UBE2T Promotes Temozolomide Resistance of Glioblastoma Through Regulating the Wnt/ß-Catenin Signaling Pathway.

Purpose: Patients with glioblastoma (GBM) have poor prognosis and limited therapeutic options, largely because of chemoresistance to temozolomide (TMZ) treatment. Ubiquitin conjugating enzyme E2 T (UBE2T) plays a key role in regulating the malignancy of various tumors, including GBM; however, its role in TMZ resistance of GBM has not been elucidated. The purpose of this study was to clarify the role of UBE2T in mediating TMZ resistance and investigate the specific underlying mechanism. Methods: Western blotting was used to detect the protein levels of UBE2T and Wnt/ß-catenin-related factors. CCK-8, flow cytometry, and colony formation assays were used to examine the effect of UBE2T on TMZ resistance. Wnt/ß-catenin signaling pathway activation was inhibited using XAV-939, and a xenograft mouse model was generated to clarify the function of TMZ in vivo. Results: UBE2T knockdown sensitized GBM cells to TMZ treatment, whereas UBE2T overexpression promoted TMZ resistance. The specific UBE2T inhibitor, M435-1279, increased the sensitivity of GBM cells to TMZ. Mechanistically, our results demonstrated that UBE2T induces ß-catenin nuclear translocation and increases the protein levels of downstream molecules, including survivin and c-Myc. Inhibition of Wnt/ß-catenin signaling using XAV-939 blocked TMZ resistance due to UBE2T overexpression in GBM cells. In addition, UBE2T was shown to facilitate TMZ resistance by inducing Wnt/ß-catenin signaling pathway activation in a mouse xenograft model. Combined treatment with TMZ and UBE2T inhibitor achieved superior tumor growth suppression relative to TMZ treatment alone. Conclusion: Our data reveal a novel role of UBE2T in mediating TMZ resistance of GBM cells via regulating Wnt/ß-catenin signaling. These findings indicate that targeting UBE2T has promising potential to overcome TMZ resistance of GBM.

Inhibition of ATG3 ameliorates liver steatosis by increasing mitochondrial function.

BACKGROUND & AIMS: Autophagy-related gene 3 (ATG3) is an enzyme mainly known for its actions in the LC3 lipidation process, which is essential for autophagy. Whether ATG3 plays a role in lipid metabolism or contributes to non-alcoholic fatty liver disease (NAFLD) remains unknown. METHODS: By performing proteomic analysis on livers from mice with genetic manipulation of hepatic p63, a regulator of fatty acid metabolism, we identified ATG3 as a new target downstream of p63. ATG3 was evaluated in liver samples from patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the livers of mice. RESULTS: ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and non-alcoholic steatohepatitis) compared with those without liver disease. Moreover, genetic knockdown of ATG3 in mice and human hepatocytes ameliorates p63- and diet-induced steatosis, while its overexpression increases the lipid load in hepatocytes. The inhibition of hepatic ATG3 improves fatty acid metabolism by reducing c-Jun N-terminal protein kinase 1 (JNK1), which increases sirtuin 1 (SIRT1), carnitine palmitoyltransferase 1a (CPT1a), and mitochondrial function. Hepatic knockdown of SIRT1 and CPT1a blunts the effects of ATG3 on mitochondrial activity. Unexpectedly, these effects are independent of an autophagic action. CONCLUSIONS: Collectively, these findings indicate that ATG3 is a novel protein implicated in the development of steatosis. LAY SUMMARY: We show that autophagy-related gene 3 (ATG3) contributes to the progression of non-alcoholic fatty liver disease in humans and mice. Hepatic knockdown of ATG3 ameliorates the development of NAFLD by stimulating mitochondrial function. Thus, ATG3 is an important factor implicated in steatosis.

Effects of E2 binding enzyme UBC9 on porcine oocyte maturation, apoptosis and embryo development.

