E2 Ubiquitin-conjugating Enzyme Research Articles

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

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

UBC Gene Family Analysis in Salvia castanea and Roles of ScUBC2/5 Genes under Abiotic Stress.

Salvia castanea Diels, a relative of the medicinal plant Salvia miltiorrhiza Bunge, belongs to the genus Salvia and family Lamiaceae. Ubiquitin-conjugating enzyme E2 (UBC) is an important ubiquitin-binding enzyme in protein ubiquitination. This study aimed to analyze the regulatory role of UBC genes, particularly ScUBC2/5, on the growth and adaptation of S. castanea to extreme environments including cold or drought stress. We identified nine UBC genes in S. castanea and found that these genes were extremely stable and more highly expressed in the roots than other tissues. This suggested that UBC genes might play a role in promoting root adaptation to cold and dry environments. Further analysis of UBC gene expression in hairy roots under cold (4 °C) and UV stress also confirmed their importance under stress. The contents of tanshinone and salvianolic acid in hairy roots with the overexpression of ScUBC2/5 were increased compared to non-transgenic wild type, and the cold and UV resistance of hairy roots was increased compared with that of wild type. Together, these findings highlighted the role of ScUBC2/5 in enhancing secondary metabolite accumulation and regulation in response to cold and ultraviolet stress in S. castanea, providing a new perspective for genetic improvement in its phytochemistry.

Knockdown of Ubiquitin-Conjugating Enzyme E2 T Abolishes the Progression of Head and Neck Squamous Cell Carcinoma by Inhibiting NF-Κb Signaling and inducing Ferroptosis.

Ubiquitin-conjugating enzyme 2T (UBE2T) has been reported to be associated with uncontrolled cell growth and tumorigenesis in multiple cancer types. However, the understanding of its regulatory role in the carcinogenesis of Head And Neck Squamous Cell Carcinoma (HNSC) is limited. UBE2T expression in HNSC patient samples and the correlation between its expression and patients' survival rates were evaluated using The Cancer Genome Atlas (TCGA) database. Cell survival and proliferation were investigated in UM-SCC1 and UM-SCC15 cells infected with control and shUBE2T lentivirus. The xenograft mouse model was established using UM-SCC15 cells to examine HNSC tumorigenesis with or without UBE2T. Western blot, qRT-PCR, and ferroptosis assays were carried out to disclose the interaction between UBE2T and NF-κB signaling and ferroptosis. The increased expression of UBE2T was noted in tumor tissues of patients with HNSC, correlating with a significantly reduced overall survival time in this patient cohort. Knockdown of UBE2T inhibited HNSC tumorigenesis and tumor growth. Mechanistically, inhibition of UBE2T suppressed NF-ΚB signaling and induced ferroptosis in HNSC. Our study underscores the multifaceted role of UBE2T in HNSC, illuminating its potential as a biomarker and therapeutic target.

Formation of functional E3 ligase complexes with UBC2 and UEV1 of Leishmania mexicana

In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation.

Abstract 401: The impact of DNA structural variants on PRKN gene function and colorectal cancer pathogenesis

