SUMOylation Assays Research Articles

Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

Abnormal SUMOylation is implicated in non-alcoholic fatty liver disease (NAFLD) progression. Forkhead box protein A1 (FoxA1) has been shown to protect liver from steatosis, which was down-regulated in NAFLD. This study elucidated the role of FoxA1 deSUMOylation in NAFLD. NAFLD models were established in high-fat diet (HFD)-induced mice and palmitate acid (PAL)-treated hepatocytes. Hepatic steatosis was evaluated by biochemical and histological methods. Lipid droplet formation was determined by BODIPY and Oil red O staining. Target molecule levels were analyzed by RT-qPCR, Western blotting, and immunohistochemistry staining. SUMOylation of FoxA1 was determined by Ni-NTA pull-down assay and SUMOylation assay Ultra Kit. Protein interaction and ubiquitination were detected by Co-IP. Gene transcription was assessed by ChIP and dual luciferase reporter assays. Liver FoxA1 knockout mice developed severe liver steatosis, which could be ameliorated by sirtuin 6 (Sirt6) overexpression. Nutritional stresses reduced Sumo2/3-mediated FoxA1 SUMOylation at lysine residue K6, which promoted lipid droplet formation by repressing fatty acid β-oxidation. Moreover, Sirt6 was a target gene of FoxA1, and Sirt6 transcription activity was restrained by deSUMOylation of FoxA1 at site K6. Furthermore, nutritional stresses-induced deSUMOylation of FoxA1 promoted the ubiquitination and degradation of FoxA1 with assistance of murine double minute 2 (Mdm2). Finally, activating FoxA1 SUMOylation delayed the progression of NAFLD in mice. DeSUMOylation of FoxA1 at K6 promotes FoxA1 degradation and then inhibits Sirt6 transcription, thereby suppressing fatty acid β-oxidation and facilitating NAFLD development. Our findings suggest that FoxA1 SUMOylation activation might be a promising therapeutic strategy for NAFLD.

DeSUMOylation of RBMX regulates exosomal sorting of cargo to promote renal tubulointerstitial fibrosis in diabetic kidney disease

IntroductionDiabetic kidney disease (DKD) has become the primary cause of chronic renal failure in China, and renal tubulointerstitial fibrosis plays a central role in DKD progression. Urinary exosomes, which reflect kidney changes, are largely influenced by RNA-binding proteins (RBPs) in their miRNA content. ObjectivesOur research aimed to determine the effect of the RNA-binding protein RBMX on exosomal miRNA in DKD. MethodsWe introduced a higher level of Rbmx into diabetic mice using an adenoassociated virus and isolated exosomes from their kidney tissue through advanced centrifugation techniques and specialized kits. We then conducted a series of tests, including qRT-PCR, Western blot, MitoSOX, ATP luminescence, coimmunoprecipitation, SUMOylation assays, RNA immunoprecipitation, and confocal microscopy. ResultsRBMX is found in higher levels in DKD and contributes to worsening kidney fibrosis, mitochondrial damage, and miRNA mismanagement in exosomes. It specifically binds with miR-26a, miR-23c, and miR-874 within the exosomes. This dysfunction may be linked to changes in RBMX SUMOylation. These miRNAs seem to protect against mitochondrial damage in kidney cells by targeting CERS6. ConclusionDeSUMOylation of RBMX plays a crucial role in determining the makeup of miRNAs in kidney cell exosomes, impacting the protective miRNAs which regulate mitochondrial damage through their interaction with CERS6 mRNA, ultimately affecting mitochondrial health in DKD.

The transmembrane domain of the rice small protein OsS1Fa1 is responsible for subcellular localization and drought tolerance.

