Search

Human APOBEC3G (A3G) is a virus restriction factor that inhibits HIV-1 replication and triggers lethal hypermutation on viral reverse transcripts. HIV-1 viral infectivity factor (Vif) breaches this host A3G immunity by hijacking a cellular E3 ubiquitin ligase complex to target A3G for ubiquitination and degradation. The molecular mechanism of A3G targeting by Vif-E3 ligase is unknown, limiting the antiviral efforts targeting this host-pathogen interaction crucial for HIV-1 infection. Here, we report the cryo-electron microscopy structures of A3G bound to HIV-1 Vif in complex with T cell transcription cofactor CBF-β and multiple components of the Cullin-5 RING E3 ubiquitin ligase. The structures reveal unexpected RNA-mediated interactions of Vif with A3G primarily through A3G's noncatalytic domain, while A3G's catalytic domain is poised for ubiquitin transfer. These structures elucidate the molecular mechanism by which HIV-1 Vif hijacks the host ubiquitin ligase to specifically target A3G to establish infection and offer structural information for the rational development of antiretroviral therapeutics.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

The timing and amplitude of plant signaling are frequently regulated through posttranslational modification of key signaling sectors, which facilitates rapid and flexible responses. Protein ubiquitination can serve as a degradation marker, influence subcellular localization, alter protein-protein interactions, and affect protein activity. Identification of polyubiquitinated proteins has been challenging due to their rapid degradation by the proteasome or removal of modifications by deubiquitination enzymes (DUBs). Tandem ubiquitin binding entities (TUBEs) are based on ubiquitin-associated domains and protect against both proteasomal degradation and DUBs. Here, we provide a protocol for purification of ubiquitinated plant proteins using TUBEs after transient expression in Nicotiana benthamiana. This protocol can also be applied to other plants to purify multiple ubiquitinated proteins or track ubiquitination of a target protein. This methodology provides an effective method for identification of ubiquitin ligase substrates and can be coupled with TUBEs targeting specific ubiquitination linkages.

The TINCR (Terminal differentiation-Induced Non-Coding RNA) gene is selectively expressed in epithelium tissues and is involved in the control of human epidermal differentiation and wound healing. Despite its initial report as a long non-coding RNA, the TINCR locus codes for a highly conserved ubiquitin-like microprotein associated with keratinocyte differentiation. Here we report the identification of TINCR as a tumor suppressor in squamous cell carcinoma (SCC). TINCR is upregulated by UV-induced DNA damage in a TP53-dependent manner in human keratinocytes. Decreased TINCR protein expression is prevalently found in skin and head and neck squamous cell tumors and TINCR expression suppresses the growth of SCC cells in vitro and in vivo. Consistently, Tincr knockout mice show accelerated tumor development following UVB skin carcinogenesis and increased penetrance of invasive SCCs. Finally, genetic analyses identify loss-of-function mutations and deletions encompassing the TINCR gene in SCC clinical samples supporting a tumor suppressor role in human cancer. Altogether, these results demonstrate a role for TINCR as protein coding tumor suppressor gene recurrently lost in squamous cell carcinomas.

The post-translational modification of proteins by ubiquitin-like modifiers (UbLs), such as SUMO, ubiquitin, and Nedd8, regulates a vast array of cellular processes. Dedicated UbL deconjugating proteases families reverse these modifications. During bacterial infection, effector proteins, including deconjugating proteases, are released to disrupt host cell defenses and promote bacterial survival. NopD, an effector protein from rhizobia involved in legume nodule symbiosis, exhibits deSUMOylation activity and, unexpectedly, also deubiquitination and deNeddylation activities. Here, we present two crystal structures of Bradyrhizobium (sp. XS1150) NopD complexed with either Arabidopsis SUMO2 or ubiquitin at 1.50 Å and 1.94 Å resolution, respectively. Despite their low sequence similarity, SUMO and ubiquitin bind to a similar NopD interface, employing a unique loop insertion in the NopD sequence. In vitro binding and activity assays reveal specific residues that distinguish between deubiquitination and deSUMOylation. These unique multifaceted deconjugating activities against SUMO, ubiquitin, and Nedd8 exemplify an optimized bacterial protease that disrupts distinct UbL post-translational modifications during host cell infection. The unique structural features of the NopD catalytic domain, an effector protease from symbiotic bacteria rhizobia, grant a surprising versatility to remove various regulatory tags (SUMO, ubiquitin, Nedd8) from host plant proteins. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics. [ABSTRACT FROM AUTHOR]

