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• BMSC-exosomes inhibit ferroptosis and alleviate spinal cord injury in rats. • BMSC-exosomes inhibit ferroptosis of neuronal cells PC12. • BMSC-exosomes inhibits ferroptosis in PC12 cells by regulating NRF2. • BMSC- exosomes regulates NRF2 protein stability through miR-219-5p. • UBE2Z plays a role in the regulation of NRF2 by miR-219-5p. The aim of this study was to investigate the molecular mechanism of exosomal miR-219-5p derived from bone marrow mesenchymal stem cells (BMSCs) in the treatment of spinal cord injury (SCI). Basso Beattie Bresnahan (BBB) score and tissue staining were used to assess SCI and neuronal survival in rats. The contents of Fe2+, malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were detected by a kit. The expression levels of ubiquitin-conjugating enzyme E2 Z (UBE2Z), nuclear factor erythroid 2-related Factor 2 (NRF2) and ferroptosis-related proteins were detected by Western blotting. In addition, the ability of BMSC-derived exosomes to inhibit ferroptosis in neuronal cells in rats with SCI was validated by in vivo injection of ferroptosis inhibitors/inducers. In this study, we found that miR-219-5p-rich BMSC-derived exosomes inhibited ferroptosis in SCI rats and that the alleviating effect of BMSC-Exos on SCI was achieved by inhibiting the ferroptosis signaling pathway and that NRF2 played a key role in this process. Our study confirmed that BMSC exosome-specific delivery of miR-219-5p can target UBE2Z to regulate its stability and that overexpression of UBE2Z reverses miR-219-5p regulation of NRF2. In addition, in vivo experiments showed that BMSC exosomes alleviated ferroptosis in neuronal SCI progression, and inhibiting the expression of miR-219-5p in BMSCs reduced the alleviating effect of exosomes on ferroptosis in neuronal cells and SCI. miR-219-5p in BMSC-derived exosomes can repair the injured spinal cord. In addition, miR-219-5p alleviates ferroptosis in neuronal cells induced by SCI through the UBE2Z/NRF2 pathway. [ABSTRACT FROM AUTHOR]

The ubiquitination is crucial for controlling cellular homeostasis and protein modification, in which ubiquitin-conjugating enzyme (E2) acts as the central player in the ubiquitination system. Ubiquitin-conjugating enzymes, which have special domains that catalyse substrates, have sequence discrepancies and modulate various pathophysiological processes in different cells of multiple organisms. E2s take part in the mitosis of primordial germ cells, meiosis of spermatocytes and the formation of mature haploid spermatids to maintain normal male fertility. In this review, we summarize the various types of E2s and their functions during distinct stages of spermatogenesis. [ABSTRACT FROM AUTHOR]

Legionella pneumophila, the bacteria responsible for Legionnaires' disease, is able to evade the host's defense mechanisms by modifying the ubiquitin landscape on the Legionella-containing vacuole (LCV). Two recent studies published in Nature Communications have revealed the cooperative action between two Legionella effectors, Sdc and Sde, in decorating the LCV with mixed ubiquitin chains. These modifications prevent the recognition of the LCV by autophagy receptors, allowing Legionella to establish a replicative niche within host cells. The findings highlight the sophisticated strategies employed by Legionella to manipulate the host ubiquitin system and evade autophagic clearance. Further research is needed to understand the temporal and spatial regulation of these effectors and their interplay with other bacterial effectors. The discovery of this mechanism opens up new avenues for studying host-pathogen interactions and developing therapeutic strategies against infections. [Extracted from the article]

Protein homeostasis depends on many fundamental processes including mRNA synthesis, translation, post-translational modifications, and proteolysis. In the late 70s and early 80s the discovery that the small 76 amino acid protein ubiquitin could be attached to target proteins via amulti-stage process involving ubiquitin-activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases, revealed an exciting new post-translational mechanism to regulate protein degradation. This cellular system was uncovered using biochemical methods by Avram Hershko, who would later won the Nobel prize for this discovery; however, the biological functions of ubiquitin ligases remained unknown for many years. It was initially described that ubiquitin modifies proteins at one or more lysine residues and once a long ubiquitin chain was assembled, proteins were degraded by the proteasome. Now we know that proteins can be mono-, multimono-, homotypic poly-, or heterotypic poly-ubiquitylated, each of which confers a specific signal that goes beyond protein degradation regulating additional key cellular functions such as signal transduction, protein localization, recognition of damaged proteins, etc. [ABSTRACT FROM AUTHOR]

Ubiquitination is an evolutionary, ancient system of post-translational modification of proteins that occurs through a cascade involving ubiquitin activation, transfer, and conjugation. The maturation of this system has followed two main pathways. The first is the conservation of a universal structural fold of ubiquitin and ubiquitin-like proteins, which are present in both Archaea and Bacteria, as well as in multicellular Eukaryotes. The second is the rise of the complexity of the superfamily of ligases, which conjugate ubiquitin-like proteins to substrates, in terms of an increase in the number of enzyme variants, greater variation in structural organization, and the diversification of their catalytic domains. Here, we examine the diversity of the ubiquitination system among different organisms, assessing the variety and conservation of the key domains of the ubiquitination enzymes and ubiquitin itself. Our data show that E2 ubiquitin-conjugating enzymes of metazoan phyla are highly conservative, whereas the homology of E3 ubiquitin ligases with human orthologues gradually decreases depending on "molecular clock" timing and evolutionary distance. Surprisingly, Chordata and Echinodermata, which diverged over 0.5 billion years ago during the Cambrian explosion, share almost the same homology with humans in the amino acid sequences of E3 ligases but not in their adaptor proteins. These observations may suggest that, firstly, the E2 superfamily already existed in its current form in the last common metazoan ancestor and was generally not affected by purifying selection in metazoans. Secondly, it may indicate convergent evolution of the ubiquitination system and highlight E3 adaptor proteins as the "upper deck" of the ubiquitination system, which plays a crucial role in chordate evolution. [ABSTRACT FROM AUTHOR]

Parkin is an E3 ubiquitin ligase implicated in early-onset forms of Parkinson's disease. It catalyzes a transthiolation reaction by accepting ubiquitin (Ub) from an E2 conjugating enzyme, forming a short-lived thioester intermediate, and transfers Ub to mitochondrial membrane substrates to signal mitophagy. A major impediment to the development of Parkinsonism therapeutics is the lack of structural and mechanistic detail for the essential, short-lived transthiolation intermediate. It is not known how Ub is recognized by the catalytic Rcat domain in parkin that enables Ub transfer from an E2~Ub conjugate to the catalytic site and the structure of the transthiolation complex is undetermined. Here, we capture the catalytic intermediate for the Rcat domain of parkin in complex with ubiquitin (Rcat-Ub) and determine its structure using NMR-based chemical shift perturbation experiments. We show that a previously unidentified a-helical region near the Rcat domain is unmasked as a recognition motif for Ub and guides the C-terminus of Ub toward the parkin catalytic site. Further, we apply a combination of guided AlphaFold modeling, chemical cross-linking, and single turnover assays to establish and validate a model of full-length parkin in complex with UbcH7, its donor Ub, and phosphoubiquitin, trapped in the process of transthiolation. Identification of this catalytic intermediate and orientation of Ub with respect to the Rcat domain provides important structural insights into Ub transfer by this E3 ligase and explains how the previously enigmatic Parkinson's pathogenic mutation T415N alters parkin activity. [ABSTRACT FROM AUTHOR]