Abstract Background: Cancer is caused by somatic DNA alterations, such as single nucleotide variants, somatic copy number alterations and structural variants (SVs). PRKN is among the genes most frequently affected by SVs in colorectal cancer (CRC), with 37% of primary cancers and 56% of metastatic lesions having focal deletions in PRKN. PRKN encodes the E3 ubiquitin ligase Parkin, which regulates the biological activity and degradation of substrates via post-translational modifications. The genomic location of PRKN is within a common fragile site. Therefore, it remains unclear whether the high frequency of SVs in PRKN are passenger events due to genomic instability or driver events of CRC pathogenesis. Aim: Our study aims to investigate the impact of SVs on the functionality of Parkin in CRC pathogenesis. Methods: We used publicly available DNA deep whole genome sequencing (WGS) and tumor-matched RNA sequencing data (n=394) to estimate the impact of SVs in PRKN on tumor biology. For this, the transcriptome-wide p-value distribution generated by differential expression analysis was used as a measure of biological impact. The HCT116-MLH1-repaired cell line and patient-derived adenoma organoids were genetically modified using CRISPR/Cas9 to mimic the SVs observed in patient material and analyze their impact. RNA-sequencing, whole proteome and ubiquitin remnant-specific proteomics were used to assess the effect of SVs on gene function and identify altered biological processes. Results: Analysis of WGS and RNA sequencing of patient material showed that SVs in PRKN have a large impact on CRC biology, comparable to the impact of mutations in TP53 (impact score 0.379 vs 0.332). The most pronounced effects are seen in the ubiquitin-like, RING0 and RING1 domains. These domains are important for recognition and/or binding to ubiquitin, E2 ubiquitin-conjugating enzymes and substrates. In the HCT116-MLH1-repaired CRC cell line, hemizygous introduction of an SV in PRKN had minimal effects on genome-wide gene expression. In contrast, homozygous deletion altered the expression of nearly 2000 genes. Analyses of ubiquitin motif remnants showed differential ubiquitination of proteins involved in DNA repair following the introduction of an SV in PRKN in the CRC cell line. These proteins include TOP2A (Log2 fold change (L2FC) = -3.69, P = 5.71−5), RAD18 (L2FC = -4.55, P = 1.35−4) and Spartan (SPRTN; L2FC = -2.57, P = 7.43−4). Conclusion: SVs in PRKN have a high impact on tumor biology and result in aberrant gene expression and post-translational modifications of proteins related to DNA repair. Further research will expand to the patient-derived adenoma organoids and focus on understanding if these recurrent somatic alterations contribute to genomic instability and the development of CRC pathogenesis. Citation Format: Elisabeth J. van Bree, Soufyan Lakbir, Carmen Rubio-Alarcón, Renske de Wit, Anne Bolijn, Pien Delis-van Diemen, Marianne Tijssen, Ellen Stelloo, Harma Feitsma, Gerrit A. Meijer, Beatriz Carvalho, Sanne Abeln, Remond J. Fijneman. The impact of DNA structural variants on PRKN gene function and colorectal cancer pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 401.

Oligomerization mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor.

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognize ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerization, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, and assemble into a high molecular weight oligomeric complex, the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerization of DTX3L is important for the DTX3L-PARP9 complex to read mono-ADP-ribosylation on a ligand-regulated transcription factor.

Chronic social defeat stress induces the down-regulation of the Nedd4L-GLT-1 ubiquitination pathway in the prefrontal cortex of mice.

Stressful life events contribute to the onset of major depressive disorder (MDD). We recently demonstrated abnormalities in ubiquitination in the pathophysiology of MDD. However, the underlying molecular mechanisms remain unclear. We investigated the involvement of the ubiquitination system-mediated glutamatergic dysfunction in social impairment induced by chronic social defeat stress (CSDS). Adult C57BL/6J mice were exposed to aggressor ICR male mice for 10 consecutive days. Social impairment was induced by CSDS in the social interaction test 1 days after the last stress exposure. In terms of brain microdialysis, CSDS reduced depolarization-evoked glutamate release in the prefrontal cortex (PFC), which was reversed by a glutamate transporter 1 (GLT-1) inhibitor. Interestingly, the expression of ubiquitinated, but not total GLT-1, was decreased in the PFC of mice exposed to CSDS. The expression of neural precursor cells expressing developmentally downregulated gene 4-like (Nedd4L: E3 ligase for GLT-1), and ubiquitin-conjugating enzyme E2D2 (Ube2d2: E2 ubiquitin-conjugating enzyme for Nedd4L) was also reduced in CSDS mice. Furthermore, the downregulation of the Nedd4L-GLT-1 ubiquitination pathway decreased SIT ratio, but up-regulation increased it even in non-CSDS mice. Taken together, the decrease in GLT-1 ubiquitination may reduce the release of extracellular glutamate induced by high-potassium stimulation, which may lead to social impairment, while we could not find differences in GLT-1 ubiquitination between susceptible and resistant CSDS mice. In conclusion, GLT-1 ubiquitination could play a crucial role in the pathophysiology of MDD and is an attractive target for the development of novel antidepressants.

Loss of UBE2S causes meiosis I arrest with normal spindle assembly checkpoint dynamics in mouse oocytes.

The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.

