Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

Over expression of ubiquitin-conjugating enzyme E2O in bone marrow mesenchymal stromal cells partially attenuates acute myeloid leukaemia progression.

Bone marrow mesenchymal stromal cells (BM-MSCs) are implicated in the pathogenesis of acute myeloid leukaemia (AML). However, due to the high heterogeneity of AML the mechanism underlying the cross-talk between MSCs and leukaemia cells is not well understood. We found that mixed-lineage leukaemia-AF9 (MLL-AF9)-induced AML mice-derived MSCs had higher proliferative viability compared to wild-type mice-derived MSCs with ubiquitin-conjugating enzyme E2O (Ube2o) down-regulation. After overexpression of UBE2O in AML-derived MSCs, the growth capacity of MSCs was reduced with nuclear factor kappa B subunit 1 (NF-κB) pathway deactivation. In vitro co-culture assay revealed that UBE2O-overexpression MSCs suppressed the proliferation and promoted apoptosis of AML cells by direct contact. In vivo results revealed that the leukaemia burden was reduced and the overall survival of AML mice was prolonged, with decreased dissemination of leukaemia cells in BM, spleen, liver and peripheral blood. Additionally, subcutaneous tumorigenesis revealed that tumour growth was also suppressed in the UBE2O-overexpression MSCs group. In conclusion, UBE2O was expressed at a low level in MLL-AF9-induced AML mice-derived MSCs. Overexpression of UBE2O in MSCs suppressed their proliferation through NF-κB pathway deactivation, which resulted in AML suppression. Our study provides a theoretical basis for a BM microenvironment-based therapeutic strategy to control disease progression.

UBE2O and USP7 co-regulate RECQL4 ubiquitinylation and homologous recombination-mediated DNA repair.

The human RecQ DNA helicase, RECQL4, plays a pivotal role in maintaining genomic stability by regulating the DNA double-strand breaks (DSBs) repair pathway, and is, thus, involved in the regulation of aging and cancer onset. However, the regulatory mechanisms of RECQL4, especially its post-translational modifications, have not been fully illustrated. Here, we report that the E2/E3 hybrid ubiquitin-conjugating enzyme, UBE2O, physically interacts with RECQL4, and mediates the multi-monoubiquitinylation of RECQL4, subsequently leading to its proteasomal degradation. Functionally, we showed that UBE2O inhibits homologous recombination (HR)-mediated DSBs repair, and this inhibition depends on its E2 catalytic activity and RECQL4 expression. Mechanistically, we showed that UBE2O attenuates the interaction of RECQL4 and DNA damage repair proteins, the MRE11-RAD50-NBS1 complex and CtIP. Furthermore, we show that deubiquitinylase USP7 interacts with both UBE2O and RECQL4, and in that it antagonizes UBE2O-mediated regulation of RECQL4 stability and function. Collectively, we found a novel regulatory mechanism of ubiquitin-mediated regulation of RECQL4 in HR-mediated DSBs repair process.

UBE2O ubiquitinates PTRF/CAVIN1 and inhibits the secretion of exosome-related PTRF/CAVIN1.

BACKGROUND: Exosomes are small vesicles released by cells, which have crucial functions in intercellular communication. Exosomes originated from cell membrane invagination and are released followed by multivesicular bodies (MVBs) fused with the cell membrane. It is known that Polymerase I and Transcript Release Factor (PTRF, also known as Caveolin-associated Protein-1, CAVIN1) plays an important role in caveolae formation and exosome secretion. And PTRF in exosomes has been identified as a potential biomarker in multiple malignancies such as glioma and renal cell carcinoma. However, the mechanisms of how to regulate the secretion of exosome-related PTRF remain unknown. METHODS: We performed exogenous and endogenous immunoprecipitation assays to investigate the interaction between ubiquitin-conjugating enzyme E2O (UBE2O) and PTRF. We identified UBE2O ubiquitinated PTRF using ubiquitination assays. Then, exosomes were isolated by ultracentrifugation and identified by transmission electronic microscopy, western blot and nanoparticle tracking analysis. The effect of UBE2O on the secretion of exosome-related PTRF was analyzed by western blot, and the effect of UBE2O on exosome secretion was evaluated by exosome markers and the total protein content of exosomes. RESULTS: Here, we showed that UBE2O interacts with PTRF directly and ubiquitinates PTRF. Functionally, we found that UBE2O inhibited the effects of PTRF on exosome secretion via decreasing caveolae formation. Importantly, UBE2O decreased exosome secretion, resulting in downregulating PTRF secretion via exosomes. Our study also identified Serum Deprivation Protein Response (SDPR, also known as Caveolin-associated Protein-2, CAVIN2) interacted with both UBE2O and PTRF. Furthermore, we found that SDPR promotes PTRF expression in exosomes. Interestingly, even in the presence of SDPR, UBE2O still inhibited the secretion of exosome-related PTRF. CONCLUSIONS: Our study demonstrated that UBE2O downregulated exosome release and controlled the secretion of exosome-related PTRF through ubiquitinating PTRF. Since exosomes play an important role in malignant tumor growth and PTRF included in exosomes is a biomarker for several malignant tumors, increasing UBE2O expression in cells has the potential to be developed as a novel approach for cancer treatment. Video Abstract.

