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Menon, Manoj B., Christopher Tiedje, Juri Lafera та ін. "Endoplasmic reticulum-associated ubiquitin-conjugating enzyme Ube2j1 is a novel substrate of MK2 (MAPKAP kinase-2) involved in MK2-mediated TNFα production". Biochemical Journal 456, № 2 (2013): 163–72. http://dx.doi.org/10.1042/bj20130755.
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The protein kinase MK2 phosphorylates the endoplasmic reticulum-associated ubiquitin-conjugating enzyme Ube2j1 under various stress conditions and during the innate immune response in macrophages. Although its apparent enzyme activity stays unaltered, Ube2j1 contributes to MK2-dependent biosynthesis of tumour necrosis factor α.
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Pasupala, Nagesh, Marie E. Morrow, Lauren T. Que, Barbara A. Malynn, Averil Ma, and Cynthia Wolberger. "OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination." Journal of Biological Chemistry 293, no. 47 (2018): 18285–95. http://dx.doi.org/10.1074/jbc.ra118.004677.
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OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.
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Liu, Tingting, and Jianshe Wei. "Validation of a Novel Cuproptosis–Related Prognostic Gene Marker and Differential Expression Associated with Lung Adenocarcinoma." Current Issues in Molecular Biology 45, no. 10 (2023): 8502–18. http://dx.doi.org/10.3390/cimb45100536.
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Background: Cuproptosis induction is seen as a promising alternative for immunotherapies and targeted therapies in breast cancer. The objective of this research was to examine the prognostic and biological importance of cuproptosis-related genes (CRGs) in lung adenocarcinoma (LUAD). Methods: The following methods were used: GSE10072 dataset and TCGA database analysis, differential expression analysis of CRGs, and biological function (BP) and signaling pathway enrichment analysis, prognostic analysis and clinical analysis of CRGs, construction of the prognostic signature and RNA modified genes and miRNA analysis of CRGs in LUAD, immunoinfiltration analysis and immunohistochemical staining of DβH, UBE2D3, SOD1, UBE2D1 and LOXL2. Results: AOC1, ATOX1, CCL8, CCS, COX11, CP, LOXL2, MAP2K2, PDK1, SCO2, SOD1, UBE2D1, UBE2D3 and VEGFA showed significantly higher expression, while ATP7B, DβH, PDE3B, SLC31A2, UBE2D2, UBE2D4 and ULK2 showed lower expression in LUAD tissues than normal tissues. We also found that ATP7B (4%), AOC1 (3%) PDE3B (2%), DβH (2%), CP (1%), ULK2 (1%), PDK1 (1%), LOXL2 (1%) and UBE2D3 (1%) showed higher mutation frequencies. The univariate Cox analysis was used to identify CRGs that have prognostic value. It identified 21 genes that showed significant prognostic value, containing DβH, UBE2D3, SOD1, UBE2D1 and LOXL2. Patients with DβH up–expression have a longer survival time and patients with UBE2D3, SOD1, UBE2D1 and LOXL2 down–expression also have a longer survival time. hsa–miR–29c–3p, hsa–miR–29a–3p, hsa–miR–181c–5p, hsa–miR–1245a, etc., play an important role in the miRNA regulatory network, and in LUAD, miR–29a, miR–29c and miR–181c high expression survival was longer, and miR–1245a low expression survival was longer. We also performed an analysis to examine the relationships between DβH, LOXL2, SOD1, UBE2D1 and UBE2D3 and immune infiltration in LUAD, including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and DCs. Conclusion: DβH, UBE2D3, SOD1, UBE2D1, and LOXL2 are potential candidates implicated in LUAD and can be further explored for their application as diagnostic, prognostic, and therapeutic biomarkers for LUAD.
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Magaziner, Samuel J., Jason C. Collins, Brecca Miller, Kelly V. Ruggles, Achim Werner, and David B. Beck. "The Mechanism of Cell Autonomous Inflammation in VEXAS Syndrome Is Mediated By Proteotoxic Stress." Blood 144, Supplement 1 (2024): 187. https://doi.org/10.1182/blood-2024-202567.
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VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a severe, multi-system disease caused by mutations in the UBA1 gene acquired in hematopoietic stem cells (HSCs) affecting 1/4000 men over the age of 50 (Beck DB, NEJM 2020; Beck DB, JAMA 2023). UBA1 is the E1 enzyme responsible for initiating the majority of ubiquitylation and has two isoforms: a nuclear isoform, UBA1a, and a cytoplasmic isoform, UBA1b. UBA1 mutations in VEXAS occur predominantly at the Met41 hotspot, lead to loss of the cytoplasmic isoform UBA1b, and are restricted to the myeloid lineage. We have previously shown that UBA1 mutations generate a bottleneck in transferring ubiquitin to cytoplasmic E2 enzymes and UBA1b isoform levels correlate with patient outcome (Collins J, EMBO 2024; Ferrada M, Blood 2022). However, how a reduction in cytoplasmic E2 charging and ubiquitylation causes inflammation is poorly understood. Here, we identify the cell autonomous mechanisms that initiate and drive inflammation in VEXAS. First, we investigated the impact of defective cytoplasmic ubiquitylation on protein homeostasis by analyzing total proteomes of CD14+ monocytes isolated from healthy donors or VEXAS patients. Pathway analysis revealed upregulation of not only inflammation but also substrates associated with protein processing in the endoplasmic reticulum and the unfolded protein response (UPR). To validate these findings, we constructed a THP1, human monocyte, model of VEXAS utilizing a conditional Cas9-mediated knock-out of endogenous UBA1 in the background of constitutive lentivirally incorporated wild-type (UBA1WT) or VEXAS disease associated UBA1 (UBA1M41V). When induced, the UBA1M41V line generated vacuoles, showed poly-ubiquitin defects, and produced an inflammatory response. We then performed unbiased proteomics and again observed upregulation of conserved pathways in ER protein processing, UPR, and inflammation. Immunoblot and transcriptomic analyses of both patient CD14+ monocytes and the cell model confirmed upregulated markers of UPR and inflammation including BiP, p-eIF2α, XBP-1s, ATF4, and p-STAT1. To examine whether UPR activation is driven by loss of ubiquitin conjugation to a specific E2, we performed an unbiased molecular screen with the Chinese Hamster Ovary (CHO) line ts20. At restrictive temperatures endogenous UBA1 is lost. By complementing the system with UBA1WT or UBA1M41V we were able to screen all 28 E2 enzymes assayable in CHO cells. While most E2 enzymes showed a mild loss of E2~Ub thioester species when cytoplasmic UBA1b is lost due to UBA1M41V, only two, UBE2J1 and UBE2G2, showed substantial loss of ubiquitin conjugation. These same E2s were found to be disproportionately undercharged in both our THP1 VEXAS model and patient cells when assayed by immunoblotting. This demonstrates the E2 defects to be conserved and specific to Met41 mutations across both model cell lines and patient monocytes. UBE2J1, UBE2J2, and UBE2G2 are cognate members of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway. Importantly, UBE2J1 and UBE2J2 are the only cytoplasmic restricted E2s in the entire proteome. Thus, we hypothesized that loss of cytoplasmic ubiquitylation might specifically inhibit ERAD. To examine this, we used an ERAD substrate reporter, CD3δ-GFP, and were able to show that in the THP-1 UBA1M41V model, ERAD substrate processing is defective. Furthermore, selective chemical inhibition of a cognate E3 of ERAD, HRD1, as well as siRNA-mediated depletion of the ERAD E2s phenocopied ERAD defects as well UPR activation and inflammation. Finally, we posited that ERAD failure drives chronic UPR activation and inappropriate inflammation. Indeed, when we utilized UPR-specific small molecule inhibitors targeting ATF6, IRE1α, and PERK, in the settings of both the THP-1 UBA1M41V model and CD14+ patient cells, we were able to dramatically reduce the inflammatory phenotype. In summary, we identified that VEXAS mutations in the Met41 hotspot in UBA1 lead to an undercharging of the cytoplasmic-resident E2 enzymes of ERAD, subsequent ERAD failure, the accumulation of unfolded proteins, and chronic activation of the UPR leading to inflammation capable of being modulated by UPR-targeting small molecule drugs. These results identify the mechanisms initiating and propagating inflammation in VEXAS syndrome and highlight multiple avenues for therapeutic intervention.
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Shirasaki, Ryosuke, Ricardo De Matos Simoes, Shizuka Yamano, et al. "Functional Genomic Characterization of Endoplasmic Reticulum-Associated Dependencies in Multiple Myeloma - Biologic and Therapeutic Implications." Blood 136, Supplement 1 (2020): 3–4. http://dx.doi.org/10.1182/blood-2020-143117.
