SUMMARYThis study systematically explored the role of NEDD8 in pediatric acute myeloid leukemia (AML) through patient sample analysis, database mining, and in vitro experiments. Our results demonstrated that NEDD8 was significantly overexpressed in newly diagnosed pediatric AML patients and was associated with poor survival outcomes. Functional enrichment analysis of the TARGET database further revealed a strong correlation between NEDD8 and cancer-related pathways. In vitro experiments showed that NEDD8 knockdown significantly inhibited the proliferation of AML cells (THP-1 and MV4-11) and induced cell cycle arrest. Collectively, these findings highlight the critical role of NEDD8 in pediatric AML pathogenesis and suggest its potential as both a prognostic biomarker and a therapeutic target.

Ubiquitination represents one of the most critical post-translational modifications, comprising a multi-stage enzyme process that plays a pivotal role in a myriad of cellular biological activities. The deregulation of the processes of ubiquitination and deubiquitination is associated with the development of cancers and other diseases. This typescript reviews the impact of ubiquitination on metabolic processes, elucidating the regulatory functions of ubiquitination on pivotal enzymes within metabolic pathways in pathological contexts. It underscores the role of ubiquitination-driven metabolism disorders in the etiology of cancers, and oncogenesis, and highlights the potential therapeutic efficacy of targeting ubiquitination-driven enzymes in cancer metabolism, their combination with immune checkpoint inhibitors, and their clinical applications.

Ubiquitination is a type of post-translational modification, referring to the process in which the small molecular protein ubiquitin covalently binds to target proteins under the catalysis of a series of enzymes. The process of ubiquitination is vital in the onset and progression of breast cancer. The use of the ubiquitin-protease system is expected to be a new way to treat human breast cancer. This research aimed to investigate the evolution patterns, key areas of interest, and future directions of ubiquitination in breast cancer via bibliometric analysis.

Research articles on ubiquitination modifications in breast cancer were sourced from the Web of Science Core Collection database and analyzed via Microsoft Excel 2021, Bibliometrix, VOSviewer, and Citespace software for thorough bibliometrics.

From 2005-2024, 1850 English articles published in 405 journals by 1842 institutions/universities from 61 countries were included in the study. Keywords, research fields, co-cited literature and other information were included. Research on ubiquitination modifications has focused on breast cancer, expression, protein, activation, degradation, ubiquitination, phosphorylation, etc. Notably, the keywords that broke out in the past five years have focused on "triple-negative breast cancer", "promotion", and "metabolism". These findings suggest that key areas of current research are metabolism, immunity, survival, and prognosis in triple-negative breast cancer.

Our findings indicate that research on triple-negative breast cancer, as well as its immunological and metabolic aspects, is a burgeoning and promising area. Our work offers valuable guidance and fresh perspectives on the relationship between breast cancer and ubiquitin modification.

As an E3 ubiquitin ligase and an Fc receptor, tripartite motif-containing protein 21 (TRIM21) plays a crucial role in immune defense, signal transduction, and cellular regulation. TRIM21 is widely expressed in various tissues, but it is particularly abundant in cardiovascular tissues and is involved in the pathogenesis of various cardiovascular diseases (CVDs). However, although TRIM21 is involved in the regulation of several key molecular pathways in the immune system, its specific role in CVD remains unclear. In this review, we comprehensively summarize the regulatory role of TRIM21 in signaling pathways and discuss the function of TRIM21 in CVD, to provide a systematic understanding of this important protein in CVD and offer insights for further research into the pathogenesis of CVD and its potential applications.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide, with a prevalence as high as 32.4%. MASLD encompasses a spectrum of liver pathologies, ranging from steatosis to metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, and, in some cases, progression to end-stage liver disease (cirrhosis and hepatocellular carcinoma). A comprehensive understanding of the pathogenesis of this highly prevalent liver disease may facilitate the identification of novel targets for the development of improved therapies. E3 ubiquitin ligases and deubiquitinases (DUBs) are key regulatory components of the ubiquitin‒proteasome system (UPS), which plays a pivotal role in maintaining intracellular protein homeostasis. Emerging evidence implicates that aberrant expression of E3 ligases and DUBs is involved in the progression of MASLD. Here, we review abnormalities in E3 ligases and DUBs by (1) discussing their targets, mechanisms, and functions in MASLD; (2) summarizing pharmacological interventions targeting these enzymes in preclinical and clinical studies; and (3) addressing challenges and future therapeutic strategies. This review synthesizes current evidence to highlight the development of novel therapeutic strategies based on the UPS for MASLD and progressive liver disease.

Adaptation to changes in amino acid availability is crucial for cellular homeostasis, which requires an intricate orchestration of involved pathways. Some cancer cells can maintain cellular fitness upon amino acid shortage, which has a poorly understood mechanistic basis. Leveraging a genome-wide CRISPR-Cas9 screen, we find that superoxide dismutase 2 (SOD2) has a previously unrecognized dismutase-independent function. We demonstrate that SOD2 regulates global proteasomal protein degradation and promotes cell survival under conditions of metabolic stress in malignant cells through the E3 ubiquitin ligases UBR1 and UBR2. Consequently, inhibition of SOD2-mediated protein degradation highly sensitizes different cancer entities, including patient-derived xenografts, to amino acid depletion, highlighting the pathophysiological relevance of our findings. Our study reveals that SOD2 is a regulator of proteasomal protein breakdown upon starvation, which serves as an independent catabolic source of amino acids, a mechanism co-opted by cancer cells to maintain cellular fitness.