UBE2O promotes hepatocellular carcinoma cell proliferation and invasion by regulating the AMPKα2/mTOR pathway.

The ubiquitin-conjugating enzyme (E2) is a critical component of the ubiquitin-proteasome system and regulates hepatocarcinogenesis by controlling protein degradation. Ubiquitin-conjugating enzyme E2 O (UBE2O), a member of the E2 family, functions as an oncogene in human cancers. Nevertheless, the role of UBE2O in hepatocellular carcinoma (HCC) remains unknown yet. Here, we demonstrated that the UBE2O level was markedly upregulated in HCC compared with adjacent noncancerous tissues. UBE2O overexpression was also confirmed in HCC cell lines. UBE2O overexpression was prominently associated with advanced tumor stage, high tumor grade, venous infiltration, and reduced HCC patients' survivals. UBE2O knockdown inhibited the migration, invasion, and proliferation of HCCLM3 cells. UBE2O overexpression enhanced the proliferation and mobility of Huh7 cells. Mechanistically, UBE2O mediated the ubiquitination and degradation of AMP-activated protein kinase α2 (AMPKα2) in HCC cells. UBE2O silencing prominently increased AMPKα2 level and reduced phosphorylated mechanistic target of rapamycin kinase (p-mTOR), MYC, Cyclin D1, HIF1α, and SREBP1 levels in HCCLM3 cells. UBE2O depletion markedly activated the AMPKα2/mTOR pathway in Huh7 cells. Moreover, AMPKα2 silencing reversed UBE2O downregulation-induced mTOR pathway inactivation. Rapamycin, an inhibitor of mTOR, remarkably abolished UBE2O-induced mTOR phosphorylation and HCC cell proliferation and mobility. To conclude, UBE2O was highly expressed in HCC and its overexpression conferred to the poor clinical outcomes of patients. UBE2O contributed to the malignant behaviors of HCC cells, including cell proliferation, migration, and invasion, by reducing AMPKα2 stability and activating the mTOR pathway.

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

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

Ubiquitin-conjugating enzyme UBE2O regulates cellular clock function by promoting the degradation of the transcription factor BMAL1.

Dysregulation of the circadian rhythm is associated with many diseases, including diabetes, obesity, and cancer. Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Arntl or Bmal1) is the only clock gene whose loss disrupts circadian locomotor behavior in constant darkness. BMAL1 levels are affected by proteasomal inhibition and by several enzymes in the ubiquitin-proteasome system, but the exact molecular mechanism remains unclear. Here, using immunoprecipitation and MS analyses, we discovered an interaction between BMAL1 and ubiquitin-conjugating enzyme E2 O (UBE2O), an E3-independent E2 ubiquitin-conjugating enzyme (i.e. hybrid E2/E3 enzyme). Biochemical experiments with cell lines and animal tissues validated this specific interaction and uncovered that UBE2O expression reduces BMAL1 levels by promoting its ubiquitination and degradation. Moreover, UBE2O expression/knockdown diminished/increased, respectively, BMAL1-mediated transcriptional activity but did not affect BMAL1 gene expression. Bioluminescence experiments disclosed that UBE2O knockdown elevates the amplitude of the circadian clock in human osteosarcoma U2OS cells. Furthermore, mapping of the BMAL1-interacting domain in UBE2O and analyses of BMAL1 stability and ubiquitination revealed that the conserved region 2 (CR2) in UBE2O significantly enhances BMAL1 ubiquitination and decreases BMAL1 protein levels. A Cys-to-Ser substitution experiment identified the critical Cys residue in the CR2 domain responsible for BMAL1 ubiquitination. This work identifies UBE2O as a critical regulator in the ubiquitin-proteasome system, which modulates BMAL1 transcriptional activity and circadian function by promoting BMAL1 ubiquitination and degradation under normal physiological conditions.

