Protein SUMOylation Research Articles

Dysregulation of protein SUMOylation networks in Huntington's disease R6/2 mouse striatum.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat mutation in the Huntingtin (HTT) gene. The mutation impacts neuronal protein homeostasis and cortical/striatal circuitry. SUMOylation is a post-translational modification with broad cellular effects including via modification of synaptic proteins. Here, we used an optimised SUMO protein-enrichment and mass spectrometry method to identify the protein SUMOylation/SUMO interaction proteome in the context of HD using R6/2 transgenic and non-transgenic (NT) mice. Significant changes in enrichment of SUMOylated and SUMO-interacting proteins were observed, including those involved in presynaptic function, cytomatrix at the active zone scaffolding, cytoskeleton organization, and glutamatergic signaling. Mitochondrial and RNA-binding proteins also showed altered enrichment. Modified SUMO-associated pathways in HD tissue include clathrin-mediated endocytosis signaling, synaptogenesis signaling, synaptic long-term potentiation, and SNARE signaling. To evaluate how modulation of SUMOylation might influence functional measures of neuronal activity in HD cells in vitro, we utilised primary neuronal cultures from R6/2 and NT mice. A receptor internalization assay for the metabotropic glutamate receptor 7 (mGLUR7), a SUMO enriched protein in the mass spec, showed decreased internalization in R6/2 neurons compared to NT. siRNA-mediated knockdown of the E3 SUMO ligase Protein Inhibitor of Activated STAT1 (Pias1), which can SUMO modify mGLUR7, prevented this HD phenotype. In addition, microelectrode array analysis of primary neuronal cultures indicated early hyperactivity in HD cells, while later timepoints demonstrated deficits in several measurements of neuronal activity within cortical neurons. HD phenotypes were rescued at selected timepoints following knockdown of Pias1. Collectively, our results provide a mouse brain SUMOome resource and show that significant alterations occur within the post-translational landscape of SUMO-protein interactions of synaptic proteins in HD mice, suggesting that targeting of synaptic SUMO networks may provide a proteostatic systems-based therapeutic approach for HD and other neurological. Disorders.

The SUMO gene MrSmt3 is involved in SUMOylation, conidiation and stress response in Metarhizium robertsii.

Smt3, as a small ubiquitin-like modifier (SUMO), play an essential role in the regulation of protein SUMOylation, and thus this process can affect various important biological functions. Here, we investigated the roles of MrSmt3 (yeast SUMO/Smt3 homologs) in the entomopathogenic fungus Metarhizium robertsii. Our results of subcellular localization assays demonstrated that MrSmt3 was present in the cytoplasm and nucleus, whereas MrSmt3 was largely localized in the nucleus during oxidative stress. Importantly, disruption of MrSmt3 significantly decreased the level of protein SUMOylation under heat stress. Deletion of MrSmt3 led to a significant decrease in conidial production, and increased sensitivity to various stresses, including heat, oxidative, and cell wall-disturbing agents. However, bioassays of direct injection and topical inoculation demonstrated that deletion of MrSmt3 did not affect fungal virulence. Furthermore, RNA-seq analysis identified 1,484 differentially expressed genes (DEGs) of the WT and ΔMrSmt3 during conidiation, including 971 down-regulated DEGs and 513 up-regulated DEGs, and further analysis showed that the expression level of several classical conidiation-associated genes, such as transcription factor AbaA (MAA_00694), transcription factor bZIP (MAA_00888) and transcription factor Ste12 (MAA_10450), was down-regulated in the ΔMrSmt3 mutant. Specifically, the major downregulated DEGs were mainly associated with a variety of metabolic regulatory processes including metabolic process, organic substance metabolic process and primary metabolic process. Collectively, our findings highlight the important roles of the SUMO gene MrSmt3 in modulating SUMOylation, conidiation and stress response in M. robertsii.

Functional characterization of protein SUMOylation in the miRNA transcription regulation during heat stress in Arabidopsis.