Neuronal morphogenesis depends on extracellular guidance cues accurately instructing intracellular cytoskeletal remodeling. Here, we describe a novel role for the actin binding protein Coronin 1A (Coro1A) in neuronal morphogenesis, where it mediates responses to the axon guidance cue netrin-1. We found that Coro1A localizes to growth cones and filopodial structures and is required for netrindependent axon turning, branching, and corpus callosum development. We previously discovered that Coro1A interacts with TRIM67, a brain enriched E3 ubiquitin ligase that interacts with a netrin receptor and is also required for netrin-mediated neuronal morphogenesis. Loss of Coro1A and loss of TRIM67 shared similar phenotypes, suggesting that they may function together in the same netrin pathway. A Coro1A mutant deficient in binding TRIM67 was not able to rescue loss of Coro1A phenotypes, indicating that the interaction between Coro1A and TRIM67 is required for netrin responses. Together, our findings reveal that Coro1A is required for proper neuronal morphogenesis, where it collaborates with TRIM67 downstream of netrin.

The advent of targeted protein degradation technologies, particularly Proteolysis-Targeting Chimeras (PROTACs), enable the selective elimination of target proteins and open up new avenues for the treatment of various diseases. This review delves into the pivotal role of mass spectrometry (MS) in the advancement of PROTACs. MS-based methodologies serve as invaluable tools for identifying PROTAC targets, validating their efficacy, and elucidating ubiquitination sites and protein degradation dynamics. These insights profoundly enrich our comprehension of the mechanisms of action and facilitate the rational design of PROTACs. Furthermore, this review discusses the role of MS in the structural analysis of proteins and the formation of ternary complexes crucial for the activity of PROTACs. The synergy between MS and PROTAC technology holds the promise of groundbreaking advancements in drug discovery by deepening our understanding of the underlying mechanisms that govern PROTAC drug action, thereby promoting the development of innovative strategies for disease treatment.

Cullin-RING ligases (CRLs) are central regulators of environmental and cellular stress responses, orchestrating diverse processes through the ubiquitination of substrate proteins. As modular complexes, CRLs employ substrate-specific adaptors to target proteins for degradation and other ubiquitin-mediated processes, enabling dynamic adaptation to environmental cues. Recent advances have highlighted the largest CRL subfamily SCF (Skp1-cullin-F-box) in environmental sensing, a role historically underappreciated for SCF ubiquitin ligases. Notably, emerging evidence suggests that the F-box domain, a 50-amino acid motif traditionally recognized for mediating protein-protein interactions, can act as a direct environmental sensor due to its ability to bind heavy metals. Despite these advances, the roles of many CRL components in environmental sensing remain poorly understood. This review provides an overview of CRLs in stress response regulation and emphasizes the emerging functions of F-box proteins in environmental adaptation.

Lenvatinib is a prevalent treatment for hepatocellular carcinoma (HCC), yet resistance to the drug significantly limits its effectiveness. This study investigates the role of FBXO32 (F-Box Protein 32) in HCC progression and lenvatinib resistance. Methods: We utilized the GSE211850 and GSE46408 datasets to identify an E3 ubiquitin ligase that is highly expressed in both lenvatinib-resistant HCC cells and HCC tissues. The expression and clinical relevance of this E3 ubiquitin ligase were further validated using lenvatinib-resistant HCC cells, online databases, and HCC clinical tissue samples. The phenotype was verified by cell and animal experiments. Techniques such as RNA sequencing, western blotting, immunofluorescence, Co-immunoprecipitation (Co‑IP), Ubiquitination, and cycloheximide (CHX) chase assay reveal the mechanism. FBXO32 is highly expressed in both lenvatinib-resistant HCC cells and HCC tissues. High FBXO32 expression correlated with increased ALT, AFP levels, larger tumors, and advanced TNM stages, serving as an independent risk factor for overall survival (OS) and recurrence-free survival (RFS). Functional assays demonstrated that FBXO32 overexpression enhanced cell proliferation, stemness, apoptosis resistance, and lenvatinib resistance, while knockdown had opposing effects. KEGG enrichment analysis indicated a link between FBXO32 and the Hedgehog signaling pathway. FBXO32-mediated degradation of SUFU, a Hedgehog pathway inhibitor, activated this pathway. Inhibiting Hedgehog signaling counteracted FBXO32's impact on HCC growth and resistance. Conclusion: FBXO32 is a critical marker for lenvatinib efficacy and HCC prognosis, suggesting that targeting FBXO32 or the Hedgehog pathway could provide innovative strategies for overcoming lenvatinib resistance in HCC.