The Ubiquitin-Conjugating Enzyme E2 O (UBE2O) and Its Therapeutic Potential in Human Leukemias and Solid Tumors.

Protein degradation is a biological phenomenon essential for cellular homeostasis and survival. Selective protein degradation is performed by the ubiquitination system which selectively targets proteins that need to be eliminated and leads them to proteasome degradation. In this narrative review, we focus on the ubiquitin-conjugating enzyme E2 O (UBE2O) and highlight the role of UBE2O in many biological and physiological processes. We further discuss UBE2O's implications in various human diseases, particularly in leukemias and solid cancers. Ultimately, our review aims to highlight the potential role of UBE2O as a therapeutic target and offers new perspectives for developing targeted treatments for human cancers.

Age-Associated UBE2O Reduction Promotes Neuronal Death in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease leading to dementia in the elderly. Ubiquitin proteasome system (UPS) is critical for protein homeostasis, while the functional decline of UPS with age contributes to the pathogenesis of AD. Ubiquitin-conjugating enzyme E2O (UBE2O), an E2-E3 hybrid enzyme, is a major component of UPS. However, its role in AD pathogenesis has not been fully defined. OBJECTIVE: We aimed to identify the age-associated expression of UBE2O and its role AD pathogenesis. METHODS: Western blot analysis were used to assess expression of UBE2O in organs/tissues and cell lines. Immunofluorescence staining was performed to examine the cellular distribution of UBE2O. Neuronal death was determined by the activity of lactate dehydrogenase. RESULTS: UBE2O is highly expressed in the cortex and hippocampus. It is predominantly expressed in neurons but not in glial cells. The peak expression of UBE2O is at postnatal day 17 and 14 in the cortex and hippocampus, respectively. Moreover its expression is gradually reduced with age. Importantly, UBE2O is significantly reduced in both cortex and hippocampus of AD mice. Consistently, overexpression of amyloid-ß protein precursor (AßPP) with a pathogenic mutation (AßPPswe) for AD reduces the expression of UBE2O and promotes neuronal death, while increased expression of UBE2O rescues AßPPswe-induced neuronal death. CONCLUSION: Our study indicates that age-associated reduction of UBE2O may facilitates neuronal death in AD, while increasing UBE2O expression or activity may be a potential approach for AD treatment by inhibiting neuronal death.

Effect of genetic variants in UBE2O and TPK1 on facial morphology of Koreans.

Human face is a highly heritable and complex trait. Many genome-wide analyses have identified genetic variants influencing facial morphology. Genome-wide association studies (GWASs) investigating facial morphologies of different populations provide a comprehensive insight into the genetic basis of the human face. Here, we report a GWAS of normal facial variation in Koreans using an array optimized for the Korean population (KoreanChip). We found that novel genetic variants encompassing four loci reached the genome-wide significance threshold. They include LOC107984547, UBE2O, TPK1, and LINC01148 loci associated with facial angle, brow ridge protrusion, nasal height, and eyelid curvature. Our results also validated previously published genetic loci, including FAT4, SOX9, and TBX3 loci. All confirmed genetic variants showed phenotypic differences involving each facial trait based on the effect of the minor allele. The present study highlights genetic signals associated with normal human facial variation and provides candidates for functional studies. Key points: GWAS of normal facial variation in the Korean population was conducted using a Korean genome chip.Previously reported genetic signals associated with FAT4, SOX9, and TBX3 loci were replicated in the Korean populations.Genetic signals in UBE2O and TPK1 loci were identified as novel variants for corresponding facial features.

Renal Klotho safeguards platelet lifespan in advanced chronic kidney disease through restraining Bcl-xL ubiquitination and degradation.

