Deubiquitinating enzymes: potential regulators of the tumor microenvironment and implications for immune evasion.

Ubiquitination and deubiquitination are important forms of posttranslational modification that govern protein homeostasis. Deubiquitinating enzymes (DUBs), a protein superfamily consisting of more than 100 members, deconjugate ubiquitin chains from client proteins to regulate cellular homeostasis. However, the dysregulation of DUBs is reportedly associated with several diseases, including cancer. The tumor microenvironment (TME) is a highly complex entity comprising diverse noncancerous cells (e.g., immune cells and stromal cells) and the extracellular matrix (ECM). Since TME heterogeneity is closely related to tumorigenesis and immune evasion, targeting TME components has recently been considered an attractive therapeutic strategy for restoring antitumor immunity. Emerging studies have revealed the involvement of DUBs in immune modulation within the TME, including the regulation of immune checkpoints and immunocyte infiltration and function, which renders DUBs promising for potent cancer immunotherapy. Nevertheless, the roles of DUBs in the crosstalk between tumors and their surrounding components have not been comprehensively reviewed. In this review, we discuss the involvement of DUBs in the dynamic interplay between tumors, immune cells, and stromal cells and illustrate how dysregulated DUBs facilitate immune evasion and promote tumor progression. We also summarize potential small molecules that target DUBs to alleviate immunosuppression and suppress tumorigenesis. Finally, we discuss the prospects and challenges regarding the targeting of DUBs in cancer immunotherapeutics and several urgent problems that warrant further investigation.

Exploring the regulatory role of FBXL19-AS1 in triple-negative breast cancer through the miR-378a-3p/OTUB2 axis.

The regulatory potential of long noncoding RNA (lncRNA) FBXL19-AS1 has been highlighted in various cancers, but its effect on triple-negative breast cancer (TNBC) remains unclear. Here, we aimed to elucidate the role of FBXL19-AS1 in TNBC and its underlying mechanism. RT-qPCR was employed to detect the expressions of FBXL19-AS1 and miR-378a-3p in tissues and cells. Immunohistochemical staining and western blot were utilized to detect the expression levels of proteins. Cell activities were detected using flow cytometry, CCK-8, and transwell assay. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were deployed to investigate interactions of different molecules. Protein-protein interaction (PPI) network, gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathways were used to analyze the downstream pathway. In vivo xenograft model was conducted to detect the effect of FBXL19-AS1 on tumor growth. FBXL19-AS1 was overexpressed in TNBC tissues and cell lines compared with counterparts. FBXL19-AS1 knockdown suppressed TNBC cell activities, whereas its overexpression exhibited the opposite effect. Mechanistically, FBXL19-AS1 was found to interact with miR-378a-3p. Further analysis revealed that miR-378a-3p exerted tumor-suppressive effects in TNBC cells. Additionally, miR-378a-3p targeted and downregulated the expression of ubiquitin aldehyde binding 2 (OTUB2), a deubiquitinase associated with TNBC progression. In vivo experiments substantiated the inhibitory effects of FBXL19-AS1 knockdown on TNBC tumorigenesis, and a miR-378a-3p inhibitor partially rescued these effects. The downstream pathway of the miR-378a-3p/OTUB2 axis was explored, revealing connections with proteins involved in modifying other proteins, removing ubiquitin molecules, and influencing signaling pathways, including the Hippo signaling pathway. Western blot analysis confirmed changes in YAP and TAZ expression levels, indicating a potential regulatory network. In summary, FBXL19-AS1 promotes exacerbation in TNBC by suppressing miR-378a-3p, leading to increased OTUB2 expression. The downstream mechanism may be related to the Hippo signaling pathway. These findings propose potential therapeutic targets for TNBC treatment.

OTUB1 contributes to the stability and function of Influenza A virus NS2.

