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Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen that causes acute diarrhoea, dehydration and high mortality rates in suckling pigs. Tripartite motif 8 (TRIM8) has been shown to play multiple roles in the host's defence against viral infections. However, the functions of TRIM8 in regulating PEDV infection are still not well understood. In our study, we found a significant upregulation of TRIM8 following PEDV infection. We created TRIM8 knockout and overexpression cell lines and discovered that TRIM8 can inhibit PEDV replication within host cells. Co-immunoprecipitation assays revealed that TRIM8 directly interacts with the nucleocapsid protein (N) of PEDV, specifically within the coiled-coil structural domain of TRIM8. Furthermore, TRIM8 was shown to reduce the expression of the PEDV N protein in a dose-dependent manner. Mechanistically, TRIM8 inhibits the expression of PEDV N through K48-linked ubiquitin proteasome degradation. Transcriptomics analysis revealed that TRIM8 facilitates the expression of genes associated with several pathways, including the IL-17 signalling pathway, chemokine signalling pathway, and cytokine-cytokine receptor interaction. This suggests that TRIM8 plays a crucial role in boosting antiviral immune responses against PEDV infection. Our findings provide new insights into the functions and mechanisms of TRIM8 in regulating PEDV infection and highlight its potential as a molecular target for the prevention and control of this virus., Competing Interests: Declarations. Ethics approval and consent to participate: This article does not involve any studies involving human participants or animals conducted by any of the authors. Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

Intracellular proteins take part in almost every body function; thus, protein homeostasis is of utmost importance. The ubiquitin proteasome system (UPS) has a fundamental role in protein homeostasis. Its main role is to selectively eradicate impaired or misfolded proteins, thus halting any damage that could arise from the accumulation of these malfunctioning proteins. Proteasomes have a critical role in controlling protein homeostasis in all cell types, including stem cells. We will discuss the role of UPS enzymes as well as the 26S proteasome complex in stem cell biology from several angles. First, we shall overview common trends of proteasomal activity and gene expression of different proteasomal subunits and UPS enzymes upon passaging and differentiation of stem cells toward various cell lineages. Second, we shall explore the effect of modulating proteasomal activity in stem cells and navigate through the interrelation between proteasomes' activity and various proteasome-related transcription factors. Third, we will shed light on curated microRNAs and long non-coding RNAs using various bioinformatics tools that might have a possible role in regulating UPS in stem cells and possibly, upon manipulation, can enhance the differentiation process into different lineages and/or delay senescence upon cell passaging. This will help to decipher the role played by individual UPS enzymes and subunits as well as various interrelated molecular mediators in stem cells' maintenance and/or differentiation and open new avenues in stem cell research. This can ultimately provide a leap toward developing novel therapeutic interventions related to proteasome dysregulation., (© 2025 International Union of Biochemistry and Molecular Biology.)

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis and antioxidative defenses, and it may play a critical role in Parkinson's disease (PD). F-box/WD repeat domain-containing protein (FBXW7), an E3 protein ligase, promotes the degradation of substrate proteins through the ubiquitin-proteasome system (UPS) and leads to the clearance of PGC-1α. Here, we elucidate a novel post-translational mechanism for regulating PGC-1α levels in neurons. We show that enhancing chaperone-mediated autophagy (CMA) activity promotes the CMA-mediated degradation of FBXW7 and consequently increases PGC-1α. We confirm the relevance of this pathway in vivo by showing decreased FBXW7 and increased PGC-1α as a result of boosting CMA selectively in dopaminergic (DA) neurons by overexpressing lysosomal-associated membrane protein 2A (LAMP2A) in TH-Cre-LAMP2-loxp conditional mice. We further demonstrate that these mice are protected against MPTP-induced oxidative stress and neurodegeneration. These results highlight a novel regulatory pathway for PGC-1α in DA neurons and suggest targeted increasing of CMA or decreasing FBXW7 in DA neurons as potential neuroprotective strategies in PD., (© 2024 Federation of European Biochemical Societies.)

Ubiquitination is one of the most important post-translational modifications (PTMs) and involves the covalent attachment of ubiquitin to a lysine residue on a target protein. Despite ubiquitination playing a crucial role in regulating cellular processes, the ubiquitinated proteome has not been studied extensively in recombinant Chinese hamster ovary (CHO) cells. Moreover, ubiquitination modification in CHO cells is likely to have an impact on protein function related to the efficient productivity of biopharmaceuticals. In this chapter, we describe a comprehensive protocol for ubiquitin di-Glycine (diGly) peptide enrichment using an immunoprecipitation method from recombinant CHO cell proteins followed by Liquid chromatography-Mass spectrometry (LC-MS) analysis of the ubiquitinated proteome. The methods described are also applicable to differential ubiquitinated proteomic studies., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Detecting strain-specific barcodes with mass spectrometry can facilitate the screening of genetically engineered bacterial libraries. Here, we introduce intact protein barcoding, a method to measure protein-based library barcodes and metabolites using flow injection mass spectrometry (FI-MS). Protein barcodes are based on ubiquitin with N-terminal tags of six amino acids. We demonstrate that FI-MS detects intact ubiquitin proteins and identifies the mass of N-terminal barcodes. In the same analysis, we measured relative concentrations of primary metabolites. We constructed six ubiquitin-barcoded CRISPR interference (CRISPRi) strains targeting metabolic enzymes and analyzed their metabolic profiles and ubiquitin barcodes. FI-MS detected barcodes and distinct metabolome changes in CRISPRi-targeted pathways. We demonstrate the scalability of intact protein barcoding by measuring 132 ubiquitin barcodes in microtiter plates. These results show that intact protein barcoding enables fast and simultaneous detection of library barcodes and intracellular metabolites, opening up new possibilities for mass spectrometry-based barcoding., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The airborne fungus Aspergillus fumigatus is a major pathogen that poses a serious health threat to humans by causing aspergillosis. Azole antifungals inhibit sterol 14-demethylase (encoded by cyp51A), an enzyme crucial for fungal cell survival. However, the most common mechanism of azole resistance in A. fumigatus is associated with the mutations in cyp51A and tandem repeats in its promoter, leading to reduced drug-enzyme interaction and overexpression of cyp51A. It remains unknown whether post-translational modifications of Cyp51A contribute to azole resistance. In this study, we report that the Cyp51A expression is highly induced upon exposure to itraconazole, while its ubiquitination level is significantly reduced by itraconazole. Loss of the ubiquitin-conjugating enzyme Ubc7 confers resistance to multiple azole antifungals but hinders hyphal growth, conidiation, and virulence. Western blot and immunoprecipitation assays show that deletion of ubc7 reduces Cyp51A degradation by impairing its ubiquitination, thereby leading to drug resistance. Most importantly, the overexpression of ubc7 in common environmental and clinical azole-resistant cyp51A isolates partially restores azole sensitivity. Our findings demonstrate a non-cyp51A mutation-based resistance mechanism and uncover a novel role of post-translational modification in contributing to azole resistance in A. fumigatus., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ling Lu reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The human ataxin-3 protein contains an N-terminal Josephin domain, composed of a papain-like cysteine protease with a helical hairpin insertion, and a C-terminal region with two or three ubiquitin interacting motifs and a polyglutamine tract. Expansion of the polyglutamine tract leading to protein aggregation and neuronal degradation has been linked to Machado-Joseph disease/spinocerebellar ataxia type 3, the most common form of dominantly inherited ataxia. In this study, we performed sequence self-homology dot plot analysis and compared orthologous proteins to analyze the architecture of ataxin-3 during the evolution of Filozoa. This analysis uncovered up to three additional repetitions of the ubiquitin binding motif in ataxin-3, including the helical hairpin insertion in the Josephin domain, and revealed a highly conserved multimodular architecture that is broadly preserved throughout the Filozoa. Overall, a set of 78 putative ubiquitin binding repeats from 18 exemplar proteins were identified. Apparent neofunctionalization events could also be recognized, including modification of repeat 5 which gave rise to the disease-linked polyglutamine tract, just before the Sarcopterygian divergence. This model provides a unifying principle for the ataxin-3 protein architecture and can potentially provide new insights into the role of molecular interactions in ataxin-3 function and Machado-Joseph disease/spinocerebellar ataxia type 3 disease mechanisms., (© 2024 The Protein Society.)

