Aberrant SUMO2/3 modification of RUNX1 upon SENP1 inhibition is linked to the development of diabetic retinopathy in mice.

Our previous report established that RUNX family transcription factor 1 (RUNX1) promotes proliferation of mouse retinal microvascular endothelial cells (mRMECs) and exacerbates diabetic retinopathy (DR). However, the mechanism behind the upregulation of RUNX1 remains unclear. This study aims to investigate the possible correlation between histone SUMOylation and RUNX1 in DR, as well as the involved molecules. A mouse model of diabetes was induced by streptozotocin (STZ). These mice had increased retinal thickness and elevated production of inflammatory cytokines. Additionally, they showed elevated levels of SUMO1 and SUMO2/3, but reduced levels of SUMO specific peptidase 1 (SENP1) in retinal tissues. Co-immunoprecipitation and Western blot assays revealed that the RUNX1 protein was primarily modified by SUMO2/3, and SENP1 inhibited SUMO2/3 modification, thereby reducing RUNX1 expression. Overexpression of SENP1 alleviated symptoms in mice and alleviated inflammation. In vitro experiments demonstrated that the SENP1 overexpression suppressed the proliferation, migration, and angiogenesis of high-glucose-induced mRMECs. However, further overexpression of RUNX1 counteracted the alleviating effects of SENP1 both in vivo and in vitro. In conclusion, this study demonstrates that the downregulation of SENP1 in DR leads to SUMO2/3-dependent activation of RUNX1. This activation promotes proliferation of mRMECs and exacerbates DR symptoms in mice.

SUMO2/3 promotes the progression and oxaliplatin resistance of colorectal cancer through facilitating the SUMOylation at Ku80-K307.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide and is typically treated with the FOLFOX regimen (folinic acid, 5-fluorouracil, and oxaliplatin). However, oxaliplatin resistance remains a serious clinical problem. In the present study, we found that SUMO2/3 was overexpressed in CRC tissues and exogenous overexpression of SUMO2/3 promoted CRC cell proliferation, extension, and invasion and positively regulated the cell cycle. In contrast, SUMO2/3 gene knockdowns inhibited migration and repressed cell viability in vitro and in vivo. In addition, we found that SUMO2/3 was recruited to the cell nucleus and suppressed oxaliplatin-induced apoptosis of CRC cells. Moreover, Ku80, a DNA-binding protein essential for the repair of DNA double-strand breaks, was confirmed to bind with SUMO2/3. Notably, Ku80 undergoes SUMOylation at K307 by SUMO2/3 and this correlated with apoptosis in CRC cells suffering oxaliplatin stress. Collectively, we found that SUMO2/3 plays a specific role in CRC tumorigenesis and acts through Ku80 SUMOylation which is linked with the development of CRC-oxaliplatin resistance.

miR-212-5p Suppresses Glioma Development via Targeting SUMO2.

Recently, increasing studies have suggested that miRNAs play a significant role in the occurrence and development of glioma. More researches are needed to explore the role of miRNAs in glioma, which will help to find new therapeutic targets. miR-212-5p has been reported to be involved in the progression in many cancers. However, whether miR-212-5p has a regulative effect on glioma remains un clear. In this study, we aimed to explore the effect of miR-212-5p on glioma development and its mechanism. Here, we demonstrated that miR-212-5p was lowly expressed in glioma cell. miR-212-5p suppressed the glioma cell proliferation, inhibited the migratory and invasive capabilities and promoted apoptosis in glioma cells. Besides, miR-212-5p also inhibited tumor growth in vivo. We found small ubiquitin-like modifier 2 (SUMO2) was the target of miR-212-5p, and miR-212-5p suppressed SUMO2 expression to regulate the proliferation, migration, and apoptosis of glioma cells. These findings indicated that miR-212-5p may be a possible therapeutic target for the treatment for glioma.

SUMO2-mediated SUMOylation of SH3GLB1 promotes ionizing radiation-induced hypertrophic cardiomyopathy through mitophagy activation.

