WRN inhibition leads to its chromatin-associated degradation via the PIAS4-RNF4-p97/VCP axis.

Synthetic lethality provides an attractive strategy for developing targeted cancer therapies. For example, cancer cells with high levels of microsatellite instability (MSI-H) are dependent on the Werner (WRN) helicase for survival. However, the mechanisms that regulate WRN spatiotemporal dynamics remain poorly understood. Here, we used single-molecule tracking (SMT) in combination with a WRN inhibitor to examine WRN dynamics within the nuclei of living cancer cells. WRN inhibition traps the helicase on chromatin, requiring p97/VCP for extraction and proteasomal degradation in a MSI-H dependent manner. Using a phenotypic screen, we identify the PIAS4-RNF4 axis as the pathway responsible for WRN degradation. Finally, we show that co-inhibition of WRN and SUMOylation has an additive toxic effect in MSI-H cells and confirm the in vivo activity of WRN inhibition using an MSI-H mouse xenograft model. This work elucidates a regulatory mechanism for WRN that may facilitate identification of new therapeutic modalities, and highlights the use of SMT as a tool for drug discovery and mechanism-of-action studies.

Suppression of interferon α and γ response by Huwe1-mediated Miz1 degradation promotes SARS-CoV-2 replication.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been demonstrated to limit the host interferon response; however, the underlying mechanism remains unclear. Here, we found that SARS-CoV-2 infection upregulated the E3 ubiquitin ligase Huwe1, which in turn facilitated the degradation of the transcription factor Miz1. The degradation of Miz1 hampered interferon alpha and gamma responses, consequently fostering viral replication and impeding viral clearance. Conversely, silencing or inhibiting Huwe1 enhanced the interferon responses, effectively curbing viral replication. Consistently, overexpressing Miz1 augmented the interferon responses and limited viral replication, whereas silencing Miz1 had the opposite effect. Targeting Huwe1 or overexpressing Miz1 elicited transcriptomic alterations characterized by enriched functions associated with bolstered antiviral response and diminished virus replication. Further study revealed Miz1 exerted epigenetic control over the transcription of specific interferon signaling molecules, which acted as common upstream regulators responsible for the observed transcriptomic changes following Huwe1 or Miz1 targeting. These findings underscore the critical role of the Huwe1-Miz1 axis in governing the host antiviral response, with its dysregulation contributing to the impaired interferon response observed during COVID-19.

dsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe.

The E3 SUMO ligase PIAS2 is expressed at high levels in differentiated papillary thyroid carcinomas but at low levels in anaplastic thyroid carcinomas (ATC), an undifferentiated cancer with high mortality. We show here that depletion of the PIAS2 beta isoform with a transcribed double-stranded RNA-directed RNA interference (PIAS2b-dsRNAi) specifically inhibits growth of ATC cell lines and patient primary cultures in vitro and of orthotopic patient-derived xenografts (oPDX) in vivo. Critically, PIAS2b-dsRNAi does not affect growth of normal or non-anaplastic thyroid tumor cultures (differentiated carcinoma, benign lesions) or cell lines. PIAS2b-dsRNAi also has an anti-cancer effect on other anaplastic human cancers (pancreas, lung, and gastric). Mechanistically, PIAS2b is required for proper mitotic spindle and centrosome assembly, and it is a dosage-sensitive protein in ATC. PIAS2b depletion promotes mitotic catastrophe at prophase. High-throughput proteomics reveals the proteasome (PSMC5) and spindle cytoskeleton (TUBB3) to be direct targets of PIAS2b SUMOylation at mitotic initiation. These results identify PIAS2b-dsRNAi as a promising therapy for ATC and other aggressive anaplastic carcinomas.

A UL26-PIAS1 complex antagonizes anti-viral gene expression during Human Cytomegalovirus infection.

