Coronaviral PLpro proteases and the immunomodulatory roles of conjugated versus free Interferon Stimulated Gene product-15 (ISG15).

Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.

Drug resistance mechanisms and treatment strategies mediated by Ubiquitin-Specific Proteases (USPs) in cancers: new directions and therapeutic options.

Drug resistance represents a significant obstacle in cancer treatment, underscoring the need for the discovery of novel therapeutic targets. Ubiquitin-specific proteases (USPs), a subclass of deubiquitinating enzymes, play a pivotal role in protein deubiquitination. As scientific research advances, USPs have been recognized as key regulators of drug resistance across a spectrum of treatment modalities, including chemotherapy, targeted therapy, immunotherapy, and radiotherapy. This comprehensive review examines the complex relationship between USPs and drug resistance mechanisms, focusing on specific treatment strategies and highlighting the influence of USPs on DNA damage repair, apoptosis, characteristics of cancer stem cells, immune evasion, and other crucial biological functions. Additionally, the review highlights the potential clinical significance of USP inhibitors as a means to counter drug resistance in cancer treatment. By inhibiting particular USP, cancer cells can become more susceptible to a variety of anti-cancer drugs. The integration of USP inhibitors with current anti-cancer therapies offers a promising strategy to circumvent drug resistance. Therefore, this review emphasizes the importance of USPs as viable therapeutic targets and offers insight into fruitful directions for future research and drug development. Targeting USPs presents an effective method to combat drug resistance across various cancer types, leading to enhanced treatment strategies and better patient outcomes.

Genome-wide identification and expression analysis of Ubiquitin-specific protease gene family in maize (Zea mays L.).

BACKGROUND: Ubiquitin-specific proteases (UBPs) are a large family of deubiquitinating enzymes (DUBs). They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristics of the UBP gene family in the important staple crop, maize (Zea mays L.). RESULTS: In this study, we performed a bioinformatic analysis of the entire maize genome and identified 45 UBP genes. Phylogenetic analysis indicated that 45 ZmUBP genes can be divided into 15 subfamilies. Analysis of evolutionary patterns and divergence levels indicated that ZmUBP genes were present before the isolation of dicotyledons, were highly conserved and subjected to purifying selection during evolution. Most ZmUBP genes exhibited different expression levels in different tissues and developmental stages. Based on transcriptome data and promoter element analysis, we selected eight ZmUBP genes whose promoters contained a large number of plant hormones and stress response elements and were up-regulated under different abiotic stresses for RT-qPCR analysis, results showed that these genes responded to abiotic stresses and phytohormones to varying degrees, indicating that they play important roles in plant growth and stress response. CONCLUSIONS: In this study, the structure, location and evolutionary relationship of maize UBP gene family members were analyzed for the first time, and the ZmUBP genes that may be involved in stress response and plant growth were identified by combining promoter element analysis, transcriptome data and RT-qPCR analysis. This study informs research on the involvement of maize deubiquitination in stress response.

Ubiquitin-specific proteases (USPs) in leukemia: a systematic review.

BACKGROUND: Leukemia, a type of blood cell cancer, is categorized by the type of white blood cells affected (lymphocytes or myeloid cells) and disease progression (acute or chronic). In 2020, it ranked 15th among the most diagnosed cancers and 11th in cancer-related deaths globally, with 474,519 new cases and 311,594 deaths (GLOBOCAN2020). Research into leukemia's development mechanisms may lead to new treatments. Ubiquitin-specific proteases (USPs), a family of deubiquitinating enzymes, play critical roles in various biological processes, with both tumor-suppressive and oncogenic functions, though a comprehensive understanding is still needed. AIM: This systematic review aimed to provide a comprehensive review of how Ubiquitin-specific proteases are involved in pathogenesis of different types of leukemia. METHODS: We systematically searched the MEDLINE (via PubMed), Scopus, and Web of Science databases according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA) to identify relevant studies focusing on the role of USPs in leukemia. Data from selected articles were extracted, synthesized, and organized to present a coherent overview of the subject matter. RESULTS: The review highlights the crucial roles of USPs in chromosomal aberrations, cell proliferation, differentiation, apoptosis, cell cycle regulation, DNA repair, and drug resistance. USP activity significantly impacts leukemia progression, inhibition, and chemotherapy sensitivity, suggesting personalized diagnostic and therapeutic approaches. Ubiquitin-specific proteases also regulate gene expression, protein stability, complex formation, histone deubiquitination, and protein repositioning in specific leukemia cell types. CONCLUSION: The diagnostic, prognostic, and therapeutic implications associated with ubiquitin-specific proteases (USPs) hold significant promise and the potential to transform leukemia management, ultimately improving patient outcomes.

[Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells].

OBJECTIVE: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro. METHODS: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo. RESULTS: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type Ⅰ (COLⅠ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLⅠ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLⅠ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group. CONCLUSION: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo, and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.

Synthesis and structure-activity relationships of USP48 deubiquitinylase inhibitors.

Ubiquitin-specific proteases represent a family of enzymes that catalyze the cleavage of ubiquitin from specific substrate proteins to regulate their activity. USP48 is a rarely studied USP, which has recently been linked to inflammatory signaling via regulation of the transcription factor nuclear factor kappa B. Nonetheless, a crystal structure of USP48 has not yet been resolved and potent inhibitors are not known. We screened a set of 14 commercially available USP inhibitors for their activity against USP48 and identified the USP2 inhibitor "ML364" as a candidate for further optimization. Using a ligand-based approach, we derived and synthesized a series of ML364 analogs. The IC50 concentrations of the new compounds to inhibit USP48 were determined in a deubiquitinylase activity assay by measuring the fluorescence intensity using tetra-ubiquitin rhodamine110 as substrate. A compound containing a carboxylic acid functionalization (17e) inhibited USP48 activity toward tetra-ubiquitin rhodamine110 with an IC50 of 12.6 µM. Further structure-based refinements are required to improve the inhibition activity and specificity.

USP37 Deubiquitinates CDC73 in HPT-JT Syndrome.

The CDC73/HRPT2 gene, a defect which causes hyperparathyroidism-jaw tumor (HPT-JT) syndrome, encodes CDC73/parafibromin. We aimed to investigate whether CDC73 would be a target for ubiquitin-proteasome degradation. We cloned full-length cDNAs encoding a family of 58 ubiquitin-specific deubiquitinating enzymes (DUBs), also known as ubiquitin-specific proteases (USPs). Use of the yeast two-hybrid system then enabled us to identify USP37 as interacting with CDC73. The biochemical interaction between the USP37 and CDC73 and their reciprocal binding domains were studied. Co-localization of CDC73 and USP37 was observed in cells. CDC73 was found to be polyubiquitinated, and polyubiquitination of CDC73 was prominent in mutants. CDC73 was deubiquitinated via K48-specific ubiquitin chains by USP37, but not by the catalytically inactive USP37C350S mutant. Observation of the binding between deletion mutants of CDC73 and USP37 revealed that the ß-catenin binding site of CDC73 and the ubiquitin-interacting motifs 2 and 3 (UIM2 and 3) of USP37 were responsible for the interaction between the two proteins. Moreover, these two enzymes co-existed within the nucleus of COS7 cells. We conclude that USP37 is a DUB for CDC73 and that the two proteins interact through specific domains, suggesting that USP37 is responsible for the stability of CDC73 in HPT-JT syndrome.

A Quantitative Proteomics Study of Early Heat-Regulated Proteins by Two-Dimensional Difference Gel Electrophoresis Identified OsUBP21 as a Negative Regulator of Heat Stress Responses in Rice.

To understand the early heat shock (HS)-regulated cellular responses that influence the tolerance of rice plant to high environmental temperatures, two-dimensional difference gel electrophoresis (2D-DIGE) is performed to explore the early HS-regulated proteome. Multiple proteins that show abundance changes after 1 and 5 min of HS treatment are identified. Of the early HS-regulated proteins identified, the abundance of a ubiquitin-specific protease, OsUBP21, and its Arabidopsis homolog, AtUBP13, is found to be upregulated by 5 min of HS treatment. Further, knocking the expression of OsUBP21 or AtUBP13 down or out increases the tolerance of rice and Arabidopsis plants to HS stress, suggesting that the function of these ubiquitin-specific proteases in regulating plant HS responses is conserved between monocots and dicots. 2D-DIGE showed a group of proteins are differentially regulated in wild-type and ubp21 mutant after 30 min of HS treatment. Among these proteins, 11 are found to interact directly with OsUBP21; thus, they may be targets of OsUBP21. Future analyses of the roles of these OsUBP21-interacting proteins in plant HS responses will help reveal the protein ubiquitination/deubiquitination-regulated cellular responses induced by HS in rice.

[Effects of ubiquitin-specific proteases 2-69 on proliferation of breast cancer cells].

