Ubiquitin-specific Protease Research Articles

USP7 facilitates deubiquitination of LRRC42 in colorectal cancer to accelerate tumorigenesis and augment Wnt/β-catenin signaling

Colorectal cancer is a prevalent malignancy with an increasing incidence worldwide. Leucine-rich repeat-containing protein 42 (LRRC42) is known to be dysregulated in tumor tissues, yet its role in colorectal cancer remains largely unexplored. Herein, the function of LRRC42 in colorectal cancer was investigated using clinical samples, cellular experiments, animal models, and multiple omics techniques. The results demonstrated that LRRC42 was highly expressed in colorectal cancer tissues and was associated with poor clinical outcomes. Silencing LRRC42 suppressed cell proliferation, induced G0/G1 phase arrest, and promoted apoptosis by reducing Bcl2 expression while elevating the expression of Bax, cleaved PARP and cleaved caspase 3. Conversely, LRRC42 overexpression exhibited the opposite effects. Consistent findings were observed in vivo. Additionally, ubiquitin specific peptidase 7 was identified as a potential LRRC42-interacting protein through immunoprecipitation-mass spectrometry, with ubiquitin specific peptidase 7 stabilizing LRRC42 expression by promoting its deubiquitination. Notably, LRRC42 overexpression partially reversed the effects of ubiquitin specific peptidase 7 silencing on tumor cell proliferation and apoptosis. mRNA sequencing analysis revealed that differentially expressed genes in LRRC42 overexpressing cells were linked to Wnt signaling pathway, suggesting that LRRC42 overexpression may activate this pathway. Furthermore, LRRC42 was proved to elevate the levels of ki67, cyclin D1 and WNT3, while reducing the level of p-β-catenin. These findings suggest that LRRC42 perhaps serve as a potential oncogenic factor in colorectal cancer, regulated by ubiquitin specific peptidase 7 and capable of activating Wnt/β-catenin signaling pathway.

Deubiquitinase USP5 regulates RIPK1 driven pyroptosis in response to myocardial ischemic reperfusion injury

BackgroundGasdermin D (GSDMD) mediated pyroptosis plays a significant role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. However, the precise mechanisms regulating pyroptosis remain unclear. In the study, we aimed to investigate the underlying mechanism of pyroptosis in myocardial I/R injury.MethodsIn the present study, we analyzed the effects of USP5 on the RIPK1 kinase activity mediated pyroptosis in vitro after H/R (hypoxia/reoxygenation) and in vivo in a MI/R mouse model. TTC and Evan’s blue dye, Thioflavin S and immunohistochemistry staining were performed in wild-type, RIPK1flox/flox Cdh5-Cre and USP5 deficiency mice. CMEC cells were transfected with si-USP5. HEK293T cells were transfected with USP5 and RIPK1 overexpression plasmid or its mutants. The levels of USP5, RIPK1, Caspase-8, FADD and GSDMD were determined by Western blot. Protein interactions were evaluated by immunoprecipitation. The protein colocalization in cells was monitored using a confocal microscope.ResultsIn this study, our data demonstrate that RIPK1 is essential for limiting cardiac endothelial cell (CMEC) pyroptosis mediated by caspase-8 in response to myocardial I/R. Additionally, we investigate the role of ubiquitin-specific protease 5 (USP5) as a deubiquitinase for RIPK1. Mechanistically, USP5 interacts with RIPK1, leading to its deubiquitination and stabilization.ConclusionsThese findings offer new insights into the role of USP5 in regulating RIPK1-induced pyroptosis.

Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells.