Salinity is a major abiotic stress that adversely affects rice growth and production. However, the detailed regulatory mechanisms of salt stress response in rice remain largely unexplored. In this study, we established that the RING-type E3 ubiquitin ligase OsRFI2 plays a negative role in salt tolerance in rice. Knockout mutants of OsRFI2 (Osrfi2) exhibited high tolerance, whereas OsRFI2-overexpressed transgenic lines (OE-OsRFI2) were more sensitive to salt stress. OsRFI2 that has E3 ligase activity interacts with ascorbate peroxidase OsAPX8 in chloroplast, and catalyzes its ubiquitination and degradation through the 26 S proteasome pathway. The Osapx8 mutants, like OE-OsRFI2 lines, showed high sensitivity to high salt concentrations, accumulating greater amounts of MDA, H2O2 and O2-, which lead to compromised cell permeability and ROS accumulation. Thus, the OsRFI2-OsAPX8 module adds novel clues for better understanding the regulatory mechanism of salt stress response in rice.

Alternative splicing can produce a variety of splicing isoforms to increase protein diversity, participate in the regulation of gene expression and the occurrence and development of diseases, and thus play an important role in innate immunity. Ubiquitin like with PHD and ring finger domains 2 (Uhrf2) protein is associated with cell proliferation, inflammation, tumors, and cancer, and is currently the focus of medical immunology research, but there is little research on alternative splicing of the Uhrf2 gene. In this study, we identified two different splicing isoforms of Uhrf2 in Miichthys miiuy through Sanger sequencing, dual-luciferase reporter gene assay, qRT-PCR, subcellular localization experiments, and named them Uhrf2-α and Uhrf2-β. Subcellular localization experiments found that Uhrf2-α was mainly located in the nucleus, while Uhrf2-β was mainly located in the cytoplasm. Although their localization was different, both could significantly inhibit the activation of IRF3 and NF-κB signaling pathways, and effectively inhibit the levels of inflammatory cytokines. These results indicate that Uhrf2-α and Uhrf2-β play important negative regulatory roles in innate immune responses in fish.

High-risk human papillomavirus E6 oncoprotein is a model system for the recognition and degradation of cellular p53 tumor suppressor protein. There remains a gap in the understanding of the ubiquitin transfer reaction, including placement of the E6AP catalytic HECT domain of the ligase concerning the p53 substrate and how E6 itself is protected from ubiquitination. We determined the cryoelectron microscopy (cryo-EM) structure of the E6AP/E6/p53 complex, related the structure to in vivo modeling of the tri-molecular complex, and identified structural interactions associated with activation of the ubiquitin ligase function. The structure reveals that the N-terminal ordered domain (NOD) in E6AP has a terminal alpha helix that mediates the interaction of the NOD with the HECT domain of E6AP and protects the HPV-E6 protein from ubiquitination. In addition, this NOD helix is required for E6AP ligase function by contributing to the affinity of the E6-E6AP association, modulating E6 substrate recognition, while displacing UbcH7.

One way cells control the speed and specificity of protein degradation is by regulating the activity of ubiquitin ligases. Upon proteotoxic stress in yeast, the intrinsically disordered protein Roq1 binds the ubiquitin ligase Ubr1 as a pseudosubstrate, thereby modulating the degradation of substrates of the N-degron pathway and promoting the elimination of misfolded proteins. The mechanism underlying this reprograming of Ubr1 is unknown. Here, we show that Roq1 controls Ubr1 by means of two cooperating multifunctional motifs. The N-terminal arginine and a short hydrophobic motif of Roq1 interact with Ubr1 as part of a heterobivalent binding mechanism. Via its N-terminal arginine, Roq1 regulates the ubiquitination of various N-degron substrates and folded proteins. Via its hydrophobic motif, Roq1 accelerates the ubiquitination of misfolded proteins. These findings reveal how a small, intrinsically disordered protein with a simple architecture engages parallel channels of communication to reprogram a functionally complex ubiquitin ligase.

Diabetic kidney disease (DKD) is a prevalent complication associated with diabetes in which podocyte dysfunction significantly contributes to the development and progression of the condition. Ring finger protein 183 (RNF183) is an ER-localized, transmembrane ring finger protein with classical E3 ligase activity. However, whether RNF183 is involved in glomerular podocyte dysfunction, which is the mechanism of action of DKD, is still poorly understood. In this study, we first demonstrated that RNF183 expression in glomerular podocytes of patients with DKD decreased as the disease progressed. Additionally, our transcriptome sequencing analysis of kidney tissues from diabetic mice revealed a significant reduction in RNF183 expression within the kidney cortex. Similarly, the expression of RNF183 was significantly reduced both in the kidneys of diabetic mice and in human podocytes exposed to high glucose conditions. The downregulation of RNF183 resulted in a suppression of autophagic activity, an increase in apoptotic cell death, and reduced expression of cellular markers in HPC cells. We found that RNF183 was modified via N6-methyladenosine (m6A) RNA methylation. Meanwhile, treatment with meclofenamic acid 2 (MA2), an m6A demethylase inhibitor, resulted in the upregulation of RNF183 expression in HPC cells cultured in high glucose conditions. Furthermore, high glucose treatment decreased the transcription and protein levels in both the m6A writer methyltransferaselike3 (METTL3) and the m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). IGF2BP2 assisted with METTL3, which is jointly involved in the transcription of RNF183. Furthermore, we confirmed that RNF183 directly ubiquitinates M2 pyruvate kinase (PKM2) through co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometry (LC-MS) experiments. The level of PKM2 ubiquitination was increased following RNF183 overexpression, leading to enhanced PKM2 protein degradation and subsequently alleviating high glucose-induced podocyte damage. The results of this study indicated that RNF183 was regulated via m6A methylation modification and that RNF183 expression was reduced in HPC cells treated with high glucose, which resulted in decreased PKM2 ubiquitination levels and subsequently aggravated podocyte injury. The findings suggest that RNF183 may serve as a potential therapeutic target for diabetic kidney injury, offering new insights into its role in the progression of DKD.