Viral disruption of innate immune signaling is a critical determinant of productive infection. The Human Cytomegalovirus (HCMV) UL26 protein prevents anti-viral gene expression during infection, yet the mechanisms involved are unclear. We used TurboID-driven proximity proteomics to identify putative UL26 interacting proteins during infection to address this issue. We find that UL26 forms a complex with several immuno-regulatory proteins, including several STAT family members and various PIAS proteins, a family of E3 SUMO ligases. Our results indicate that UL26 prevents STAT phosphorylation during infection and antagonizes transcriptional activation induced by either interferon α (IFNA) or tumor necrosis factor α (TNFα). Additionally, we find that the inactivation of PIAS1 sensitizes cells to inflammatory stimulation, resulting in an anti-viral transcriptional environment similar to ΔUL26 infection. Further, PIAS1 is important for HCMV cell-to-cell spread, which depends on the presence of UL26, suggesting that the UL26-PIAS1 interaction is vital for modulating intrinsic anti-viral defense.

PIAS3 acts as a zinc sensor under zinc deficiency and plays an important role in myocardial ischemia/reperfusion injury.

Alterations in zinc transporter expression in response to zinc loss protect cardiac cells from ischemia/reperfusion (I/R) injury. However, the underlying molecular mechanisms how cardiac cells sense zinc loss remains unclear. Here, we found that zinc deficiency induced ubiquitination and degradation of the protein inhibitor of activated STAT3 (PIAS3), which can alleviate myocardial I/R injury by activating STAT3 to promote the expression of ZIP family zinc transporter genes. The RING finger domain within PIAS3 is vital for PIAS3 degradation, as PIAS3-dRing (missing the RING domain) and PIAS3-Mut (zinc-binding site mutation) were resistant to degradation in the setting of zinc deficiency. Meanwhile, the RING finger domain within PIAS3 is critical for the inhibition of STAT3 activation. Moreover, PIAS3 knockdown increased cardiac Zn2+ levels and reduced myocardial infarction in mouse hearts subjected to I/R, whereas wild-type PIAS3 overexpression, but not PIAS3-Mut, reduced cardiac Zn2+ levels, and exacerbated myocardial infarction. These findings elucidate a unique mechanism of zinc sensing, showing that fast degradation of the zinc-binding regulatory protein PIAS3 during zinc deficiency can correct zinc dyshomeostasis and alleviate reperfusion injury.

Miz1 represses type I interferon production and limits viral clearance during influenza A virus infection.

Type I interferons (IFNs) are critical for the antiviral immune response, and fine-tuning type I IFN production is critical to effectively clearing viruses without causing harmful immunopathology. We showed that the transcription factor Miz1 epigenetically repressed the expression of genes encoding type I IFNs in mouse lung epithelial cells by recruiting histone deacetylase 1 (HDAC1) to the promoters of Ifna and Ifnb. Loss of function of Miz1 resulted in augmented production of these type I IFNs during influenza A virus (IAV) infection, leading to improved viral clearance in vitro and in vivo. IAV infection induced Miz1 accumulation by promoting the cullin-4B (CUL4B)-mediated ubiquitylation and degradation of the E3 ubiquitin ligase Mule (Mcl-1 ubiquitin ligase E3; also known as Huwe1 or Arf-BP1), which targets Miz1 for degradation. As a result, Miz1 accumulation limited type I IFN production and favored viral replication. This study reveals a previously unrecognized function of Miz1 in regulating antiviral defense and a potential mechanism for influenza viruses to evade host immune defense.

Disrupting PIAS3-mediated SUMOylation of MLK3 ameliorates poststroke neuronal damage and deficits in cognitive and sensorimotor behaviors.

