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Background: Inflammatory diseases are often initiated by the activation of inflammasomes triggered by pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), which mediate pyroptosis. Although pyroptosis resulting from aberrant inflammasome triggering in thyroid follicular cells (TFCs) has been observed in Hashimoto's thyroiditis (HT) patients, the underlying mechanisms remain largely unknown. Given the extensive involvement of protein ubiquitination and deubiquitination in inflammatory diseases, we aimed to investigate how deubiquitinating enzymes regulate thyroid follicular cell pyroptosis and HT pathogenesis. Methods: Our study specifically investigated the role of Ubiquitin-specific peptidase 1 (USP1), a deubiquitinase (DUB), in regulating the inflammasome components NLRP3 and AIM2, which are crucial in pyroptosis. We conducted a series of experiments to elucidate the function of USP1 in promoting pyroptosis associated with inflammasomes and the progression of HT. These experiments involved techniques such as USP1 knockdown or inhibition, measurement of key pyroptosis indicators including caspase-1, caspase-1 p20, and GSDMD-N, and examination of the effects of USP1 abrogation on HT using a mouse model. Furthermore, we explored the impact of USP1 on NLRP3 transcription and its potential interaction with p65 nuclear transportation. Results: Our findings provide compelling evidence indicating that USP1 plays a pivotal role in promoting inflammasome-mediated pyroptosis and HT progression by stabilizing NLRP3 and AIM2 through deubiquitination. Furthermore, we discovered that USP1 modulates the transcription of NLRP3 by facilitating p65 nuclear transportation. Knockdown or inhibition of USP1 resulted in weakened cell pyroptosis, as evidenced by reduced levels of caspase-1 p20 and GSDMD-N, which could be restored upon AIM2 overexpression. Remarkably, USP1 abrogation significantly ameliorated HT in the mice model, likely to that treating mice with pyroptosis inhibitors VX-765 and disulfiram. Conclusions: Our study highlights a regulatory mechanism of USP1 on inflammasome activation and pyroptosis in TFCs during HT pathogenesis. These findings expand our understanding of HT and suggest that inhibiting USP1 may be a potential treatment strategy for managing HT. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammatory diseases are often initiated by the activation of inflammasomes triggered by pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), which mediate pyroptosis. Although pyroptosis resulting from aberrant inflammasome triggering in thyroid follicular cells (TFCs) has been observed in Hashimoto's thyroiditis (HT) patients, the underlying mechanisms remain largely unknown. Given the extensive involvement of protein ubiquitination and deubiquitination in inflammatory diseases, we aimed to investigate how deubiquitinating enzymes regulate thyroid follicular cell pyroptosis and HT pathogenesis. Methods Our study specifically investigated the role of Ubiquitin-specific peptidase 1 (USP1), a deubiquitinase (DUB), in regulating the inflammasome components NLRP3 and AIM2, which are crucial in pyroptosis. We conducted a series of experiments to elucidate the function of USP1 in promoting pyroptosis associated with inflammasomes and the progression of HT. These experiments involved techniques such as USP1 knockdown or inhibition, measurement of key pyroptosis indicators including caspase-1, caspase-1 p20, and GSDMD-N, and examination of the effects of USP1 abrogation on HT using a mouse model. Furthermore, we explored the impact of USP1 on NLRP3 transcription and its potential interaction with p65 nuclear transportation. Results Our findings provide compelling evidence indicating that USP1 plays a pivotal role in promoting inflammasome-mediated pyroptosis and HT progression by stabilizing NLRP3 and AIM2 through deubiquitination. Furthermore, we discovered that USP1 modulates the transcription of NLRP3 by facilitating p65 nuclear transportation. Knockdown or inhibition of USP1 resulted in weakened cell pyroptosis, as evidenced by reduced levels of caspase-1 p20 and GSDMD-N, which could be restored upon AIM2 overexpression. Remarkably, USP1 abrogation significantly ameliorated HT in the mice model, likely to that treating mice with pyroptosis inhibitors VX-765 and disulfiram. Conclusions Our study highlights a regulatory mechanism of USP1 on inflammasome activation and pyroptosis in TFCs during HT pathogenesis. These findings expand our understanding of HT and suggest that inhibiting USP1 may be a potential treatment strategy for managing HT.

