Ubiquitin-conjugating Enzyme E2 Variant Research Articles

Role of Ubiquitin-conjugating enzyme E2 (UBE2) in two immune-mediated inflammatory skin diseases: a mendelian randomization analysis

Psoriasis vulgaris (PV) and Atopic dermatitis (AD) are the two major types of immune-mediated inflammatory skin disease (IMISD). Limited studies reported the association between Ubiquitin-conjugating enzyme E2 (UBE2) and IMISD. We employed a two-sample Mendelian randomization (MR) study to assess the causality between UBE2 and PV & AD. UBE2 association genome-wide association study (GWAS) data were collected. The inverse variance weighted (IVW) method was utilized as the principal method in our Mendelian randomization (MR) study, with additional using the MR-Egger, weighted median, simple mode, and weighted mode methods. The MR-Egger intercept test, Cochran’s Q test, MR-Pleiotropy RESidual Sum and Outlier (MR-PRESSO) and leave-one-out analysis were conducted to identify heterogeneity and pleiotropy, colocalization analysis was also performed. The results showed that Ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) was causally associated with PV (OR = 0.909, 95% CI: 0.830–0.996, P = 0.040), Ubiquitin-conjugating enzyme E2 L3 (UBE2L3) was causally associated with AD (OR = 0.799, 95% CI: 0.709–0.990, P < 0.001). Both UBE2V1 and UBE2L3 may play protective roles in patients with PV or AD, respectively. No other significant result has been investigated. No heterogeneity or pleiotropy was observed. This study provided new evidence of the relationship between UBE2V1 and PV, UBE2L3 and AD. Our MR suggested that UBE2V1 plays an inhibitory role in PV progression, UBE2L3 plays an inhibitory role in AD. These could be novel and effective ways to treat PV and AD.

Identification of reference genes for RT-qPCR analysis across kiwifruit species under iron deficiency conditions

Selecting appropriate reference genes is crucial for the accuracy of gene expression analysis. Although rootstock species play vital roles in kiwifruit production, the reference genes has not been identified in kiwifruit roots and across kiwifruit species. Additionally, iron (Fe) deficiency has been frequently observed in kiwifruit orchards, but the reference genes is not validated. Here, we assessed ten candidate reference genes: actin (ACT), actin-depolymerizing factor family protein (ADF), ADP-ribosylation factor (ARF), eukaryotic translation initiation factor 5A (eIF5A), ribosomal protein L19 (RPL19), 40S ribosomal protein S17 (RPS17), suppressor of G2 allele of SKP1 (SGT1), translationally-controlled tumor protein (TCTP), translation initiation factor 2-γ (TIF2-γ) and ubiquitin-conjugating enzyme E2 variant (UBE2V), for their suitability as reliable internal controls in roots, stems and leaves of four kiwifruit species (Actinidia chinensis, A. macrosperma, A. polygama, and A. valvata) under direct and bicarbonate-induced Fe deficiency conditions. The expression stability of the candidate genes was evaluated by four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder). Based on the comprehensive ranking, we recommended TCTP + UBE2V as the best reference genes for roots, UBE2V + ACT for stems, RPL19 + ARF for leaves, and UBE2V + RPL19 for all the tissues. The reliability of the recommended reference genes was verified by six target genes involved in Fe uptake and transport. These results provide a basis for improving the accuracy of gene expression analysis and elucidating the molecular mechanisms of Fe deficiency adaptation in kiwifruit species.

Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination.

Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined. To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryABR120G) and identify how Ube2v1 exerts its effect. Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryABWT) or CryABR120G. Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryABR120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryABWT and CryABR120G-expressing neonatal rat ventricular cardiomyocytes. Ubiquitin proteasome system performance was analyzed using a ubiquitin proteasome system reporter protein. Ube2v1 knockdown improved ubiquitin proteasome system performance and promoted the degradation of insoluble ubiquitinated proteins in CryABR120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1's function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using AAV9 (adeno-associated virus 9) to deliver multiplexed single guide RNAs against Ube2v1 in cardiac-specific Cas9 mice alleviated CryABR120G-induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo. Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryABR120G-induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

Urinary miRNA-27b-3p and miRNA-1228-3p correlate with the progression of Kidney Fibrosis in Diabetic Nephropathy