MicroRNAs (miRNAs) play an essential role as non-coding-RNA-type epigenetic regulators in response to high-temperature stress in plants. There are crucial roles for global transcriptional regulation under SUMO (small ubiquitin-related MOdifier) stress response (SSR). However, the molecular mechanisms underlying its downstream regulation remain unclear. In this study, SUMO-specific chromatin immunoprecipitation sequencing analysis detected specific binding in the promoter region of miRNAs under high-temperature stress. A correlation analysis between this binding and miRNA profiling revealed that the location of SUMO on the chromosome was correlated with the expression pattern of miRNAs, particularly miR398a and miR824a. In contrast, knockout mutants of the SSR-dependent SUMO E3 ligase SAP AND MIZ 1 in Arabidopsis exhibited opposing trends in target gene expression for the SUMO-related miRNAs compared to the wild type. Multi-omics correlation analyses identified 34 SUMO-candidate proteins that might be involved in the regulation of miRNA response to high-temperature stress. Therefore, we propose a potential model whereby high-temperature exposure induces nuclear entry of SUMO molecules, modifying specific transcription factors that bind to miRNA gene promoters and potentially regulate miRNA expression.

Highly sensitive site-specific SUMOylation proteomics in Arabidopsis.

SUMOylation-the attachment of a small ubiquitin-like modifier (SUMO) to target proteins-plays roles in controlling plant growth, nutrient signalling and stress responses. SUMOylation studies in plants are scarce because identifying SUMOylated proteins and their sites is challenging. To date, only around 80 SUMOylation sites have been identified. Here we introduce lysine-null SUMO1 into the Arabidopsis sumo1 sumo2 mutant and establish a two-step lysine-null SUMO enrichment method. We identified a site-specific SUMOylome comprising over 2,200 SUMOylation sites from 1,300 putative acceptors that function in numerous nuclear processes. SUMOylation marks occur on several motifs, differing from the canonical ψKxE motif in distant eukaryotes. Quantitative comparisons demonstrate that SUMOylation predominantly enhances the stability of SUMO1 acceptors. Our study delivers a highly sensitive and efficient method for site-specific SUMOylome studies and provides a comprehensive catalogue of Arabidopsis SUMOylation, serving as a valuable resource with which to further explore how SUMOylation regulates protein function.

Protein SUMOylation and Its Functional Role in Nuclear Receptor Control

Post-translational protein modifications (PTMs) significantly enhance the functional diversity of proteins and are therefore important for the expansion and the dynamics of the cell’s proteome. In addition to structurally simpler PTMs, substrates also undergo modification through the reversible attachment of small proteins. The best understood PTM of this nature to date is the covalent conjugation of ubiquitin and ubiquitin-like proteins (UBLs) to their substrates. The protein family of small ubiquitin-like modifier (SUMO) is one of these UBLs that has received increasing scientific attention. The pathway of SUMOylation is highly conserved in all eukaryotic cells and is crucial for their survival. It plays an essential role in many biological processes, such as the maintenance of genomic integrity, transcriptional regulation, gene expression, and the regulation of intracellular signal transduction, and thereby influences DNA damage repair, immune responses, cell cycle progression, and apoptosis. Several studies have already shown that in this context protein SUMOylation is involved in the control mechanisms of various cellular receptors. This article unites data from different studies focusing on the investigation of the strictly conserved three-step enzyme cascade of protein SUMOylation and the functional analysis of the involved proteins E1, E2, and E3 and SUMOylation target proteins. Furthermore, this review highlights the role of nuclear receptor SUMOylation and its importance for the cellular functionality and disease development arising from defects in correct protein SUMOylation.