Rab GTPases are representative targets of manipulation by intracellular bacterial pathogens for hijacking membrane trafficking. Legionella pneumophila recruits many Rab GTPases to its vacuole and exploits their activities. Here, we found that infection-associated regulation of Rab10 dynamics involves ubiquitin signaling cascades mediated by the SidE and SidC families of Legionella ubiquitin ligases. Phosphoribosyl-ubiquitination of Rab10 catalyzed by the SidE ligases is crucial for its recruitment to the bacterial vacuole. SdcB, the previously uncharacterized SidC-family effector, resides on the vacuole and contributes to retention of Rab10 at the late stages of infection. We further identified MavC as a negative regulator of SdcB. By the transglutaminase activity, MavC crosslinks ubiquitin to SdcB and suppresses its function, resulting in elimination of Rab10 from the vacuole. These results demonstrate that the orchestrated actions of many L. pneumophila effectors fine-tune the dynamics of Rab10 during infection. [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]

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment. Here, Xian et al. use phosphoproteomics to identify that the Shigella effector OspG interacts with a regulator of Cullin-RING ubiquitin ligases to promote the ubiquitination of septins and consequent inhibition of septin cage formation. [ABSTRACT FROM AUTHOR]

The application of artificial intelligence (AI) in the analysis of molecular biology data is becoming increasingly widespread. The Western Blot (WB) technique, a cornerstone in proteomic research, facilitates the identification and analysis of proteins, such as the frameshift mutant of ubiquitin B (UBB+1). In our study, we attempted to assess the potential of four different AI models—Gemini, Gemini Advanced, Microsoft Copilot, and ChatGPT 4—in the analysis of WB imagery containing UBB+1, derived from peripheral blood studies of patients suffering from schizophrenia. Participants, all male and diagnosed with schizophrenia, were recruited from the Specialist Psychiatric Care Team of Babinski Hospital in Lodz. After obtaining their informed consent, blood samples were collected and transported to the laboratory of the Department of Medical Biochemistry at the Medical University of Lodz. The samples were processed, synthesis of Ub-48UBB+1 dimers was performed, and the WB technique was applied. The result of the WB analysis, in the form of a photograph with basic labels but without a legend (JPG format), was implemented into ChatGPT 4, Microsoft Copilot, Gemini and Gemini Advanced. Following the implementation of the image, the command 'Could you analyze the attached photo?' was added, along with the protocol from Sample Preparation and Synthesis of Ub-48UBB+1 Dimers. The AI models effectively analyzed and interpreted WB images, with variations in their approaches and depth. Gemini excelled in detailing the WB process and biological significance of bands, while Gemini Advanced focused on specific band identification, especially Ub-48UBB+1 dimers. Microsoft Copilot provided a basic overview with less technicality, and ChatGPT 4 offered comprehensive band interpretations, linking them to patient samples and standards, thus confirming the hypothesis about the differing capabilities of these models. This discovery demonstrates the advanced capabilities of ChatGPT 4 and highlights the growing role of AI in scientific research, including the interpretation of results. [ABSTRACT FROM AUTHOR]

Ubiquitin regulates various cellular functions by posttranslationally modifying substrates with diverse ubiquitin codes. Recent discoveries of new ubiquitin chain topologies, types of bonds, and non-protein substrates have substantially expanded the complexity of the ubiquitin code. Here, we describe the ubiquitin system covering the basic principles and recent discoveries related to mechanisms, technologies, and biological importance. [ABSTRACT FROM AUTHOR]

Lipids have key functions in the assembly of cell structures, serve as an important energy source, and are precursors for signaling molecules. Ubiquitination is one of the most common post-translational modifications in eukaryotes and is essential for cellular and tissue homeostasis. Ubiquitination is central in regulating lipid metabolism by controlling the turnover of proteins and lipids involved in lipid metabolism. Dysregulation of ubiquitination contributes to lipid abnormalities in divergent disorders, and targeting the ubiquitin-proteasome system serves as an interesting therapeutic approach. Lipids are essential molecules that play key roles in cell physiology by serving as structural components, for storage of energy, and in signal transduction. Hence, efficient regulation and maintenance of lipid homeostasis are crucial for normal cellular and tissue function. In the past decade, increasing research has shown the importance of ubiquitination in regulating the stability of key players in different aspects of lipid metabolism. This review describes recent insights into the regulation of lipid metabolism by ubiquitin signaling, discusses how ubiquitination can be targeted in diseases characterized by lipid dysregulation, and identifies areas that require further research. [ABSTRACT FROM AUTHOR]

Introduction Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in both adult civilian and military populations. Currently, diagnostic and prognostic methods are limited to imaging and clinical findings. Biomarker measurements offer a potential method to assess head injuries and help predict outcomes, which has a potential benefit to the military, particularly in the deployed setting where imaging modalities are limited. We determine how biomarkers such as ubiquitin C-terminal hydrolase-L1 (UCH-L1), glial fibrillary acidic protein (GFAP), S100B, neurofilament light chain (NFL), and tau proteins can offer important information to guide the diagnosis, acute management, and prognosis of TBI, specifically in military personnel. Materials and Methods We performed a narrative review of peer-reviewed literature using online databases of Google Scholar and PubMed. We included articles published between 1988 and 2022. Results We screened a total of 73 sources finding a total of 39 original research studies that met inclusion for this review. We found five studies that focused on GFAP, four studies that focused on UCH-L1, eight studies that focused on tau proteins, six studies that focused on NFL, and eight studies that focused on S100B. The remainder of the studies included more than one of the biomarkers of interest. Conclusions TBI occurs frequently in the military and civilian settings with limited methods to diagnose and prognosticate outcomes. We highlighted several promising biomarkers for these purposes including S100B, UCH-L1, NFL, GFAP, and tau proteins. S100B and UCH-L1 appear to have the strongest data to date, but further research is necessary. The robust data that explain the optimal timing and, more importantly, trending of these biomarker measurements are necessary before widespread application. [ABSTRACT FROM AUTHOR]