The influenza A virus (IAV) consists of 8 single-stranded, negative-sense viral RNA (vRNA) segments. After infection, vRNA is transcribed, replicated, and wrapped by viral nucleoprotein (NP) to form viral ribonucleoprotein (vRNP). The transcription, replication, and nuclear export of the viral genome are regulated by the IAV protein, NS2, which is translated from spliced mRNA transcribed from viral NS vRNA. This splicing is inefficient, explaining why NS2 is present in low abundance after IAV infection. The levels of NS2 and its subsequent accumulation are thought to influence viral RNA replication and vRNP nuclear export. Here we show that NS2 is ubiquitinated at the K64 and K88 residues by K48-linked and K63-linked polyubiquitin (polyUb) chains, leading to the degradation of NS2 by the proteasome. Additionally, we show that a host deubiquitinase, OTUB1, can remove polyUb chains conjugated to NS2, thereby stabilizing NS2. Accordingly, knock down of OTUB1 by siRNA reduces the nuclear export of vRNP, and reduces the overall production of IAV. These results collectively demonstrate that the levels of NS2 in IAV-infected cells are regulated by a ubiquitination-deubiquitination system involving OTUB1 that is necessary for optimal IAV replication.

E3 ligases and DUBs target ferroptosis: A potential therapeutic strategy for neurodegenerative diseases.

Ferroptosis is a form of cell death mediated by iron and lipid peroxidation (LPO). Recent studies have provided compelling evidence to support the involvement of ferroptosis in the pathogenesis of various neurodegenerative diseases (NDDs), such as Alzheimer's disease (AD), Parkinson's disease (PD). Therefore, understanding the mechanisms that regulate ferroptosis in NDDs may improve disease management. Ferroptosis is regulated by multiple mechanisms, and different degradation pathways, including autophagy and the ubiquitinproteasome system (UPS), orchestrate the complex ferroptosis response by directly or indirectly regulating iron accumulation or lipid peroxidation. Ubiquitination plays a crucial role as a protein posttranslational modification in driving ferroptosis. Notably, E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs) are key enzymes in the ubiquitin system, and their dysregulation is closely linked to the progression of NDDs. A growing body of evidence highlights the role of ubiquitin system enzymes in regulating ferroptosis sensitivity. However, reports on the interaction between ferroptosis and ubiquitin signaling in NDDs are scarce. In this review, we first provide a brief overview of the biological processes and roles of the UPS, summarize the core molecular mechanisms and potential biological functions of ferroptosis, and explore the pathophysiological relevance and therapeutic implications of ferroptosis in NDDs. In addition, reviewing the roles of E3s and DUBs in regulating ferroptosis in NDDs aims to provide new insights and strategies for the treatment of NDDs. These include E3- and DUB-targeted drugs and ferroptosis inhibitors, which can be used to prevent and ameliorate the progression of NDDs.

A protein sequence-based deep transfer learning framework for identifying human proteome-wide deubiquitinase-substrate interactions.

Protein ubiquitination regulates a wide range of cellular processes. The degree of protein ubiquitination is determined by the delicate balance between ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination. In comparison to the E3-substrate interactions, the DUB-substrate interactions (DSIs) remain insufficiently investigated. To address this challenge, we introduce a protein sequence-based ab initio method, TransDSI, which transfers proteome-scale evolutionary information to predict unknown DSIs despite inadequate training datasets. An explainable module is integrated to suggest the critical protein regions for DSIs while predicting DSIs. TransDSI outperforms multiple machine learning strategies against both cross-validation and independent test. Two predicted DUBs (USP11 and USP20) for FOXP3 are validated by "wet lab" experiments, along with two predicted substrates (AR and p53) for USP22. TransDSI provides new functional perspective on proteins by identifying regulatory DSIs, and offers clues for potential tumor drug target discovery and precision drug application.

OTUD5 promotes the growth of hepatocellular carcinoma by deubiquitinating and stabilizing SLC38A1.