SUMOylation is a dynamic post-translational modification process. However, the function of small ubiquitin-like modifiers (SUMOs) in the maturation of porcine oocytes and embryo growth is not well known. Therefore, the aim of this study was to investigate the effect of E2 binding enzyme UBC9 on the expression of SUMO-1 protein during the in vitro maturation of porcine oocytes and embryo development after in vitro fertilization. Four groups were used: 0 (Control), 5, 10 and 15 µg/ml UBC9. Western blotting, flow cytometry and RT-qPCR were used to detect the in vitro maturation of porcine oocytes, SUMO-1 content, viability and the expression of apoptotic genes. Compared to those in the control treatment, the maturation rate (p < .05) and viability (p < .01) of oocytes in the 5 µg/ml treatment group decreased significantly. SUMO-1 protein markers appeared at 59 and 71 kDa and the content of SUMO-1 protein in the 10 µg/ml treatment group decreased significantly (p < .05). In the expression of apoptosis-related genes, Bcl-2 gene expression was significantly downregulated in the 10 µg/ml treatment group (p < .05). However, Bax and Caspase-3 were significantly upregulated in the 5 µg/ml treatment group (p < .05). During embryonic development, the cleavage rate of oocytes in the 10 µg/ml treatment group was significantly reduced (p < .05), whereas blastocyst formation rate in the 5 µg/ml treatment group was significantly reduced. UBC9 regulates SUMO-1 content in mature pig oocytes in vitro, which affects oocyte maturation rate, viability, apoptotic genes expression and embryo development after fertilization.

Saliva exosomes-derived UBE2O mRNA promotes angiogenesis in cutaneous wounds by targeting SMAD6.

BACKGROUND: Enhancing angiogenesis is critical for accelerating wound healing. Application of different types of exosomes (Exos) to promote angiogenesis represents a novel strategy for enhanced wound repair. Saliva is known to accelerate wound healing, but the underlying mechanisms remain unclear. RESULTS: Our results have demonstrated that saliva-derived exosomes (saliva-Exos) induce human umbilical vein endothelial cells (HUVEC) proliferation, migration, and angiogenesis in vitro, and promote cutaneous wound healing in vivo. Further experiments documented that Ubiquitin-conjugating enzyme E2O (UBE2O) is one of the main mRNAs of saliva-Exos, and activation of UBE2O has effects similar to those of saliva-Exos, both in vitro and in vivo. Mechanistically, UBE2O decreases the level of SMAD family member 6 (SMAD6), thereby activating bone morphogenetic protein 2 (BMP2), which, in turn, induces angiogenesis. CONCLUSIONS: The present work suggests that administration of saliva-Exos and UBE2O represents a promising strategy for enhancing wound healing through promotion of angiogenesis.

Inhibition of UVSSA ubiquitination suppresses transcription-coupled nucleotide excision repair deficiency caused by dissociation from USP7.

Transcription-coupled nucleotide excision repair (TC-NER) is a subpathway of nucleotide excision repair that efficiently removes transcription-blocking DNA damage from the transcribed strands of active genes. UVSSA is a causative gene for UV-sensitive syndrome (UVS S), which is an autosomal recessive disorder characterized by hypersensitivity to UV light and deficiency in TC-NER. UV-stimulated scaffold protein A (UVSSA), the product of UVSSA, forms a complex with ubiquitin-specific peptidase 7 (USP7) and is stabilized by interaction with USP7. The central region of UVSSA, which contains the tumor necrosis factor receptor-associated factor (TRAF)-binding motif, is required for the interaction with the N-terminal TRAF domain of USP7. Here, we showed that UVSSA is mono-ubiquitinated in vitro and identified a lysine residue (Lys414 ) in UVSSA as the target of ubiquitination. The deubiquitination activity of USP7 was inhibited by the ubiquitin-conjugating enzyme UbcH6. Lys414 was also modified by poly-ubiquitin chains in vivo. UVSSA deficient in the interaction with USP7 is ubiquitinated and degraded by the proteasome, and the degradation leads to deficiency in TC-NER. The substitution of Lys414 by Arg of UVSSA inhibited its degradation and thereby suppressed the deficiency in TC-NER.

UbcH10 overexpression increases carcinogenesis and blocks ALLN susceptibility in colorectal cancer.