The maternal-to-zygotic transition (MZT) is a conserved developmental process where the maternally-derived protein and mRNA cache is replaced with newly made zygotic gene products. We have previously shown that in Drosophila the deposited RNA-binding proteins ME31B, Cup, and Trailer Hitch are ubiquitylated by the CTLH E3 ligase and cleared. However, the organization and regulation of the CTLH complex remain poorly understood in flies because Drosophila lacks an identifiable substrate adaptor, and the mechanisms restricting the degradation of ME31B and its cofactors to the MZT are unknown. Here, we show that the developmental regulation of the CTLH complex is multi-pronged, including transcriptional control by OVO and autoinhibition of the E3 ligase. One major regulatory target is the subunit Muskelin, which we demonstrate is a substrate adaptor for the Drosophila CTLH complex. Finally, we find that Muskelin has few targets beyond the three known RNA-binding proteins, showing exquisite target specificity. Thus, multiple levels of integrated regulation restrict the activity of the embryonic CTLH complex to early embryogenesis, during which time it regulates three important RNA-binding proteins.

The Cancer Testis Antigens (CTAs) are a group of germ cell proteins that are absent from normal somatic cells yet aberrantly expressed in many cancer cells. When mis-expressed in cancer cells, many CTAs promote tumorigenic characteristics including genome instability, DNA damage tolerance and therapy resistance. Here we highlight some of the CTAs for which their roles in genome maintenance in cancer cells are well established. We consider three broad CTA categories: (1) Melanoma Antigens (MAGEs) (2) Mitotic CTAs and (3) CTAs with roles in meiotic homologous recombination. Many cancer cells rely on CTAs to tolerate intrinsic and therapy-induced genotoxic stress. Therefore, CTAs represent molecular vulnerabilities of cancer cells and may provide opportunities for therapy. Owing to their high-level expression in tumors and absence from normal somatic cells, CTA-directed therapies could have a high level of specificity and would likely be devoid of side-effect toxicity.

WD-repeat containing protein 26 (WDR26) is an essential component of the CTLH E3 ligase complex. Mutations in WDR26 lead to Skraban-Deardorff, an intellectual disability syndrome with clinical features resembling other disorders arising from defects in transcriptional regulation and chromatin structure. However, the role of WDR26 and its associated CTLH complex in regulating chromatin or transcription has not been elucidated. Here, we assessed how loss of WDR26 affects chromatin accessibility and gene expression. Transcriptome analysis of WDR26 knockout HeLa cells revealed over 2000 differentially expressed genes, while ATAC-Seq analysis showed over 32,000 differentially accessible chromatin regions, the majority mapping to intergenic and intronic regions and 13 % mapping to promoters. Above all, we found that WDR26 loss affected expression of genes regulated by AP-1 and NF-1 transcription factors and resulted in dramatic changes in their chromatin accessibility. Overall, our analyses implicate WDR26 and the CTLH complex in chromatin regulation.

Protein degradation orchestrated by SKP1·CUL1·F-box protein (SCF) ubiquitin ligases is a fundamental process essential for cellular and organismal function. The dynamic assembly of SCFs, facilitated by CAND1, ensures timely ubiquitination of diverse SCF target proteins. As a homolog of CAND1, CAND2 alone has been implicated in various human diseases, yet its functional mechanisms remain elusive. Here, we investigate the role of CAND2 in human cells and its distinct mode of action compared to CAND1. Using an array of quantitative assays, we demonstrate that CAND2 promotes SCF-mediated protein degradation as an F-box protein exchange factor. While CAND2 binds CUL1 with structure and affinity comparable to CAND1, it exhibits lower efficiency in exchanging F-box proteins. Kinetic measurements reveal a significantly higher KM for CAND2-catalyzed SCF disassembly than CAND1, which explains the lower exchange efficiency of CAND2 and is likely due to conformations of the CAND2·SCF exchange intermediate complex being less favorable for F-box protein dissociation. Our study provides mechanistic insights into the biochemical and structural properties of CAND2, as well as its role in regulating cellular dynamics of SCFs, laying a foundation for understanding contributions of CAND2 to healthy and diseased human cells.

E3 ubiquitin ligases (E3s) confer specificity of protein degradation through ubiquitination of substrate proteins. Yet, the vast majority of the >600 human E3s have no known substrates. To identify proteolytic E3-substrate pairs at scale, we developed combinatorial mapping of E3 targets (COMET), a framework for testing the role of many E3s in degrading many candidate substrates within a single experiment. We applied COMET to SCF ubiquitin ligase subunits that mediate degradation of target substrates (6,716 F-box-ORF [open reading frame] combinations) and E3s that degrade short-lived transcription factors (TFs) (26,028 E3-TF combinations). Our data suggest that many E3-substrate relationships are complex rather than 1:1 associations. Finally, we leverage deep learning to predict the structural basis of E3-substrate interactions and probe the strengths and limits of such models. Looking forward, we consider the practicality of transposing this framework, i.e., computational structural prediction of all possible E3-substrate interactions, followed by multiplex experimental validation.