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Background: Endoplasmic reticulum (ER)-associated degradation (ERAD) for unfolded proteins represents an important biological vulnerability for multiple myeloma (MM) cells, given the high proteostatic stress in these cells due to immunoglobulin secretion. This helps explain the efficacy of proteasome inhibitors in MM compared to most other neoplasms. Aim: To systematically investigate ER-associated genes that mediate the essential role of ERAD in MM. Methods: We integrated results from (i) genome-scale studies for CRISPR-gene knockout in 19 MM cell lines (Avana library) and CRISPR-activation (Calabrese library) in 5 MM lines, to define ER-associated dependencies and drivers of increased MM cell growth; (ii) publicly-available CRISPR-based studies for genes regulating ERAD of substrates located in the luminal (ERAD-L), membrane (ERAD-M) or cytosolic (ERAD-C) side of the ER in non-MM models; (iii) functional characterization of candidate genes in MM models in vitro and in vivo. Results: While ER function in general is globally required for all normal and malignant cells, a surprisingly large proportion of ER-related genes are not "pan-essential" genes (e.g. CERES scores <-0.4 are for <80% of human cell lines): in fact CRISPR-mediated disruption of many ER genes elicits no growth disadvantage for most non-MM tumor cell lines. Notably, though, several of these latter non-"pan- essential" genes are recurrently essential for MM cell lines, or even preferential dependencies for MM compared to other cancers. By overlaying the patterns of CRISPR essentiality in MM lines vs. the functional modules of ER function, we observe that recurrent or preferential ER-associated dependencies for MM cells include genes involved in (a) ER membrane protein complexes mediating dislocation of misfolded ER proteins to the cytosol (e.g. HERPUD1, SEL1L, AUP1 for ERAD-L substrates; and AMFR and RNF139 for ERAD-M and -C substrates,) and associated ER-specific E2/E3 enzymes (SYVN1, UBE2J1, UBE2J2, and UBE2G2); (b) upstream of the ER-to-cytosol dislocation complexes, several enzymes (e.g. DPM1, DPM3, ALG3, ALG9, ALG12, ALG6, ALG8) required for N-glycan-dependent surveillance of quality control for luminal ER glycoproteins; (c) chaperones (e.g. DNAJB11, DNAJBC3) for BiP complexes with misfolded proteins; (d) the known ER stress-sensor IRE1a (ERN1) and its downstream transcription factor XBP1. CRISPR-activation of nearly all these recurrent/MM-preferential ER-associated genes led to no significant growth advantage for MM cell lines. Several of the MM-preferential ER-associated genes (HERPUD1, SEL1L, SYVN1, UBE2J1) are expressed at significantly higher levels in MM vs. non-MM cell lines (CCLE) or patient derived samples (CoMMpass study vs. TCGA) and are proximal to binding sites of key transcription factors for plasma cell identity such as IRF4 and PRDM1. By contrast, CRISPR-mediated LOF of such genes in MM cells decreases their growth in vitro (e.g. competition assays vs. CRISPR LOF of non-essential genes not expressed in MM cells) and in vivo for MM cells growing in a bone marrow-like scaffold system with a "humanized" mesenchymal bone marrow stromal cell compartment. Knockout of MM-preferential ER-associated genes induces markers of ER stress/unfolded protein response (e.g. BiP induction by UBE2J1 or HERPUD1 LOF); and sensitizes MM cells to proteasome inhibition. Accordingly, pharmacological inhibition (LS-102) of SYVN1 (Synovolin-1) an E3 ligase which helps remove unfolded proteins from the cytosol into the ER was more effective at a lower drug concentration in MM compared to non-MM-cell lines. Conclusions: Our results provide functional insights into the role of ERAD in MM cells and nominate specific mediators which are not broadly essential across all cancers, but are recurrent or even preferentially essential for MM. Our study indicates a previously underappreciated role for the ER-to-cytosol dislocon complex that includes HERPUD1, SEL1L, SYVN1 and UBE2J1; distinct enzymes of the N-glycan-dependent ER glycoproteins quality control system; and ER chaperones in MM. These findings provide a rationale for development of agents selectively targeting these key regulators of ER protein folding processes in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Tango Therapeutics: Consultancy; Cedilla Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:EMD Serono: Research Funding; Abbvie: Research Funding; Karyopharm: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; FIMECS: Consultancy, Honoraria; Takeda: Other: employment of a relative; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Arch Oncology: Research Funding.
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Napolitano, Luisa M., Ellis G. Jaffray, Ronald T. Hay, and Germana Meroni. "Functional interactions between ubiquitin E2 enzymes and TRIM proteins." Biochemical Journal 434, no. 2 (2011): 309–19. http://dx.doi.org/10.1042/bj20101487.
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The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM–UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets.
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Koenig, Paul-Albert, Peter K. Nicholls, Florian I. Schmidt, et al. "The E2 Ubiquitin-conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice." Journal of Biological Chemistry 289, no. 50 (2014): 34490–502. http://dx.doi.org/10.1074/jbc.m114.604132.
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Wei, Yingbing, Shuaibin Pei, Yiru Huang, et al. "UBE2J1 suppresses interferon signaling by facilitating the ubiquitination and degradation of IRF7." Aquaculture 595 (January 2025): 741640. http://dx.doi.org/10.1016/j.aquaculture.2024.741640.
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Yang, Dexin, Xin Ma, Jie Xu, Ke Jia, Xiaoli Liu, and Ping Zhang. "Zfx-induced upregulation of UBE2J1 facilitates endometrial cancer progression via PI3K/AKT pathway." Cancer Biology & Therapy 22, no. 3 (2021): 238–47. http://dx.doi.org/10.1080/15384047.2021.1883186.
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Cremer, Tom, Marlieke L. M. Jongsma, Fredrik Trulsson, Alfred C. O. Vertegaal, Jacques Neefjes, and Ilana Berlin. "The ER-embedded UBE2J1/RNF26 ubiquitylation complex exerts spatiotemporal control over the endolysosomal pathway." Cell Reports 34, no. 3 (2021): 108659. http://dx.doi.org/10.1016/j.celrep.2020.108659.
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Burr, Marian L., Florencia Cano, Stanislava Svobodova, Louise H. Boyle, Jessica M. Boname, and Paul J. Lehner. "HRD1 and UBE2J1 target misfolded MHC class I heavy chains for endoplasmic reticulum-associated degradation." Proceedings of the National Academy of Sciences 108, no. 5 (2011): 2034–39. http://dx.doi.org/10.1073/pnas.1016229108.
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Le, Thanh Thi, Johanna Ainsworth, Cristian Polo Rivera, Thomas Macartney, and Karim P. M. Labib. "Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes." Biochemical Journal 478, no. 14 (2021): 2825–42. http://dx.doi.org/10.1042/bcj20210315.
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Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.
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Elangovan, Muthukumar, Hae Kwan Chong, Jin Hee Park, Eui Ju Yeo, and Yung Joon Yoo. "The role of ubiquitin-conjugating enzyme Ube2j1 phosphorylation and its degradation by proteasome during endoplasmic stress recovery." Journal of Cell Communication and Signaling 11, no. 3 (2017): 265–73. http://dx.doi.org/10.1007/s12079-017-0386-6.
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Schneider, Constanze, Shan Lin, Angela H. Su, Gabriela Alexe, and Kimberly Stegmaier. "The UBE2J2/UBE2K-MARCH5 Ubiquitination Machinery Regulates Apoptosis in Response to Venetoclax in Acute Myeloid Leukemia." Blood 142, Supplement 1 (2023): 2785. http://dx.doi.org/10.1182/blood-2023-182312.