Diabetic Nephropathy (DN) is a chronic complication of diabetes and the primary cause of end stage renal disease. Differential diagnosis for DN requires invasive histological investigation, thus there is need for non-invasive biomarkers to discriminate among different histological lesions in diabetic patients. With the aim to identify a pattern of differentially expressed miRNAs in kidney biopsies of DN patients, we assayed miRNA expression in kidney biopsies from DN patients, diabetic patients with membranous nephropathy and patients with normal histology. Nine miRNAs were differentially expressed among the three groups, and 2 miRNAs (miR-27b-3p and miR-1228-3p) showed interaction with an ubiquitin-conjugating E2 enzyme variant (UBE2v1). UBE2v1 mediates the formation of lysine 63-linked ubiquitin chains, a mechanism we previously showed as involved in DN kidney fibrosis. Both miRNAs were validated as down-regulated in biopsies and urines of DN patients, possibly affected by DNA methylation. Interestingly, the urinary levels of both miRNAs could also discriminate among different degrees of renal fibrosis. Finally, we showed that the combined urinary expression of both miRNAs was also able to discriminate DN patients from other glomerulonephritides in diabetic patients. In conclusion we identified two miRNAs potentially useful as candidate biomarkers of tubular-interstitial fibrosis in diabetic patients with DN.

Abstract LB-284: miRNAs/Ago2/YY1/PcG protein/DNMT complex mediates epigenetic silencing in bortezomib-induced apoptosis of leukemia

Abstract miRNAs are a class of non-coding RNAs, which bind to the 3'-untranslated regions of their target mRNAs to repress translation. However, the details of miRNA-mediated epigenetic regulation targeting genomic DNA remained largely unknown. Here, we demonstrated that transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, is a highly selective reversible inhibitor for leukemia treatment, and resulting in caspase activation and apoptosis. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 feedback to suppress the transcription of CEBPD itself and genes adjacent to its 5'-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Additionally, we previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for mediating epigenetic silencing of the CEBPD gene; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. Moreover, we revealed that miRNA binding plays a key role for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. Altogether, we provided new insights for miRNA-mediated epigenetic regulation in the case of bortezomib-induced cell death in leukemia. Citation Format: Ju-Ming Wang. miRNAs/Ago2/YY1/PcG protein/DNMT complex mediates epigenetic silencing in bortezomib-induced apoptosis of leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-284.

Cowpea and abiotic stresses: identification of reference genes for transcriptional profiling by qPCR

BackgroundDue to cowpea ability to fix nitrogen in poor soils and relative tolerance to drought and salt stresses, efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Real-time quantitative PCR (qPCR) has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. In the present study, nine candidate reference genes were rigorously tested for their application in normalization of qPCR data onto roots of four distinct cowpea accessions under two abiotic stresses: root dehydration and salt (NaCl, 100 mM). In addition, the regulation of four target transcripts, under the same referred conditions was also scrutinized.ResultsgeNorm, NormFinder, BestKeeper, and ΔCt method results indicated a set of three statistically validated RGs for each stress condition: (I) root dehydration (actin, ubiquitin-conjugating enzyme E2 variant 1D, and a Phaseolus vulgaris unknown gene—UNK), and (II) salt (ubiquitin-conjugating enzyme E2 variant 1D, F-box protein, and UNK). The expression profile of the target transcripts suggests that flavonoids are important players in the cowpea response to the abiotic stresses analyzed, since chalcone isomerase and chalcone synthase were up-regulated in the tolerant and sensitive accessions. A lipid transfer protein also participates in the cowpea tolerance mechanisms to root dehydration and salt stress. The referred transcript was up-regulated in the two tolerant accessions and presented no differential expression in the sensitive counterparts. Chitinase B, in turn, generally related to plant defense, was an important target transcript under salt stress, being up-regulated at the tolerant, and down-regulated in the sensitive accession.ConclusionsReference genes suitable for qPCR analyses in cowpea under root dehydration and salt stress were identified. This action will lead to a more accurate and reliable analysis of gene expression on this species. Additionally, the results obtained in this study may guide future research on gene expression in cowpea under other abiotic stress types that impose osmotic imbalance. The target genes analyzed, in turn, deserve functional evaluation due to their transcriptional regulation under stresses and biotechnological potential.

