SUMOylation of YTHDF2 promotes mRNA degradation and cancer progression by increasing its binding affinity with m6A-modified mRNAs.

N 6-Methyladenosine (m6A) is the most abundant modification within diverse RNAs including mRNAs and lncRNAs and is regulated by a reversible process with important biological functions. Human YTH domain family 2 (YTHDF2) selectively recognized m6A-RNAs to regulate degradation. However, the possible regulation of YTHDF2 by protein post-translational modification remains unknown. Here, we show that YTHDF2 is SUMOylated in vivo and in vitro at the major site of K571, which can be induced by hypoxia while reduced by oxidative stress and SUMOylation inhibitors. SUMOylation of YTHDF2 has little impact on its ubiquitination and localization, but significantly increases its binding affinity of m6A-modified mRNAs and subsequently results in deregulated gene expressions which accounts for cancer progression. Moreover, Disease-free survival analysis of patients with lung adenocarcinoma derived from TCGA dataset reveals that higher expression of YTHDF2 together with higher expression of SUMO1 predicts poor prognosis. Our works uncover a new regulatory mechanism for YTHDF2 recognition of m6A-RNAs and highlight the importance of YTHDF2 SUMOylation in post-transcriptional gene expression regulation and cancer progression.

PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington's disease.

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.

Ginkgoic acid impedes gastric cancer cell proliferation, migration and EMT through inhibiting the SUMOylation of IGF-1R.

The imbalance of SUMOylation is related to different cancers, including gastric cancer (GC). Ginkgolic acid (GA) inhibits the growth and invasion of many cancer cells, and it has been reported to restrain SUMOylation. However, the role of GA in GC and whether it functions through SUMOylation remains to be clarified. Our research revealed that GA (15:1) inhibited cell proliferation, migration, epithelial-mesenchymal transition (EMT) and overall protein SUMOylation in BGC823 and HGC27 cells. In addition, knockdown of SUMO1 (small ubiquitin-like modifier) instead of SUMO2/3 played a similar role to GA in cell behaviors. Besides, nuclear IGF-1R (insulin-like growth factor 1 receptor) expression was markedly upregulated in GC cells compared to normal gastric epithelial cells. GA prevented IGF-1R from binding to SUMO1, thereby suppressing its nuclear accumulation. Further research found that IGF-1R directly bound to SNAI2 (snail family zinc finger 2) promoter. The interference of IGF-1R downregulated the mRNA and protein levels of SNAI2, while the overexpression of SUMO1, IGF-1R and UBC9 (SUMO-conjugating enzyme) played the opposite role. Furthermore, the co-transfection of SUMO1, UBC9 and IGF-1R vectors or the overexpression of SNAI2 reversed the inhibitory effects of GA on cell proliferation, migration and EMT. Finally, GA impeded the growth of GC xenografts and decreased the expression of nuclear IGF-1R and SNAI2 in vivo. In conclusion, these findings demonstrated that GA hindered the progression of GC by inhibiting the SUMOylation of IGF-1R. Thus, GA might be a promising therapeutic for GC.

The SUMO pathway in pancreatic cancer: insights and inhibition.

An urgent medical need to develop novel treatment strategies for patients with pancreatic ductal adenocarcinoma (PDAC) exists. However, despite various efforts in the histopathological and molecular subtyping of PDAC, novel targeted or specific therapies have not been established. Posttranslational modifications (PTMs) with ubiquitin-like proteins, including small ubiquitin-like modifiers (SUMOs), mediate numerous processes that can contribute to the fitness and survival of cancer cells. The contribution of SUMOylation to transcriptional control, DNA repair pathways, mitotic progression, and oncogenic signalling has been described. Here we review functions of the SUMO pathway in PDAC, with a special focus on its connection to an aggressive subtype of the disease characterised by high MYC activity, and discuss SUMOylation inhibitors under development for precise PDAC therapies.

Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites.

Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.

SUMOylation inhibitors synergize with FXR agonists in combating liver fibrosis.

