Protein Inhibitor Of Activated STAT Y Research Articles

Downregulation of cardiac PIASy inhibits Cx43 SUMOylation and ameliorates ventricular arrhythmias in a rat model of myocardial ischemia/reperfusion injury.

Dysfunction of the gap junction channel protein connexin 43 (Cx43) contributes to myocardial ischemia/reperfusion (I/R)-induced ventricular arrhythmias. Cx43 can be regulated by small ubiquitin-like modifier (SUMO) modification. Protein inhibitor of activated STAT Y (PIASy) is an E3 SUMO ligase for its target proteins. However, whether Cx43 is a target protein of PIASy and whether Cx43 SUMOylation plays a role in I/R-induced arrhythmias are largely unknown. Male Sprague-Dawley rats were infected with PIASy short hairpin ribonucleic acid (shRNA) using recombinant adeno-associated virus subtype 9 (rAAV9). Two weeks later, the rats were subjected to 45 min of left coronary artery occlusion followed by 2 h reperfusion. Electrocardiogram was recorded to assess arrhythmias. Rat ventricular tissues were collected for molecular biological measurements. Following 45 min of ischemia, QRS duration and QTc intervals statistically significantly increased, but these values decreased after transfecting PIASy shRNA. PIASy downregulation ameliorated ventricular arrhythmias induced by myocardial I/R, as evidenced by the decreased incidence of ventricular tachycardia and ventricular fibrillation, and reduced arrythmia score. In addition, myocardial I/R statistically significantly induced PIASy expression and Cx43 SUMOylation, accompanied by reduced Cx43 phosphorylation and plakophilin 2 (PKP2) expression. Moreover, PIASy downregulation remarkably reduced Cx43 SUMOylation, accompanied by increased Cx43 phosphorylation and PKP2 expression after I/R. PIASy downregulation inhibited Cx43 SUMOylation and increased PKP2 expression, thereby improving ventricular arrhythmias in ischemic/reperfused rats heart.

Cardiac-targeted PIASy gene silencing mediates deSUMOylation of caveolin-3 and prevents ischemia/reperfusion-induced Nav1.5 downregulation and ventricular arrhythmias

BackgroundAbnormal myocardial Nav1.5 expression and function cause lethal ventricular arrhythmias during myocardial ischemia–reperfusion (I/R). Protein inhibitor of activated STAT Y (PIASy)-mediated caveolin-3 (Cav-3) SUMO modification affects Cav-3 binding to the voltage-gated sodium channel 1.5 (Nav1.5). PIASy activity is increased after myocardial I/R, but it is unclear whether this is attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias.MethodsUsing recombinant adeno-associated virus subtype 9 (AAV9), rat cardiac PIASy was silenced using intraventricular injection of PIASy short hairpin RNA (shRNA). After two weeks, rat hearts were subjected to I/R and electrocardiography was performed to assess malignant arrhythmias. Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurements.ResultsPIASy was upregulated by I/R (P < 0.01), with increased SUMO2/3 modification of Cav-3 and reduced membrane Nav1.5 density (P < 0.01). AAV9-PIASy shRNA intraventricular injection into the rat heart downregulated PIASy after I/R, at both mRNA and protein levels (P < 0.05 vs. Scramble-shRNA + I/R group), decreased SUMO-modified Cav-3 levels, enhanced Cav-3 binding to Nav1.5, and prevented I/R-induced decrease of Nav1.5 and Cav-3 co-localization in the intercalated disc and lateral membrane. PIASy silencing in rat hearts reduced I/R-induced fatal arrhythmias, which was reflected by a modest decrease in the duration of ventricular fibrillation (VF; P < 0.05 vs. Scramble-shRNA + I/R group) and a significantly reduced arrhythmia score (P < 0.01 vs. Scramble-shRNA + I/R group). The anti-arrhythmic effects of PIASy silencing were also evidenced by decreased episodes of ventricular tachycardia (VT), sustained VT and VF, especially at the time 5–10 min after ischemia (P < 0.05 vs. Scramble-shRNA + IR group). Using in vitro human embryonic kidney 293 T (HEK293T) cells and isolated adult rat cardiomyocyte models exposed to hypoxia/reoxygenation (H/R), we confirmed that increased PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R. Mutation of SUMO consensus lysine sites in Cav-3 (K38R or K144R) altered the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells.ConclusionsI/R-induced cardiac PIASy activation increased Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5-related ventricular arrhythmias. Cardiac-targeted PIASy silencing mediated Cav-3 deSUMOylation and partially prevented I/R-induced Nav1.5 downregulation in the plasma membrane of cardiomyocytes, and subsequent ventricular arrhythmias in rats. PIASy was identified as a potential therapeutic target for life-threatening arrhythmias in patients with ischemic heart diseases.