In this issue of Molecular Cell, Sulter et al.1 describe a high-throughput method named COMET (combinatorial mapping of E3 targets) that enables direct screening for interactions between E3 ubiquitin ligases and their proteolytic substrate proteins.

Indole-3-carbinol (I3C) is a metabolic derivative of glucobrassicin found in cruciferous vegetables. Known for its anticarcinogenic properties, I3C has been shown to target the NEDD4 family HECT E3 ligases, NEDD4-1 and WWP1, yet in vitro confirmation for the latter is lacking. Here, we characterize the interactions of I3C and a set of 17 derivatives with WWP1 and its homologue, WWP2. Saturation transfer difference (STD) NMR analysis confirmed strong interaction of I3C with WWP1 but weaker with WWP2. However, while autoubiquitination activity assays revealed weak inhibition of WWP1, the I3C condensation product, 3,3'-diindolylmethane (DIM), was more potent (IC50 111.2 μM; 95% CI = 85.1, 145.8). Molecular modeling of DIM to the ubiquitin exosite of both enzymes suggested the WW2 domain makes hydrophobic interactions with the ligand that may contribute to inhibitory action. Taken together, our results suggest future drug lead development should focus on the SAR between WWP1 and DIM.

The loss of functions of tumor suppressor (TS) genes plays a key role in not only tumor initiation but also tumor progression leading to poor prognosis. While therapeutic inhibition of oncogene-encoded kinases has shown clinical success, restoring TS functions remains challenging due to conceptual and technical limitations. E3 ubiquitin ligases that ubiquitinate TS proteins for accelerated degradation in cancers emerge as promising therapeutic targets. Unlike proteasomal inhibitors with a broad spectrum, inhibitors of an E3 ligase would offer superior selectivity and efficacy in enhancing expression of its substrate TS proteins as far as the TS proteins retain wild-type structures. Recent advances in developing E3 inhibitors, including MDM2 inhibitors, highlight their potential and ultimately guide the framework to establish E3 inhibition as effective strategies to treat specific types of cancers. This review explores E3 ligases that negatively regulate bona fide TS proteins, the developmental status of E3 inhibitors, and their promise and pitfalls as therapeutic agents for anti-cancer precision medicine.

We report targeted protein degradation through the site-specific recruitment of native ubiquitin ligases to a protein of interest via conjugation of E3 ligase ligands. Direct comparison of degradation ability of proteins displaying the corresponding bioconjugation handle at different regions of protein surfaces was explored. We demonstrate the benefit of proximal lysine residues and investigate flexibility in linker length for the design of optimal degraders. Two proteins without known small molecule ligands, EGFP and DUSP6, were differentially degraded when modified at different locations on their protein surfaces. Further, the cereblon-mediated degradation of the known PROTAC target ERRα was improved through the recruitment of the E3 ligase to regions different from the known ligand binding site. This new methodology will provide insight into overall protein degradability, even in the absence of a known small molecule ligand and inform the process of new ligand and PROTAC development to achieve optimal protein degradation. Furthermore, this approach represents a new, small molecule-based conditional OFF switch of protein function with complete genetic specificity. Importantly, the protein of interest is only modified with a minimal surface modification (<200 Da) and does not require any protein domain fusions.

The goal of precision oncology is to find effective therapeutics for every patient. Through the inclusion of emerging therapeutics in a high-throughput drug screening platform, our functional genomics pipeline inverts the common paradigm to identify patient populations that are likely to benefit from novel therapeutic strategies.

Utilizing drug screening data across a panel of 46 cancer cell lines from 11 tumor lineages, we identified an ovarian cancer-specific sensitivity to the first-in-class CRL4 inhibitors KH-4-43 and 33-11. CRL4 (i.e., Cullin-4 RING E3 ubiquitin ligase) is known to be dysregulated in a variety of cancer contexts, making it an attractive therapeutic target. Unlike proteasome inhibitors that are associated with broad toxicity, CRL4 inhibition offers the potential for tumor-specific effects.

We observed that CRL4 inhibition negatively regulates core gene signatures that are upregulated in ovarian tumors and significantly slowed tumor growth as compared to the standard of care, cisplatin, in OVCAR8 xenografts. Building on this, we performed combination drug screening in conjunction with proteomic and transcriptomic profiling to identify ways to improve the antitumor effects of CRL4 inhibition in ovarian cancer models. CRL4 inhibition consistently resulted in activation of the mitogen-activated protein kinase (MAPK) signaling cascade at both the transcriptomic and protein levels, suggesting that survival signaling is induced in response to CRL4 inhibition. These observations were concordant with the results of the combination drug screens in seven ovarian cancer cell lines that showed CRL4 inhibition cooperates with MEK inhibition. Preclinical studies in OVCAR8 and A2780 xenografts confirmed the therapeutic potential of the combination of KH-4-43 and trametinib, which extended overall survival and slowed tumor progression relative to either single agent or the standard of care.