Abstract 238: Ube2v1 Promotes Mutant αB-crystallin-induced Protein Aggregation

Background: Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. An unbiased genome-wide short hairpin RNA screen was performed to seek novel genes that could prevent or clear proteotoxic aggregates in a model of desmin-related cardiomyopathy, caused by the R120G mutation of αB-crystallin (CryAB R120G ). Among a total of 236 hits, we validated a candidate gene named ubiquitin-conjugating E2 enzyme variant 1 (ube2v1). Ube2v1 expression is detectable in the heart and the literature states that it must bind to ube2n to form a heterodimer. The heterodimer then catalyzes the synthesis of Lys63-linked poly-ubiquitin chains, participating in DNA repair and the damage response. Ube2v1 protein levels were increased in CryAB R120G transgenic hearts. However, the function of ube2v1 in cardiac proteotoxicity is completely unknown. To assess whether and how ube2v1 impacts cardiac protein aggregation catalyzed by cardiomyocyte-specific expression of mutated CryAB. Methods and Results: Neonatal rat ventricular cardiomyocytes (NRVMs) were infected with adenoviruses expressing either wild-type CryAB or CryAB R120G . Subsequently, loss- and gain-of-function experiments were performed using ube2v1 siRNA or recombinant adenovirus mediated ube2v1 overexpression. Knockdown of ube2v1 decreased aggregate accumulation and attenuated cytotoxicity caused by CryAB R120G expression, while overexpressing ube2v1 enhanced aggregate formation. Ubiquitin proteasome system (UPS) function was analyzed using a UPS reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein (GFPu). Knockdown of ube2v1 improved proteasomal function and promoted the degradation of insoluble ubiquitinated proteins in CryAB R120G cardiomyocytes, but did not alter autophagic flux. Conclusions: Inhibition of ube2v1 improves CryAB R120G -induced aggregate formation through enhancing proteasome activity. It provides a novel therapeutic target to treat cardiac proteinopathies.

Ube2v1-mediated ubiquitination and degradation of Sirt1 promotes metastasis of colorectal cancer by epigenetically suppressing autophagy

BackgroundUbiquitination is a basic post-translational modification for cellular homeostasis, and members of the conjugating enzyme (E2) family are the key components of the ubiquitin–proteasome system. However, the role of E2 family in colorectal cancer (CRC) is largely unknown. Our study aimed to investigate the role of Ube2v1, one of the ubiquitin-conjugating E2 enzyme variant proteins (Ube2v) but without the conserved cysteine residue required for the catalytic activity of E2s, in CRC.MethodsImmunohistochemistry and real-time RT-PCR were used to study the expressions of Ube2v1 at protein and mRNA levels in CRC, respectively. Western blotting and immunofluorescence, transmission electron microscopy, and in vivo rescue experiments were used to study the functional effects of Ube2v1 on autophagy and EMT program. Quantitative mass spectrometry, immunoprecipitation, ubiquitination assay, western blotting, and real-time RT-PCR were used to analyze the effects of Ube2v1 on histone H4 lysine 16 acetylation, interaction with Sirt1, ubiquitination of Sirt1, and autophagy-related gene expression.ResultsUbe2v1 was elevated in CRC samples, and its increased expression was correlated with poorer survival of CRC patients. Ube2v1 promoted migration and invasion of CRC cells in vitro and tumor growth and metastasis of CRC cells in vivo. Interestingly, Ube2v1suppressed autophagy program and promoted epithelial mesenchymal transition (EMT) and metastasis of CRC cells in an autophagy-dependent pattern in vitro and in vivo. Moreover, both rapamycin and trehalose attenuated the enhanced Ube2v1-mediated lung metastasis by inducing the autophagy pathway in an orthotropic mouse xenograft model of lung metastasis. Mechanistically, Ube2v1 promoted Ubc13-mediated ubiquitination and degradation of Sirt1 and inhibited histone H4 lysine 16 acetylation, and finally epigenetically suppressed autophagy gene expression in CRC.ConclusionsOur study functionally links Ube2v1, an E2 member in the ubiquitin–proteasome system, to autophagy program, thereby shedding light on developing Ube2v1 targeted therapy for CRC patients.