The article discusses the impact of endocrine-disrupting chemicals (EDCs) on human health. EDCs are exogenous compounds that disrupt the synthesis, secretion, and elimination of natural hormones, leading to various health issues such as asthma, cardiovascular diseases, obesity, infertility, and cancers. The article highlights the diverse mechanisms of action of EDCs, including disruption of ligand-receptor interaction, interference of signal transduction, and alteration of epigenetic landscapes. It also presents several studies that explore the effects of specific EDCs on male fertility, testicular function, reproductive health, liver injuries, brain development, behavioral disorders, and cancer development. The research emphasizes the need for updated toxicological approaches to assess the low-dose effects of EDCs and calls for further investigation into the potential risks posed by these chemicals. [Extracted from the article]

Seed size (SS) constitutes a pivotal trait in watermelon breeding. In this study, we present findings from an examination of two watermelon accessions, namely, BW85 and F211. Seeds from BW85 exhibited a significant enlargement compared to those of F211 at 13 days after pollination (DAP), with the maximal disparity in seed length and width manifesting at 17 DAP. A comprehensive study involving both metabolic and transcriptomic analyses indicated a significant enrichment of the ubiquinone and other terpenoid-quinone biosynthesis KEGG pathways. To detect the genetic region governing seed size, a BSA-seq analysis was conducted utilizing the F2 (BW85 × F211) population, which resulted in the identification of two adjacent QTLs, namely, SS6.1 and SS6.2, located on chromosomes 6. SS6.1 spanned from Chr06:4847169 to Chr06:5163486, encompassing 33 genes, while SS6.2 ranged from Chr06:5379337 to Chr06:5419136, which included only one gene. Among these genes, 11 exhibited a significant differential expression between BW85 and F211 according to transcriptomic analysis. Notably, three genes (Cla97C06G113960, Cla97C06G114180, and Cla97C06G114000) presented a differential expression at both 13 and 17 DAP. Through annotation, Cla97C06G113960 was identified as a ubiquitinconjugating enzyme E2, playing a role in the ubiquitin pathway that mediates seed size control. Taken together, our results provide a novel candidate gene influencing the seed size in watermelon, shedding light on the mechanism underlying seed development. [ABSTRACT FROM AUTHOR]

Objective To investigate the expression level of serum ubiquitin-conjugating enzyme 2C (UBE2C) and tripartite motif-containing protein 27 (TRIM27) in patients with endometrial cancer (EC) and their correlation with pathological parameters. Methods A total of 96 EC patients from March 2020 to March 2023 were selected as EC group; 65 patients with endometrial atypical hyperplasia (EAH) as EAH group, and 80 healthy subjects as the control group. Enzyme linked immunosorbent assay was applied to analyze the expression level of serum UBE2C and TRIM27. The relationship between serum UBE2C, TRIM27, and pathological data was analyzed; receiver operating characteristic was applied to evaluate the predictive value of serum UBE2C and TRIM27 level for EC. Results The level of serum UBE2C and TRIM27 of EC patients was obviously higher than that in healthy subjects (P < 0.05), and was correlated with tumor diameter, tumor differentiation, lymph node metastasis, FIGO stage, muscle layer invasion depth, cervical involvement, estrogen receptor expression, and progestogen receptor expression (P < 0.05). There was positive correlation between serum UBE2C and TRIM27 (r = 0.475, P < 0.001); and the level of serum UBE2C and TRIM27 was positively correlated with tumor diameter, lymph node metastasis, FIGO stage, and depth of musclular invasion, but negatively with tumor differentiation, estrogen receptor expression, and progestogen receptor expression (P < 0.05). The combination of UBE2C and TRIM27 had obviously higher AUC in evaluating EC than single detection (ZUBE2C-combination=3.406, P < 0.001, ZTRIM27-combination = 3.285, P = 0.001). Conclusion The expression level of UBE2C and TRIM27 in serum of EC patients is up-regulated, which is closely related to pathological parameters. The level of serum UBE2C and TRIM27 can provide reference for early diagnosis of EC. [ABSTRACT FROM AUTHOR]

Ubiquitin (Ub) is a small, highly conserved protein essential for eukaryotic biology, and is unique in its formation of polyubiquitin chains by conjugation to one of its seven lysine side chains. Here we report that atomic tailoring of Ub side chains – i. e. the insertion, deletion, or replacement of specific atoms – has significant and unexpected consequences on the enzymatic conjugation of Ub oligomers by isopeptide bond formation mediated by E2 conjugating enzymes. These studies employed chemical synthesis and ligation methods to prepare numerous specifically tailored Ub monomers on multi‐milligram scales. While some modifications including N‐terminal acylation and methionine replacement did not affect protein folding or Ub chain formation with Ube2 K, other modifications had a pronounced effect of oligomerization with Ubc13/Mms2. We observed that Ala46Hse mutation obliterates the ability of this Ub monomer to accept another Ub at Lys63 in Ubc13‐mediated conjugations. Exhaustive replacement of all seven lysines with shorter surrogates Orn, Dab, or Dap essentially blocks Ub chain formation, and in the case of Dap, precludes proper folding of the Ub protein. [ABSTRACT FROM AUTHOR]

(1) Background: Tree peonies display extensive cultivar diversity due to widespread hybridization, resulting in a complex genetic architecture. This complexity complicates the selection of universal reference genes across different cultivars for qRT-PCR analyses. Paeonia suffruticosa 'Doulv', notable for its unique green blooms in China, exhibits chlorosis post-flowering and features petaloid stamens and pistils. (2) Methods: Based on published literature and RNA-seq data from 'Doulv', nine candidate reference genes—ACT (Actin), TUB (β-Tubulin), UBC (Ubiquitin Conjugating Enzyme), UBQ (Ubiquitin), UPL (Ubiquitin Protein Ligase), PP2A (Protein Phosphatase 2A), PP2C (Protein Phosphatase 2C), MBF1A (Multiprotein Bridging Factor 1A), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase)—were selected. Their expression stability was assessed across various tissues and developmental stages of 'Doulv' flowers using qRT-PCR, with evaluations conducted via GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0. Gene cloning and expression analyses of PsCUC3, including its subcellular localization, were performed. (3) Results: GAPDH and ACT were identified as the most stable reference genes in petaloid stamens across various developmental stages of 'Doulv', whereas UBC and MBF1A were optimal across different tissues. Notably, specific conserved amino acids in PsCUC3 from 'Doulv' diverged from those in NAM/CUC3 proteins of other species, impacting its protein structure. PsCUC3 expression analysis revealed no correlation with chlorophyll content in petaloid stamens but an association with petaloid organ development. Furthermore, PsCUC3 was predominantly localized in the nucleus. (4) Conclusions: This study comprehensively evaluated suitable reference genes using GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0 software, establishing a robust qRT-PCR detection system for 'Doulv' peony. These results provide a solid experimental foundation for further research on 'Doulv' peony. Building on this experimental foundation, the functional analysis of the PsCUC3 gene was conducted. The findings suggest a potential association between the PsCUC3 gene and floral morphology alterations in 'Doulv', identifying PsCUC3 as crucial for understanding the molecular mechanisms influencing floral structure in tree peonies. [ABSTRACT FROM AUTHOR]