Background: As an actin cytoskeleton interactor, PDZ (postsynaptic density 65-discs large-zonula occludens 1) and LIM (abnormal cell lineage 11-isket 1-mechanosensory abnormal 3) domain protein 7 (PDLIM7) was supposed to play an essential role modulating cytoskeleton. Focal adhesions (FAs), which are located at the cytomembrane terminus of actin cytoskeleton, function as a force sensor and can transform the mechanical signal to a biochemical signal. Focal adhesion kinase (FAK) localizes to and regulates signal transduction in FAs, which play an essential role in cell polarity, migration, and invasion. However, whether PDLIM7 is involved in FAs-associated signal transduction and its role in tumor invasion and metastasis remains largely unknown. Methods: A cohort of 80 patients with papillary thyroid carcinoma (PTC) at The Second Affiliated Hospital of Guilin Medical University, as well as 512 PTC samples from The Cancer Genome Atlas thyroid cancer database was utilized to analyze the expression of PDLIM7 and its association with prognosis. Survival data were assessed using Kaplan-Meier curves, whereas clinicopathological characteristics such as age, sex, tumor size, multilocality, extrathyroidal extension, lymph metastases, Hashimoto's thyroiditis, distant metastasis, and TNM stage were considered. Functional assays were performed in vitro and in a xenograft mouse model to assess the role of PDLIM7 in PTC cell lines. The colocalization of PDLIM7 with FAK and integrin alpha V (ITGAV) was determined using immunofluorescence assay and immunoprecipitation assay. Protein expression levels in cell and tissue biopsies were measured through Western blotting and immunohistochemistry. Results: (1) The PDLIM7 protein frequently upregulated in both PTC tissues and cells, and overexpression of PDLIM7 is associated with advanced clinicopathological characteristics. (2) Knockdown of PDLIM7 effectively inhibits cell proliferation, migration, and invasion in PTC cell lines in vitro. (3) Knockdown of PDLIM7 hinders the growth and metastasis of TPC-1 xenografts in vivo. (4) PDLIM7 demonstrates colocalization with both FAK and the FA molecule ITGAV and the knockdown of PDLIM7 resulted in disassembly of FAs and proteosome-dependent degradation of FAK in PTC cell lines. Conclusions: PDLIM7 function as an oncoprotein in PTC to promote metastasis, and a novel underlying mechanism is proposed that PDLIM7 keeps FAK protein from proteosome-dependent degradation. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress of hepatocytes plays a causative role in non-alcoholic fatty liver disease (NAFLD). Reduced expression of hepatic nuclear factor 4α (HNF4α) is a critical event in the pathogenesis of NAFLD and other liver diseases. Whether ER stress regulates HNF4α expression remains unknown. The aim of this study was to delineate the machinery of HNF4α protein degradation and explore a therapeutic strategy based on protecting HNF4α stability during NAFLD progression. Correlation of HNF4α and tribbles homologue 3 (TRIB3), an ER stress sensor, was evaluated in human and mouse NAFLD tissues. RNA–sequencing, mass spectrometry analysis, co-immunoprecipitation, in vivo and in vitro ubiquitination assays were used to elucidate the mechanisms of TRIB3-mediated HNF4α degradation. Molecular docking and co-immunoprecipitation analyses were performed to identify a cell-penetrating peptide that ablates the TRIB3–HNF4α interaction. TRIB3 directly interacts with HNF4α and mediates ER stress-induced HNF4α degradation. TRIB3 recruits tripartite motif containing 8 (TRIM8) to form an E3 ligase complex that catalyzes K48-linked polyubiquitination of HNF4α on lysine 470. Abrogating the degradation of HNF4α attenuated the effect of TRIB3 on a diet-induced NAFLD model. Moreover, the TRIB3 gain-of-function variant p.Q84R is associated with NAFLD progression in patients, and induces lower HNF4α levels and more severe hepatic steatosis in mice. Importantly, disrupting the TRIB3–HNF4α interaction using a cell-penetrating peptide restores HNF4α levels and ameliorates NAFLD progression in mice. Our findings unravel the machinery of HNF4α protein degradation and indicate that targeting TRIB3–TRIM8 E3 complex-mediated HNF4α polyubiquitination may be an ideal strategy for NAFLD therapy. Reduced expression of hepatic nuclear factor 4α (HNF4α) is a critical event in the pathogenesis of NAFLD and other liver diseases. However, the mechanism of HNF4α protein degradation remains unknown. Herein, we reveal that TRIB3–TRIM8 E3 ligase complex is responsible for HNF4α degradation during NAFLD. Inhibiting the TRIB3–HNF4α interaction effectively stabilized HNF4α protein levels and transcription factor activity in the liver and ameliorated TRIB3-mediated NAFLD progression. Our findings demonstrate that disturbing the TRIM8–TRIB3–HNF4α interaction may provide a novel approach to treat NAFLD and even other liver diseases by stabilizing the HNF4α protein. [Display omitted] • TRIB3 is elevated in fatty liver and potentiates NAFLD progression. • TRIB3 interacts with HNF4α and promotes its K48-linked ubiquitination. • TRIB3–TRIM8 complex catalyzes ubiquitination of HNF4α at lysine 470. • Blocking TRIB3-HNF4α interaction alleviates NASH progression by inhibiting HNF4α degradation. [ABSTRACT FROM AUTHOR]