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Evasion of apoptosis is crucial for the growth, survival and chemoresistance of many cancer types, including acute myeloid leukemia (AML); thus, the reactivation of apoptosis can be exploited as a therapeutic approach. Apoptosis induction is mainly controlled by the balance between anti-apoptotic and pro-apoptotic BCL2 family proteins on the mitochondrial membrane. Venetoclax, a selective inhibitor antagonizing the anti-apoptotic protein BCL2, has emerged as a promising therapy in AML. Despite high response rates in combination with hypomethylating agents, however, some patients display upfront resistance, and most patients will ultimately relapse. Therefore, identification of synergistic targets for combination therapies with venetoclax is important for improving the clinical application of this drug. To systematically identify key genes that can modulate the venetoclax effect, we performed a genome-scale CRISPR-Cas9 screen in the AML cell line MV4-11. Consistent with previous reports, loss of the apoptosis effector BAX or the pro-apoptotic gene NOXA ( PMAIP1) caused venetoclax resistance, while sgRNAs targeting the anti-apoptotic genes BCLXL ( BCL2L1), BCL2L2 and BCL2A1 were significantly more depleted in venetoclax-treated cells. Furthermore, depletion of the E2 ubiquitin-conjugating enzymes, UBE2J2 and UBE2K, ranked highly among the venetoclax sensitizers. We validated that deletion of either UBE2J2 or UBE2K increased induction of apoptosis in multiple AML cell lines and patient-derived xenograft (PDX) cells upon venetoclax treatment and was deleterious as single gene perturbation in some models. Exploiting the Broad Institute Cancer DepMap dataset, which includes genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, revealed that dependency on UBE2J2 or UBE2K significantly correlated with a dependency on the E3 ligase MARCH5. We previously identified the repression of MARCH5 as a strong inducer of apoptosis in AML. Since E2s coordinate with ubiquitin E3 ligases to execute ubiquitination, this DepMap result suggested that UBE2J2 and UBE2K serve as MARCH5 E2 partners. To test this hypothesis, we utilized NanoBiT technology, a structural complementation reporter system, to detect protein interactions between MARCH5 and the E2 candidates. LgBIT and SmBIT, two split subunits of luciferase, were fused with MARCH5 and the E2 proteins, respectively. The luminescent signal was activated upon the co-expression of LgBIT-MARCH5 with either of the SmBIT-tagged E2 proteins but not an empty vector, suggesting that MARCH5 and UBE2J2/UBE2K constitute ubiquitination machinery that regulates apoptosis in AML. We next tested the possible redundancy between the two E2s and showed that the double knockout of UBE2J2/ UBE2K further enhanced the venetoclax sensitivity. MARCH5 depletion results in increased NOXA expression, an important node in dictating venetoclax response. Several reports indicated that NOXA is also a critical downstream mediator of MARCH5. In contrast, we previously showed that MARCH5 can regulate apoptosis independently of NOXA in AML. To reassess the role of NOXA and other pro-apoptotic proteins in MARCH5-mediated apoptosis, we conducted an unbiased CRISPR rescue screen in a MARCH5-dTAG degradation system derived from PDX17-14, a complex karyotype, MLL-AF10 PDX model. BAX was the top rescuing target, emphasizing that apoptosis induction is the main mechanism accounting for the growth inhibition of MARCH5-depleted cells. However, depletion of other pro-apoptotic BCL2 members, including NOXA and BIM, did not rescue MARCH5 depletion in this screen consistent with our previously published data. Here, we confirmed that NOXA KO does not rescue MARCH5 depletion in additional AML models. Similarly, KO of UBE2J2 or UBE2K can repress AML cell growth and increase venetoclax sensitivity even in the context of NOXA KO. Our study highlights that UBE2J2 and UBE2K are two important functional partners of MARCH5 in regulating apoptosis in AML cells and can serve as additional targets for enhancing venetoclax efficacy. Unbiased screening and low-throughput target validation further emphasize that the MARCH5 ubiquitination machinery regulates apoptosis in AML cells largely in a NOXA-independent manner. Additional studies are needed to dissect the MARCH5/UBE2J2/UBE2K complex-mediated apoptosis regulation in AML.
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Vriend, Jerry, Thatchawan Thanasupawat, Namita Sinha, and Thomas Klonisch. "Ubiquitin Proteasome Gene Signatures in Ependymoma Molecular Subtypes." International Journal of Molecular Sciences 23, no. 20 (2022): 12330. http://dx.doi.org/10.3390/ijms232012330.
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The ubiquitin proteasome system (UPS) is critically important for cellular homeostasis and affects virtually all key functions in normal and neoplastic cells. Currently, a comprehensive review of the role of the UPS in ependymoma (EPN) brain tumors is lacking but may provide valuable new information on cellular networks specific to different EPN subtypes and reveal future therapeutic targets. We have reviewed publicly available EPN gene transcription datasets encoding components of the UPS pathway. Reactome analysis of these data revealed genes and pathways that were able to distinguish different EPN subtypes with high significance. We identified differential transcription of several genes encoding ubiquitin E2 conjugases associated with EPN subtypes. The expression of the E2 conjugase genes UBE2C, UBE2S, and UBE2I was elevated in the ST_EPN_RELA subtype. The UBE2C and UBE2S enzymes are associated with the ubiquitin ligase anaphase promoting complex (APC/c), which regulates the degradation of substrates associated with cell cycle progression, whereas UBE2I is a Sumo-conjugating enzyme. Additionally, elevated in ST_EPN_RELA were genes for the E3 ligase and histone deacetylase HDAC4 and the F-box cullin ring ligase adaptor FBX031. Cluster analysis demonstrated several genes encoding E3 ligases and their substrate adaptors as EPN subtype specific genetic markers. The most significant Reactome Pathways associated with differentially expressed genes for E3 ligases and their adaptors included antigen presentation, neddylation, sumoylation, and the APC/c complex. Our analysis provides several UPS associated factors that may be attractive markers and future therapeutic targets for the subtype-specific treatment of EPN patients.
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Geisler, Sven, Stefanie Vollmer, Sonia Golombek, and Philipp J. Kahle. "The ubiquitin-conjugating enzymes UBE2N, UBE2L3 and UBE2D2/3 are essential for Parkin-dependent mitophagy." Journal of Cell Science 127, no. 15 (2014): 3280–93. http://dx.doi.org/10.1242/jcs.146035.
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Sievers, Quinlan L., Jessica A. Gasser, Glenn S. Cowley, Eric S. Fischer, and Benjamin L. Ebert. "Genome-wide screen identifies cullin-RING ligase machinery required for lenalidomide-dependent CRL4CRBN activity." Blood 132, no. 12 (2018): 1293–303. http://dx.doi.org/10.1182/blood-2018-01-821769.
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Key Points Genome-scale CRISPR knockout screen identifies cullin-RING ligase regulators as top mediators of lenalidomide resistance. The E2 ubiquitin-conjugating enzymes, UBE2D3 and UBE2G1, play distinct roles in lenalidomide-induced substrate ubiquitination by CRL4CRBN.
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Liang, Xue-hai, Joshua G. Nichols, Dario Tejera, and Stanley T. Crooke. "Perinuclear positioning of endosomes can affect PS-ASO activities." Nucleic Acids Research 49, no. 22 (2021): 12970–85. http://dx.doi.org/10.1093/nar/gkab1198.
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Abstract Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs that act on cellular RNAs must enter cells and be released from endocytic organelles to elicit antisense activity. It has been shown that PS-ASOs are mainly released by late endosomes. However, it is unclear how endosome movement in cells contributes to PS-ASO activity. Here, we show that PS-ASOs in early endosomes display Brownian type motion and migrate only short distances, whereas PS-ASOs in late endosomes (LEs) move linearly along microtubules with substantial distances. In cells with normal microtubules and LE movement, PS-ASO-loaded LEs tend to congregate perinuclearly. Disruption of perinuclear positioning of LEs by reduction of dynein 1 decreased PS-ASO activity, without affecting PS-ASO cellular uptake. Similarly, disruption of perinuclear positioning of PS-ASO-LE foci by reduction of ER tethering proteins RNF26, SQSTM1 and UBE2J1, or by overexpression of P50 all decreased PS-ASO activity. However, enhancing perinuclear positioning through reduction of USP15 or over-expression of RNF26 modestly increased PS-ASO activity, indicating that LE perinuclear positioning is required for ensuring efficient PS-ASO release. Together, these observations suggest that LE movement along microtubules and perinuclear positioning affect PS-ASO productive release.
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Lin, Chih-Yi, Chung-Jen Yu, Chun-Yu Liu, et al. "Abstract P2-26-14: CDK4/6 inhibitors modulate the ubiquitin–proteasome pathway in ER+ breast cancer by downregulation of UBE2C/S/T." Cancer Research 83, no. 5_Supplement (2023): P2–26–14—P2–26–14. http://dx.doi.org/10.1158/1538-7445.sabcs22-p2-26-14.
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Abstract Despite significant improvement in therapeutic development in the past decades, breast cancer remains a formidable cause of death for women worldwide. The hormone positive subtype (HR(+)) (also known as luminal type) is the most prevalant category of breast cancer, comprising ~70% of patients. The clinical success of the three CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib has revolutionized the treatment of choice for metastatic HR(+) breast cancer. Accumulating evidence demonstrate that the properties of CDK4/6 inhibitors extend beyond inhibition of the cell cycle, including modulation of immune function, sensitizing PI3K inhibitors, metabolism reprogramming, kinome rewiring, modulation of the proteosome, and many others. The ubiquitin–proteasome pathway (UPP) is a crucial cellular proteolytic system that maintains the homeostasis and turnover of proteins. By transcriptional profiling of the HR(+) breast cancer cell lines MCF7 and T47D treated with Palbociclib, we have uncovered a novel mechanism that demonstrate the CDK4/6 inhibitors suppress the expression of three ubiquitin conjugating enzymes UBE2C, UBE2S, UBE2T. Further validation in the HR(+) cell lines show that Palbociclib and ribociclib decrease UBE2C at both the mRNA and protein level, but this phenomenon was not shared with abemaciclib. These three E2 enzymes modulate several E3 ubiquitin ligases, including the APC/C complex which plays a role in G1/S progression. We further demonstrate the UBE2C/UBE2T expression levels are associated with breast cancer survival, and HR(+) breast cancer cells demonstrate dependence on the UBE2C. Our study suggests a novel link between CDK4/6 inhibitor and UPP pathway, adding to the potential mechanisms of their clinical efficacy in cancer. Citation Format: Chih-Yi Lin, Chung-Jen Yu, Chun-Yu Liu, Ta-Chung Chao, Chi-Cheng Huang, Ling-Ming Tseng, Jiun-I Lai. CDK4/6 inhibitors modulate the ubiquitin–proteasome pathway in ER+ breast cancer by downregulation of UBE2C/S/T [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-14.