BACKGROUND: Thrombosis and hemorrhage as two opposite pathologies are prevalent within the chronic kidney disease (CKD) population. Platelet homeostasis, which positions centrally in their pathogenesis, varies among the CKD population, while the underlying mechanism is poorly understood. OBJECTIVE: To investigate the change character and mechanism of platelet homeostasis in CKD and its association with renal Klotho deficiency. METHODS: The change character of platelet homeostasis and its association with renal Klotho deficiency were determined based on a cohort study as well as CKD mice and Klotho-deficient mice with CKD. The effects on thrombopoiesis and platelet lifespan were examined by flow cytometry and platelet transfer. The underlying mechanism was explored by proteomics, flow cytometry, western blot, and immunoprecipitation. RESULTS: We show that platelet count declines both in patient and mouse models with advanced CKD (Adv-CKD) and is positively associated with circulating Klotho levels. Mechanistically, we identify that ubiquitin ligase UBE2O governs Bcl-xL ubiquitination and degradation in platelets, whereas Adv-CKD-induced oxidative stress in platelets stimulates p38MAPK to promote Bcl-xL phosphorylation, which facilitates UBE2O binding to Bcl-xL and subsequent Bcl-xL degradation. Consequently, platelet lifespan is shortened in Adv-CKD, culminating in platelet count decline. However, kidney-secreted soluble Klotho protein restricts oxidative stress in platelets, thereby preserving Bcl-xL expression and platelet lifespan. CONCLUSIONS: Our findings uncover the mechanism of platelet count decline in Adv-CKD and identify renal Klotho as a long-range regulator of platelet lifespan, which not only provide a molecular mechanism underlying CKD-associated thrombocytopenia and hemorrhage but also offer a promising therapy choice.

Identification of novel non-HFE mutations in Chinese patients with hereditary hemochromatosis.

BACKGROUNDS: Hereditary hemochromatosis (HH) is mainly caused by homozygous p.C282Y mutations in HFE in the Caucasians. We recently reported non-HFE mutations constitute the major cause of HH in Chinese. However, there is still a relatively high proportion of cases with primary iron overload from unexplained causes. We aimed to explore novel non-HFE mutations in Chinese patients with primary iron overload. METHODS: Whole exome sequence was conducted to screen mutations in novel HH-related genes in the 9 cases with unexplained primary iron overload. Then the representative candidate genes were screened for mutations in another cohort of 18 HH cases. The biological function of the selected genes and variants were analyzed in vitro. RESULTS: Whole exome sequencing of 9 cases with unexplained primary iron overload identified 42 missense variants in 40 genes associated with iron metabolism pathway genes such as UBE2O p.K689R and PCSK7 p.R711W. Subsequent Sanger sequencing of the UBE2O and PCSK7 genes in the 27 cases with primary iron overload identified p.K689R in UBE2O, p.R711W and p.V143F in PCSK7 at frequency of 2/27,1/27 and 2/27 respectively. In vitro siRNA interference of UBE2O and PCSK7 resulted in down-regulated HAMP mRNA expression. Adenovirus generation of UBE2O p.K689R in cell lines resulted in increased expression of SMAD6 and SMAD7 and downregulation of p-SMAD1/5 and HAMP expression, and the reduction of hepcidin level. CONCLUSIONS: Our study identified a series of novel candidate non-HFE mutations in Chinese patients with HH. These may provide insights into the genetic basis of unexplained primary iron overload.

Regulatory roles of an atypical ubiquitin ligase UBE2O in orphans of multiprotein complexes for degradation.

UBE2O as an atypical ubiquitin-conjugating enzyme possesses an E2-E3 hybrid enzyme activity. It can regulate substrate levels or transcriptional activities by cooperating with other E3 ubiquitin ligases or forming homomeric complexes displaying intrinsic E2 and E3 activities. UBE2O controls the quality of cell proteome including protein degradation, modification, transport and location. Recent studies reveal that UBE2O plays a vital role in intracellular protein ubiquitination processes by regulating BMP/SMAD, TRAF/NF-κB, mTOR/HIF1a and IL-1ß/IRAK4 signaling pathways, c-Maf stability and BAP1 subcellular location, which is proposed as a quality control supervisor of multiprotein complexes for degradation. Its abnormality leads to a variety of physical activity disorders and even occurrence of cancer. UBE2O is entirely distinct in molecular structure and functions from other E2 ubiquitin ligase. Exploring and elucidating regulatory mechanism of UBE2O may identify novel crucial molecular targets so as to pave therapeutic approaches for ubiquitination-associated metabolic disorders and diseases. Here, we particularly feature regulatory pathways of UBE2O in orphans of multiprotein complexes for degradation and its potential application.

Arborinine from Glycosmis parva leaf extract inhibits clear-cell renal cell carcinoma by inhibiting KDM1A/UBE2O signaling.

Background: Arborinine is a natural product isolated from Globigerina parva (G. parva) leaf extract that shows strong anticancer activity with its role in clear-cell renal cell carcinoma (ccRCC) unreported. Objective: We aim to evaluate the role of Arborinine in ccRCC. Design: Arborinine was tested for its effects in ccRCC cell lines in vitro and in silico. Results: Arborinine conferred inhibitory effect to ccRCC cells at reasonable doses. Arborinine showed inhibitory effects on Lysine Demethylase 1A (KDM1A) in ccRCC cells and decreased levels of KDM1A outputs and on epithelial mesenchymal transition (EMT) markers. Arborinine significantly inhibited proliferation, apoptosis, and cell cycle progression and migration of ccRCC cells. Using in silico ChIP analysis and luciferase activity validation, we identified Ubiquitin-conjugating enzyme E2O (UBE2O) as an active transcription target downstream of KDM1A. UBE2O expression was not only correlated with KDM1A expression but also associated with worsened prognosis in ccRCC. Overexpression of UBE2O abrogated cancer-inhibitory effect of Arborinine. Discussion: Arborinine holds promise as an additive in the treatment of ccRCC. Conclusions: We have shown for the first time that Arborinine showed inhibitory effect on ccRCC via KDM1A/UBE2O signaling.

E2-E3 ubiquitin enzyme pairing - partnership in provoking or mitigating cancers.

The ubiquitin-proteasome system (UPS) modulates carcinogenesis through ubiquitination of cancer-related target proteins, leading to their degradation in the proteasome. This may deactivate tumor suppressors or activate tumor promoters- either way causing homeostatic imbalance. As major components of the UPS, the E2 and E3 enzymes are recognized as pivotal determinants of substrate recognition and ubiquitination. Identification of E2-E3 pairing selectivity is particularly pertinent to early diagnosis and potential development of targeted cancer therapeutics. This review is motivated by recent findings and new insights into the molecular dynamics of ubiquitination triggered by specific E2-E3 pairing, leading to cancer initiation and progression if cancer suppressors are degraded or cancer suppression (if cancer promoters are degraded), respectively. We provide an overview of strategies employed in screening for E2-E3 interactions based on up-to-date studies focusing on the E2-E3 interface motifs. Of considerable recent interest is how E2 and E3 might switch their functional partnerships via UBE2O, which suggests an emerging significance on how UBE2O might influence E2-E3 pairing. Thus, a reflection on the role of UBE2O is included. Finally, we deliberate on the rational and cautious development of anti-cancer cocktail drugs which specifically target E2-E3 interacting residues for precision in cancer-killing with minimal side-effects. To this end, a list of potential future research is proposed.

The Positive Correlations between the Expression of Histopathological Ubiquitin-Conjugating Enzyme 2O Staining and Prostate Cancer Advancement.

BACKGROUND: The mTOR signaling pathway is inactivated by AMPK's tumor-suppressing function. It is recognized that ubiquitin conjugating enzyme 2O (UBE2O), which directly targets AMPK for ubiquitination and degradation, is intensified in human cancers. METHODS: This study investigated the clinical data about prostate cancer. Examination was also carried out into tissue microarrays (TMA) of human prostate cancer (n = 382) and adjacent non-neoplastic tissues around prostate cancer (n = 61). The TMA slides were incubated with antibodies against UBE2O, and the cores were scored by the pathologist blind to cancer results. RESULTS: Very strong positive correlations were identified between the expression of UBE2O staining and high PSA and pathological stage of prostate cancer. Cox's proportional hazard analysis established correlations between the following: (1) positive surgical margin and biochemical recurrence free survival, (2) PSA grade and clinical recurrence free survival, (3) regional lymph node positive and clinical recurrence free survival, (4) adjuvant treatment and overall survival, and (5) pathological T stage and overall survival. CONCLUSION: There is a positive correlation between the expression of UBE2O staining and prognosis for prostate cancer. Thus, a prostate cancer prognosis can be assessed with the expression of UBE2O staining.