Mysterin is a large intracellular protein harboring a RING finger ubiquitin ligase domain and is also referred to as RING finger protein 213 (RNF213). The author performed the first molecular cloning of the mysterin gene as the final step in genetic exploration of cerebrovascular moyamoya disease (MMD) and initiated the next round of exploration to understand its molecular and cellular functions. Although much remains unknown, accumulating findings suggest that mysterin functions in cells by targeting massive intracellular structures, such as lipid droplets (LDs) and various invasive pathogens. In the latter case, mysterin appears to directly surround and ubiquitylate the surface of pathogens and stimulate cell-autonomous antimicrobial reactions, such as xenophagy and inflammatory response. To date, multiple mutations causing MMD have been identified within and near the RING finger domain of mysterin ; however, their functional relevance remains largely unknown. Besides the RING finger, mysterin harbors a dynein-like ATPase core and an RZ finger, another ubiquitin ligase domain unique to mysterin, while functional exploration of these domains has also just commenced. In this review, the author attempts to summarize the core findings regarding the molecular structure and function of the mysterin protein, with an emphasis on the perspective of MMD research. [ABSTRACT FROM AUTHOR]

Recently, the development of protein degraders (protein-degrading compounds) has prominently progressed. There are two remarkable classes of protein degraders: proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs). Almost 70 years have passed since thalidomide was initially developed as a sedative-hypnotic drug, which is currently recognized as one of the most well-known MGDs. During the last two decades, a myriad of PROTACs and MGDs have been developed, and the molecular mechanism of action (MOA) of thalidomide was basically elucidated, including identifying its molecular target cereblon (CRBN). CRBN forms a Cullin Ring Ligase 4 with Cul4 and DDB1, whose substrate specificity is controlled by its binding ligands. Thalidomide, lenalidomide and pomalidomide, three CRBN-binding MGDs, were clinically approved to treat several intractable diseases (including multiple myeloma). Several other MGDs and CRBN-based PROTACs (ARV-110 and AVR-471) are undergoing clinical trials. In addition, several new related technologies regarding PROTACs and MGDs have also been developed, and achievements of protein degraders impact not only therapeutic fields but also basic biological science. In this article, I introduce the history of protein degraders, from the development of thalidomide to the latest PROTACs and related technologies. [ABSTRACT FROM AUTHOR]

Mitochondria are essential eukaryotic organelles that produce ATP as well as synthesize various macromolecules. They also participate in signalling pathways such as the innate immune response and apoptosis. These diverse functions are performed by >1,000 different mitochondrial proteins. Although mitochondria are continuously exposed to potentially damaging conditions such as reactive oxygen species, proteases/peptidases localized in different mitochondrial subcompartments, termed mitoproteases, maintain mitochondrial quality and integrity. In addition to processing incoming precursors and degrading damaged proteins, mitoproteases also regulate metabolic reactions, mitochondrial protein half-lives and gene transcription. Impaired mitoprotease function is associated with various pathologies. In this review, we highlight recent advances in our understanding of mitochondrial quality control regulated by autophagy, ubiquitin–proteasomes and mitoproteases. [ABSTRACT FROM AUTHOR]

In mammals, hearing loss is irreversible due to the lack of the regenerative capacity of the auditory epithelium. However, stem/progenitor cells in mammalian cochleae may be a therapeutic target for hearing regeneration. The ubiquitin proteasome system plays an important role in cochlear development and maintenance. In this study, we investigated the role of ubiquitin C-terminal hydrolase L1 (UCHL1) in the process of the transdifferentiation of auditory supporting cells (SCs) into hair cells (HCs). The expression of UCHL1 gradually decreased as HCs developed and was restricted to inner pillar cells and third-row Deiters' cells between P2 and P7, suggesting that UCHL1-expressing cells are similar to the cells with Lgr5-positive progenitors. UCHL1 expression was decreased even under conditions in which supernumerary HCs were generated with a γ-secretase inhibitor and Wnt agonist. Moreover, the inhibition of UCHL1 by LDN-57444 led to an increase in HC numbers. Mechanistically, LDN-57444 increased mTOR complex 1 activity and allowed SCs to transdifferentiate into HCs. The suppression of UCHL1 induces the transdifferentiation of auditory SCs and progenitors into HCs by regulating the mTOR pathway. [ABSTRACT FROM AUTHOR]