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Zhang, Zebo, Haiyan Qian, Li Wang, et al. "Construction of a circRNA-miRNA-mRNA Regulatory Network for Coronary Artery Disease by Bioinformatics Analysis." Cardiology Research and Practice 2022 (February 16, 2022): 1–10. http://dx.doi.org/10.1155/2022/4017082.
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Background. Circular RNAs (circRNAs) were known to be related to the pathogenesis of many diseases through competing endogenous RNA (ceRNA) regulatory mechanisms. However, the function of circRNA in coronary artery disease (CAD) remains unclear. In this study, we aim to construct a circRNA-related competing endogenous RNA (ceRNA) network in CAD. Methods. The gene expression profiles of CAD were obtained from Gene Expression Omnibus datasets. Bioinformatics analysis was performed to construct a ceRNA regulatory network, from which the hub genes involved were identified through protein-protein interaction (PPI) networks leading to the identification of the circRNA-miRNA-hub gene subnetwork. In addition, function enrichment analysis was performed to detect the potential biological mechanism in which circRNA might be involved. Results. A total of 115 DEcircRNAs (differentially expressed circRNAs), 17 DEmiRNAs (differentially expressed microRNAs), and 790 DEmRNAs (differentially expressed mRNAs) were identified between CAD and control groups from microarray datasets. Functional enrichment analysis showed that DEmRNAs were significantly involved in inflammation-related pathways and ubiquitin-protein ligase binding. After identifying 20 DEcircRNA-DEmiRNA pairs and 561 DEmiRNA-DEmRNA pairs, we obtained a circRNA-miRNA-mRNA regulatory network. PPI network analysis showed that eight hub genes were closely related to CAD, leading to the identification of a circRNA-miRNA-hub gene subnetwork consisting of nine circRNAs (hsa_circ_0020275, hsa_circ_0020387, hsa_circ_0020417, hsa_circ_0045512, hsa_circ_0047336, hsa_circ_0069094, hsa_circ_0071326, hsa_circ_0071330, and hsa_circ_0085340), four miRNAs (hsa-miR-136-5p, hsa-miR-376c-3p, hsa-miR-411-5p, and hsa-miR-654-5p), and eight mRNAs (MKRN1, UBE2H, UBE2W, UBE2D1, UBE2F, BE2J1, ZNRF1, and SIAH2). In addition, we discovered these hub genes were enriched in the ubiquitin-mediated proteolysis pathway, suggesting circRNAs may be involved in the pathogenesis of CAD through this pathway. Conclusions. This study may deepen our understanding of the potential role of circRNA-miRNA-mRNA regulation network in CAD and suggest novel diagnostic biomarkers and therapeutic targets for CAD.
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Menon, M. B., C. Tiedje, J. Lafera та ін. "Endoplasmic reticulum-associated ubiquitin-conjugating enzyme Ube2j1 is a novel substrate of MK2 (MAPKAP kinase-2) involved in MK2-mediated TNFα production". Biochemical Journal 457, № 1 (2013): 229. http://dx.doi.org/10.1042/bj4570229.
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Fletcher, Adam J., Donna L. Mallery, Ruth E. Watkinson, Claire F. Dickson, and Leo C. James. "Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21." Proceedings of the National Academy of Sciences 112, no. 32 (2015): 10014–19. http://dx.doi.org/10.1073/pnas.1507534112.
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Tripartite motif (TRIM) 21 is a cytosolic antibody receptor that neutralizes antibody-coated viruses that penetrate the cell and simultaneously activates innate immunity. Here we show that the conjugation of TRIM21 with K63-linked ubiquitin (Ub-63Ub) catalyzed by the sequential activity of nonredundant E2 Ub enzymes is required for its dual antiviral functions. TRIM21 is first labeled with monoubiquitin (monoUb) by the E2 Ube2W. The monoUb is a substrate for the heterodimeric E2 Ube2N/Ube2V2, resulting in TRIM21-anchored Ub-63Ub. Depletion of either E2 abolishes Ub-63Ub and Ub-48Ub conjugation of TRIM21, NF-κB signaling, and virus neutralization. The formation of TRIM21-Ub-63Ub precedes proteasome recruitment, and we identify an essential role for the 19S-resident and degradation-coupled deubiquitinase Poh1 in TRIM21 neutralization, signaling, and cytokine induction. This study elucidates a complex mechanism of step-wise ubiquitination and deubiquitination activities that allows contemporaneous innate immune signaling and neutralization by TRIM21.
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El-Mallawany, Nader, Janet Ayello, Nancy Day, et al. "Global Proteomic Profiling of Endemic Versus Sporadic Epstein-Barr Virus (EBV) Positive Burkitt Lymphoma (BL)." Blood 114, no. 22 (2009): 4779. http://dx.doi.org/10.1182/blood.v114.22.4779.4779.
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Abstract Abstract 4779 Background EBV infection of normal B-cells is commonly associated with the pathogenesis of BL (Brady et al, Clin Path, 2007). Endemic BL (eBL) is characteristically positive (100%) for EBV, contrasting with sporadic BL (sBL), where approximately 30% of cases are positive for EBV. eBL vs. sBL have significantly different breakpoint regions within c-myc (Shiramizu/Magrath et al, Blood, 1991). Overexpression of c-myc is the sine quae non of BL. C-myc interactions with other genes/proteins is multilayered and complex (Basso/Della-Favera, Nat Gen, 2005). Apoptotic pathway disruption is propelled by EBV and is critically important in c-myc deregulation and subsequent lymphomagenesis that occurs in EBV+ eBL vs. sBL (Ruf et al, J Vir, 2000). Global analysis of proteins expressed in EBV+ eBL vs. sBL may provide insights into biologic, pathogenetic, and molecular differences between the two subtypes of lymphoma, and potentially identify targets for the development of therapeutic agents. Objectives To compare the proteomic expression profile and signal transduction pathways of EBV+ eBL vs. sBL. Methods Whole cell lysates obtained from the EBV+ eBL cell line Raji and the EBV+ sBL cell line NC37 were digested and labeled with iTRAQ” labeling reagents, following manufacturer's protocol. The peptides were resolved by 2D-LC technique (off-line Strong cation exchange followed by on-line reverse-phase liquid chromatography). Data-dependant High energy C-trap Dissociation MS/MS spectra were acquired using an Orbitrap XL Tandem Mass Spectrometer (ThermoFisher). The MS/MS data was searched using X!Tandem/TPP software suite against human IPI database (v3.50) appended with decoy (reverse) sequences. iTRAQ” ratios of proteins (ProteinProphet probability of >0.9) were normalized and differentially expressed proteins were determined through Mixture Modeling. Protein interactions were further analyzed using the GoMiner and Ingenuity pathway analysis tools. Results Over 400 proteins were identified as being differentially expressed by a ≥ 1.25 fold change between the two cell lines. We identified differentially expressed proteins in both cell lines that are involved in a wide array of cellular processes as exhibited in Figure 1. Cellular processes uniquely involved by proteins over-expressed in eBL included immune response, hematopoiesis, cell proliferation, heat shock, and B-cell activation, while those uniquely identified in sBL included cell division, response to virus, and NF-kB cascade proteins. Specific cell-regulatory pathways implicated by the differential protein profile expressions (with associated proteins in parentheses) included the p53 apoptosis pathway (PCNA, MSH6, C1QBP, MAP4, and BAX), the caspase network of apoptosis (HCLS1, ACIN1, and AIFM1), the tumor suppressor protein RB network (MCM7, PA2G4, and API5), general apoptotic pathways (HSP90 and PDCD4), B-cell differentiation and proliferation pathways (TPD52 and IKBKG), and the ubiquitin-proteasome pathway (UBE2J1, UBE2C, and UBE2S). Seven of these proteins are c-myc target genes. Ingenuity protein network analysis revealed nine proteins identified in the experiment with interactions connected through the p53, caspase, and tumor necrosis factor apoptosis pathways. Conclusion Proteomic profile analysis of EBV+ eBL and sBL revealed over and under-expression of multiple proteins that may be implicated in the multi-factorial nature of disease pathogenesis. This is the first MS-based direct proteomic comparison of eBL and sBL. Our results suggest that there are potentially different mechanisms driving cell proliferation and resistance to apoptosis in eBL versus sBL and that EBV infection may be involved in the processes that drive lymphomagenesis. Ultimately, identification of proteins unique to the distinct disease subtypes will serve to establish tumor markers that may enable development of new diagnostic, prognostic, and therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.
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Hunt, Liam C., Michelle Curley, Kudzai Nyamkondiwa, et al. "The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity." PLOS Biology 23, no. 1 (2025): e3002998. https://doi.org/10.1371/journal.pbio.3002998.