Long Noncoding RNA HCG18 Promotes Malignant Phenotypes of Breast Cancer Cells via the HCG18/miR-103a-3p/UBE2O/mTORC1/HIF-1α-Positive Feedback Loop.

In recent years, an increasing number of studies have reported that long noncoding RNAs (lncRNAs) play crucial roles in breast cancer (BC) progression and metastasis. Another study group of our research center reported that lncRNA HCG18 was one of the 30 upregulated lncRNAs in BC tissues compared with normal tissues in The Cancer Genome Atlas database. However, the exact biological roles of HCG18 in BC remain unclear. In this study, we demonstrated that HCG18 is significantly upregulated in BC tissues and cells and that BC patients with high HCG18 expression tend to have poor prognosis. In vitro assays indicated that HCG18 promotes BC cell proliferation and invasion and endows BC cells with cancer stemness properties. In vivo assays revealed that reducing HCG18 expression in the BC cell line MDA-MB-231 markedly decreased tumor growth and lung metastasis in xenograft mouse models. In terms of mechanism, we found that HCG18 positively regulated the expression of BC-related ubiquitin-conjugating enzyme E2O (UBE2O) by sponging miR-103a-3p, and our previous research verified that UBE2O could promote the malignant phenotypes of BC cells through the UBE2O/AMPKα2/mTORC1 axis. Furthermore, as a downstream target of the HCG18/miR-103a-3p/UBE2O/mTORC1 axis, hypoxia-inducible factor 1α transcriptionally promoted HCG18 expression and then formed a positive feedback loop in BC. Taken together, these results confirm that HCG18 plays an oncogenic role in BC and might serve as a prognostic biomarker and a potential therapeutic target for BC treatment.

Diverse roles of the E2/E3 hybrid enzyme UBE2O in the regulation of protein ubiquitination, cellular functions, and disease onset.

The ubiquitin-proteasome system is an important regulatory machinery involved in proteostasis and cellular signaling. Proteins are ubiquitinated via the concerted action of E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. Although most of the studies to date focus on the significance of E3 ubiquitin ligases in disease development and therapeutic treatment, recent discoveries suggest that E2 ubiquitin-conjugating enzymes might also be potential drug targets. The ubiquitin-conjugating enzyme E2 O (UBE2O), an E3-independent E2 (i.e. an E2/E3 hybrid enzyme), can directly mediate the ubiquitination of many substrates. These include 5'-AMP-activated protein kinase catalytic subunit α2 (AMPKα2), tumor suppressor ubiquitin carboxyl-terminal hydrolase BAP1, mixed-lineage leukemia (MLL) protein, SMAD family member 6 (SMAD6), transcription factor c-Maf and aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL or BMAL1), and free ribosomal proteins, which are ubiquitinated in distinct ways, thereby associating UBE2O with a variety of biological functions. Furthermore, UBE2O is frequently amplified or mutated in multiple cancers, and its high expression is associated with low survival rate of gastric, lung, breast, and prostate cancer patients. However, the molecular mechanisms by which UBE2O contributes to tumor initiation and progression are not fully elucidated. This review focuses on emerging insights from genetics, biochemistry, and cell biology to explore the biological functions of UBE2O and its therapeutic potential.

New Insights into the Role of E2s in the Pathogenesis of Diseases: Lessons Learned from UBE2O.

Intracellular communication via ubiquitin (Ub) signaling impacts all aspects of cell biology and regulates pathways critical to human development and viability; therefore aberrations or defects in Ub signaling can contribute to the pathogenesis of human diseases. Ubiquitination consists of the addition of Ub to a substrate protein via coordinated action of E1-activating, E2-conjugating and E3-ligating enzymes. Approximately 40 E2s have been identified in humans, and most are thought to be involved in Ub transfer; although little information is available regarding the majority of them, emerging evidence has highlighted their importance to human health and disease. In this review, we focus on recent insights into the pathogenetic roles of E2s (particularly the ubiquitin-conjugating enzyme E2O [UBE2O]) in debilitating diseases and cancer, and discuss the tantalizing prospect that E2s may someday serve as potential therapeutic targets for human diseases.

The ubiquitin-conjugating enzyme UBE2O modulates c-Maf stability and induces myeloma cell apoptosis.

BACKGROUND: UBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown. METHODS: Mass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively. RESULTS: UBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway. CONCLUSIONS: UBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.