Autophagy is an intracellular degradation mechanism by which cytoplasmic materials are delivered to and degraded in the lysosome-fused autophagosome (autolysosome) and proposed to have been established at an early stage of eukaryotic evolution. Dinoflagellates harboring endosymbiotic diatoms (so-called "dinotoms"), which retain their own nuclei and mitochondria in addition to plastids, have been investigated as an intermediate toward the full integration of a eukaryotic phototroph into the host-controlled organelle (i.e., plastid) through endosymbiosis. Pioneering studies systematically evaluated the degree of host governance on several metabolic pathways in the endosymbiotic diatoms (ESDs). However, little attention has been paid to the impact of the endosymbiotic lifestyle on the autophagy operated in the ESDs. In this study, we searched for ATG3, ATG4, ATG5, ATG7, ATG8, ATG10, and ATG12, which are required for autophagosome formation, in the RNA-seq data from dinotoms Durinskia baltica and Kryptoperidinium foliaceum. We detected two evolutionally distinct sets of the ATG proteins in the dinotom species, one affiliated with the dinoflagellate homologs and the other with the diatom homologs in phylogenetic analyses. The results suggest that the ATG proteins descended from the diatom taken up by the dinoflagellate host persist for autophagosome formation and, most likely, autophagy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Ubiquitination is one kind of crucial protein post-translational modification (PTM) in eukaryotic cells, forming sorts of types of polyubiquitin chain linkages on the substrates. Ubiquitin conjugating enzymes (E2s) play an essential role in the formation of ubiquitin chains. However, the mechanism of the formation of atypical chain (such as K27) and the identification of the related substrates are not well understood. Previously we developed an orthogonal ubiquitin transfer (OUT) pathway to identify the substrates of a specific ubiquitin ligase (E3). In OUT pathway, a ubiquitin mutant (xUb) is transferred to the substrates through an engineered xE1-xE2-xE3 cascade. In this study, we reengineered a new OUT pathway for the transfer of xUb-K27, an isoform of xUb with only one lysine at Lys27 resident. The newly designed xUba1-xUbe2D2 (xE1-xE2) pairs can transfer xUb-K27 to downstream wild type E3s and form K27 linkages. Ube2D2 is known for its versatility as it forms all sorts of polyubiquitin chains on substrates. Therefore, the xE1-xE2 pairs are empowered to transfer other ubiquitin mutants, for instance, xUb-K6 and xUb-K11. The new xE1-xE2 pairs also deepen the understanding of structural information about the E1-E2 interaction, and provide further insights into the mechanism of chain formation mediated by E2., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The ubiquitin-conjugating E2 enzymes play a central role in ubiquitin transfer. Disruptions to the ubiquitin system are implicated in multiple diseases, and as a result, molecules that modulate the activity of the ubiquitin system are of interest. E2 enzyme function relies on interactions with partner proteins, and the disruption of these is an effective way to modulate activity. Here, we report the discovery of ubiquitin variants (UbVs) that inhibit the E2 enzyme, Ube2d2 (UbcH5b). The six UbVs identified inhibit ubiquitin chain building, and the structural and biophysical characterisation of two of these demonstrate they bind to Ube2d2 with low micromolar affinity and high specificity. Both characterised UbVs bind at a site that overlaps with E1 binding, while the more inhibitory UbV has an additional binding site that blocks a critical non-covalent ubiquitin-binding site on the E2 enzyme. The discovery of novel protein-based ubiquitin derivatives that inhibit protein-protein interactions is an important step towards discovering small molecules that inhibit the activity of E2 enzymes. Furthermore, the specificity of the UbVs within the Ube2d family suggests that it may be possible to develop tools to selectively inhibit highly related E2 enzymes., (© 2024 Federation of European Biochemical Societies.)