Glioma is a central nervous system (CNS) malignant tumor with high heterogeneity and mortality, which severely threatens the health of patients. The overall survival of glioma patients is relatively short and it is critical to identify new molecular targets for developing effective treatment strategies. UBE2K is a ubiquitin conjugating enzyme with oncogenic function in several malignant tumors. However, whether UBE2K participates in gliomas remains unknown. Herein, in glioma cells, UBE2K was found highly expressed in U87 and U251 cells. Subsequently, U87 and U251 cells were transfected with si‐UBE2K to silence UBE2K, with the si‐NC transfection as the negative control. In both U87 and U251 cells, the cell viability was sharply reduced by transfecting si‐UBE2K for 48 and 72 h. Markedly decreased colony number, reduced number of migrated cells and invaded cells, and declined relative wound healing rate were observed in si‐UBE2K transfected U87 and U251 cells. Moreover, the Bcl‐2 level was markedly reduced, while the Bax and cleaved‐caspase‐3 levels were sharply increased in U87 and U251 cells after the si‐UBE2K transfection. Furthermore, the p62 level was signally declined, while the Beclin‐1 and LC‐3 II/I levels were greatly increased in U87 and U251 cells by the si‐UBE2K transfection. Furthermore, the facilitating effect of si‐UBE2K on the apoptosis and autophagy in U87 and U251 cells was abolished by the coculture of 3‐MA, an inhibitor of autophagy. Collectively, UBE2K facilitated the in vitro growth of glioma cells, possibly by inhibiting the autophagy‐related apoptosis, which might be a promising target for treating glioma. [ABSTRACT FROM AUTHOR]

Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation. Ubiquitin system factors are critical regulators of DNA repair pathways. Through a functional genetic screen, the authors identify a novel role of ubiquitin-conjugating enzyme UBE2D3 in promoting DNA repair at deprotected telomeres by limiting the accumulation of RNF168 and promoting ample ATM-dependent phosphorylation of KAP1. [ABSTRACT FROM AUTHOR]

This article, titled "Elucidating the Role of a Ubiquitin-Conjugating Enzyme Bendless during Drosophila Hematopoiesis," explores the role of the ubiquitin-conjugating enzyme Bendless in Drosophila hematopoiesis. The study investigates the expression of Bendless in different cellular subsets of the larval lymph gland and examines the effects of Bendless loss of function mutants on hematopoiesis. The results indicate that Bendless plays a role in regulating niche size and differentiation, and its overexpression leads to the activation of the JNK pathway and lamellocyte differentiation. The study suggests that Bendless may regulate multiple signaling pathways involved in Drosophila hematopoiesis. [Extracted from the article]

Objectives: This study aimed to reveal the role and possible mechanism of the ubiquitin-conjugating enzyme 2T (UBE2T) in the biological activities of breast cancer stem cells (BCSCs). Methods: The specific protein and gene expression were quantified by Western blotting and quantitative real-time polymerase chain reaction, the proportion of BCSCs was examined by flow cytometry, and the self-renewal and proliferation of BCSCs were verified by serial sphere formation and soft agar. Results: Increasing expression of UBE2T was drastically found in breast cancer than that in adjacent tissues. Furthermore, UBE2T overexpression significantly increased the proportion of BCSCs in breast cancer cells and promoted their self-renewal and proliferation. Silent UBE2T exhibited the opposite functions. UBE2T increased the levels of the mammalian target of rapamycin and the phosphorylated mammalian target of rapamycin. Mammalian target of rapamycin (mTOR) inhibitor rapamycin inhibited the function of UBE2T in BCSCs. Conclusion: UBE2T plays a role in BCSCs through mTOR pathway and may suggest a novel therapeutic strategy for breast cancer. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic β-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic β-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of β-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation and subsequent presentation of the PPIB10-18 autoantigen. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future T1D therapies. [ABSTRACT FROM AUTHOR]

Most cellular ubiquitin signaling is initiated by UBA1, which activates and transfers ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic) syndrome. Despite extensive clinical investigation into this lethal disease, little is known about the underlying molecular mechanisms. Here, by dissecting VEXAS-causing UBA1 mutations, we discovered that p.Met41 mutations alter cytoplasmic isoform expression, whereas other mutations reduce catalytic activity of nuclear and cytoplasmic isoforms by diverse mechanisms, including aberrant oxyester formation. Strikingly, non-p.Met41 mutations most prominently affect transthioesterification, revealing ubiquitin transfer to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. A similar E2 charging bottleneck exists in some lung cancer-associated UBA1 mutations, but not in spinal muscular atrophy-causing UBA1 mutations, which instead, render UBA1 thermolabile. Collectively, our results highlight the precision of conformational changes required for faithful ubiquitin transfer, define distinct and shared mechanisms of UBA1 inactivation in diverse diseases, and suggest that specific E1-E2 modules control different aspects of tissue differentiation and maintenance. Synopsis: Mutations in the ubiquitin-activating enzyme UBA1 cause autoinflammation (VEXAS syndrome), lung cancer in never-smokers (LCINS), and spinal muscular atrophy (SMA). Here, systematic mutation profiling defines mechanisms of UBA1 inactivation that are shared and distinct in these diseases. Identification of non-canonical VEXAS-causing UBA1 mutations that lead to aberrant ubiquitin adenylation, thiolation, and intramolecular oxyester formation. Non-canonical VEXAS mutations most prominently affect ubiquitin transfer to E2s. Two classes of UBA1 disease mutations either create a bottleneck at the E2 transfer step and are deficient in E2 charging and polyubiquitylation in cells, or render UBA1 activity thermolabile and exhibit subtle cellular defects. While non-canonical VEXAS mutations only fall in class 1 and SMA-associated mutations in class 2, LCNIS-associated mutations comprise class 1 and 2. Different classes of mutations in the ubiquitin-activating enzyme cause either autoinflammatory VEXAS syndrome, spinal muscular atrophy SMA, or lung cancer in never-smokers (LCINS). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ETHYLENE RESPONSE FACTOR 1 (ERF1) plays an important role in integrating hormone crosstalk and stress responses. Previous studies have shown that ERF1 is unstable in the dark and its degradation is mediated by UBIQUITIN‐CONJUGATING ENZYME 18. However, whether there are other enzymes regulating ERF1's stability remains unclear. Here, we use various in vitro and in vivo biochemical, genetic and stress‐tolerance tests to demonstrate that both CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and SUMO‐CONJUGATING ENZYME 1 (SCE1) regulate the stability of ERF1. We also performed transcriptomic analyses to understand their common regulatory pathways. We show that COP1 mediates ERF1 ubiquitination in the dark while SCE1 mediates ERF1 sumoylation in the light. ERF1 stability is positively regulated by SCE1 and negatively regulated by COP1. Upon abiotic stress, SCE1 plays a positive role in stress defence by regulating the expression of ERF1's downstream stress‐responsive genes, whereas COP1 plays a negative role in stress response. Moreover, ERF1 also promotes photomorphogenesis and the expression of light‐responsive genes. Our study reveals the molecular mechanism of how COP1 and SCE1 counteract to regulate ERF1's stability and light‐stress signalling crosstalk. Summary statement: ETHYLENE RESPONSE FACTOR 1 (ERF1) plays an important role in biotic and abiotic stress response. This study shows that both COP1 and SCE1 interact with ERF1 and regulate its stability by ubiquitination and sumoylation, respectively. COP1‐ERF1‐SCE1 module mediates the crosstalk between light and stress signallings. [ABSTRACT FROM AUTHOR]

