Search Results (29)

Ubiquitin-specific protease 1 (USP1) is an emerging target for poly(ADP-ribose) polymerase 1 (PARP1) inhibitor-resistant and BRCA1/BRCA2 mutant tumors. USP1 is a deubiquitylating enzyme responsible for the removal of the mono-ubiquitin mark on FANCD2, PARP1, and the replication factor proliferating cell nuclear antigen (PCNA), among other proteins. USP1 facilitates proper PCNA-mediated polymerase switching from error-prone trans-lesion synthesis DNA polymerases to replicative DNA polymerases. Due to the critical role of USP1 in DNA synthesis and DNA repair, and the discovery that USP1 deubiquitylates PARP1, USP1 inhibitors (USP1i) were found to have a synthetic lethal relationship with PARP1 inhibitors (PARPi), suggesting a mechanistic link between poly(ADP-ribose) (PAR) dynamics and USP1-mediated ubiquitin hydrolysis. However, the relationship between USP1 inhibition and inhibitors of poly(ADP-ribose) glycohydrolase (PARGi), the primary enzyme responsible for PAR hydrolysis, has not been resolved. Using cell cytotoxicity, synergy, PCNA-ubiquitin, and PAR analyses, it is demonstrated herein that PARG inhibition, combined with USP1 inhibition, leads to increased levels of mono-ubiquitinated PCNA, decreased PAR accumulation, and synergistic cytotoxicity between ML323, a potent USP1i, and PDD00017273, a model PARGi. Future studies will focus on the mechanism that contributes to USP1/PARG synthetic lethality, the mechanism of cell death, and the impact of USP1 on PAR/ubiquitin dynamics and replication stress signaling. Full article

SARS-CoV-2 virus and its variants remain a global health threat, due to their capacity for rapid evolution. Variants throughout the COVID-19 pandemic exhibited variations in virulence, impacting vaccine protection and disease severity. Investigating nonstructural protein variants is critical to understanding viral evolution and manipulation of host protein interactions. We focus on nonstructural protein 3 (nsp3), with multiple domains with different activities, including viral polyprotein cleavage, host deubiquitylation, de-ISGylation, and double-membrane vesicle formation. Using affinity purification–mass spectrometry (AP-MS), we identify differential protein interactions in nsp3 caused by mutations found in variants identified between 2019 and 2024: Alpha 20I, Beta 20H, Delta 21I, Delta 21J, Gamma 20J, Kappa 21B, Lambda 21G, Omicron 21K, and Omicron 21L. A small set of amino acid substitutions in the N-terminal region of nsp3 (nsp3.1) could be traced to increased interactions with RNA-binding proteins, which are vital in viral replication. Meanwhile, variants of the central region of nsp3 (nsp3.2) were found to share interactions with protein quality control machinery, including ER-associated degradation. In this construct, shared trends in interactor enrichment are observed between Omicron 21K and Delta 21I. These results underscore how minor mutations reshape host interactions, emphasizing the evolutionary arms race between the host and virus. We provide a roadmap to track the interaction changes driven by SARS-CoV-2 variant evolution. Full article

Ubiquitin-specific protease 32 (USP32), a deubiquitylating enzyme that controls the ubiquitin process, is overexpressed in multiple cancers and serves as a promising therapeutic target for cancer therapy. Drugs targeting ferroptosis have exhibited promising anticancer activity. Lycobetaine (LBT), a natural alkaloid, holds promise against various cancers, yet its specific targets and anticancer mechanisms remain unclear. In this study, we show that LBT induced ferroptosis in lung squamous cell carcinoma (LUSC) cells, accompanied by glutathione depletion and the accumulation of lipid peroxidation, malondialdehyde, and ferrous iron. Mechanistically, drug affinity responsive target stability-based mass spectrometry analysis, molecular dynamics simulations, and a cellular thermal shift assay confirmed that USP32 is a potential target of LBT in LUSC cells. Moreover, a strong interaction between USP32 and nuclear factor erythroid 2-related factor 2 (NRF2) was found via immunoprecipitation–mass spectrometry and co-immunoprecipitation. In addition, the ubiquitination assay results demonstrated that LBT treatment significantly increased NRF2 ubiquitination and degradation by targeting USP32. Importantly, USP32 overexpression effectively attenuated the effects of LBT on proliferation and ferroptosis in LUSC cells. In orthotopic LUSC xenografts, the administration of LBT significantly inhibited tumor growth and metastasis and induced ferroptosis by targeting the USP32–NRF2 signaling axis. Taken together, these data suggest that LBT exerts its anticancer effects by inhibiting USP32-mediated NRF2 deubiquitination to induce ferroptosis and that LBT may serve as a prospective USP32-targeting agent for LUSC treatment. Full article