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Ubiquitin-conjugating enzymes (E2s) are key for protein turnover and quality control via ubiquitination. Some E2s also physically interact with the proteasome, but it remains undetermined which E2s maintain proteostasis during aging. Here, we find that E2s have diverse roles in handling a model aggregation-prone protein (huntingtin-polyQ) in the Drosophila retina: while some E2s mediate aggregate assembly, UBE2D/effete (eff) and other E2s are required for huntingtin-polyQ degradation. UBE2D/eff is key for proteostasis also in skeletal muscle: eff protein levels decline with aging, and muscle-specific eff knockdown causes an accelerated buildup in insoluble poly-ubiquitinated proteins (which progressively accumulate with aging) and shortens lifespan. Mechanistically, UBE2D/eff is necessary to maintain optimal proteasome function: UBE2D/eff knockdown reduces the proteolytic activity of the proteasome, and this is rescued by transgenic expression of human UBE2D2, an eff homolog. Likewise, human UBE2D2 partially rescues the lifespan and proteostasis deficits caused by muscle-specific effRNAi and re-establishes the physiological levels of effRNAi-regulated proteins. Interestingly, UBE2D/eff knockdown in young age reproduces part of the proteomic changes that normally occur in old muscles, suggesting that the decrease in UBE2D/eff protein levels that occurs with aging contributes to reshaping the composition of the muscle proteome. However, some of the proteins that are concertedly up-regulated by aging and effRNAi are proteostasis regulators (e.g., chaperones and Pomp) that are transcriptionally induced presumably as part of an adaptive stress response to the loss of proteostasis. Altogether, these findings indicate that UBE2D/eff is a key E2 ubiquitin-conjugating enzyme that ensures protein quality control and helps maintain a youthful proteome composition during aging.
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de Carvalho, Luiz Gustavo Almeida, Tatiana Takahasi Komoto, Daniel Antunes Moreno, et al. "USP15-USP7 Axis and UBE2T Differential Expression May Predict Pathogenesis and Poor Prognosis in De Novo Myelodysplastic Neoplasm." International Journal of Molecular Sciences 24, no. 12 (2023): 10058. http://dx.doi.org/10.3390/ijms241210058.
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The aim of this study was to evaluate the expression of USP7, USP15, UBE2O, and UBE2T genes in Myelodysplastic neoplasm (MDS) to identify possible targets of ubiquitination and deubiquitination in MDS pathobiology. To achieve this, eight datasets from the Gene Expression Omnibus (GEO) database were integrated, and the expression relationship of these genes was analyzed in 1092 MDS patients and healthy controls. Our results showed that UBE2O, UBE2T, and USP7 were upregulated in MDS patients compared with healthy individuals, but only in mononucleated cells collected from bone marrow samples (p < 0.001). In contrast, only the USP15 gene showed a downregulated expression compared with healthy individuals (p = 0.03). Additionally, the upregulation of UBE2T expression was identified in MDS patients with chromosomal abnormalities compared with patients with normal karyotypes (p = 0.0321), and the downregulation of UBE2T expression was associated with MDS hypoplastic patients (p = 0.033). Finally, the USP7 and USP15 genes were strongly correlated with MDS (r = 0.82; r2 = 0.67; p < 0.0001). These findings suggest that the differential expression of the USP15-USP7 axis and UBE2T may play an important role in controlling genomic instability and the chromosomal abnormalities that are a striking characteristic of MDS.
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Choi, Yun-Seok, Yun-Ju Lee, Seo-Yeon Lee, et al. "Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities." Journal of Biological Chemistry 290, no. 4 (2014): 2251–63. http://dx.doi.org/10.1074/jbc.m114.624809.
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Paubel, Agathe, Sylviane Marouillat, Audrey Dangoumau, et al. "Dynamic Expression of Genes Encoding Ubiquitin Conjugating Enzymes (E2s) During Neuronal Differentiation and Maturation: Implications for Neurodevelopmental Disorders and Neurodegenerative Diseases." Genes 15, no. 11 (2024): 1381. http://dx.doi.org/10.3390/genes15111381.
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Background: The ubiquitination process plays a crucial role in neuronal differentiation and function. Numerous studies have focused on the expression and functions of E3 ligases during these different stages, far fewer on E2 conjugating enzymes. In mice, as in humans, these E2s belong to 17 conjugating enzyme families. Objectives: We analyzed by real-time PCR the expression dynamics of all known E2 genes during an in vitro differentiation of mouse hippocampal neuronal cultures, and after, we analyzed their stimulation with N-methyl-D-aspartate (NMDA). Results: We found that 36 of the 38 E2 genes were expressed in hippocampal neurons. Many were up-regulated during neuritogenesis and/or synaptogenesis stages, such as Ube2h, Ube2b, and Aktip. Rapid and delayed responses to NMDA stimulation were associated with the increased expression of several E2 genes, such as Ube2i, the SUMO-conjugating E2 enzyme. We also observed similar expression profiles within the same E2 gene family, consistent with the presence of similar transcription factor binding sites in their respective promoter sequences. Conclusions: Our study indicates that specific expression profiles of E2 genes are correlated with changes in neuronal differentiation and activity. A better understanding of the regulation and function of E2s is needed to better understand the role played by the ubiquitination process in physiological mechanisms and pathophysiological alterations involved in neurodevelopmental or neurodegenerative diseases.
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Ghilarducci, Kim, Valérie C. Cabana, Camille Desroches, et al. "Functional interaction of ubiquitin ligase RNF167 with UBE2D1 and UBE2N promotes ubiquitination of AMPA receptor." FEBS Journal 288, no. 16 (2021): 4849–68. http://dx.doi.org/10.1111/febs.15796.
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Shirasaki, Ryosuke, Sondra L. Downey-Kopyscinski, Ricardo De Matos Simoes, et al. "Interactions with a "Humanized" Mesenchymal Bone Marrow Stromal Niche In Vivo Modify the Patterns of Essential Genes for Myeloma Cells: Therapeutic Implications." Blood 136, Supplement 1 (2020): 40. http://dx.doi.org/10.1182/blood-2020-143151.
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Background: The biology and treatment response of human multiple myeloma (MM) cells in vivo is influenced by interactions with mesenchymal bone marrow stromal cells (BMSCs). For several key BMSC-derived cytokines (including IL-6) only the human, not murine, form optimally interacts with their respective receptor(s) on human MM cells. To better simulate the treatment responses of human MM cells in the BM, "humanized" BM-like niches in vivo have been engineered with biocompatible ceramic scaffolds "functionalized" via osteogenic differentiation of human mesenchymal BMSCs and implanted subcutaneously in immunocompromised mice. Aim: To determine if the patterns of genetic dependencies elucidated through in vitro CRISPR-based functional genomic studies are recapitulated when human MM cells are grown in mice within BM-like scaffolds with "humanized" mesenchymal stromal compartment. Methods: Cas9+ MM cell lines KMS11 and XG7 were transduced with a library of 1372 single-guide RNAs (sgRNAs) targeting 184 genes of interest (4 sgRNAs/gene), including 89 genes preferentially essential for MM cell lines compared to other neoplasms in vitro; broad-spectrum oncogenic targets (e.g. KRAS, MYC); tumor suppressor genes (e.g. PTEN); genes with limited in vitro essentiality in MM cells, but significantly higher expression vs. non-MM lines (e.g. ZFP91, ZBP1); and 155 olfactory receptor (OR) genes (typically not expressed or biologically inactive in tumor cells) as "DNA cutting" control sgRNAs. MM cells transduced with this focused CRISPR knockout (KO) library were injected into "humanized" scaffolds implanted subcutaneously (s.c.) in NSG mice. Tumors were collected when mice reached criteria for euthanasia and next-generation sequencing quantified the changes in sgRNA distribution at the end vs. start of experiment. Results: A large majority of genes defined by in vitro CRISPR KO screens as MM-preferential dependencies were also essential for MM cells in BM-like scaffolds. Among 57 MM-preferential dependencies in vitro which were plausible dependencies for KMS11 cells (e.g. CERES scores &lt;-0.4), 50 genes were essential for KMS11 cells in BM-like scaffolds in vivo (average log2fold change&lt;-1.0 and depletion of 3+ of 4 sgRNAs relative to the 99% confidence interval for control sgRNAs). These genes included key transcription factors/cofactors (e.g. IRF4, PRDM1, POU2AF1, RELB, MAF); epigenetic regulators (e.g. CARM1, MTA2, DOT1L); kinases upstream of NFkappaB (CHUK, IKBKB); ER regulators (e.g. SYVN1). Most "core-essential" or broad-spectrum oncogenic dependencies (e.g. MYC, CFLAR, CDK7 on both lines; KRAS in XG7) of this sgRNA library remained essential in vivo; while PTEN KO cells were enriched. Notably, several genes had more pronounced essentiality in vivo vs. invitro (e.g. BCL2, PIM2); or converted from non-essential in vitro to essential in vivo. For instance, among 95 genes of this library which are not likely dependencies in vitro (CERES scores &gt;-0.4) for KMS11 cells, 29 genes exhibited in vivo essentiality for both KMS11 and XG7 cells: several of these latter "in vivo dependencies" are recurrently essential for other MM lines in vitro (e.g. ZBTB38, UBE2J1, TCF3, DNAJB11), while also others have limited if any in vitro essentiality (e.g. ZBP1, PYGO2) across MM despite significantly higher transcript levels vs. other neoplasias. Notably, several genes with increased essentiality in the "humanized" BM scaffolds vs. in vitro also had stronger in vivo dependency in the BM scaffolds vs. when growth of the same MM cells as s.c. plasmacytomas (e.g. BCL2, PIM2, UBE2J1, SYVN1, ALG9, AMFR). Co-culture with BMSCs or IL-6 treatment induces several of these transcripts in MM cells suggesting that increased dependency of MM on these genes in the "humanized" BM model is due at least partly to its human cytokines. Conclusions: This study provides evidence for significant impact of the "humanized" BM-like niche on the patterns of genetic dependencies for human MM cells. Most genes preferentially essential for MM cells in vitro remain essential for their in vivo growth in the "humanized" BM model. Notably, several genes that do not meet criteria for dependency in vitro show such metrics in "humanized" BM scaffolds, but not sc plasmacytomas. These observations highlight important implications of the "humanized" BM-like in vivo model for current and future efforts to define and prioritize therapeutic targets for MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Tango Therapeutics: Consultancy; Cedilla Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Janssen/Johnson & Johnson: Research Funding; Arch Oncology: Research Funding; TEVA: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; EMD Serono: Research Funding; Abbvie: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.