OsS1Fa1, a homologue of spinach S1Fa, is a small protein in rice that contains four distinct conserved motifs and participates in drought tolerance. However, the biological functions of these conserved motifs have not been characterized to date. Therefore, we investigated the roles of these conserved domains in the localization and cellular function of OsS1Fa1. We analysed the subcellular localization of OsS1Fa1 using confocal laser scanning microscopy (CLSM), following particle bombardment and bacterial infiltration. An E.coli in vivo reconstituted sumoylation assay was conducted to investigate sumoylation of OsS1Fa1. We characterized the function of the transmembrane domain of OsS1Fa1 in drought tolerance using transgenic Arabidopsis plants. Fluorescence analysis showed that OsS1Fa1 localized to the nuclear and cytoplasmic membranes. Mutation and cell fractionation analyses revealed that the membrane localization domain determined the subcellular localization of OsS1Fa1. The rice homologue OsS1Fa2 and Arabidopsis orthologs AtS1Fa1, AtS1Fa2, and AtS1Fa3 also exhibited similar localization patterns as OsS1Fa1. Sumoylation analysis demonstrated that OsS1Fa1 was conjugated with the small ubiquitin-related modifier (SUMO). Transgenic analysis showed that overexpression of OsS1Fa1(TMm1), a mutant form of the transmembrane domain of OsS1Fa1, in Arabidopsis did not enhance drought stress tolerance, whereas OsS1Fa1 overexpression improved the drought tolerance of transgenic Arabidopsis. Our data indicate that rice and Arabidopsis S1Fa1 proteins localize in the nuclear and cytoplasmic membranes, and that transmembrane domain determines subcellular localization and plays an important role in drought stress tolerance.

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells

BackgroundThe eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown.MethodsSUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6–SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1–GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student’s t-test, or one-way or two-way analysis of variance (ANOVA).ResultsWe found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells.ConclusionsSUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.Graphical

Interplay between SUMO1-related SUMOylation and phosphorylation of p65 promotes hepatocellular carcinoma progression

The nuclear factor kappaB (NF-κB) subunit p65, plays an important role in the progression of hepatocellular carcinoma (HCC). Phosphorylation of p65 is considered as an important mechanism for the positive regulation of NF-κB activity. According to our previous data, p65 can be SUMOylated by small ubiquitin-related modifier 1 (SUMO1) protein, and SUMO1 promotes p65 nuclear import and HCC progression. However, the effect of SUMO1-related p65 SUMOylation on NF-κB transcriptional activity and the relationship between phosphorylation and SUMOylation of p65 remain obscure. Here, we found that phosphorylated p65 level was increased in cancer tissues of HCC patients, and similar phenomenon was found for SUMO1 expression but not for SUMO2/3. Further clinical data showed a positive correlation between SUMO1 and phosphorylated p65. We also verified that overexpression of SUMO1 upregulated phosphorylated p65 levels. Next, we verified SUMO1-related p65 SUMOylation with in vitro SUMOylation assay, constructed mutants of p65 SUMOylation and phosphorylation, and found that SUMO1-related p65 SUMOylation promoted p65 nuclear import and increased NF-κB activity. Both SUMO1-related p65 SUMOylation and p65 phosphorylation (especially at S276 site) increased the viability and invasion of hepatoma cells, and decreased the apoptosis of hepatoma cells. At last, we found that the phosphorylation of p65 promoted the level of SUMO1-related p65 SUMOylation, and SUMO1-related p65 SUMOylation upregulated phosphorylated p65 (at S276 site). Our study contributes to the exploration of the oncogenic mechanism of p65, which is the important protein in HCC.

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

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). 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-β) signaling pathway in combination with SUMOylation assay. 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. 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.

MiR‑122/SENP1 axis confers stemness and chemoresistance to liver cancer through Wnt/β‑catenin signaling.

The property of inherent stemness of tumor cells coupled with the development of chemoresistance results in a poor prognosis for patients with liver cancer. Therefore, the present study focused on microRNA (miR)-122, a potential tumor suppressor, the expression of which has been previously shown to be significantly decreased and negatively associated with cancer cell stemness in liver cancer. The present study aimed to identify the molecular targets of miR-122 whilst uncovering the mechanism underlying chemoresistance and stemness of HepG2 cells in liver cancer. Bioinformatics online tools, such as ENCORI, coupled with dual-luciferase reporter assays in HepG2 cells, were used to identify and validate small ubiquitin-like modifier (SUMO) specific peptidase 1 (SENP1) as a potential target of miR-122 in liver cancer. The liver cancer stem cell population was determined using sphere formation assays and flow cytometry, whilst stem cell markers (Oct3/4, Nanog, B lymphoma Mo-MLV insertion region 1 homolog and Notch1) were detected by reverse transcription-quantitative PCR. Chemoresistance, cell proliferation and migratory ability of HepG2 cells were monitored using Cell Counting Kit-8, colony formation and Transwell assays, respectively. The overexpression of miR-122 by mimic transfection led to a significant decrease in the number spheres, downregulation of stem cell marker expression, the number of CD24+ cells, drug-resistance protein levels (P-glycoprotein and multidrug resistance protein), impaired chemoresistance, proliferation and migration of HepG2 cells. The transfection of SENP1 overexpression vector resulted in contrasting functions to miR-122 mimics, by partially reversing the effects induced by miR-122 mimic transfection in HepG2 cells. Wnt/β-catenin signaling has been proven to be involved in cancer stemness and malignant behavior. Western blotting analysis in HepG2 cells showed that the expression levels of both Wnt1 and β-catenin were significantly reduced after overexpressing miR-122, but increased after overexpressing SENP1. Co-transfection with the SENP1 overexpression vector reversed the suppression induced by the miR-122 mimics on Wnt1 and β-catenin expression. Co-immunoprecipitation, SUMOylation and half-life assays showed SENP1 interacted with β-catenin and decreased the SUMOylation of β-catenin, thereby enhancing its stability. Finally, tumor xenograft analyses revealed that HepG2 cells transfected with Agomir-122 exerted significantly lower tumor initiation frequency and growth rate, and a superior response to DOX in vivo, compared with those transfected with Agomir NC. Taken together, data from the present study miR-122/SENP1 axis can regulate β-catenin stability through de-SUMOylation, thereby promoting stemness and chemoresistance in liver cancer.