Jacaranone derived from Senecio scandens, a traditional Chinese medicine used for centuries, has been documented to exhibit anti-inflammatory and antiproliferative properties in various tumor cell lines. However, the mechanism of action and relationship between inflammation and apoptosis induced by jacaranone remain inadequately elucidated. In this study, the targets of jacaranone and cancer were identified from various databases, while potential targets and pathways were predicted through the analysis of the protein–protein interactions (PPI) network and pathway enrichment. Through a comprehensive network pharmacology analysis and corroborating experimental findings, we revealed that jacaranone induces tumor cell death by fine-tuning the tumor necrosis factor receptor 1 (TNFR1) downstream signaling pathway. TNFR1 serves as a key node that assembles into complexes I and II, regulating pathways including the nuclear factor (NF)-κB signaling pathway and the cell apoptosis pathway, which play crucial roles in cellular life activities. Jacaranone successfully guides survival signaling pathways to apoptotic mechanisms by inhibiting the assembly of complex I and promoting the formation of complex II. In particular, the main action mechanism of jacaranone lies in inducing the degradation of the inhibitor of apoptosis protein (cIAP)-2. cIAP-2 serves as an E3 ubiquitin ligase that ubiquitinates receptor-interacting serine/threonine-protein kinase 1 (RIPK1), thereby hindering the formation of complex I and effectively reducing the phosphorylation of Inhibitor of κB kinase (IKK) β. When the deubiquitylation process of RIPK1 is triggered, it may promote the formation of complex II, which ultimately leads to cell apoptosis. This fully demonstrates the key role of jacaranone in regulating TNFR1 complexes, especially through the degradation of cIAP-2. Taken together, jacaranone hinders the assembly of TNFR1 complex I and promotes the formation of complex II to induce apoptosis of cancer cells. Our findings unveil a novel mechanism underlying jacaranone, while also presenting a fresh approach for the development of new pharmaceuticals. Full article

The gaseous hormone ethylene plays pivotal roles in plant growth and development. The rate-limiting enzyme of ethylene biosynthesis in seed plants is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). ACS proteins are encoded by a multigene family and the expression of ACS genes is highly regulated, especially at a post-translational level. AtACS7, the only type III ACS in Arabidopsis, is degraded in a 26S proteasome-dependent pathway. Here, by using liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) analysis, two lysine residues of AtACS7, lys285 (K285) and lys366 (K366), were revealed to be ubiquitin-modified in young, light-grown Arabidopsis seedlings but not in etiolated seedlings. Deubiquitylation-mimicking mutations of these residues significantly increased the stability of the AtACS7^K285RK366R mutant protein in cell-free degradation assays. All results suggest that K285 and K366 are the major ubiquitination sites on AtACS7, providing deeper insights into the post-translational regulation of AtACS7 in Arabidopsis. Full article

Tubeimoside-1 (TBMS-1), a traditional Chinese medicinal herb, is commonly used as an anti-cancer agent. In this study, we aimed to investigate its effect on the sensitization of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Our results revealed that even though monotherapy using TBMS-1 or TRAIL at sublethal concentrations did not affect cancer cell death, combination therapy using TBMS-1 and TRAIL increased apoptotic cell death. Mechanistically, TBMS-1 destabilized c-FLIP expression by downregulating STAMBPL1, a deubiquitinase (DUB). Specifically, when STAMBPL1 and c-FLIP bound together, STAMBPL1 deubiquitylated c-FLIP. Moreover, STAMBPL1 knockdown markedly increased sensitivity to TRAIL by destabilizing c-FLIP. These findings were further confirmed in vivo using a xenograft model based on the observation that combined treatment with TBMS-1 and TRAIL decreased tumor volume and downregulated STAMBPL1 and c-FLIP expression levels. Overall, our study revealed that STAMBPL1 is essential for c-FLIP stabilization, and that STAMBPL1 depletion enhances TRAIL-mediated apoptosis via c-FLIP downregulation. Full article