Cyclins are essential for cell proliferation, the cell cycle and tumorigenesis in all eukaryotes. UbcH10 regulates the degradation of cyclins in a ubiquitin-dependent manner. Here, we report that UbcH10 is likely involved in tumorigenesis. We found that cancer cells exposed to n-acetyl-leu-leu-norleucinal (ALLN) treatment and UbcH10 depletion exhibit a synergistic therapeutic effect. Abundant expression of UbcH10 drives resistance to ALLN-induced cell death, while cells deficient in UbcH10 were susceptible to ALLN-induced cell death. The depletion of UbcH10 hindered tumorigenesis both in vitro and in vivo, as assessed by colony formation, growth curve, soft agar and xenograft assays. These phenotypes were efficiently rescued through the introduction of recombinant UbcH10. In the UbcH10-deficient cells, alterations in the expression of cyclins led to cell cycle changes and subsequently decreases in tumorigenesis. The tumorigenesis of xenograft tumors from UbcH10-deficient cells treated with ALLN was decreased relative to wild-type cells treated with ALLN in nude mice. On the molecular level, we observed that UbcH10 deficiency enhances the activation of caspase 8 and caspase 3 but not caspase 9 to impair cell viability upon ALLN treatment. Collectively, our results suggest that, as an oncogene, UbcH10 is a potential drug target for the treatment of colorectal cancer.

SUMO, Ubiquitin, UBL Proteins: Implications For Human Diseases - Fifth International Conference.

The fifth international conference on SUMO, Ubiquitin, UBL Proteins: Implications for Human Diseases, held in Houston, included topics covering the latest advances and new targets in the field of protein modification. This conference report highlights selected presentations on the structural characterization of ubiquitination and SUMOylation machinery; the regulation of ubiquitination enzymes, including E3 ligases; the functions and mechanism of action of SUMO-targeted ubiquitin ligases (STUbLs); the regulation of gene expression by SUMO; non-degradative functions of ubiquitin and SUMO in signal transduction; mechanisms and functions of ISG15 conjugation; the interaction of pathogens with host cell SUMOylation machinery; and stabilization of the Axin protein. Investigational drugs discussed include MLN-4924 (Millennium Pharmaceuticals Inc).

Effects of MEK5/ERK5 association on small ubiquitin-related modification of ERK5: implications for diabetic ventricular dysfunction after myocardial infarction.

Diabetes mellitus (DM) contributes to the exacerbation of left ventricle (LV) dysfunction after myocardial infarction (MI). Activation of ERK5, an atypical mitogen activated protein kinase with transcriptional activity, inhibits apoptosis and LV dysfunction after doxorubicin treatment. SUMOylation has been proposed as a negative regulator of various transcription factors. In the current study, we investigated the role of ERK5-SUMOylation in ERK5 transcriptional activity as well as on DM-mediated exacerbation of LV dysfunction and apoptosis after MI. ERK5 wild-type transcriptional activity was inhibited by Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), but not in the ERK5-SUMOylation-site defective mutant (K6R/K22R). H2O2 and high glucose, 2 well-known mediators of diabetes, induced ERK5-SUMOylation, and the K6R/K22R mutant, dominant negative form of Ubc9, and siRNA-PIAS1 reversed H2O2-mediated reduction of ERK5 transcriptional activity in cardiomyocytes, indicating the presence of SUMOylation-dependent ERK5 transcriptional repression. Constitutively active form of MEK5alpha (CA-MEK5alpha) inhibited ERK5-SUMOylation independent of kinase activity, but dependent on MEK5-ERK5 association. To investigate the pathological role of ERK5-SUMOylation in DM mice after MI, we used cardiac specific CA-MEK5alpha transgenic mice (CA-MEK5alpha-Tg). MI was induced in streptozotocin (STZ)-injected (DM+MI group) or vehicle-injected mice (MI group) by ligating the left coronary artery. The ERK5-SUMOylation was increased in the DM+MI, but not in the MI group. ERK5-SUMOylation, the exacerbation of LV dysfunction, and the number of TUNEL-positive cells in DM+MI was significantly inhibited in CA-MEK5alpha-Tg mice. Of note, we could not detect any difference of cardiac function after MI in non-diabetic CA-MEK5alpha-Tg and non-transgenic littermate control mice. These results demonstrated that ERK5 transcriptional activity is subject to downregulation by diabetes-dependent SUMOylation, which resulted in a proapoptotic condition contributing to poor post-MI LV function.