Farnesoid X receptor (FXR) is a promising target for nonalcoholic steatohepatitis (NASH) and fibrosis. Although various FXR agonists have shown anti-fibrotic effects in diverse preclinical animal models, the response rate and efficacies in clinical trials were not optimum. Here we report that prophylactic but not therapeutic administration of obeticholic acid (OCA) prevents hepatic stellate cell (HSC) activation and fibrogenesis. Activated HSCs show limited response to OCA and other FXR agonists due to enhanced FXR SUMOylation. SUMOylation inhibitors rescue FXR signaling and thereby increasing the efficacy of OCA against HSC activation and fibrosis. FXR upregulates Perilipin-1, a direct target gene of FXR, to stabilize lipid droplets and thereby prevent HSC activation. Therapeutic coadministration of OCA and SUMOylation inhibitors drastically impedes liver fibrosis induced by CCl4, bile duct ligation, and more importantly NASH. In conclusion, we propose a promising therapeutic approach by combining SUMOylation inhibitors and FXR agonists for liver fibrosis.

Ubiquitin, SUMO, and NEDD8: Key Targets of Bacterial Pathogens.

Manipulation of host protein post-translational modifications (PTMs) is used by various pathogens to interfere with host cell functions. Among these modifications, ubiquitin (UBI) and ubiquitin-like proteins (UBLs) constitute key targets because they are regulators of pathways essential for the host cell. In particular, these PTM modifiers control pathways that have been described as crucial for infection such as pathogen entry, replication, propagation, or detection by the host. Although bacterial pathogens lack eucaryotic-like UBI or UBL systems, many of them produce proteins that specifically interfere with these host PTMs during infection. In this review we discuss the different mechanisms used by bacteria to interfere with host UBI and the two UBLs, SUMO and NEDD8.

Small-Molecule Inhibitors Targeting Protein SUMOylation as Novel Anticancer Compounds.

SUMOylation, one of post-translational modifications, is covalently modified on lysine residues of a target protein through an enzymatic cascade reaction similar to protein ubiquitination. Along with identification of many SUMOylated proteins, protein SUMOylation has been proven to regulate multiple biologic activities including transcription, cell cycle, DNA repair, and innate immunity. The dysregulation of protein SUMOylation and deSUMOylation modification is linked with carcinogenesis and tumor progression. The SUMOylation-associated enzymes are usually elevated in various cancers, which function as cancer biomarkers to relate to poor outcomes for patients. Considering the significance of protein SUMOylation in regulating diverse biologic functions in cancer progression, numerous small-molecule inhibitors targeting protein SUMOylation pathway are developed as potentially clinical anticancer therapeutics. Here, we systematically summarize the latest progresses of associations of small ubiquitin-like modifier (SUMO) enzymes with cancers and small-molecular inhibitors against human cancers by targeting SUMOylation enzymes. We also compared the pros and cons of several special anticancer inhibitors targeting SUMO pathway. As more efforts are invested in this field, small-molecule inhibitors targeting the SUMOylation modification pathway are promising for development into novel anticancer drugs.

Silencing SUMO2 promotes protection against degradation and apoptosis of nucleus pulposus cells through p53 signaling pathway in intervertebral disc degeneration.

OBJECTIVE: Intervertebral disc degeneration (IDD), as a common cause of back pain, is related to the promotion of cellular senescence and reduction in proliferation. Based on recent studies, small ubiquitin-related modifier (SUMO) proteins have been implicated in various biological functions. Therefore, in the present study, we investigated the effects of SUMO2 on proliferation and senescence of nucleus pulposus cells (NPCs) via mediation of p53 signaling pathway in rat models of IDD. METHODS: After the establishment of rat models of IDD for the measurement of positive expression of SUMO2/3 protein, the mRNA and protein levels of SUMO2, molecular phenotype [matrix metalloproteinase-2 (MMP-2) and hypoxia-inducible factor-1α (HIF-1α)] and p53 signaling pathway-related genes [p21, murine double minute-2 (MDM2), growth arrest and DNA-damage-inducible protein 45 α (GADD45α), cyclin-dependent kinase 2/4 (CDK2/4), and CyclinB1] were determined, followed by the detection of cell proliferation, cell cycle, apoptosis, and cell senescence. RESULTS: The rat models of IDD were successfully constructed. The results obtained showed that there was a higher positive expression of SUMO2/3 protein in IDD rats. Moreover, the silencing of the SUMO2 gene decreases the levels of SUMO2, p53, p21, MDM2, GADD45α, MMP-2, and HIF-1α expressions and p53 phosphorylation level while it increases the levels of CDK2/4 and CyclinB1 expressions. In addition, SUMO2 gene silencing enhances proliferation and suppresses apoptosis and cell senescence of NPCs. CONCLUSION: In conclusion, SUMO2 gene silencing promotes proliferation, and inhibits the apoptosis and senescence of NPCs in rats with IDD through the down-regulation of the p53 signaling pathway. Thus, SUMO2 is a potential target in the treatment of IDD.