BACKGROUND: Deubiquitinating enzymes (DUBs) cleave ubiquitin on substrate molecules to maintain protein stability. DUBs reportedly participate in the tumorigenesis and tumour progression of hepatocellular carcinoma (HCC). OTU deubiquitinase 5 (OTUD5), a DUB family member, has been recognized as a critical regulator in bladder cancer, breast cancer and HCC. However, the expression and biological function of OTUD5 in HCC are still controversial. RESULTS: We determined that the expression of OTUD5 was significantly upregulated in HCC tissues. High levels of OTUD5 were also detected in most HCC cell lines. TCGA data analysis demonstrated that high OTUD5 expression indicated poorer overall survival in HCC patients. OTUD5 silencing prominently suppressed HCC cell proliferation, while its overexpression markedly enhanced the proliferation of HCC cells. Mass spectrometry analysis revealed solute carrier family 38 member 1 (SLC38A1) as a candidate downstream target protein of OTUD5. Coimmunoprecipitation analysis confirmed the interaction between OTUD5 and SLC38A1. OTUD5 knockdown reduced and OTUD5 overexpression increased SLC38A1 protein levels in HCC cells. However, OTUD5 alteration had no effect on SLC38A1 mRNA expression. OTUD5 maintained SLC38A1 stability by preventing its ubiquitin-mediated proteasomal degradation. SLC38A1 silencing prominently attenuated the OTUD5-induced increase in HCC cell proliferation. Finally, OTUD5 knockdown markedly suppressed the growth of HCC cells in vivo. CONCLUSIONS: OTUD5 is an oncogene in HCC. OTUD5 contributes to HCC cell proliferation by deubiquitinating and stabilizing SLC38A1. These results may provide a theoretical basis for the development of new anti-HCC drugs.

Deubiquitination of CDC6 by OTUD6A promotes tumour progression and chemoresistance.

BACKGROUND: CDC6 is an oncogenic protein whose expression level fluctuates during the cell cycle. Although several E3 ubiquitin ligases responsible for the ubiquitin-mediated proteolysis of CDC6 have been identified, the deubiquitination pathway for CDC6 has not been investigated. METHODS: The proteome-wide deubiquitinase (DUB) screening was used to identify the potential regulator of CDC6. Immunofluorescence, protein half-life and deubiquitination assays were performed to determine the protein stability of CDC6. Gain- and loss-of-function experiments were implemented to analyse the impacts of OUTD6A-CDC6 axis on tumour growth and chemosensitivity in vitro. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced conditional Otud6a knockout (CKO) mouse model and tumour xenograft model were performed to analyse the role of OTUD6A-CDC6 axis in vivo. Tissue specimens were used to determine the association between OTUD6A and CDC6. RESULTS: OTUD6A interacts with, depolyubiquitinates and stabilizes CDC6 by removing K6-, K33-, and K48-linked polyubiquitination. Moreover, OTUD6A promotes cell proliferation and decreases sensitivity to chemotherapy by upregulating CDC6. CKO mice are less prone to BCa tumorigenesis induced by BBN, and knockdown of OTUD6A inhibits tumour progression in vivo. Furthermore, OTUD6A protein level has a positive correlation with CDC6 protein level, and high protein levels of OTUD6A and CDC6 are associated with poor prognosis in patients with bladder cancer. CONCLUSIONS: We reveal an important yet missing piece of novel DUB governing CDC6 stability. In addition, our findings propose a model for the OTUD6A-CDC6 axis that provides novel insights into cell cycle and chemosensitivity regulation, which may become a potential biomarker and promising drug target for cancer treatment.

Viral deubiquitinating proteases and the promising strategies of their inhibition.

Several viruses are now known to code for deubiquitinating proteases in their genomes. Ubiquitination is an essential post-translational modification of cellular substrates involved in many processes in the cell, including in innate immune signalling. This post-translational modification is regulated by the ubiquitin conjugation machinery, as well as various host deubiquitinating enzymes. The conjugation of ubiquitin chains to several innate immune related factors is often needed to induce downstream signalling, shaping the antiviral response. Viral deubiquitinating proteins, besides often having a primary function in the viral replication cycle by cleaving the viral polyprotein, are also able to cleave ubiquitin chains from such host substrates, in that way exerting a function in innate immune evasion. The presence of viral deubiquitinating enzymes has been firmly established for numerous animal-infecting viruses, such as some well-researched and clinically important nidoviruses, and their presence has now been confirmed in several plant viruses as well. Viral proteases in general have long been highlighted as promising drug targets, with a current focus on small molecule inhibitors. In this review, we will discuss the range of viral deubiquitinating proteases known to date, summarise the various avenues explored to inhibit such proteases and discuss novel strategies and models intended to inhibit and study these specific viral enzymes.

