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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.

Protein inhibitors of activated STAT (PIAS) are involved in the regulation of the JAK/STAT signaling pathway and have interactions with NF-κB, p73 and p53. These proteins regulate immune responses; therefore dysregulation in their expression leads to several immune-mediated disorders. In the present study, we examined expression of PIAS1-4 in peripheral blood of patients with acute/chronic inflammatory demyelinating polyradiculoneuropathy (AIDP/CIDP) compared with healthy subjects. We demonstrated down-regulation of all PIAS genes in both AIDP and CIDP cases compared with controls. Similarly, comparisons in gender-based groups revealed down-regulation of these gene0s in patients of each gender compared with gender-matched controls. There was no significant difference in expression of PIAS genes between AIDP and CIDP cases. Based on the area under the receiver operating characteristic curves, PIAS1-4 genes could distinguish between inflammatory demyelinating polyradiculoneuropathy and healthy status with accuracy values of 0.87, 0.87, 0.79 and 0.80, respectively. In differentiation between AIDP cases and healthy controls, these values were 0.92, 0.92, 0.83 and 0.86, respectively. Finally, PIAS1-4 genes could discriminate CIDP from healthy status with accuracy values of 0.82, 0.83, 0.75 and 0.75, respectively. The current study underscores the role of PIAS genes in the pathogenesis of inflammatory demyelinating polyradiculoneuropathy and their potential usage as biomarkers.

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.

Aim: Protein inhibitor of activated STAT ( PIAS) family includes transcriptional regulator proteins with SUMO E3 ligase activity. They regulate expression of several genes involved in cell proliferation, differentiation and survival. Method: We evaluated expression of PIAS1–4 genes in 54 breast cancer tissues and their paired adjacent noncancerous tissues. Results: PIAS2 and PIAS3 genes were significantly downregulated in tumoral tissues compared with adjacent noncancerous tissues. PIAS1–3 expressions were significantly lower in estrogen receptor (ER+) samples compared with ER- samples while PIAS4 had the opposite trend. PIAS3 expression was significantly higher in grade 1 samples compared with grade 2 samples. Conclusion: These findings highlight the role of PIAS genes in the pathogenesis of breast cancer and their association with determinants of response to antihormone therapies.

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.

Objectives Protein inhibitors of activated STAT (PIAS) are transcription co-regulator of the Janus kinase/signal transducer and activator of transcription signaling pathway as well as nuclear factor-κB family of transcription factors. Both of them are involved in cytokine release during inflammatory response. Patients and methods Considering the role of cytokine imbalance in the pathogenesis of multiple sclerosis (MS), we compared blood expressions of PIAS1-4 genes in 48 interferon β (IFNβ)-treated MS patients with those of healthy subjects by means of real time PCR. Results Although the expression levels of these genes were not significantly different between MS patients and healthy subjects, significant inverse correlations have been found between PIAS1 expression and age at disease onset. PIAS2 and PIAS3 expressions were inversely correlated with Expanded Disability Status Scale (EDSS) in patients. Moreover, PIAS3 expression was correlated with disease duration in patients and with age in controls. In addition, PIAS4 expression was inversely correlated with EDSS and age at disease onset while it was positively correlated with disease duration. Conclusion The present study provides evidences for altered expression of PIAS genes in IFNβ-treated MS patients compared with healthy subjects. However, future studies are needed for elaboration of their exact function in this disorder.

The protein inhibitor of activated STAT (PIAS) functions in diverse aspects, including immune response, cell apoptosis, cell differentiation, and proliferation. In the present study, thePIASin the pearl oysterPinctada fucata martensiiwas characterized. The sequence features of PmPIAS were similar to that of other PIAS sequences with PIAS typical domains, including SAP, Pro-Ile-Asn-Ile-Thr (PINIT), RLD domain, AD, and S/T-rich region. Homologous analysis showed that PmPIAS protein sequence showed the conserved primary structure compared with other species. Ribbon representation of PIAS protein sequences also showed a conserved structure among species, and the PINIT domain and RLD domain showed the conserved structure compared with the sequence ofHomo sapiens. The expression pattern ofPmPIASin different tissues showed significant high expression in the gonad.PmPIASalso exhibited a significantly higher expression in the 1 and 2 days after cold tolerance stress (17°C) and showed its potential in the cold tolerance. The SNP analysis of the exon region ofPmPIASobtained 18 SNPs, and among them, 11 SNPs showed significance among different genotypes and alleles between cold tolerance selection line and base stock, which showed their potential in the breeding for cold tolerance traits.