The ubiquitin kinase-ligase pair PINK1-PRKN recognizes and transiently labels damaged mitochondria with ubiquitin phosphorylated at Ser65 (p-S65-Ub) to mediate their selective degradation (mitophagy). Complete loss of PINK1 or PRKN function unequivocally leads to early-onset Parkinson disease, but it is debated whether impairments in mitophagy contribute to disease later in life. While the pathway has been extensively studied in cell culture upon acute and massive mitochondrial stress, basal levels of activation under endogenous conditions and especially in vivo in the brain remain undetermined. Using rodent samples, patient-derived cells, and isogenic neurons, we here identified age-dependent, brain region-, and cell type-specific effects and determined expression levels and extent of basal and maximal activation of PINK1 and PRKN. Our work highlights the importance of defining critical risk and therapeutically relevant levels of PINK1-PRKN signaling which will further improve diagnosis and prognosis and will lead to better stratification of patients for future clinical trials. [ABSTRACT FROM AUTHOR]

Wnt signaling plays a crucial role in embryonic development and homeostasis maintenance. Delicate and sensitive fine-tuning of Wnt signaling based on the proper timings and positions is required to balance cell proliferation and differentiation and maintain individual health. Therefore, homeostasis is broken by tissue hypoplasia or tumor formation once Wnt signal dysregulation disturbs the balance of cell proliferation. The well-known regulatory mechanism of Wnt signaling is the molecular reaction associated with the cytoplasmic accumulation of effector β-catenin. In addition to β-catenin, most Wnt effector proteins are also regulated by ubiquitin-dependent modification, both qualitatively and quantitatively. This review will explain the regulation of the whole Wnt signal in four regulatory phases, as well as the different ubiquitin ligases and the function of deubiquitinating enzymes in each phase. Along with the recent results, the mechanism by which RNF43 negatively regulates the surface expression of Wnt receptors, which has recently been well understood, will be detailed. Many RNF43 mutations have been identified in pancreatic and gastrointestinal cancers and examined for their functional alteration in Wnt signaling. Several mutations facilitate or activate the Wnt signal, reversing the RNF43 tumor suppressor function into an oncogene. RNF43 may simultaneously play different roles in classical multistep tumorigenesis, as both wild-type and mutant RNF43 suppress the p53 pathway. We hope that the knowledge obtained from further research in RNF43 will be applied to cancer treatment in the future despite the fully unclear function of RNF43. [ABSTRACT FROM AUTHOR]

Activity-based ubiquitin probes (Ub-ABPs) have recently been developed as effective tools for studying the capabilities of E1–E2–E3 enzymes, but most of them can only be used in cell lysates. Here, we report the first cell-penetrating Ub-Dha probes based on thiazolidine-protected cysteines, which enable successful delivery into cells confirmed by a fluorophore at the N-terminus of Ub and live-cell fluorescence microscopy. A total of 18 E1–E2–E3 enzymes in live cells were labelled and enriched in combination with label-free quantification (LFQ) mass spectrometry. This work provided a new cell-penetrating Ub tool for studying the activity and function of Ub-related enzymes. [ABSTRACT FROM AUTHOR]

Myofibrillogenesis regulator‐1 (MR‐1) is a multifunctional protein involved in the development of various human tumors. The study is the first to report the promoting effect of MR‐1 on the development and metastasis of non‐small cell lung cancer (NSCLC). MR‐1 is upregulated in NSCLC and positively associated with poor prognosis. The overexpression of MR‐1 promotes the metastasis of NSCLC cells by stabilizing the expression of Notch3‐ICD (NICD3) in the cytoplasm through enrichment analysis, in vitro and in vivo experimental researches. And Notch3 signaling can upregulate many genes related to metastasis. The stabilizing effect of MR‐1 on NICD3 is achieved through the mono‐ubiquitin lysosomal pathway and the specific E3 ubiquitin ligase is Itchy homolog (ITCH). There is a certain interaction between MR‐1 and NICD3. Elevated MR‐1 can affect the level of ITCH phosphorylation, reduce its E3 enzyme activity, and thus lead to reduce the ubiquitination and degradation of NICD3. Interference with the interaction between MR‐1 and NICD3 can increase the degradation of NICD3 and impair the metastatic ability of NSCLC cells, which is a previously overlooked treatment option in NSCLC. In summary, interference with the interaction between MR‐1 and NICD3 in the progression of lung cancer may be a promising therapeutic target. [ABSTRACT FROM AUTHOR]

Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission. [ABSTRACT FROM AUTHOR]