Together, these data demonstrate the prospective utility of functional modeling pipelines for therapeutic development and underscore the clinical potential of CRL4 inhibition in the ovarian cancer context.

A precision medicine pipeline identifies ovarian cancer sensitivity to CRL4 inhibitors. CRL4 inhibition induces activation of MAPK signalling as identified by RNA sequencing, proteomics, and phosphoproteomics. Inhibitor combinations that target both CRL4 and this CRL4 inhibitor-induced survival signalling enhance ovarian cancer sensitivity to treatment.

In addition to adaptive immune checkpoint of PD-1/PD-L1, the innate immune checkpoint SIRPα/CD47 plays an important role in regulation of tumor immune escape. However, the mechanism of CD47 ubiquitination on tumor immune escape remains unclear. Here it is found that TRAF2 bound to the C-terminal of CD47 cytoplasmic fragment and induced its ubiquitination, leading to inhibition of CD47 autophagic degradation by disrupting its binding to LC3, which in turn inhibited macrophage phagocytosis and promoted tumor immune escape. In contrast, loss of TRAF2 facilitated CD47 autophagic degradation and inhibited tumor immune escape. Moreover, autophagy induction promoted CD47 degradation and enhanced the efficacy of CD47 antibody anti-tumor immunotherapy. These findings revealed a novel mechanism of ubiquitination of CD47 on tumor immune escape.

Osteoporosis is a systemic skeletal disorder characterized by reduced bone density and an increased risk of fractures, particularly prevalent in the aging population. Osteoporotic complications, including vertebral compression fractures, hip fractures, and distal forearm fractures, affect over 8.9 million individuals globally, placing a significant economic strain on healthcare systems. Recent advances have expanded our understanding of the mechanisms underlying osteoporosis, particularly the intricate regulatory networks involved in bone metabolism. A central player in these processes is ubiquitin-mediated proteasomal degradation, a crucial post-translational modification system that involves ubiquitin, the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin ligase (E3), deubiquitinating enzymes, and the proteasome. Among the various E3 ligases, the NEDD4 family has emerged as a key regulator of both bone development and osteoporotic pathology. This review delineates the role of NEDD4 family in osteoporosis and identifies potential drug targets within these pathways, offering insights into novel therapeutic approaches for osteoporosis through targeted intervention.

Hakai protein (CBLL1 gene) was identified as an E3 ubiquitin ligase of E-cadherin complex, inducing its ubiquitination and degradation, thus inducing epithelial-to-mesenchymal transition. Most of the knowledge about the protein was associated to its E3 ubiquitin ligase canonical role. However, important recent published research has highlighted the noncanonical role of Hakai, independent of its E3 ubiquitin ligase activity, underscoring its involvement in the N6-methyladenosine (m6A) writer complex and its impact on the methylation of RNA. The involvement of Hakai in this mRNA modification process has renewed the relevance of this protein as an important contributor in cancer. Moreover, Hakai potential as a cancer biomarker and its prognostic value in malignant disease also emphasize its untapped potential in precision medicine, which would also be discussed in detail in our review. The development of the first small-molecule inhibitor that targets its atypical substrate binding domain is a promising step that could eventually lead to patient benefit, and we would cover its discovery and ongoing efforts toward its use in clinic.

Spermatogenesis in Lepidoptera holds significant importance due to its unique process of dichotomous spermatogenesis, yielding eupyrene and apyrene spermatozoa through a complex molecular mechanism. While E3 ubiquitin ligases are known to play vital roles in spermatogenesis across various processes, their functions in dichotomous spermatogenesis remain less known. We utilized the RNAi, biochemical and microscopic procedures to unravel the function of ScE3 Siah in dichotomous spermatogenesis of adult Sitotroga cerealella. In S. cerealella E3 ligase Siah predominantly expressed in adult tissues. Knockdown of ScE3 Siah leads to disruptions in testes and sperm morphology, affecting the structure of eupyrene and apyrene sperm bundles and causing defective ultrastructure in eupyrene sperm. This disruption results in a reduction in the number of dichotomous sperms and significantly reduces their motility. Moreover, ScE3 Siah knockdown inhibits the transfer and motility of dichotomous sperm, impacting spermatophore formation in females and ultimately reducing egg production. Understanding the role of ScE3 Siah is not only crucial for comprehending the complex processes involved in dichotomous spermatogenesis and fertilization but also provides an avenue for sustainable pest control management.

Ubiquitination is cells' second most abundant posttranslational protein modification after phosphorylation. The ubiquitin-proteasome system (UPS) is critical in maintaining essential life processes such as cell cycle control, DNA damage repair, and apoptosis. Mutations in ubiquitination pathway genes are strongly linked to the development and spread of multiple cancers since several of the UPS family members possess oncogenic or tumor suppressor activities. This comprehensive review delves into understanding the ubiquitin code, shedding light on its role in cancer cell biology and immune evasion. Furthermore, we highlighted recent advances in the field for targeting the UPS pathway members for effective therapeutic intervention against human cancers. We also discussed the recent update on small-molecule inhibitors and PROTACs and their progress in preclinical and clinical trials.