Dominant negative OTULIN-related autoinflammatory syndrome.

OTU deubiquitinase with linear linkage specificity (OTULIN) regulates inflammation and cell death by deubiquitinating linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC). Biallelic loss-of-function mutations causes OTULIN-related autoinflammatory syndrome (ORAS), while OTULIN haploinsuffiency has not been associated with spontaneous inflammation. However, herein, we identify two patients with the heterozygous mutation p.Cys129Ser in OTULIN. Consistent with ORAS, we observed accumulation of linear ubiquitin chains, increased sensitivity to TNF-induced death, and dysregulation of inflammatory signaling in patient cells. While the C129S mutation did not affect OTULIN protein stability or binding capacity to LUBAC and linear ubiquitin chains, it did ablate OTULIN deubiquitinase activity. Loss of activity facilitated the accumulation of autoubiquitin chains on LUBAC. Altered ubiquitination of LUBAC inhibits its recruitment to the TNF receptor signaling complex, promoting TNF-induced cell death and disease pathology. By reporting the first dominant negative mutation driving ORAS, this study expands our clinical understanding of OTULIN-associated pathology.

Novel insights into the role of ubiquitination in osteoarthritis.

Ubiquitination (Ub) and deubiquitination are crucial post-translational modifications (PTMs) that precisely regulate protein degradation. Under the catalysis of a cascade of E1-E2-E3 ubiquitin enzymes, ubiquitination extensively regulates protein degradation exerting direct impact on various cellular processes, while deubiquitination opposes the effect of ubiquitination and prevents proteins from degradation. Notably, such dynamic modifications have been widely investigated to be implicated in cell cycle, transcriptional regulation, apoptosis and so on. Therefore, dysregulation of ubiquitination and deubiquitination could lead to certain diseases through abnormal protein accumulation and clearance. Increasing researches have revealed that the dysregulation of catalytic regulators of ubiquitination and deubiquitination triggers imbalance of cartilage homeostasis that promotes osteoarthritis (OA) progression. Hence, it is now believed that targeting on Ub enzymes and deubiquitinating enzymes (DUBs) would provide potential therapeutic pathways. In the following sections, we will summarize the biological role of Ub enzymes and DUBs in the development and progression of OA by focusing on the updating researches, with the aim of deepening our understanding of the underlying molecular mechanism of OA pathogenesis concerning ubiquitination and deubiquitination, so as to explore novel potential therapeutic targets of OA treatment.

The mechanism of USP43 in the development of tumor: a literature review.

Ubiquitination of the proteins is crucial for governing protein degradation and regulating fundamental cellular processes. Deubiquitinases (DUBs) have emerged as significant regulators of multiple pathways associated with cancer and other diseases, owing to their capacity to remove ubiquitin from target substrates and modulate signaling. Consequently, they represent potential therapeutic targets for cancer and other life-threatening conditions. USP43 belongs to the DUBs family involved in cancer development and progression. This review aims to provide a comprehensive overview of the existing scientific evidence implicating USP43 in cancer development. Additionally, it will investigate potential small-molecule inhibitors that target DUBs that may have the capability to function as anti-cancer medicines.

Applications of protein ubiquitylation and deubiquitylation in drug discovery.

The ubiquitin (Ub)-proteasome system (UPS) is the major machinery mediating specific protein turnover in eukaryotic cells. By ubiquitylating unwanted, damaged, or harmful proteins and driving their degradation, UPS is involved in many important cellular processes. Several new UPS-based technologies, including molecular glue degraders and PROTACs (proteolysis-targeting chimeras) to promote protein degradation, and DUBTACs (deubiquitinase-targeting chimeras) to increase protein stability, have been developed. By specifically inducing the interactions between different Ub ligases and targeted proteins that are not otherwise related, molecular glue degraders and PROTACs degrade targeted proteins via the UPS; in contrast, by inducing the proximity of targeted proteins to deubiquitinases, DUBTACs are created to clear degradable poly-Ub chains to stabilize targeted proteins. In this review, we summarize the recent research progress in molecular glue degraders, PROTACs, and DUBTACs and their applications. We discuss immunomodulatory drugs, sulfonamides, cyclin-dependent kinase-targeting molecular glue degraders, and new development of PROTACs. We also introduce the principle of DUBTAC and its applications. Finally, we propose a few future directions of these three technologies related to targeted protein homeostasis.

