taken together, these findings indicate that IFN-γ promotes LXR-α degradation through a SUMO-ubiquitin-dependent pathway, which may inhibit cholesterol efflux mediated by ABCG1 from macrophages and promote the development of atherosclerosis.

Sirtuin 1 (SIRT1) has been reported to be involved in the mechanisms underlying longevity and has also been indicated as a valuable regulator of age-related neurological disorders. Some natural products increase SIRT1 activity and stimulate deacetylation of various proteins. In the present study, SIRT1 overexpression by genetic modification or treatment with SIRT1 activators significantly inhibited the secretion of nitric oxide and expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory mediator-interleukin 1β-in microglia. SIRT1 activation also decreased the levels of K379 acetyl-p53 and the protein inhibitor of activated Stat 1 expression in microglial cells. In addition, it dramatically promoted M2 polarization of microglia, which enhanced cell motility and altered phagocytic ability. We also used minocycline, a well-known inhibitor of microglial activation, to study the mechanism of SIRT1 signaling. Minocycline treatment decreased neuroinflammatory responses and promoted M2 polarization of microglia. It also reduced the acetyl-p53 level in the brain tissues in an inflammatory mouse model. Our findings demonstrated that SIRT1 participates in the maintenance of microglial polarization homeostasis and that minocycline exerts regulatory effects on SIRT1 activation. Therefore, our results indicate that SIRT1 activation may be a useful therapeutic target for the treatment of neuroinflammation-associated disorders.

The protein inhibitor of activated STAT (PIAS) proteins are important signal transduction modulator family and regulate the innate immune signaling pathway induced by certain transcription factors, including NF-κB, IRF3, and JAK/STAT. The PIAS protein mechanism that regulates innate immune response in mammals has been well described in the literature; however, whether the PIAS gene exists in ducks as well as the role of PIAS in duck IFN-β expression is still unclear. Here, we cloned duck PIAS (duPIAS), finding PIAS2 could repress IFN-β production. DuPIAS2 contains SAP-PINIT-RLD-S/T characteristic domains, and its overexpression could inhibit virus-induced IFN-β promoter activation. Moreover, duPIAS2 interacts with duck interferon regulatory factor 7 (IRF7) and inhibits IFN-β promoter activation induced by duck IRF7. Additionally, its inhibitory function does not rely on its SUMO E3 ligase activity but rather its C-terminal portion. The above results demonstrate that duPIAS2 is a repressor of IFN-β production induced by duck IRF7.

Ribosome biogenesis is critical for proliferating cells and requires the coordinated activities of three eukaryotic RNA polymerases. We recently showed that the small ubiquitin-like modifier (SUMO) system controls the global level of RNA polymerase II (Pol II)–controlled transcription in mammalian cells by regulating cyclin-dependent kinase 9 activity. Here, we present evidence that the SUMO system also plays a critical role in the control of Pol I transcription. Using an siRNA-based knockdown approach, we found that multiple SUMO E3 ligases of the PIAS (protein inhibitor of activated STAT) family are involved in SUMO-mediated repression of ribosomal DNA (rDNA) gene transcription. We demonstrate that endogenous SUMO represses rDNA transcription primarily by repressing upstream-binding factor and proto-oncogene c-Myc expression and that ectopic overexpression of SUMO-associated enzymes additionally represses rDNA transcription via c-Myc SUMOylation and its subsequent degradation. The results of our study reveal a critical role of SUMOylation in the control of rDNA transcription, uncover the underlying mechanisms involved, and indicate that the SUMO system coordinates Pol I– and Pol II–mediated transcription in mammalian cells.

Reactive oxygen species (ROS) increase during adipogenesis and in obesity. Oxidants react with cysteine residues of proteins to form glutathione (GSH) adducts, S-glutathionylation, that are selectively removed by glutaredoxin-1 (Glrx). We have previously reported that Glrx knockout mice had increased protein S-glutathionylation and developed obesity by an unknown mechanism. In this study, we demonstrated that 3T3L1 adipocytes differentiation increased ROS and protein S-glutathionylation. Glrx ablation elevated protein S-glutathionylation and lipid content in 3T3L1 cells. Glrx replenishment decreased the lipid content of Glrx KO 3T3L1 cells. Glrx KO also increased protein expression and protein S-glutathionylation of the adipogenic transcription factor CCAAT enhancer-binding protein (C/EBP) β. Protein S-glutathionylation decreased the interaction of C/EBPβ and protein inhibitor of activated STAT (PIAS) 1, a small ubiquitin-related modifier (SUMO) E3 ligase that facilitates C/EBPβ degradation. Experiments with truncated mutant C/EBPβ demonstrated that PIAS1 interacted with the liver-enriched inhibitory protein (LIP) region of C/EBPβ. Furthermore, mass spectrometry analysis identified protein S-glutathionylation of Cys201 and Cys296 in the LIP region of C/EBPβ. The C201S, C296S double mutant C/EBPβ prevented protein S-glutathionylation and preserved the interaction with PIAS1. In summary, Glrx ablation stimulated 3T3L1 cell differentiation and adipogenesis via increased protein S-glutathionylation of C/EBPβ, stabilizing and increasing C/EBPβ protein levels.