Introduction: The Anaphase Promoting Complex (APC/C), an E3 ubiquitin ligase, plays a key role in cell cycle control, but it is also thought to operate in postmitotic neurons. Most studies linking APC/C function to neuron biology employed perturbations of the APC/C activators, cell division cycle protein 20 (Cdc20) and Cdc20 homologue 1 (Cdh1). However, multiple lines of evidence indicate that Cdh1 and Cdc20 can function in APC/C-independent contexts, so that the effects of their perturbation cannot strictly be linked to APC/C function. Methods: We therefore deleted the gene encoding Anaphase Promoting Complex 4 (APC4), a core APC/C component, in neurons cultured from conditional knockout (cKO) mice. Results: Our data indicate that several previously published substrates are actually not APC/C substrates, whereas ubiquitin specific peptidase 1 (USP1) protein levels are altered in APC4 knockout (KO) neurons. We propose a model where the APC/C ubiquitylates USP1 early in development, but later ubiquitylates a substrate that directly or indirectly stabilizes USP1. We further discovered a novel role of the APC/C in regulating the number of neurites exiting somata, but we were unable to confirm prior data indicating that the APC/C regulates neurite length, neurite complexity, and synaptogenesis. Finally, we show that APC4 SUMOylation does not impact the ability of the APC/C to control the number of primary neurites or USP1 protein levels. Discussion: Our data indicate that perturbation studies aimed at dissecting APC/C biology must focus on core APC/C components rather than the APC/C activators, Cdh20 and Cdh1. [ABSTRACT FROM AUTHOR]

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1β (Nsp1β) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1β protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense. [ABSTRACT FROM AUTHOR]

The circadian clock is generated by a molecular timekeeping mechanism coordinating daily oscillations of physiology and behaviors in mammals. In the mammalian circadian clockwork, basic helix–loop–helix ARNT‐like protein 1 (BMAL1) is a core circadian component whose defects lead to circadian disruption and elicit behavioral arrhythmicity. To identify previously unknown regulators for circadian clocks, we searched for genes influencing BMAL1 protein level by using a CRISPR/Cas9‐based genome‐wide knockout library. As a result, we found that the deubiquitinase ubiquitin carboxyl‐terminal hydrolase 1 (USP1) positively affects BMAL1 protein abundance. Overexpression of wild‐type USP1, but not a deubiquitinase‐inactive mutant USP1, upregulated BMAL1 protein level, whereas genetic ablation of USP1 downregulated BMAL1 protein level in U2OS cells. Furthermore, treatment with USP1 inhibitors led to significant downregulation of BMAL1 protein in U2OS cells as well as mouse tissues. Subsequently, genetic ablation or pharmacological inhibition of USP1 resulted in reduced mRNA levels of a panel of clock genes and disrupted circadian rhythms in U2OS cells. Mechanistically, USP1 was able to de‐ubiquitinate BMAL1 and inhibit the proteasomal degradation of BMAL1. Interestingly, the expression of Usp1 was much higher than the other two deubiquitinases of BMAL1 (Usp2 and Usp9X) in the mouse heart, implying a tissue‐specific function of USP1 in the regulation of BMAL1 stability. Our work thus identifies deubiquitinase USP1 as a previously unknown regulator of the mammalian circadian clock and highlights the potential of genome‐wide CRISPR screens in the identification of regulators for the circadian clock. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 belongs to the subgenus Sarbecovirus, which universally encodes the accessory protein ORF6. SARS-CoV-2 ORF6 is an antagonist of the interferon (IFN)-mediated antiviral response and plays an important role in viral infections. However, the mechanism by which the host counteracts the function of ORF6 to restrict viral replication remains unclear. In this study, we found that most ORF6 proteins encoded by sarbecoviruses could be ubiquitinated and subsequently degraded via the proteasome pathway. Through extensive screening, we identified that the deubiquitinase USP1, which effectively and broadly deubiquitinates sarbecovirus ORF6 proteins, stabilizes ORF6 proteins, resulting in enhanced viral replication. Therefore, ubiquitination and deubiquitination of ORF6 are important for antagonizing IFN-mediated antiviral signaling and influencing the virulence of SARS-CoV-2. These findings highlight an essential molecular mechanism and may provide a novel target for therapeutic interventions against viral infections. IMPORTANCE The ORF6 proteins encoded by sarbecoviruses are essential for effective viral replication and infection and are important targets for developing effective intervention strategies. In this study, we confirmed that sarbecovirus ORF6 proteins are important antagonists of the host immune response and identified the regulatory mechanisms of ubiquitination and deubiquitination of most sarbecovirus ORF6 proteins. Moreover, we revealed that DUB USP1 prevents the proteasomal degradation of all ORF6 proteins, thereby promoting the virulence of SARS-CoV-2. Thus, impeding ORF6 function is helpful for attenuating the virulence of sarbecoviruses. Therefore, our findings provide a deeper understanding of the molecular mechanisms underlying sarbecovirus infections and offer potential new therapeutic targets for the prevention and treatment of these infections. [ABSTRACT FROM AUTHOR]