Crosstalk between PKA and PIAS3 regulates cardiac Kv4 channel SUMOylation

Post-translational SUMOylation of nuclear and cytosolic proteins maintains homeostasis in eukaryotic cells and orchestrates programmed responses to changes in metabolic demand or extracellular stimuli. In excitable cells, SUMOylation tunes the biophysical properties and trafficking of ion channels. Ion channel SUMOylation status is determined by the opposing enzyme activities of SUMO ligases and deconjugases. Phosphorylation also plays a permissive role in SUMOylation. SUMO deconjugases have been identified for several ion channels, but their corresponding E3 ligases remain unknown. This study shows PIAS3, a.k.a. KChAP, is a bona fide SUMO E3 ligase for Kv4.2 and HCN2 channels in HEK cells, and endogenous Kv4.2 and Kv4.3 channels in cardiomyocytes. PIAS3-mediated SUMOylation at Kv4.2-K579 increases channel surface expression through a rab11a-dependent recycling mechanism. PKA phosphorylation at Kv4.2-S552 reduces the current mediated by Kv4 channels in HEK293 cells, cardiomyocytes, and neurons. This study shows PKA mediated phosphorylation blocks Kv4.2-K579 SUMOylation in HEK cells and cardiomyocytes. Together, these data identify PIAS3 as a key downstream mediator in signaling cascades that control ion channel surface expression.

TOPORS E3 ligase mediates resistance to hypomethylating agent cytotoxicity in acute myeloid leukemia cells

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are commonplace. To address this, we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identify TOPORS as a loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable, we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML.

Pepper SUMO protease CaDeSI2 positively modulates the drought responses via deSUMOylation of clade A PP2C CaAITP1.

Posttranslational modification of multiple ABA signaling components is an essential process for the adaptation and survival of plants under stress conditions. In our previous study, we established that the pepper group A PP2C protein CaAITP1, one of the core components of ABA signaling, undergoes ubiquitination mediated by the RING-type E3 ligase CaAIRE1. In this study, we discovered an additional form of regulation mediated via the SUMOylation of CaAITP1. Pepper plants subjected to drought stress were characterized by reductions in both the stability and SUMOylation of CaAITP1 protein. Moreover, we identified a SUMO protease, Capsicum annuum DeSUMOylating Isopeptidase 2 (CaDeSI2), as a new interacting partner of CaAITP1. In vitro and in vivo analyses revealed that CaAITP1 is deSUMOylated by CaDeSI2. Silencing of CaDeSI2 in pepper plants led to drought-hypersensitive and ABA-hyposensitive phenotypes, whereas overexpression of CaDeSI2 in transgenic Arabidopsis plants resulted in the opposite phenotypes. Importantly, we found that the CaAITP1 protein was stabilized in response to the silencing of CaDeSI2, and CaDeSI2 and CaAITP1 co-silenced pepper plants were characterized by drought-tolerant phenotypes similar to those observed in CaAITP1-silenced pepper. Collectively, our findings indicate that CaDeSI2 reduces the stability of CaAITP1 via deSUMOylation, thereby positively regulating drought tolerance.

RREB1-mediated SUMOylation enhancement promotes chemoresistance partially by transcriptionally upregulating UBC9 in colorectal cancer.

Chemoresistance is a main cause of chemotherapy failure and tumor recurrence. The effects of global protein SUMOylation on chemoresistance in colorectal cancer (CRC) remains to be investigated. Herein, we have proposed that the elevated SUMO2/3-modified proteins confer 5-fluorouracil (5-FU) chemoresistance acquisition in CRC. The SUMOylation levels of global proteins in CRC cell lines were elevated compared with normal colon cell line NCM460. 5-FU treatment obviously reduced SUMOylation of global proteins in 5-FU-sensitive CRC cells including HT29, HCT116 and HCT-8. However, in 5-FU-resistant HCT-8/5-FU cells, the expression level of SUMO2/3-modified proteins was increased under 5-FU exposure in a concentration-dependent manner. 5-FU treatment combined with SUMOylation inhibitor ML-792 significantly increased the sensitivity of 5-FU-resistant cells to 5-FU and reduced colony formation numbers in HCT-8/5-FU cells. And UBC9-mediated SUMOylation elevation contributes to 5-FU resistance in HCT116 cells. Moreover, we also identified RREB1 as a regulator of SUMOylation profiling of global cellular proteins via directly binding to the promoter of UBC9. Overexpression of RREB1 promoted 5-FU resistance in CRC, which was partially abolished by treatment of inhibitor ML-792. In conclusion, RREB1-enhanced protein SUMOylation contributes to 5-FU resistance acquisition in CRC.