Hengshun Aromatic Vinegar Ameliorates Vascular Endothelial Injury via Regulating PKCζ-Mediated Oxidative Stress and Apoptosis.

Vascular endothelial injury (VEI) is an early event of atherosclerosis, and reversing endothelial dysfunction has become a new trend in the prevention and treatment of cardiovascular diseases. Hengshun aromatic vinegar (HSAV), a traditional vinegar, has been reported to have many pharmacological activities, but its effect against VEI and the molecular mechanism are still unknown. In this study, effects of HSAV on VEI were evaluated in H2O2-induced human umbilical vein endothelial cells (HUVECs) and methionine-induced VEI in rats. Results showed that HSAV significantly increased cell viability, inhibited apoptosis, and reduced the generation of reactive oxygen species (ROS) in H2O2-induced HUVECs. Meanwhile, HSAV decreased serum homocysteine (Hcy), endothelin 1 (ET-1), and oxidized low-density lipoprotein (ox-LDL) levels, increased nitric oxide (NO) and endothelin nitric oxide synthase (eNOS) levels, ameliorated pathological changes in rats with VEI induced by methionine. In parallel, HSAV relieved oxidative stress by decreasing malondialdehyde (MDA) level and increasing superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) levels in rats with VEI. Mechanism studies indicated that HSAV markedly downregulated the expression of protein kinase C zeta (PKCζ), and consequently regulated sirtuin 1 (Sirt1)-mediated oxidative stress signal pathway, and protein inhibitor of activated STATy (PIASy)-mediated apoptosis pathway, leading to the alleviation of oxidative stress and inhibition of apoptosis. These regulative effects of HSAV were further validated by knockdown and overexpression of PKCζ in vitro. In conclusion, HSAV showed protective effect against VEI by inhibiting PKCζ and, thereby, ameliorating oxidative stress and inhibiting apoptosis. This study not only provides guidance for the consumption of vinegar in daily life but also promotes the development of diet supplement for disease prevention.

Homeoprotein Msx1-PIASy Interaction Inhibits Angiogenesis.

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.

Perturbation of maternal PIASy abundance disrupts zygotic genome activation and embryonic development via SUMOylation pathway.

ABSTRACTDuring the maternal-to-zygotic transition (MZT), mRNAs and proteins stored in oocytes are degraded and zygotic genes are activated. We have previously shown that the ubiquitin-proteasome system (UPS)-mediated degradation of maternal proteins plays a role in the onset of zygotic transcription. However, it is still unclear which maternal proteins should be degraded for zygotic genome activation and ensuring subsequent embryonic development. In this study, we screen for these maternal factors that are degraded via the UPS. We thus identified a maternal protein PIASy (protein inhibitor of activated STATy), which is an E3 SUMO ligase. The overexpression of PIASy in fertilized embryos causes developmental arrest at the two-cell stage due to severe abnormal chromosome segregation and impaired zygotic transcription. We find that this developmental role of PIASy is related to its SUMOylation activity. Moreover, PIASy overexpression leads to increased trimethylation of histone H3 lysine 9 (H3K9me3) in two-cell nuclei and enhanced translocation of H3K9me3 methyltransferase to the pronucleus. Hence, PIASy is a maternal factor that is degraded after fertilization and may be important for the proper induction of zygotic genome activation and embryonic development.