A transposon insertion in the promoter of OsUBC12 enhances cold tolerance during japonica rice germination

Low-temperature germination (LTG) is an important agronomic trait for rice (Oryza sativa). Japonica rice generally has greater capacity for germination at low temperatures than the indica subpopulation. However, the genetic basis and molecular mechanisms underlying this complex trait are poorly understood. Here, we report that OsUBC12, encoding an E2 ubiquitin-conjugating enzyme, increases low-temperature germinability in japonica, owing to a transposon insertion in its promoter enhancing its expression. Natural variation analysis reveals that transposon insertion in the OsUBC12 promoter mainly occurs in the japonica lineage. The variation detected in eight representative two-line male sterile lines suggests the existence of this allele introgression by indica-japonica hybridization breeding, and varieties carrying the japonica OsUBC12 locus (transposon insertion) have higher low-temperature germinability than varieties without the locus. Further molecular analysis shows that OsUBC12 negatively regulate ABA signaling. OsUBC12-regulated seed germination and ABA signaling mainly depend on a conserved active site required for ubiquitin-conjugating enzyme activity. Furthermore, OsUBC12 directly associates with rice SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 1.1 (OsSnRK1.1), promoting its degradation. OsSnRK1.1 inhibits LTG by enhancing ABA signaling and acts downstream of OsUBC12. These findings shed light on the underlying mechanisms of UBC12 regulating LTG and provide genetic reference points for improving LTG in indica rice.

Recruitment of ubiquitin E2 enzymes is determined jointly by the U-box domains and substrates of E3 ligases.

Ubiquitination is a cascade reaction involving E1, E2, and E3 enzymes. The orthogonal ubiquitin transfer (OUT) method has been previously established to identify potential substrates of E3 ligases. In this study, we verified the ubiquitination of five substrates mediated by the E3 ligases CHIP and E4B. To further explore the activity of U-box domains of E3 ligases, two mutants with the U-box domains interchanged between CHIP and E4B were generated. They exhibited a significantly reduced ubiquitination ability. Additionally, different E3s recruited similar E2 ubiquitin-conjugating enzymes when ubiquitinating the same substrates, highlighting that U-box domains determined the E2 recruitment, while the substrate determined the E2 selectivity. This study reveals the influence of substrates and U-box domains on E2 recruitment, providing a novel perspective on the function of U-box domains of E3 ligases.

Identification and validation of UBE2B as a prognostic biomarker promoting the development of esophageal carcinomas.

Ubiquitination is a crucial biological mechanism in humans, essential for regulating vital biological processes, and has been recognized as a promising focus for cancer therapy. Our objective in this research was to discover potential enzymes associated with ubiquitination that may serve as therapeutic targets for individuals with esophageal carcinoma (ESCA). To identify genes linked to the prognosis of ESCA, we examined mRNA sequencing data from patients with ESCA in the TCGA database. Further investigation into the role of the candidate gene in ESCA was conducted through bioinformatic analyses. Subsequently, we carried out biological assays to assess its impact on ESCA development. Through univariate Cox regression analysis, we identified Ubiquitin Conjugating Enzyme E2 B (UBE2B) as a potential gene associated with the prognosis of ESCA. UBE2B exhibited significant upregulation and was found to be correlated with survival outcomes in ESCA as well as other cancer types. Additionally, UBE2B was observed to be involved in various biological pathways linked to the development of ESCA, including TNF-a signaling via NF-κB, epithelial-mesenchymal transition, inflammatory response, and hypoxia. Moreover, immune-related pathways like B cell activation (GO: 0042113), B cell receptor signaling pathway (GO: 0050853) and B cell mediated immunity (GO:0019724) were also involved. It was found that high expression of UBE2B was correlated with the increase of several kinds of T cells (CD8 T cells, Th1 cells) and macrophages, while effector memory T cell (Tem) and Th17 cells decreased. Furthermore, UBE2B showed potential as a prognostic biomarker for ESCA, displaying high sensitivity and specificity. Notably, proliferation and migration in ESCA cells were effectively suppressed when the expression of UBE2B was knocked down. To summarize, this study has made a discovery regarding the importance of gaining new insights into the role of UBE2B in ESCA. UBE2B might be an oncogene with good ability in predicting and diagnosing ESCA. Consequently, this discovery highlights the feasibility of targeting UBE2B as a viable approach for treating patients with ESCA.