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Burge, Rebecca J., Andreas Damianou, Anthony J. Wilkinson, Boris Rodenko, and Jeremy C. Mottram. "Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex." PLOS Pathogens 16, no. 10 (2020): e1008784. http://dx.doi.org/10.1371/journal.ppat.1008784.
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Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.
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Ardley, Helen Clare, Terry Patrick Moynihan, Alexander Fred Markham, and Philip Alan Robinson. "Promoter analysis of the human ubiquitin-conjugating enzyme gene family UBE2L1–4, including UBE2L3 which encodes UbcH7." Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1491, no. 1-3 (2000): 57–64. http://dx.doi.org/10.1016/s0167-4781(00)00024-5.
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Behera, Adaitya Prasad, Pritam Naskar, Shubhangi Agarwal, Prerana Agarwal Banka, Asim Poddar, and Ajit B. Datta. "Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications." Biochemical Journal 475, no. 9 (2018): 1569–82. http://dx.doi.org/10.1042/bcj20170909.
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RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.
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Xu, Na, James Gulick, Hanna Osinska, et al. "Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination." Circulation Research 126, no. 7 (2020): 907–22. http://dx.doi.org/10.1161/circresaha.119.316444.
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Rationale: 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. Objective: To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryAB R120G ) and identify how Ube2v1 exerts its effect. Methods and Results: Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryAB WT ) or CryAB R120G . Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryAB R120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryAB WT and CryAB R120G -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 CryAB R120G 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 CryAB R120G -induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo. Conclusions: Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryAB R120G -induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.
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FUJIOKA, Yoshie A., Asuka ONUMA, Wataru FUJII, Koji SUGIURA, and Kunihiko NAITO. "Contributions of UBE2C and UBE2S to meiotic progression of porcine oocytes." Journal of Reproduction and Development 64, no. 3 (2018): 253–59. http://dx.doi.org/10.1262/jrd.2018-006.
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Bhardwaj, Abhishek, Robert S. Banh, Wei Zhang, Sachdev S. Sidhu, and Benjamin G. Neel. "MMD-associated RNF213 SNPs encode dominant-negative alleles that globally impair ubiquitylation." Life Science Alliance 5, no. 5 (2022): e202000807. http://dx.doi.org/10.26508/lsa.202000807.
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Single-nucleotide polymorphisms (SNPs) in RNF213, which encodes a 591-kD protein with AAA+ ATPase and RING E3 domains, are associated with a rare, autosomal dominant cerebrovascular disorder, moyamoya disease (MMD). MMD-associated SNPs primarily localize to the C-terminal region of RNF213, and some affect conserved residues in the RING domain. Although the autosomal dominant inheritance of MMD could most easily explained by RNF213 gain-of-function, the type of ubiquitylation catalyzed by RNF213 and the effects of MMD-associated SNPs on its E3 ligase activity have remained unclear. We found that RNF213 uses the E2-conjugating enzymes UBE2D2 and UBE2L3 to catalyze distinct ubiquitylation events. RNF213-UBED2 catalyzes K6 and, to a lesser extent, K48-dependent poly-ubiquitylation in vitro, whereas RNF213-UBE2L3 catalyzes K6-, K11-, and K48-dependent poly-ubiquitylation events. MMD-associated SNPs encode proteins with decreased E3 activity, and the most frequent MMD allele, RNF213R4810K, is a dominant-negative mutant that decreases ubiquitylation globally. By contrast, MMD-associated RNF213 SNPs do not affect ATPase activity. Our results suggest that decreased RNF213 E3 ligase activity is central to MMD pathogenesis.
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Lim, Key-Hwan, and Jae-Yeol Joo. "Predictive Potential of Circulating Ube2h mRNA as an E2 Ubiquitin-Conjugating Enzyme for Diagnosis or Treatment of Alzheimer’s Disease." International Journal of Molecular Sciences 21, no. 9 (2020): 3398. http://dx.doi.org/10.3390/ijms21093398.
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Neurodegenerative disorders are caused by neuronal cell death, miscommunications between synapse, and abnormal accumulations of proteins in the brain. Alzheimer’s disease (AD) is one of the age-related disorders, which are the most common degenerative disorders today, and strongly affects memory consolidation and cognitive function in the brain. Amyloid-β and tau proteins are triggers for AD pathogenesis, and usually used as AD candidate biomarkers in the clinical research. Especially, clinical exam, brain imaging and molecular biological methods are being used to diagnosis for AD. Genome-wide association study (GWAS) is a new biomedical method, and its use contributes to understanding many human diseases, including brain diseases. Here, we identified ubiquitin conjugating enzyme E2 (Ube2) gene expression in neurons through GWAS. The subfamilies of Ube2’s genetic expression and inborn errors affect the ubiquitin proteasome system (UPS), leading to protein degradation in the brain. We found that only Ube2h mRNA transcription was significantly increased in the blood from AD, however we did not find any change of Ube2 subfamily genes’ expression in the blood and brain tissue. These data may provide information for diagnosis or clinical approach, and suggest that cell-free circulating Ube2h mRNA is a novel potential biomarker for AD.
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Fuentes-Antrás, Jesús, Ana Lucía Alcaraz-Sanabria, Esther Cabañas Morafraile, et al. "Mapping of Genomic Vulnerabilities in the Post-Translational Ubiquitination, SUMOylation and Neddylation Machinery in Breast Cancer." Cancers 13, no. 4 (2021): 833. http://dx.doi.org/10.3390/cancers13040833.
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The dysregulation of post-translational modifications (PTM) transversally impacts cancer hallmarks and constitutes an appealing vulnerability for drug development. In breast cancer there is growing preclinical evidence of the role of ubiquitin and ubiquitin-like SUMO and Nedd8 peptide conjugation to the proteome in tumorigenesis and drug resistance, particularly through their interplay with estrogen receptor signaling and DNA repair. Herein we explored genomic alterations in these processes using RNA-seq and mutation data from TCGA and METABRIC datasets, and analyzed them using a bioinformatic pipeline in search of those with prognostic and predictive capability which could qualify as subjects of drug research. Amplification of UBE2T, UBE2C, and BIRC5 conferred a worse prognosis in luminal A/B and basal-like tumors, luminal A/B tumors, and luminal A tumors, respectively. Higher UBE2T expression levels were predictive of a lower rate of pathological complete response in triple negative breast cancer patients following neoadjuvant chemotherapy, whereas UBE2C and BIRC5 expression was higher in luminal A patients with tumor relapse within 5 years of endocrine therapy or chemotherapy. The transcriptomic signatures of USP9X and USP7 gene mutations also conferred worse prognosis in luminal A, HER2-enriched, and basal-like tumors, and in luminal A tumors, respectively. In conclusion, we identified and characterized the clinical value of a group of genomic alterations in ubiquitination, SUMOylation, and neddylation enzymes, with potential for drug development in breast cancer.
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Alpi, Arno F., Viduth Chaugule, and Helen Walden. "Mechanism and disease association of E2-conjugating enzymes: lessons from UBE2T and UBE2L3." Biochemical Journal 473, no. 20 (2016): 3401–19. http://dx.doi.org/10.1042/bcj20160028.