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.

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.

Proinflammatory cytokine activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signal transduction pathway is a critical event in the pathogenesis and progression of rheumatoid arthritis. Under normal conditions, JAK/STAT signaling reflects the influence of negative regulators of JAK/STAT, exemplified by the suppressor of cytokine signaling and protein inhibitor of activated STAT. However, in rheumatoid arthritis (RA) both of these regulators are dysfunctional. Thus, continuous activation of JAK/STAT signaling in RA synovial joints results in the elevated level of matrix metalloproteinase gene expression, increased frequency of apoptotic chondrocytes and most prominently ‘apoptosis resistance’ in the inflamed synovial tissue. Tofacitinib, a JAK small molecule inhibitor, with selectivity for JAK2/JAK3 was approved by the United States Food and Drug Administration (US FDA) for the therapy of RA. Importantly, tofacitinib has demonstrated significant clinical efficacy for RA in the post-US FDA-approval surveillance period. Of note, the success of tofacitinib has spurred the development of JAK1, JAK2 and other JAK3-selective small molecule inhibitors, some of which have also entered the clinical setting, whereas other JAK inhibitors are currently being evaluated in RA clinical trials.

Rationale: Colitis-associated colorectal cancer (CAC) usually exhibits an accelerated disease progression, an increased resistance to therapeutic drugs and a higher mortality rate than sporadic colorectal cancer (CRC). PIAS3 is a member of the protein inhibitor of activated STAT (PIAS) family; however, little is known about the expression and biological functions of PIAS3 in CAC. The aim of our study was to investigate the biological mechanisms of PIAS3 in CAC. Methods: PIAS3 expression was examined in colon tissues of CAC/CRC patients and azoxymethane-dextran sulfate sodium (AOM-DSS)-induced mice. The role of PIAS3 was studied using a series of in vitro, in vivo and clinical approaches. Results: Downregulated PIAS3 expression, upregulated miR-18a expression and highly activated NF-κB and STAT3 were observed in colon tissues of CAC/CRC patients and AOM-DSS-induced mice. In vitro experiments revealed that PIAS3 significantly inhibited the activation of NF-κB and STAT3 and demonstrated that activated NF-κB and STAT3 transcriptionally regulated miR-18a level, and up-regulation of miR-18a expression led to defective PIAS3 expression. Moreover, PIAS3-mediated autoregulatory feedback loops (PIAS3/NF-κB/miR-18a and PIAS3/STAT3/miR-18a) were verified in vitro and were found to regulate cell proliferation. Additionally, modulation of the feedback loops via overexpression of PIAS3 or knockdown of miR-18a significantly inhibited cell proliferation in a mouse CRC xenograft model. Furthermore, upregulation of PIAS3 by intracolonic administration of PIAS3 lentivirus or anti-miR-18a lentivirus in AOM-DSS-induced mice led to dramatically reduced tumor sizes/numbers, whereas knockdown of PIAS3 in CAC mice significantly promoted tumor growth. Conclusion: Our data clearly show that PIAS3-mediated feedback loops control cell proliferation and function as robust driving forces for CAC progression. Targeting these highly activated feedback loops might offer promising therapeutic strategies for CAC.

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.

Misregulation of transcription factors, including signal transducer and activator of transcription (STAT) proteins, leads to inappropriate gene expression patterns that can promote tumor initiation and progression. Under physiologic conditions, STAT signaling is stimulus-dependent and tightly regulated by endogenous inhibitors, namely suppressor of cytokine signaling (SOCS) proteins, phosphatases, and protein inhibitor of activated STAT (PIAS) proteins. However, in tumorigenesis, STAT proteins become constitutively active and promote the expression of pro-growth and pro-survival genes. Although STAT activation has been widely implicated in cancer, therapeutic STAT inhibitors are still largely absent from the clinic. This review dissects the mechanisms of action of two families of endogenous STAT inhibitors, the SOCS and PIAS families, to potentially inform the development of novel therapeutic inhibitors.

Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic inflammation in synovial tissues. Hyperplasia of synovial tissues leads to the formation of pannus that invades the joint cartilage and bone, resulting in joint destruction. Fas ligand (FasL), which is a member of the tumor necrosis factor superfamily, contributes to the pathogenesis of autoimmune diseases, including RA. The current study attempted to identify genes whose expressions in rheumatoid fibroblast-like synoviocytes (RA-FLS) were regulated by FasL, using cDNA microarray. A total of four individual lines of primary cultured RA-FLS were incubated either with recombinant human FasL protein or PBS as an unstimulated control for 12 h. Gene expression was detected using a microarray assay. The results revealed the expression profiles of genes in RA-FLS regulated by Fas and investigated the functions of the genes that were regulated. Among the genes in this profile, the mRNA expression changes of the following genes were indicated to be of note using RT-qPCR: Dual specificity phosphatase 6, epiregulin, interleukin 11, angiopoietin-like 7, protein inhibitor of activated STAT 2 and growth differentiation factor 5. These genes may affect the pathogenesis of RA by affecting apoptosis, proliferation, cytokine production, cytokine-induced inflammation, intracellular signaling, angiogenesis, bone destruction and chondrogenesis. To the best of our knowledge, the current study is the first study to reveal the expression profile of genes in RA-FLS regulated by FasL. The data demonstrated that FasL may regulate the expression of a number of key molecules in RA-FLS, thus affecting RA pathogenesis. Further studies of the genes detected may improve the understanding of RA pathogenesis and provide novel treatment targets for RA.

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.

Nutrient restriction, also known as caloric restriction, has been extensively examined for its positive impact on lifespan, immune system boost, and aging. In addition, nutrient restriction is implicated in decreasing cancer initiation and progression. Given the phenotypic changes associated with nutrient restriction, we hypothesized significant protein expression alterations must be associated with caloric restriction. To compare the molecular and phenotypic changes caused by glucose restriction and fetal bovine serum (FBS) restriction there is need for an efficient model system. We establish three dimensional cell culture models, known as spheroids, in the HCT 116 colorectal cancer cell line as a high throughput model for studying the proteomic changes associated with nutrient restriction. Flow cytometry was used to assess apoptosis and autophagy levels in the spheroids under nutrient restriction. Isobaric tags for relative and absolute quantification (iTRAQ) and liquid chromatography tandem mass spectrometry were used to determine differential protein abundances between the nutrient restriction conditions. We identified specific proteins that have implications in cancer progression and metastasis that are differentially regulated by restriction of either glucose or serum. These proteins include the up regulation of sirtuin 1 (SIRT1) and protein inhibitor of activated STAT 1 (PIAS1) and down regulation of multi-drug resistance protein (MRP1) and Zinc finger and BTB domain-containing protein 7A (ZBTB7). The results indicate nutrient restriction causes lower apoptotic and higher autophagy rates in HCT 116 spheroids. In addition, proteins shown to be differentially regulated by both glucose and serum restriction were similarly regulated.

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.