OTUD1 enhances gastric cancer aggressiveness by deubiquitinating EBV-encoded protein BALF1 to stabilize the apoptosis inhibitor Bcl-2.

The Epstein-Barr virus (EBV) is implicated in several cancers, including EBV-associated gastric cancer (EBVaGC). This study focuses on EBV-encoded BALF1 (BamH1 A fragment leftward reading frame 1), a key apoptosis regulator in EBV-related cancers, whose specific impact on EBVaGC was previously unknown. Our findings indicate that BALF1 overexpression in gastric cancer cells significantly enhances their proliferation, migration, and resistance to chemotherapy-induced apoptosis, confirming BALF1's oncogenic potential. A novel discovery is that BALF1 undergoes degradation via the ubiquitin-proteasome pathway. Through analysis of 69 deubiquitinating enzymes (DUBs), ovarian tumor protease (OTU) domain-containing protein 1 (OTUD1) emerged as a vital regulator for maintaining BALF1 protein stability. Furthermore, BALF1 was found to play a role in regulating the stability of the B-cell lymphoma-2 (Bcl-2) protein, increasing its levels through deubiquitination. This mechanism reveals BALF1's multifaceted oncogenic role in gastric cancer, as it contributes both directly and indirectly to cancer progression, particularly by stabilizing Bcl-2, known for its anti-apoptotic characteristics. These insights significantly deepen our understanding of EBV's involvement in the pathogenesis of gastric cancer. The elucidation of OTUD1's role in BALF1 regulation and its influence on Bcl-2 stabilization provide new avenues for therapeutic intervention in EBVaGC, bridging the gap between viral oncogenesis and cellular protein regulation and offering a more holistic view of gastric cancer development under the influence of EBV.

The role of MrUbp4, a deubiquitinase, in conidial yield, thermotolerance, and virulence in Metarhizium robertsii.

Ubiquitin-specific proteases (UBPs), the largest subfamily of deubiquitinating enzymes, regulate ubiquitin homeostasis and play diverse roles in eukaryotes. Ubp4 is essential for the growth, development, and pathogenicity of various fungal pathogens. However, its functions in the growth, stress responses, and virulence of entomopathogenic fungi remain unclear. In this study, we elucidated the role of the homolog of Ubp4, MrUbp4, in the entomopathogenic fungus Metarhizium robertsii. Deletion of MrUbp4 led to a notable increase in ubiquitination levels, demonstrating the involvement of MrUbp4 in protein deubiquitination. Furthermore, the ΔMrUbp4 mutant displayed a significant reduction in conidial yield, underscoring the pivotal role of MrUbp4 in conidiation. Additionally, the mutant exhibited heightened resistance to conidial heat treatment, emphasizing the role of MrUbp4 in thermotolerance. Notably, insect bioassays unveiled a substantial impairment in the virulence of the ΔMrUbp4 mutant. This was accompanied by a notable decrease in cuticle penetration ability and appressorium formation upon further analysis. In summary, our findings highlight the essential role of MrUbp4 in regulating the conidial yield, thermotolerance, and contributions to the virulence of M. robertsii.

Non-canonical deubiquitination of OTUB1 induces IFNγ-mediated cell cycle arrest via regulation of p27 stability.

The deubiquitinase OTUB1, implicated as a potential oncogene in various tumors, lacks clarity in its regulatory mechanism in tumor progression. Our study investigated the effects and underlying mechanisms of OTUB1 on the breast cancer cell cycle and proliferation in IFNγ stimulation. Loss of OTUB1 abrogated IFNγ-induced cell cycle arrest by regulating p27 protein expression, whereas OTUB1 overexpression significantly enhanced p27 expression even without IFNγ treatment. Tyr26 phosphorylation residue of OTUB1 directly bound to p27, modulating its post-translational expression. Furthermore, we identified crucial lysine residues (K134, K153, and K163) for p27 ubiquitination. Src downregulation reduced OTUB1 and p27 expression, suggesting that IFNγ-induced cell cycle arrest is mediated by the Src-OTUB1-p27 signaling pathway. Our findings highlight the pivotal role of OTUB1 in IFNγ-induced p27 expression and cell cycle arrest, offering therapeutic implications.

