DNA damage-induced proteasome phosphorylation controls substrate recognition and facilitates DNA repair.

Upon DNA damage, numerous proteins are targeted for ubiquitin-dependent proteasomal degradation, which is an integral part of the DNA repair program. Although details of the ubiquitination processes have been intensively studied, little is known about whether and how the 26S proteasome is regulated in the DNA damage response (DDR). Here, we show that human Rpn10/PSMD4, one of the three ubiquitin receptors of the 26S proteasome, is rapidly phosphorylated in response to different types of DNA damage. The phosphorylation occurs at Rpn10-Ser266 within a conserved SQ motif recognized by ATM/ATR/DNA-PK. Blockade of S266 phosphorylation attenuates homologous recombination-mediated DNA repair and sensitizes cells to genotoxic insults. In vitro and in cellulo experiments indicate that phosphorylation of S266, located in the flexible linker between the two ubiquitin-interacting motifs (UIMs) of Rpn10, alters the configuration of UIMs, and actually reduces ubiquitin chain (substrate) binding. As a result, essential DDR proteins such as BRCA1 are spared from premature degradation and allowed sufficient time to engage in DNA repair, a scenario supported by proximity labeling and quantitative proteomic studies. These findings reveal an inherent self-limiting mechanism of the proteasome that, by controlling substrate recognition through Rpn10 phosphorylation, fine-tunes protein degradation for optimal responses under stress.

SelK promotes glioblastoma cell proliferation by inhibiting ß-TrCP1 mediated ubiquitin-dependent degradation of CDK4.

BACKGROUND: Glioblastoma (GB) is recognized as one of the most aggressive brain tumors, with a median survival of 14.6 months. However, there are still some patients whose survival time was greater than 3 years, and the biological reasons behind this clinical phenomenon arouse our research interests. By conducting proteomic analysis on tumor tissues obtained from GB patients who survived over 3 years compared to those who survived less than 1 year, we identified a significant upregulation of SelK in patients with shorter survival times. Therefore, we hypothesized that SelK may be an important indicator related to the occurrence and progression of GBM. METHODS: Proteomics and immunohistochemistry from GB patients were analyzed to investigate the correlation between SelK and clinical prognosis. Cellular phenotypes were evaluated by cell cycle analysis, cell viability assays, and xenograft models. Immunoblots and co-immunoprecipitation were conducted to verify SelK-mediated ubiquitin-dependent degradation of CDK4. RESULTS: SelK was found to be significantly upregulated in GB samples from short-term survivors (≤ 1 year) compared to those from long-term survivors (≥ 3 years), and its expression levels were negatively correlated with clinical prognosis. Knocking down of SelK expression reduced GB cell viability, induced G0/G1 phase arrest, and impaired the growth of transplanted glioma cells in nude mice. Down-regulation of SelK-induced ER stress leads to a reduction in the expression of SKP2 and an up-regulation of ß-TrCP1 expression. Up-regulation of ß-TrCP1, thereby accelerating the ubiquitin-dependent degradation of CDK4 and ultimately inhibiting the malignant proliferation of the GB cells. CONCLUSION: This study discovered a significant increase in SelK expression in GB patients with poor prognosis, revealing a negative correlation between SelK expression and patient outcomes. Further mechanistic investigations revealed that SelK enhances the proliferation of GB cells by targeting the endoplasmic reticulum stress/SKP2/ß-TrCP1/CDK4 axis.

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate.

Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code.

Method to Purify Phase-Separated p62 Bodies Using Fluorescence-Activated Particle Sorting.

p62 bodies are ubiquitin-positive cytoplasmic condensates formed by liquid-liquid phase separation. They are targeted by selective autophagy and play important roles in intracellular quality control and stress responses. However, little is known about their constituents. In this chapter, we describe a method for purifying p62 bodies using fluorescence-activated particle sorting. This method contributes to the identification of novel components of p62 bodies under various physiological and stress conditions.

ATAD5 functions as a regulatory platform for Ub-PCNA deubiquitination.

Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.

A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates.

Over 600 E3 ligases in humans execute ubiquitination of specific target proteins in a spatiotemporal manner to elicit desired signaling effects. Here, we developed a ubiquitin-specific proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich bona fide substrates of a ligase using a one-step streptavidin pulldown under denaturing conditions. We applied our method, which we named Ub-POD, to the really interesting new gene (RING) E3 ligase RAD18 and identified proliferating cell nuclear antigen and several other critical players in the DNA damage repair pathway. Furthermore, we successfully applied Ub-POD to the RING ubiquitin ligase tumor necrosis factor receptor-associated factor 6 and a U-box-type E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein. We anticipate that our method could be widely adapted to all classes of ubiquitin ligases to identify substrates.