Immunosuppression with tacrolimus improved implantation and rescued expression of uterine progesterone receptor and its co-regulators FKBP52 and PIASy at nidation in the obese and diabetic mice: Comparative studies with metformin

Diabesity is often associated with subfertility and recurrent miscarriages. Evidence links systemic and local uterine cytotoxicity to the pathogenesis of implantation failure (IF) in diabetes. Immunosuppression with tacrolimus improved pregnancy outcomes in obese and diabetic mice and repeated IF in women with elevated Th1/Th2 blood cell ratios. However the mode of action of tacrolimus in protecting against IF and the molecular mechanisms associated with recurrent miscarriages in the obese and diabetic subjects are yet to be elucidated. Here we administered tacrolimus (FK506) (0.1 mg/kg) for four consecutive weeks to the NONcNZO10/LtJ mice, a model of human PCOS, chronically fed with 60% kCal fat for 16 consecutive weeks to simulate human obesity-associated T2DM. Compared to those immunosuppressed with tacrolimus and their normative controls, high-fat fed (HFD) diabetic NONcNZO mice exhibited higher rates of peri- and post-implantation resorption and had aberrant expression of uterine IFNγ and progesterone receptor (PGR) and its immunophilin co-chaperone FKBP52 at nidation. Immature uterodomes and lack of activation of uterine STAT3 and NFκB at implantation were characteristics of IF in the HFD-dNONcNZO dams also low in the deciduogenic factors IL11 and GM-CSF. Therapeutic interventions with tacrolimus or metformin normalized the expression of decidual IFNγ, PGR and FKBP52, increased co-localization of protein inhibitor of activated STATy (PIASy) to PGR and resulted in the upregulation of uterine IL11and LIF. Rescued phosphorylation of STAT3 and NFκBp65 and uterodome maturation at nidation defined implantation success in treated dams. To our knowledge this is the first report to show that the impact of HFD on the hemochorial implantation is at least in part mediated through disruption of PGR signaling at nidation and that immunosuppression with tacrolimus or treatment with metformin restores PGR-mediated influences during implantation in the obese and diabetic subjects.

Protein Inhibitor of Activated STAT Y (PIASy) Regulates Insulin Secretion by Interacting with LIM Homeodomain Transcription Factor Isl1.

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.

SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity

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.

SUMOylation and Ubiquitylation Circuitry Controls Pregnane X Receptor Biology in Hepatocytes.

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.

PIASy-Mediated Sumoylation of SREBP1c Regulates Hepatic Lipid Metabolism upon Fasting Signaling

SREBP1c is a key transcription factor that regulates de novo lipogenesis during anabolic periods. However, the molecular mechanisms involved in the suppression of SREBP1c under nutritional deprivation are largely unknown. In this study, we demonstrate that the small ubiquitin-related modifier (SUMO) E3 ligase, a protein inhibitor of activated STAT Y (PIASy), sumoylates SREBP1c at Lys98, leading to suppression of the hepatic lipogenic program upon fasting-induced signals. In primary hepatocytes, ablation of PIASy stimulated intracellular lipid accumulation through the induction of SREBP1c and its target genes. Given that protein kinase A (PKA) plays important roles in catabolic responses, activated PKA enhances the sumoylation of SREBP1c and potentiates the interaction between SREBP1c and PIASy. Notably, overexpression of PIASy in obese db/db mice ameliorated hepatic steatosis, while suppression of PIASy in lean (wild-type) mice stimulated hepatic lipogenesis with increased expression of SREBP1c target genes. Furthermore, PKA-mediated SREBP1c phosphorylation augmented SREBP1c sumoylation, subsequently leading to degradation of SREBP1c via ubiquitination. Together, these data suggest that PKA-induced SREBP1c sumoylation by PIASy is a key regulatory mechanism to turn off hepatic lipogenesis during nutritional deprivation.