Activated small ubiquitin-like modifiers (SUMOs) have been implicated in neuropathological processes following ischemic stroke. However, the target proteins of SUMOylation and their contribution to neuronal injury remain to be elucidated. MLK3 (mixed-lineage kinase 3), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, is a critical regulator of neuronal lesions following cerebral ischemia. Here, we found that SUMOylation of MLK3 increases in both global and focal ischemic rodent models and primary neuronal models of oxygen and glucose deprivation (OGD). SUMO1 conjugation at the Lys401 site of MLK3 promoted its activation, stimulated its downstream p38/c-Jun N-terminal kinase (JNK) cascades, and led to cell apoptosis. The interaction of MLK3 with PIAS3, a SUMO ligase, was elevated following ischemia and reperfusion. The PINIT domain of PIAS3 was involved in direct interactions with MLK3. Overexpression of the PINIT domain of PIAS3 disrupted the MLK3-PIAS3 interaction, inhibited SUMOylation of MLK3, suppressed downstream signaling, and reduced cell apoptosis and neurite damage. In rodent ischemic models, the overexpression of the PINIT domain reduced brain lesions and alleviated deficits in learning, memory, and sensorimotor functions. Our findings demonstrate that brain ischemia-induced MLK3 SUMOylation by PIAS3 is a potential target against poststroke neuronal lesions and behavioral impairments.

Integrative analysis revealed a correlation of PIAS family genes expression with prognosis, immunomodulation and chemotherapy.

BACKGROUND: Protein inhibitor of activated STATs (PIAS) has pleiotropic biological effects, such as protein post-translational modification, transcriptional coregulation and gene editing. It is reported that PIAS family genes are also correlated with immune cells infiltration in cancers that highlights their unnoticed biological role in tumor progression. However, the relationship of their expression with prognosis, immune cell infiltration, tumor microenvironment, and immunotherapy in pan-cancer has been rarely reported. METHODS: The multi-omics data were used to investigate the expression level of PIAS family members in pan-cancer, and the prognostic value of their expression in different tumors was analyzed by univariate Cox regression and Kaplan-Meier. Correlation analysis was used to investigate the relationship of PIAS gene expression with tumor microenvironment, immune infiltrating subtypes, stemness score and drug sensitivity. In addition, we also used wound healing and transwell assays to verify the biological effects of PIAS family gene expression on invasion and metastasis of HCC cells. RESULTS: We found that PIAS family genes expression is significantly heterogeneous in tumors by multi-genomic analysis, and associated with poor prognosis in patients with multiple types of cancer. Furthermore, we also found that genetic alterations of PIAS family genes were not only common in different types of human tumors, but were also significantly associated with disease-free survival (DFS) across pan-cancer. Single-cell analysis revealed that PIAS family genes were mainly distributed in monocytes/macrophages. Additionally, we also found that their expression was associated with tumor microenvironment (including stromal cells and immune cells) and stemness score (DNAss and RNAss). Drug sensitivity analysis showed that PIAS family genes were able to predict the response to chemotherapy and immunotherapy. PIAS family genes expression is closely related to tumor metastasis, especially PIAS3. High PIAS3 expression significantly promotes the migration and invasion of liver cancer cell lines (HCC-LM3 and MHCC97-H). CONCLUSIONS: Taking together, these findings contribute to determine whether the PIAS family genes are a potential oncogenic target gene, which have important contribution for the development of cancer immunotherapy.

Multi-omics integration identifies cell-state-specific repression by PBRM1-PIAS1 cooperation.

PBRM1 is frequently mutated in cancers of epithelial origin. How PBRM1 regulates normal epithelial homeostasis, prior to cancer initiation, remains unclear. Here, we show that PBRM1's gene regulatory roles differ drastically between cell states, leveraging human skin epithelium (epidermis) as a research platform. In progenitors, PBRM1 predominantly functions to repress terminal differentiation to sustain progenitors' regenerative potential; in the differentiation state, however, PBRM1 switches toward an activator. Between these two cell states, PBRM1 retains its genomic binding but associates with differential interacting proteins. Our targeted screen identified the E3 SUMO ligase PIAS1 as a key interactor. PIAS1 co-localizes with PBRM1 on chromatin to directly repress differentiation genes in progenitors, and PIAS1's chromatin binding drastically diminishes in differentiation. Furthermore, SUMOylation contributes to PBRM1's repressive function in progenitor maintenance. Thus, our findings highlight PBRM1's cell-state-specific regulatory roles influenced by its protein interactome despite its stable chromatin binding.

SUMOylation of the m6A reader YTHDF2 by PIAS1 promotes viral RNA decay to restrict EBV replication.

YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) is a member of the YTH protein family that binds to N6-methyladenosine (m6A)-modified RNA, regulating RNA stability and restricting viral replication, including Epstein-Barr virus (EBV). PIAS1 is an E3 small ubiquitin-like modifier (SUMO) ligase known as an EBV restriction factor, but its role in YTHDF2 SUMOylation remains unclear. In this study, we investigated the functional regulation of YTHDF2 by PIAS1. We found that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues (K281, K571, and K572). Importantly, PIAS1 synergizes with wild-type YTHDF2, but not a SUMOylation-deficient mutant, to limit EBV lytic replication. Mechanistically, YTHDF2 lacking SUMOylation exhibits reduced binding to EBV transcripts, leading to increased viral mRNA stability. Furthermore, PIAS1 mediates SUMOylation of YTHDF2's paralogs, YTHDF1 and YTHDF3, to restrict EBV replication. These results collectively uncover a unique mechanism whereby YTHDF family proteins control EBV replication through PIAS1-mediated SUMOylation, highlighting the significance of SUMOylation in regulating viral mRNA stability and EBV replication.IMPORTANCEm6A RNA modification pathway plays important roles in diverse cellular processes and viral life cycle. Here, we investigated the relationship between PIAS1 and the m6A reader protein YTHDF2, which is involved in regulating RNA stability by binding to m6A-modified RNA. We found that both the N-terminal and C-terminal regions of YTHDF2 interact with PIAS1. We showed that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues. We also demonstrated that PIAS1 enhances the anti-EBV activity of YTHDF2. We further revealed that PIAS1 mediates the SUMOylation of other YTHDF family members, namely, YTHDF1 and YTHDF3, to limit EBV replication. These findings together illuminate an important regulatory mechanism of YTHDF proteins in controlling viral RNA decay and EBV replication through PIAS1-mediated SUMOylation.

Upregulation of tumor suppressor PIAS3 by Honokiol promotes tumor cell apoptosis via selective inhibition of STAT3 tyrosine 705 phosphorylation.

The natural product Honokiol exhibits robust antitumor activity against a range of cancers, and it has also received approval to undergo phase I clinical trial testing. We confrmed that honokiol can promote the apoptotic death of tumor cells through cell experiments. Then siRNA constructs specific for PIAS3, PIAS3 overexpression plasmid and the mutation of the STAT3 Tyr705 residue were used to confirm the mechanism of Honokiol-induced apoptosis. Finally, we confrmed that honokiol can promote PIAS3 upregulation, in turn suppressing STAT3 Tyr705 phosphorylation through the in vivo and in vitro experiments. Honokiol was ultimately found to reduce tumor cell viability by promoting apoptosis through a mechanism dependent on the ability of Honokiol to promote PIAS3 upregulation and the selective inhibition of p-STAT3 (Tyr705) without affecting p-STAT3 (Ser727) or p-STAT1 (Tyr701) levels. PIAS3 knockdown and overexpression in tumor cells altered STAT3 activation and associated DNA binding activity through the control of Tyr705 phosphorylation via PIAS3-STAT3 complex formation, ultimately shaping Honokiol-induced tumor cell apoptosis. Honokiol was also confirmed to significantly prolong the survival of mice bearing xenograft tumors in a PIAS3-dependent fashion. Together, these findings highlight a novel pathway through which Honokiol can promote PIAS3 upregulation, in turn suppressing STAT3 Tyr705 phosphorylation and promoting the apoptotic death of tumor cells.

SUMOylation and coupling of eNOS mediated by PIAS1 contribute to maintenance of vascular homeostasis.