Objective: To investigate the expression and significance of ubiquitin-specific proteases 2-69(USP2-69) in invasive ductal carcinoma of breast. Methods: Twenty-four cases of human breast tissue with invasive ductal carcinoma diagnosed at Huanshan Hospital, Fudan University from 2013 to 2015 were collected, and the expression of USP2-69 mRNA and protein was detected by molecular hybridization, Western blot and immunohistochemistry. USP2-69 was over-expressed in cultured human breast cancer cell line MCF-7 by USP2-69 plasmid transfection. The cellular proliferative activity was investigated in vitro. Results: The USP2-69 mRNA and protein were highly expressed in breast invasive ductal carcinoma, compared to adjacent normal tissues (P<0.01). Ki-67 protein expression was also increased in cases with high USP2-69 protein level. Western blot showed significantly higher USP2-69 protein level in cancer tissue compared to the adjacent normal tissue. In the cultured tumor cells, there was increased S phase fraction, cellular proliferation rate, flat positive clones, cyclin D1 expression and decreased p27 expression in USP2-69-transfected MCF-7 cells. Conclusions: USP2-69 is over-expressed in breast invasive ductal carcinoma, and is closely related to proliferation promoting effects. The data provide an important experimental basis for further study on the molecular mechanism of breast cancer cell proliferation.

UBIQUITIN-SPECIFIC PROTEASES function in plant development and stress responses.

KEY MESSAGE: UBIQUITIN-SPECIFIC PROTEASES play important roles in plant development and stress responses. Protein ubiquitination and deubiquitination are reversible processes, which can modulate the stability, activity as well as subcellular localization of the substrate proteins. UBIQUITIN-SPECIFIC PROTEASE (UBP) protein family participates in protein deubiquitination. Members of UBP family are involved in a variety of physiological processes in plants, as evidenced by their functional characterization in model plant Arabidopsis and other plants. UBPs are conserved in plants and distinct UBPs function in different regulatory processes, although functional redundancies exist between some members. Here we briefly reviewed recent advances in understanding the biological functions of UBP protein family in Arabidopsis, particularly the molecular mechanisms by which UBPs regulate plant development and stress responses. We believe that elucidation of UBPs function and regulation in Arabidopsis will provide new insights about protein deubiquitination and might shed light on the understanding of the mechanistic roles of UBPs in general, which will definitely contribute to crop improvement in agriculture.

Ubiquitin-specific proteases: From biological functions to potential therapeutic applications in gastric cancer.

Deubiquitination, a post-translational modification regulated by deubiquitinases, is essential for cancer initiation and progression. Ubiquitin-specific proteases (USPs) are essential elements of the deubiquitinase family, and are overexpressed in gastric cancer (GC). Through the regulation of several signaling pathways, such as Wnt/ß-Catenin and nuclear factor-κB signaling, and the promotion of the expression of deubiquitination- and stabilization-associated proteins, USPs promote the proliferation, metastasis, invasion, and epithelial-mesenchymal transition of GC. In addition, the expression of USPs is closely related to clinicopathological features, patient prognosis, and chemotherapy resistance. USPs therefore could be used as prognostic biomarkers. USP targeting small molecule inhibitors have demonstrated strong anticancer activity. However, they have not yet been tested in the clinic. This article provides an overview of the latest fundamental research on USPs in GC, aiming to enhance the understanding of how USPs contribute to GC progression, and identifying possible targets for GC treatment to improve patient survival.

Exploring Ubiquitin-specific proteases as therapeutic targets in Glioblastoma.

Glioblastoma (GB) remains a formidable challenge and requires new treatment strategies. The vital part of the Ubiquitin-proteasome system (UPS) in cellular regulation has positioned it as a potentially crucial target in GB treatment, given its dysregulation oncolines. The Ubiquitin-specific proteases (USPs) in the UPS system were considered due to the garden role in the cellular processes associated with oncolines and their vital function in the apoptotic process, cell cycle regulation, and autophagy. The article provides a comprehensive summary of the evidence base for targeting USPs as potential factors for neoplasm treatment. The review considers the participation of the UPS system in the development, resulting in the importance of p53, Rb, and NF-κB, and evaluates specific goals for therapeutic administration using midnight proteasomal inhibitors and small molecule antagonists of E1 and E2 enzymes. Despite the slowed rate of drug creation, recent therapeutic discoveries based on USP system dynamics hold promise for specialized therapies. The review concludes with an analysis of future wanderers and the feasible effects of targeting USPs on personalized GB therapies, which can improve patient hydration in this current and unattractive therapeutic landscape. The manuscript emphasizes the possibility of USP oncogene therapy as a promising alternative treatment line for GB. It stresses the direct creation of research on the medical effectiveness of the approach.

Corrigendum: Roles of ubiquitin-specific proteases in inflammatory diseases.

[This corrects the article DOI: 10.3389/fimmu.2024.1258740.].

Roles of ubiquitin-specific proteases in inflammatory diseases.

