Rg3 promotes the SUMOylation of SERCA2a and corrects cardiac dysfunction in heart failure.

SUMOylation of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) has been shown to play a critical role in the abnormal Ca2+ cycle of heart failure. Ginsenoside Rg3 (Rg3), the main active constituent of Panax ginseng, exerts a wide range of pharmacological effects in cardiovascular diseases. However, the effect of Rg3 on abnormal Ca2+ homeostasis in heart failure has not been reported. In this study, we showed a novel role of Rg3 in the abnormal Ca2+ cycle in cardiomyocytes of mice with heart failure. Among mice undergoing transverse aortic constriction, animals that received Rg3 showed improvements in cardiac function and Ca2+ homeostasis, accompanied by increases in the SUMOylation level and SERCA2a activity. In an isoproterenol (ISO)-induced cell hypertrophy model, Rg3 reduced the ISO-induced Ca2+ overload in HL-1 cells. Gene knockout of SUMO1 in mice inhibited the cardioprotective effect of Rg3, and SUMO1 knockout mice that received Rg3 did not exhibit improved Ca2+ homeostasis in cardiomyocytes. Additionally, mutation of the SUMOylation sites of SERCA2a blocked the positive effect of Rg3 on the ISO-induced abnormal Ca2+ cycle in HL-1 cells, and was accompanied by an abnormal endoplasmic reticulum stress response and generation of ROS. Our data demonstrated that Rg3 has a positive effect on the abnormal Ca2+ cycle in the cardiomyocytes of mice with heart failure. SUMO1 is an important factor that mediates the protective effect of Rg3. Our findings suggest that drug intervention by regulating the SUMOylation of SERCA2a can provide a novel therapeutic strategy for the treatment of heart failure.

The SUMO E3 ligase SIZ1 partially regulates STOP1 SUMOylation and stability in Arabidopsis thaliana.

The zinc finger transcription factor STOP1 plays a crucial role in aluminum (Al) resistance and low phosphate (Pi) response. Al stress and low Pi availability do not affect STOP1 mRNA expression but are able to induce STOP1 protein accumulation by post-transcriptional regulatory mechanisms. We recently reported that STOP1 can be mono-SUMOylated at K40, K212, or K395 sites, and deSUMOylated by the SUMO protease ESD4. SUMOylation of STOP1 is important for the regulation of STOP1 protein function and Al resistance. In the present study, we further characterized the role of the SUMO E3 ligase SIZ1 in STOP1 SUMOylation, Al resistance and low Pi response. We found that mutation of SIZ1 reduced but not eliminated STOP1 SUMOylation, suggesting that SIZ1-dependent and -independent pathways are involved in the regulation of STOP1 SUMOylation. The STOP1 protein levels were decreased in siz1 mutants. Nevertheless, the expression of STOP1-target gene AtALMT1 was increased instead of reduced in siz1 mutants. The mutants showed enhanced Al resistance and low Pi response. Our results suggest that SIZ1 regulates Al resistance and low Pi response likely through the modulation of AtALMT1 expression.

Rice Bran Extract Protected against LPS-Induced Neuroinflammation in Mice through Targeting PPAR-γ Nuclear Receptor.

PPAR-γ anti-inflammatory functions have received significant attention since its agonists have been shown to exert a wide range of protective effects in many experimental models of neurologic diseases. Rice bran is very rich in polyunsaturated fatty acids, which are reported to act as PPAR-γ partial agonists. Herein, the anti-inflammatory effect of rice bran extract (RBE) through PPAR-γ activation was evaluated in LPS-induced neuroinflammatory mouse model in comparison to pioglitazone (PG) using 80 Swiss albino mice. RBE (100 mg/kg) and PG (30 mg/kg) were given orally for 21 days and LPS (0.25 mg/kg) was injected intraperitoneally for the last 7 days. TNF-α and COX-2 brain contents were evaluated by real-time PCR and immunohistochemical analysis. In addition, NFκB binding to its response element was evaluated alongside with the effect of treatments on IκB gene expression. Furthermore, PPAR-γ sumoylation was also studied. Finally, histopathological examination was performed for different brain areas. RBE administration was found to protect against the LPS-induced inflammatory effects by decreasing the inflammatory mediator expression in mice brains. It also decreased PPAR-γ sumoylation without significant effect on IκB expression or NFκB binding to its response element. The majority of the effects were attenuated in presence of PPAR-γ antagonist (GW9662). Level of significance was set to P < 0.05. Such findings highlight the agonistic effect of RBE component(s) on PPAR-γ and support the hypothesis of involvement of PPAR-γ activation in its neuroprotective effect.