Ubiquitin‐conjugating enzyme (UBC) is a crucial component of the ubiquitin‐proteasome system, which contributes to plant growth and development. While some UBCs have been identified as potential regulators of abiotic stress responses, the underlying mechanisms of this regulation remain poorly understood. Here, we report a cotton (Gossypium hirsutum) UBC gene, GhUBC10‐2, which negatively regulates the salt stress response. We found that the gain of function of GhUBC10‐2 in both Arabidopsis (Arabidopsis thaliana) and cotton leads to reduced salinity tolerance. Additionally, GhUBC10‐2 interacts with glutathione S‐transferase (GST) U17 (GhGSTU17), forming a heterodimeric complex that promotes GhGSTU17 degradation. Intriguingly, GhUBC10‐2 can be self‐polyubiquitinated, suggesting that it possesses E3‐independent activity. Our findings provide new insights into the PTM of plant GST‐mediated salt response pathways. Furthermore, we found that the WRKY transcription factor GhWRKY13 binds to the GhUBC10‐2 promoter and suppresses its expression under salt conditions. Collectively, our study unveils a regulatory module encompassing GhWRKY13–GhUBC10‐2–GhGSTU17, which orchestrates the modulation of reactive oxygen species homeostasis to enhance salt tolerance. Summary statement: GhUBC10‐2 negatively regulates salt tolerance by targeting glutathione S‐transferase U17 (GhGSTU17) for its degradation. GhWRKY13 binds the W‐box of the GhUBC10‐2 promoter to repress GhUBC10‐2 transcription. The GhWRKY13–GhUBC10‐2–GhGSTU17 module offers a guidance for cotton breeding. [ABSTRACT FROM AUTHOR]

Using a bioinformatics approach to identify binding pockets between proteins is a preferable method before modifying the genome to delineate host interactions with viruses. Based on extensive proteomics data in numerous databases, several interaction prediction methods are available to identify binding sites between viruses and hosts at the individual residue level, but little is known about the interaction prediction strategy for plant viruses. Begomoviruses, belonging to the family Geminiviridae, constitute a group of circular single-stranded (ss) DNA viruses that encode multifunctional proteins responsible for viral replication, causing severe diseases in multiple host plants. These viruses usually escape through plant defense mechanism overcoming physical and chemical barriers to trigger the infection with all possible combinations of interaction in the target host protein partners. Here, we have applied our computational approach for plant virus interaction at domain level. Previous study showed that myristoylation-like motif in Begomovirus cotton leaf curl Multan associated betasatellite protein βC1 (CLCuMB- βC1) played an important role in interaction with ubiquitin conjugating enzyme protein (UBC3) in tomato. This kind of binding at residue level has been validated using in vivo and in vitro molecular approach. Here, an in silico approach was utilized which is a combinatorial source of previous and recent protein prediction methods to determine all possible identified interface sites between βC1 and UBC3. This molecular interaction of CLCuMB- βC1 was further verified in the actual host i.e. cotton using a bimolecular fluorescence complementation system and yeast two-hybrid assay. This computational and molecular data will help to identify the interaction between virus and host before using any expensive and time-consuming molecular techniques. [ABSTRACT FROM AUTHOR]

Soil salinity is one of the most brutal environmental factors that affect crop growth and productivity. Chenopodium quinoa is known as one of the essential food crops in the future due to its agronomic and nutritive value and its strong adaptability to stress environments and soil conditions. However, the molecular aspects of salt tolerance of quinoa remain not well known. Therefore, the expression study of candidate genes related to salt tolerance has become one of the most important features for the identification of their functions. However, one of the most crucial points in Quantitative PCR (qPCR) data analysis is the selection of appropriate reference gene that should be stable and unaffected in a given condition. In this study, six candidate reference genes were analysed in order to select the most stable one under salt stress conditions. The expression stability was assessed using three different algorithms: geNorm, NormFinder and BestKeeper. The most stable and appropriate reference genes screened in this study whose expression was confirmed to be constant in different salt treated and untreated quinoa plant were Monensin sensitivity1 (MON1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Factor elongation 1-alpha (EF-1α), while Ubiquitin-conjugating enzyme (UBC) and Ubiquitin-protein ligase 7 (UPL7) had the worst stability. However, the data obtained by BestKeeper demonstrated slight differences compared to those from geNorm and NormFinder. Overall, we designated MON1 and GAPDH as the best candidates and the most stable housekeeping gene under salt stress conditions and their geometric means would provide accurate normalization factor for expression data in Chenopodium quinoa. [ABSTRACT FROM AUTHOR]

Background: Ubiquitination is an important regulatory step of selective protein degradation in the plant UPS (ubiquitin–proteasome system), which is involved in various biological processes in eukaryotes. Ubiquitin-conjugating enzymes play an intermediate role in the process of protein ubiquitination reactions and thus play an essential role in regulating plant growth and response to adverse environmental conditions. However, a genome-wide analysis of the UBC gene family in wheat (Triticum aestivum L.) has not yet been performed. Results: In this study, the number, physiochemical properties, gene structure, collinearity, and phylogenetic relationships of TaUBC family members in wheat were analyzed using bioinformatics methods. The expression pattern of TaUBC genes in different tissues/organs and developmental periods, as well as the transcript levels under abiotic stress treatment, were analyzed using RNA-Seq data and qRT-PCR. Meanwhile, favorable haplotypes of TaUBC25 were investigated based on wheat resequencing data of 681 wheat cultivars from the Wheat Union Database. The analyses identified a total of 93 TaUBC family members containing a UBC domain in wheat genome. These genes were unevenly distributed across 21 chromosomes, and numerous duplication events were observed between gene members. Based on phylogenetic analysis, the TaUBC family was divided into 13 E2 groups and a separate UEV group. We investigated the expression of TaUBC family genes under different tissue/organ and stress conditions by quantitative real-time PCR (qRT-PCR) analysis. The results showed that some TaUBC genes were specifically expressed in certain tissues/organs and that most TaUBC genes responded to NaCl, PEG6000, and ABA treatment with different levels of expression. In addition, we performed association analysis for the two haplotypes based on key agronomic traits such as thousand-kernel weight (TKW), kernel length (KL), kernel weight (KW), and kernel thickness (KT), examining 122 wheat accessions at three environmental sites. The results showed that TaUBC25-Hap II had significantly higher TKW, KL, KW, and KT than TaUBC25-Hap I. The distribution analysis of haplotypes showed that TaUBC25-Hap II was preferred in the natural population of wheat. Conclusion: Our results identified 93 members of the TaUBC family in wheat, and several genes involved in grain development and abiotic stress response. Based on the SNPs detected in the TaUBC sequence, two haplotypes, TaUBC25-Hap I and TaUBC25-Hap II, were identified among wheat cultivars, and their potential value for wheat breeding was validated by association analysis. The above results provide a theoretical basis for elucidating the evolutionary relationships of the TaUBC gene family and lay the foundation for studying the functions of family members in the future. [ABSTRACT FROM AUTHOR]