Ubiquitin-specific proteases (USPs), as one of the deubiquitinating enzymes (DUBs) families, regulate the fate of proteins and signaling pathway transduction by removing ubiquitin chains from the target proteins. USPs are essential for the modulation of a variety of physiological processes, such as DNA repair, cell metabolism and differentiation, epigenetic modulations as well as protein stability. Recently, extensive research has demonstrated that USPs exert a significant impact on innate and adaptive immune reactions, metabolic syndromes, inflammatory disorders, and infection via post-translational modification processes. This review summarizes the important roles of the USPs in the onset and progression of inflammatory diseases, including periodontitis, pneumonia, atherosclerosis, inflammatory bowel disease, sepsis, hepatitis, diabetes, and obesity. Moreover, we highlight a comprehensive overview of the pathogenesis of USPs in these inflammatory diseases as well as post-translational modifications in the inflammatory responses and pave the way for future prospect of targeted therapies in these inflammatory diseases.

USP36 regulates the proliferation, survival, and differentiation of hFOB1.19 osteoblast.

BACKGROUND: Effective bone formation relies on osteoblast differentiation, a process subject to intricate post-translational regulation. Ubiquitin-specific proteases (USPs) repress protein degradation mediated by the ubiquitin-proteasome pathway. Several USPs have been documented to regulate osteoblast differentiation, but whether other USPs are involved in this process remains elusive. METHODS: In this study, we conducted a comparative analysis of 48 USPs in differentiated and undifferentiated hFOB1.19 osteoblasts, identifying significantly upregulated USPs. Subsequently, we generated USP knockdown hFOB1.19 cells and evaluated their osteogenic differentiation using Alizarin red staining. We also assessed cell viability, cell cycle progression, and apoptosis through MTT, 7-aminoactinomycin D staining, and Annexin V/PI staining assays, respectively. Quantitative PCR and Western blotting were employed to measure the expression levels of osteogenic differentiation markers. Additionally, we investigated the interaction between the USP and its target protein using co-immunoprecipitation (co-IP). Furthermore, we depleted the USP in hFOB1.19 cells to examine its effect on the ubiquitination and stability of the target protein using immunoprecipitation (IP) and Western blotting. Finally, we overexpressed the target protein in USP-deficient hFOB1.19 cells and evaluated its impact on their osteogenic differentiation using Alizarin red staining. RESULTS: USP36 is the most markedly upregulated USP in differentiated hFOB1.19 osteoblasts. Knockdown of USP36 leads to reduced viability, cell cycle arrest, heightened apoptosis, and impaired osteogenic differentiation in hFOB1.19 cells. USP36 interacts with WD repeat-containing protein 5 (WDR5), and the knockdown of USP36 causes an increased level of WDR5 ubiquitination and accelerated degradation of WDR5. Excessive WDR5 improved the impaired osteogenic differentiation of USP36-deficient hFOB1.19 cells. CONCLUSIONS: These observations suggested that USP36 may function as a key regulator of osteoblast differentiation, and its regulatory mechanism may be related to the stabilization of WDR5.

Therapeutic cysteine protease inhibitors: a patent review (2018-present).

INTRODUCTION: Cysteine proteases are involved in a broad range of biological functions, ranging from extracellular matrix turnover to immunity. Playing an important role in the onset and progression of several diseases, including cancer, immune-related and neurodegenerative disease, viral and parasitic infections, cysteine proteases represent an attractive drug target for the development of therapeutic tools. AREAS COVERED: Recent scientific and patent literature focusing on the design and study of cysteine protease inhibitors with potential therapeutic application has been reviewed. EXPERT OPINION: The discovery of a number of effective structurally diverse cysteine protease inhibitors opened up new challenges and opportunities for the development of therapeutic tools. Mechanistic studies and the availability of X-ray crystal structures of some proteases, alone and in complex with inhibitors, provide crucial information for the rational design and development of efficient and selective cysteine protease inhibitors as preclinical candidates for the treatment of different diseases.

Targeting ubiquitin specific proteases (USPs) in cancer immunotherapy: from basic research to preclinical application.

Tumors have evolved in various mechanisms to evade the immune system, hindering the antitumor immune response and facilitating tumor progression. Immunotherapy has become a potential treatment strategy specific to different cancer types by utilizing multifarious molecular mechanisms to enhance the immune response against tumors. Among these mechanisms, the ubiquitin-proteasome system (UPS) is a significant non-lysosomal pathway specific to protein degradation, regulated by deubiquitinating enzymes (DUBs) that counterbalance ubiquitin signaling. Ubiquitin-specific proteases (USPs), the largest DUB family with the strongest variety, play critical roles in modulating immune cell function, regulating immune response, and participating in antigen processing and presentation during tumor progression. According to recent studies, the expressions of some USP family members in tumor cells are involved in tumor immune escape and immune microenvironment. This review explores the potential of targeting USPs as a new approach for cancer immunotherapy, highlighting recent basic and preclinical studies investigating the applications of USP inhibitors. By providing insights into the structure and function of USPs in cancer immunity, this review aims at assisting in developing new therapeutic approaches for enhancing the immunotherapy efficacy.