Hypertrophic cardiomyopathy (HC) is characterized by the enlargement of individual cardiomyocytes, which is a typical pathophysiological process that occurs in various cardiovascular diseases. Ionizing radiation (IR) is an important independent risk factor for hypertrophic cardiomyopathy, but the underlying molecular mechanism is still unclear. In the present study, we aimed to clarify the role of IR in promoting cardiac hypertrophy and investigate the mechanism by which the SUMO2-mediated SUMOylation of SH3GLB1 affects mitophagy in IR-induced cardiac hypertrophy. In vivo, IR promoted cardiac hypertrophy by activating mitophagy. In vitro, IR upregulated PINK1 and Parkin protein expression and damaged mitochondrial morphological structure. We further demonstrated that SH3GLB1 deficiency inhibited mitophagy activation and restored mitochondrial cristae, revealing a regulatory role of SH3GLB1 in cardiac hypertrophy. IR promoted interactions between SH3GLB1 and mitochondrial membrane proteins, such as MFN1/2, TOM20 and Drp1, further indicating that the mechanism by which SH3GLB1 functions in cardiac hypertrophy might involve mitophagy. A bioinformatics prediction found that SUMO2 could SUMOylate SH3GLB1 at position K82. Consistent with this finding, both co-IP assays and laser confocal microscopy showed that IR promoted the interaction and colocalization of SUMO2 and SH3GLB1. In summary, our study identifies IR as an important factor that promotes hypertrophic cardiomyopathy by accelerating the activation of mitophagy through the SUMO2-mediated SUMOylation of SH3GLB1; thus, IR exerts dual therapeutic effects in the treatment of thoracic tumours with long-term radiotherapy. Additionally, this study provides novel treatment strategies and targets for preventing the hypertrophic cardiomyopathy caused by thoracic tumour radiotherapy. Furthermore, SH3GLB1 may be a promising experimental target for the development of strategies for treating cardiovascular diseases caused by IR.

MxA Mediates SUMO-Induced Resistance to Vesicular Stomatitis Virus.

UNLABELLED: Multiple cellular pathways are regulated by small ubiquitin-like modifier (SUMO) modification, including ubiquitin-mediated proteolysis, signal transduction, innate immunity, and antiviral defense. In the study described in this report, we investigated the effects of SUMO on the replication of two members of the Rhabdoviridae family, vesicular stomatitis virus (VSV) and rabies virus (RABV). We show that stable expression of SUMO in human cells confers resistance to VSV infection in an interferon-independent manner. We demonstrate that SUMO expression did not alter VSV entry but blocked primary mRNA synthesis, leading to a reduction of viral protein synthesis and viral production, thus protecting cells from VSV-induced cell lysis. MxA is known to inhibit VSV primary transcription. Interestingly, we found that the MxA protein was highly stabilized in SUMO-expressing cells. Furthermore, extracts from cells stably expressing SUMO exhibited an increase in MxA oligomers, suggesting that SUMO plays a role in protecting MxA from degradation, thus providing a stable intracellular pool of MxA available to combat invading viruses. Importantly, MxA depletion in SUMO-expressing cells abrogated the anti-VSV effect of SUMO. Furthermore, SUMO expression resulted in interferon-regulatory factor 3 (IRF3) SUMOylation, subsequently decreasing RABV-induced IRF3 phosphorylation and interferon synthesis. As expected, this rendered SUMO-expressing cells more sensitive to RABV infection, even though MxA was stabilized in SUMO-expressing cells, since its expression did not confer resistance to RABV. Our findings demonstrate opposing effects of SUMO expression on two viruses of the same family, intrinsically inhibiting VSV infection through MxA stabilization while enhancing RABV infection by decreasing IFN induction. IMPORTANCE: We report that SUMO expression reduces interferon synthesis upon RABV or VSV infection. Therefore, SUMO renders cells more sensitive to RABV but unexpectedly renders cells resistant to VSV by blocking primary mRNA synthesis. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed restriction factors. Among the various anti-VSV restriction factors, only MxA is known to inhibit VSV primary transcription, and we show here that its expression does not alter RABV infection. Interestingly, MxA depletion abolished the inhibition of VSV by SUMO, demonstrating that MxA mediates SUMO-induced intrinsic VSV resistance. Furthermore, MxA oligomerization is known to be critical for its protein stability, and we show that higher levels of oligomers were formed in cells expressing SUMO than in wild-type cells, suggesting that SUMO may play a role in protecting MxA from degradation, providing a stable intracellular pool of MxA able to protect cells from viral infection.