Abstract 358: miRNAs/Ago2/YY1/PcG protein/DNMT complex mediates epigenetic silencing of locus genes and associates with bortezomib-induced apoptosis of leukemia

Abstract miRNAs are a class of non-coding RNAs, which bind to the 3'-untranslated regions of their target mRNAs to repress translation. However, the details of miRNA-mediated epigenetic regulation targeting genomic DNA remained largely unknown. Here, we demonstrated that transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, is a highly selective reversible inhibitor for leukemia treatment, and resulting in caspase activation and apoptosis. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 feedback to suppress the transcription of CEBPD itself and genes adjacent to its 5'-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Additionally, we previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for mediating epigenetic silencing of the CEBPD gene; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. Moreover, we revealed that miRNA binding plays a key role for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. Altogether, we provided new insights for miRNA-mediated epigenetic regulation in the case of bortezomib-induced cell death in leukemia. Citation Format: Ju-Ming Wang, Yu-Yi Chu, Chiung-Yuan Ko, Shao-Ming Wang, Pin-I Lin, Han-Ying Wang, Wen-Chi Lin, Dong-Yu Wu. miRNAs/Ago2/YY1/PcG protein/DNMT complex mediates epigenetic silencing of locus genes and associates with bortezomib-induced apoptosis of leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 358.

Abstract A48: Ube2v1 promotes EMT and metastasis by suppression of autophagy

Abstract Ubiquitination is one of the basic post-translational modifications for cellular homeostasis. The conjugating enzyme (E2) family plays as a bridge linking the first step mediated by E1 with the final step mediated by E3 in the ubiquitin-proteasome system. However, the role of Ube2v1, one of ubiquitin-conjugating E2 enzyme variant proteins (Ube2v), in colorectal cancer (CRC) and autophagy is unclear. Here, we found that Ube2v1 is elevated in tumor samples of CRC patients and correlated with poorer survival of CRC patients. Furthermore, Ube2v1 promotes migration and invasion of CRC cells in vitro and boosts tumor growth and metastasis of CRC cells in vivo. Interestingly, Ube2v1 suppresses autophagy program and promotes EMT and metastasis of CRC cells in an autophagy-dependent pattern in vitro and in vivo. Moreover, rapamycin attenuates the enhanced in vitro migration and invasion and in vivo lung metastasis of Ube2v1 overexpression by inducing the autophagy pathway. Mechanistically, Ube2v1 increases histone H4 lysine 16 acetylation by downregulating expression of Sirt1 and epigenetically suppresses gene expression of autophagy genes in colorectal cancer. In conclusion, Ube2v1 functions as a global regulator for autophagy by epigenetically transcriptional suppression of autophagy, and consequently promotes EMT and metastasis of CRC. Our study functionally links Ube2v1, one member of E2 family in the ubiquitin-proteasome system to autophagy program in CRC progression and metastasis. Citation Format: Tong Shen, Lingdong Cai, Yuhong Liu, Jianming Li. Ube2v1 promotes EMT and metastasis by suppression of autophagy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A48.

O-GlcNAcylation confers protection against Staphylococcus aureus infection in Caenorhabditis elegans through ubiquitination.

Glycosylation is one of the most prevalent post-translational modifications in biological systems. In Caenorhabditis elegans, O-GlcNAcylation has been shown to be actively involved in the regulation of dauer formation and detoxification of toxins secreted by invading pathogens. On this backdrop, the present study is focused on understanding the role of O-GlcNAcylation in C. elegans during Staphylococcus aureus infection using a gel based proteomic approach. Results of time course killing assays with wild-type and mutants of glycosylation and comparison of results revealed an increase in the survival of the C. elegans oga-1 mutant when compared to wild-type N2 and the ogt-1 mutant. Increased survival of C. elegans N2 upon S. aureus infection in the presence of O-(2-acetamido-2-deoxy-d-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc-an OGA inhibitor) further confirmed the involvement of O-GlcNAcylation in protecting C. elegans from infection. The two-dimensional gel-based proteomic analysis of the control and S. aureus infected C. elegans oga-1 mutant followed by mass spectrometric identification of differentially expressed proteins has been carried out. The results revealed that key proteins involved in ubiquitination such as Cullin-1 (CUL-1), Cullin-3 (CUL-3), BTB and MATH domain-containing protein 15 (BATH-15), ubiquitin-conjugating enzyme E2 variant 3 (UEV-3) and probable ubiquitin-conjugating enzyme E2 7 (UBC-7) are upregulated. Real-time PCR analysis further confirms the upregulation of genes encoding the above-mentioned proteins which are involved in the ubiquitin-mediated pathways in C. elegans. In addition, treatment of C. elegans wild-type N2 and the oga-1 mutant with PUGNAc + suramin and suramin (an ubiquitination inhibitor), respectively has resulted in increased sensitivity to S. aureus infection. Hence, it is presumed that upregulation of proteins involved in the ubiquitination pathway could be the key regulatory mechanism responsible for the enhanced survival of the oga-1 mutant during S. aureus infection.