[Display omitted] Hypertensive nephropathy is second only to diabetes for the causation of chronic kidney disease worldwide. As the mortality and morbidity of hypertensive nephropathy keep increasing, it is important to elucidate its pathogenesis and develop new treatment strategies. In this study, an angiotensin II (Ang II)-induced renal cell system was established, and the expression of ubiquitin specific peptidase 1 (USP1) in human kidney (HK-2) cells was found to be regulated by Ang II treatment through quantitative RT-PCR and Western blot assay. The detection of glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and lipid reactive oxygen species (ROS) levels revealed that interference with USP1 reversed Ang II-induced oxidative stress and ferroptosis, which was enhanced by overexpression of USP1. Subsequently, USP1 inhibitor SJB3-019A loaded in MIL-100 and PEGTK was modified to fabricate SJB3-019A@MIL-PEGTK nanoparticles, which was confirmed to exhibit excellent alleviation of hypertension-induced oxidative stress and ferroptosis in renal cells both in vitro and in vivo. Our study identified an important pathogenesis of hypertensive nephropathy and SJB3-019A@MIL-PEGTK nanoparticle was used to develop an effective clinical treatment for hypertensive nephropathy. [ABSTRACT FROM AUTHOR]

Reversible protein ubiquitination represents an essential determinator of cellular homeostasis, and the ubiquitin-specific enzymes, particularly deubiquitinases (DUBs), are emerging as promising targets for drug development. DUBs are composed of seven different subfamilies, out of which ubiquitin-specific proteases (USPs) are the largest family with 56 members. One of the well-characterized USPs is USP1, which contributes to several cellular biological processes including DNA damage response, immune regulation, cell proliferation, apoptosis, and migration. USP1 levels and activity are regulated by multiple mechanisms, including transcription regulation, phosphorylation, autocleavage, and proteasomal degradation, ensuring that the cellular function of USP1 is performed in a suitably modulated spatio-temporal manner. Moreover, USP1 with deregulated expression and activity are found in several human cancers, indicating that targeting USP1 is a feasible therapeutic approach in anti-cancer treatment. In this review, we highlight the essential role of USP1 in cancer development and the regulatory landscape of USP1 activity, which might provide novel insights into cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Nonunion is a failure of fracture healing and a major complication after fractures. Ubiquitin-specific protease 1 (USP1) is a deubiquitinase that involved in cell differentiation and cell response to DNA damage. Herein we investigated the expression, function and mechanism of USP1 in nonunion. Methods and results Clinical samples were used to detect the USP1 expression in nonunion. ML323 was selected to inhibit USP1 expression throughout the study. Rat models and mouse embryonic osteoblasts cells (MC3T3-E1) were used to investigate the effects of USP1 inhibition on fracture healing and osteogenesis in vivo and in vitro, respectively. Histological changes were examined by micro-computerized tomography (Micro-CT), hematoxylin & eosin (H&E) staining and Masson staining. Alkaline phosphatase (ALP) activity detection and alizarin red staining were used for osteogenic differentiation observation. The expression of related factors was detected by quantitative real-time PCR, western blot or immunohistochemistry (IHC). It was shown that USP1 was highly expressed in nonunion patients and nonunion rats. USP1 inhibition by ML323 promoted fracture healing in nonunion rats and facilitated the expression of osteogenesis-related factors and the signaling of PI3K/Akt pathway. In addition, USP1 inhibition accelerated osteogenic differentiation and promoting PI3K/Akt signaling in MC3T3-E1 cells. Conclusions USP1 inhibition plays a promotive role in coordinating osteogenic differentiation and fracture healing during nonunion. PI3K/Akt may be the downstream pathway of USP1.