CoREST family of transcriptional co-repressors regulates gene expression and cell fate determination during development. CoREST co-repressors recruit with different affinity the histone demethylase LSD1 (KDM1A) and the deacetylases HDAC1/2 to repress with variable strength the expression of target genes. CoREST protein levels are differentially regulated during cell fate determination and in mature tissues. However, regulatory mechanisms of CoREST co-repressors at the protein level have not been studied. Here, we report that CoREST (CoREST1, RCOR1) and its homologs CoREST2 (RCOR2) and CoREST3 (RCOR3) interact with PIASγ (protein inhibitor of activated STAT), a SUMO (small ubiquitin-like modifier)-E3-ligase. PIASγ increases the stability of CoREST proteins and facilitates their SUMOylation by SUMO-2. Interestingly, the SUMO-conjugating enzyme, Ubc9 also facilitates the SUMOylation of CoREST proteins. However, it does not change their protein levels. Specificity was shown using the null enzymatic form of PIASγ (PIASγ-C342A) and the SUMO protease SENP-1, which reversed SUMOylation and the increment of CoREST protein levels induced by PIASγ. The major SUMO acceptor lysines are different and are localized in nonconserved sequences among CoREST proteins. SUMOylation-deficient CoREST1 and CoREST3 mutants maintain a similar interaction profile with LSD1 and HDAC1/2, and consequently maintain similar repressor capacity compared with wild-type counterparts. In conclusion, CoREST co-repressors form protein complexes with PIASγ, which acts both as SUMO E3-ligase and as a protein stabilizer for CoREST proteins. This novel regulation of CoREST by PIASγ interaction and SUMOylation may serve to control cell fate determination during development.

As a conserved posttranslational modification, SUMOylation has been shown to play important roles in chromatin-related biological processes including transcription. However, how the SUMOylation machinery associates with chromatin is not clear. Here, we present evidence that multiple SUMOylation machinery components, including SUMO E1 proteins SAE1 and SAE2 and the PIAS (protein inhibitor of activated STAT) family SUMO E3 ligases, are primarily associated with the nuclear matrix rather than with chromatin. We show using nuclease digestion that all PIAS family proteins maintain nuclear matrix association in the absence of chromatin. Of importance, we identify multiple histones including H3 and H2A.Z as directly interacting with PIAS1 and demonstrate that this interaction requires the PIAS1 SAP (SAF-A/B, Acinus, and PIAS) domain. We demonstrate that PIAS1 promotes SUMOylation of histones H3 and H2B in both a SAP domain- and an E3 ligase activity-dependent manner. Furthermore, we show that PIAS1 binds to heat shock-induced genes and represses their expression and that this function also requires the SAP domain. Altogether, our study reveals for the first time the nuclear matrix as the compartment most enriched in SUMO E1 and PIAS family E3 ligases. Our finding that PIAS1 interacts directly with histone proteins also suggests a molecular mechanism as to how nuclear matrix-associated PIAS1 is able to regulate transcription and other chromatin-related processes.

Protein inhibitor of activated Stat 3 (Pias3) is implicated in guiding specification of rod and cone photoreceptors through post-translational modification of key retinal transcription factors. To investigate its role during retinal development, we deleted exon 2-5 of the mouse Pias3 gene, which resulted in complete loss of the Pias3 protein. Pias3−/− mice did not show any overt phenotype, and retinal lamination appeared normal even at 18 months. We detected reduced photopic b-wave amplitude by electroretinography following green light stimulation of postnatal day (P)21 Pias3−/− retina, suggesting a compromised visual response of medium wavelength (M) cones. No change was evident in response of short wavelength (S) cones or rod photoreceptors until 7 months. Increased S-opsin expression in the M-cone dominant dorsal retina suggested altered distribution of cone photoreceptors. Transcriptome profiling of P21 and 18-month-old Pias3−/− retina revealed aberrant expression of a subset of photoreceptor genes. Our studies demonstrate functional redundancy in SUMOylation-associated transcriptional control mechanisms and identify a specific, though limited, role of Pias3 in modulating spatial patterning and optimal function of cone photoreceptor subtypes in the mouse retina., Summary: Loss of Pias3 in mice results in altered dorso-ventral patterning of retinal cone photoreceptors by modulating the expression of a subset of genes, but does not affect rod development.

SUMOylation modifies the interactome, localization, activity, and lifespan of its target proteins. This process regulates several cellular machineries, including transcription, DNA damage repair, cell-cycle progression, and apoptosis. Accordingly, SUMOylation is critical in maintaining cellular homeostasis, and its deregulation leads to the corruption of a plethora of cellular processes that contribute to disease states. Among the proteins involved in SUMOylation, the protein inhibitor of activated STAT (PIAS) E3-ligases were initially described as transcriptional coregulators. Recent findings also indicate that they have a role in regulating protein stability and signaling transduction pathways. PIAS proteins interact with up to 60 cellular partners affecting several cellular processes, most notably immune regulation and DNA repair, but also cellular proliferation and survival. Here, we summarize the current knowledge about their role in tumorigenesis and cancer-related processes. Cancer Res; 77(7); 1542–7. ©2017 AACR.

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway as a negative regulator in mammals and Drosophila, but the function of PIAS in crustaceans is still unclear. In this study, a PIAS termed LvPIAS was cloned and characterized from Litopenaeus vannamei. The full length of LvPIAS was 3065 bp, including a 2361 bp open reading frame (ORF) coding for a protein of 786 aa. LvPIAS expression was most abundant in muscle and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly I: C and white spot syndrome virus (WSSV). LvPIAS could be induced by the transcription factor LvSTAT, but LvPIAS could inhibit the transcriptional activity of LvSTAT to the LvPIAS promoter conversely, which indicated that there was a negative feedback loop between LvSTAT and LvPIAS. Furthermore, RNAi-mediated knockdown of LvPIAS shrimps showed higher survival rate to WSSV infection than those in the control group (dsGFP injection), suggesting that LvPIAS may play a negatively role against WSSV infection.