Crosstalk of ubiquitin system and non-coding RNA in fibrosis.

Chronic tissue injury triggers changes in the cell type and microenvironment at the site of injury and eventually fibrosis develops. Current research suggests that fibrosis is a highly dynamic and reversible process, which means that human intervention after fibrosis has occurred has the potential to slow down or cure fibrosis. The ubiquitin system regulates the biological functions of specific proteins involved in the development of fibrosis, and researchers have designed small molecule drugs to treat fibrotic diseases on this basis, but their therapeutic effects are still limited. With the development of molecular biology technology, researchers have found that non-coding RNA (ncRNA) can interact with the ubiquitin system to jointly regulate the development of fibrosis. More in-depth explorations of the interaction between ncRNA and ubiquitin system will provide new ideas for the clinical treatment of fibrotic diseases.

Crosstalk among Alternative Polyadenylation, Genetic Variants and Ubiquitin Modification Contribute to Lung Adenocarcinoma Risk.

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

Ubiquitin: Not just a one-way ticket to the proteasome, but a therapeutic dial to fine-tune the molecular landscape of disease.

Recently, there is a rise in studies that recognize the importance of targeting ubiquitin and related molecular machinery in various therapeutic contexts. Here we briefly discuss the history of ubiquitin, its biological roles in protein degradation and beyond, as well as the current state of ubiquitin-targeting therapeutics across diseases. We conclude that targeting ubiquitin machinery is approaching a renaissance, and tapping its full potential will require embracing a wholistic perspective of ubiquitin's multifaceted roles.

Autophagy and ubiquitin-dependent proteolysis processes in left ventricular mass loss in pulmonary arterial hypertension.

The goal of this study was to evaluate the intensity of autophagy and ubiquitin-dependent proteolysis processes occurring in myocardium of left ventricle (LV) in subsequent stages of pulmonary arterial hypertension (PAH) to determine mechanisms responsible for LV mass loss in a monocrotaline-induced PAH rat model. LV myocardium samples collected from 32 Wistar rats were analyzed in an early PAH group (n = 8), controls time-paired (n = 8), an end-stage PAH group (n = 8), and their controls (n = 8). Samples were subjected to histological analyses with immunofluorescence staining, autophagy assessment by western blotting, and evaluation of ubiquitin-dependent proteolysis in the LV by immunoprecipitation of ubiquitinated proteins. Echocardiographic, hemodynamic, and heart morphometric parameters were assessed regularly throughout the experiment. Considerable morphological and hemodynamic remodeling of the LV was observed over the course of PAH. The end-stage PAH was associated with significantly impaired LV systolic function and a decrease in LV mass. The LC3B-II expression in the LV was significantly higher in the end-stage PAH group compared to the early PAH group (p = 0.040). The measured LC3B-II/LC3B-I ratios in the end-stage PAH group were significantly elevated compared to the controls (p = 0.039). Immunofluorescence staining showed a significant increase in the abundance of LC3 puncta in the end-stage PAH group compared to the matched controls. There were no statistically significant differences in the levels of expression of all ubiquitinated proteins when comparing both PAH groups and matched controls. Autophagy may be considered as the mechanism behind the LV mass loss at the end stage of PAH.

The Role of Ubiquitination in Osteosarcoma Development and Therapies.

The ubiquitin-proteasome system (UPS) maintains intracellular protein homeostasis and cellular function by regulating various biological processes. Ubiquitination, a common post-translational modification, plays a crucial role in the regulation of protein degradation, signal transduction, and other physiological and pathological processes, and is involved in the pathogenesis of various cancers, including osteosarcoma. Osteosarcoma, the most common primary malignant bone tumor, is characterized by high metastatic potential and poor prognosis. It is a refractory bone disease, and the main treatment modalities are surgery combined with chemotherapy. Increasing evidence suggests a close association between UPS abnormalities and the progression of osteosarcoma. Due to the complexity and pleiotropy of the ubiquitination system, each step in the ubiquitination process can be targeted by drugs. In recent years, research and development of inhibitors targeting the ubiquitin system have increased gradually, showing great potential for clinical application. This article reviews the role of the ubiquitination system in the development and treatment of osteosarcoma, as well as research progress, with the hope of improving the therapeutic effects and prognosis of osteosarcoma patients by targeting effective molecules in the ubiquitination system.