Lin28A is an oncoprotein overexpressed in several cancer types such as testicular, ovarian, colon, breast and lung cancers. As a pluripotency factor that promotes tumorigenesis, Lin28A is associated with more undifferentiated and aggressive tumors phenotypes. Moreover, Lin28A is a highly stable protein that is difficult to downregulate. The compound resveratrol (RSV) has anticancer effects. The present study aimed to elucidate the mechanisms underlying the downregulation of Lin28A protein expression by RSV in the NCCIT cell line. NCCIT cells were treated with different concentrations of RSV to investigate its effects on Lin28A expression. The mRNA expression levels of Lin28A and ubiquitin-specific protease 28 (USP28) were assessed using reverse transcription-quantitative PCR. Western blot analysis was employed to evaluate the protein levels of Lin28A, USP28 and phosphorylated Lin28A. In addition, in some experiments, cells were treated with a MAPK/ERK pathway inhibitor, and other experiments involved transfecting cells with small interfering RNAs targeting USP28. The results demonstrated that RSV significantly reduced Lin28A expression by destabilizing the protein; this effect was mediated by the ability of RSV to suppress the expression of USP28, a deubiquitinase that normally protects Lin28A from ubiquitination and degradation. Additionally, RSV inhibited phosphorylation of Lin28A via the MAPK/ERK pathway; this phosphorylation event has previously been shown to enhance the stability of Lin28A by increasing its half-life. This resulted in Lin28A degradation through the proteasomal pathway in NCCIT cells. The results provide further evidence of the anticancer activity of RSV, and identified Lin28A and USP28 as promising therapeutic targets. As a stable oncoprotein, downregulating Lin28A expression is challenging. However, the present study demonstrated that RSV can overcome this hurdle by inhibiting USP28 expression and MAPK/ERK signaling to promote Lin28A degradation. Furthermore, elucidating these mechanisms provides avenues for developing targeted cancer therapies.

USP36 promotes colorectal cancer progression through inhibition of p53 signaling pathway via stabilizing RBM28.

Colorectal cancer (CRC) stands as the second most common cause of cancer-related mortality globally and p53, a widely recognized tumor suppressor, contributes to the development of CRC. Ubiquitin-specific protease 36 (USP36), belonging to the deubiquitinating enzyme family, is involved in tumor progression across multiple cancers. However, the underlying molecular mechanism in which USP36 regulates p53 signaling pathway in CRC is unclear. Here, our study revealed that USP36 was increased in CRC tissues and associated with unfavorable prognosis. Functionally, elevated USP36 could promote proliferation, migration, and invasion of CRC cells in vitro and in vivo. Mechanistically, USP36 could interact with and stabilize RBM28 via deubiquitination at K162 residue. Further, upregulated RBM28 could bind with p53 to suppress its transcriptional activity and therefore inactivate p53 signaling pathway. Collectively, our investigation identified the novel USP36/RBM28/p53 axis and its involvement in promoting cell proliferation and metastasis in CRC, which presents a promising therapeutic strategy for CRC treatment.

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay.

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

Loss of USP10 promotes hepatocellular carcinoma proliferation by regulating the serine synthesis pathway through inhibition of LKB1 activity.

Metabolic dysregulation is emerging as a critical factor in tumorigenesis, and reprogramming of serine metabolism has been identified as an essential factor in the progression of hepatocellular carcinoma (HCC). Studies have shown that LKB1 deficiency can activate mTOR to upregulate the serine synthesis pathway (SSP) and promote tumor progression. Our team discovered that ubiquitin-specific protease 10 (USP10) can inhibit HCC proliferation through mTOR, but its relationship with SSP needs further investigation. The metabolite assays revealed a significant increase in serine content in HCC tissues. Through the LKB1/mTOR/activating transcription factor 4 (ATF4) axis, loss of USP10 may increase serine biosynthesis and promote the proliferation of HCC invitro and invivo. Furthermore, it was found that USP10 could activate LKB1 through deubiquitination. Analyzing clinical HCC tissues revealed a positive correlation between USP10 and LKB1. Additionally, those with high expression of USP10 in HCC tissues showed a better degree of tumor differentiation and longer overall survival time. Moreover, we found increased expression of both serine and its synthase in liver tumor tissues of USP10 liver-specific KO mice. Loss of USP10 inhibits the activity of LKB1, contributing to the stimulation of the mTOR/ATF4 axis and SSP and then promoting the proliferation of HCC. This work presents a novel approach for serine-targeted treatment in HCC.