The Protein Inhibitor of Activated STAT 1 (PIAS1) is an E3 SUMO ligase that plays important roles in various cellular pathways, including STAT signaling, p53 pathway, and the steroid hormone signaling pathway. PIAS1 can SUMOylate PML (at Lys-65 and Lys-160) and PML-RARα promoting their ubiquitin-mediated degradation. Increasing evidence shows that PIAS1 is overexpressed in various human malignancies, including prostate and lung cancers. To understand the mechanism of action of PIAS1, we developed a quantitative SUMO proteomic approach to identify potential substrates of PIAS1 in a system-wide manner. Our analyses enabled the profiling of 983 SUMO sites on 544 proteins, of which 204 SUMO sites on 123 proteins were identified as putative PIAS1 substrates. These substrates are involved in different cellular processes, such as transcriptional regulation, DNA binding and cytoskeleton dynamics. Further functional studies on Vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, revealed that PIAS1 exerts its effects on cell migration and cell invasion through the SUMOylation of VIM at Lys-439 and Lys-445 residues. VIM SUMOylation was necessary for its dynamic disassembly, and cells expressing a non-SUMOylatable VIM mutant showed reduced levels of proliferation and migration. Our approach not only provides a novel strategy for the identification of E3 SUMO ligase substrates, but also yields valuable biological insights into the unsuspected role of PIAS1 and VIM SUMOylation on cell motility.

Excessive nutrition promotes the pathogenesis of non-alcoholic steatohepatitis (NASH), characterized by the accumulation of pro-inflammation mediators in the liver. In the present study we investigated the regulation of pro-inflammatory transcription in hepatocytes by protein inhibitor of activated STAT 4 (PIAS4) in this process and the underlying mechanisms. We report that expression of the class III deacetylase SIRT1 was down-regulated in the livers of NASH mice accompanied by a simultaneous increase in the expression and binding activity of PIAS4. Exposure to high glucose stimulated the expression PIAS4 in cultured hepatocytes paralleling SIRT1 repression. Estrogen, a known NASH-protective hormone, ameliorated SIRT1 trans-repression by targeting PIAS4. Over-expression of PIAS4 enhanced, while PIAS4 knockdown alleviated, repression of SIRT1 transcription by high glucose. Lentiviral delivery of short hairpin RNA (shRNA) targeting PIAS4 attenuated hepatic inflammation in NASH mice by restoring SIRT1 expression. Mechanistically, PIAS4 promoted NF-κB-mediated pro-inflammatory transcription in a SIRT1 dependent manner. In conclusion, our study indicates that PIAS4 mediated SIRT1 repression in response to nutrient surplus contributes to the pathogenesis of NASH. Therefore, targeting PIAS4 might provide novel therapeutic strategies in the intervention of NASH.

Lear et al. report a novel molecular pathway in which Fibrosis Inducing E3 Ligase 1 (FIEL1) regulates TGFβ and fibrosis pathway through SUMO-E3 ligase PIAS4. They also develop a small molecule inhibitor toward FIEL1 that is highly effective in ameliorating fibrosis in mice., The E3 small ubiquitin-like modifier (SUMO) protein ligase protein inhibitor of activated STAT 4 (PIAS4) is a pivotal protein in regulating the TGFβ pathway. In this study, we discovered a new protein isoform encoded by KIAA0317, termed fibrosis-inducing E3 ligase 1 (FIEL1), which potently stimulates the TGFβ signaling pathway through the site-specific ubiquitination of PIAS4. FIEL1 targets PIAS4 using a double locking mechanism that is facilitated by the kinases PKCζ and GSK3β. Specifically, PKCζ phosphorylation of PIAS4 and GSK3β phosphorylation of FIEL1 are both essential for the degradation of PIAS4. FIEL1 protein is highly expressed in lung tissues from patients with idiopathic pulmonary fibrosis (IPF), whereas PIAS4 protein levels are significantly reduced. FIEL1 overexpression significantly increases fibrosis in a bleomycin murine model, whereas FIEL1 knockdown attenuates fibrotic conditions. Further, we developed a first-in-class small molecule inhibitor toward FIEL1 that is highly effective in ameliorating fibrosis in mice. This study provides a basis for IPF therapeutic intervention by modulating PIAS4 protein abundance.

Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms.

The signal transducers and activators of the transcription (STAT) family play an important role in regulatory and cellular functions by regulating the expression of a variety of genes, including cytokines and growth factors. In the present study, a Pinctada fucata STAT protein, termed PfSTAT, was described. The deduced amino acid sequence of PfSTAT contains the conserved STAT_bind domain and the SH2 domain, and the additional Bin/Amphiphysin/Rvs (BAR) domain, but does not have STAT_alpha and STAT_int domains. Multiple sequence alignments revealed that PfSTAT showed relatively low identity with vertebrate and other invertebrate STATs, and phylogenetic analysis indicated that the evolution of STAT may have been more complex and ancient. Gene expression analysis revealed that PfSTAT is involved in the immune response to polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus insertion operation. This study contributes to a better understanding of PfSTAT in protecting the pearl oyster from disease or injury caused by grafting.

The human telomeric protein TRF2 protects chromosome ends by facilitating their organization into the protective capping structure. Here we show that the stability of TRF2 is regulated via modification by the small ubiquitin-like modifiers (SUMO). TRF2 specifically interacts with and is sumoylated by PIAS1 in mammalian cells. The proteasome inhibitor stabilizes SUMO-conjugated TRF2 without affecting the level of unmodified TRF2, suggesting that SUMO conjugation is required for proteasomal degradation of TRF2. We also show that RNF4, a mammalian SUMO-targeted ubiquitin ligase, interacts with TRF2 in a SUMO-dependent manner and preferentially targets SUMO-conjugated TRF2 for ubiquitination. Collectively, our data demonstrate that the PIAS1-mediated sumoylation status of TRF2 serves as a molecular switch that controls the level of TRF2 at telomeres.