Reconstitution of the in vitro STUB1-mediated ubiquitination reaction system for α-1 antitrypsin mutant Z protein

The α-1 antitrypsin Z-mutant protein (ATZ) is the primary cause of α-1 antitrypsin deficiency (AATD). Studying the ubiquitination modification and degradation of ATZ protein is importance for developing treatments for AATD. STUB1 is an important E3 ubiquitin ligase that regulates ubiquitination modification of various proteins. However, whether STUB1 in involved in the ubiquitination modification of ATZ has not been fully elucidated. In this study, the ATZ and STUB1 coding genes were first cloned into the pET28a plasmid, constructing 2 protein expression plasmids. The recombinant plasmids were then transferred into the Escherichia coli for expression. With the optimization of induction temperature and IPTG dosage, the recombinant proteins were successfully expressed. The target proteins were then efficiently purified from cell lysates using metal-chelating affinity chromatography, and the accuracy of the amino acid sequence was verified through protein mass spectrometry analysis. Using the purified ATZ and STUB1, we established an in vitro ubiquitination reaction system. Experimental results showed that, in the presence of ATP, E1 ubiquitin-activating enzyme, and E2 ubiquitin-conjugating enzyme, STUB1 catalyzed the ubiquitination modification of ATZ. This study provides a method for obtaining the ATZ protein in vitro, elucidates the mechanism of STUB1 mediating ATZ ubiquitination, thereby advancing our understanding of the intracellular degradation mechanism of the α-1 antitrypsin Z-mutant.

A Survey on the Expression of the Ubiquitin Proteasome System Components HECT- and RBR-E3 Ubiquitin Ligases and E2 Ubiquitin-Conjugating and E1 Ubiquitin-Activating Enzymes during Human Brain Development.

Human brain development involves a tightly regulated sequence of events that starts shortly after conception and continues up to adolescence. Before birth, neurogenesis occurs, implying an extensive differentiation process, sustained by changes in the gene expression profile alongside proteome remodeling, regulated by the ubiquitin proteasome system (UPS) and autophagy. The latter processes rely on the selective tagging with ubiquitin of the proteins that must be disposed of. E3 ubiquitin ligases accomplish the selective recognition of the target proteins. At the late stage of neurogenesis, the brain starts to take shape, and neurons migrate to their designated locations. After birth, neuronal myelination occurs, and, in parallel, neurons form connections among each other throughout the synaptogenesis process. Due to the malfunctioning of UPS components, aberrant brain development at the very early stages leads to neurodevelopmental disorders. Through deep data mining and analysis and by taking advantage of machine learning-based models, we mapped the transcriptomic profile of the genes encoding HECT- and ring-between-ring (RBR)-E3 ubiquitin ligases as well as E2 ubiquitin-conjugating and E1 ubiquitin-activating enzymes during human brain development, from early post-conception to adulthood. The inquiry outcomes unveiled some implications for neurodevelopment-related disorders.

Inhibition of miR-143-3p Restores Blood-Testis Barrier Function and Ameliorates Sertoli Cell Senescence.

Due to the increasing trend of delayed childbirth, the age-related decline in male reproductive function has become a widely recognized issue. Sertoli cells (SCs) play a vital role in creating the necessary microenvironment for spermatogenesis in the testis. However, the mechanism underlying Sertoli cell aging is still unclear. In this study, senescent Sertoli cells showed a substantial upregulation of miR-143-3p expression. miR-143-3p was found to limit Sertoli cell proliferation, promote cellular senescence, and cause blood-testis barrier (BTB) dysfunction by targeting ubiquitin-conjugating enzyme E2 E3 (UBE2E3). Additionally, the TGF-β receptor inhibitor SB431542 showed potential in alleviating age-related BTB dysfunction, rescuing testicular atrophy, and reversing the reduction in germ cell numbers by negatively regulating miR-143-3p. These findings clarified the regulatory pathways underlying Sertoli cell senescence and suggested a promising therapeutic approach to restore BTB function, alleviate Sertoli cell senescence, and improve reproductive outcomes for individuals facing fertility challenges.

Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases

E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL–E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s.

Transcriptome analysis of Chinese mitten crabs (Eriocheir sinensis) gills in response to ammonia stress.