Endothelial dysfunction (ED) is commonly considered a crucial initiating step in the pathogenesis of numerous cardiovascular diseases. The coupling of endothelial nitric oxide synthase (eNOS) is important in maintaining normal endothelial functions. However, it still remains elusive whether and how eNOS SUMOylation affects the eNOS coupling. In the study, we investigate the roles and possible action mechanisms of protein inhibitor of activated STAT 1 (PIAS1) in ED. Human umbilical vein endothelial cells (HUVECs) treated with palmitate acid (PA) in vitro and ApoE-/- mice fed with high-fat diet (HFD) in vivo were constructed as the ED models. Our in vivo data show that PIAS1 alleviates the dysfunction of vascular endothelium by increasing nitric oxide (NO) level, reducing malondialdehyde (MDA) level, and activating the phosphatidylinositol 3-kinase-protein kinase B-endothelial nitric oxide synthase (PI3K-AKT-eNOS) signaling in ApoE-/- mice. Our in vitro data also show that PIAS1 can SUMOylate eNOS under endogenous conditions; moreover, it antagonizes the eNOS uncoupling induced by PA. The findings demonstrate that PIAS1 alleviates the dysfunction of vascular endothelium by promoting the SUMOylation and inhibiting the uncoupling of eNOS, suggesting that PIAS1 would become an early predictor of atherosclerosis and a new potential target of the hyperlipidemia-related cardiovascular diseases.

SUMOylation of SMAD4 by PIAS1 in Conjunction with Vimentin Upregulation Promotes Migration Potential in Non-Small Cell Lung Cancer.

BACKGROUND: The expression of vimentin as a marker of epithelial-to-mesenchymal transition (EMT) has been speculated to be associated with tissue heterogeneity and metastases of non-small cell lung cancer (NSCLC). METHODS: This study utilized in vitro co-immunoprecipitation with small interfering RNAs (siRNAs) against protein inhibitors of STAT system type 1 (PIAS1) or SMAD4 in transforming growth factor-beta (TGF-ß) signaling pathway in combination with SUMOylation assay. RESULTS: We successfully demonstrated that PIAS1 enhanced SUMOylation of SMAD4 by forming a complex PIAS1-SUMO1-SMAD4 protein complex. This, in accordance with subsequently increased production of vimentin microfilaments, led to enhanced migration ability of non-small cell lung cancer (NSCLC) A549 line, observed from wound healing assay. CONCLUSIONS: Our results further supported the positive correlation of SUMOylated SMAD4 mediated by PIAS1 and downstream overexpression of vimentin. In addition, the observation that overexpression of vimentin in this certain cell line was not necessarily linked with accelerated relative wound closure raised concerns that further exploration will be needed to confirm if the causal relationship exists between vimentin expression and the metastases of NSCLC, and if so, to what extent vimentin contributes to it.

Methylation Levels in the Promoter Region of FHIT and PIAS1 Genes Associated with Mastitis Resistance in Xinjiang Brown Cattle.

Mastitis causes serious economic losses in the dairy industry, but there are no effective treatments or preventive measures. In this study, the ZRANB3, PIAS1, ACTR3, LPCAT2, MGAT5, and SLC37A2 genes in Xinjiang brown cattle, which are associated with mastitis resistance, were identified using a GWAS. Pyrosequencing analysis showed that the promoter methylation levels of the FHIT and PIAS1 genes in the mastitis group were higher and lower, respectively, than those in the healthy group (65.97 ± 19.82% and 58.00 ± 23.52%). However, the methylation level of the PIAS1 gene promoter region in the mastitis group was lower than that in the healthy group (11.48 ± 4.12% and 12.17 ± 4.25%). Meanwhile, the methylation levels of CpG3, CpG5, CpG8, and CpG15 in the promoter region of the FHIT and PIAS1 genes in the mastitis group were significantly higher than those in the healthy group (p < 0.01), respectively. RT-qPCR showed that the expression levels of the FHIT and PIAS1 genes were significantly higher in the healthy group than those in the mastitis group (p < 0.01). Correlation analysis showed that the promoter methylation level of the FHIT gene was negatively correlated with its expression. Hence, increased methylation in the promoter of the FHIT gene reduces the mastitis resistance in Xinjiang brown cattle. Finally, this study provides a reference for the molecular-marker-assisted selection of mastitis resistance in dairy cattle.

Mmu_circ_0000037 inhibits the progression of acute pancreatitis by miR-92a-3p/Pias1 axis.