Gastrodin Improves the Activity of the Ubiquitin-Proteasome System and the Autophagy-Lysosome Pathway to Degrade Mutant Huntingtin.

Gastrodin (GAS) is the main chemical component of the traditional Chinese herb Gastrodia elata (called "Tianma" in Chinese), which has been used to treat neurological conditions, including headaches, epilepsy, stroke, and memory loss. To our knowledge, it is unclear whether GAS has a therapeutic effect on Huntington's disease (HD). In the present study, we evaluated the effect of GAS on the degradation of mutant huntingtin protein (mHtt) by using PC12 cells transfected with N-terminal mHtt Q74. We found that 0.1-100 µM GAS had no effect on the survival rate of Q23 and Q74 PC12 cells after 24-48 h of incubation. The ubiquitin-proteasome system (UPS) is the main system that clears misfolded proteins in eukaryotic cells. Mutated Htt significantly upregulated total ubiquitinated protein (Ub) expression, decreased chymotrypsin-like, trypsin-like and caspase-like peptidase activity, and reduced the colocalization of the 20S proteasome with mHtt. GAS (25 µM) attenuated all of the abovementioned pathological changes, and the regulatory effect of GAS on mHtt was found to be abolished by MG132, a proteasome inhibitor. The autophagy-lysosome pathway (ALP) is another system for misfolded protein degradation. Although GAS downregulated the expression of autophagy markers (LC3II and P62), it increased the colocalization of LC3II with lysosomal associated membrane protein 1 (LAMP1), which indicates that ALP was activated. Moreover, GAS prevented mHtt-induced neuronal damage in PC12 cells. GAS has a selective effect on mHtt in Q74 PC12 cells and has no effect on Q23 and proteins encoded by other genes containing long CAGs, such as Rbm33 (10 CAG repeats) and Hcn1 (>30 CAG repeats). Furthermore, oral administration of 100 mg/kg GAS increased grip strength and attenuated mHtt aggregates in B6-hHTT130-N transgenic mice. This is a high dose (100 mg/kg GAS) when compared with experiments on HD mice with other small molecules. We will design more doses to evaluate the dose-response relationship of the inhibition effect of GAS on mHtt in our next study. In summary, GAS can promote the degradation of mHtt by activating the UPS and ALP, making it a potential therapeutic agent for HD.

Research progress of ubiquitin and ubiquitin-like signaling in Toxoplasma gondii.

Toxoplasmosis, a zoonotic parasitic disease caused by Toxoplasma gondii (T. gondii), is prevalent worldwide. The fact should be emphasized that a considerable proportion of individuals infected with T. gondii may remain asymptomatic; nevertheless, the condition can have severe implications for pregnant women or immunocompromised individuals. The current treatment of toxoplasmosis primarily relies on medication; however, traditional anti-toxoplasmosis drugs exhibit significant limitations in terms of efficacy, side effects, and drug resistance. The life cycles of T. gondii are characterized by distinct stages and its body morphology goes through dynamic alterations during the growth cycle that are intricately governed by a wide array of post-translational modifications (PTMs). Ubiquitin (Ub) signaling and ubiquitin-like (Ubl) signaling are two crucial post-translational modification pathways within cells, regulating protein function, localization, stability, or interactions by attaching Ub or ubiquitin-like proteins (Ubls) to target proteins. While these signaling mechanisms share some functional similarities, they have distinct regulatory mechanisms and effects. T. gondii possesses both Ub and Ubls and plays a significant role in regulating the parasite's life cycle and maintaining its morphology through PTMs of substrate proteins. Investigating the role and mechanism of protein ubiquitination in T. gondii will provide valuable insights for preventing and treating toxoplasmosis. This review explores the distinctive characteristics of Ub and Ubl signaling in T. gondii, with the aim of inspiring research ideas for the identification of safer and more effective drug targets against toxoplasmosis.

Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair.

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

Targeted Protein Degradation (TPD) for Immunotherapy: Understanding Proteolysis Targeting Chimera-Driven Ubiquitin-Proteasome Interactions.

Targeted protein degradation or TPD, is rapidly emerging as a treatment that utilizes small molecules to degrade proteins that cause diseases. TPD allows for the selective removal of disease-causing proteins, including proteasome-mediated degradation, lysosome-mediated degradation, and autophagy-mediated degradation. This approach has shown great promise in preclinical studies and is now being translated to treat numerous diseases, including neurodegenerative diseases, infectious diseases, and cancer. This review discusses the latest advances in TPD and its potential as a new chemical modality for immunotherapy, with a special focus on the innovative applications and cutting-edge research of PROTACs (Proteolysis TArgeting Chimeras) and their efficient translation from scientific discovery to technological achievements. Our review also addresses the significant obstacles and potential prospects in this domain, while also offering insights into the future of TPD for immunotherapeutic applications.

Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Capturing the catalytic intermediates of parkin ubiquitination.

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

Floxuridine supports UPS independent of germline signaling and proteostasis regulators via involvement of detoxification in C. elegans.

The ubiquitin-proteasome system (UPS) is critical for maintaining proteostasis, influencing stress resilience, lifespan, and thermal adaptability in organisms. In Caenorhabditis elegans, specific proteasome subunits and activators, such as RPN-6, PBS-6, and PSME-3, are associated with heat resistance, survival at cold (4°C), and enhanced longevity at moderate temperatures (15°C). Previously linked to improving proteostasis, we investigated the impact of sterility-inducing floxuridine (FUdR) on UPS functionality under proteasome dysfunction and its potential to improve cold survival. Our findings reveal that FUdR significantly enhances UPS activity and resilience during proteasome inhibition or subunit deficiency, supporting worms' normal lifespan and adaptation to cold. Importantly, FUdR effect on UPS activity occurs independently of major proteostasis regulators and does not rely on the germ cells proliferation or spermatogenesis. Instead, FUdR activates a distinct detoxification pathway that supports UPS function, with GST-24 appearing to be one of the factors contributing to the enhanced activity of the UPS upon knockdown of the SKN-1-mediated proteasome surveillance pathway. Our study highlights FUdR unique role in the UPS modulation and its crucial contribution to enhancing survival under low-temperature stress, providing new insights into its mechanisms of action and potential therapeutic applications.

Inhibition of the ubiquitin-proteasome system reduces the abundance of pyruvate dehydrogenase kinase 1 in cultured myotubes.

Pyruvate dehydrogenase kinase (PDK), which phosphorylates the pyruvate dehydrogenase complex, regulates glucose metabolism in skeletal muscle. PDK1, an isozyme whose expression is controlled by hypoxia-inducible factor-1α (HIF-1α), is thought to play a role in muscle adaptation to hypoxia. While transcriptional upregulation of PDK1 by HIF-1α is well characterised, mechanisms controlling proteolysis of PDK1 in skeletal muscle have not been thoroughly investigated. Proteasome inhibitor MG132 paradoxically reduced the abundance of PDK1 in human cancer cells and rat L6 myotubes, suggesting that MG132 might direct PDK1 towards autophagic degradation. The objectives of our current study were to determine (1) whether MG132 suppresses PDK1 levels in primary human myotubes, (2) whether chloroquine, an inhibitor of autophagy, prevents MG132-induced suppression of PDK1 in L6 myotubes, and (3) whether PYR-41, an inhibitor of ubiquitination, suppresses PDK1 in L6 myotubes. Using qPCR and/or immunoblotting, we found that despite markedly upregulating HIF-1α protein, MG132 did not alter the PDK1 expression in cultured primary human myotubes, while it suppressed both PDK1 mRNA and protein in L6 myotubes. The PDK1 levels in L6 myotubes were suppressed also during co-treatment with chloroquine and MG132. PYR-41 markedly increased the abundance of HIF-1α in primary human and L6 myotubes, while reducing the abundance of PDK1. In L6 myotubes treated with PYR-41, chloroquine increased the abundance of the epidermal growth factor receptor, but did not prevent the suppression of PDK1. Collectively, our results suggest that cultured myotubes degrade PDK1 via a pathway that cannot be inhibited by MG132, PYR-41, and/or chloroquine.

Ubiquitination and deubiquitination in cancer: from mechanisms to novel therapeutic approaches.

Ubiquitination, a pivotal posttranslational modification of proteins, plays a fundamental role in regulating protein stability. The dysregulation of ubiquitinating and deubiquitinating enzymes is a common feature in various cancers, underscoring the imperative to investigate ubiquitin ligases and deubiquitinases (DUBs) for insights into oncogenic processes and the development of therapeutic interventions. In this review, we discuss the contributions of the ubiquitin-proteasome system (UPS) in all hallmarks of cancer and progress in drug discovery. We delve into the multiple functions of the UPS in oncology, including its regulation of multiple cancer-associated pathways, its role in metabolic reprogramming, its engagement with tumor immune responses, its function in phenotypic plasticity and polymorphic microbiomes, and other essential cellular functions. Furthermore, we provide a comprehensive overview of novel anticancer strategies that leverage the UPS, including the development and application of proteolysis targeting chimeras (PROTACs) and molecular glues.