Drought stress poses a persistent threat to field crops and significantly limits global agricultural productivity. Plants employ ubiquitin‐dependent degradation as a crucial post‐translational regulatory mechanism to swiftly adapt to changing environmental conditions. JUL1 is a RING‐type E3 ligase related to drought stress in Arabidopsis. In this study, we explored the function of BnaJUL1 (a homologous gene of JUL1 in Brassica napus) and discovered a novel gene BnaTBCC1 participating in drought tolerance. First, we utilised BnaJUL1‐cri materials through the clustered regularly interspaced short palindromic repeats (CRISPR)‐CRISPR‐associated protein 9 system. Second, we confirmed that BnaJUL1 regulated drought tolerance through the drought tolerance assay and transcriptome analysis. Then, we identified a series of proteins interacting with BnaJUL1 through yeast library screening, including BnaTBCC1 (a tubulin binding cofactor C domain‐containing protein); whose homologous gene TBCC1 knockdown mutants (tbcc1‐1) exhibited ABA‐sensitive germination in Arabidopsis, we then confirmed the involvement of BnaTBCC1 in drought tolerance in both Arabidopsis and Brassica. Finally, we established that BnaJUL1 could ubiquitinate and degrade BnaTBCC1 to regulate drought tolerance. Consequently, our study unveils BnaJUL1 as a novel regulator that ubiquitinates and degrades BnaTBCC1 to modulate drought tolerance and provided desirable germplasm for further breeding of drought tolerance in rapeseed. Summary statement: E3 ligase BnaJUL1 ubiquitinates and degrades a tubulin binding cofactor C domain‐containing protein (BnaTBCC1) to regulate drought tolerance in Brassica napus L. [ABSTRACT FROM AUTHOR]

Functional consequences of protein ubiquitination have gone far beyond the degradation regulation as was initially imagined during its discovery 40 years back. The state-of-theart has revealed the plethora of signaling pathways that are largely regulated by ubiquitination process in eukaryotes. To no surprise, ubiquitination is often dysregulated in many human diseases, including cancer, neurodegeneration and infection. Hence it has become a major focus with high-gain research value for many investigators to unravel new proteoforms, that are the targets of this ubiquitination modification. Despite many biochemical or proteomic approaches available for ubiquitination detection, mass-spectrometry stood out to be the most efficient and transformative technology to read this complex modification script. Here in this review, we have discussed how different ubiquitin codes can be decoded qualitatively and quantitatively following various sequential proteomic approaches to date reported and indicated the current limitations with scope for improvements. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system (UPS) is a pivotal cellular mechanism responsible for the selective degradation of proteins, playing an essential role in proteostasis, protein quality control, and regulating various cellular processes, with ubiquitin marking proteins for degradation through a complex, multi-stage process. The shuttle proteins family is a very unique group of proteins that plays an important role in the ubiquitin-proteasome system. Ddi1, Dsk2, and Rad23 are shuttle factors that bind ubiquitinated substrates and deliver them to the 26S proteasome. Besides mediating the delivery of ubiquitinated proteins, they are also involved in many other biological processes. Ddi1, the least-studied shuttle protein, exhibits unique physicochemical properties that allow it to play non-canonical functions in the cells. It regulates cell cycle progression and response to proteasome inhibition and defines MAT type of yeast cells. The Ddi1 contains UBL and UBA domains, which are crucial for binding to proteasome receptors and ubiquitin respectively, but also an additional domain called RVP. Additionally, much evidence has been provided to question whether Ddi1 is a classical shuttle protein. For many years, the true nature of this protein remained unclear. Here, we highlight the recent discoveries, which shed new light on the structure and biological functions of the Ddi1 protein. [ABSTRACT FROM AUTHOR]

Proteolysis-targeting chimeras (PROTACs) describe compounds that bind to and induce degradation of a target by simultaneously binding to a ubiquitin ligase. More generally referred to as bifunctional degraders, PROTACs have led the way in the field of targeted protein degradation (TPD), with several compounds currently undergoing clinical testing. Alongside bifunctional degraders, single-moiety compounds, or molecular glue degraders (MGDs), are increasingly being considered as a viable approach for development of therapeutics, driven by advances in rational discovery approaches. This review focuses on drug discovery with respect to bifunctional and molecular glue degraders within the ubiquitin proteasome system, including analysis of mechanistic concepts and discovery approaches, with an overview of current clinical and pre-clinical degrader status in oncology, neurodegenerative and inflammatory disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

MAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. Here, we employed NMR and AlphaFold2 (AF) to elucidate the interaction mode between RAD18 and MAGEA4, and reveal that the RAD6-binding domain (R6BD) of RAD18 occupies a groove in the C-terminal winged-helix subdomain of MAGEA4. We found that MAGEA4 partially displaces RAD6 from the RAD18 R6BD and inhibits degradative RAD18 autoubiquitination, which could be countered by a competing peptide of the RAD18 R6BD. AlphaFold2 and cross-linking mass spectrometry (XL-MS) also revealed an evolutionary invariant intramolecular interaction between the catalytic RING and the DNA-binding SAP domains of RAD18, which is essential for PCNA mono-ubiquitination. Using interaction proteomics, we found that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a manner similar to the MAGEA4/RAD18 complex, suggesting that the MAGEA4 peptide-binding groove also serves as a ligase-binding cleft in other type-I MAGEs. Our data provide new insights into the mechanism and regulation of RAD18-mediated PCNA mono-ubiquitination. Synopsis: MAGE proteins bind RING ubiquitin ligases and often promote pro-oncogenic pathways. Through structural and biochemical approaches, this study gives insight into how MAGEA4 binds and regulates the RING E3 RAD18, which is crucial for DNA repair. MAGEA4 utilizes a C-terminal peptide binding groove (PBG) to bind and regulate RAD18. The PBG is important for RING ligase binding in other MAGE proteins. MAGEA4 protects RAD18 from degradative autoubiquitination. The RAD18 RING and SAP domains interact together to mediate monoubiquitination of the substrate PCNA. The interaction mode is conserved in another MAGE/RING pair, MAGE-C2 and TRIM28. MAGEA4 partly displaces RAD6 from the key DNA repair E3 RAD18, thereby protecting it from degradative autoubiquitination. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of the coronavirus family and caused severe economic losses to the global swine industry. Previous studies have established that p53 is a host restriction factor for PEDV infection, and p53 degradation occurs in PEDV-infected cells. However, the underlying molecular mechanisms through which PEDV viral proteins regulate p53 degradation remain unclear. In this study, we found that PEDV infection or expression of the nucleocapsid protein downregulates p53 through a post-translational mechanism: increasing the ubiquitination of p53 and preventing its nuclear translocation. We also show that the PEDV N protein functions by recruiting the E3 ubiquitin ligase COP1 and suppressing COP1 selfubiquitination and protein degradation, thereby augmenting COP1-mediated degradation of p53. Additionally, COP1 knockdown compromises N-mediated p53 degradation. Functional mapping using truncation analysis showed that the Nterminal domains of N protein were responsible for interacting with COP1 and critical for COP1 stability and p53 degradation. The results presented here suggest the COP1-dependent mechanism for PEDV N protein to abolish p53 activity. This study significantly increases our understanding of PEDV in antagonizing the host antiviral factor p53 and will help initiate novel antiviral strategies against PEDV. [ABSTRACT FROM AUTHOR]

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and posttranslational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin--like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GβL (G protein β-subunit--like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GβL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR--Raptor (regulatory protein associated with mTOR) and mTOR--Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GβL but not SUMOylation-defective KR mutant GβL promotes mTOR signaling in GβL-depleted cells. Taken together, we report for the very first time that SUMO modifies GβL, in- fluences the assembly of mTOR protein complexes, and regulates mTOR activity. [ABSTRACT FROM AUTHOR]

BACKGROUND: Impairment of the ubiquitin‐proteasome system (UPS) has been implicated in abnormal protein accumulation in Alzheimer's disease. It remains unclear if genetic variation affects the intrinsic properties of neurons that render some individuals more vulnerable to UPS impairment. METHODS: Induced pluripotent stem cell (iPSC)‐derived neurons were generated from over 50 genetically variant and highly characterized participants of cohorts of aging. Proteomic profiling, proteasome activity assays, and Western blotting were employed to examine neurons at baseline and in response to UPS perturbation. RESULTS: Neurons with lower basal UPS activity were more vulnerable to tau accumulation following mild UPS inhibition. Chronic reduction in proteasome activity in human neurons induced compensatory elevation of regulatory proteins involved in proteostasis and several proteasome subunits. DISCUSSION: These findings reveal that genetic variation influences basal UPS activity in human neurons and differentially sensitizes them to external factors perturbing the UPS, leading to the accumulation of aggregation‐prone proteins such as tau. Highlights: Polygenic risk score for AD is associated with the ubiquitin‐proteasome system (UPS) in neurons.Basal proteasome activity correlates with aggregation‐prone protein levels in neurons.Genetic variation affects the response to proteasome inhibition in neurons.Neuronal proteasome perturbation induces an elevation in specific proteins involved in proteostasis.Low basal proteasome activity leads to enhanced tau accumulation with UPS challenge. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection poses a significant burden on global public health. Unfortunately, current treatments cannot fully alleviate this burden as they have limited effect on the transcriptional activity of the tenacious covalently closed circular DNA (cccDNA) responsible for viral persistence. Consequently, the HBV life cycle should be further investigated to develop new anti‐HBV pharmaceutical targets. Our previous study discovered that the host gene TMEM203 hinders HBV replication by participating in calcium ion regulation. The involvement of intracellular calcium in HBV replication has also been confirmed. In this study, we found that transient receptor potential vanilloid 4 (TRPV4) notably enhances HBV reproduction by investigating the effects of several calcium ion‐related molecules on HBV replication. The in‐depth study showed that TRPV4 promotes hepatitis B core/capsid protein (HBc) protein stability through the ubiquitination pathway and then promotes the nucleocapsid assembly. HBc binds to cccDNA and reduces the nucleosome spacing of the cccDNA‐histones complex, which may regulate HBV transcription by altering the nucleosome arrangement of the HBV genome. Moreover, our results showed that TRPV4 promotes cccDNA‐dependent transcription by accelerating the methylation modification of H3K4. In conclusion, TRPV4 could interact with HBV core protein and regulate HBV during transcription and replication. These data suggest that TRPV4 exerts multifaceted HBV‐related synergistic factors and may serve as a therapeutic target for CHB. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have unique characteristics where they can both contribute to all three germ layers in vivo and self-renewal indefinitely in vitro. Post-translational modifications of proteins, particularly by the ubiquitin proteasome system (UPS), control cell pluripotency, self-renewal, and differentiation. A significant number of UPS members (mainly ubiquitin ligases) regulate pluripotency and influence ESC differentiation with key elements of the ESC pluripotency network (including the "master" regulators NANOG and OCT4) being controlled by ubiquitination. To further understand the role of the UPS in pluripotency, we performed an RNAi screen during induction of cellular reprogramming and have identified FBXO9 as a novel regulator of pluripotency associated protein DPPA5. Our findings indicate that FBXO9 silencing facilitates the induction of pluripotency through decreased proteasomal degradation of DPPA5. These findings identify FBXO9 as a key regulator of pluripotency. [ABSTRACT FROM AUTHOR]