Protein SUMOylation promotes cAMP-independent EPAC1 activation

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

MODL-03. THERAPEUTIC EFFICACY OF IMIPIRIDONES IN LYNCH SYNDROME-ASSOCIATED GLIOMAS

Abstract INTRODUCTION Lynch Syndrome, a hereditary cancer syndrome linked to DNA mismatch repair gene mutations (MMRD), predisposes individuals to various cancers, including high grade gliomas (Lynch-HGG). Effective treatments for Lynch-HGGs are scarce, and suitable pre-clinical models are needed to identify novel therapeutic combinations. METHODS In this study, we generated patient-derived cell line models of Lynch-HGGs (n=3), characterized them through Western blot analysis for MMRD gene markers, and conducted genome and transcriptomic analyses. Murine models were then generated via intracranial injection of cultured cells expressing mCherry-luciferase reporter genes. Drug screening based on transcriptomic analysis was performed to identify potential therapeutic agents. RESULTS Our findings showed loss of MMRD protein consistent with the primary mutation, and a constant loss of P53, a tumor suppressor protein. One cell line, 7316-3058, exhibited an absence of NF1 expression, another critical tumor suppressor protein. Transcriptomic analysis unveiled upregulation in cell cycle processes and SUMOylation of DNA damage response and repair proteins. In vitro treatment with imipiridone compounds (ONC201 and ONC206) demonstrated varying degrees of drug response, with 7316-3058 displaying high sensitivity to the targeted inhibitor. Unfortunately, this therapeutic efficacy did not translate to the in vivo murine model, suggesting underlying mechanisms that impede drug efficiency. CONCLUSION In summary, we developed effective pre-clinical models of Lynch-HGG that can be used to screen different drug combinations targeted to treat these tumors. Furthermore, despite promising in-vitro data, we demonstrated the limited efficacy of imipiridones in treating Lynch-HGG in vivo, emphasizing the challenges in translating in vitro findings to in vivo contexts. This study contributes valuable insights to the development of effective therapeutic strategies for Lynch Syndrome-associated gliomas.

DIPG-16. THE BIG AND SMALL OF PROTEIN POST-TRANSLATIONAL MODIFICATIONS: TARGETING SUMOYLATION AND CITRULLINATION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Protein post-translational modifications (PTMs) alter the properties of protein through proteolytic cleavage, or the addition of modifying groups to one or more amino acids. Histones are major targets of PTMs, including through SUMOylation of lysine residues and citrullination of arginine residues, with both processes able to regulate gene transcription. We aimed to delineate the potential role of SUMOylation and citrullination in the epigenetic dysregulation that drives Diffuse Midline Glioma (DMG) tumorigenicity. METHODS AND RESULTS DMG tumours demonstrated an upregulation of SUMOylation machinery pathway components and increased expression of peptidylarginine deiminases (PADs), the enzymes responsible for citrullination. Nanomolar doses of the SUMOylation inhibitor TAK-981 blocks DMG cell cycle G2/M transition and results in potent decreases in cell viability and clonogenic potential, triggering ‘viral mimicry’ in DMG cells, resulting in increased expression of interferon stimulated genes and inflammatory mediators including IL-1β, IL-6 and TNFα, whilst also causing a pro-inflammatory M1 macrophage response. These data suggest that SUMOylation inhibition may lead to a double-punch effect against DMG by inhibiting tumorgenicity and stimulating an immune response. In further studies, we have shown that pharmacological inhibition of PAD activity in DMG decreases histone citrullination and restores histone methylation – considered the Achilles heel of DMG. This was accompanied by induction of reactive oxygen species and an unfolded protein response, resulting in DMG cell death in a dose dependent manner. In addition to fully characterising the epigenetic and global changes within DMG initiated through SUMOylation or PAD inhibition, we are currently performing preliminary drug efficacy studies in orthotopic DMG models, providing the necessary pre-clinical research pipeline towards an effective treatment for DMG. CONCLUSIONS Protein SUMOylation and citrullination are previously unrecognised therapeutic targets in DMG and potentially play key roles in tumour initiation and pathogenesis. Our data suggests a novel therapeutic approach for children with DMG.