Identification and functional characterization of the SUMO system in sweet potato under salt and drought stress

Sumoylation is a crucial post-translation modification (PTM) that is the covalent attachment of SUMO molecules to the substrate catalyzed by enzyme cascade. Sumoylation is essential in almost every physiological process of plants, particularly in response to abiotic stress. However, little is known about sumoylation in sweet potato (Ipomoea batatas), the world’s seventh most important food crop. In this study, 17 sweet potato SUMO system genes have been cloned and functionally characterized. Multiple sequence alignment and phylogenetic analysis showed sweet potato SUMO system proteins had conserved domains and activity sites. IbSUMOs, IbSAE1, and IbSCE1 were localized in the cytoplasm and nucleus. E3 SUMO ligases showed nuclear or punctate localization. In vitro sumoylation assay confirmed the catalytic activity of sweet potato SUMO system components. Heterologous expression of IbSIZ1 genes in Arabidopsis atsiz1 mutant rescued the defective germination and growth phenotype. IbSCE1a/b and IbSIZ1a/b/c were salt and drought responsive genes. Heterologous expression of IbSCE1a/b/c improved the drought tolerance of Arabidopsis thaliana, while IbSIZ1a/b/c significantly enhanced the salt and drought tolerance. Our findings define that the SUMO system in sweet potato shared with conserved function but also possessed specific characterization. The resources presented here would facilitate uncovering the significance of sumoylation in sweet potato.

Dynamic SUMOylation of MORC2 orchestrates chromatin remodelling and DNA repair in response to DNA damage and drives chemoresistance in breast cancer.

Rationale: SUMOylation regulates a plethora of biological processes, and its inhibitors are currently under investigation in clinical trials as anticancer agents. Thus, identifying new targets with site-specific SUMOylation and defining their biological functions will not only provide new mechanistic insights into the SUMOylation signaling but also open an avenue for developing new strategy for cancer therapy. MORC family CW-type zinc finger 2 (MORC2) is a newly identified chromatin-remodeling enzyme with an emerging role in the DNA damage response (DDR), but its regulatory mechanism remains enigmatic. Methods: In vivo and in vitro SUMOylation assays were used to determine the SUMOylation levels of MORC2. Overexpression and knockdown of SUMO-associated enzymes were used to detect their effects on MORC2 SUMOylation. The effect of dynamic MORC2 SUMOylation on the sensitivity of breast cancer cells to chemotherapeutic drugs was examined through in vitro and in vivo functional assays. Immunoprecipitation, GST pull-down, MNase, and chromatin segregation assays were used to explore the underlying mechanisms. Results: Here, we report that MORC2 is modified by small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 at lysine 767 (K767) in a SUMO-interacting motif dependent manner. MORC2 SUMOylation is induced by SUMO E3 ligase tripartite motif containing 28 (TRIM28) and reversed by deSUMOylase sentrin-specific protease 1 (SENP1). Intriguingly, SUMOylation of MORC2 is decreased at the early stage of DNA damage induced by chemotherapeutic drugs that attenuate the interaction of MORC2 with TRIM28. MORC2 deSUMOylation induces transient chromatin relaxation to enable efficient DNA repair. At the relatively late stage of DNA damage, MORC2 SUMOylation is restored, and SUMOylated MORC2 interacts with protein kinase CSK21 (casein kinase II subunit alpha), which in turn phosphorylates DNA-PKcs (DNA-dependent protein kinase catalytic subunit), thus promoting DNA repair. Notably, expression of a SUMOylation-deficient mutant MORC2 or administration of SUMO inhibitor enhances the sensitivity of breast cancer cells to DNA-damaging chemotherapeutic drugs. Conclusions: Collectively, these findings uncover a novel regulatory mechanism of MORC2 by SUMOylation and reveal the intricate dynamics of MORC2 SUMOylation important for proper DDR. We also propose a promising strategy to sensitize MORC2-driven breast tumors to chemotherapeutic drugs by inhibition of the SUMO pathway.