Background Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family and the pestivius virus group. BVDV is responsible for significant economic loss in cattle industry worldwide because of reducing reproductive performance, increasing incidence of other diseases and mortality among young stock. The core (C) protein of the Flaviviridae family member is involved in host antiviral immune response through activation of related signaling pathways that affect the viral replication. However, the influence of C protein-interaction partners in BVDV infections is poorly defined. Methods To explore C-protein-interacting partners, yeast two-hybrid was used to screen the interaction protein of C protein using bovine peripheral blood mononuclear cell (PBMC) cDNA library. The co-immunoprecipitation and confocal assays were manipulated to determine the interaction between potential partners and C protein. Knockdown and overexpression of the partner were used to examine whether the C-protein-interacting partner plays a role in BVDV proliferation and virulence. Meanwhile, qRT-PCR and western blot assays were used to investigate the effect of C protein and C-protein-interacting partner on the immune response of host cells. Results We identified protein inhibitor of activated STAT 4 (PIAS4) as a novel interacting partner of the BVDV C protein. Co-immunoprecipitation and confocal assays demonstrated a strong interaction between C protein and PIAS4. Silencing of PIAS4 with small interfering RNA suppressed C protein expression and BVDV growth, while overexpression of PISA4 increased C protein expression and BVDV growth. The overexpression of PIAS4 increased the cell apoptosis. Meanwhile, the expressions of STAT4, SOCS3, IFITM, IFN-α were negatively regulated by the expression of PIAS4. The expression of C protein suppressed the antiviral proteins expression, and the inhibition effect was enhanced by interaction of PIAS4 and C protein. These results highlighted the beneficial properties of cellular PIAS4 for BVDV protein expression and growth. Conclusions This study provides reliable clues for understanding the roles of PIAS4 in the regulation of BVDV growth.

Background: Hematoporphyrin derivative (HPD) has a sensibilization effect in lung adenocarcinoma. This study was conducted to identify the target genes of HPD in lung adenocarcinoma. Methods: RNA sequencing was performed using the lung adenocarcinoma cell line A549 after no treatment or treatment with X-ray or X-ray + HPD. The differentially expressed genes (DEGs) were screened using Mfuzz package by noise-robust soft clustering analysis. Enrichment analysis was carried out using “BioCloud” online tool. Protein–protein interaction (PPI) network and module analyses were performed using Cytoscape software. Using WebGestalt tool and integrated transcription factor platform (ITFP), microRNA target and transcription factor (TF) target pairs were separately predicted. An integrated regulatory network was visualized with Cytoscape software. Results: A total of 815 DEGs in the gene set G1 (continuously dysregulated genes along with changes in processing conditions [untreated—treated with X-ray—X-ray + treated with HPD]) and 464 DEGs in the gene set G2 (significantly dysregulated between X-ray + HPD-treated group and untreated/X-ray-treated group) were screened. The significant module identified from the PPI network for gene set G1 showed that ribosomal protein L3 (RPL3) gene could interact with heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1). TFs AAA domain containing 2 (ATAD2) and protein inhibitor of activated STAT 1 (PIAS1) were separately predicted for the genes in gene set G1 and G2, respectively. In the integrated network for gene set G2, ubiquitin-specific peptidase 25 (USP25) was targeted by miR-200b, miR-200c, and miR-429. Conclusion: RPL3, HSP90AA1, ATAD2, and PIAS1 as well as USP25, which is targeted by miR-200b, miR-200c, and miR-429, may be the potential targets of HPD in lung adenocarcinoma.

Background The Slug-E-cadherin axis plays a critical role in non-small-cell lung cancers (NSCLCs) where aberrant upregulation of Slug promotes cancer metastasis. Now, the post-translational modifications of Slug and their regulation mechanisms still remain unclear in lung cancer. Hence, exploring the protein linkage map of Slug is of great interest for investigating the scenario of how Slug protein is regulated in lung cancer metastasis. Methods The Slug associated proteins, Ubc9 and SUMO-1, were identified using yeast two-hybrid screening; and in vitro SUMOylation assays combined with immunoprecipitation and immunoblotting were performed to explore the detail events and regulations of Slug SUMOylation. The functional effects of SUMOylation on Slug proteins were examined by EMSA, reporter assay, ChIP assay, RT-PCR, migration and invasion assays in vitro, tail vein metastatic analysis in vivo, and also evaluated the association with clinical outcome of NSCLC patients. Results Slug protein could interact with Ubc9 and SUMO-1 and be SUMOylated in cells. Amino acids 130–212 and 33–129 of Slug are responsible for its binding to Ubc9 and protein inhibitor of activated STAT (PIAS)y, respectively. SUMOylation could enhance the transcriptional repression activity of Slug via recruiting more HDAC1, resulting in reduced expression of downstream Slug target genes and enhanced lung cancer metastasis. In addition, hypoxia could increase Slug SUMOylation through attenuating the interactions of Slug with SENP1 and SENP2. Finally, high expression Slug and Ubc9 levels were associated with poor overall survival among NSCLC patients. Conclusions Ubc9/PIASy-mediated Slug SUMOylation and subsequent HDAC1 recruitment may play a crucial role in hypoxia-induced lung cancer progression, and these processes may serve as therapeutic targets for NSCLC. Electronic supplementary material The online version of this article (10.1186/s13046-018-0996-8) contains supplementary material, which is available to authorized users.