The tripartite-motif protein 56 (TRIM56) is a RING-type E3 ubiquitin ligase whose functions were recently beginning to be unveiled. While the physiological role(s) of TRIM56 remains unclear, emerging evidence suggests this protein participates in host innate defense mechanisms that guard against viral infections. Interestingly, TRIM56 has been shown to pose a barrier to viruses of distinct families by utilizing its different domains. Apart from exerting direct, restrictive effects on viral propagation, TRIM56 is implicated in regulating innate immune signaling pathways that orchestrate type I interferon response or autophagy, through which it indirectly impacts viral fitness. Remarkably, depending on viral infection settings, TRIM56 either operates in a canonical, E3 ligase-dependent fashion or adopts an enzymatically independent, non-canonical mechanism to bolster innate immune signaling. Moreover, the recent revelation that TRIM56 is an RNA-binding protein sheds new light on its antiviral mechanisms against RNA viruses. This review summarizes recent advances in the emerging roles of TRIM56 in innate antiviral immunity. We focus on its direct virus-restricting effects and its influence on innate immune signaling through two critical pathways: the endolysosome-initiated, double-stranded RNA-sensing TLR3-TRIF pathway and the cytosolic DNA-sensing, cGAS-STING pathway. We discuss the underpinning mechanisms of action and the questions that remain. Further studies understanding the complexity of TRIM56 involvement in innate immunity will add to critical knowledge that could be leveraged for developing antiviral therapeutics.

Degrons are short amino acid sequences that can facilitate the degradation of protein substrates. They can be classified as either ubiquitin-dependent or -independent based on their interactions with the ubiquitin proteasome system (UPS). These amino acid sequences are often found in exposed regions of proteins serving as either a tethering point for an interaction with an E3 ligase or initiating signaling for the direct degradation of the protein. Recent advancements in the protein degradation field have shown the therapeutic potential of both classes of degrons through leveraging their degradative effects to engage specific protein targets. This review explores what targeted protein degradation applications degrons can be used in and how they have inspired new degrader technology to target a wide variety of protein substrates.

Wound healing is a complex biological process involving multiple cellular and molecular mechanisms. Ubiquitination, a crucial post-translational modification, plays a vital role in regulating various aspects of wound healing through protein modification and degradation. This review comprehensively examines the molecular mechanisms of ubiquitination in wound healing, focusing on its regulation of inflammatory responses, macrophage polarization, angiogenesis, and the activities of fibroblasts and keratinocytes. We discuss how ubiquitination modifies key signaling pathways, including TGF-β/Smad3, NF-κB, and HIF-α, which are essential for proper wound healing. Understanding these mechanisms provides insights into potential therapeutic strategies for treating impaired wound healing, particularly in conditions such as diabetes. The review highlights recent advances in understanding ubiquitination's role in wound healing and discusses future research directions for developing targeted therapeutic approaches.

Antiviral innate immunity is a complicated system initiated by the induction of type I interferon (IFN-I) and downstream interferon-stimulated genes (ISGs) and is finely regulated by numerous positive and negative factors at different signaling adaptors. During this process, posttranslational modifications, especially ubiquitination, are the most common regulatory strategy used by the host to switch the antiviral innate signaling pathway and are mainly controlled by E3 ubiquitin ligases from different protein families. A comprehensive understanding of the regulatory mechanisms and a novel discovery of regulatory factors involved in the IFN-I signaling pathway are important for researchers to identify novel therapeutic targets against viral infectious diseases based on innate immunotherapy. In this section, we use the E3 ubiquitin ligase as an example to guide the identification of a protein belonging to the RING Finger (RNF) family that regulates the RIG-I-mediated IFN-I pathway through ubiquitination.

Membrane-associated Ring-CH 5 (MARCH5) is a mitochondrial E3 ubiquitin ligase playing a key role in the regulation of mitochondrial dynamics. In mammals, MARCH5 negatively regulates mitochondrial antiviral signaling (MAVS) protein aggregation during viral infection and hampers downstream type I interferon signaling to prevent excessive immune activation. However, its precise functional role in the teleost immune system remains unclear. This study investigated the molecular characteristics and immune response of the MARCH5 ortholog in Amphiprion clarkii (A. clarkii; AcMARCH5). The predicted AcMARCH5 protein sequence consists of 287 amino acids with a molecular weight of 32.02 kDa and a theoretical isoelectric point of 9.11. It contains four C-terminal transmembrane (TM) domains and an N-terminal RING cysteine-histidine (CH) domain, which directly regulates ubiquitin transfer. Multiple sequence alignment revealed a high level of conservation between AcMARCH5 and its orthologs in other vertebrate species. Under normal physiological conditions, AcMARCH5 showed the highest mRNA expression in the muscle, brain, and kidney tissues of A. clarkii. Upon stimulation with polyinosinic:polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi, AcMARCH5 expression was drastically modulated. Functional assays showed that overexpression of AcMARCH5 in fathead minnow (FHM) cells downregulated antiviral gene expression, accompanied by enhanced viral hemorrhagic septicemia virus (VHSV) replication. In murine macrophages, AcMARCH5 overexpression markedly reduced the production of pro-inflammatory cytokines in response to poly I:C treatment. Additionally, AcMARCH5 exhibited an anti-apoptotic effect in H2O2-treated FHM cells. Collectively, these results suggest that AcMARCH5 may play a role in maintaining cellular homeostasis under disease and stress conditions in A. clarkii.

By the ubiquitin-proteasomes, cellular proteins are structurally degraded and turnover. Many essential functions and regulations of cells are regulated and controlled by these proteins. Recent studies indicated that many cancer types have been associated with aberrations in the ubiquitination pathway, which involves three enzymatic steps. Dietary phytochemicals have been identified as having the potential to inhibit carcinogenesis recently. As part of this group of phytochemicals, curcumin can play a crucial role in suppressing carcinogenesis by changing many reactions affected by the ubiquitin-proteasome pathway. Due to its ability to change some biological processes such as NF-κB, inhibit some cyclins, and induce apoptosis, it can be used as a drug in cancer treatment.

In addition to the tumor suppressor role of Cullin 7 (Cul7), one of the proteins belonging to the Cullin (Cul) family, studies have also suggested that Cul7 may act as an oncogene under certain conditions. The role of the Cul7 molecule in breast cancer is still unclear, and understanding its function could have significant implications for identifying novel therapeutic targets or improving diagnostic strategies in breast cancer management. In this study, the levels of the Cul7 molecule in plasma and noninvasive material saliva were investigated, and its possibility as a marker for breast cancer was discussed. Protein levels of blood and saliva samples taken from breast cancer patients and a healthy control group were measured by the ELISA (Enzyme-Linked Immunosorbent Assay) method. Gene expression levels between the two groups were analyzed by the qPCR (quantitative Polymerase Chain Reaction) method. In our study, Cul7 mRNA and protein expression levels were examined in 60 breast cancer patients and 20 healthy female controls, and a statistically insignificant difference was found between the patient and control groups in both plasma and saliva samples (p > 0.05). No correlation was found between the clinical characteristics of the patients and plasma and saliva Cul7 gene expression and protein levels (p > 0.05). Considering the possibility of Cul7 being a biomarker at the protein and mRNA levels, plasma is thought to be a better study material for Cul7. Our findings suggest that in the context of a study on salivary material, the expression of Cul7 at the mRNA level may have better potential utility as a biomarker.

The Pro/N-degron recognizing C-terminal to LisH (CTLH) complex is an E3 ligase of emerging interest in the developmental biology field and for targeted protein degradation (TPD) modalities. The human CTLH complex forms distinct supramolecular ring-shaped structures dependent on the multimerization of WDR26 or muskelin β-propeller proteins. Here, we find that, in HeLa cells, CTLH complex E3 ligase activity is dictated by an interplay between WDR26 and muskelin in tandem with muskelin autoregulation. Proteomic experiments revealed that complex-associated muskelin protein turnover is a major ubiquitin-mediated degradation event dependent on the CTLH complex in unstimulated HeLa cells. We observed that muskelin and WDR26 binding to the scaffold of the complex is interchangeable, indicative of the formation of separate WDR26 and muskelin complexes, which correlated with distinct proteomes in WDR26 and muskelin knockout cells. We found that mTOR inhibition-induced degradation of Pro/N-degron containing protein HMGCS1 is distinctly regulated by a muskelin-specific CTLH complex. Finally, we found that mTOR inhibition also activated muskelin degradation, likely as an autoregulatory feedback mechanism to regulate CTLH complex activity. Thus, rather than swapping substrate receptors, the CTLH E3 ligase complex controls substrate selectivity through the differential association of its β-propeller oligomeric subunits WDR26 and muskelin.

Regulation of protein longevity via the ubiquitin (Ub) - proteasome pathway is fundamental to eukaryotic biology. Ubiquitin E3 ligases (E3s) interact with substrate proteins and provide specificity to the pathway. A small subset of E3s bind to specific exposed N-termini (N-degrons) and promote the ubiquitination of the bound protein. Collectively these E3s, and other N-degron binding proteins, are known as N-recognins. There is considerable functional divergence between fungi, animal, and plant N-recognins. In plants, at least three proteins (PRT1, PRT6, and BIG) participate in the Arg/N-degron pathway. PRT1 has demonstrated E3 ligase activity, whereas PRT6 and BIG are candidate E3s. The Arg/N-degron pathway plays a central role in plant development, germination, and submersion tolerance. The pathway has been manipulated both to improve crop performance and for conditional protein degradation. A more detailed structural and biochemical understanding of the Arg/N-recognins and their substrates is required to fully realise the biotechnological potential of the pathway. This perspective focuses on the structural and molecular details of substrate recognition and ubiquitination in the plant Arg/N-degron pathway. While PRT1 appears to be plant specific, the PRT6 and BIG proteins are similar to UBR1 and UBR4, respectively. Analysis of the cryo-EM structures of Saccharomyces UBR1 suggests that the mode of ubiquitin conjugating enzyme (E2) and substrate recruitment is conserved in PRT6, but regulation of the two N-recognins may be significantly different. The structurally characterised domains from human UBR4 are also likely to be conserved in BIG, however, there are sizeable gaps in our understanding of both proteins.