Identification and expression analysis of a new small ubiquitin-like modifier from Taenia pisiformis

The small ubiquitin-like modifier (SUMO) plays important roles, with the SUMOylation pathway as one of its core components. In the present work, a single SUMO gene was initially identified from Taenia pisiformis and designated as TpSUMO. Bioinformatic analysis showed that the TpSUMO gene contained a 309 bp open reading frame (ORF), encoding 102 amino acids, and had a predicted molecular weight of ∼12 kDa. The amino acid sequence of TpSUMO was deduced and it shared 44.00% identity with human SUMO2 (HsSUMO2) and exhibited more than 97.78% identity with SUMOs from Taenia and Echinococcus. TpSUMO possessed a putative non-consensus site (FK11MG) within its N-terminus and a typical di-glycine (GG) motif at the C-terminus. Basic local alignment search tool (BLAST) analysis showed that only a single SUMO-related ortholog was present in each set of known genome data for fourteen tapeworm species. The precursor His-TpSUMO-FL, mature His-TpSUMO-GG and mutant His-TpSUMO-GGK11R proteins (∼18 kDa) were expressed in Escherichia coli Rosseta (DE3), and rabbit polyclonal anti-TpSUMO was generated with a high titer of 1.28 × 105. In vitro SUMOylation assay results showed that TpSUMO multimer formation in the His-TpSUMO-GG reaction could be catalyzed by the human SAE1/SAE2 and UBC9 conjugation system, but K11R mutation disrupted TpSUMO chain synthesis. Quantitative real-time PCR (qRT-PCR) further revealed that TpSUMO was ubiquitously expressed in different stages of T. pisiformis and in higher levels during an early development phase (day 14) of adult worms. Immunofluorescence localization showed that TpSUMO was detected in the bladder wall of cysticerci, in the testis in immature segment, and within eggs in the gravid proglottids. These findings indicated that TpSUMO is a new member of the SUMO protein family and may play a vital role in regulation of functions within proteins involved in worm growth and development.

Epac1 Regulates Cellular SUMOylation by Promoting The Formation of SUMO‐activating Nuclear Condensates

Protein SUMOylation plays an essential role in maintaining cellular homeostasis when cells are under stress. However, how SUMOylation is regulated, and a molecular mechanism linking cellular stress to SUMOylation remains elusive. We hypothesized that cAMP, a major stress‐response second messenger, acts through exchange protein directly activated by cAMP (Epac1) to regulate cellular SUMOylation. Proteomic analysis reveals that Epac1 associated proteome is highly‐enriched with major components of the SUMOylation machinery and well‐known SUMO target proteins. Activation of Epac1 by intracellular cAMP in Human Umbilical Vein Endothelial Cells (HUVECs) promotes cellular SUMOylation and activates cellular SUMO‐activating enzyme E1 (AOS1/UBA2). Conversely, silencing Epac1 leads to a reduced cellular SUMOylation and SUMO E1 activation. However, in vitro SUMOylation assay using purified recombinant components of SUMOylation machinery shows that Epac1 does not directly activate SUMO E1 and SUMO‐conjugating enzyme E2 (UBC9). Moreover, Epac1‐mediated cellular SUMOylation is independent on its guanine nucleotide exchange activity, suggesting that Epac1 promotes cellular SUMOylation through an unconventional mechanism. Co‐immunofluorescence staining and Structured Illumination Microscopy (SIM) super‐resolution imaging analyses reveal that Epac1 activation promotes the formation of Epac1 nuclear condensates that co‐localize with nuclear UBA2/UBC9/SUMO2/3. The discovery of Epac1‐based biomolecular condensates is consistent with the facts that Epac1 contains intrinsically disordered regions (IDRs) and undergoes salt‐ and cAMP‐dependent phase separation (PS). Importantly, the formation of Epac1 nuclear condensates is required for Epac1‐mediated cellular SUMOylation, as a Δ (1‐148) Epac1 mutant incapable of PS and forming nuclear condensates fails to promote cellular SUMOylation. Furthermore, genetic knockout of Epac1 obliterates oxidized low‐density lipoprotein (oxLDL) induced cellular SUMOylation in macrophages, leading to suppression of foam cell formation. Our findings represent a major conceptual advance in our understanding of cell stress responses by providing a direct connection between protein SUMOylation and cAMP/Epac1 signaling pathway, two major cellular stress processes. The ability of cAMP to directly modulate the dynamics of cellular condensate provides the first experimental evidence that protein phase separation can be regulated by an endogenous ligand.