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Ubiquitin signalling is a fundamental eukaryotic regulatory system, controlling diverse cellular functions. A cascade of E1, E2, and E3 enzymes is required for assembly of distinct signals, whereas an array of deubiquitinases and ubiquitin-binding modules edit, remove, and translate the signals. In the centre of this cascade sits the E2-conjugating enzyme, relaying activated ubiquitin from the E1 activating enzyme to the substrate, usually via an E3 ubiquitin ligase. Many disease states are associated with dysfunction of ubiquitin signalling, with the E3s being a particular focus. However, recent evidence demonstrates that mutations or impairment of the E2s can lead to severe disease states, including chromosome instability syndromes, cancer predisposition, and immunological disorders. Given their relevance to diseases, E2s may represent an important class of therapeutic targets. In the present study, we review the current understanding of the mechanism of this important family of enzymes, and the role of selected E2s in disease.
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Hou, Liyan, Yingbo Li, Ying Wang, et al. "UBE2D1 RNA Expression Was an Independent Unfavorable Prognostic Indicator in Lung Adenocarcinoma, but Not in Lung Squamous Cell Carcinoma." Disease Markers 2018 (October 21, 2018): 1–8. http://dx.doi.org/10.1155/2018/4108919.
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In this study, we investigated the potential prognostic value of ubiquitin-conjugating enzyme E2D1 (UBE2D1) RNA expression in different histological subtypes of non-small-cell lung cancer (NSCLC). A retrospective study was performed by using molecular, clinicopathological, and survival data in the Cancer Genome Atlas (TCGA)—Lung Cancer. Results showed that both lung adenocarcinoma (LUAD) (N=514) and lung squamous cell carcinoma (LUSC) (N=502) tissues had significantly elevated UBE2D1 RNA expression compared to the normal tissues (p<0.001 and p=0.036, respectively). UBE2D1 RNA expression was significantly higher in LUAD than in LUSC tissues. Increased UBE2D1 RNA expression was independently associated with shorter OS (HR: 1.359, 95% CI: 1.031–1.791, p=0.029) and RFS (HR: 1.842, 95% CI: 1.353–2.508, p<0.001) in LUAD patients, but not in LUSC patients. DNA amplification was common in LUAD patients (88/551, 16.0%) and was associated with significantly upregulated UBE2D1 RNA expression. Based on these findings, we infer that UBE2D1 RNA expression might only serve as an independent prognostic indicator of unfavorable OS and RFS in LUAD, but not in LUSC.
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Sarkari, Feroz, Keith Wheaton, Anthony La Delfa, et al. "Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1." Journal of Biological Chemistry 288, no. 23 (2013): 16975–85. http://dx.doi.org/10.1074/jbc.m113.469262.
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Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme found in all eukaryotes that catalyzes the removal of ubiquitin from specific target proteins. Here, we report that UbE2E1, an E2 ubiquitin conjugation enzyme with a unique N-terminal extension, is a novel USP7-interacting protein. USP7 forms a complex with UbE2E1 in vitro and in vivo through the ASTS USP7 binding motif within its N-terminal extension in an identical manner with other known USP7 binding proteins. We show that USP7 attenuates UbE2E1-mediated ubiquitination, an effect that requires the N-terminal ASTS sequence of UbE2E1 as well as the catalytic activity of USP7. Additionally, USP7 is critical in maintaining the steady state levels of UbE2E1 in cells. This study reveals a new cellular mechanism that couples the opposing activities of the ubiquitination machinery and a deubiquitinating enzyme to maintain and modulate the dynamic balance of the ubiquitin-proteasome system.
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Mokarram, Pooneh, Fatemeh Shakiba-Jam, Soudabeh Kavousipour, Mostafa Moradi Sarabi, and Atefeh Seghatoleslam. "Promoter Methylation Status of Two Novel Human Genes, UBE2Q1 and UBE2Q2, in Colorectal Cancer: a New Finding in Iranian Patients." Asian Pacific Journal of Cancer Prevention 16, no. 18 (2016): 8247–52. http://dx.doi.org/10.7314/apjcp.2015.16.18.8247.
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Li, Yu, and Vincent K. Tsiagbe. "Failure of vSAg29-responsive CD4+CD25+ Treg cells to inhibit CD4+CD25− T cells resides in their differential regulation of cytokine, chemokine and toll-like receptor family genes (50.1)." Journal of Immunology 178, no. 1_Supplement (2007): S90. http://dx.doi.org/10.4049/jimmunol.178.supp.50.1.
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Abstract Endogenous mammary tumor virus (Mtv29)-encoded superantigen (vSAg29) mediates “reverse immune surveillance” that characterizes SJL lymphoma (RCS) development. CD4+Vβ16+ vSAg29-responsive T cells provide growth factors (notably IL-5 and IL-4) for RCS growth. The vSAg29-responsive CD4+ T cells belong to both CD4+CD25+ (Treg) and CD4+CD25− (non-Treg) populations. Unlike the known inhibitory ability of Treg cell on non-Treg cell responses to nominal antigens, Treg cells responding to vSAg29 fail to inhibit response of vSAg29-responsive non-Treg cells. This unique behavior of vSAg29-responsive T cells was found to be linked to their differential expression of cytokine, chemokine and toll-like receptor family genes. Treg cells responding to vSAg29 significantly inhibited the production of IL-4, IL-5, IL-10, IFN-γ, CCR4, CCL17, TOLLIP, UBE2N, NFKBIB, by CD4+CD25− cells. On the other hand, the addition of CD4+ Treg cells to non-Treg cells responding to vSAg29, resulted in enhanced production of mRNA for MIF, IL-10R, BCL6, CXCL4 (PF4), MAPK8 (JNK1), UBE2V1. Research funded by CA10273 from NCI.
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Yin, Lina, Yuanyuan Xue, Qiannan Shang, et al. "Pharmaceutical Inhibition of Neddylation as Promising Treatments for Various Cancers." Current Topics in Medicinal Chemistry 19, no. 12 (2019): 1059–69. http://dx.doi.org/10.2174/1568026619666190311110646.
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Background: Neddylation is an important post-translational modification of proteins, in which a NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is covalently introduced onto the substrate proteins to regulate their functions and homeostasis. As neddylation is frequently up-regulated in various cancers, its interference was proposed as a promising therapy of related diseases. Objective: The recent advances in developing neddylation interfering agents were summarized to provide an overview of current achievements and perspectives for future development. Methods: Reports on neddylation interfering agents were acquired from Pubmed as well as the EPO and clinicaltrials.gov websites, which were subsequently analyzed and summarized according to targets, chemical structures and biological activities. Results: Neddylation as a sophisticated procedure comprises proteolytic processing of NEDD8 precursor, deploying conjugating enzymes E1 (NAE), E2 (UBE2M and UBE2F) and various E3, as well as translocating NEDD8 along these conjugating enzymes sequentially and finally to substrate proteins. Among these nodes, NAE, UBE2M and the interaction between UBE2M-DCN1 have been targeted by small molecules, metal complexes, peptides and RNAi. A NAE inhibitor pevonedistat (MLN4924) is currently under evaluation in clinical trials for the treatment of various cancers. Conclusion: With multiple inhibitory approaches of neddylation being introduced, the development of neddylation interference as a novel cancer therapy is significantly boosted recently, although its efficacy and the best way to achieve that are still to be demonstrated in clinical trials.
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Barreyro, Laura, Avery Sampson, Kathleen Hueneman, et al. "Therapeutic Targeting of the Ubiquitin Conjugating Enzyme UBE2N in Myeloid Malignancies." Blood 132, Supplement 1 (2018): 4050. http://dx.doi.org/10.1182/blood-2018-99-115359.