Bortezomib-induced miRNAs direct epigenetic silencing of locus genes and trigger apoptosis in leukemia

MicroRNAs (miRNAs) have been suggested to repress transcription via binding the 3′-untranslated regions of mRNAs. However, the involvement and details of miRNA-mediated epigenetic regulation, particularly in targeting genomic DNA and mediating epigenetic regulation, remain largely uninvestigated. In the present study, transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, a clinical and highly selective drug for leukemia treatment, and contributed to bortezomib-induced cell death. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 could target CpG islands in the 5′-flanking region of the CEBPD gene. We previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for CCAAT/enhancer binding protein delta (CEBPD) gene inactivation; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. The miRNA/Ago2/YY1/PcG group protein/DNMT complex linked the inactivation of CEBPD and genes adjacent to its 5′-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Moreover, we revealed that miRNA binding is necessary for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. The present study successfully characterized the interactions of the miRNA/Ago2/YY1/PcG group protein/DNMT complex and provided new insights for miRNA-mediated epigenetic regulation in bortezomib-induced leukemic cell arrest and cell death.

Abstract 855: Ube2v1 promotes epithelial mesenchymal transition and metastasis in colorectal cancer by epigenetically transcriptional suppression of autophagy

Abstract Ubiquitination is one of the basic post-translational modifications for cellular homeostasis. The conjugating enzyme (E2) family plays as a bridge linking the first step mediated by E1 with the final step mediated by E3 in the ubiquitin-proteasomesystem. However, the role of Ube2v1, one of Ubiquitin-conjugating E2 enzyme variant proteins (Ube2v), in colorectal cancer (CRC) and autophagy is unclear. Here, we found that Ube2v1 is elevated in tumor samples of CRC patients and correlated with poorer survival of CRC patients. Furthermore, Ube2v1 promotes migration and invasion of CRC cells in vitro and boosts tumor growth and metastasis of CRC cells in vivo. Interestingly, Ube2v1 suppresses autophagy program and promotes EMT and metastasis of CRC cells in an autophagy dependent pattern in vitro and in vivo. Moreover, Rapamycin attenuates the enhanced in vitro migration and invasion and in vivo lung metastasis of Ube2v1 overexpression by inducing the autophagy pathway. Mechanistically, Ube2v1 increases histone H4 lysine 16 acetylation by down-regulating expression of Sirt1 and epigenetically suppresses gene expression of autophagy genes in colorectal cancer. In conclusion, Ube2v1 functions as a globalregulator for autophagy by epigenetically transcriptional suppression of autophagy, and consequently promotes EMT and metastasis of CRC. Our study functionally links Ube2v1, one member of E2 family in the ubiquitin-proteasome system toautophagy program in CRC progression and metastasis. Note: This abstract was not presented at the meeting. Citation Format: Tong Shen, Dong Ling Cai, Hong Yu Liu, Juan Wen Gan, Ming Li, Ru Jing Wang, Da Peng Guo, Qun Zhou, Xing Xing Lu, Shi Li, Na Li Sun, Ming Jian Li. Ube2v1 promotes epithelial mesenchymal transition and metastasis in colorectal cancer by epigenetically transcriptional suppression of autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 855. doi:10.1158/1538-7445.AM2017-855

Identification of New Lead Molecules Against UBE2NL Enzyme for Cancer Therapy.

Cancer is characterized by abnormal growth of cells. Targeting ubiquitin proteins in the discovery of new anticancer therapeutics is an attractive strategy. The present study uses the structure-based drug discovery methods to identify new lead structures, which are selective to the putative ubiquitin-conjugating enzyme E2N-like (UBE2NL). The 3D structure of the UBE2NL was evaluated using homology modeling techniques. The model was validated using standard in silico methods. The hydrophobic pocket of UBE2NL that aids in binding with its natural receptor ubiquitin-conjugating enzyme E2 variant (UBE2V) was identified through protein-protein docking study. The binding site region of the UBE2NL was identified using active site prediction tools. The binding site of UBE2NL which is responsible for cancer cell progression is considered for docking study. Virtual screening study with the small molecular structural database was carried out against the active site of UBE2NL. The ligand molecules that have shown affinity towards UBE2NL were considered for ADME prediction studies. The ligand molecules that obey the Lipinski's rule of five and Jorgensen's rule of three pharmacokinetic properties like human oral absorption etc. are prioritized. The resultant ligand molecules can be considered for the development of potent UBE2NL enzyme inhibitors for cancer therapy.

Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy

The purpose of our study was to evaluate how hyperglycemia (HG) influences Lys63 protein ubiquitination and its involvement in tubular damage and fibrosis in diabetic nephropathy (DN). Gene and protein expression of UBE2v1, a ubiquitin-conjugating E2-enzyme variant that mediates Lys63-linked ubiquitination, and Lys63-ubiquitinated proteins increased in HK2 tubular cells under HG. Matrix-assisted laser desorption/ionization-time of flight/tandem mass spectrometry identified 30 Lys63-ubiquitinated proteins, mainly involved in cellular organization, such as β-actin, whose Lys63 ubiquitination increased under HG, leading to cytoskeleton disorganization. This effect was reversed by the inhibitor of the Ubc13/UBE2v1 complex NSC697923. Western blot analysis confirmed that UBE2v1 silencing in HK2 under HG, restored Lys63-β-actin ubiquitination levels to the basal condition. Immunohistochemistry on patients with type 2 diabetic (T2D) revealed an increase in UBE2v1- and Lys63-ubiquitinated proteins, particularly in kidneys of patients with DN compared with control kidneys and other nondiabetic renal diseases, such as membranous nephropathy. Increased Lys63 ubiquitination both in vivo in patients with DN and in vitro, correlated with α-SMA expression, whereas UBE2v1 silencing reduced HG-induced α-SMA protein levels, returning them to basal expression. In conclusion, UBE2v1- and Lys63-ubiquitinated proteins increase in vitro under HG, as well as in vivo in T2D, is augmented in patients with DN, and may affect cytoskeleton organization and influence epithelial-to-mesenchymal transition. This process may drive the progression of tubular damage and interstitial fibrosis in patients with DN.-Pontrelli, P., Conserva, F., Papale, M., Oranger, A., Barozzino, M., Vocino, G., Rochetti, M. T., Gigante, M., Castellano, G., Rossini, M., Simone, S., Laviola, L., Giorgino, F., Grandaliano, G., Di Paolo, S., Gesualdo, L. Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy.

Anti-centrosome antibodies in breast cancer are the expression of autoimmunity.

Centrosome abnormalities have been observed in nearly all human solid tumors, but their role in tumorigenesis is unclear. We have demonstrated that autoantibodies reacting with antigens in centrosomes are frequently found in BC sera. In this work, we attempted to characterize the centrosome antigens associated with BC. We immunoscreened a T7 cDNA library of BC proteins with BC sera, and the autoantigens identified were printed as a microarray and hybridized with BC and control sera. We used immunohistochemistry (IHC) to investigate the expression of the cloned autoantigens in BC tissue. Immunoscreening with BC sera led to the cloning of autoantibodies recognizing epitopes developing in a family of proteins located on centrosomes such as peri-centriolar material-1, isomorph CRA, stathmin1, HS actin gamma1, SUMO/sentrin peptidase 2, and ubiquitin-conjugating enzyme E2 variant 1. Antibody reactivity to these proteins that are associated with centrosome assembly and/or microtubule function was highly associated with the diagnosis of BC. IHC staining of formalin-fixed paraffin-embedded sections with specific antibodies showed that aurora and stathmin are expressed in BC. The discovery of autoantibodies to important centrosome antigens associated with BC suggests that this immune reactivity could be related to autoimmunity developing in BC. Our finding that some of these antibodies are also present in a group of healthy women suggests that breakdown of tolerance to centrosome proteins may occur early in breast carcinogenesis and that autoantibodies to centrosome antigens might be biomarkers of early BC.

Ube2W conjugates ubiquitin to α-amino groups of protein N-termini

The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13–UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation.