The Chinese mitten crab (Eriocheir sinensis) is an important commercial species in China. E. sinensis is typically farmed in rice-crab symbiosis, as an important ecological farming model. However, E. sinensis is often exposed to a high ammonia environment due to the application of nitrogen fertilizers essential for rice growth. We investigated the molecular mechanisms in the gills of E. sinensis exposed to high ammonia at transcriptional and histological levels. We randomly assigned E. sinensis to two groups (control group, CG; ammonia stress group, AG), and gill samples were excised from the CG and AG groups for histopathological and transcriptome analyses. The histopathological evaluation revealed that ammonia stress damaged the gills of E. sinensis. The transcriptome analysis showed that some essential genes, including Xanthine dehydrogenase (XDH), Ubiquitin C-terminal hydrolase-L3 (UCHL3), O-linked N-acetylglucosamine transferase (OGT), Cathepsin B (CTSB), and Ubiquitin-conjugating enzyme E2 W (UBE2W) changed significantly during ammonia exposure. These genes are related to ammonia detoxification, the immune response, and apoptosis. This study demonstrated the molecular response mechanism of E. sinensis gills to ammonia stress at the transcriptional and histological levels. This study provides insight for further study on the molecular mechanism of ammonia stress in crustaceans and supplies technical support for rice crab symbiosis.

The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination

Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases.

IRE1 endoribonuclease signaling promotes myeloid cell infiltration in glioblastoma.

Intrinsic or environmental stresses trigger the accumulation of improperly folded proteins in the endoplasmic reticulum (ER), leading to ER stress. To cope with this, cells have evolved an adaptive mechanism named the unfolded protein response (UPR) which is hijacked by tumor cells to develop malignant features. Glioblastoma (GB), the most aggressive and lethal primary brain tumor, relies on UPR to sustain growth. We recently showed that IRE1 alpha (referred to IRE1 hereafter), 1 of the UPR transducers, promotes GB invasion, angiogenesis, and infiltration by macrophage. Hence, high tumor IRE1 activity in tumor cells predicts a worse outcome. Herein, we characterized the IRE1-dependent signaling that shapes the immune microenvironment toward monocytes/macrophages and neutrophils. We used human and mouse cellular models in which IRE1 was genetically or pharmacologically invalidated and which were tested in vivo. Publicly available datasets from GB patients were also analyzed to confirm our findings. We showed that IRE1 signaling, through both the transcription factor XBP1s and the regulated IRE1-dependent decay controls the expression of the ubiquitin-conjugating E2 enzyme UBE2D3. In turn, UBE2D3 activates the NFκB pathway, resulting in chemokine production and myeloid infiltration in tumors. Our work identifies a novel IRE1/UBE2D3 proinflammatory axis that plays an instrumental role in GB immune regulation.

Comparative proteomics reveal the humoral immune rejection of pearl oyster Pinctada fucata to xenograft from Pinctada maxima

To investigate the mechanism underlying immune rejection in pearl oyster Pinctada fucata following allograft (P. fucata mantle pieces) and xenograft (P. maxima mantle pieces) transplantations, we analyzed serum samples collected at multiple time points (6, 12, 24, 48, 96, 144, and 192 h) after transplantation using a proteomics approach. We identified 2127 proteins with high confidence according to the selected peptides (false discovery rate = 0.01), among which 1148 exhibited quantitative differences. Functional enrichment analysis revealed significant changes in protein functional categories, including signal transduction, translation, cytoskeleton, and post-translational modifications. Immune-related proteins, such as the ubiquitin-conjugating enzyme E2, apoptosis-linked gene, heat shock protein, and apoptosis inhibitor 5 were associated with allografts and xenografts. Notably, the PI3K-Akt signaling, focal adhesion, and extracellular matrix (ECM)-receptor interaction pathways were enriched among upregulated proteins (xenograft vs. allograft), while the ribosome pathway was enriched among downregulated proteins. These findings suggest that implantation stress may affect protein biosynthesis or activate multiple molecular pathways involved in key immune proteins. Furthermore, the xenograft group demonstrated greater sensitivity in recognizing and responding to foreign substances compared to the allograft group. This heightened responsiveness enabled the xenograft group to rapidly activate humoral immune factors, which triggered the signaling pathway responsible for suppressing the inflammatory response. This study provides novel insights into the immune reactions of host oysters to grafted mantle pieces, particularly xenografts, and may offer strategies to improve xenograft transplantation technologies in the pearl culture industry.