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.

S mall u biquitin-like mo difier (SUMO) is used by the intrinsic antiviral immune response to restrict viral pathogens, such as herpes simplex virus 1 (HSV-1). Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined. We show that unconjugated SUMO levels are largely maintained throughout infection regardless of the presence of ICP0, the HSV-1 SUMO-targeted ubiquitin ligase. Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate. These data highlight the continued importance for SUMO signaling throughout infection. We show that the SUMO ligase protein inhibitor of activated STAT 4 (PIAS4) is upregulated during HSV-1 infection and localizes to nuclear domains that contain viral DNA. PIAS4 is recruited to sites associated with HSV-1 genome entry through SUMO interaction motif (SIM)-dependent mechanisms that are destabilized by ICP0. In contrast, PIAS4 accumulates in replication compartments through SIM-independent mechanisms irrespective of ICP0 expression. Depletion of PIAS4 enhances the replication of ICP0-null mutant HSV-1, which is susceptible to restriction by the intrinsic antiviral immune response. The mechanisms of PIAS4-mediated restriction are synergistic with the restriction mechanisms of a characterized intrinsic antiviral factor, promyelocytic leukemia protein, and are antagonized by ICP0. We provide the first evidence that PIAS4 is an intrinsic antiviral factor. This novel role for PIAS4 in intrinsic antiviral immunity contrasts with the known roles of PIAS proteins as suppressors of innate immunity. IMPORTANCE Posttranslational modifications with s mall u biquitin-like mo difier (SUMO) proteins regulate multiple aspects of host immunity and viral replication. The p rotein i nhibitor of a ctivated S TAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of innate immune signaling. We now identify a unique and contrasting role for PIAS proteins as positive regulators of the intrinsic antiviral immune response to herpes simplex virus 1 (HSV-1) infection. We show that PIAS4 relocalizes to nuclear domains that contain viral DNA throughout infection. Depletion of PIAS4, either alone or in combination with the intrinsic antiviral factor promyelocytic leukemia protein, significantly impairs the intrinsic antiviral immune response to HSV-1 infection. Our data reveal a novel and dynamic role for PIAS4 in the cellular-mediated restriction of herpesviruses and establish a new functional role for the PIAS family of SUMO ligases in the intrinsic antiviral immune response to DNA virus infection.

The JAK/STAT pathway is induced by the binding of a cytokine to its cognate receptor. The receptor’s engagement with the cytokine recruits a JAK protein, which activates itself via auto/trans-phosphorylation. In turn, the activated JAKs recruit and phosphorylate STAT proteins. The phosphorylated STAT proteins form a dimer, translocate to the cell nucleus and acts as a transcription factor to induce gene expression. In this way, the JAK/STAT pathway can mediate a cell’s response to extracellular signals. The proteins ultimately induced by the JAK/STAT pathway contribute to processes such as inflammatory response, differentiation, proliferation, and apoptosis. When the JAK/STAT pathway becomes dysregulated, proto-oncogenes and/or tumor-suppressor genes are often inappropriately expressed, commonly resulting in oncogenesis. This review discusses how SOCS, PIAS, and PTPS proteins modulate the JAK/STAT pathway ensuring that it remains cyclic and transient. The use of jakibins, STAT inhibitors, decoy oligonucleotides, RNA interference and genome editing to synthetically regulate a dysregulated JAK/STAT pathway in cancer cells are also considered.

The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) is a critical factor of cartilage destruction in rheumatoid arthritis (RA). Increased FLSs migration and subsequent degradation of the extracellular matrix are essential to the pathology of RA. Protein inhibitor of activated STAT (PIAS), whose family members include PIAS1, PIAS2 (PIASx), PIAS3, and PIAS4 (PIASy), play important roles in regulating various cellular events, such as cell survival, migration, and signal transduction in many cell types. However, whether PIAS proteins have a role in the pathogenesis of RA is unclear. In this study, we evaluated the role of PIAS proteins in FLSs migration, invasion, and matrix metalloproteinases (MMPs) expression in RA. We observed increased expression of PIAS3, but not PIAS1, PIAS2, or PIAS4, in FLSs and synovial tissues from patients with RA. We found that PIAS3 knockdown by short hairpin RNA reduced migration, invasion, and MMP-3, MMP-9, and MMP-13 expression in FLSs. In addition, we demonstrated that PIAS3 regulated lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of PIAS3 knockdown on Rac1/PAK1 and JNK activation. Our results indicated that PIAS3-mediated SUMOylation of Rac1 controlled its activation and modulated the Rac1 downstream activity of PAK1 and JNK. Furthermore, inhibition of Rac1, PAK1, or JNK decreased migration and invasion of RA FLSs. Thus, our observations suggest that PIAS3 suppression may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, and activation.

Previous studies have shown that protein inhibitor of activated STAT (PIAs)xα is crucial in protein sumoylation and is associated with cancer cell progression. However, the mechanism underlying the inhibitory effect on cancer cells, which may assist in developing novel treatment strategies in cancer remains to be elucidated. In present study, the expression levels of PIAsxα from tissue samples of osteosarcoma and adjacent tissues from 25 patients were analyzed using reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemical analyses. In addition, techniques using an overexpression vector and small interfering (si)RNAs were used to examine the effect of PIAsxα on osteosarcoma cells. Finally, using xenograft U2-OS osteosarcoma cells overexpressing PIAsxα, the effect of PIAsxα on osteosarcoma formation was determined. The results revealed low expression of PIAsxα in osteosarcoma tissues. In addition, following overexpression of PIAsxα, the apoptotic rates were significantly increased. The rate of G2/M arrest was at the highest level in the overexpression group, compared with other groups assessed. Furthermore, the expression levels of cyclin D1 and cyclin D3 were inhibited following PIAsxα increase, indicating the repressive effects of PIAsxα on cell cycle. Accordingly, cyclin D kinase (CDK) genes, including CDK4, CDK6 and CDK8, increased markedly following treatment with PIAsxα siRNAs. The expression levels of CDK4, CDK6 and CDK8 decreased significantly in the overexpression group, compared to the other groups. Furthermore, high expression levels of PIAsxα inhibited tumor formation in a nude mouse model. Taken together, these findings provide evidence for the effects of PIAsxα and its mechanism on osteosarcoma progression, which offers novel insight into sumoylation and the cell cycle in osteosarcoma.

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.

Obesity is associated with chronic low-level inflammation, especially in fat tissues, which contributes to insulin resistance and type 2 diabetes mellitus (T2DM). Protein inhibitor of activated STAT 1 (PIAS1) modulates a variety of cellular processes such as cell proliferation and DNA damage responses. Particularly, PIAS1 functions in the innate immune system and is a key regulator of the inflammation cascade. However, whether PIAS1 is involved in the regulation of insulin sensitivity remains unknown. Here, we demonstrated that PIAS1 expression in white adipose tissue (WAT) was downregulated by c-Jun N-terminal kinase in prediabetic mice models. Overexpression of PIAS1 in inguinal WAT of prediabetic mice significantly improved systemic insulin sensitivity, whereas knockdown of PIAS1 in wild-type mice led to insulin resistance. Mechanistically, PIAS1 inhibited the activation of stress-induced kinases and the expression of nuclear factor-κB target genes in adipocytes, mainly including proinflammatory and chemotactic factors. In doing so, PIAS1 inhibited macrophage infiltration in adipose tissue, thus suppressing amplification of the inflammation cascade, which in turn improved insulin sensitivity. These results were further verified in a fat transplantation model. Our findings shed light on the critical role of PIAS1 in controlling insulin sensitivity and suggest a therapeutic potential of PIAS1 in T2DM.