#2743 Impact of fatty acid-binding protein 4 on SUMOylation balance and renal tubular epithelial cell senescence in acute kidney injury

Abstract Background and Aims Dysregulated lipid metabolism and cellular senescence intricately interact in the context of acute kidney injury (AKI). Fatty acid-binding protein 4 (FABP4), a critical enzyme regulating lipid metabolism, potentially induces cell senescence through lipotoxicity in AKI. However, the specific mechanisms remain unclear. Method We established an ischemia-reperfusion-induced AKI (IRI-AKI) model in C57 mice and quantified and localized FABP4 expression in renal tissues using Western blotting (WB), qPCR, and immunofluorescence (IF). Oil Red O staining and beta-galactosidase staining were employed to assess lipid deposition and cellular senescence in tubular cells. The same measurements were repeated in IRI-AKI by knocking out Fabp4. Additionally, bulk mRNA sequencing and single-cell sequencing were conducted on renal tissues to analyze key molecules and related mechanisms. Results In vivo, we observed a significant decline in renal function, severe tubular epithelial damage, and abundant inflammation in the IRI-AKI group, which was noticeably alleviated in the Fabp4 knockout group (Fig. 1). Furthermore, we detected upregulation of FABP4 expression in the kidneys of IRI-AKI, primarily localized in the tubular epithelial cells. Fabp4 knockout effectively reduced tubular lipid accumulation and cellular senescence (Fig. 3). Through integrating mRNA and single-cell sequencing data, UBC9, an E2 enzyme involved in protein SUMOylation, emerged as a potential key regulator of cell senescence and AKI. WB and qPCR both confirmed the upregulation of UBC9 expression in IRI-AKI and its downregulation after Fabp4 knockout. Conversely, we observed that downregulated SUMOylation in IRI-AKI was upregulated in the Fabp4 knockout group (Fig. 4). Conclusion In conclusion, FABP4 modulates the expression of UBC9 and SUMOylation, thereby mediating cellular senescence and causing renal tubular cell damage in IRI-AKI. The mechanisms underlying FABP4’s regulation of UBC9 and the opposing trend between UBC9 and SUMOylation require further elucidation.

SUMO-specific protease 1 regulates germinal center B cell response through deSUMOylation of PAX5

Humoral immunity depends on the germinal center (GC) reaction where B cells are tightly controlled for class-switch recombination and somatic hypermutation and finally generated into plasma and memory B cells. However, how protein SUMOylation regulates the process of the GC reaction remains largely unknown. Here, we show that the expression of SUMO-specific protease 1 (SENP1) is up-regulated in GC B cells. Selective ablation of SENP1 in GC B cells results in impaired GC dark and light zone organization and reduced IgG1-switched GC B cells, leading to diminished production of class-switched antibodies with high-affinity in response to a TD antigen challenge. Mechanistically, SENP1 directly binds to Paired box protein 5 (PAX5) to mediate PAX5 deSUMOylation, sustaining PAX5 protein stability to promote the transcription of activation-induced cytidine deaminase. In summary, our study uncovers SUMOylation as an important posttranslational mechanism regulating GC B cell response.

Knocking Down PIAS3 Reduces H2O2-induced Oxidative Stress Injury in HT22 Cells.