Background: Cystic echinococcosis (CE) is a widespread zoonosis caused by the infection with Echinococcus granulosus sensu lato (E. granulosus s.l.). CE cysts mainly develop in the liver of intermediate hosts, characterized by the fibrotic tissue that separates host organ from parasite. However, precise mechanism underlying the formation of fibrotic tissue in CE remains unclear. Methods: To investigate the potential impact of ubiquitin-conjugating enzymes on liver fibrosis formation in CE, two members of ubiquitin-conjugating (UBC) enzyme of Echinococcus granulosus (EgE2D2 and EgE2N) were recombinantly expressed in Escherichia coli and analyzed for bioinformatics, immunogenicity, localization, and enzyme activity. In addition, the secretory pathway and their effects on the formation of liver fibrosis were also explored. Results: Both rEgE2D2 and rEgE2N possess intact UBC domains and active sites, exhibiting classical ubiquitin binding activity and strong immunoreactivity. Additionally, EgE2D2 and EgE2N were widely distributed in protoscoleces and germinal layer, with differences observed in their distribution in 25-day strobilated worms. Further, these two enzymes were secreted to the hydatid fluid and CE-infected sheep liver tissues via a non-classical secretory pathway. Notably, TGFβ1-induced LX-2 cells exposed to rEgE2D2 and rEgE2N resulted in increasing expression of fibrosis-related genes, enhancing cell proliferation, and facilitating cell migration. Conclusions: Our findings suggest that EgE2D2 and EgE2N could secrete into the liver and may interact with hepatic stellate cells, thereby promoting the formation of liver fibrosis. [ABSTRACT FROM AUTHOR]

Summary: Eukaryotes have evolved sophisticated post‐translational modifications to regulate protein function and numerous biological processes, including ubiquitination controlled by the coordinated action of ubiquitin‐conjugating enzymes and deubiquitinating enzymes (Dubs). However, the function of deubiquitination in pathogenic fungi is largely unknown.Here, the distribution of Dubs in the fungal kingdom was surveyed and their functions were systematically characterized using the phytopathogen Fusarium graminearum as the model species, which causes devastating diseases of all cereal species world‐wide.Our findings demonstrate that Dubs are critical for fungal development and virulence, especially the ubiquitin‐specific protease 15 (Ubp15). Global ubiquitome analysis and subsequent experiments identified three important substrates of Ubp15, including the autophagy‐related protein Atg8, the mitogen‐activated protein kinase Gpmk1, and the mycotoxin deoxynivalenol (DON) biosynthetic protein Tri4. Ubp15 regulates the deubiquitination of the Atg8, thereby impacting its subcellular localization and the autophagy process. Moreover, Ubp15 also modulates the deubiquitination of Gpmk1 and Tri4. This modulation subsequently influences their protein stabilities and further affects the formation of penetration structures and the biosynthetic process of DON, respectively.Collectively, our findings reveal a previously unknown regulatory pathway of a deubiquitinating enzyme for fungal virulence and highlight the potential of Ubp15 as a target for combating fungal diseases. [ABSTRACT FROM AUTHOR]

This editorial discusses the role of posttranslational modifications (PTMs) in cancer biology. It highlights various articles published in the journal "Frontiers in Oncology" that explore PTMs of key proteins and their association with cancer pathogenesis. The articles cover topics such as phosphorylation, ubiquitination, and acetylation, and their impact on protein functionality and cancer progression. The findings emphasize the importance of understanding PTMs in order to develop novel biomarkers and therapeutic targets for cancer treatment. [Extracted from the article]

At each stage of the HIV life cycle, host cellular proteins are hijacked by the virus to establish and enhance infection. We adapted the virus packageable HIV-CRISPR screening technology at a genome-wide scale to comprehensively identify host factors that affect HIV replication in a human T cell line. Using a smaller, targeted HIV Dependency Factor (HIVDEP) sublibrary, we then performed screens across HIV strains representing different clades and with different biological properties to define which T cell host factors are important across multiple HIV strains. Nearly 90% of the genes selected across various host pathways validated in subsequent assays as bona fide host dependency factors, including numerous proteins not previously reported to play roles in HIV biology, such as UBE2M, MBNL1, FBXW7, PELP1, SLC39A7, and others. Our ranked list of screen hits across diverse HIV-1 strains form a resource of HIV dependency factors for future investigation of host proteins involved in HIV biology. IMPORTANCE With a small genome of ~9.2 kb that encodes 14 major proteins, HIV must hijack host cellular machinery to successfully establish infection. These host proteins necessary for HIV replication are called "dependency factors." Whole-genome, and then targeted screens were done to try to comprehensively identify all dependency factors acting throughout the HIV replication cycle. Many host processes were identified and validated as critical for HIV replication across multiple HIV strains.

The selection of stable reference genes for the normalization of reverse transcription quantitative real-time PCR (RT-qPCR) is generally overlooked despite being the crucial element in determining the accuracy of the relative expression of genes. In the present study, the stability of seven candidate reference genes: actin (act), α-tubulin (atub), β-tubulin (btub), translation elongation factor 1-α (ef1), elongation factor 2 (ef2), ubiquitin-conjugating enzyme (ubc) and 40S ribosomal protein S3A (ws21) in Phytophthora capsici has been validated. The validation was performed at six infection time points during its interaction with its susceptible host Piper nigrum, two developmental stages, and for the combined dataset. Four algorithms: geNorm, NormFinder, BestKeeper, and the ΔCt method were compared, and a comprehensive ranking order was produced using RefFinder. The overall analysis revealed that ef1, ws21, and ubc were identified as the three most stable genes in the combined dataset, ef1, ws21, and act were the most stable at the infection stages, and, ef1, btub, and ubc were most stable during the developmental stages. These findings were further corroborated by validating the P. capsici pathogenesis gene NPP1 expression. The findings are significant as this is the first study addressing the stability of reference genes for P. capsici–P. nigrum interaction studies. [ABSTRACT FROM AUTHOR]

Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0–6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility. The structure of the nucleosome-bound human Bre1 complex reveals that its two RING domains bind the acidic patch and nucleosomal DNA, directing the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. The binding mode suggests a possible regulatory mechanism through nucleosomal DNA flexibility. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: 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). Methods: 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. Results: 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-kB, 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

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. Japonica rice generally has greater capacity for low temperatures germination (LTG) than indica rice. Here, the authors report an E2 ubiquitin-conjugating enzyme-encoding gene OsUBC12 can increase LTG in japonica rice by negatively regulate ABA signaling via promoting the proteasomal degradation of OsSnRK1.1. [ABSTRACT FROM AUTHOR]

PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation. The peroxisomal protein shuttling receptor PEX5 transports newly synthesised proteins from the cytosol to the peroxisome, where it is monoubiquitinated at cysteine 11 so it can be extracted back to the cytosol by ATP. These authors show that longer ubiquitin chains attached to this cysteine are rapidly removed by the ubiquitin-conjugating enzyme E2D3, ensuring the correct functioning of this protein import machinery. [ABSTRACT FROM AUTHOR]

Cell cycle dysregulation is a defining feature of breast cancer. Here, 1‐methyl‐nicotinamide (1‐MNA), metabolite of nicotinamide N‐methyltransferase(NNMT) is identified, as a novel driver of cell‐cycle progression in breast cancer. NNMT, highly expressed in breast cancer tissues, positively correlates with tumor grade, TNM stage, Ki‐67 index, and tumor size. Ablation of NNMT expression dramatically suppresses cell proliferation and causes cell‐cycle arrest in G0/G1 phase. This phenomenon predominantly stems from the targeted action of 1‐MNA, resulting in a specific down‐regulation of p27 protein expression. Mechanistically, 1‐MNA expedites the degradation of p27 proteins by enhancing cullin‐1 neddylation, crucial for the activation of Cullin‐1‐RING E3 ubiquitin ligase(CRL1)—an E3 ubiquitin ligase targeting p27 proteins. NNMT/1‐MNA specifically up‐regulates the expression of UBC12, an E2 NEDD8‐conjugating enzyme required for cullin‐1 neddylation. 1‐MNA showes high binding affinity to UBC12, extending the half‐life of UBC12 proteins via preventing their localization to lysosome for degradation. Therefore, 1‐MNA is a bioactive metabolite that promotes breast cancer progression by reinforcing neddylation pathway‐mediated p27 degradation. The study unveils the link between NNMT enzymatic activity with cell‐cycle progression, indicating that 1‐MNA may be involved in the remodeling of tumor microenvironment. [ABSTRACT FROM AUTHOR]

Genome-wide association studies have identified several gene polymorphisms, including UBE2E2 , associated with type 2 diabetes. Although UBE2E2 is one of the ubiquitin-conjugating enzymes involved in the process of ubiquitin modifications, the pathophysiological roles of UBE2E2 in metabolic dysfunction are not yet understood. Here, we showed upregulated UBE2E2 expression in the islets of a mouse model of diet-induced obesity. The diabetes risk allele of UBE2E2 (rs13094957) in noncoding regions was associated with upregulation of UBE2E 2 mRNA in the human pancreas. Although glucose-stimulated insulin secretion was intact in the isolated islets, pancreatic β-cell–specific UBE2E2 -transgenic (TG) mice exhibited reduced insulin secretion and decreased β-cell mass. In TG mice, suppressed proliferation of β-cells before the weaning period and while receiving a high-fat diet was accompanied by elevated gene expression levels of p21 , resulting in decreased postnatal β-cell mass expansion and compensatory β-cell hyperplasia, respectively. In TG islets, proteomic analysis identified enhanced formation of various types of polyubiquitin chains, accompanied by increased expression of Nedd4 E3 ubiquitin protein ligase. Ubiquitination assays showed that UBE2E2 mediated the elongation of ubiquitin chains by Nedd4. The data suggest that UBE2E2-mediated ubiquitin modifications in β-cells play an important role in regulating glucose homeostasis and β-cell mass. [ABSTRACT FROM AUTHOR]

Ubiquitination often generates lysine 48-linked polyubiquitin chains that signal proteolytic destruction of the protein target. A significant subset of ubiquitination proceeds by a priming/extending mechanism, in which a substrate is first monoubiquitinated with a priming E2-conjugating enzyme or a set of E3 ARIH/E2 enzymes specific for priming. This is then followed by ubiquitin (Ub) chain extension catalyzed by an E2 enzyme capable of elongation. This report provides further insights into the priming/extending mechanism. We employed reconstituted ubiquitination systems of substrates CK1α (casein kinase 1α) and β-catenin by Cullin-RING E3 Ub ligases (CRLs) CRL4CRBN and CRL1βTrCP, respectively, in the presence of priming E2 UbcH5c and elongating E2 Cdc34b (cell division cycle 34b). We have established a new "apyrase chase" strategy that uncouples priming from chain elongation, which allows accurate measurement of the decay rates of the ubiquitinated substrate with a defined chain length. Our work has revealed highly robust turnover of monoubiquitinated β-catenin that empowers efficient polyubiquitination. The results of competition experiments suggest that the interactions between the ubiquitinated β-catenin and CRL1βTrCP are highly dynamic. Moreover, ubiquitination of the Ub-modified β-catenin appeared more resistant to inhibition by competitors than the unmodified substrate, suggesting tighter binding with CRL1βTrCP. These findings support a role for conjugated Ub in enhancing interactions with E3. [ABSTRACT FROM AUTHOR]

Recent research reveals the critical roles of endoplasmic reticulum (ER)-associated protein degradation (ERAD)-related ubiquitin-conjugating enzyme AtUBC32 orthologs and their partnering E3 ligases, which play dual roles in enhancing both crop yield and stress resistance. These findings open avenues for breeding high-yield, stress-tolerant crops and inspire further exploration of the ERAD pathway in agricultural innovation. [ABSTRACT FROM AUTHOR]

The mechanism of genome DNA replication in circular single-stranded DNA viruses is currently a mystery, except for the fact that it undergoes rolling-circle replication. Herein, we identified SUMOylated porcine nucleophosmin-1 (pNPM1), which is previously reported to be an interacting protein of the viral capsid protein, as a key regulator that promotes the genome DNA replication of porcine single-stranded DNA circovirus. Upon porcine circovirus type 2 (PCV2) infection, SUMO2/3 were recruited and conjugated with the K263 site of pNPM1's C-terminal domain to SUMOylate pNPM1, subsequently, the SUMOylated pNPM1 were translocated in nucleoli to promote the replication of PCV2 genome DNA. The mutation of the K263 site reduced the SUMOylation levels of pNPM1 and the nucleolar localization of pNPM1, resulting in a decrease in the level of PCV2 DNA replication. Meanwhile, the mutation of the K263 site prevented the interaction of pNPM1 with PCV2 DNA, but not the interaction of pNPM1 with PCV2 Cap. Mechanistically, PCV2 infection increased the expression levels of Ubc9, the only E2 enzyme involved in SUMOylation, through the Cap-mediated activation of ERK signaling. The upregulation of Ubc9 promoted the interaction between pNPM1 and TRIM24, a potential E3 ligase for SUMOylation, thereby facilitating the SUMOylation of pNPM1. The inhibition of ERK activation could significantly reduce the SUMOylation levels and the nucleolar localization of pNPM1, as well as the PCV2 DNA replication levels. These results provide new insights into the mechanism of circular single-stranded DNA virus replication and highlight NPM1 as a potential target for inhibiting PCV2 replication. Author summary: Different types of DNA viruses employ different mechanisms to replicate their genome DNA. Porcine circovirus type 2 (PCV2) is the most representative circular single-stranded DNA virus that harms the pig industry all over the world. In this study, we found that the PCV2 Cap interacting protein pNPM1 also interacts with PCV2 DNA in a SUMOylated form to promote PCV2 DNA replication. The SUMOylation of pNPM1 at the conserved K263 site is critical for the interaction of pNPM1 with PCV2 DNA and the replication of PCV2 DNA. Furthermore, we found that PCV2 infection promotes the SUMO2/3 mediated SUMOylation of pNPM1, while does not significantly alter the expression level of pNPM1. PCV2 Cap is the major component that promotes pNPM1 SUMOylation by activating ERK/Ubc9/TRIM24 signalings. These results contribute to a better understanding of the replication mechanism of circular single-stranded DNA viruses, particularly PCV2. [ABSTRACT FROM AUTHOR]