Tricyclic antidepressants target FKBP51 SUMOylation to restore glucocorticoid receptor activity.

FKBP51 is an important inhibitor of the glucocorticoid receptor (GR) signaling. High FKBP51 levels are associated to stress-related disorders, which are linked to GR resistance. SUMO conjugation to FKBP51 is necessary for FKBP51's inhibitory action on GR. The GR/FKBP51 pathway is target of antidepressant action. Thus we investigated if these drugs could inhibit FKBP51 SUMOylation and therefore restore GR activity. Screening cells using Ni2+ affinity and in vitro SUMOylation assays revealed that tricyclic antidepressants- particularly clomipramine- inhibited FKBP51 SUMOylation. Our data show that clomipramine binds to FKBP51 inhibiting its interaction with PIAS4 and therefore hindering its SUMOylation. The inhibition of FKBP51 SUMOylation decreased its binding to Hsp90 and GR facilitating FKBP52 recruitment, and enhancing GR activity. Reduction of PIAS4 expression in rat primary astrocytes impaired FKBP51 interaction with GR, while clomipramine could no longer exert its inhibitory action. This mechanism was verified in vivo in mice treated with clomipramine. These results describe the action of antidepressants as repressors of FKBP51 SUMOylation as a molecular switch for restoring GR sensitivity, thereby providing new potential routes of antidepressant intervention.

A proximity ligation assay (PLA) based sensing platform for the ultrasensitive detection of P53 protein-specific SUMOylation

Protein SUMOylation is a kind of post-translational modification that closely involves numerous physiological and pathological processes. Because of the important role of SUMOylation in living cells, sensitivity characterization of SUMOylation is urgent for the understanding of SUMOylatin-mediated cellular function. Due to the low- expression of SUMOylated substrate in living cells, a sensitive and selective sensing platform for the assay SUMOylation modification events is highly desired. In this work, we implemented SUMOylation using purified p53 by using the proximity ligation assay (PLA) method. GST-tagged SUMO 1 was employed for the SUMOylation assay. Using the sensing platform, we obtain P53 SUMOylation with ultrasensitivity and selectivity, which equally 83 copy P53 in 1 μL solution. Other forms of SUMOylation are also amenable to profiling by this well-designed strategy. Our PLA sensing platform will also provide a more coherent referential experience on the cellular protein-protein interaction responses that mainly measure expression levels of individual proteins or protein-protein interactions.

Root responses to aluminium and iron stresses require the SIZ1 SUMO ligase to modulate the STOP1 transcription factor.

SUMMARYSTOP1, an Arabidopsis transcription factor favouring root growth tolerance against Al toxicity, acts in the response to iron under low Pi (−Pi). Previous studies have shown that Al and Fe regulate the stability and accumulation of STOP1 in roots, and that the STOP1 protein is sumoylated by an unknown E3 ligase. Here, using a forward genetics suppressor screen, we identified the E3 SUMO (small ubiquitin‐like modifier) ligase SIZ1 as a modulator of STOP1 signalling. Mutations in SIZ1 increase the expression of ALMT1 (a direct target of STOP1) and root growth responses to Al and Fe stress in a STOP1‐dependent manner. Moreover, loss‐of‐function mutations in SIZ1 enhance the abundance of STOP1 in the root tip. However, no sumoylated STOP1 protein was detected by Western blot analysis in our sumoylation assay in Escherichia coli, suggesting the presence of a more sophisticated mechanism. We conclude that the sumo ligase SIZ1 negatively regulates STOP1 signalling, at least in part by modulating STOP1 protein in the root tip. Our results will allow a better understanding of this signalling pathway.