Background: The anti-estrogen tamoxifen is a highly effective hormonal therapy for hormonal-positive (HR+) breast cancer patients; however, the estrogen receptor-negative, progesterone receptor-positive (ER–/PR+) subtype does not give the benefits of tamoxifen. Therefore ER–/PR+ breast cancer has a poor clinical outcome, and novel drug therapy for ER–/PR+ breast cancer could benefit these patients. Methods: 53,805 gene expressions were characterized into HR+ BC and triple-negative breast cancer (TNBC) and analyzed through Breast Cancer Gene Expression Miner in 4319 breast cancer patient samples. The clinical outcomes including overall survival, distant metastasis-free survival, and relapse-free survival were obtained from the PrognoScan database containing 1190 human breast cancer patient samples. To determine the function of ERα and inflammation-related genes such as USP1, CDC20, and CASP1, we used the CRISPR–Cas9 system or gene knockdown (KD) system. To check tumor cell proliferation and migration of ERα KO breast cancer cell line, we used tamoxifen and the inflammation inhibitor Ac-YVAD-CHO. For further confirmation, cancer growth was checked with the inflammation inhibitor in ERα KO breast cancer cell line using a three-dimensional (3D) organoid tissue culture system (ex vivo). Results: We found that gene expression in ER−/PR+ hormonal-positive breast cancer is positively related to ER–/PR– very similar to TNBC, not other HR+ breast cancer using a 4319 breast cancer patient database. Especially, inflammation-related genes, USP1, CDC20, and CASP1, which are highly expressed in TNBC, are also upregulated in ER−/PR+ HR+ breast cancer. Suppression of USP1, CDC20, and CASP1 inhibited tumor cell growth and metastasis in ERα KO (ER–/PR +) cell lines. Interestingly, loss of ERα in HR+ cell lines is not responsive to tamoxifen, but highly sensitive to the inflammation inhibitor, Ac-YVAD-CHO. In in vitro and ex vivo (3D organoid) models, inflammation inhibitor-specific blocks ER–/PR+ tumor proliferation and migration. Conclusions: These findings suggest that an inflammation inhibitor might be a potential option for therapy for ER–/PR+ HR breast cancer patients. [ABSTRACT FROM AUTHOR]

Background: Nonunion is a failure of fracture healing and a major complication after fractures. Ubiquitin-specific protease 1 (USP1) is a deubiquitinase that involved in cell differentiation and cell response to DNA damage. Herein we investigated the expression, function and mechanism of USP1 in nonunion. Methods and results: Clinical samples were used to detect the USP1 expression in nonunion. ML323 was selected to inhibit USP1 expression throughout the study. Rat models and mouse embryonic osteoblasts cells (MC3T3-E1) were used to investigate the effects of USP1 inhibition on fracture healing and osteogenesis in vivo and in vitro, respectively. Histological changes were examined by micro-computerized tomography (Micro-CT), hematoxylin & eosin (H&E) staining and Masson staining. Alkaline phosphatase (ALP) activity detection and alizarin red staining were used for osteogenic differentiation observation. The expression of related factors was detected by quantitative real-time PCR, western blot or immunohistochemistry (IHC). It was shown that USP1 was highly expressed in nonunion patients and nonunion rats. USP1 inhibition by ML323 promoted fracture healing in nonunion rats and facilitated the expression of osteogenesis-related factors and the signaling of PI3K/Akt pathway. In addition, USP1 inhibition accelerated osteogenic differentiation and promoting PI3K/Akt signaling in MC3T3-E1 cells. Conclusions: USP1 inhibition plays a promotive role in coordinating osteogenic differentiation and fracture healing during nonunion. PI3K/Akt may be the downstream pathway of USP1. [ABSTRACT FROM AUTHOR]

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., (© 2024 Wiley Periodicals LLC.)

Vascular calcification (VC) poses significant challenges in cardiovascular health. This study employs single-cell transcriptome sequencing to dissect cellular dynamics in this process. We identify distinct cell subgroups, notably in vascular smooth muscle cells (VSMCs), and observe differences between calcified atherosclerotic cores and adjacent regions. Further exploration reveals ID3 as a key gene regulating VSMC function. In vitro experiments demonstrate ID3's interaction with USP1 and E12, modulating cell proliferation and osteogenic differentiation. Animal models confirm the critical role of the USP1/ID3/E12/P21 axis in VC. This study sheds light on a novel regulatory mechanism, offering potential therapeutic targets., Competing Interests: Declaration of competing interest The author declares no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Methods: First, GSEA strategy and WGCNA analysis were used to screen for anti-ovarian cancer drugs and furthern optimal module, respectively. In addition, functional enrichments of module genes were realized by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Kaplan-Meier was then employed to analyze the prognostic impact of USP1 expression on OV patients. Cell proliferation and cell cycle assays were used to confirm biological functions of USP1 in the final. Results: Through the forementioned methods, we obtained five candidate drugs against OV from 353 anticancer drugs, and proposed ML323 as a novel anti-OV drug. As our hypothesized, ML323 significantly inhibited the proliferation of OV cells. Combined with WGCNA and KEGG analysis, the turquoise module was related to ML323, together with cell cycle. USP1 was subsequently identified as a target of ML323 and according to the TCGA database, USP1 negatively correlated with prognosis in OV, and its reduction and ML323-treatment both inhibited the proliferation of OV cells, blocking the S phase of cell cycle in vitro. Conclusion: Taken together, ML323 exerts its inhibitory effect on the proliferation of OV cells by targeting USP1-regulated cell cycle, providing a therapeutical strategy and potential target against OV. [ABSTRACT FROM AUTHOR]

BCAT2-mediated branched-chain amino acid (BCAA) catabolism is critical for pancreatic ductal adenocarcinoma (PDAC) development, especially at an early stage. However, whether a high-BCAA diet promotes PDAC development in vivo , and the underlying mechanism of BCAT2 upregulation, remain undefined. Here, we find that a high-BCAA diet promotes pancreatic intraepithelial neoplasia (PanIN) progression in LSL-KrasG12D/+ ; Pdx1-Cre (KC) mice. Moreover, we screened with an available deubiquitylase library which contains 31 members of USP family and identified that USP1 deubiquitylates BCAT2 at the K229 site. Furthermore, BCAA increases USP1 protein at the translational level via the GCN2-eIF2α pathway both in vitro and in vivo. More importantly, USP1 inhibition recedes cell proliferation and clone formation in PDAC cells and attenuates pancreas tumor growth in an orthotopic transplanted mice model. Consistently, a positive correlation between USP1 and BCAT2 is found in KC; LSL-KrasG12D/+ ; p53flox/+ ; Pdx1-Cre mice and clinical samples. Thus, a therapeutic targeting USP1-BCAT2-BCAA metabolic axis could be considered as a rational strategy for treatment of PDAC and precisive dietary intervention of BCAA has potentially translational significance. [ABSTRACT FROM AUTHOR]