Protein inhibitor of activated STAT (PIAS) proteins are activation-suppressing proteins for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. The inhibitory mechanism of PIAS proteins in the Janus kinase (JAK)/STAT signaling pathway has been well studied in mammals and Drosophila. However, the roles of PIAS in crustaceans are unclear. In the present study, we identified PIAS in kuruma shrimp Marsupenaeus japonicus and found that its relative expression could be induced by Vibrio anguillarum stimulation. To explore the function of PIAS in shrimp infected with V. anguillarum, we performed an RNA interference assay. After knockdown of PIAS expression in shrimp subjected to V. anguillarum infection, bacterial clearance was enhanced and the survival rate increased compared with those in the control shrimp (dsGFP injection). Simultaneously, the expression levels of antimicrobial peptides (AMPs), including anti-lipopolysaccharide factor (ALF) A1, C1, C2, and CruI-1, increased. Further study revealed that knockdown of PIAS also enhanced STAT phosphorylation and translocation. Pulldown assay indicated that PIAS interacts with activated STAT in shrimp. In conclusion, PIAS negatively regulates JAK/STAT signaling by inhibiting the phosphorylation and translocation of STAT through the interaction between PIAS and STAT, which leads to the reduction of AMP expression in shrimp. Our results revealed a new mechanism of PIAS-mediated gene regulation of the STAT signal pathway.

Cobalt protoporphyrin (CoPP) is a potent HO-1 inducer and generally known to be an antioxidant in various cell types. Little is known about the CoPP-induced cyclooxygenase-2 (COX-2) expression and its downstream signaling in microglial cells. In current study, CoPP caused concentration- and time-dependent increases in COX-2 expression in microglial cells. Furthermore, activation of apoptosis signal-regulating kinase (ASK) 1/MAP kinase involved in CoPP-induced COX-2 expression in microglia. CoPP also induced P2X7 receptor activation, and treatment of P2X7 inhibitors effectively reduced CoPP-induced COX-2 expression. Protein inhibitor of activated STAT (PIAS) 1 is reported to be involved in modulating anti-inflammatory response through negative regulation of transcription factors. Interestingly, treatment with CoPP markedly induced PIAS1 degradation which is regulated by PI3K, Akt, and glycogen synthase kinase 3α/β (GSK3α/β) signaling pathways. These results suggest that CoPP induces COX-2 expression through activating P2X7 receptors and ASK1/MAP kinases as well as PIAS1 degradation signaling pathways. Our study provides a new insight into the regulatory effect of CoPP on neuroinflammation in microglial cells.

The development of the follicle and competent oocyte is highly coordinated, requiring interplay among several systems. These implicate endocrine, immune, and metabolic signals, intrafollicular paracrine factors from theca, mural, and cumulus granulosa cells, and the oocyte itself. Granulosa cells play a key role in their interaction. COV434 is one of the few human granulosa cell lines that can be used as an in vitro model for ovarian research. We aimed to evaluate the possible activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway by IL-6-type cytokines leukemia inhibitory factor (LIF) and oncostatin M (OSM) in COV434 cells. Expression of GP130 (glycoprotein 130), STAT3 (signal transducer and activators of transcription 3), PIAS3 (protein inhibitor of activated STAT 3), and SOCS3 (suppressor of cytokine signaling 3) genes after stimulation with LIF or OSM was assessed using RT-qPCR (real-time PCR). GP130 transcripts were significantly upregulated after incubation with LIF or OSM for 24h. Expression of the STAT3 gene was stimulated only after incubation with LIF, but not OSM. SOCS3 showed significant upregulation for all time periods and the levels of PIAS3 were initially down- and after 24h upregulated. Furthermore, the major signaling components of the JAK/STAT pathway, GP130 and STAT3, and the kinase activation patterns of STAT3, were examined at protein level. We found constitutive protein expression for GP130, STAT3, pSTAT3(ser727) and upregulation of pSTAT3(tyr705) by LIF and OSM. Our results demonstrate the activation of the JAK/STAT pathway by LIF and OSM in human granulosa cells.

Protein modifications with small ubiquitin–like modifier (SUMO) have been found to play a key role in regulating the formation of atherosclerosis.1–3 SUMO proteins covalently modify certain residues of specific target substrates and change the function of these substrates. It has been well established that the protein inhibitor of activated STAT (PIAS) family of proteins has not only SUMO E3 ligase activity, but also transrepression activity.4 Lerchenmuller et al5 have now reported a critical role for PIAS1 in regulating S100A6-mediated endothelial cell proliferation. In particular, they show that S100A6 induces PIAS1 expression and, consequently, increases the entry and progression of cell cycle in endothelial cells via inhibiting STAT1-mediated induction of IFITM1 (interferon-inducible transmembrane protein 1; Figure A). It has been reported that the direct inhibition of Thr55 phosphorylation of p53 induced by IFITM1 stabilizes p53 expression and upregulates p53-p21 expression, leading to cell cycle inhibition.9 Therefore, Lerchenmuller et al5 assume that this may be one of the regulatory mechanisms by which IFITM1 can inhibit endothelial cell proliferation. These data suggest that in response to vascular endothelial growth factor-A and subsequent S100A6 induction, PIAS1 plays a crucial role in regulating endothelial proliferation. In this editorial, the role of the PIAS family will be briefly reviewed by focusing on the regulation of endothelial functions, including inflammation, proliferation, and Kruppel like factor 2/endothelial nitric oxide synthase expression. Figure. A , Multiple functions of PIAS1. B , The domain structure of PIAS1. PIAS1 S90 site lie in the NF-κB (nuclear factor kappa-light chain-enhancer of activated B cells) interacting region, whereas PIAS1 S522 site does not lie in any of PIAS1’s functional domains involved in NF-κB binding or its SUMO E3 ligase activity (RLD) domain.6 Reprinted from Le …