Targeting USP14/UCHL5: A Breakthrough Approach to Overcoming Treatment-Resistant FLT3-ITD-Positive AML.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations in acute myeloid leukemia (AML) are associated with poor prognosis and therapy resistance. This study aimed to demonstrate that inhibiting the deubiquitinating enzymes ubiquitin-specific peptidase 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5) (USP14/UCHL5) with b-AP15 or the organogold compound auranofin (AUR) induces apoptosis in the ITD-transformed human leukemia cell line MV4-11 and mononuclear leukocytes derived from patients with FLT3-ITD-positive AML. This study included patients diagnosed with AML at Tokyo Medical and Dental University Hospital between January 2018 and July 2024. Both treatments blocked downstream FLT3 pathway events, with the effects potentiated by USP14 knockdown. Both treatments inhibited FLT3 deubiquitination via K48 and disrupted translation initiation via 4EBP1, a downstream FLT3 target. FLT3 was downregulated in the leukemic cells, with the associated activation of stress-related MAP kinase pathways and increased NF-E2-related factor 2. Furthermore, the overexpression of B-cell lymphoma-extra-large and myeloid cell leukemia-1 prevented the cell death caused by b-AP15 and AUR. These results suggest that inhibiting USP14/UCHL5, which involves multiple regulatory mechanisms, is a promising target for novel therapies for treatment-resistant FLT3-ITD-positive AML.

Features of Usp1 expression and localization in different types of malignant neoplasms

Aim. Malignant neoplasms pose a significant challenge in medicine, requiring a deep understanding of the molecular mechanisms underlying their pathogenesis. This study aimed to investigate the expression profile and subcellular localization of ubiquitin-specific protease 1 (USP1) in various types of cancer. Methods. Using the TCGA database, an analysis of USP1 expression was conducted across different cancer types. Immunofluorescence analysis with confocal microscopy was utilized to determine the subcellular localization of USP1 in chronic myeloid leukemia (CML) and prostate cancer cells. Results. Bioinformatic analysis revealed heterogeneous USP1 expression, with increased levels observed in acute myeloid leukemia, esophageal carcinoma, and glioblastoma multiforme. Immunofluorescence analysis confirmed nuclear localization of USP1 in CML cells and showed that inhibiting USP1 interaction with UAF1 using ML323 disrupts its transport to the nucleus. Prostate cancer cells exhibited both nuclear and atypical cytoplasmic USP1 localization, suggesting alterations in its expression, interaction with UAF1, and disruption of signaling pathways. Conclusions. USP1 plays a multifaceted role in cancer processes, with dysregulation of its expression and localization associated with tumorigenesis. Targeting USP1 holds therapeutic potential for cancer treatment.

USP22 promotes gefitinib resistance and inhibits ferroptosis in non-small cell lung cancer by deubiquitination of MDM2.

The emergence of chemoresistance markedly compromised the treatment efficiency of human cancer, including non-small cell lung cancer (NSCLC). In the present study, we aimed to explore the effects of ubiquitin-specific peptidase 22 (USP22) and murine double minute 2 (MDM2) in gefitinib resistance in NSCLC. Immunohistochemistry (IHC) assay, quantitative real-time polymerase chain reaction (qRT-PCR) assay and western blot assay were carried out to determine the expression of USP22 and MDM2. Transwell assay and flow cytometry analysis were performed to evaluate cell migration and apoptosis. Cell Counting Kit-8 (CCK-8) assay was employed to assess gefitinib resistance. The phenomenon of ferroptosis was estimated by related commercial kits. The oxidized C11-BODIPY fluorescence intensity by C11-BODIPY staining. The relation between USP22 and MDM2 was analyzed by ubiquitination assay and co-immunoprecipitation (Co-IP) assay. USP22 was abnormally upregulated in NSCLC tissues and cells, and USP22 silencing markedly repressed NSCLC cell migration and facilitated apoptosis and ferroptosis. Moreover, our results indicated that ferroptosis could enhance the suppressive effect of gefitinib on NSCLC cells. Besides, USP22 overexpression enhanced gefitinib resistance and ferroptosis protection in NSCLC cells. Mechanically, USP22 stabilized MDM2 and regulated MDM2 expression through deubiquitination of MDM2. MDM2 deficiency partially restored the effects of USP22 on gefitinib resistance and ferroptosis in NSCLC cells. Of note, we validated the promotional effect of USP22 on gefitinib resistance in NSCLC in vivo through establishing the murine xenograft model. USP22/MDM2 promoted gefitinib resistance and inhibited ferroptosis in NSCLC, which might offer a novel strategy for overcoming gefitinib resistance in NSCLC.

USP1-mediated deubiquitination of KDM1A promotes the malignant progression of triple-negative breast cancer.