Several nuclear receptor (NR) superfamily members are known to be the molecular target of either the small ubiquitin-related modifier (SUMO) or ubiquitin-signaling pathways. However, little is currently known regarding how these two post-translational modifications interact to control NR biology. We show that SUMO and ubiquitin circuitry coordinately modifies the pregnane X receptor (PXR, NR1I2) to play a key role in regulating PXR protein stability, transactivation capacity, and transcriptional repression. The SUMOylation and ubiquitylation of PXR is increased in a ligand- and tumor necrosis factor alpha -: dependent manner in hepatocytes. The SUMO-E3 ligase enzymes protein inhibitor of activated signal transducer and activator of transcription-1 (STAT1) STAT-1 (PIAS1) and protein inhibitor of activated STAT Y (PIASy) drive high levels of PXR SUMOylation. Expression of protein inhibitor of activated stat 1 selectively increases SUMO(3)ylation as well as PXR-mediated induction of cytochrome P450, family 3, subfamily A and the xenobiotic response. The PIASy-mediated SUMO(1)ylation imparts a transcriptionally repressive function by ameliorating interaction of PXR with coactivator protein peroxisome proliferator-activated receptor gamma coactivator-1-alpha. The SUMO modification of PXR is effectively antagonized by the SUMO protease sentrin protease (SENP) 2, whereas SENP3 and SENP6 proteases are highly active in the removal of SUMO2/3 chains. The PIASy-mediated SUMO(1)ylation of PXR inhibits ubiquitin-mediated degradation of this important liver-enriched NR by the 26S proteasome. Our data reveal a working model that delineates the interactive role that these two post-translational modifications play in reconciling PXR-mediated gene activation of the xenobiotic response versus transcriptional repression of the proinflammatory response in hepatocytes. Taken together, our data reveal that the SUMOylation and ubiquitylation of the PXR interface in a fundamental manner directs its biologic function in the liver in response to xenobiotic or inflammatory stress.

Growth hormone (GH) plays a key role regulating somatic growth and in controlling metabolism and other physiological processes in humans and other animal species. GH acts by binding to the extracellular part of its transmembrane receptor, leading to induction of multiple intracellular signal transduction pathways that culminate in changes in gene and protein expression. A key agent in GH-stimulated growth is the latent transcription factor signal transducer and activator of transcription (STAT) 5B, one of four STAT proteins induced by the GH receptor in cultured cells and in vivo. As shown by genetic and biochemical studies, GH-activated STAT5B promotes transcription of the gene encoding the critical growth peptide, insulin-like growth factor-I (IGF-I), and natural null mutations of STAT5B in humans lead to growth failure accompanied by diminished IGF-I expression. Here we have examined the possibility that other GH-activated STATs can enhance IGF-I gene transcription, and thus potentially contribute to GH-regulated somatic growth. We find that human STAT5A is nearly identical to STAT5B in its biochemical and functional responses to GH but that STAT1 and STAT3 show a weaker profile of in vitro binding to STAT DNA elements from the IGF-I gene than STAT5B, and are less potent inducers of gene transcription through these elements. Taken together, our results offer a molecular explanation for why STAT5B is a key in vivo mediator of GH-activated IGF-I gene transcription and thus of GH-regulated somatic growth.

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 long-term objective of this study was to produce STAT synthetic protein in chicken during growth period resulting from the increase of growth hormone (GH) as growth promoter. This study used ten male chicken Lohman from PT. Multibreeder Indonesia. The chicken were kept within batteried cage, with a capacity of one chicken in each cage. The chickens were fed twice a day, at 6 a.m. and 6 p.m. with the amount of feed 10% less than standard. On day 21 the chicken were slaughtered to obtain the samples, i.e., adipose, liver and muscles for the following examinations (1) isolation of STAT-3 signaling protein from adipose, liver and muscles of the chicken, (2) analysis of STAT-3 signaling protein using SDS-PAGE method, and (3) identification of STAT-3 signaling protein using Western blot method by means of protein detection using electrophoresis with polyacrylamide gels. Results of examination on protein in hepatic, muscle and adipose of chickens in growth period revealed that STAT protein was positively pre...