Ubiquitin and ubiquitin-like (UUL) modifications play pleiotropic functions and are subject to fine regulatory mechanisms frequently altered in cancer. However, the comprehensive impact of UUL modification on breast cancer remains unclear. Transcriptomic and clinical data of breast cancer were downloaded from TCGA and GEO databases. Molecular subtyping of breast cancer was conducted using the NMF and CIBERSORT algorithms. Prognostic genes were identified via univariate, lasso and multivariate Cox regression analyses. Clinical pathological features, immune cell infiltration, immune therapeutic response and chemotherapy drug sensitivity were compared between groups using the Wilcoxon test. Survival analysis was performed using the Kaplan-Meier method and log-rank test. A total of 63 UUL modification-related genes were differentially expressed, with 29 up-regulated and 34 down-regulated genes. These genes were used to generate two UUL modification patterns that exhibited significant differences in prognostic features and immune cell infiltration. The UUL modification patterns were associated with 2038 differentially expressed genes that were significantly enriched in nuclear division, chromosome segregation, neuroactive ligand-receptor interaction, cell cycle, and other biological processes. Of these genes, 425 were associated with breast cancer prognosis, which enabled the classification of breast cancer into two clusters with significantly distinct prognoses. We developed a prognostic model, UULscore, which comprised nine genes and showed a significant correlation with partial immune cell infiltration. Furthermore, UULscore demonstrated potential predictive value in breast cancer overall survival prediction, immune therapeutic response, and chemotherapy drug sensitivity. UULscore, stage, radiotherapy, and chemotherapy were identified as independent prognostic factors for breast cancer. Based on these factors, a nomogram model was constructed, which demonstrated exceptional prognostic predictive performance. The present study identified two UUL modification-derived molecular subtypes in breast cancer, and have successfully constructed a risk-scoring model that holds potential value in prognosis, immune infiltration, immune therapeutic response, and chemotherapy sensitivity.

Automation of protein purification methods can increase researchers' efficiency in life sciences. However, currently reported automated protein purification methods require cost-intensive fast protein liquid chromatography systems, such as ÄKTA pure and ÄKTA explorer, without any reported application to the more cost-efficient entry-level system, ÄKTA go. To fill this gap, here we propose a fast, efficient, and versatile automated protein purification strategy for the ÄKTA go. Straightforward integration of two additional accessories, a column valve and a sample loop, into the default ÄKTA go system and making minor rearrangements of flow lines, enabled automation of multi-step protein purification processes. Utilizing this established system, we demonstrate the automated purification of three distinct types of proteins: ubiquitin, polyhistidine-tagged talin, and GST-tagged human rhinovirus 14 3C protease. The described automation strategy is suitable even for small budget-conscious laboratories operating on ÄKTA go systems, thus reducing researchers' time and efforts spent on routine sample preparation tasks of their investigations., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency., (© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.)

The covalent attachment of Ub (ubiquitin) to target proteins (ubiquitylation) represents one of the most versatile PTMs (post-translational modifications) in eukaryotic cells. Substrate modifications range from a single Ub moiety being attached to a target protein to complex Ub chains that can also contain Ubls (Ub-like proteins). Ubiquitylation plays pivotal roles in most aspects of eukaryotic biology, and cells dedicate an orchestrated arsenal of enzymes to install, translate, and reverse these modifications. The entirety of this complex system is coined the Ub code. Deciphering the Ub code is challenging due to the difficulty in reconstituting enzymatic machineries and generating defined Ub/Ubl-protein conjugates. This Review provides a comprehensive overview of recent advances in using GCE (genetic code expansion) techniques to study the Ub code. We highlight strategies to site-specifically ubiquitylate target proteins and discuss their advantages and disadvantages, as well as their various applications. Additionally, we review the potential of small chemical PTMs targeting Ub/Ubls and present GCE-based approaches to study this additional layer of complexity. Furthermore, we explore methods that rely on GCE to develop tools to probe interactors of the Ub system and offer insights into how future GCE-based tools could help unravel the complexity of the Ub code.

Cells respond to invading pathogens and danger signals from the environment by adapting gene expression to meet the need for protective effector molecules. While this innate immune response is required for the cell and the organism to recover, excess immune activation may lead to loss of homeostasis, thereby promoting chronic inflammation and cancer progression. The molecular basis of innate immune defence is comprised of factors promoting survival and proliferation, such as cytokines, antimicrobial peptides and anti-apoptotic proteins. As the molecular mechanisms regulating innate immune responses are conserved through evolution, the fruit fly Drosophila melanogaster serves as a convenient, affordable and ethical model organism to enhance understanding of immune signalling. Fly immunity against bacterial infection is built up by both cellular and humoral responses, where the latter is regulated by the Imd and Toll pathways activating NF-κB transcription factors Relish, Dorsal and Dif, as well as JNK activation and JAK/STAT signalling. As in mammals, the Drosophila innate immune signalling pathways are characterised by ubiquitination of signalling molecules followed by ubiquitin receptors binding to the ubiquitin chains, as well as by rapid changes in protein levels by ubiquitin-mediated targeted proteasomal and lysosomal degradation. In this review, we summarise the molecular signalling pathways regulating immune responses to pathogen infection in Drosophila, with a focus on ubiquitin-dependent control of innate immunity and inflammatory signalling., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

It is estimated that there are 544.9 million people suffering from chronic respiratory diseases in the world, which is the third largest chronic disease. Although there are various clinical treatment methods, there is no specific drug for chronic pulmonary diseases, including chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD) and idiopathic pulmonary fibrosis (IPF). Therefore, it is urgent to clarify the pathological mechanism and medication development. Single-cell transcriptome data of human and mouse from GEO database were integrated by "Harmony" algorithm. The data was standardized and normalized by using "Seurat" package, and "SingleR" algorithm was used for cell grouping annotation. The "Findmarker" function is used to find differentially expressed genes (DEGs), which were enriched and analyzed by using "clusterProfiler", and a protein interaction network was constructed for DEGs, and four algorithms are used to find the hub genes. The expression of hub genes were analyzed in independent human and mouse single-cell transcriptome data. Bulk RNA data were used to integrate by the "SVA" function, verify the expression levels of hub genes and build a diagnostic model. The L1000FWD platform was used to screen potential drugs. Through exploring the similarities and differences by integrated single-cell atlas, we found that the lung parenchymal cells showed abnormal oxidative stress, cell matrix adhesion and ubiquitination in COPD, corona virus disease 2019 (COVID-19), ILD and IPF. Meanwhile, the lung resident immune cells showed abnormal Toll-like receptor signals, interferon signals and ubiquitination. However, unlike acute pneumonia (COVID-19), chronic pulmonary disease shows enhanced ubiquitination. This phenomenon was confirmed in independent external human single-cell atlas, but unfortunately, it was not confirmed in mouse single-cell atlas of bleomycin-induced pulmonary fibrosis model and influenza virus-infected mouse model, which means that the model needs to be optimized. In addition, the bulk RNA-Seq data of COVID-19, ILD and IPF was integrated, and we found that the immune infiltration of lung tissue was enhanced, consistent with the single-cell level, UBA52, UBB and UBC were low expressed in COVID-19 and high expressed in ILD, and had a strong correlation with the expression of cell matrix adhesion genes. UBA52 and UBB have good diagnostic efficacy, and salermide and SSR-69071 can be used as their candidate drugs. Our study found that the disorder of protein ubiquitination in chronic pulmonary diseases is an important cause of pathological phenotype of pulmonary fibrosis by integrating scRNA-Seq and bulk RNA-Seq, which provides a new horizons for clinicopathology, diagnosis and treatment., (© 2024. The Author(s).)

Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants., (© The Author(s) 2024. Published by Oxford University Press.)

Thermal variations due to global climate change are expected to modify the distributions of marine ectotherms, with potential pathogen translocations. This is of particular concern at high latitudes where cold-adapted stenothermal fish such as the Notothenioids occur. However, little is known about the combined effects of thermal fluctuations and immune challenges on the balance between cell damage and repair processes in these fish. The aim of this study was to determine the effect of thermal variation on specific genes involved in the ubiquitination and apoptosis pathways in two congeneric Notothenioid species, subjected to simulated bacterial and viral infections. Adult fish of Harpagifer bispinis and Harpagifer antarcticus were collected from Punta Arenas (Chile) and King George Island (Antarctica), respectively, and distributed as follows: injected with PBS (control), LPS (2.5 mg/kg) or Poly I:C (2 mg/kg) and then submitted to 2, 5 and 8 °C. After 1 week, samples of gills, liver and spleen were taken to evaluate the expression by real-time PCR of specific genes involved in ubiquitination (E3-ligase enzyme) and apoptosis (BAX and SMAC/DIABLO). Gene expression was tissue-dependent and increased with increasing temperature in the gills and liver while showing an opposite pattern in the spleen. Studying a pair of sister species that occur across the Antarctic Polar Front can help us understand the particular pressures of intertidal lifestyles and the effect of temperature in combination with biological stressors on cell damage and repair capacity in a changing environment., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Ubiquitin modification and alternative polyadenylation play crucial roles in the onset and progression of cancer. Hence, this study aims to comprehensively and deeply understand gene regulation and associated biological processes in lung adenocarcinoma (LUAD) by integrating both mechanisms. Alternative polyadenylation (APA)-related E3 ubiquitin ligases in LUAD were identified through multiple databases, and the association between selected genetic loci influencing gene expression (apaQTL-SNPs) and LUAD risk were evaluated through the GWAS database of the Female Lung Cancer Consortium in Asia (FLCCA). Subsequently, the interaction between RNF213 and ZBTB20, as well as their functional mechanisms in LUAD, were investigated using bioinformatics analysis, Western blot, co-immunoprecipitation, and colony formation experiments. A total of five apaQTL-SNPs (rs41301932, rs4494603, rs9890400, rs56066320, and rs41301932), located on RNF213, were significantly associated with LUAD risk ( p < 0.05), and they inhibit tumor growth through ubiquitin-mediated degradation of ZBTB20.

Background: Ubiquitin/ubiquitin-like (Ub/UBL)-related genes have been reported to be associated with the survival of osteosarcoma patients but have not yet been systematically explored., Methods: The prognostic value of Ub/UBL-related genes, immune cell infiltration and clinicopathological features of patients were explored by Cox and LASSO regression analyses. A prognostic model was established and then validated in the GSE21257 dataset. The differential expression of hub genes in osteosarcoma was confirmed by qRT-PCR, western blotting and immunohistochemistry., Results: Tripartite Motif Containing 8 (TRIM8) and Ubiquitin Like With PHD And Ring Finger Domains 2 (UHRF2) were screened as genes with prognostic value in osteosarcoma. Kaplan-Meier analysis and scatter plots indicated that patients in the high gene significance score group tended to have a worse prognosis. The concordance index, calibration analysis and receiver operating characteristic analysis suggested that the model had good prediction accuracy and high sensitivity and specificity. Decision curve analysis revealed that patients could obtain greater net benefit from this model. Functional analyses of the differentially expressed genes indicated that they were involved in important functions and pathways. TRIM8 and UHRF2 were confirmed to be highly expressed in osteosarcoma cell lines and tissues., Conclusions: TRIM8 and UHRF2 are potential prognostic genes in osteosarcoma, and these results provide insights into the roles of these genes and their implications for patient outcomes., (© 2024. The Author(s).)

Deciphering ubiquitin proteoform signaling and its role in disease has been a long-standing challenge in the field. The effects of ubiquitin modifications, its relation to ubiquitin-related machineries, and its signaling output has been particularly limited by its reconstitution and means of characterization. Advances in genetic code expansion have contributed towards addressing these challenges by precision incorporation of unnatural amino acids through site selective codon suppression. This review discusses recent advances in studying the 'writers', 'readers', and 'erasers' of the ubiquitin code using genetic code expansion. Highlighting strategies towards genetically encoded protein ubiquitination, ubiquitin phosphorylation, acylation, and finally surveying ubiquitin interactions, we strive to bring attention to this unique approach towards addressing a widespread proteoform problem., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Eukaryotic transcription is a finely orchestrated process and it is controlled by transcription factors as well as epigenetic regulators. Transcription factors and epigenetic regulators undergo different types of posttranslational modifications including ubiquitination to control transcription process. Ubiquitination, traditionally associated with protein degradation, has emerged as a crucial contributor to the regulation of chromatin structure through ubiquitination of histone and chromatin remodelers. Ubiquitination introduces new layers of intricacy to the regulation of transcription initiation through controlling the equilibrium between euchromatin and heterochromatin states. Nucleosome, the fundamental units of chromatin, spacing in euchromatin and heterochromatin states are regulated by histone modification and chromatin remodeling complexes. Chromatin remodeling complexes actively sculpt the chromatin architecture and thereby influence the transcriptional states of genes. Therefore, understanding the dynamic behavior of nucleosome spacing is critical as it impacts various cellular functions through controlling gene expression profiles. In this comprehensive review, we discussed the intricate interplay between ubiquitination and transcription initiation, and illuminated the underlying molecular mechanisms that occur in a variety of biological contexts. This exploration sheds light on the complex regulatory networks that govern eukaryotic transcription, providing important insights into the fine orchestration of gene expression and chromatin dynamics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ligase complex in which the ubiquitin-like (UBL) domains of SHARPIN and HOIL-1L interact with HOIP to determine the structural stability of LUBAC. The interactions between subunits within LUBAC have been a topic of extensive research. However, the impact of the LTM motif on the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP remains unclear. Here, we discover that the absence of the LTM motif in the AlphaFold2-predicted LUBAC structure alters the HOIP-UBA structure. We employ GeoPPI to calculate the changes in binding free energy (ΔG) caused by single-point mutations between subunits, simulating their protein-protein interactions. The results reveal that the presence of the LTM motif decreases the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP, leading to a decrease in the structural stability of LUBAC. Furthermore, using the AlphaFold2-predicted results, we find that HOIP (629‒695) and HOIP-UBA bind to both sides of HOIL-1L-UBL, respectively. The experiments of Gromacs molecular dynamics simulations, SPR and ITC demonstrate that the elongated domain formed by HOIP (629‒695) and HOIP-UBA, hereafter referred to as the HOIP (466‒695) structure, interacts with HOIL-1L-UBL to form a structurally stable complex. These findings illustrate the collaborative interaction between HOIP-UBA and HOIP (629‒695) with HOIL-1L-UBL, which influences the structural stability of LUBAC.

Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer., (© 2024 The Author(s).)

The nuclear pore complex (NPC) mediates the selective exchange of macromolecules between the nucleus and the cytoplasm. Neurodegenerative diseases such as amyotrophic lateral sclerosis are characterized by mislocalization of nucleoporins (Nups), transport receptors, and Ras-related nuclear proteins into nucleoplasmic or cytosolic aggregates, underscoring the importance of precise assembly of the NPC. The assembly state of large protein complexes is strictly monitored by the protein quality control system. The ubiquitin-proteasome system may eliminate aberrant, misfolded, and/or orphan components; however, the involvement of the ubiquitin-proteasome system in the degradation of nonnative Nups in the NPC remains unclear. Here, we show that in Saccharomyces cerevisiae, although Nup1 (the FG-Nup component of the central core of the NPC) was stable, C-terminally green fluorescent protein-tagged Nup1, which had been incorporated into the NPC, was degraded by the proteasome especially under heat stress conditions. The degradation was dependent on the San1 ubiquitin ligase and Cdc48/p97, as well as its cofactor Doa1. We also demonstrate that San1 weakly but certainly contributes to the degradation of nontagged endogenous Nup1 in cells defective in NPC biogenesis by the deletion of NUP120. In addition, the overexpression of SAN1 exacerbated the growth defect phenotype of nup120Δ cells, which may be caused by excess degradation of defective Nups due to the deletion of NUP120. These biochemical and genetic data suggest that San1 is involved in the degradation of nonnative Nups generated by genetic mutation or when NPC biogenesis is impaired., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFβ signaling., Methods: Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments., Results: rs34195470 genotype associates with differential methylation at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140., Conclusions: As far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFβ signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk., (© 2024. The Author(s).)

The ubiquitin signaling cascade plays a crucial role in human cells. Consistent with this, malfunction of ubiquitination and deubiquitination is implicated in the initiation and progression of numerous human diseases, including cancer. Therefore, the development of potent and specific modulators of ubiquitin signal transduction has been at the forefront of drug development. In the past decade, a structure-based combinatorial protein-engineering approach has been used to generate ubiquitin variants (UbVs) as protein-based modulators of multiple components in the ubiquitin-proteasome system. Here, we review the design and generation of phage-displayed UbV libraries, including the processes of binder selection and library improvement. We also provide a comprehensive overview of the general in vitro and cellular methodologies involved in characterizing UbV binders. Finally, we describe two recent applications of UbVs for developing molecules with therapeutic potential., (© 2024 Cold Spring Harbor Laboratory Press.)

The E2 ubiquitin conjugator Rad6 is required for DNA damage bypass in budding yeast but remain functionally unknown in filamentous fungi. Here, we report pleiotropic effect of Rad6 ortholog in Beauveria bassiana, a wide-spectrum fungal insecticide. Global ubiquitination signal was greatly attenuated in the absence of rad6. The blocked ubiquitination led to severe growth defect, blocked asexual development, and abolished infectivity/insect pathogenicity, which correlated with compromised conidial quality (including viability, hydrophobicity, adherence to insect cuticle, and thermotolerance) and blocked secretion of cuticle-degrading enzymes including Pr1 family proteases. Importantly, Rad6 played much greater role in photoreactivation of UVB-impaired conidia by a 3- or 5-h light plus 9- or 7-h dark incubation than in dark reactivation of those impaired conidia by a 12-h dark incubation. The high activity of Rad6 in photoreactivation in vivo was derived from its link to a protein complex cored by the photolyase regulators WC1 and WC2 via the strong interactions of Rad6 with the E3 partner Rad18 and Rad18 with WC2 revealed in yeast two-hybrid assays. Transcriptomic analysis resulted in identification of 2700 differentially regulated genes involved in various function categories and metabolism pathways, indicating a regulatory role of Rad6-mediated ubiquitination in gene expression networks and genomic stability. Conclusively, Rad6 is required for asexual and insect-pathogenic lifecycles, solar UV damage repair, and genomic expression of B. bassiana. The primary dependence of its strong anti-UV role on photoreactivation in vivo unveils a scenario distinct from the core role of its yeast ortholog in DNA damage bypass., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1 , show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin-binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome-Rpn1, Rpn10, and Rpn13-and the Ubl domains of Rad23 and Dsk2 are critical under different condensate-inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes., Competing Interests: Competing interests statement:The authors declare no competing interest.

K63-linked ubiquitin chains attached to plasma membrane proteins serve as tags for endocytosis and endosome-to-lysosome sorting. USP8 is an essential deubiquitinase for the maintenance of endosomal functions. Prolonged depletion of USP8 leads to cell death, but the major effects on cellular signaling pathways are poorly understood. Here, we show that USP8 depletion causes aberrant accumulation of K63-linked ubiquitin chains on endosomes and induces immune and stress responses. Upon USP8 depletion, two different decoders for K63-linked ubiquitin chains, TAB2/3 and p62, were recruited to endosomes and activated the TAK1-NF-κB and Keap1-Nrf2 pathways, respectively. Oxidative stress, an environmental stimulus that potentially suppresses USP8 activity, induced accumulation of K63-linked ubiquitin chains on endosomes, recruitment of TAB2, and expression of the inflammatory cytokine. The results demonstrate that USP8 is a gatekeeper of misdirected ubiquitin signals and inhibits immune and stress response pathways by removing K63-linked ubiquitin chains from endosomes., (© 2024 Endo et al.)