Previous research has indicated the highly expressed lysine-specific histone demethylase 1A (KDM1A) in several human malignancies, including triple-negative breast cancer (TNBC). However, its detailed mechanisms in TNBC development remain poorly understood. The mRNA levels of KDM1A and Yin Yang 1 (YY1) were determined by RT-qPCR analysis. Western blot was performed to measure KDM1A and ubiquitin-specific protease 1 (USP1) protein expression. Cell proliferation, apoptosis, invasion, migration and stemness were evaluated by MTT assay, EdU assay, flow cytometry, transwell invasion assay, wound-healing assay and sphere-formation assay, respectively. ChIP and dual-luciferase reporter assays were conducted to determine the relationship between YY1 and KDM1A. Xenograft tumor experiment and IHC were carried out to investigate the roles of USP1 and KDM1A in TNBC development in vivo. The highly expressed KDM1A was demonstrated in TNBC tissues and cells, and KDM1A knockdown significantly promoted cell apoptosis, and hampered cell proliferation, invasion, migration, and stemness in TNBC cells. USP1 could increase the stability of KDM1A via deubiquitination, and USP1 depletion restrained the progression of TNBC cells through decreasing KDM1A expression. Moreover, YY1 transcriptionally activated KDM1A expression by directly binding to its promoter in TNBC cells. Additionally, USP1 inhibition reduced KDM1A expression to suppress tumor growth in TNBC mice in vivo. In conclusion, YY1 upregulation increased KDM1A expression via transcriptional activation. USP1 stabilized KDM1A through deubiquitination to promote TNBC progression.

USP15-Mediated Deubiquitination of FKBP 5 and Activation of the αIIbβ3 Signaling Pathway Regulate Thrombosis in Mice.

Platelets have the hemostatic function, and their aberrant activation is associated with occlusive thrombus formation. Plasma exosomes are rich in platelets containing ubiquitin-specific peptidase 15 (USP15). Herein, we aim to explore the effect of USP15 on thrombosis, as well as expounding whether USP15 acts as an upstream target of FK506 binding protein 5 (FKBP5) to regulate occlusive thrombus formation. Washed human platelets were treated with thrombin for measurement of USP15 and FKBP5 expressions. USP15 loss/gain-of-function variant in HEK293 cells was performed by cell transfection, and the interaction between USP15 and FKBP5 was examined using immunoprecipitation and ubiquitination assays. Mice with USP15-knockout platelets (Plt USP15-/-) were modeled, and subjected to calculation of bleeding time, artery thrombosis imaging and clot retraction assay. FKBP5 expression and the inhibitor of nuclear factor kappa B kinase subunit epsilon (IKBKE)/phosphatidylinositol 3-kinase (PI3K)/Rap1 pathway in wild-type and Plt USP15-/- mice-derived platelets were detected using Western blot. The activation of αIIbβ3 in washed platelets was analyzed using flow cytometry. USP15 and FKBP5 expressions were upregulated in platelets after thrombin treatment. Following transfection of USP15 knockdown and USP15 overexpression plasmids into HEK293 cells, FKBP5 protein expression was downregulated by USP15 knockdown while being upregulated by USP15 overexpression. USP15 bound to FKBP5 and protected FKBP5 against ubiquitination. Knockdown of platelet USP15 prolonged bleeding time, inhibited arterial thrombosis and delayed clot retraction in mice. Knockdown of platelet USP15 also decreased protein expressions of FKBP5, IKBKE and Rap1, p-PI3K/PI3K ratio, and activation of αIIbβ3 in mice. USP15 knockdown in platelets affects thrombosis in mice by promoting the instability of FKBP5 to repress the activation of IKBKE/PI3K/Rap1 pathway-mediated αIIbβ3.

Lung-Targeted Lipid Nanoparticle-Delivered siUSP33 Attenuates SARS-CoV-2 Replication and Virulence by Promoting Envelope Degradation.