Endometrial receptivity defects and impaired implantation in diabetic NOD mice.

Implantation failure is a major hurdle to a successful pregnancy. The high rate of postimplantation fetal loss in nonobese diabetic (NOD) mice is believed to be related to an abnormal decidual production of interferon (IFN)gamma. To address whether diabetes alters the natural events associated with successful implantation, certain morphological and molecular features of uterine receptivity in diabetic NOD (dNOD) mice were examined in normally mated pregnancy and in concanavalin A (ConA)-induced pseudopregnancy. As opposed to normoglycemic NOD (cNOD) mice, dNOD mice expressed retarded maturation of their uterine pinopodes and overexpressed MUC1 mucin at implantation sites (P < 0.001). Uterine production of leukemia inhibitory factor (LIF) and phosphorylation of uterine NFkappaBp65 and STAT3-Ty705 were found to be low (P < 0.01) during Day 4.5 postcoitum, whereas IFNgamma was aberrantly overexpressed. Loss of temporal regulation of progesterone receptor A (PR A) and PR B, together with aberrantly increased expression of the protein inhibitor of activated STAT-y (PIASy) (P < 0.01) and reduced recruitment (P < 0.01) of the latter to nuclear progesterone receptor sites were prominent features of decidualization failure occurring at peri-implantation in dNOD mice. In conclusion, the aberrant expression of endometrial IFNgamma in dNOD mice is associated with a nonreceptive endometrial milieu contributing to peri-implantation embryo loss in type 1 diabetes.

CT7 (MAGE-C1) Promotes Survival and Interacts with STAT1 and PIASy in Multiple Myeloma Cells

Abstract 1807The type I Melanoma Antigen GEnes MAGE-A3 and CT7 (MAGE-C1) are detected in more than 75% of primary multiple myeloma specimens, and their expression is correlated with proliferation and progression of disease. We previously showed that MAGE-A3 inhibits apoptosis in human myeloma cell lines (HMCL) and primary cells in part through ubiquitinylation of the prototypical tumor suppressor p53, which targets it for proteasomal degradation and inhibits its pro-apoptotic transcriptional program (Nardiello et al, Clin Cancer Res, 2011; 17:4309). However, silencing of MAGE-A3 in HMCL and primary cells that lacked functional p53, either through deletions or mutations, also resulted in apoptosis. Silencing of CT7 alone did not affect survival of HMCL that lacked p53, but it did increase their sensitivity to chemotherapy-induced apoptosis. Many type I MAGE proteins, including MAGE-A3, bind to the RING domain protein TRIM28/Kap1 through their highly conserved MAGE Homology Domain (MHD) to form E3 ubiquitin ligase complexes, but it was unknown if CT7 also associated with TRIM28. These results lead to the hypothesis that CT7 is a survival factor for myeloma cells that can act through p53-independent mechanisms. To investigate the biochemical activity of CT7 and identify non-p53 pathways regulated by type I MAGE in myeloma cells, we analyzed protein-protein interactions with CT7 by two methods. We immunoprecipitated (IP’ed) CT7 from lysates of HMCL followed by sequencing of co-IP’ed proteins by mass spectroscopy and we performed yeast two-hybrid screening with a bait construct containing the MHD of CT7. CT7 was reciprocally co-IP’ed from HMCL lysates with TRIM28, demonstrating that it, too, associated with this RING domain protein. These methods also revealed two novel interactions with CT7. CT7 reciprocally co-IP’ed with STAT1, a transcription factor that plays a critical role in receptor-mediated signaling for cytokines such as interferon α/β. CT7 also interacted with Protein Inhibitor of Activated STAT y (PIASy), a modified RING domain protein that negatively regulates STAT proteins through its Small Ubiquitin-like MOdifier (SUMO) ligase activity, which sequesters target proteins out of the nucleus and into the cytoplasm. Surprisingly, phosphorylated STAT1 (pSTAT1) was detected in lysates from unstimulated HMCL, indicating a tonic level of activation, but most or all of the pSTAT1 was in the cytoplasm, suggesting that its transcriptional activity was being blocked through exclusion from the nucleus. Phospho-STAT1 also appeared to preferentially associate with the CT7/TRIM28 complex. Phospho-STAT1 did not appear to be ubiquitinylated in these HMCL, but SUMO2/3 modification of pSTAT1 was detected. These results suggest that CT7 negatively regulates pSTAT1 activity in HMCL by sequestering the transcription factor out of the nucleus through SUMO modification. This may be a direct result of CT7-mediated SUMOylation in partnership with PIASy, or indirectly due to CT7/TRIM28-mediated ubiquitinylation events that result in activation of a SUMO ligase such as PIASy. These findings suggest the possibility that complexes containing type I MAGE proteins may also have SUMO ligase activity and identify STAT1 as a novel non-p53 biochemical pathway regulated by these genes. Disclosures:No relevant conflicts of interest to declare.