A key signalling pathway required for clearance of viruses from host cells relies on the receptor protein, retinoic acid-inducible gene I (RIG-I). The activity of RIG-I is tightly controlled, and once bound to viral dsRNA, addition of lysine 63-linked ubiquitin chains activates signalling. Meanwhile, the addition of lysine 48-linked ubiquitin chains to RIG-I is required to terminate signalling when the infection has been resolved. Really interesting new gene (RING) finger protein 125 (RNF125) is the E3 ligase responsible for addition of the ubiquitin chains that terminate signalling, with disruption of its function associated with Tenorio syndrome. Here we describe a novel RNF125 gene variant in an individual with clinical symptoms including intellectual disability, macrocephaly and congenital heart disease, consistent with Tenorio syndrome. The newly identified Tenorio syndrome-associated variant [(NM&#95;017831.4):c.670G>C p.Glu224Gln] is the first to be found in the ubiquitin interaction motif (UIM) of RNF125. While the E3 ligase activity of this RNF125 variant is retained, it has an impaired ability to interact with lysine 63-linked ubiquitin chains. The function of the UIM in RNF125 is uncertain; however, this study suggests that the UIM binds lysine 63-linked ubiquitin chains, and that this interaction is required for the normal function of RNF125., (© 2023 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

N 6 methyladenosine (m 6 A) is the most prevalent RNA epigenetic modification, regulated by methyltransferases and demethyltransferases and recognized by methylation-related reading proteins to impact mRNA splicing, translocation, stability, and translation efficiency. It significantly affects a variety of activities, including stem cell maintenance and differentiation, tumor formation, immune regulation, and metabolic disorders. Ubiquitination refers to the specific modification of target proteins by ubiquitin molecule in response to a series of enzymes. E3 ligases connect ubiquitin to target proteins and usually lead to protein degradation. On the contrary, deubiquitination induced by deubiquitinating enzymes (DUBs) can separate ubiquitin and regulate the stability of protein. Recent studies have emphasized the potential importance of ubiquitination and deubiquitination in controlling the function of m 6 A modification. In this review, we discuss the impact of ubiquitination and deubiquitination on m 6 A functional molecules in diseases, such as metabolism, cellular stress, and tumor growth., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Aim: Circular ubiquitin-like, containing PHD and ring finger domains 1 (circUHRF1) is aberrantly upregulated in human hepatocellular carcinoma (HCC) tissues. However, the underlying molecular mechanisms remain obscure. The present study aimed at elucidating the interactive function of circUHRF1-G9a-ubiquitin-like, containing PHD and ring finger domains 1 (UHRF1) mRNA-eukaryotic translation initiation factor 4A3 (EIF4A3)-PDZ and LIM domain 1 (PDLIM1) network in HCC., Methods: Expression of circUHRF1, mRNAs of G9a, UHRF1, PDLIM1, epithelial-mesenchymal transition (EMT)-related proteins, and Hippo-Yap pathway components was determined by quantitative polymerase chain reaction (Q-PCR), immunofluorescence, or Western blot analysis. Tumorigenic and metastatic capacities of HCC cells were examined by cellular assays including Cell Counting Kit-8, colony formation, wound healing, and transwell assays. Molecular interactions between EIF4A3 and UHRF1 mRNA were detected by RNA pull-down experiment. Complex formation between UHRF1 and PDLIM1 promoter was detected by chromatin immunoprecipitation assay. Co-immunoprecipitation was performed to examine the binding between UHRF1 and G9a., Results: Circular ubiquitin-like, containing PHD and ring finger domains 1, G9a, and UHRF1 were upregulated, while PDLIM1 was downregulated in HCC tissue samples and cell lines. Cellular silencing of circUHRF1 repressed HCC proliferation, invasion, migration, and EMT. G9a formed a complex with UHRF1 and inhibited PDLIM1 transcription., Conclusion: Eukaryotic translation initiation factor 4A3 regulated circUHRF1 expression by binding to UHRF1 mRNA promoter. circUHRF1 increased the stability of G9a and UHRF1 mRNAs through recruiting EIF4A3. Overexpression of circUHRF1 aggravated HCC progression through Hippo-Yap pathway and PDLIM1 inhibition. By elucidating the molecular function of circUHRF1-G9a-UHRF1 mRNA-EIF4A3-PDLIM1 network, our data shed light on the HCC pathogenesis and suggest a novel therapeutic strategy for future HCC treatment., (© 2023 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48–UN complex and its assembly. The Lys48-linked polyubiquitin-mediated proteasomal degradation in yeast depends on Cdc48 and its cofactors Ufd1 and Npl4. Here, the authors present crystal structures of Npl4 bound to Lys48-linked diubiquitin and the Npl4-binding motif of Ufd1, providing insights into the reaction mechanism of the Cdc48- Ufd1/Npl4 complex. [ABSTRACT FROM AUTHOR]