Delayed administration of pituitary adenylate cyclase-activating polypeptide 38 ameliorates renal ischemia/reperfusion injury in mice by modulating Toll-like receptors

We investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) ameliorates kidney injury after ischemia/reperfusion (IR) by modulating Toll-like receptor (TLR)-associated signaling pathways. Male C57BL/6 mice were subjected to bilateral renal ischemia for 45min. PACAP38, 20μg in 100μl of saline, was administered i.p. at 24 and 48h after IR, and mice were euthanized at 72h. In IR mice, PACAP38 maintained serum creatinine near control levels (0.81±0.08 vs. 0.69±0.17mg/dl in controls, p=NS, vs. 1.8±0.03 in saline-treated IR mice, p<0.01) and significantly reduced the expression of kidney injury biomarkers. PACAP38 significantly reduced the levels of apoptosis and neutrophil infiltration, and protected against tubular damage. With PCR arrays, 59 of 83 TLR-related genes significantly changed their expression after IR. TLR2 increased 162 fold, followed by Fas-associated death domain (37 fold) and TLR6 (24 fold), while ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) decreased 55 fold. PACAP38 given 24 and 48h after IR injury significantly reversed these changes in 56 genes, including TLR2, TLR3, TLR4, TLR6, and genes in the NF-κB pathways. The alterations in TLR2, TLR3, TLR6, and UBE2V1 were confirmed by RT-PCR. After IR, PACAP38 also suppressed protein levels of TLR-associated cytokines. PACAP38 reversed the changes in IR-activated TLR-associated NF-κB signaling pathways even when treatment was delayed 24h. Therefore, PACAP38 could be an effective therapeutic for unexpected IR-mediated renal injury. The prominently IR-induced TLR-related genes identified in this study could be novel drug targets.

Interfacial water molecules in SH3 interactions: a revised paradigm for polyproline recognition

In spite of its biomedical relevance, polyproline recognition is still not fully understood. The disagreement between the current description of SH3 (Src homology 3) complexes and their thermodynamic behaviour calls for a revision of the SH3-binding paradigm. Recently, Abl-SH3 was demonstrated to recognize its ligands by a dual binding mechanism involving a robust network of water-mediated hydrogen bonds that complements the canonical hydrophobic interactions. The systematic analysis of the SH3 structural database in the present study reveals that this dual binding mode is universal to SH3 domains. Tightly bound buried-interfacial water molecules were found in all SH3 complexes studied mediating the interaction between the peptide ligand and the domain. Moreover, structural waters were also identified in a high percentage of the free SH3 domains. A detailed analysis of the pattern of water-mediated interactions enabled the identification of conserved hydration sites in the polyproline-recognition region and the establishment of relationships between hydration profiles and the sequence of both ligands and SH3 domains. Water-mediated interactions were also systematically observed in WW (protein-protein interaction domain containing two conserved tryptophan residues), UEV (ubiquitin-conjugating enzyme E2 variant) and EVH-1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] structures. The results of the present study clearly indicate that the current description of proline-rich sequence recognition by protein-protein interaction modules is incomplete and insufficient for a correct understanding of these systems. A new binding paradigm is required that includes interfacial water molecules as relevant elements in polyproline recognition.

Proteomic profiling of proteins dysregulted in Chinese esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer death in China. In the present study, proteins in tumors and adjacent normal esophageal tissues from 41 patients with ESCC were extracted, and two-dimensional electrophoresis (2-DE) was performed using the pH 3-10 and 4-7 immobilized pH gradient strips. The protein spots expressed differentially between tumors and normal tissues were identified by matrix-assisted laser desorption/ionization and liquid chromatography electrospray/ionization ion trap mass spectrometry. A total of 22 proteins differentially expressed between ESCC and normal esophageal tissues were identified, in which 17 proteins were upregulated and 5 downregulated in tumors. Biological functions of these proteins are related to cell signal transduction, cell proliferation, cell motility, glycolysis, regulation of transcription, oxidative stress processes, and protein folding. Some of the proteins obtained were confirmed by Western blotting and immunohistochemical staining. We showed that high expression of calreticulin and 78-kDa glucose-regulated protein (GRP78) were correlated with poor prognosis by Kaplan-Meier analysis and log rank analysis. Zinc finger protein 410, annexin V, similar to the ubiquitin-conjugating enzyme E2 variant 1 isoform c, mutant hemoglobin beta chain, TPM4-ALK fusion oncoprotein type 2, similar to heat shock congnate 71-kDa protein, GRP78, and pyruvate kinase M2 (M2-PK) were for the first time observed to be dysregulated in human ESCC tissues. The proteins here identified will contribute to the understanding of the tumorigenesis and progression of Chinese ESCC and may potentially provide useful markers for diagnosis or targets for therapeutic intervention and drug development.