Deubiquitinase BRCC3 promotes the migration, invasion and EMT progression of colon adenocarcinoma by stabilizing MET expression.

BACKGROUND: Breast cancer type 1 susceptibility protein/breast cancer type 2 susceptibility protein-containing complex subunit 3 (BRCC3), a deubiquitinase (DUBs), is overexpressed in various cancers. However, the underlying biological roles of BRCC3 in adenocarcinoma colon (COAD) have yet to be decrypted. OBJECTIVE: In this work, we explored the potential biological function of BRCC3 in the natural process of COAD cells. METHODS: The expression levels of BRCC3 in COAD tissues and cell lines were investigated via quantitative real time polymerase chain reaction and western blotting analyses. Meanwhile, short hairpin RNAs targeting BRCC3 (sh-BRCC3) or mesenchymal-epithelial transition factor (MET) (sh-MET) were used to investigate the biological function, including proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT) progression in COAD cells. Furthermore, the expression levels of EMT-related biomarkers were detected with western blotting analysis. Furthermore, we also performed Co-IP assay to identify the correlation between BRCC3 and MET. RESULTS: BRCC3 expression was increased in COAD tissues and cell lines. ShRNA-mediated downmodulation of BRCC3 in COAD cell lines induced EMT progression. BRCC3 knockdown resulted in decreased migration as well as invasion and increased apoptosis of SW480 and Lovo cells. Besides, MET was regulated by BRCC3 and involved in the migration, invasion, and EMT in SW480 and Lovo cells. Finally, we uncovered that the overexpressed MET reversed the effects of BRCC3 knockdown in COAD cell development. CONCLUSIONS: BRCC3 acted as a critical factor in the development of COAD by deubiquitinating and stabilizing MET, which might provide an emerging biomarker for the therapeutic and diagnosis strategy of COAD.

Deubiquitinase USP4 suppresses antitumor immunity by inhibiting IRF3 activation and tumor cell-intrinsic interferon response in colorectal cancer.

Despite the approval of immune checkpoint blockade (ICB) therapy for various tumor types, its effectiveness is limited to only approximately 15% of patients with microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR) colorectal cancer (CRC). Approximately 80%-85% of CRC patients have a microsatellite stability (MSS) phenotype, which features a rare T-cell infiltration. Thus, elucidating the mechanisms underlying resistance to ICB in patients with MSS CRC is imperative. In this study, we demonstrate that ubiquitin-specific peptidase 4 (USP4) is upregulated in MSS CRC tumors and negatively regulates the immune response against tumors in CRC. Additionally, USP4 represses the cellular interferon (IFN) response and antigen presentation and impairs PRR signaling-mediated cell death. Mechanistically, USP4 impedes the nuclear localization of interferon regulator Factor 3 (IRF3) by deubiquitinating the K63-polyubiquitin chain of TRAF6 and IRF3. Knockdown of USP4 enhances the infiltration of T cells in CRC tumors and overcomes ICB resistance in an MC38 syngeneic mouse model. Moreover, published datasets revealed that patients showing higher USP4 expression exhibited decreased responsiveness to anti-PD-L1 therapy. These findings highlight an essential role of USP4 in the suppression of antitumor immunity in CRC.

A novel proteomic signature of osteoclast differentiation unveils the deubiquitinase UCHL1 as a necessary osteoclastogenic driver.