Hereditary spastic paraplegias refer to a heterogeneous group of neurodegenerative disorders resulting from degeneration of the corticospinal tract. Clinical characterization of patients with hereditary spastic paraplegias represents progressive spasticity, exaggerated reflexes and muscular weakness. Here, to expand on the increasingly broad pools of previously unknown hereditary spastic paraplegia causative genes and subtypes, we performed whole exome sequencing for six affected and two unaffected individuals from two unrelated Chinese families with an autosomal dominant hereditary spastic paraplegia and lacking mutations in known hereditary spastic paraplegia implicated genes. The exome sequencing revealed two stop-gain mutations, c.247_248insGTGAATTC (p.I83Sfs*11) and c.526G>T (p.E176*), in the ubiquitin-associated protein 1 (UBAP1) gene, which co-segregated with the spastic paraplegia. We also identified two UBAP1 frameshift mutations, c.324_325delCA (p.H108Qfs*10) and c.425_426delAG (p.K143Sfs*15), in two unrelated families from an additional 38 Chinese pedigrees with autosomal dominant hereditary spastic paraplegias and lacking mutations in known causative genes. The primary disease presentation was a pure lower limb predominant spastic paraplegia. In vivo downregulation of Ubap1 in zebrafish causes abnormal organismal morphology, inhibited motor neuron outgrowth, decreased mobility, and shorter lifespan. UBAP1 is incorporated into endosomal sorting complexes required for transport complex I and binds ubiquitin to function in endosome sorting. Patient-derived truncated form(s) of UBAP1 cause aberrant endosome clustering, pronounced endosome enlargement, and cytoplasmic accumulation of ubiquitinated proteins in HeLa cells and wild-type mouse cortical neuron cultures. Biochemical and immunocytochemical experiments in cultured cortical neurons derived from transgenic Ubap1flox mice confirmed that disruption of UBAP1 leads to dysregulation of both early endosome processing and ubiquitinated protein sorting. Strikingly, deletion of Ubap1 promotes neurodegeneration, potentially mediated by apoptosis. Our study provides genetic and biochemical evidence that mutations in UBAP1 can cause pure autosomal dominant spastic paraplegia. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved mechanism to overcome various stresses and recycle cytoplasmic components and organelles. Ubiquitin-like (UBL) protein Atg12 is a key protein involved in autophagosome formation through stimulation of Atg8 conjugation to phosphatidylethanolamine. Here, we describe the identification of the autophagy-related gene Acatg12, encoding an Atg12 homologous protein in the cephalosporin C producing fungus Acremonium chrysogenum. Disruption of Acatg12 impaired the delivery and degradation of eGFP-Atg8, indicating that the autophagic process was blocked. Meanwhile, conidiation was dramatically reduced in the Acatg12 disruption mutant (∆Acatg12). In contrast, cephalosporin C production was increased twofold in ∆Acatg12, but fungal growth was reduced after 6 days fermentation. Consistent with these results, the transcriptional level of the cephalosporin biosynthetic genes was increased in ∆Acatg12. The results extend our understanding of autophagy in filamentous fungi. [ABSTRACT FROM AUTHOR]

Lung adenocarcinoma (LUAD) is the most common pathological type of lung cancer, but the early diagnosis rate is low. The RNA-binding ubiquitin ligase MEX3C promotes tumorigenesis in several cancers but its mechanism of action in LUAD is unclear. In this study, the biological activity of MEX3C was assessed in LUAD. MEX3C and RUNX3 mRNA levels in the tissues of LUAD patients were determined using reverse transcription‑quantitative PCR. The involvement of MEX3C in the growth and metastasis of LUAD cells was measured by EdU assay, CCK-8, colony formation, Transwell assay, TUNEL, and flow cytometry. Expression of apoptosis and epithelial-mesenchymal transition related proteins were determined using western blotting analysis. LUAD cells transfected with si-MEX3C were administered to mice subcutaneously to monitor tumor progression and metastasis. We found that MEX3C is strongly upregulated in LUAD tissue sections, and involved in proliferation and migration. A549 and H1299 cells had significantly higher levels of MEX3C expression compared to control HBE cells. Knockdown of MEX3C dramatically decreased cell proliferation, migration, and invasion, and accelerated apoptosis. Mechanistically, we demonstrate MEX3C induces ubiquitylation and degradation of tumor suppressor RUNX3. Moreover, RUNX3 transcriptionally represses Suv39H1, as revealed by RNA pull-down and chromatin immunoprecipitation assays. The in vivo mice model demonstrated that knockdown of MEX3C reduced LUAD growth and metastasis significantly. Collectively, we reveal a novel MEX3C-RUNX3-Suv39H1 signaling axis driving LUAD pathogenesis. Targeting MEX3C may represent a promising therapeutic strategy against LUAD., (© 2024. The Author(s).)

Parkinson's disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Loss-of-function mutations in the gene encoding the ubiquitin E3 ligase Parkin are causal for familial and juvenile PD. Among several therapeutic approaches being explored to treat or improve the prognosis of patients with PD, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy. A major barrier is the lack of high-throughput platforms for the robust and accurate quantification of Parkin activity in vitro. Here, we present two different and complementary Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/MS)-based approaches for the quantification of Parkin E3 ligase activity in vitro. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators., Competing Interests: Declaration of interests B.B. is a shareholder and CEO of Vincere Biosciences, Inc., focused on developing therapeutics targeting mitochondrial pathways. When this study was initiated, K.M. was the CSO at Vincere, accountable for drug discovery. K.M. is an advisor to The Michael J. Fox Foundation, serves on advisory panels at the National Institutes of Health, and consults for several biopharmaceutical companies. M.M.K.M. is a member of the scientific advisory board of Montara Therapeutics, Inc and scientific consultant at MSD. R.T. is an employee of the DUB Profiling Service within the MRC-PPU Reagents and Services (https://mrcppureagents.dundee.ac.uk/). C.J.H. is manager of the MRC-PPU Reagents and Services., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

E3 ubiquitin ligases play a pivotal role in ubiquitination, a crucial post-translational modification process. Anaphase-promoting complex (APC), a large cullin-RING E3 ubiquitin ligase, regulates the unidirectional progression of the cell cycle by ubiquitinating specific target proteins and triggering plant immune responses. Several E3 ubiquitin ligases have been identified owing to advancements in sequencing and annotation of the wheat genome. However, the types and functions of APC E3 ubiquitin ligases in wheat have not been reported. This study identified 14 members of the APC gene family in the wheat genome and divided them into three subgroups (CCS52B, CCS52A, and CDC20) to better understand their functions. Promoter sequence analysis revealed the presence of several cis-acting elements related to hormone and stress responses in the APC E3 ubiquitin ligases in wheat. All identified APC E3 ubiquitin ligase family members were highly expressed in the leaves, and the expression of most genes was induced by the application of methyl jasmonate (MeJA). In addition, the APC gene family in wheat may play a role in plant defense mechanisms. This study comprehensively analyzes APC genes in wheat, laying the groundwork for future research on the function of APC genes in response to viral infections and expanding our understanding of wheat immunity mechanisms.