The SEL1L-HRD1 protein complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD). Despite recent advances in both mouse models and humans, in vivo evidence for the importance of SEL1L in the ERAD complex formation and its (patho-)physiological relevance in mammals remains limited. Here we report that SEL1L variant p.Ser658Pro (SEL1LS658P) is a pathogenic hypomorphic mutation, causing partial embryonic lethality, developmental delay, and early-onset cerebellar ataxia in homozygous mice carrying the bi-allelic variant. Biochemical analyses reveal that SEL1LS658P variant not only reduces the protein stability of SEL1L, but attenuates the SEL1L-HRD1 interaction, likely via electrostatic repulsion between SEL1L F668 and HRD1 Y30 residues. Proteomic screens of SEL1L and HRD1 interactomes reveal that SEL1L-HRD1 interaction is a prerequisite for the formation of a functional HRD1 ERAD complex, as SEL1L is required for the recruitment of E2 enzyme UBE2J1 as well as DERLIN to HRD1. These data not only establish the disease relevance of SEL1L-HRD1 ERAD, but also provide additional insight into the formation of a functional HRD1 ERAD complex. The importance of the SEL1L-HRD1 interaction in vivo was unclear. Here, authors reported that SEL1L-HRD1 interaction is required to form a functional HRD1 ERAD complex by recruiting the E2 enzyme UBE2J1 and DERLIN to HRD1. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ubiquitination, catalyzed usually by a three-enzyme cascade (E1, E2, E3), regulates various eukaryotic cellular processes. E3 ligases are the most critical components of this catalytic cascade, determining both substrate specificity and polyubiquitination linkage specificity. Here, we reveal the mechanism of a naturally occurring E3-independent ubiquitination reaction of a unique human E2 enzyme UBE2E1 by solving the structure of UBE2E1 in complex with substrate SETDB1-derived peptide. Guided by this peptide sequence-dependent ubiquitination mechanism, we developed an E3-free enzymatic strategy SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized ubiquitinated sites, ubiquitin chain linkages and lengths. Notably, this strategy can also be used to generate site-specific branched ubiquitin chains or even NEDD8-modified proteins. Our work not only deepens the understanding of how an E3-free substrate ubiquitination reaction occurs in human cells, but also provides a practical approach for obtaining ubiquitinated proteins to dissect the biochemical functions of ubiquitination. Ubiquitin E3 ligases are key to accessing ubiquitinated proteins, but only a few substrates have defined E3 ligases. Here, the authors reveal the mechanism of naturally occurring E3-independent ubiquitination and develop an E3-free enzymatic strategy for the versatile generation of ubiquitinated proteins. [ABSTRACT FROM AUTHOR]

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 membraneassociated 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. IMPORTANCE Since 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. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is an extremely common genitourinary malignancy among elderly men. Many evidence have shown the efficacy of curcumin (CUR) in inhibiting the progression of PCa. However, the pharmacological function of CUR in PCa is still not quite clear. In this research, CUR was found to suppress the proliferation and enhance the apoptotic rate in in vitro PCa cell models in a dose‐ and time‐dependent manner. In a xenograft animal model, the administration of CUR contributed to a significant decrease in the growth of the xenograft tumor induced by the transplanted PC‐3 cells. Ubiquitin‐conjugating enzyme E2 C is implicated in the modulation of multiple types of cancers. In humans, the expression levels of UBE2C are significantly higher in PCa versus benign prostatic hyperplasia. Treatment with CUR decreased the expression of UBE2C, whereas it increased miR‐483‐3p expression. In contrast with the control mice, the CUR‐treated mice showed a significant reduction in UBE2C and Ki‐67 in PCa cells. The capability of proliferation, migration, and invasion of PCa cells was inhibited by the knockdown of UBE2C mediated by siRNA. Furthermore, dual luciferase reporter gene assay indicated the binding of miR‐483‐3p to UBE2C. In summary, CUR exerts its antitumor effects through regulation of the miR‐483‐3p/UBE2C axis by decreasing UBE2C and increasing miR‐483‐3p. The findings may also provide new molecular markers for PCa diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Oleuropein is the main constituent of olive leaf extract, and it has shown antioxidant and gastroprotective properties against gastric ulcers. Chitosan nanoparticles are known for their mucoadhesive abilities, and consequently, they can increase the retention time of drugs in the gastrointestinal tract. Therefore, loading oleuropein onto chitosan nanoparticles is expected to enhance its biological efficiency. Oleuropein-loaded chitosan nanoparticles were prepared and characterized for particle size, surface charge, in vitro release, and anti-inflammatory activity. Their in vivo efficacy was assessed by measuring specific inflammatory and protective biomarkers, along with histopathological examination. The optimum oleuropein chitosan nanoparticles were cationic, had a size of 174.3 ± 2.4 nm and an entrapment efficiency of 92.81%, and released 70% of oleuropein within 8 h. They recorded a lower IC50 in comparison to oleuropein solutions for membrane stabilization of RBCs (22.6 vs. 25.6 µg/mL) and lipoxygenase inhibition (7.17 vs. 15.6 µg/mL). In an ethanol-induced gastric ulcer in vivo model, they decreased IL-1β, TNF-α, and TBARS levels by 2.1, 1.7, and 1.3 fold, respectively, in comparison to increments caused by exposure to ethanol. Moreover, they increased prostaglandin E2 and catalase enzyme levels by 2.4 and 3.8 fold, respectively. Immunohistochemical examination showed that oleuropein chitosan nanoparticles markedly lowered the expression of IL-6 and caspase-3 in gastric tissues in comparison to oleuropein solution. Overall, oleuropein chitosan nanoparticles showed superior gastroprotective effects to oleuropein solution since comparable effects were demonstrated at a 12-fold lower drug dose, delineating that chitosan nanoparticles indeed enhanced the potency of oleuropein as a gastroprotective agent. [ABSTRACT FROM AUTHOR]