Effects of thioredoxin: SUMO and intein on soluble fusion expression of an antimicrobial peptide OG2 in Escherichia coli.

OG2 is a modified antimicrobial peptide of Palustrin-OG1 (OG1), which is derived from Odorrana grahami frog. OG2 has shown much higher selective antimicrobial activity and lower hemolytic activity than OG1, indicating OG2 may be a promising antimicrobial agent. In this study, we investigated three fusion partners, including thioredoxin, Mxe GyrA intein, and small ubiquitin-like modifier (SUMO), each fused with OG2, and examined their effects on the expression level and solubility of OG2 in Escherichia coli. The codon-optimized OG2 gene was cloned into pET32a (+) and pTWIN1 for fusion with thioredoxin and Mxe GyrA intein, respectively. In addition, the SUMO-OG2 gene was amplified by splice overlap extension PCR method and was cloned into pET30a (+). All recombinant plasmids were then transformed into E. coli BL21(DE3)pLysS, and the expressed fusion proteins were verified. Upon isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction, OG2 fused with thioredoxin (Trx-OG2) showed the highest yield as a soluble fusion protein (50 mg/L), followed by Mxe GyrA intein (44 mg/L) and SUMO (11 mg/L). The thioredoxin-fused protein (Trx-OG2) was then purified by nickel-nitrilotriacetic acid chromatography and desalted by Sephadex G25. The OG2 released by both tobacco etch virus protease and enterokinase from Trx-OG2 showed strong antimicrobial activity against Staphylococcus aureus ATCC25923.

Small ubiquitin-related modifier is secreted and shows cytokine-like activity.

Small ubiquitin-related modifier (SUMO) is a type I ubiquitin-like protein family member and is covalently attached to various target proteins. Through this post-translational modification, SUMO plays important roles in various cellular events. Here, we show that SUMO is secreted from cultured cells in an endoplasmic reticulum (ER)/Golgi-independent manner and that this secretion occurs without covalent binding to target proteins or chain formation. Overexpression experiments using C-terminally truncated mutants of SUMO revealed that the secretion requires the C-terminal sequence. Recombinant SUMO-3 protein was capable of binding to and promoting the proliferation of cultured cells. Thus, we propose that SUMO functions as a cytokine-like molecule extracellularly.

Sumoylated RGS-Rz proteins act as scaffolds for Mu-opioid receptors and G-protein complexes in mouse brain.

The RGSZ1 and RGSZ2 proteins, members of the RGS-Rz subfamily of GTPase-activating proteins (GAP), are involved in Mu-opioid receptor desensitization. The expression of these proteins, as well as of their main target the Gz protein, is virtually restricted to the nervous tissue. In synaptosomal membranes, these Rz proteins undergo post-translational modifications such as glycosylation and phosphorylation, and they may covalently attach to small ubiquitin-like modifier (SUMO) proteins. While RGSZ1 exists in conjugated and non-conjugated forms, RGSZ2 is mostly conjugated to SUMO-1, SUMO-2 and SUMO-3 proteins. These sumoylated forms of the GAPs readily associated with Mu-opioid receptors but they associated only poorly with Delta receptors. Furthermore, G alpha i2 and G alpha z subunits co-precipitated with the sumoylated forms of RGSZ1/Z2 proteins, but to a lesser extent with the Ser phosphorylated SUMO-free form of RGSZ1. Upon Mu-opioid receptor activation, there is a strong increase in the association of G alpha proteins with RGSZ2 proteins that persists for intervals longer than 24 h. This effect probably accounts for their role in Mu-opioid receptor desensitization. Only a moderate increase was observed with RGSZ1, the non-sumoylated form of which probably acts as an efficient GAP for these G alpha subunits. Therefore, sumoylation regulates the biological activity of RGS-Rz proteins and it is likely that it serves to switch their behavior, from that of a GAP for activated G alpha subunits to that of a scaffold protein for specific signaling proteins.