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.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (β-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. β-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Paf1 (Polymerase-associated factor 1) complex (Paf1C) is evolutionarily conserved from yeast to humans, and facilitates transcription elongation as well as co-transcriptional histone covalent modifications and mRNA 3'-end processing. Thus, Paf1C is a key player in regulation of eukaryotic gene expression. Paf1C consists of Paf1, Cdc73, Ctr9, Leo1 and Rtf1 in both yeast and humans, but it has an additional component, Ski8, in humans. The abundances of these components regulate the assembly of Paf1C and/or its functions, thus implying the mechanisms involved in regulating the abundances of the Paf1C components in altered gene expression and hence cellular pathologies. Towards finding the mechanisms associated with the abundances of the Paf1C components, we analyzed here whether the Paf1C components are regulated via targeted ubiquitylation and 26S proteasomal degradation. We find that the Paf1C components except Paf1 do not undergo the 26S proteasomal degradation in both yeast and humans. Paf1 is found to be regulated by the ubiquitin-proteasome system (UPS) in yeast and humans. Alteration of such regulation changes Paf1's abundance, leading to aberrant gene expression. Intriguingly, while the Rtf1 component of Paf1C does not undergo the 26S proteasomal degradation, it is found to be ubiquitylated, suggesting that Rtf1 ubiquitylation could be engaged in Paf1C assembly and/or functions. Collectively, our results reveal distinct UPS regulation of the Paf1C components, Paf1 and Rtf1, in a proteolysis-dependent and -independent manners, respectively, with functional implications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The β-catenin has two intrinsically disordered regions in both C- and N-terminal domains that trigger the formation of phase-separated condensates. Variants in its C-terminus are associated with familial exudative vitreoretinopathy (FEVR), yet the pathogenesis and the role of these variants in inducing abnormal condensates, are unclear. In this study, we identified a novel heterozygous frameshift variant, c.2104-2105insCC (p.Gln703ProfsTer33), in CTNNB1 from a FEVR-affected family. This variant encodes an unstable truncated protein that was unable to activate Wnt signal transduction, which could be rescued by the inhibition of proteasome or phosphorylation. Further functional experiments revealed the propensity of the Gln703ProfsTer33 variant to form cytoplasmic condensates, exhibiting a lower turnover rate after fluorescent bleaching due to enhanced interaction with AXIN1. LiCl, which specifically blocks GSK3β-mediated phosphorylation, restored signal transduction, cell proliferation, and junctional integrity in primary human retinal microvascular endothelial cells over-expressed with Gln703ProfsTer33. Finally, experiments on two reported FEVR-associated mutations in the C-terminal domain of β-catenin exhibited several functional defects similar to the Gln703ProfsTer33. Together, our findings unravel that the C-terminal region of β-catenin is pivotal for the regulation of AXIN1/β-catenin interaction, acting as a switch to mediate nucleic and cytosolic condensates formation that is implicated in the pathogenesis of FEVR., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ubiquitin specific protease 5 (USP5) is a vital deubiquitinating enzyme that regulates various physiological functions by removing ubiquitin chains from target proteins. This review provides an overview of the structural and functional characteristics of USP5. Additionally, we discuss the role of USP5 in regulating diverse cellular processes, including cell proliferation, apoptosis, DNA double-strand damage, methylation, heat stress, and protein quality control, by targeting different substrates. Furthermore, we describe the involvement of USP5 in several pathological conditions such as tumors, pathological pain, developmental abnormalities, inflammatory diseases, and virus infection. Finally, we introduce newly developed inhibitors of USP5. In conclusion, investigating the novel functions and substrates of USP5, elucidating the underlying mechanisms of USP5-substrate interactions, intensifying the development of inhibitors, and exploring the upstream regulatory mechanisms of USP5 in detail can provide a new theoretical basis for the treatment of various diseases, including cancer, which is a promising research direction with considerable potential. Overall, USP5 plays a critical role in regulating various physiological and pathological processes, and investigating its novel functions and regulatory mechanisms may have significant implications for the development of therapeutic strategies for cancer and other diseases., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2024. The Author(s).)

The serine/threonine kinase PINK1 is responsible for phosphorylating a ubiquitin (Ub)-like domain in an E3 Ub ligase Parkin protein and a Parkin-bound Ub. PINK1 works as a mitochondrial quality control by phosphorylating and activating the E3 ubiquitin ligase Parkin. Recent medicinal study has reported that mutations of Parkin and PINK1 cause defects in mitophagy and induce early-onset Parkinson's disease (EOPD). In this study, we conducted molecular dynamics simulations to investigate the structural discrepancy caused by a clinical G409V mutation in PINK1 kinase domain's A-loop. The Ub phosphorylation begins with PINK1 D362 deprotonating the hydroxyl group of the substrate Ub's S65' and PINK1's A-loop is responsible for coordinating S65'. On contrary to G409 offering structural plasticity, the replaced, bulky V409 interferes with the alignment of D362-S65', seriously hampering Ub phosphorylation, leading to the accumulation of damaged mitochondria, and ultimately EOPD. In this study, we predicted the hPINK1 WT -Ub WT binding mode and detected the structural impact brought by G409V replacement. It is expected the concluded remarks to be beneficial for developing cures to alleviate structural interference and restore PINK1 function., Competing Interests: Declaration of competing interest We have no conflict of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Huntington's disease (HD) is a neurodegenerative disorder caused by a dominant gain-of-function mutation in the huntingtin gene, resulting in an elongated polyglutamine repeat in the mutant Huntingtin (mHtt) that mediates aberrant protein interactions. Previous studies implicated the ubiquitin-proteasome system in HD, suggesting that restoring cellular proteostasis might be a key element in suppressing pathology. We applied genetic interaction tests in a Drosophila model to ask whether modulating the levels of deubiquitinase enzymes affect HD pathology. By testing 32 deubiquitinase genes we found that overexpression of Yod1 ameliorated all analyzed phenotypes, including neurodegeneration, motor activity, viability, and longevity. Yod1 did not have a similar effect in amyloid beta overexpressing flies, suggesting that the observed effects might be specific to mHtt. Yod1 overexpression did not alter the number of mHtt aggregates but moderately increased the ratio of larger aggregates. Transcriptome analysis showed that Yod1 suppressed the transcriptional effects of mHtt and restored the expression of genes involved in neuronal plasticity, vesicular transport, antimicrobial defense, and protein synthesis, modifications, and clearance. Furthermore, Yod1 overexpression in HD flies leads to the upregulation of genes involved in transcriptional regulation and synaptic transmission, which might be part of a response mechanism to mHtt-induced stress., (© 2023. The Author(s).)