SUMOylation Protects FASN Against Proteasomal Degradation in Breast Cancer Cells Treated with Grape Leaf Extract.

Existing therapeutic strategies for breast cancer are limited by tumor recurrence and drug-resistance. Antioxidant plant-derived compounds such as flavonoids reduce adverse outcomes and have been identified as a potential source of antineoplastic agent with less undesirable side effects. Here, we describe the novel regulation of fatty-acid synthase (FASN), the key enzyme in de novo fatty-acid synthesis, whereby Vitis vinifera L. cv Vermentino leaf hydroalcoholic extract lowers its protein stability that is regulated by small ubiquitin-like modifier (SUMO)ylation. The phenolic compounds characterization was performed by liquid chromatography-mass spectrometry (LC-MS), whereas mass spectrometry (LC-MS/MS), Western blotting/co-immunoprecipitation (Co-IP) and RT-PCR, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenicity assays, and FACS analysis were used to measure the expression of targets and tumorigenicity. Vermentino extract exhibits antitumorigenic effects, and we went on to determine that FASN and ubiquitin-conjugating enzyme 9 (UBC9), the sole E2 enzyme required for SUMOylation, were significantly reduced. Moreover, FASN was found SUMOylated in human breast cancer tissues and cell lines, and lack of SUMOylation caused by SUMO2 silencing reduced FASN protein stability. These results suggest that SUMOylation protects FASN against proteasomal degradation and may exert oncogenic activity through alteration of lipid metabolism, whereas Vermentino extract inhibits these effects which supports the additional validation of the therapeutic value of this compound in breast cancer.

A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver.

SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.

Development of a high-throughput screen to detect inhibitors of TRPS1 sumoylation.

Small ubiquitin-like modifier (SUMO) belongs to the family of ubiquitin-like proteins (Ubls) that can be reversibly conjugated to target-specific lysines on substrate proteins. Although covalently sumoylated products are readily detectible in gel-based assays, there has been little progress toward the development of robust quantitative sumoylation assay formats for the evaluation of large compound libraries. In an effort to identify inhibitors of ubiquitin carrier protein 9 (Ubc9)-dependent sumoylation, a high-throughput fluorescence polarization assay was developed, which allows detection of Lys-1201 sumoylation, corresponding to the major site of functional sumoylation within the transcriptional repressor trichorhino-phalangeal syndrome type I protein (TRPS1). A minimal hexapeptide substrate peptide, TMR-VVK1201TEK, was used in this assay format to afford high-throughput screening of the GlaxoSmithKline diversity compound collection. A total of 728 hits were confirmed but no specific noncovalent inhibitors of Ubc9 dependent trans-sumoylation were found. However, several diaminopyrimidine compounds were identified as inhibitors in the assay with IC50 values of 12.5 µM. These were further characterized to be competent substrates which were subject to sumoylation by SUMO-Ubc9 and which were competitive with the sumoylation of the TRPS1 peptide substrates.

Development of a high-throughput screening assay for inhibitors of small ubiquitin-like modifier proteases.

Small ubiquitin-like modifier (SUMO1-3) is a small group of proteins that are ligated to lysine residues in target proteins. SUMO conjugation is a highly dynamic process, as SUMOylated proteins are rapidly deconjugated by SUMO proteases. SUMO conjugation/deconjugation plays pivotal roles in major cellular pathways and is associated with a number of pathological conditions. It is therefore of significant clinical interest to develop new strategies to screen for compounds to specifically interfere with SUMO conjugation/deconjugation. Here, we describe a novel high-throughput screening (HTS)-compatible assay to identify inhibitors of SUMO proteases. The assay is based on AlphaScreen technology and uses His-tagged SUMO2 conjugated to Strep-tagged SUMO3 as a SUMO protease substrate. A bacterial SUMOylation system was used to generate this substrate. A three-step purification strategy was employed to yield substrate of high quality. Our data indicated that this unique substrate can be readily detected in the AlphaScreen assays in a dose-dependent manner. Cleavage reactions by SUMO protease with or without inhibitor were monitored based on AlphaScreen signals. Furthermore, the assay was adapted to a 384-well format, and the interplate and interday variability was evaluated in eight 384-well plates. The average Z' factor was 0.83 ± 0.04, confirming the suitability for HTS applications.