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications. [ABSTRACT FROM AUTHOR]

SKP2 (S-phase kinase-associated protein 2) is a member of the F-box family of substrate-recognition subunits in the SCF ubiquitin-protein ligase complexes. It is associated with ubiquitin-mediated degradation in the mammalian cell cycle components and other target proteins involved in cell cycle progression, signal transduction, and transcription. Being an oncogene in solid tumors and hematological malignancies, it is frequently associated with drug resistance and poor disease outcomes. In the current review, we discussed the novel role of SKP2 in different hematological malignancies. Further, we performed a limited in-silico analysis to establish the involvement of SKP2 in a few publicly available cancer datasets. Interestingly, our study identified Skp2 expression to be altered in a cancer-specific manner. While it was found to be overexpressed in several cancer types, few cancer showed a down-regulation in SKP2. Our review provides evidence for developing novel SKP2 inhibitors in hematological malignancies. We also investigated the effect of SKP2 status on survival and disease progression. In addition, the role of miRNA and its associated families in regulating Skp2 expression was explored. Subsequently, we predicted common miRNAs against Skp2 genes by using miRNA-predication tools. Finally, we discussed current approaches and future prospective approaches to target the Skp2 gene by using different drugs and miRNA-based therapeutics applications in translational research. [ABSTRACT FROM AUTHOR]

A cascade of three enzymes, E1−E2−E3, is responsible for transferring ubiquitin to target proteins, which controls many different aspects of cellular signaling. The role of the E2 has been largely overlooked, despite influencing substrate identity, chain multiplicity, and topology. Here we report a method—targeted charging of ubiquitin to E2 (tCUbE)—that can track a tagged ubiquitin through its entire enzymatic cascade in living mammalian cells. We use this approach to reveal new targets whose ubiquitination depends on UbcH5a E2 activity. We demonstrate that tCUbE can be broadly applied to multiple E2s and in different human cell lines. tCUbE is uniquely suited to examine E2−E3‐substrate cascades of interest and/or piece together previously unidentified cascades, thereby illuminating entire branches of the UPS and providing critical insight that will be useful for identifying new therapeutic targets in the UPS. [ABSTRACT FROM AUTHOR]

Cyanamides have emerged as privileged scaffolds in covalent inhibitors of deubiquitinating enzymes (DUBs). However, many compounds with a cyanopyrrolidine warhead show cross‐reactivity toward small subsets of DUBs or toward the protein deglycase PARK7/DJ‐1, hampering their use for the selective perturbation of a single DUB in living cells. Here, we disclose N'‐alkyl,N‐cyanopiperazines as structures for covalent enzyme inhibition with exceptional specificity for the DUB UCHL1 among 55 human deubiquitinases and with effective target engagement in cells. Notably, transitioning from 5‐membered pyrrolidines to 6‐membered heterocycles eliminated PARK7 binding and introduced context‐dependent reversibility of the isothiourea linkage to the catalytic cysteine of UCHL1. Compound potency and specificity were analysed by a range of biochemical assays and with a crystal structure of a cyanopiperazine in covalent complex with UCHL1. The structure revealed a compound‐induced conformational restriction of the cross‐over loop, which underlies the observed inhibitory potencies. Through the rationalization of specificities of different cyanamides, we introduce a framework for the investigation of protein reactivity of bioactive nitriles of this compound class. Our results represent an encouraging case study for the refining of electrophilic compounds into chemical probes, emphasizing the potential to engineer specificity through subtle chemical modifications around the warhead. [ABSTRACT FROM AUTHOR]