As a structural protein of SARS-CoV-2, the envelope (E) protein not only plays a key role in the formation of viral particles, but also forms ion channels and has pathogenic functions, including triggering cell death and inflammatory responses. The stability of E proteins is controlled by the host ubiquitin-proteasome system. By screening human deubiquitinases, it is found that ubiquitin-specific protease 33 (USP33) can enhance the stability of E proteins depending on its deubiquitinase activity, thereby promoting viral replication. In the absence of USP33, E proteins are rapidly degraded, leading to a reduced viral load and inflammation. Using lipid nanoparticle (LNP) encapsulation of siUSP33 by adjusting the lipid components (ionizable cationic lipids), siUSP33 is successfully delivered to mouse lung tissues, rapidly reducing USP33 expression in the lungs and maintaining knockdown for at least 14 days, effectively suppressing viral replication and virulence. This method of delivery allows efficient targeting of the lungs and a response to acute infections without long-term USP33 deficiency. This research, based on the deubiquitination mechanism of USP33 on the E protein, demonstrates that LNP-mediated siRNA delivery targeting USP33 plays a role in antiviral and anti-inflammatory responses, offering a novel strategy for the prevention and treatment of SARS-CoV-2.

Knockdown of USP7 alleviates atherosclerosis in ApoE-deficient mice by regulating EZH2 expression.

Atherosclerosis (AS) is a chronic vascular disease associated with lipid accumulation. Understanding the molecular mechanisms of AS is essential. Ubiquitin-specific protease 7 (USP7) is a deubiquitination enzyme involved in various cellular processes, including lipid metabolism. In this study, we aimed to elucidate the role of USP7 in AS progression and its underlying mechanism using ApoE-deficient mice. We found that USP7 ablation improved the morphological characteristics of AS in these mice. USP7 knockdown reduced inflammation, evidenced by decreases in inflammatory markers IL-6, TNF-α, and IL-1β by 35, 40, and 38%, respectively (p < 0.01). Additionally, USP7 depletion reduced oxidative stress, indicated by a 30% reduction in malondialdehyde levels and increases in superoxide dismutase and glutathione peroxidase levels by 25 and 28%, respectively (p < 0.01). Moreover, USP7 knockdown blocked lipid accumulation in aortic tissue cells. Mechanistically, USP7 knockdown inhibited enhancer of Zeste Homolog 2 (EZH2) expression, thereby suppressing AS progression. In conclusion, USP7 depletion alleviated AS progression in ApoE-deficient mice by targeting EZH2 expression. USP7 may serve as a therapeutic target for AS.

MLKL-USP7-UBA52 signaling is indispensable for autophagy in brain through maintaining ubiquitin homeostasis

ABSTRACT Individuals with genetic elimination of MLKL (mixed lineage kinase domain like pseudokinase) exhibit an increased susceptibility to neurodegenerative diseases like Alzheimer disease (AD). However, the mechanism is not yet fully understood. Here, we observed significant compromise in macroautophagy/autophagy in the brains of mlkl knockout (KO) mice, as evidenced by the downregulation of BECN1/Beclin1 and ULK1 (unc-51 like autophagy activating kinase 1). We identified UBA52 (ubiquitin A-52 residue ribosomal protein fusion product 1) as the binding partner of MLKL under physiological conditions. Loss of Mlkl induced a decrease in ubiquitin levels by preventing UBA52 cleavage. Furthermore, we demonstrated that the deubiquitinase (DUB) USP7 (ubiquitin specific peptidase 7) mediates the processing of UBA52, which is regulated by MLKL. Moreover, our results indicated that the reduction of BECN1 and ULK1 upon Mlkl loss is attributed to a decrease in their lysine 63 (K63)-linked polyubiquitination. Additionally, single-nucleus RNA sequencing revealed that the loss of Mlkl resulted in the disruption of multiple neurodegenerative disease-related pathways, including those associated with AD. These results were consistent with the observation of cognitive impairment in mlkl KO mice and exacerbation of AD pathologies in an AD mouse model with mlkl deletion. Taken together, our findings demonstrate that MLKL-USP7-UBA52 signaling is required for autophagy in brain through maintaining ubiquitin homeostasis, and highlight the contribution of Mlkl loss-induced ubiquitin deficits to the development of neurodegeneration. Thus, the maintenance of adequate levels of ubiquitin may provide a novel perspective to protect individuals from multiple neurodegenerative diseases through regulating autophagy.Abbreviations: 4HB: four-helix bundle; AAV: adeno-associated virus; AD: Alzheimer disease; AIF1: allograft inflammatory factor 1; APOE: apolipoprotein E; APP: amyloid beta precursor protein; Aβ: amyloid β; BECN1: beclin 1; co-IP: co-immunoprecipitation; DEGs: differentially expressed genes; DLG4: discs large MAGUK scaffold protein 4; DUB: deubiquitinase; EBSS: Earle’s balanced salt solution; GFAP: glial fibrillary acidic protein; HRP: horseradish peroxidase; IL1B: interleukin 1 beta; IL6: interleukin 6; IPed: immunoprecipitated; KEGG: Kyoto Encyclopedia of Genes and Genomes; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MLKL: mixed lineage kinase domain like pseudokinase; NSA: necrosulfonamide; OPCs: oligodendrocyte precursor cells; PFA: paraformaldehyde; PsKD: pseudo-kinase domain; SYP: synaptophysin; UB: ubiquitin; UBA52: ubiquitin A-52 residue ribosomal protein fusion product 1; UCHL3: ubiquitin C-terminal hydrolase L3; ULK1: unc-51 like autophagy activating kinase 1; UMAP: uniform manifold approximation and projection; UPS: ubiquitin-proteasome system; USP7: ubiquitin specific peptidase 7; USP9X: ubiquitin specific peptidase 9 X-linked.