Oxidative stress is involved in the pathological processes of many neurodegenerative diseases. Protein modification by small ubiquitin-like modifiers (SUMOs) has been implicated in oxidative stress injury. By conjugating SUMOs to their selective protein substrates, SUMO ligases play critical roles in regulating functions of proteins involved in oxidative stress injury. In this study, we screened siRNAs to knockdown the SUMO ligase PIAS3 to assess its role in H2O2-induced injury in HT22 cells. H2O2 stimulation increased total protein SUMOylation, facilitated intracellular reactive oxygen species (ROS) release, increased cleaved caspase-3 levels, promoted p38 and JNK activation (phosphorylation), upregulated apoptosis, and decreased cell viability. The siRNA against PIAS3 329-347 (siPIAS3-329) markedly downregulated the protein expression of PIAS3 and reversed these effects, whereas siNC (negative control) had no effect. Our findings demonstrate that PIAS3-mediated SUMOylation facilitates oxidative stress injury and p38/JNK-mediated cell apoptosis and that PIAS3 is a potential target to protect against oxidative stress injury.

Topotecan and Ginkgolic Acid Inhibit the Expression and Transport Activity of Human Organic Anion Transporter 3 by Suppressing SUMOylation of the Transporter.

Organic anion transporter 3 (OAT3), expressed at the basolateral membrane of kidney proximal tubule cells, facilitates the elimination of numerous metabolites, environmental toxins, and clinically important drugs. An earlier investigation from our laboratory revealed that OAT3 expression and transport activity can be upregulated by SUMOylation, a post-translational modification that covalently conjugates SUMO molecules to substrate proteins. Topotecan is a semi-synthetic derivative of the herbal extract camptothecin, approved by the FDA to treat several types of cancer. Ginkgolic acid (GA) is one of the major components in the extract of Ginkgo biloba leaves that has long been used in food supplements for preventing dementia, high blood pressure, and supporting stroke recovery. Both topotecan and GA have been shown to affect protein SUMOylation. In the current study, we tested our hypothesis that topotecan and GA may regulate OAT3 SUMOylation, expression, and transport function. Our data show that the treatment of OAT3-expressing cells with topotecan or GA significantly decreases the SUMOylation of OAT3 by 50% and 75%, respectively. The same treatment also led to substantial reductions in OAT3 expression and the OAT3-mediated transport of estrone sulfate, a prototypical substrate. Such reductions in cell surface expression of OAT3 correlated well with an increased rate of OAT3 degradation. Mechanistically, we discovered that topotecan enhanced the association between OAT3 and the SUMO-specific protease SENP2, a deSUMOylation enzyme, which contributed to the significant decrease in OAT3 SUMOylation. In conclusion, this study unveiled a novel role of topotecan and GA in inhibiting OAT3 expression and transport activity and accelerating OAT3 degradation by suppressing OAT3 SUMOylation. During comorbidity therapies, the use of topotecan or Ginkgo biloba extract could potentially decrease the transport activity of OAT3 in the kidneys, which will in turn affect the therapeutic efficacy and toxicity of many other drugs that are substrates for the transporter.

SUMOylation modification of FTO facilitates oxidative damage response of arsenic by IGF2BP3 in an m6A-dependent manner

N6-methyladenosine (m6A) is the most common form of internal post-transcriptional methylation observed in eukaryotic mRNAs. The abnormally increased level of m6A within the cells can be catalyzed by specific demethylase fat mass and obesity-associated protein (FTO) and stay in a dynamic and reversible state. However, whether and how FTO regulates oxidative damage via m6A modification remain largely unclear. Herein, by using both in vitro and in vivo models of oxidative damage induced by arsenic, we demonstrated for the first time that exposure to arsenic caused a significant increase in SUMOylation of FTO protein, and FTO SUMOylation at lysine (K)− 216 site promoted the down-regulation of FTO expression in arsenic target organ lung, and therefore, remarkably elevating the oxidative damage via an m6A-dependent pathway by its specific m6A reader insulin-like growth factor-2 mRNA-binding protein-3 (IGF2BP3). Consequently, these findings not only reveal a novel mechanism underlying FTO-mediated oxidative damage from the perspective of m6A, but also imply that regulation of FTO SUMOylation may serve as potential approach for treatment of oxidative damage.

Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation

Protein SUMOylation provides a principal driving force for cellular stress responses, including DNA–protein crosslink (DPC) repair and arsenic-induced PML body degradation. In this study, using genome-scale screens, we identified the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL), combining ubiquitin ligase activity through its RING domain with poly-SUMO binding via SUMO-interacting motifs, analogous to the STUbL RNF4. Mechanistically, TOPORS is a SUMO1-selective STUbL that complements RNF4 in generating complex ubiquitin landscapes on SUMOylated targets, including DPCs and PML, stimulating efficient p97/VCP unfoldase recruitment and proteasomal degradation. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, involving defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Our findings establish TOPORS as a STUbL whose parallel action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO–ubiquitin crosstalk.

UBA2 as a Prognostic Biomarker and Potential Therapeutic Target in Glioma.

Gliomas are characterized by aggressive behavior, leading to severe disability and high mortality. Ubiquitin-like modifier activating enzyme 2 (UBA2) is a subunit of the E1-activating enzyme involved in the SUMOylation (SUMO, small ubiquitin-related modifier) of numerous proteins. Although the abnormality of UBA2 is linked to the progression of various tumor types, the role of UBA2 in glioma is still unknown. A bioinformatic analysis using several public databases was conducted to examine the expression level, clinicopathological correlations, and prognostic significance of UBA2 in glioma. The correlation between UBA2 expression and drug sensitivity in cancers was also explored. Multiple cellular experiments were conducted to validate the role of UBA2 in glioma. Analysis of multiple databases and cellular experiments revealed that UBA2 was overexpressed in glioma tissues and cell lines, respectively. UBA2 expression in gliomas correlated with World Health Organization (WHO) grade, IDH gene status, 1p19q deletion, histological type, and immune cell infiltration in glioma. UBA2 expression in carcinomas also correlated with drug sensitivity. Kaplan-Meier analysis revealed that high expression of UBA2 predicted poorer survival in glioma patients. A nomogram model containing UBA2 expression was constructed for clinical practice. Knockdown of UBA2 was observed to suppress glioma cell progression and sensitize glioma cells to irradiation in vitro. Overall, this research showed that UBA2 might be involved not only in the development of glioma but also in the regulation of immunity, drug sensitivity, and radiosensitivity. Therefore, UBA2 may be a potential target for therapy and a candidate biomarker for glioma diagnosis and prognosis.

Rhes, a striatal enriched protein, regulates post-translational small-ubiquitin-like-modifier (SUMO) modification of nuclear proteins and alters gene expression

Rhes (Ras homolog enriched in the striatum), a multifunctional protein that regulates striatal functions associated with motor behaviors and neurological diseases, can shuttle from cell to cell via the formation of tunneling-like nanotubes (TNTs). However, the mechanisms by which Rhes mediates diverse functions remain unclear. Rhes is a small GTPase family member which contains a unique C-terminal Small Ubiquitin-like Modifier (SUMO) E3-like domain that promotes SUMO post-translational modification of proteins (SUMOylation) by promoting “cross-SUMOylation” of the SUMO enzyme SUMO E1 (Aos1/Uba2) and SUMO E2 ligase (Ubc-9). Nevertheless, the identity of the SUMO substrates of Rhes remains largely unknown. Here, by combining high throughput interactome and SUMO proteomics, we report that Rhes regulates the SUMOylation of nuclear proteins that are involved in the regulation of gene expression. Rhes increased the SUMOylation of histone deacetylase 1 (HDAC1) and histone 2B, while decreasing SUMOylation of heterogeneous nuclear ribonucleoprotein M (HNRNPM), protein polybromo-1 (PBRM1) and E3 SUMO-protein ligase (PIASy). We also found that Rhes itself is SUMOylated at 6 different lysine residues (K32, K110, K114, K120, K124, and K245). Furthermore, Rhes regulated the expression of genes involved in cellular morphogenesis and differentiation in the striatum, in a SUMO-dependent manner. Our findings thus provide evidence for a previously undescribed role for Rhes in regulating the SUMOylation of nuclear targets and in orchestrating striatal gene expression via SUMOylation.