Bone destruction, a major source of morbidity, is mediated by heightened differentiation and activity of osteoclasts (OC), highly specialized multinucleated myeloid cells endowed with unique bone-resorptive capacity. The molecular mechanisms regulating OC differentiation in the bone marrow are still partly elusive. Here, we aimed to identify new regulatory circuits and actionable targets by comprehensive proteomic characterization of OCgenesis from mouse bone marrow monocytes, adopting two parallel unbiased comparative proteomic approaches. This work disclosed an unanticipated protein signature of OCgenesis, with most gene products currently unannotated in bone-related functions, revealing broad structural and functional cellular reorganization and divergence from macrophagic immune activity. Moreover, we identified the deubiquitinase UCHL1 as the most upregulated cytosolic protein in differentiating OCs. Functional studies proved it essential, as UCHL1 genetic and pharmacologic inhibition potently suppressed OCgenesis. Furthermore, proteomics and mechanistic dissection showed that UCHL1 supports OC differentiation by restricting the anti-OCgenic activity of NRF2, the transcriptional activator of the canonical antioxidant response, through redox-independent stabilization of the NRF2 inhibitor, KEAP1. Besides offering a valuable experimental framework to dissect OC differentiation, our study discloses the essential role of UCHL1, exerted through KEAP1-dependent containment of NRF2 anti-OCgenic activity, yielding a novel potential actionable pathway against bone loss.

Relevance of mutations in protein deubiquitinases genes and TP53 in corticotroph pituitary tumors.

Introduction: Corticotroph pituitary neuroendocrine tumors (PitNETs) develop from ACTH-producing cells. They commonly cause Cushing's disease (CD), however, some remain clinically silent. Recurrent USP8, USP48, BRAF and TP53 mutations occur in corticotroph PitNETs. The aim of our study was to determine frequency and relevance of these mutations in a possibly large series of corticotroph PitNETs. Methods: Study included 147 patients (100 CD and 47 silent tumors) that were screened for hot-spot mutations in USP8, USP48 and BRAF with Sanger sequencing, while 128 of these patients were screened for TP53 mutations with next generation sequencing and immunohistochemistry. Results: USP8 mutations were found in 41% CD and 8,5% silent tumors, while USP48 mutations were found in 6% CD patients only. Both were more prevalent in women. They were related to higher rate of biochemical remission, non-invasive tumor growth, its smaller size and densely granulated histology, suggesting that these mutation may be favorable clinical features. Multivariate survival analyses did not confirm possible prognostic value of mutation in protein deubiquitinases. No BRAF mutations were found. Four TP53 mutations were identified (2 in CD, 2 in silent tumors) in tumors with size >10mm including 3 invasive ones. They were found in Crooke's cell and sparsely granulated tumors. Tumors with missense TP53 mutations had higher TP53 immunoreactivity score than wild-type tumors. Tumor with frameshift TP53 variant had low protein expression. TP53 mutation was a poor prognostic factor in CD according to uni- and multivariate survival analyses in spite of low mutations frequency. Conclusions: We confirmed high prevalence of USP8 mutations and low incidence of USP48 and TP53 mutations. Changes in protein deubiquitinases genes appear to be favorable prognostic factors in CD. TP53 mutations are rare, occur in both functioning and silent tumors and are related to poor clinical outcome in CD.

BRCC3 mediates inflammation and pyroptosis in cerebral ischemia/reperfusion injury by activating the NLRP6 inflammasome.

AIMS: Neuroinflammation and pyroptosis are key mediators of cerebral ischemia/reperfusion (I/R) injury-induced pathogenic cascades. BRCC3, the human homolog of BRCC36, is implicated in neurological disorders and plays a crucial role in neuroinflammation and pyroptosis. However, its effects and potential mechanisms in cerebral I/R injury in mice are unclear. METHODS: Cellular localization of BRCC3 and the interaction between BRCC3 and NLRP6 were assessed. Middle cerebral artery occlusion/reperfusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R) models were established in mice and HT22 cells, respectively, to simulate cerebral I/R injury in vivo and in vitro. RESULTS: BRCC3 protein expression peaked 24 h after MCAO and OGD/R. BRCC3 knockdown reduced the inflammation and pyroptosis caused by cerebral I/R injury and ameliorated neurological deficits in mice after MCAO. The effects of BRCC3 on inflammation and pyroptosis may be mediated by NLRP6 inflammasome activation. Moreover, both BRCC3 and its N- and C-terminals interacted with NLRP6, and both BRCC3 and its terminals reduced NLRP6 ubiquitination. Additionally, BRCC3 affected the interaction between NLRP6 and ASC, which may be related to inflammasome activation. CONCLUSION: BRCC3 shows promise as a novel target to enhance neurological recovery and attenuate the inflammatory responses and pyroptosis caused by NLRP6 activation in cerebral I/R injury.