The p-MYH9/USP22/HIF-1α axis promotes lenvatinib resistance and cancer stemness in hepatocellular carcinoma

Lenvatinib is a targeted drug used for first-line treatment of hepatocellular carcinoma (HCC). A deeper insight into the resistance mechanism of HCC against lenvatinib is urgently needed. In this study, we aimed to dissect the underlying mechanism of lenvatinib resistance (LR) and provide effective treatment strategies. We established an HCC model of acquired LR. Cell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were used to detect the stemness of HCC cells. Molecular and biochemical strategies such as RNA-sequencing, immunoprecipitation, mass spectrometry and ubiquitination assays were used to explore the underlying mechanisms. Patient-derived HCC models and HCC samples from patients were used to demonstrate clinical significance. We identified that increased cancer stemness driven by the hypoxia-inducible factor-1α (HIF-1α) pathway activation is responsible for acquired LR in HCC. Phosphorylated non-muscle myosin heavy chain 9 (MYH9) at Ser1943, p-MYH9 (Ser1943), could recruit ubiquitin-specific protease 22 (USP22) to deubiquitinate and stabilize HIF-1α in lenvatinib-resistant HCC. Clinically, p-MYH9 (Ser1943) expression was upregulated in HCC samples, which predicted poor prognosis and LR. A casein kinase-2 (CK2) inhibitor and a USP22 inhibitor effectively reversed LR in vivo and in vitro. Therefore, the p-MYH9 (Ser1943)/USP22/HIF-1α axis is critical for LR and cancer stemness. For the diagnosis and treatment of LR in HCC, p-MYH9 (Ser1943), USP22, and HIF-1α might be valuable as novel biomarkers and targets.

The splicing factor QKI inhibits metastasis by modulating alternative splicing of E-Syt2 in papillary thyroid carcinoma

Alternative splicing (AS) plays a crucial role in the hallmarks of cancer and can open new avenues for targeted therapies. However, the aberrant AS events and the metastatic cascade in papillary thyroid carcinoma (PTC) remain largely unclear. Here, we identify the splicing factor, quaking protein (QKI), which was significantly downregulated in PTC and correlated with poor survival outcomes in patients with PTC. Functional studies indicated that low expression of QKI promoted the PTC cell growth and metastasis in vitro and in vivo. Mechanistically, low QKI induced exon 14 retention of extended synaptotagmin 2 (E-Syt2) and produced a long isoform transcript (termed E-Syt2L) that acted as an important oncogenic factor of PTC metastasis. Notably, overexpression of long non-coding RNA eosinophil granule ontogeny transcript (EGOT) physically binds to QKI and suppressed its activity by inhibiting ubiquitin specific peptidase 25 (USP25) mediated deubiquitination and subsequent degradation of QKI. Collectively, these data demonstrate the novel mechanistic links between the splicing factor QKI and splicing event in PTC metastasis and support the potential utility of targeting splicing events as a therapeutic strategy for PTC.