SUMO2/3 modification of activating transcription factor 5 (ATF5) controls its dynamic translocation at the centrosome.

Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element-binding protein family of transcription factors. ATF5 regulates stress responses and cell survival, proliferation, and differentiation and also plays a role in viral infections, cancer, diabetes, schizophrenia, and the olfactory system. Moreover, it was found to also have a critical cell cycle-dependent structural function at the centrosome. However, the mechanism that controls the localization of ATF5 at the centrosome is unclear. Here we report that ATF5 is small ubiquitin-like modifier (SUMO) 2/3-modified at a conserved SUMO-targeting consensus site in various types of mammalian cells. We found that SUMOylation of ATF5 is elevated in the G1 phase of the cell cycle and diminished in the G2/M phase. ATF5 SUMOylation disrupted the interaction of ATF5 with several centrosomal proteins and dislodged ATF5 from the centrosome at the end of the M phase. Of note, blockade of ATF5 SUMOylation deregulated the centrosome cycle, impeded ATF5 translocation from the centrosome, and caused genomic instability and G2/M arrest in HeLa cells. Our results indicate that ATF5 SUMOylation is an essential mechanism that regulates ATF5 localization and function at the centrosome.

Sumoylation of SMAD 4 ameliorates the oxidative stress-induced apoptosis in osteoblasts.

Oxidative stress-induced mitochondrial function and cell apoptosis to osteoblasts, plays a critical role in the pathophysiology of osteoporosis. However, mechanisms underlying such process remain not yet clear. We aims in this study to investigate a possible role of SMAD (the mothers against decapentaplegic homolog 4 (SMAD4) in the oxidative stress-induced apoptosis, in homo sapiens osteoblast hFOB1.19 cells. Results demonstrated that the treatment with more than 100µM H2O2 significantly downregulated the cellular viability, whereas markedly induced apoptosis in hFOB1.19 cells. The SMAD4 was markedly reduced in both mRNA and protein levels in the H2O2 -treated hFOB1.19 cells, along with the reduction of Small ubiquitin-related modifier 1 (SUMO 1) and SUMO 2/3. The immunoprecipitation assay confirmed indicated the interaction between SUMO 1 (or SUMO 2/3) and SMAD4. Moreover, the SMAD4 overexpression markedly ameliorated the H2O2-resulted viability reduction and apoptosis induction in hFOB1.19 cells. Interestingly, such amelioration was blocked by the knockdown of SUMO 2/3. Taken together, we conclued that SMAD4 inhibits the H2O2-induced apoptosis in osteoblast hFOB1.19 cells; such inhibition might depend on the SUMOylation by SUMO 2/3. It implies a promising role of SMAD4 in oxidative stress-promoted damage to osteoblasts.

Inhibitors targeting the SUMOylation pathway: A patent review 2012­2015 (Review).

Small ubiquitin­related modifier (SUMO) proteins bind to the lysine residue of target proteins to produce functionally mature proteins. The abnormal SUMOylation of certain target proteins is associated with diseases including cancer, heart disease, diabetes, arthritis, degenerative diseases and brain ischemia/stroke. Thus, there has been growing appreciation for the potential importance of the SUMO conjugation pathway as a target for treating these diseases. This review introduces the important steps in the reversible SUMOylation pathway. The SUMO inhibitors disclosed in the patents between 2012 and 2015 are divided into different categories according to their mechanisms of action. Certain compounds disclosed in this review have also been reported in other articles for their inhibition of the SUMOylation pathway following screening in cell lines. Although there are few studies using animal models or clinical trials that have used these compounds, the application of bortezomin, a ubiquitylation inhibitor, for treating cancer indicates that SUMO inhibitors may be clinically successful.

Respiratory syncytial virus induces NRF2 degradation through a promyelocytic leukemia protein - ring finger protein 4 dependent pathway.

Respiratory syncytial virus (RSV) is the most important cause of viral acute respiratory tract infections and hospitalizations in children, for which no vaccine or specific treatments are available. RSV causes airway mucosa inflammation and cellular oxidative damage by triggering production of reactive oxygen species and by inhibiting at the same time expression of antioxidant enzymes, via degradation of the transcription factor NF-E2-related factor 2 (NRF2). RSV infection induces NRF2 deacetylation, ubiquitination, and degradation through a proteasome-dependent pathway. Although degradation via KEAP1 is the most common mechanism, silencing KEAP1 expression did not rescue NRF2 levels during RSV infection. We found that RSV-induced NRF2 degradation occurs in an SUMO-specific E3 ubiquitin ligase - RING finger protein 4 (RNF4)-dependent manner. NRF2 is progressively SUMOylated in RSV infection and either blocking SUMOylation or silencing RNF4 expression rescued both NRF2 nuclear levels and transcriptional activity. RNF4 associates with promyelocytic leukemia - nuclear bodies (PML-NBs). RSV infection induces the expression of PML and PML-NBs formation in an interferon (INF)-dependent manner and also induces NRF2 - PMN-NBs association. Inhibition of PML-NB formation by blocking IFN pathway or silencing PML expression resulted in a significant reduction of RSV-associated NRF2 degradation and increased antioxidant enzyme expression, identifying the RNF4-PML pathway as a key regulator of antioxidant defenses in the course of viral infection.

Generation of specific inhibitors of SUMO-1- and SUMO-2/3-mediated protein-protein interactions using Affimer (Adhiron) technology.

Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO-1, SUMO-2, or SUMO-3), and it regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate noncovalent interactions with SUMO. We describe the use of the Affimer system of peptide display for the rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modeling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO-1-mediated interactions or interactions mediated by either SUMO-2 or SUMO-3. Not only will these reagents enable investigation of the biological roles of SUMOylation, but the Affimer technology used to generate these synthetic binding proteins could also be exploited to design or validate reagents or therapeutics that target other protein-protein interactions.

Probing the roles of SUMOylation in cancer cell biology by using a selective SAE inhibitor.

Small ubiquitin-like modifier (SUMO) family proteins regulate target-protein functions by post-translational modification. However, a potent and selective inhibitor targeting the SUMO pathway has been lacking. Here we describe ML-792, a mechanism-based SUMO-activating enzyme (SAE) inhibitor with nanomolar potency in cellular assays. ML-792 selectively blocks SAE enzyme activity and total SUMOylation, thus decreasing cancer cell proliferation. Moreover, we found that induction of the MYC oncogene increased the ML-792-mediated viability effect in cancer cells, thus indicating a potential application of SAE inhibitors in treating MYC-amplified tumors. Using ML-792, we further explored the critical roles of SUMOylation in mitotic progression and chromosome segregation. Furthermore, expression of an SAE catalytic-subunit (UBA2) S95N M97T mutant rescued SUMOylation loss and the mitotic defect induced by ML-792, thus confirming the selectivity of ML-792. As a potent and selective SAE inhibitor, ML-792 provides rapid loss of endogenously SUMOylated proteins, thereby facilitating novel insights into SUMO biology.

Arsenic trioxide targets MTHFD1 and SUMO-dependent nuclear de novo thymidylate biosynthesis.

Arsenic exposure increases risk for cancers and is teratogenic in animal models. Here we demonstrate that small ubiquitin-like modifier (SUMO)- and folate-dependent nuclear de novo thymidylate (dTMP) biosynthesis is a sensitive target of arsenic trioxide (As2O3), leading to uracil misincorporation into DNA and genome instability. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and serine hydroxymethyltransferase (SHMT) generate 5,10-methylenetetrahydrofolate for de novo dTMP biosynthesis and translocate to the nucleus during S-phase, where they form a multienzyme complex with thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR), as well as the components of the DNA replication machinery. As2O3 exposure increased MTHFD1 SUMOylation in cultured cells and in in vitro SUMOylation reactions, and increased MTHFD1 ubiquitination and MTHFD1 and SHMT1 degradation. As2O3 inhibited de novo dTMP biosynthesis in a dose-dependent manner, increased uracil levels in nuclear DNA, and increased genome instability. These results demonstrate that MTHFD1 and SHMT1, which are key enzymes providing one-carbon units for dTMP biosynthesis in the form of 5,10-methylenetetrahydrofolate, are direct targets of As2O3-induced proteolytic degradation, providing a mechanism for arsenic in the etiology of cancer and developmental anomalies.

SUMOylation Inhibition Mediated by Disruption of SUMO E1-E2 Interactions Confers Plant Susceptibility to Necrotrophic Fungal Pathogens.

Protein modification by SUMO modulates essential biological processes in eukaryotes. SUMOylation is facilitated by sequential action of the E1-activating, E2-conjugating, and E3-ligase enzymes. In plants, SUMO regulates plant development and stress responses, which are key determinants in agricultural productivity. To generate additional tools for advancing our knowledge about the SUMO biology, we have developed a strategy for inhibiting in vivo SUMO conjugation based on disruption of SUMO E1-E2 interactions through expression of E1 SAE2UFDCt domain. Targeted mutagenesis and phylogenetic analyses revealed that this inhibition involves a short motif in SAE2UFDCt highly divergent across kingdoms. Transgenic plants expressing the SAE2UFDCt domain displayed dose-dependent inhibition of SUMO conjugation, and have revealed the existence of a post-transcriptional mechanism that regulates SUMO E2 conjugating enzyme levels. Interestingly, these transgenic plants displayed increased susceptibility to necrotrophic fungal infections by Botrytis cinerea and Plectosphaerella cucumerina. Early after fungal inoculation, host SUMO conjugation was post-transcriptionally downregulated, suggesting that targeting SUMOylation machinery could constitute a novel mechanism for fungal pathogenicity. These findings support the role of SUMOylation as a mechanism involved in plant protection from environmental stresses. In addition, the strategy for inhibiting SUMO conjugation in vivo described in this study might be applicable in important crop plants and other non-plant organisms regardless of their genetic complexity.

Specific regulation of PRMT1 expression by PIAS1 and RKIP in BEAS-2B epithelia cells and HFL-1 fibroblasts in lung inflammation.

Protein arginine methyltransferase 1 (PRMT1) catalyzes methylation of histones and other cellular proteins, and thus regulates gene transcription and protein activity. In antigen-induced pulmonary inflammation (AIPI) PRMT1 was up-regulated in the epithelium, while in chronic AIPI, increased PRMT1 shifted to fibroblasts. In this study we investigated the cell type specific regulatory mechanism of PRMT1. Epithelial cells and fibroblasts were stimulated with IL-4 or IL-1ß. Gene and protein expression were determined by RT-qPCR, immunohistochemistry staining and Western blotting. Signaling pathway inhibitors, siRNAs and shRNA were used to determine the regulatory mechanism of PRMT1. The results showed that IL-4 up-regulated PRMT1 through STAT6 signaling in epithelial cells, while IL-1ß regulated PRMT1 through NF-κB in fibroblasts. The NF-kB inhibitor protein RKIP was highly expressed in epithelial cells and blocked IL-1ß induced PRMT1 up-regulation; while the STAT6 inhibitor protein PIAS1 was expressed in fibroblasts and suppressed IL-4 induced PRMT1 expression. Furthermore, IL-4 stimulated epithelial cells to release IL-1ß which up-regulated PRMT1 expression in fibroblasts. In conclusion, the inhibitor proteins RKIP and PIAS1 regulated the cell type and signaling specific expression of PRMT1. Thus PRMT1 expression in structural lung cells in asthma can be considered as potential target for new therapeutic intervention.

Identification of new SUMO activating enzyme 1 inhibitors using virtual screening and scaffold hopping.

Sumoylation involves the enzymatic conjugation of small ubiquitin-like modifier (SUMO) protein to their substrate proteins. Sumoylation is not only crucial for maintaining normal cellular physiology but also implicated in the development of several diseases including cancer. SUMO E1, the first protein in sumoylation pathway is of particular significance due to its confirmed role in tumorogenesis. However, notwithstanding its role as potential drug target, only a few small molecule inhibitors of SUMO E1 have been identified. Here, we report the identification of pyrazole and thiazole urea containing compounds as inhibitors of SUMO E1. We have utilized 3D-shape matching, electrostatic potential similarity evaluations and molecular docking to scaffold hop from previously known aryl urea scaffold with SUMO E1 activity to thiazole and pyrazole urea based scaffolds. These two classes of compounds were found to have moderate SUMO E1 inhibitory activity and can be used as starting points for the development of highly potent lead compounds against cancer.

SENP3 regulates the global protein turnover and the Sp1 level via antagonizing SUMO2/3-targeted ubiquitination and degradation.

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.