The biological effects of cytokines are mediated by STAT proteins, a family of dimeric transcription factors. In order to elicit transcriptional activity, the STATs require activation by phosphorylation of a single tyrosine residue. Our experiments revealed that fully tyrosine-phosphorylated STAT dimers polymerize via Tyr(P)-Src homology 2 domain interactions and assemble into paracrystalline arrays in the nucleus of cytokine-stimulated cells. Paracrystals are demonstrated to be dynamic reservoirs that protect STATs from dephosphorylation. Activated STAT3 forms such paracrystals in acute phase liver cells. Activated STAT1, in contrast, does not normally form paracrystals. By reversing the abilities of STAT1 and STAT3 to be sumoylated, we show that this is due to the unique ability of STAT1 among the STATs to conjugate to small ubiquitin-like modifier (SUMO). Sumoylation had one direct effect; it obstructed proximal tyrosine phosphorylation, which led to semiphosphorylated STAT dimers. These competed with their fully phosphorylated counterparts and interfered with their polymerization into paracrystals. Consequently, sumoylation, by preventing paracrystal formation, profoundly curtailed signal duration and reporter gene activation in response to cytokine stimulation of cells. The study thus identifies polymerization of activated STAT transcription factors as a positive regulatory mechanism in cytokine signaling. It provides a unifying explanation for the different subnuclear distributions of STAT transcription factors and reconciles the conflicting results as to the role of SUMO modification in STAT1 functioning. We present a generally applicable system in which protein solubility is maintained by a disproportionately small SUMO-modified fraction, whereby modification by SUMO partially prevents formation of polymerization interfaces, thus generating competitive polymerization inhibitors.

Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity.

The Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling pathway is one of a limited number of signaling cascades that is extensively utilized for many developmental and homeostatic functions. The JAK/STAT pathway is evolutionarily conserved from insects to mammals, with homologous transduction machinery in each. Yet the mammalian pathway is composed of multiple members for each family of proteins, while flies have only a single homologue of most pathway components. This simplicity and the abundance of genetic, biochemical, and developmental tools make Drosophila an attractive model to investigate this important signaling pathway. This review provides a basic description of the Drosophila JAK/STAT cascade and summarizes currently available reagents and tools to study and manipulate the pathway.

It is known that the LIM homeodomain transcription factor Isl1 is highly expressed in all pancreatic endocrine cells and functions in regulating pancreatic development and insulin secretion. The Isl1 mutation has been found to be associated with type 2 diabetes, but the mechanism responsible for Isl1 regulation of insulin synthesis and secretion still needs to be elucidated. In the present study, the protein inhibitor of activated STAT Y (PIASy) was identified as a novel Isl1-interacting protein with a yeast two-hybrid system, and its interaction with Isl1 was further confirmed by a co-immunoprecipitation experiment. PIASy and Isl1 colocalize in human and mouse pancreas and NIT beta cells. Furthermore, PIASy and Isl1 upregulate insulin gene expression and insulin secretion in a dose-dependent manner by activating the insulin promoter. PIASy and Isl1 mRNA expression levels were also increased in type 2 diabetic db/db mice. In addition, our results demonstrate that PIASy and Isl1 cooperate to activate the insulin promoter through the Isl1 homeodomain and PIASy ring domain. These data suggest that that PIASy regulates insulin synthesis and secretion by interacting with Isl1 and provide new insight into insulin regulation, although the detailed molecular mechanism needs to be clarified in future studies.

The molecular mechanism controlling the zygotic genome activation (ZGA) in mammals remains poorly understood. The 2-cell (2C)-like cells spontaneously emerging from cultures of mouse embryonic stem cells (ESCs) share some key transcriptional and epigenetic programs with 2C-stage embryos. By studying the transition of ESCs into 2C-like cells, we identified developmental pluripotency associated 2 and 4 (Dppa2/4) as important regulators controlling zygotic transcriptional program through directly up-regulating the expression of double homeobox (Dux). In addition, we found that DPPA2 protein is sumoylated and its activity is negatively regulated by small ubiquitin-like modifier (Sumo) E3 ligase protein inhibitor of activated STAT 4 (PIAS4). PIAS4 is down-regulated during ZGA process and during transitioning of ESCs into 2C-like cells. Depleting Pias4 or overexpressing Dppa2/4 is sufficient to activate 2C-like transcriptional program, whereas depleting Dppa2/4 or forced expression of Pias4 or Sumo2–Dppa2 inhibits 2C-like transcriptional program. Furthermore, ectopic expression of Pias4 or Sumo2–Dppa2 impairs early mouse embryo development. In summary, our study identifies key molecular rivals consisting of transcription factors and a Sumo2 E3 ligase that regulate zygotic transcriptional program upstream of Dux., Analysis of cultured cells resembling those from 2-cell-stage embryos identifies transcription factors Dppa2/4 and the Sumo2 E3 ligase PIAS4 as key antagonistic regulators of the zygotic transcriptional program upstream of Dux.

It is well recognized that PIAS1, a SUMO (small ubiquitin-like modifier) E3 ligase, modulates such cellular processes as cell proliferation, DNA damage responses, and inflammation responses. Recent studies have shown that PIAS1 also plays a part in cell differentiation. However, the role of PIAS1 in adipocyte differentiation remains unknown. CCAAT/enhancer-binding protein β (C/EBPβ), a major regulator of adipogenesis, is a target of SUMOylation, but the E3 ligase responsible for the SUMOylation of C/EBPβ has not been identified. The present study showed that PIAS1 functions as a SUMO E3 ligase of C/EBPβ to regulate adipogenesis. PIAS1 expression was significantly and transiently induced on day 4 of 3T3-L1 adipocyte differentiation, when C/EBPβ began to decline. PIAS1 was found to interact with C/EBPβ through the SAP (scaffold attachment factor A/B/acinus/PIAS) domain and SUMOylate it, leading to increased ubiquitination and degradation of C/EBPβ. C/EBPβ became more stable when PIAS1 was silenced by RNA interference (RNAi). Moreover, adipogenesis was inhibited by overexpression of wild-type PIAS1 and promoted by knockdown of PIAS1. The mutational study indicated that the catalytic activity of SUMO E3 ligase was required for PIAS1 to restrain adipogenesis. Importantly, the inhibitory effect of PIAS1 overexpression on adipogenesis was rescued by overexpressed C/EBPβ. Thus, PIAS1 could play a dynamic role in adipogenesis by promoting the SUMOylation of C/EBPβ.