Stereo-selectivity of enantiomeric inhibitors to ubiquitin-specific protease 7 (USP7) dissected by molecular docking, molecular dynamics simulations, and binding free energy calculations

Stereo-selectivity of enantiomeric inhibitors to ubiquitin-specific protease 7 (USP7) dissected by molecular docking, molecular dynamics simulations, and binding free energy calculations

Polyubiquitinated PCNA triggers SLX4-mediated break-induced replication in alternative lengthening of telomeres (ALT)cancer cells.

Replication stresses are the major source of break-induced replication (BIR). Here, we show that in alternative lengthening of telomeres (ALT) cells, replication stress-induced polyubiquitinated proliferating cell nuclear antigen (PCNA) (polyUb-PCNA) triggers BIR at telomeres and the common fragile site (CFS). Consistently, depleting RAD18, a PCNA ubiquitinating enzyme, reduces the occurrence of ALT-associated promyelocytic leukemia (PML) bodies (APBs) and mitotic DNA synthesis at telomeres and CFS, both of which are mediated by BIR. In contrast, inhibiting ubiquitin-specific protease 1 (USP1), an Ub-PCNA deubiquitinating enzyme, results in an increase in the above phenotypes in a RAD18- and UBE2N (the PCNA polyubiquitinating enzyme)-dependent manner. Furthermore, deficiency of ATAD5, which facilitates USP1 activity and unloads PCNAs, augments recombination-associated phenotypes. Mechanistically, telomeric polyUb-PCNA accumulates SLX4, a nuclease scaffold, at telomeres through its ubiquitin-binding domain and increases telomere damage. Consistently, APB increase induced by Ub-PCNA depends on SLX4 and structure-specific endonucleases. Taken together, our results identified the polyUb-PCNA-SLX4 axis as a trigger for directing BIR.

Inhibition of ubiquitin-specific protease 7 ameliorates ferroptosis-mediated myocardial infarction by contrasting oxidative stress: An in vitro and in vivo analysis

BackgroundOur prior research determined that USP7 exacerbates myocardial injury. Additionally, existing studies indicate a strong connection between USP7 and ferroptosis. However, the influence of USP7 on ferroptosis-mediated myocardial infarction (MI) remains unclear. Given these findings, we are particularly interested in USP7's regulatory role in ferroptosis-mediated MI and its underlying mechanisms. MethodsIn this study, we established MI models and lentivirus-transfected groups to inhibit USP7 expression both in vivo and in vitro. Cardiac function was detected with Echocardiography. TTC and HE staining were employed to assess myocardial alterations. The expression of ferroptosis markers (PTGS2, ACSL4, GPX4) were analyzed by RT-qPCR and Western blotting. Flow cytometry and ELISA were used for measuring Fe2+, lipid ROS, GSH, and GSSG levels. TEM and Prussian blue staining were used to observe mitochondrial alterations and iron deposition. RT-qPCR, Western blotting, and immunofluorescence were conducted to analyze Keap1, Nrf2, and nuclear Nrf2 expression in vitro and in vivo. ResultsIn the MI model group, USP7 expression significantly increased, worsening ferroptosis-mediated MI. Conversely, in the USP7-inhibited group, activation of the Keap1-Nrf2 signaling pathway improved ferroptosis-mediated MI outcomes. In vitro, the MI model exhibited a marked decline in cardiomyocyte viability and notable mitochondrial damage. However, these issues improved in the USP7-inhibited groups. In vivo, USP7 intensified MI and iron deposition within the MI model group, with decreased values of LVEF, LVFS, SV, LVAWd, and LVPWs, all of which showed improvement in the USP7-inhibited group, except for LVPWd and LVPWs, which showed no significant variation. Importantly, both the in vitro and in vivo experiments revealed analogous results: a reduction in Keap1 expression and an increase in both Nrf2 and nuclear Nrf2 post USP7 inhibition. Additionally, GPX4 expression decreased while PTGS2 and ACSL4 expressions increased. Notably, concentrations of Fe2+, lipid ROS, GSH, and GSSG significantly decreased. ConclusionIn vitro and in vivo studies have found that inhibition of USP7 attenuates iron deposition and suppresses oxidative stress, resulting in amelioration of ferroptosis-induced MI.

USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication

Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks.