Multisite phosphorylation dictates selective E2-E3 pairing as revealed by Ubc8/UBE2H-GID/CTLH assemblies.

Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryoelectron microscopy (cryo-EM), biochemistry, and cell biology reveal this exquisitely specific E3-E2 pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 multisite phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data show that phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, rigidifies the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.

A heterozygous mutation in UBE2H in a patient with developmental delay leads to an aberrant brain development in zebrafish.

BACKGROUND: Ubiquitin-related rare diseases are generally characterized by developmental delays and mental retardation, but the exact incidence or prevalence is not yet fully understood. The clinical application of next-generation sequencing for pediatric seizures and developmental delay of unknown causes has become common in studies aimed at identification of a causal gene in patients with ubiquitin-related rare diseases that cannot be diagnosed using conventional fluorescence in situ hybridization or chromosome microarray tests. Our study aimed to investigate the effects of ubiquitin-proteasome system on ultra-rare neurodevelopmental diseases, through functional identification of candidate genes and variants. METHODS: In our present work, we carried out genome analysis of a patient with clinical phenotypes of developmental delay and intractable convulsion, to identify causal mutations. Further characterization of the candidate gene was performed using zebrafish, through gene knockdown approaches. Transcriptomic analysis using whole embryos of zebrafish knockdown morphants and additional functional studies identified downstream pathways of the candidate gene affecting neurogenesis. RESULTS: Through trio-based whole-genome sequencing analysis, we identified a de novo missense variant of the ubiquitin system-related gene UBE2H (c.449C>T; p.Thr150Met) in the proband. Using zebrafish, we found that Ube2h is required for normal brain development. Differential gene expression analysis revealed activation of the ATM-p53 signaling pathway in the absence of Ube2h. Moreover, depletion of ube2h led to induction of apoptosis, specifically in the differentiated neural cells. Finally, we found that a missense mutation in zebrafish, ube2h (c.449C>T; p.Thr150Met), which mimics a variant identified in a patient with neurodevelopmental defects, causes aberrant Ube2h function in zebrafish embryos. CONCLUSION: A de novo heterozygous variant in the UBE2H c.449C>T (p.Thr150Met) has been identified in a pediatric patient with global developmental delay and UBE2H is essential for normal neurogenesis in the brain.

Molecular characterization of ubiquitin-conjugating enzyme gene ube2h and siRNA-mediated regulation on targeting p53 in turbot, Scophthalmus maximus.

Ubiquitin-conjugating enzymes are key factors in the ubiquitin proteasome pathway (UPP), which play key roles in ubiquitination. These enzymes affect the efficiency of UPP during stress conditions. P53 has important control of cell cycle arrest and apoptosis in response to cellular stress; these modifications are critical for the stability and transcriptional activity of p53 as the protein activates downstream target genes that dictate the cellular response. However, few studies have investigated the effects of thermal stress in turbot (Scophthalmus maximus), specifically the UPP signaling pathway, and the crosstalk between the ube2h and p53. In this study, the rapid amplification of cDNA ends was used to obtain a full-length cDNA of the turbot UBE2H gene (Sm-ube2h) and perform bioinformatics analysis. Our results showed that the cDNA of the Sm-ube2h was 718 bp in length, encoding a 189 amino acid protein, with a theoretical isoelectric point of 4.77. It also contained a catalytic (UBCc) domain. Expression of Sm-ube2h in different tissues was detected and quantified by qPCR, which was highest in the spleen and lowest in the liver. We also investigated the Sm-ube2h expression profiles in the liver and heart after thermal stress, and changes in Sm-ube2h and p53 under thermal stress, upon RNA interference. Our data speculated that Sm-ube2h and p53 exhibited antagonistic effects under normal temperature conditions after ube2h interference, but displayed synergistic effects under thermal stress, suggesting the crosstalk between UPP and p53 signaling pathway. Our results improved our understanding of the underlying molecular mechanism of thermal tolerance in turbot.

Predictive Potential of Circulating Ube2h mRNA as an E2 Ubiquitin-Conjugating Enzyme for Diagnosis or Treatment of Alzheimer's Disease.

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-ß 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.

Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation.

The development of haematopoietic stem cells into mature erythrocytes - erythropoiesis - is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed maturation stage-dependent assemblies of topologically homologous RANBP9- and RANBP10-CTLH complexes. Moreover, protein abundance of CTLH's cognate E2 ubiquitin conjugating enzyme UBE2H increased during terminal differentiation, and UBE2H expression depended on catalytically active CTLH E3 complexes. CRISPR-Cas9-mediated inactivation of CTLH E3 assemblies or UBE2H in erythroid progenitors revealed defects, including spontaneous and accelerated erythroid maturation as well as inefficient enucleation. Thus, we propose that dynamic maturation stage-specific changes of UBE2H-CTLH E2-E3 modules control the orderly progression of human erythropoiesis.

Ubiquitin Conjugating Enzyme E2 H (UBE2H) Is Linked to Poor Outcomes and Metastasis in Lung Adenocarcinoma.

The prognosis of patients with metastatic lung adenocarcinoma (LUAD) is poor. Although novel lung cancer treatments have been developed for metastatic LUAD, not all patients are fit to receive these treatments. The present study aimed to identify the novel regulatory genes in metastatic LUAD. Because the pleural cavity is a frequent metastasis site of LUAD, the adjacent non-tumor tissue, primary tumor tissue, and metastatic lung tumor tissue in the pleura of a single patient with LUAD were collected. The gene expression profiles of the collected samples were further analyzed via RNA sequencing and bioinformatic analysis. A high expression level of ubiquitin conjugating enzyme E2 H (UBE2H), a hypoxia-mediated gene, was identified in the metastatic malignant pleural tumor. After accessing the survival data in patients with lung adenocarcinoma through online databases, a high UBE2H expression was associated with poor survival for LUAD. UBE2H knockdown in two lung adenocarcinoma cell lines suppressed the cell migration capacity and reversed the epithelial-mesenchymal transition (EMT) signaling pathway. A high expression of UBE2H-targeting microRNAs, including miR-101, miR-30a, miR-30b, miR-328, and miR-497, were associated with a favorable prognosis. Moreover, the UBE2H expression revealed a significant correlation with the copy number variation. Taken together, the presence of UBE2H regulated the EMT program and metastasis in LUAD.