BACKGROUND: Acute pancreatitis (AP) is an inflammatory disease with high mortality. Previous study has suggested that circular RNAs are dysregulated and involved in the regulation of inflammatory responses in AP. This study aimed to investigate the function and regulatory mechanism underlying mmu_circ_0000037 in caerulein-induced AP cellular model. METHODS: Caerulein-treated MPC-83 cells were used as an in vitro cellular model for AP. The expression levels of mmu_circ_0000037, microRNA (miR)-92a-3p, and protein inhibitor of activated STAT1 (Pias1) were detected by quantitative real-time polymerase chain reaction. Cell viability, amylase activity, apoptosis, and inflammatory response were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Amylase Assay Kit, flow cytometry, and enzyme-linked immunosorbent assays. The protein level was quantified by western blot analysis. The target interaction between miR-92a-3p and mmu_circ_0000037 or Pias1 were predicted by StarbaseV3.0 and validated by dual-luciferase reporter assay and RNA immunoprecipitation assay. RESULTS: Mmu_circ_0000037 and Pias1 levels were decreased, whereas miR-92a-3p expression was elevated in caerulein-induced MPC-83 cells. Overexpression of mmu_circ_0000037 protected MPC-83 cells from caerulein-induced the decrease of cell viability, as well as the promotion of amylase activity, apoptosis and inflammation. MiR-92a-3p was targeted by mmu_circ_0000037, and miR-92a-3p overexpression rescued the effect of mmu_circ_0000037 on caerulein-induced MPC-83 cell injury. Pias1 was confirmed as a target of miR-92a-3p and mmu_circ_0000037 regulated the expression of Pias1 by sponging miR-92a-3p. CONCLUSION: Mmu_circ_0000037 relieves caerulein-induced inflammatory injury in MPC-83 cells by targeting miR-92a-3p/Pias1 axis, providing a theoretical basis for the treatment of AP.

SUMO and PIAS repress NF-κB activation in a basal chordate.

Small ubiquitin-like modifier (SUMO) regulates various biological processes, including the MyD88/TICAMs-IRAKs-TRAF6-NF-κB pathway, one of the core immune pathways. However, its functions are inconsistent between invertebrates and vertebrates and have rarely been investigated in lower chordates, including amphioxus and fishes. Here, we investigated the SUMOylation gene system in the amphioxus, a living basal chordate. We found that amphioxus has a SUMOylation system that has a complete set of genes and preserves several ancestral traits. We proceeded to study their molecular functions using the mammal cell lines. Both amphioxus SUMO1 and SUMO2 were shown to be able to attach to NF-κB Rel and to inhibit NF-κB activation by 50-75% in a dose-dependent fashion. The inhibition by SUMO2 could be further enhanced by the addition of the SUMO E2 ligase UBC9. In comparison, while human SUMO2 inhibited RelA, human SUMO1 slightly activated RelA. We also showed that, similar to human PIAS1-4, amphioxus PIAS could serve as a SUMO E3 ligase and promote its self-SUMOylation. This suggests that amphioxus PIAS is functionally compatible in human cells. Moreover, we showed that amphioxus PIAS is not only able to inhibit NF-κB activation induced by MyD88, TICAM-like, TRAF6 and IRAK4 but also able to suppress NF-κB Rel completely in the presence of SUMO1/2 in a dose-insensitive manner. This suggests that PIAS could effectively block Rel by promoting Rel SUMOylation. In comparison, in humans, only PIAS3, but not PIAS1/2/4, has been reported to promote NF-κB SUMOylation. Taken together, the findings from amphioxus, together with those from mammals and other species, not only offer insights into the functional volatility of the animal SUMO system, but also shed light on its evolutionary transitions from amphioxus to fish, and ultimately to humans.

Increased Small Ubiquitin-like Modifier-Activating Enzyme SAE1 Promotes Hepatocellular Carcinoma by Enhancing mTOR SUMOylation.

SUMOylation, one of the most important posttranslational modifications of proteins, plays an essential role in various biological processes; however, enzymes that control SUMOylation in hepatocellular carcinoma (HCC) are still unclear. Comprehensive exploration of the expression and clinical significance of SUMO enzymes in HCC would be of great value. Here, we obtained the gene expression profile of each small ubiquitin-like modifier (SUMO) protein and the corresponding clinical information from The Cancer Genome Atlas. We found that all SUMO enzymes were significantly increased in HCC tissues compared with that in adjacent nontumorous tissues. We identified a 6-gene prognostic signature, including SAE1, PIAS2, PIAS3, SENP3, SENP5, and UBC9, that could effectively predict the overall survival in patients with HCC. Specifically, SAE1 was the most valuable prognostic indicator. In 282 clinical samples, we found that SAE1 was closely related to the clinicopathologic parameters and prognosis of patients with HCC. In vitro and in vivo studies showed that SAE1 knockdown inhibits the proliferation, migration, and invasion of HCC cells. Mechanistically, we confirmed that SAE1 plays a role in driving HCC progression, which is largely dependent on the SUMOylation of mTOR signaling. In conclusion, our study revealed that the expression of SUMO enzymes, especially SAE1, is highly associated with HCC development and acts as a promising prognostic predictor.

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

BACKGROUND: Abnormal 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. METHODS: Using 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. RESULTS: PIASy 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. CONCLUSIONS: I/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.

PIAS1 Alleviates Hepatic Ischemia-Reperfusion Injury in Mice through a Mechanism Involving NFATc1 SUMOylation.

Recently, attentions have come to the alleviatory effect of protein inhibitor of activated STAT1 (PIAS1) in hepatic ischemia-reperfusion injury (HIRI), but the underlying molecular mechanistic actions remain largely unknown, which were illustrated in the present study. Microarray-based analysis predicted a possible regulatory mechanism involving the PIAS1/NFATc1/HDAC1/IRF-1/p38 MAPK signaling axis in HIRI. Then, growth dynamics of hypoxia/reoxygenation- (H/R-) exposed hepatocytes and liver injury of HIRI-like mice were delineated after the alteration of the PIAS1 expression. We validated that PIAS1 downregulation occurred in H/R-exposed hepatocytes and HIRI-like mice, while the expression of NFATc1, HDAC1, and IRF-1 and phosphorylation levels of p38 were increased. PIAS1 inactivated p38 MAPK signaling by inhibiting HDAC1-mediated IRF-1 through NFATc1 SUMOylation, thereby repressing the inflammatory response and apoptosis of hepatocytes in vitro, and alleviated liver injury in vivo. Collectively, the NFATc1/HDAC1/IRF-1/p38 MAPK signaling axis is highlighted as a promising therapeutic target for potentiating hepatoprotective effects of PIAS1 against HIRI.

Suppression of Allergic Asthma by Loss of Function of Miz1-mediated Th1 Skewing.

Asthma is the most prevalent chronic respiratory disease worldwide. There is currently no cure, and it remains an important cause of morbidity and mortality. Here we report that lung-specific loss of function of the transcription factor Miz1 (c-Myc-interacting zinc finger protein-1) upregulates the pro-T-helper cell type 1 cytokine IL-12. Upregulation of IL-12 in turn stimulates a Th1 response, thereby counteracting T-helper cell type 2 response and preventing the allergic response in mouse models of house dust mite- and OVA (ovalbumin)-induced asthma. Using transgenic mice expressing Cre under a cell-specific promoter, we demonstrate that Miz1 acts in lung epithelial cells and dendritic cells in asthma. Chromatin immunoprecipitation coupled with high-throughput DNA sequencing or quantitative PCR reveals the binding of Miz1 on the Il12 promoter indicating direct repression of IL-12 by Miz1. In addition, HDAC1 (histone deacetylase 1) is recruited to the Il12 promoter in a Miz1-depdenent manner, suggesting epigenetic repression of Il12 by Miz1. Furthermore, Miz1 is upregulated in the lungs of asthmatic mice. Our data together suggest that Miz1 is upregulated during asthma, which in turn promotes asthma pathogenesis by preventing Th1 skewing through the transcriptional repression of IL-12.