Distinct phylogeny and substrate specificities suggest that 12 Arabidopsis Ovarian Tumor domain-containing (OTU) deubiquitinases participate in conserved or plant-specific functions. The otu5-1 null mutant displayed a pleiotropic phenotype, including early flowering, mimicking that of mutants harboring defects in subunits (e.g., ARP6) of the SWR1 complex (SWR1c) involved in histone H2A.Z deposition. Transcriptome and RT-qPCR analyses suggest that downregulated FLC and MAF4-5 are responsible for the early flowering of otu5-1. qChIP analyses revealed a reduction and increase in activating and repressive histone marks, respectively, on FLC and MAF4-5 in otu5-1. Subcellular fractionation, GFP-fusion expression, and MNase treatment of chromatin showed that OTU5 is nucleus-enriched and chromatin-associated. Moreover, OTU5 was found to be associated with FLC and MAF4-5. The OTU5-associated protein complex(es) appears to be distinct from SWR1c, as the molecular weights of OTU5 complex(es) were unaltered in arp6-1 plants. Furthermore, the otu5-1 arp6-1 double mutant exhibited synergistic phenotypes, and H2A.Z levels on FLC/MAF4-5 were reduced in arp6-1 but not otu5-1. Our results support the proposition that Arabidopsis OTU5, acting independently of SWR1c, suppresses flowering by activating FLC and MAF4-5 through histone modification. Double-mutant analyses also indicate that OTU5 acts independently of the HUB1-mediated pathway, but it is partially required for FLC-mediated flowering suppression in autonomous pathway mutants and FRIGIDA-Col. Full article

Coronavirus nonstructural protein 3 (nsp3) is a multi-functional protein, playing a critical role in viral replication and in regulating host antiviral innate immunity. In this study, we demonstrate that nsp3 from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and avian coronavirus infectious bronchitis virus (IBV) directly interacts with melanoma differentiation-associated gene 5 (MDA5), rendering an inhibitory effect on the MDA5-mediated type I interferon (IFN) response. By the co-expression of MDA5 with wild-type and truncated nsp3 constructs, at least three interacting regions mapped to the papain-like protease (PLpro) domain and two other domains located at the N- and C-terminal regions were identified in SARS-CoV-2 nsp3. Furthermore, by introducing point mutations to the catalytic triad, the deubiquitylation activity of the PLpro domain from both SARS-CoV-2 and IBV nsp3 was shown to be responsible for the suppression of the MDA5-mediated type I IFN response. It was also demonstrated that both MDA5 and nsp3 were able to interact with ubiquitin and ubiquitinated proteins, contributing to the interaction between the two proteins. This study confirms the antagonistic role of nsp3 in the MDA5-mediated type I IFN signaling, highlighting the complex interaction between a multi-functional viral protein and the innate immune response. Full article

The endosymbiotic Wolbachia bacteria frequently cause cytoplasmic incompatibility (CI) in their insect hosts, where Wolbachia-infected males cross with uninfected females, leading to no or fewer progenies, indicating a paternal modification by Wolbachia. Recent studies have identified a Wolbachia protein, CidB, containing a DUB (deubiquitylating enzyme) domain, which can be loaded into host sperm nuclei and involved in CI, though the DUB activity is not necessary for CI in Drosophila melanogaster. To investigate whether and how Wolbachia affect protein ubiquitination in testes of male hosts and are thus involved in male fertility, we compared the protein and ubiquitinated protein expressions in D. melanogaster testes with and without Wolbachia. A total of 643 differentially expressed proteins (DEPs) and 309 differentially expressed ubiquitinated proteins (DEUPs) were identified to have at least a 1.5-fold change with a p-value of <0.05. Many DEPs were enriched in metabolic pathway, ribosome, RNA transport, and post-translational protein modification pathways. Many DEUPs were involved in metabolism, ribosome, and proteasome pathways. Notably, 98.1% DEUPs were downregulated in the presence of Wolbachia. Four genes coding for DEUPs in ubiquitin proteasome pathways were knocked down, respectively, in Wolbachia-free fly testes. Among them, Rpn6 and Rpn7 knockdown caused male sterility, with no mature sperm in seminal vesicles. These results reveal deubiquitylating effects induced by Wolbachia infection, suggesting that Wolbachia can widely deubiquitinate proteins that have crucial functions in male fertility of their hosts, but are not involved in CI. Our data provide new insights into the regulatory mechanisms of endosymbiont/host interactions and male fertility. Full article

Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow, we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling) that in combination with DIA MS methods, allowed for the consistent profiling of 35–40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day. Full article

Cisplatin-based chemotherapy is the standard treatment for bladder urothelial carcinoma (UC). Most patients experience chemoresistance, the primary cause of treatment failure, which leads to disease relapse. The underlying mechanism of chemoresistance involves reduced apoptosis. In this study, we investigated the antitumor effect of the deubiquitylating enzyme inhibitor PR-619 in cisplatin-resistant bladder UC. Deubiquitinase (ubiquitin-specific protease 14 (USP14) and USP21) immunohistochemical staining demonstrated that deubiquitination is related to chemoresistance in patients with metastatic UC and may be a target for overcoming chemoresistance. Cytotoxicity and apoptosis were assessed using fluorescence-activated flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay, and PR-619 was found to enhance the cytotoxic and apoptotic effects of cisplatin in cisplatin-resistant T24/R cells. Mitigated cisplatin chemoresistance was associated with the concurrent suppression of c-Myc expression in T24/R cells. Moreover, the expression of c-Myc was upregulated in human bladder UC specimens from patients with chemoresistance. Experiments in a xenograft nude mouse model confirmed that PR-619 enhanced the antitumor effects of cisplatin. These results are promising for the development of therapeutic strategies to prevent UC chemoresistance through the combined use of chemotherapeutic agents/deubiquitination inhibitors (PR-619) by targeting the c-Myc pathway. Full article

Usp22 overexpression is observed in several human cancers and is correlated with poor patient outcomes. The molecular basis underlying this correlation is not clear. Usp22 is the catalytic subunit of the deubiquitylation module in the SAGA histone-modifying complex, which regulates gene transcription. Our previous work demonstrated that the loss of Usp22 in mice leads to decreased expression of several components of receptor tyrosine kinase and TGFβ signaling pathways. To determine whether these pathways are upregulated when Usp22 is overexpressed, we created a mouse model that expresses high levels of Usp22 in all tissues. Phenotypic characterization of these mice revealed over-branching of the mammary glands in females. Transcriptomic analyses indicate the upregulation of key pathways involved in mammary gland branching in mammary epithelial cells derived from the Usp22-overexpressing mice, including estrogen receptor, ERK/MAPK, and TGFβ signaling. However, Usp22 overexpression did not lead to increased tumorigenesis in any tissue. Our findings indicate that elevated levels of Usp22 are not sufficient to induce tumors, but it may enhance signaling abnormalities associated with oncogenesis. Full article

Hepatic steatosis is a consequence of distorted lipid storage and plays a vital role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). This study aimed to explore the role of the COP9 signalosome (CSN) in the development of hepatic steatosis and its interplay with the deubiquitylating enzyme (DUB) cylindromatosis (CYLD). CSN occurs as CSN^CSN7A and CSN^CSN7B variants regulating the ubiquitin proteasome system. It is a deneddylating complex and associates with other DUBs. CYLD cleaves Lys63-ubiquitin chains, regulating a signal cascade that mitigates hepatic steatosis. CSN subunits CSN1 and CSN7B, as well as CYLD, were downregulated with specific siRNA in HepG2 cells and human primary hepatocytes. The same cells were transfected with Flag-CSN7A or Flag-CSN7B for pulldowns. Hepatic steatosis in cell culture was induced by palmitic acid (PA). Downregulation of CSN subunits led to reduced PPAR-γ expression. Flag-pulldowns in both LiSa-2 and HepG2 cells and human primary hepatocytes revealed binding of CYLD preferentially to CSN^CSN7A. This was influenced by PA treatment. Silencing of CSN^CSN7B blocked lipid droplet formation caused a compensatory increase of CSN^CSN7A stabilizing CYLD. Our results demonstrate that CSN^CSN7A-mediated CYLD stabilization impedes hepatic steatosis. Therefore, stabilizing CSN^CSN7A-CYLD interaction might be a strategy to retard hepatic steatosis. Full article