Post‐translational modifications of Chmp1A with ubiquitin or sumo and subsequent function in pancreatic cancer growth

Background ESCRT families have been shown to contain ubiquitin-interacting motifs (UIMs) that interact with ubiquitinated membrane proteins for sorting into Multi-vesicular bodies (MVBs). Through an extensive database search, we found that Chmp1A of ESCRT-III contains a putative UIM and SUMO-interacting motifs (SIMs), both which overlap with the nuclear localization signal (NLS) domain. The goal of this project is to test whether Chmp1A binds and undergoes modification with ubiquitin or sumo proteins via its UIM or SIMs, and to examine the effect of these processes on pancreatic cancer development. Methodology Immunoprecipitation, Ubiquitination and SUMOylation assays were carried out to examine the interaction or modification of Chmp1A with ubiquitin or SUMO proteins. NLS- deleted Chmp1A was used to determine the significance of the UIM or SIMs in the interaction of Chmp1A with ubiquitin or SUMOs. New deletion mutants in UIM and SIMs of Chmp1A were generated to corroborate the data obtained from NLS- deleted Chmp1A. With the new deletion mutants, IP assays were performed to examine the interaction between Chmp1A and SUMO proteins. Subcellular localization of these deletion mutants was determined by immunocytochemistry. Results Our data showed that Chmp1A associated with ubiquitin and underwent ubiquitination. Full-length but not the NLS- deleted Chmp1A showed an interaction with ubiquitin, suggesting that the UIM is required for association with ubiquitin. The data showed that the full-length but not the NLS- deleted Chmp1A interacted with SUMO protein. Since two of the three SIMs are contained in the NLS domain, this data suggests the importance of SIMs for the interaction of Chmp1A with SUMO. We detected higher molecular weight bands of SUMO- bound full-length Chmp1A, which suggests modification of Chmp1A with SUMO. This data was confirmed partly with UIM or SIM deletion mutants of Chmp1A. Conclusion We identified UIMs and SIMs of Chmp1A and verified their binding and modification with ubiquitin or SUMO proteins. These motifs overlap with the NLS, which is required for the inhibition of pancreatic tumor growth. Thus, this study provides new insights into yet undiscovered mechanisms of Chmp1A in pancreatic tumor suppression.

Suppression of E4BP4 SUMOylation sensitize mice to HFD‐induced fatty liver disease by promoting lipid droplet formation

Background As the most common chronic liver disease, non-alcoholic fatty liver disease (NAFLD) affects 10-30% of the general population. Characterized by excessive lipid droplet formation in hepatocytes, fatty liver may progress to nonalcoholic steatohepatitis (NASH), eventually leading to cirrhosis and liver failure. Currently, there have been no effective therapeutic or preventive measures for NAFLD. Thus, there is an urgent need to identify hepatic mediators that promote lipid droplet accumulation as well as the underlying molecular mechanisms in the liver during the pathogenesis of NAFLD. We previously reported that E4 promoter-binding protein 4 (E4BP4) is an insulin-induced stabilizer of the lipogenic factor SREBP-1c and promotes the SREBP-1c-mediated lipogenesis in hepatocytes. Our most recent mass spectrometry data revealed that E4BP4 may undergo SUMOylation, a reversible post-translational modification that was shown to regulate E4BP4-dependent NK cell development. In this study, we set out to determine how hepatic E4BP4 regulates lipid droplet formation and liver steatosis a high-fat diet-induced NAFLD mouse model and investigate how E4BP4 SUMOylation impacts the process. Results Compared with E4bp4flox/flox mice, E4bp4 liver-specific KO (E4bp4-LKO) mice exhibit decreased lipid accumulation in the liver despite similar body weight after 12 weeks of high-fat diet feeding. Our microarray analysis showed a reduction in the expression of lipid droplet biding genes such as Cidea, Cidec (Fsp27), Plin4 in the liver of E4bp4-LKO mice. Restoring E4bp4 expression in E4bp4-LKO primary hepatocytes was sufficient to induce lipid droplet formation and Fsp27β expression, while overexpression of Fsp27β increased lipid droplets and triglycerides in E4bp4-LKO primary mouse hepatocytes and promoted lipid accumulation HFD-fed E4bp4-LKO mice. Meanwhile, we generated an E4BP4 mutant, namely E4BP4-5KR, which contains five lysine to arginine (5KR) substitutions in the five SUMOylation motifs. Our in vitro SUMOylation assay confirmed the SUMOylation of E4BP4-WT but not E4BP4-5KR mutant in the presence of E1/2, SUMO2/3, and ATP. We detected more lipid droplet formation in hepa1 cells transfected with E4bp4-5KR vs. E4bp4-WT. Lastly, the abundance of SUMOylated E4BP4 was reduced in the liver of high-fat diet-fed mice vs. regular chow-fed mice. Conclusion All together, our data demonstrated that E4bp4 drives lipid droplet formation and live steatosis in high-fat diet-fed mice likely through its regulation of lipid-droplet binding genes. Our study also highlights the critical role of deSUMOylation of hepatic E4BP4 in promoting NAFLD and suggests that E4BP4 SUMOylation in the liver could be targeted for treating NAFLD.

Sumoylation of the Carboxy-Terminal of Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Attenuates Viral DNA Replication.

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV DNA polymerase processivity factor UL44 plays an essential role in viral DNA replication. To better understand the biology of UL44, we performed a yeast two-hybrid screen for host proteins that could interact with UL44. The most frequently isolated result was the SUMO-conjugating enzyme UBC9, a protein involved in the sumoylation pathway. The UBC9-UL44 interaction was confirmed by in vitro His-tag pull-down and in vivo co-immunoprecipitation assays. Using deletion mutants of UL44, we mapped two small regions of UL44, aa 11–16, and 260–269, which might be critical for the interaction with UBC9. We then demonstrated that UL44 was a target for sumoylation by in vitro and in vivo sumoylation assays, as well as in HCMV-infected cells. We further confirmed that 410lysine located within a ψKxE consensus motif on UL44 carboxy-terminal was the major sumoylation site of UL44. Interestingly, although 410lysine had no effects on subcellular localization or protein stability of UL44, the removal of 410lysine sumoylation site enhanced both viral DNA synthesis in transfection-replication assays and viral progeny production in infected cells for HCMV, suggesting sumoylation can attenuate HCMV replication through targeting UL44. Our results suggest that sumoylation plays a key role in regulating UL44 functions and viral replication, and reveal the crucial role of the carboxy-terminal of UL44, for which little function has been known before.

Abstract PS17-33: Sumo-modified androgen receptors support the metastatic phenotype of endocrine-resistant hormone receptor-positive breast cancer

Abstract Background: Conventional endocrine therapy is the first-line treatment for hormone receptor positive (HR+) breast cancer (BCa) subtype. Frequently, intrinsic and acquired ET resistance (ET-R) persists to support a disease with poor prognosis and limited therapy than the ET-sensitive carcinoma. High levels of androgen receptors (AR) in HR+ BCa correlate with insensitivity to ET tamoxifen and greater susceptibility to cancer metastasis. However, targeting AR with antagonist enzalutamide (Enz) in ET-R HR+ BCa present conflicting results. In the current report, we present novel findings of a constitutively active modified AR population that accumulates in the ET-R HR+ BCa cells to drive cell migration and metastatic phenotype. Methods: Cell-free protein modification tests, in-house SUMOylation assays, and PLA imaging were used to determine the AR protein profile in multiple ET-sensitive, intrinsic-, and acquired-ET HR+-BCa lines. Reporter assay and targeted transcriptome studies evaluate the genomic activity of native and modified-AR mimetic. Finally, the inhibitory effect of Enz with or without combination therapy was determined using migration and spheroid growth studies. Results: In acquired and intrinsic ET-R BCa cell lines, a constitutively active, higher molecular weight SUMO-modified AR (SUMO-AR) persists at the chromatin. The SUMO-AR is resilient to ubiquitin-mediated proteasomal degradation. We now report that the canonical AR chaperone protein HSPB1/Hsp27 functions as a novel SUMO-E3 ligase for AR. SUMO-AR interacts with a unique repertoire of biomolecules as compared to unmodified AR in the same cell lines. An increase in the SUMO protein and global SUMO-modified proteome supports this multimeric complex. The ligand-independent SUMO-AR activity promotes a gene-expression profile that favors epithelial-mesenchymal transition. Treatment with Enz alone or in combination with a SUMO inhibitor attenuates migration and metastatic phenotype of ET-R HR+ BCa. Conclusion: SUMO-AR dictates metastatic susceptibility and Enz responsiveness of ET-R HR+ BCa. Consistently, targeting both unmodified and SUMO-modified AR serves as a better therapeutic strategy for advanced HR+ BCa. Citation Format: Tasneem Bawa-Khalfe, Shaymaa Bahnassy, Hariprasad Thangavel, Maram Quttina, Ashfia Fatima Khan, Dhanya Dhanyalayam, Joan Ritho, Samaneh Karami, Jing Ren. Sumo-modified androgen receptors support the metastatic phenotype of endocrine-resistant hormone receptor-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-33.

SUMOylation of IGF2BP2 promotes vasculogenic mimicry of glioma via regulating OIP5-AS1/miR-495-3p axis.

Rationale: Glioma is the most common primary malignant tumor of human central nervous system, and its rich vascular characteristics make anti-angiogenic therapy become a therapeutic hotspot. However, the existence of glioma VM makes the anti-angiogenic therapy ineffective. SUMOylation is a post-translational modification that affects cell tumorigenicity by regulating the expression and activity of substrate proteins.Methods: The binding and modification of IGF2BP2 and SUMO1 were identified using Ni2+-NTA agarose bead pull-down assays, CO-IP and western blot; and in vitro SUMOylation assays combined with immunoprecipitation and immunofluorescence staining were performed to explore the detail affects and regulations of the SUMOylation on IGF2BP2. RT-PCR and western blot were used to detect the expression levels of IGF2BP2, OIP5-AS1, and miR-495-3p in glioma tissues and cell lines. CCK-8 assays, cell transwell assays, and three-dimensional cell culture methods were used for evaluating the function of IGF2BP2, OIP5-AS1, miR-495-3p, HIF1A and MMP14 in biological behaviors of glioma cells. Meantime, RIP and luciferase reporter assays were used for inquiring into the interactions among IGF2BP2, OIP5-AS1, miR-495-3p, HIF1A and MMP14. Eventually, the tumor xenografts in nude mice further as certained the effects of IGF2BP2 SUMOylation on glioma cells.Results: This study proved that IGF2BP2 mainly binds to SUMO1 and was SUMOylated at the lysine residues K497, K505 and K509 sites, which can be reduced by SENP1. SUMOylation increased IGF2BP2 protein expression and blocked its degradation through ubiquitin-proteasome pathway, thereby increasing its stability. The expressions of IGF2BP2 and OIP5-AS1 were up-regulated and the expression of miR-495-3p was down-regulated in both glioma tissues and cells. IGF2BP2 enhances the stability of OIP5-AS1, thereby increasing the binding of OIP5-AS1 to miR-495-3p, weakening the binding of miR-495-3p to the 3'UTR of HIF1A and MMP14 mRNA, and ultimately promoting the formation of VM in glioma.Conclusions: This study first revealed that SUMOylation of IGF2BP2 regulated OIP5-AS1/miR-495-3p axis to promote VM formation in glioma cells and xenografts growth in nude mice, providing a new idea for molecular targeted therapy of glioma.

Heat Shock Protein 27 Enhances SUMOylation of Heat Shock Protein B8 to Accelerate the Progression of Breast Cancer

Heat shock proteins (HSPs) are emerging as valuable potential molecular targets in breast cancer therapy owing to their diverse functions in cancer cells. This study investigated the potential role of heat shock protein 27 (HSP27, also known as HSPB1) in breast cancer through heat shock protein B8 (HSPB8). The correlation between HSP27 and HSPB8 was identified by using co-immunoprecipitation, immunoprecipitation, and SUMOylation assays. Through gain- and loss-of-function approaches in MCF-7 cells, the effect of HSP27 on HSPB8 expression, SUMOylation level, and protein stability of HSPB8, as well as on cell proliferation, migration, and stemness, was elucidated. A mouse xenograft model of breast cancer cells was established to verify the function of HSP27 invivo. Results indicate that HSP27 and HSPB8 were highly expressed in breast cancer tissues and MCF-7 cells. HSP27 was also found to induce the SUMOylation of HSPB8 at the 106 locus and subsequently increased its protein stability, which resulted in accelerated proliferation, migration, and stemness of breast cancer cells invitro along with increased tumor metastasis of breast cancer invivo. However, these results could bereversed by the knockdown of HSPB8. Overall, HSP27 induces SUMOylation of HSPB8 to promote HSPB8 expression, thereby endorsing proliferation and metastasis of breast cancer cells. This study mayprovide insight for the development of new targets for breast cancer.