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Abstract Inflammatory and innate immune signaling pathways are activated in leukemic stem and progenitor cells and contribute to the pathogenesis of hematologic malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). UBE2N is a ubiquitin (Ub) conjugating enzyme that catalyzes lysine 63 (K63)-linked Ub chains on substrates that are critical for signal transduction of broad innate immune signaling pathways. Here we report that UBE2N is required for leukemic cell function by mediating oncogenic innate immune signaling, and identified a novel chemical class of small molecule inhibitors that target UBE2N enzymatic activity. Upon UBE2N downregulation with two lentivirally expressed shRNAs, MOLM-13 and THP-1 cells lose their clonogenic potential and undergo cell death. Unlike for AML cells, UBE2N was dispensable for colony formation and viability of healthy cord blood CD34+ cells. The active site of UBE2N contains a cysteine (Cys) at position 87, which is essential for binding and transfer of Ub to its substrates. We performed in silico structure- and in vitro cell-based screens to identify small molecules that dock to the active site of UBE2N and covalently bind to Cys-87, as an approach to inhibit Ub transfer to substrates. Two structurally-related candidates, UC-764864 and UC-764865, emerged as inhibitors of UBE2N, as they specifically blocked the E1-UBE2N thioester transfer in vitro. Treatment of MDS/AML cell lines and patient-derived primary cells with UC-764864 and UC-764865 suppressed innate immune signaling and induced cytotoxic effects in MDS/AML cell lines and primary cells while sparing healthy hematopoietic cells in vitro and in vivo. To identify the molecular basis of UBE2N inhibition, we performed a global Ub screen for changes in ubiquitinated substrates by mass spectrometry and evaluated changes in gene expression by RNA-seq in MOLM-13 cells treated with vehicle control or the newly derived UBE2N inhibitors. RNA-seq of MOLM-13 cells treated with UC-764864 revealed that inhibition of UBE2N in leukemic cells targets oncogenic innate immune pathways, including NF-kB and Type I interferon signaling networks. UC-764864 and UC-764865 reduced the ubiquitination status of UBE2N, and altered the ubiquitination of proteins involved in innate immune signaling and the DNA damage response by primarily reducing K63-linked Ub modifications. Two substrates identified by the Ub screen, DDB1 and UBE2M, are components of the CUL4-CRBN E3 ligase complex and a target of the anti-leukemic therapy, Lenalidomide (LEN). LEN has shown encouraging results in del(5q) MDS patients; however, its effects are limited in other cytogenetic subtypes of MDS or AML. Therefore, the identification of molecular targets that can enhance or extend the use of LEN in a broader spectrum of patients is desired. As such, we explored the possibility of a cooperative effect of LEN and UBE2N inhibitors on MDS/AML cells. As compared to individual treatments, the combination of LEN and UC-764864, UC-764865 or UBE2N shRNAs significantly suppressed the function and viability of MDS/AML cell lines and patient samples in vitro. More striking, treatment of LEN and UBE2N inhibitors impaired MDS/AML cells that are refractory to treatment of LEN or UBE2N inhibitors alone. These findings suggest that UBE2N is a promising target to extend the use of LEN to other subtypes of MDS or AML. In summary, we implicate the Ub conjugating enzyme UBE2N as a target in MDS/AML, and identified novel small molecule inhibitors that target UBE2N and modify the function of Ub E3 ligases that are important for UBE2N-associated diseases, including autoinflammatory and autoimmune disorders, and hematologic malignancies. Disclosures No relevant conflicts of interest to declare.
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Ben‐Eliezer, Inbal, Yael Pomerantz, Dalia Galiani, Nava Nevo, and Nava Dekel. "Appropriate expression of Ube2C and Ube2S controls the progression of the first meiotic division." FASEB Journal 29, no. 11 (2015): 4670–81. http://dx.doi.org/10.1096/fj.15-274522.
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Xu, Muyu, and Jiying Zhang. "A siRNA screening of UBE2 family demonstrated that UBE2R1 had a high repressive effect on HIV Tat protein." Biochemistry and Biophysics Reports 32 (December 2022): 101366. http://dx.doi.org/10.1016/j.bbrep.2022.101366.
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Ouyang, Weiming, and David M. Frucht. "Erk1/2 Inactivation-Induced c-Jun Degradation Is Regulated by Protein Phosphatases, UBE2d3, and the C-Terminus of c-Jun." International Journal of Molecular Sciences 22, no. 8 (2021): 3889. http://dx.doi.org/10.3390/ijms22083889.
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Constitutive photomorphogenic 1 (COP1) is the ubiquitin E3 ligase that mediates degradation of c-Jun protein upon Erk1/2 inactivation. It remains unknown how this protein degradation pathway is regulated. In this study, we investigated the roles of protein phosphatases, ubiquitin-conjugating E2 enzymes (UBE2), and an intrinsic motif of c-Jun in regulating this degradation pathway. By using pharmacological inhibitors and/or gene knockdown techniques, we identified protein phosphatase 1 (PP1) and PP2A as the phosphatases and UBE23d as the UBE2 promoting c-Jun degradation, triggered by Erk1/2 inactivation. In addition, we report that the C-terminus of c-Jun protein facilitates its degradation. The addition of a C-terminal tag or deletion of the last four amino acid residues from the C-terminus of c-Jun protects it from degradation under Erk1/2-inactivating conditions. Taken together, this study reveals that the Erk1/2 inactivation-triggered and COP1-mediated c-Jun degradation is extrinsically and intrinsically regulated, providing a new understanding of the mechanisms underlying this protein degradation pathway.
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Li, Can, Erik B. Wendlandt, Benjamin Darbro, et al. "Genetic Analysis of Multiple Myeloma Identifies Cytogenetic Alterations Implicated in Disease Complexity and Progression." Cancers 13, no. 3 (2021): 517. http://dx.doi.org/10.3390/cancers13030517.
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Multiple myeloma (MM) is a genetically heterogeneous disease characterized by genomic chaos making it difficult to distinguish driver from passenger mutations. In this study, we integrated data from whole genome gene expression profiling (GEP) microarrays and CytoScan HD high-resolution genomic arrays to integrate GEP with copy number variations (CNV) to more precisely define molecular alterations in MM important for disease initiation, progression and poor clinical outcome. We utilized gene expression arrays from 351 MM samples and CytoScan HD arrays from 97 MM samples to identify eight CNV events that represent possible MM drivers. By integrating GEP and CNV data we divided the MM into eight unique subgroups and demonstrated that patients within one of the eight distinct subgroups exhibited common and unique protein network signatures that can be utilized to identify new therapeutic interventions based on pathway dysregulation. Data also point to the central role of 1q gains and the upregulated expression of ANP32E, DTL, IFI16, UBE2Q1, and UBE2T as potential drivers of MM aggressiveness. The data presented here utilized a novel approach to identify potential driver CNV events in MM, the creation of an improved definition of the molecular basis of MM and the identification of potential new points of therapeutic intervention.
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Claustre, Agnès, Mélodie Malige, Maëlys Macheton, et al. "Structure predictions of MuRF1‐UBE2 complexes identify amino acid residues governing interaction selectivity for each MuRF1‐E2 pair." FEBS Journal, February 10, 2025. https://doi.org/10.1111/febs.70017.
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The RING‐type E3 ubiquitin–protein ligase MuRF1 (also known as TRIM63) plays an important role in skeletal muscle atrophy by targeting contractile proteins. In cellulo, MuRF1 can alternatively interact with four E2 enzymes (UBE2E1, UBE2J1, UBE2J2, or UBE2L3), suggesting different functions or targets for the four MuRF1‐E2 complexes. In this article, we studied the interface of these MuRF1‐UBE2 complexes based on AlphaFold2 and AlphaFold3 predictions. These predictions revealed the involvement of different residues at the interface of each complex. We confirmed this overall interface difference by the differential sensitivity of MuRF1‐E2 complexes to regenerating solutions in surface plasmon resonance experiments. We further confirmed several predictions individually by affinity measurements with point‐mutant E2 enzymes and truncated MuRF1. We used the interaction‐induced fluorescence change approach with fluorescent MuRF1. Besides canonical E2‐RING‐type E3 interactions, we were able to identify selective contact points between MuRF1 and its UBE2 partners. Furthermore, in the case of the MuRF1‐E2E1 pair, unlike the other MuRF1‐E2 pairs, the interaction may also be governed by a domain outside the RING domain. Since the function of RING‐type E3s is regulated by E2 enzymes, deciphering the mechanisms of selective recruitment of E2s by MuRF1 paves the way for the development of targeted therapeutics to fight muscle atrophy.
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Kumari, Smita, and Pravir Kumar. "Identification and characterization of putative biomarkers and therapeutic axis in Glioblastoma multiforme microenvironment." Frontiers in Cell and Developmental Biology 11 (July 19, 2023). http://dx.doi.org/10.3389/fcell.2023.1236271.
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Non-cellular secretory components, including chemokines, cytokines, and growth factors in the tumor microenvironment, are often dysregulated, impacting tumorigenesis in Glioblastoma multiforme (GBM) microenvironment, where the prognostic significance of the current treatment remains unsatisfactory. Recent studies have demonstrated the potential of post-translational modifications (PTM) and their respective enzymes, such as acetylation and ubiquitination in GBM etiology through modulating signaling events. However, the relationship between non-cellular secretory components and post-translational modifications will create a research void in GBM therapeutics. Therefore, we aim to bridge the gap between non-cellular secretory components and PTM modifications through machine learning and computational biology approaches. Herein, we highlighted the importance of BMP1, CTSB, LOX, LOXL1, PLOD1, MMP9, SERPINE1, and SERPING1 in GBM etiology. Further, we demonstrated the positive relationship between the E2 conjugating enzymes (Ube2E1, Ube2H, Ube2J2, Ube2C, Ube2J2, and Ube2S), E3 ligases (VHL and GNB2L1) and substrate (HIF1A). Additionally, we reported the novel HAT1-induced acetylation sites of Ube2S (K211) and Ube2H (K8, K52). Structural and functional characterization of Ube2S (8) and Ube2H (1) have identified their association with protein kinases. Lastly, our results found a putative therapeutic axis HAT1-Ube2S(K211)-GNB2L1-HIF1A and potential predictive biomarkers (CTSB, HAT1, Ube2H, VHL, and GNB2L1) that play a critical role in GBM pathogenesis.