Ubiquitin specific peptidases and prostate cancer.

Protein ubiquitination is an important post-translational modification mechanism, which regulates protein stability and activity. The ubiquitination of proteins can be reversed by deubiquitinating enzymes (DUBs). Ubiquitin-specific proteases (USPs), the largest DUB subfamily, can regulate cellular functions by removing ubiquitin(s) from the target proteins. Prostate cancer (PCa) is the second leading type of cancer and the most common cause of cancer-related deaths in men worldwide. Numerous studies have demonstrated that the development of PCa is highly correlated with USPs. The expression of USPs is either high or low in PCa cells, thereby regulating the downstream signaling pathways and causing the development or suppression of PCa. This review summarized the functional roles of USPs in the development PCa and explored their potential applications as therapeutic targets for PCa.

Ubiquitin-specific proteases: Vital regulatory molecules in bone and bone-related diseases.

Stabilization of bone structure and function involves multiple cell-to-cell and molecular interactions, in which the regulatory functions of post-translational modifications such as ubiquitination and deubiquitination shouldn't be underestimated. As the largest family of deubiquitinating enzymes, the ubiquitin-specific proteases (USPs) participate in the development of bone homeostasis and bone-related diseases through multiple classical osteogenic and osteolytic signaling pathways, such as BMP/TGF-ß pathway, NF-κB/p65 pathway, EGFR-MAPK pathway and Wnt/ß-catenin pathway. Meanwhile, USPs may also broadly regulate regulate hormone expression level, cell proliferation and differentiation, and may further influence bone homeostasis from gene fusion and nuclear translocation of transcription factors. The number of patients with bone-related diseases is currently enormous, making exploration of their pathogenesis and targeted therapy a hot topic. Pathological increases in the levels of inflammatory mediators such as IL-1ß and TNF-α lead to inflammatory bone diseases such as osteoarthritis, rheumatoid arthritis and periodontitis. While impaired body metabolism greatly increases the probability of osteoporosis. Abnormal physiological activity of bone-associated cells results in a variety of bone tumors. The regulatory role of USPs in bone-related disease has received particular attention from academics in recent studies. In this review, we focuse on the roles and mechanisms of USPs in bone homeostasis and bone-related diseases, with the expectation of informing targeted therapies in the clinic.

Ubiquitin-Specific Proteases as Potential Therapeutic Targets in Bladder Cancer-In Vitro Evaluation of Degrasyn and PR-619 Activity Using Human and Canine Models.

BACKGROUND: The inhibition of ubiquitin-specific proteases (USPs) is a novel and promising direction in the development of molecularly targeted therapies in oncology. The aim of the present study was to examine whether Degrasyn could be a potential therapeutic agent against bladder cancer (BC). Also, we aimed to determine whether Degrasyn is more effective in terms of anti-cancer activity compared to the non-selective DUB inhibitor PR-619. To facilitate the translational value of the obtained results, our experiments were performed using both human and canine in vitro models of BC. METHODS: Human T24 (urothelial grade III BC) and SV-HUC-1 (non-tumorigenic urothelial cell line), as well as canine K9TCC-PU-NK and RDSVS-TCC1 (both derived from invasive grade III urothelial bladder tumors) cell lines, were used in the present study. Cell proliferation was determined using the MTT assay and Ki-67 proliferation assay, and the level of apoptosis induced by Degrasyn and PR-619 was evaluated by Annexin V-FITC staining and caspase 3/7 activation assay. Western blot was used to assess DNA damage and key proteins involved in apoptosis. RESULTS: Degrasyn inhibited the proliferation of all BC cell lines in a concentration- and time-dependent manner. Lower concentrations of Degrasyn were more potent against human and canine BC cell lines compared to PR-619. Degrasyn induced caspase-dependent apoptosis and triggered DNA damage. PR-619 did not show a significant pro-apoptotic effect. CONCLUSIONS: Our results demonstrate that Degrasyn significantly impairs the growth of in vitro models of human and canine BC. Selective USP inhibition with Degrasyn seems to be more effective in reducing BC cell proliferation and inducing apoptosis and DNA damage than non-selective USP inhibition with PR-619.