JAK/STAT is a conserved signaling pathway in higher eukaryotes that plays a critical role in different processes of ontogenesis. This article reviews the pathway structure at the molecular level, mainly in Drosophila as a model organism. The review is focused on the data concerning the relationship between this signaling pathway and transcription machinery of higher eukaryotes.

The JAK/STAT signaling pathway plays a critical role in different processes of ontogenesis in higher eukaryotes. The fruit fly drosophila is a convenient model used to study this pathway, since, in this system, its major components are represented by unique factors. This work describes the use of Drosophila melanogaster S2 cells to study the activation of JAK/STAT target genes. It was shown that S2 cells contain large quantities of STAT protein, which migrates into the nucleus in response to the treatment of the cell with pervanadate. In response to pervanadate, STAT, along with other transcription factors, is recruited onto the regulatory sequences of target genes and activates their transcription.

Protein expression abnormalities have been implicated in the pathophysiology of schizophrenia, but the underlying cause of these changes is not known. We sought to investigate ubiquitin and ubiquitin-like (UBL) systems (SUMOylation, NEDD8ylation, and Ufmylation) as putative mechanisms underlying protein expression abnormalities seen in schizophrenia. For this, we performed western blot analysis of total ubiquitination, free ubiquitin, K48- and K63-linked ubiquitination, and E1 activases, E2 conjugases, and E3 ligases involved in ubiquitination and UBL post-translational modifications in postmortem brain tissue samples from persons with schizophrenia (n=13) and comparison subjects (n=13). We studied the superior temporal gyrus (STG) of subjects from the Mount Sinai Medical Center brain collection that were matched for age, tissue pH, and sex. We found an overall reduction of protein ubiquitination, free ubiquitin, K48-linked ubiquitination, and increased K63 polyubiquitination in schizophrenia. Ubiquitin E1 activase UBA (ubiquitin activating enzyme)-6 and E3 ligase Nedd (neural precursor cell-expressed developmentally downregulated)-4 were decreased in this illness, as were E3 ligases involved in Ufmylation (UFL1) and SUMOylation (protein inhibitor of activated STAT 3, PIAS3). NEDD8ylation was also dysregulated in schizophrenia, with decreased levels of the E1 activase UBA3 and the E3 ligase Rnf7. This study of ubiquitin and UBL systems in schizophrenia found abnormalities of ubiquitination, Ufmylation, SUMOylation, and NEDD8ylation in the STG in this disorder. These results suggest a novel approach to the understanding of schizophrenia pathophysiology, where a disruption in homeostatic adaptation of the cell underlies discreet changes seen at the protein level in this illness.

Liver X receptors (LXRs) are nuclear receptors that function to modulate lipid metabolism as well as immune and inflammatory responses. Upon activation by their ligands, LXRs up-regulate a spectrum of gene transcription programs involved in cholesterol and fatty acid homeostasis. However, the mechanisms by which LXR-mediated transcriptional activation is regulated remain incompletely understood. Here, we show that PIAS1, a member of the protein inhibitor of the activated STAT family of proteins with small ubiquitin-like modifier (SUMO) E3 ligase activity, acts to suppress LXR ligand-dependent transcriptional activation of the lipogenic program in hepatocytes. We found that liver mRNA expression levels of Pias1 and Pias3 were inversely associated with those of genes involved in lipogenesis in mouse models with diet-induced or genetic obesity. Overexpression of PIAS1 in primary hepatocytes resulted in a reduction of LXR ligand-induced fatty acid synthesis and suppression of the expression of lipogenic genes, including Srebp1c and Fas. Moreover, PIAS1 was able to interact with LXRβ and repress its transcriptional activity upon ligand stimulation, which did not require PIAS1-promoted SUMO modification of LXRβ. In addition, PIAS1 could also interact with PGC-1β and attenuate its association with LXRβ, blunting the ability of PGC-1β to co-activate LXRβ. Importantly, PIAS1 impaired LXRβ binding to its target DNA sequence. Taken together, our results suggest that PIAS1 may serve as a lipogenic regulator by negatively modulating LXRs in a SUMOylation-independent manner.

Interstitial fibrosis represents a key pathological process in non-alcoholic steatohepatitis (NASH). In the liver, fibrogenesis is primarily mediated by activated hepatic stellate cells (HSCs) transitioning from a quiescent state in response to a host of stimuli. The molecular mechanism underlying HSC activation is not completely understood. Here we report that there was a simultaneous up-regulation of PIAS4 expression and down-regulation of SIRT1 expression accompanying increased hepatic fibrogenesis in an MCD-diet induced mouse model of NASH. In cultured primary mouse HSCs, stimulation with high glucose activated PIAS4 while at the same time repressed SIRT1. Over-expression of PIAS4 directly repressed SIRT1 promoter activity. In contrast, depletion of PIAS4 restored SIRT1 expression in HSCs treated with high glucose. Estrogen, a known NASH-protective hormone, antagonized HSC activation by targeting PIAS4. Lentivirus-mediated delivery of short hairpin RNA (shRNA) targeting PIAS4 in mice ameliorated MCD diet induced liver fibrosis by normalizing SIRT1 expression in vivo. PIAS4 promoted HSC activation in a SIRT1-dependent manner in vitro. Mechanistically, PIAS4 mediated SIRT1 repression led to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters. Taken together, our data suggest SIRT1 trans-repression by PIAS4 plays an important role in HSC activation and liver fibrosis.