TRIM32 Protein Sensitizes Cells to Tumor Necrosis Factor (TNFα)-induced Apoptosis via Its RING Domain-dependent E3 Ligase Activity against X-linked Inhibitor of Apoptosis (XIAP)

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.

PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow-mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow-mediated peroxynitrite (ONOO(-)) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53-Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO(-) increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301-410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ-PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

Disturbed flow: p53 SUMOylation in the turnover of endothelial cells

Disturbed blood flow induces apoptosis of vascular endothelial cells, which causes atherosclerosis. In this issue, Heo et al. (2011. J. Cell Biol. doi:10.1083/jcb.201010051) sheds light on p53’s role in this phenomenon. Disturbed flow induces peroxynitrite production, which activates protein kinase C ζ and it’s binding to the E3 SUMO (small ubiquitin-like modifier) ligase PIASy (protein inhibitor of activated STATy). This leads to p53 SUMOylation and its export to the cytosol, where it binds to the antiapoptotic protein Bcl-2 to induce apoptosis.

Caveolin-3 Undergoes SUMOylation by the SUMO E3 Ligase PIASy: SUMOYLATION AFFECTS G-PROTEIN-COUPLED RECEPTOR DESENSITIZATION

Caveolin (Cav) proteins in the plasma membrane have numerous binding partners, but the determinants of these interactions are poorly understood. We show here that Cav-3 has a small ubiquitin-like modifier (SUMO) consensus motif (ΨKX(D/E, where Ψ is a hydrophobic residue)) near the scaffolding domain and that Cav-3 is SUMOylated in a manner that is enhanced by the SUMO E3 ligase PIASy (protein inhibitor of activated STAT-y). Site-directed mutagenesis revealed that the consensus site lysine is the preferred SUMOylation site but that mutation of all lysines is required to abolish SUMOylation. Co-expression of a SUMOylation-deficient mutant of Cav-3 with β-adrenergic receptors (βARs) alters the expression level of β(2)ARs but not β(1)ARs following agonist stimulation, thus implicating Cav-3 SUMOylation in the mechanisms for β(2)AR but not β(1)AR desensitization. Expression of endothelial nitric-oxide synthase (NOS3) was not altered by the SUMOylation-deficient mutant. Thus, SUMOylation is a covalent modification of caveolins that influence the regulation of certain signaling partners.

Inducible SUMO modification of TANK alleviates its repression of TLR7 signalling

Adaptor proteins allow temporal and spatial coordination of signalling. In this study, we show SUMOylation of the adaptor protein TANK and its interacting kinase TANK-binding kinase 1 (TBK1). Modification of TANK by the small ubiquitin-related modifier (SUMO) at the evolutionarily conserved Lys 282 is triggered by the kinase activities of IκB kinase ɛ (IKKɛ) and TBK1. Stimulation of TLR7 leads to inducible SUMOylation of TANK, which in turn weakens the interaction with IKKɛ and thus relieves the negative function of TANK on signal propagation. Reconstitution experiments show that an absence of TANK SUMOylation impairs inducible expression of distinct TLR7-dependent target genes, providing a molecular mechanism that allows the control of TANK function.

Logical network of genotoxic stress-induced NF-kappaB signal transduction predicts putative target structures for therapeutic intervention strategies

Genotoxic stress is induced by a broad range of DNA-damaging agents and could lead to a variety of human diseases including cancer. DNA damage is also therapeutically induced for cancer treatment with the aim to eliminate tumor cells. However, the effectiveness of radio- and chemotherapy is strongly hampered by tumor cell resistance. A major reason for radio- and chemotherapeutic resistances is the simultaneous activation of cell survival pathways resulting in the activation of the transcription factor nuclear factor-kappa B (NF-κB). Here, we present a Boolean network model of the NF-κB signal transduction induced by genotoxic stress in epithelial cells. For the representation and analysis of the model, we used the formalism of logical interaction hypergraphs. Model reconstruction was based on a careful meta-analysis of published data. By calculating minimal intervention sets, we identified p53-induced protein with a death domain (PIDD), receptor-interacting protein 1 (RIP1), and protein inhibitor of activated STAT y (PIASy) as putative therapeutic targets to abrogate NF-κB activation resulting in apoptosis. Targeting these structures therapeutically may potentiate the effectiveness of radio-and chemotherapy. Thus, the presented model allows a better understanding of the signal transduction in tumor cells and provides candidates as new therapeutic target structures.

Sumoylation of Smad3 stimulates its nuclear export during PIASy-mediated suppression of TGF-β signaling

Sma- and MAD-related protein 3 (Smad3) plays crucial roles in the transforming growth factor-β (TGF-β)-mediated signaling pathway, which produce a variety of cellular responses, including cell proliferation and differentiation. In our previous study, we demonstrated that protein inhibitor of activated STATy (PIASy) suppresses TGF-β signaling by interacting with and sumoylating Smad3. In the present study, we examined the molecular mechanisms of Smad3 sumoylation during PIASy-mediated suppression of TGF-β signaling. We found that small-interfering RNA-mediated reduction of endogenous PIASy expression enhanced TGF-β-induced gene expression. Importantly, coexpression of Smad3 with PIASy and SUMO1 affected the DNA-binding activity of Smad3. Furthermore, coexpression of Smad3 with PIASy and SUMO1 stimulated the nuclear export of Smad3. Finally, fluorescence resonance energy transfer analyses revealed that Smad3 interacted with SUMO1 in the cytoplasm. These results suggest that PIASy regulates TGF-β/Smad3-mediated signaling by stimulating sumoylation and nuclear export of Smad3.

PIASy mediates NEMO sumoylation and NF-κB activation in response to genotoxic stress

Protein modification by SUMO (small ubiquitin-like modifier) is an important regulatory mechanism for multiple cellular processes. SUMO-1 modification of NEMO (NF-kappaB essential modulator), the IkappaB kinase (IKK) regulatory subunit, is critical for activation of NF-kappaB by genotoxic agents. However, the SUMO ligase, and the mechanisms involved in NEMO sumoylation, remain unknown. Here, we demonstrate that although small interfering RNAs (siRNAs) against PIASy (protein inhibitor of activated STATy) inhibit NEMO sumoylation and NF-kappaB activation in response to genotoxic agents, overexpression of PIASy enhances these events. PIASy preferentially stimulates site-selective modification of NEMO by SUMO-1, but not SUMO-2 and SUMO-3, in vitro. PIASy-NEMO interaction is increased by genotoxic stress and occurs in the nucleus in a manner mutually exclusive with IKK interaction. In addition, hydrogen peroxide (H2O2) also increases PIASy-NEMO interaction and NEMO sumoylation, whereas antioxidants prevent these events induced by DNA-damaging agents. Our findings demonstrate that PIASy is the first SUMO ligase for NEMO whose substrate specificity seems to be controlled by IKK interaction, subcellular targeting and oxidative stress conditions.