To thrive in harsh environments, tardigrades have evolved the ability to enter the quiescent state of cryptobiosis, often characterized by transition into a so-called 'tun'. Here, we investigate osmobiosis, a substate of cryptobiosis induced by rising osmolyte concentrations. We follow the behaviour and morphology of Ramazzottius varieornatus during transfer from freshwater conditions into 3 Osmol/kg sucrose solution. The tardigrades easily survive the extreme change in external osmolality. During gradual exposure, they initiate tun formation at 0.2 Osmol/kg, with most specimens fully contracted into a tun at 0.5 Osmol/kg. The first transcriptomic profiling of osmobiotic tuns in comparison to active tardigrades reveals a modest shift, with 16% of the 3322 differentially expressed transcripts having a |log2 fold change| > 1. A gene ontology enrichment analysis shows enrichment within protein homeostasis and neurohormonal signalling, with a ubiquitin-conjugating enzyme and neurotransmitter receptor transcripts being down- and upregulated, respectively. A putative Hsp70 is upregulated, whereas transcripts related to eutardigrade-specific proteins, antioxidant defence and DNA repair show minor fold changes. Among putative membrane transporters, a monocarboxylate and two amino acid transporters are downregulated. Our findings are in agreement with previous studies in Ramazzottius indicating that cryptobiosis and tun formation involve no change or modest change in transcription. [ABSTRACT FROM AUTHOR]

As an important posttranslational modification, SUMOylation plays critical roles in almost all biological processes. Although it has been well-documented that SUMOylated proteins are mainly localized in the nucleus and have roles in chromatin-related processes, we showed recently that the SUMOylation machinery is actually enriched in the nuclear matrix rather than chromatin. Here, we provide compelling biochemical, cellular imaging and proteomic evidence that SUMOylated proteins are highly enriched in the nuclear matrix. We demonstrated that inactivation of SUMOylation by inhibiting SUMO-activating E1 enzyme or KO of SUMOconjugating E2 enzyme UBC9 have only mild effect on nuclear matrix composition, indicating that SUMOylation is neither required for nuclear matrix formation nor for targeting proteins to nuclear matrix. Further characterization of UBC9 KO cells revealed that loss of SUMOylation did not result in significant DNA damage, but led to mitotic arrest and chromosome missegregation. Altogether, our study demonstrates that SUMOylated proteins are selectively enriched in the nuclear matrix and suggests a role of nuclear matrix in mediating SUMOylation and its regulated biological processes. [ABSTRACT FROM AUTHOR]

Ischemic stroke is an acute serious cerebrovascular disease with high mortality and disability. Ferroptosis is an important regulated cell death (RCD) in ischemic stroke. 15‐Hydroxyprostaglandin dehydrogenase (15‐PGDH), a degrading enzyme of prostaglandin E2 (PGE2), is shown to regulate RCD such as autophagy and apoptosis. The study aimed to determine whether 15‐PGDH regulates ferroptosis and ischemic stroke, and further the exact mechanism. We demonstrated that overexpression of 15‐PGDH in the brain tissues or primary cultured neurons significantly aggravated cerebral injury and neural ferroptosis in ischemic stroke. While inhibition of 15‐PGDH significantly protected against cerebral injury and neural ferroptosis, which benefits arise from the activation of the PGE2/PGE2 receptor 4 (EP4) axis. While the impact of 15‐PGDH was abolished with glutathione peroxidase 4 (GPX4) deficiency. Then, 15‐PGDH inhibitor was found to promote the activation of cAMP‐response element‐binding protein (CREB) and nuclear factor kappa‐B (NF‐κB) via the PGE2/EP4 axis, subsequently transcriptionally upregulate the expression of GPX4. In summary, our study indicates that inhibition of 15‐PGDH promotes the activation PGE2/EP4 axis, subsequently transcriptionally upregulates the expression of GPX4 via CREB and NF‐κB, and then protects neurons from ferroptosis and alleviates the ischemic stroke. Therefore, 15‐PGDH may be a potential therapeutic target for ischemic stroke. [ABSTRACT FROM AUTHOR]

This article is an editorial published in Frontiers in Cell & Developmental Biology. It discusses the role of protein kinase inhibitors in neurodegeneration and cancer targeted therapies. The editorial highlights the importance of protein kinases in various cellular functions and their association with cancer development and neurodegenerative disorders. It explores the involvement of kinases in the initiation and progression of these conditions and suggests avenues for future research. The editorial also provides specific examples of protein kinases, such as p90 ribosomal S6 kinase, receptor tyrosine kinase-like orphan receptor 1, interleukin-1 receptor-associated kinase 4, activin receptor-like kinase 3, and putative markers in glioblastoma multiforme microenvironment. [Extracted from the article]

Background: Epithelial ovarian cancer (OC) is the fourth leading cause of cancer-related deaths in women, with a 5-year survival rate of 30%-50%. Platinum resistance is the chief culprit for the high recurrence and mortality rates. Several studies confirm that the metabolic regulation of ubiquitinating enzymes plays a vital role in platinum resistance in OC. Methods: In this study, we selected ubiquitin-conjugating enzyme E2S (UBE2S) as the candidate gene for validation. The levels of UBE2S expression were investigated using TCGA, GTEx, UALCAN, and HPA databases. In addition, the correlation between UBE2S and platinum resistance in OC was analyzed using data from TCGA. Cisplatin-resistant OC cell lines were generated and UBE2S was knocked down; the transfection efficiency was verified. Subsequently, the effects of knockdown of UBE2S on the proliferation and migration of cisplatin-resistant OC cells were examined through the CCK8, Ki-67 immunofluorescence, clone formation, wound healing, and transwell assays. In addition, the UBE2S gene was also validated in vivo by xenograft models in nude mice. Finally, the relationship between the UBE2S gene and autophagy and the possible underlying regulatory mechanism was preliminarily investigated through MDC and GFP-LC3-B autophagy detection and western blotting experiments. Most importantly, experimental validation of mTOR agonist reversion (the rescuse experiments) was also performed. Results: UBE2S was highly expressed in OC at both nucleic acid and protein levels. The results of immunohistochemistry showed that the level of UBE2S expression in platinum-resistant samples was significantly higher relative to the platinum-sensitive samples. By cell transfection experiments, knocking down of the UBE2S gene was found to inhibit the proliferation and migration of cisplatin-resistant OC cells. Moreover, the UBE2S gene could inhibit autophagy by activating the PI3K/AKT/mTOR signaling pathway to induce cisplatin resistance in OC in vivo and in vitro. Conclusion: In conclusion, we discovered a novel oncogene, UBE2S, which was associated with platinum response in OC, and examined its key role through bioinformatics and preliminary experiments. The findings may open up a new avenue for the evaluation and treatment of OC patients at high risk of cisplatin resistance. [ABSTRACT FROM AUTHOR]

Ubiquitination is a post-translational modification that targets specific proteins on their lysine residues. Depending on the type of ubiquitination, this modification ultimately regulates the stability or degradation of the targeted proteins. Ubiquitination is mediated by three different classes of enzymes: the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes and, most importantly, the E3 ubiquitin ligases. E3 ligases are responsible for the final step of the ubiquitin cascade, interacting directly with the target proteins. E3 ligases can also be involved in DNA repair, cell cycle regulation and response to stress; alteration in their levels can be involved in oncogenic transformation and cancer progression. Of all the six hundred E3 ligases of the human genome, only three of them are specific to the mitochondrion: MARCH5, RNF185 and MUL1. Their alterations (that reflect on the alteration of the mitochondria functions) can be related to cancer progression, as underlined by the increasing research performed in recent years on these three mitochondrial enzymes. This review will focus on the function and mechanisms of the mitochondrial E3 ubiquitin ligases, as well as their important targets, in cancer development and progression, also highlighting their potential use for cancer therapy. [ABSTRACT FROM AUTHOR]