The mechanism by which MORPHEUS' MOLECULE1 (MOM1) contributes to transcriptional gene silencing has remained elusive since the gene was first identified and characterized. Here, we report that two Arabidopsis thaliana PIAS (PROTEIN INHIBITOR OF ACTIVATED STAT)-type SUMO E3 ligase-like proteins, PIAL1 and PIAL2, function redundantly to mediate transcriptional silencing at MOM1 target loci. PIAL1 and PIAL2 physically interact with each other and with MOM1 to form a high molecular mass complex. In the absence of either PIAL2 or MOM1, the formation of the high molecular mass complex is disrupted. We identified a previously uncharacterized IND (interacting domain) in PIAL1 and PIAL2 and demonstrated that IND directly interacts with MOM1. The CMM2 (conserved MOM1 motif 2) domain of MOM1 was previously shown to be required for the dimerization of MOM1. We demonstrated that the CMM2 domain is also required for the interaction of MOM1 with PIAL1 and PIAL2. We found that although PIAL2 has SUMO E3 ligase activity, the activity is dispensable for PIAL2's function in transcriptional silencing. This study suggests that PIAL1 and PIAl2 act as components of the MOM1-containing complex to mediate transcriptional silencing at heterochromatin regions.

Signal transducer and activator of transcription (STAT) proteins are key signaling molecules in response to cytokines and in regulating T cell biology. However, there are contradicting reports on whether STAT is involved in T-cell antigen receptor (TCR) signaling. To better define the role of STAT in TCR signaling, we activated the CD4/CD8-associated Lck kinase by co-crosslinking TCR and CD4/CD8 co-receptors in human peripheral blood T cells. Sequential STAT1, STAT3 and STAT5 activation was observed 1 hour after TCR stimulation suggesting that STAT proteins are not the immediate targets in the TCR complex. We further identified interferon-gamma as the key cytokine in STAT1 activation upon TCR engagement. In contrast to transient STAT activation in cytokine response, this autocrine/paracrine-induced STAT activation was sustained. It correlated with the absence of two suppressor of cytokine signaling (SOCS) proteins, SOCS3 and cytokine-inducible SH2 containing protein, that are negative feedback regulators of STAT signaling. Moreover, enforced expression of SOCS3 inhibited tyrosine phosphorylation of zeta-associated protein kinase of 70 kD in TCR-stimulated human Jurkat T cells. This is the first report demonstrating delayed and prolonged STAT activation coordinated with the loss of SOCS expression in human primary T cells after co-crosslinking of TCR and CD4/CD8 co-receptors.

ErbB4 is a receptor tyrosine kinase implicated in the development and homeostasis of the heart, central nervous system, and mammary gland. Cleavable isoforms of ErbB4 release a soluble intracellular domain (ICD) that can translocate to the nucleus and function as a transcriptional coregulator. In search of regulatory mechanisms of ErbB4 ICD function, we identified PIAS3 as a novel interaction partner of ErbB4 ICD. In keeping with the small ubiquitin-like modifier (SUMO) E3 ligase function of protein inhibitor of activated STAT (PIAS) proteins, we showed that the ErbB4 ICD is modified by SUMO, and that PIAS3 stimulates the SUMOylation. Upon overexpression of PIAS3, the ErbB4 ICD generated from the full-length receptor accumulated into the nucleus in a manner that was dependent on the functional nuclear localization signal of ErbB4. In the nucleus, ErbB4 colocalized with PIAS3 and SUMO-1 in promyelocytic leukemia nuclear bodies, nuclear domains involved in regulation of transcription. Accordingly, PIAS3 overexpression had an effect on the transcriptional coregulatory activity of ErbB4, repressing its ability to coactivate transcription with Yes-associated protein. Finally, knockdown of PIAS3 with siRNA partially rescued the inhibitory effect of the ErbB4 ICD on differentiation of MDA-MB-468 breast cancer and HC11 mammary epithelial cells. Our findings illustrate that PIAS3 is a novel regulator of ErbB4 receptor tyrosine kinase, controlling its nuclear sequestration and function.

Janus kinase 1/signal transducers and activators of transcription 3 (JAK1/STAT3) pathway is one of the recognized oncogenic signaling pathways that frequently overactivated in a variety of human tumors. Despite rapid progress in elucidating the molecular mechanisms of activation of JAK/STAT pathway, the processes that regulate JAK/STAT deactivation need to be further clarified. Here we demonstrate that CUE domain-containing 2 (CUEDC2) inhibits cytokine-induced phosphorylation of JAK1 and STAT3 and the subsequent STAT3 transcriptional activity. Further analysis by a yeast two-hybrid assay showed that CUEDC2 could engage in a specific interaction with a key JAK/STAT inhibitor, SOCS3 (suppressors of cytokine signaling 3). The interaction between CUEDC2 and SOCS3 is required for the inhibitory effect of CUEDC2 on JAK1 and STAT3 activity. Additionally, we found CUEDC2 functions collaboratively with SOCS3 to inhibit JAK1/STAT3 signaling by increasing SOCS3 stability via enhancing its association with Elongin C. Therefore, our findings revealed a new biological activity for CUEDC2 as the regulator of JAK1/STAT3 signaling and paved the way to a better understanding of the mechanisms by which SOCS3 has been linked to suppression of the JAK/STAT pathway.

In their journey to acquire the ability to fertilize the egg, numerous intracellular signaling systems are activated in spermatozoa, leading to an increase in protein tyrosine phosphorylation. Although the JAK/STAT signaling pathway is usually associated with the activation of transcription of specific genes, our laboratory previously demonstrated the presence of the IL6 receptor (IL6R) and the Janus kinase 1 (JAK1) in human spermatozoa, a cell that is mostly transcriptionally inactive. In order to determine the importance of the JAK/STAT signaling pathway, our objectives were to identify and characterize the mediators of this system in human sperm. Cell fractionation and surface biotinylation assays clearly demonstrated that IL6R is expressed at the sperm membrane surface. The kinase JAK1 is enriched in membrane fractions and is activated during human sperm capacitation as suggested by its increase in phosphotyrosine content. Many signal transducer and activator of transcription (STAT) proteins are expressed in human sperm, including STAT1, STAT3, STAT4, STAT5, and STAT6. Among them, only STAT1 and STAT5 were detected in the cytosolic fraction. All the detected STAT proteins were enriched in the cytoskeletal structures. STAT4 was present in the perinuclear theca, whereas JAK1, STAT1, and STAT5 were detected in the fibrous sheath. Indirect immunofluorescence studies showed that JAK1 and STAT1 colocalized in the neck region and that STAT4 is present at the equatorial segment and flagella. The presence of STAT proteins in sperm structural components suggests that their role is different from their well-known transcription factor activity in somatic cells, but further investigations are required to determine their role in sperm function.

Metastasis tumor antigen 1 (MTA1), a component of the Mi-2·nucleosome remodeling and deacetylase complex, plays a crucial role in gene transcription, but the mechanism involved remains largely unknown. Here, we report that MTA1 is a substrate for small ubiquitin-related modifier 2/3 (SUMO2/3) in vivo. Protein inhibitor of activated STAT (PIAS) proteins enhance SUMOylation of MTA1 and may participate in paralog-selective SUMOylation, whereas sentrin/SUMO-specific protease 1 (SENP1) and 2 may act as deSUMOylation enzymes for MTA1. Moreover, MTA1 contains a functional SUMO-interacting motif (SIM) at its C terminus, and SIM is required for the efficient SUMOylation of MTA1. SUMO conjugation on Lys-509, which is located within the SUMO consensus site, together with SIM synergistically regulates the co-repressor activity of MTA1 on PS2 transcription, probably by recruiting HDAC2 onto the PS2 promoter. Interestingly, MTA1 may up-regulate the expression of SUMO2 via interaction with RNA polymerase II and SP1 at the SUMO2 promoter. These findings not only provide novel mechanistic insights into the regulation of the transcriptional repressor function of MTA1 by SUMOylation and SIM but also uncover a potential function of MTA1 in modulating the SUMOylation pathway.

Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins. A Dd-PIAS null mutant strain displays a normal terminal morphology but with accelerated development once cells are aggregated. In contrast, Dd-PIAS overexpressor strains demonstrate delayed aggregation, almost no slug phototaxis, impaired slug motility, and a prolonged slug migration period. This strain is a near phenocopy of the Dd-STATa null mutant, although it eventually forms a fruiting body, albeit inefficiently. The expression of several Dd-STATa-activated genes is upregulated in the Dd-PIAS null mutant and there is ectopic expression of pstAB makers. The concentration of a PIAS-green fluorescent protein (GFP) fusion protein, expressed under the PIAS promoter, is greatest in the pstO cells and gradually decreases with proximity to the tip of the slug and culminant: a pattern diametrically opposite to that of Dd-STATa. Our results suggest a functional interrelationship between Dd-PIAS and Dd-STATa that influences gene expression and development.

Originally identified as cytokine inhibitors, protein inhibitors of activated STAT (PIAS) are shown to regulate activities of a plethora of proteins and influence diverse processes such as immune response, cancer formation, and cell cycle progression. However, the roles of PIAS during vertebrate embryogenesis are less understood. In this study, we report isolation and initial characterization of all four PIAS genes from Xenopus laevis. The Xenopus PIAS genes are expressed throughout early development and have overlapping and distinct expression patterns, with, for example, high levels of PIAS2 in the notochord and strong expression of PIAS4 in the neural and neural crest derivatives. Overexpression of PIAS disrupts mesoderm induction and impairs body axis formation. PIAS proteins have differential ability to regulate signals from the growth factors activin, bone morphogenetic protein 4 (BMP4), and Wnt8. Our data suggest that Xenopus PIAS play important roles in mesodermal induction and patterning during early frog development.

The small ubiquitin-related modifier (SUMO) system of higher eukaryotes plays important roles in normal cell division, especially in chromosome segregation. However, only a few mitotic SUMO substrates have been identified in mammals. Here, we show that the mitotic kinase Aurora-B can be modified by SUMO. The E3 SUMO-protein ligase PIAS3 [protein inhibitor of activated STAT (signal transducer and activator of transcription)] dramatically enhanced poly-SUMO-2/3 conjugation of Aurora-B, whereas the SUMO-specific isopeptidase SENP2 (Sentrin/SUMO-specific protease) specifically deconjugated SUMO from Aurora-B. The Lys-202 residue on human Aurora-B was preferentially modified by SUMO, and enhancement of SUMOylation in cells facilitated Aurora-B autophosphorylation, which is essential for its activation. Conversely, SENP2-mediated deSUMOylation of Aurora-B down-regulated its autophosphorylation in cells and also impaired its re-activation in Aurora inhibitor VX-680-treated mitotic cells. Poly-SUMO-2 conjugation of Aurora-B occurred during the M phase of the cell cycle, and both SUMO-2 and PIAS3 were localized adjacent to Aurora-B in the kinetochores in early mitosis. Based on these results, we propose that Aurora-B is a novel mitotic SUMO substrate and that its kinase activity is fine-tuned by the SUMO system.

Discovery of emerging REGγ-regulated proteins has accentuated the REGγ-proteasome as an important pathway in multiple biological processes, including cell growth, cell cycle regulation, and apoptosis. However, little is known about the regulation of the REGγ-proteasome pathway. Here we demonstrate that REGγ can be SUMOylated in vitro and in vivo by SUMO-1, SUMO-2, and SUMO-3. The SUMO-E3 protein inhibitor of activated STAT (PIAS)1 physically associates with REGγ and promotes SUMOylation of REGγ. SUMOylation of REGγ was found to occur at multiple sites, including K6, K14, and K12. Mutation analysis indicated that these SUMO sites simultaneously contributed to the SUMOylation status of REGγ in cells. Posttranslational modification of REGγ by SUMO conjugation was revealed to mediate cytosolic translocation of REGγ and to cause increased stability of this proteasome activator. SUMOylation-deficient REGγ displayed attenuated ability to degrade p21(Waf//Cip1) due to reduced affinity of the REGγ SUMOylation-defective mutant for p21. Taken together, we report a previously unrecognized mechanism regulating the activity of the proteasome activator REGγ. This regulatory mechanism may enable REGγ to function as a more potent factor in protein degradation with a broader substrate spectrum.