Abstract Background Hepatocellular carcinoma (HCC) remains a medical challenge due to its high proliferation and metastasis. Although deubiquitinating enzymes (DUBs) play a key role in regulating protein degradation, their pathological roles in HCC have not been fully elucidated. Methods By using biomass spectrometry, co-immunoprecipitation, western blotting and immunofluorescence assays, we identify ribosomal protein S16 (RPS16) as a key substrate of ubiquitin-specific peptidase 1 (USP1). The role of USP1-RPS16 axis in the progression of HCC was evaluated in cell cultures, in xenograft mouse models, and in clinical observations. Results We show that USP1 interacts with RPS16. The depletion of USP1 increases the level of K48-linked ubiquitinated-RPS16, leading to proteasome-dependent RPS16 degradation. In contrast, overexpression of USP1-WT instead of USP1-C90A (DUB inactivation mutant) reduces the level of K48-linked ubiquitinated RPS16, thereby stabilizing RPS16. Consequently, USP1 depletion mimics RPS16 deficiency with respect to the inhibition of growth and metastasis, whereas transfection-enforced re-expression of RPS16 restores oncogenic-like activity in USP1-deficient HCC cells. Importantly, the high expression of USP1 and RPS16 in liver tissue is a prognostic factor for poor survival of HCC patients. Conclusions These findings reveal a previously unrecognized role for the activation of USP1-RPS16 pathway in driving HCC, which may be further developed as a novel strategy for cancer treatment.

Introduction and Objectives: Ubiquitin-specific proteases (USPs) act as proto-oncogenes or tumor suppressors in a wide variety of cancers. In this study, we intended to explore the role of USP1 in hepatocellular carcinoma (HCC). Materials and Methods: The clinical significance of USP1 in HCC was analyzed based on The Cancer Genome Atlas (TCGA) data and immunohistochemical staining. siRNAs and lentivirus were used to knock down and overexpress indicated genes, respectively. qRT-PCR and immunoblotting were performed to examine mRNA and protein expression, respectively. CCK8, colony formation and PI/Annexin V-APC staining were performed to examine cellular function. Immunoprecipitation, coomassie blue staining, mass spectrum and immunoblotting were conducted to evaluate the interaction between USP1 and c-kit. Results: USP1 was over-expressed in HCC patients. Patients with high expression of USP1 had shorter overall and disease free survival than those with low expression of USP1. Functional results showed that USP1 was critical for HCC cell growth and proliferation. Immunoprecipitation and immunoblotting results suggested that USP1 interacted with c-kit and promoted the stability of c-kit, which is an important target of lenvatinib in HCC. Knockdown of c-kit reversed the oncogenic function of USP1 on HCC cell growth. Lastly, USP1 upregulation conferred higher sensitivity of HCC cells to lenvatinib treatment. Conclusions: Our study demonstrated that USP1 acted as an oncogene in HCC. It also promoted lenvatinib efficacy by stabilizing c-kit. [ABSTRACT FROM AUTHOR]

The deubiquitinating enzyme USP1 contains highly conserved motifs forming its catalytic center. Recently, the COSMIC mutation database identified a mutation in USP1 at Asp‐199 in endometrial cancer. Here, we investigated the role of Asp‐199 for USP1 function. The mutation of aspartic acid to alanine (D199A) resulted in failure of USP1 to undergo autocleavage and form a complex with ubiquitin, indicating D199A Usp1 is catalytically inactive. The D199A mutation did not affect the interaction with Uaf1. Moreover, D199A Usp1 had defects in deubiquitination of FANCD2 and PCNA and displayed reduced FANCD2 foci formation and DNA repair efficiency. Furthermore, mutation of Asp‐199 to glutamic acid resulted in phenotypes similar to the D199A mutation. Collectively, our findings demonstrate the importance of Asp‐199 for USP1 activity and suggest the implications of USP1 downregulation in cancer. [ABSTRACT FROM AUTHOR]

Endothelial barrier integrity is important for vascular homeostasis, and hyperpermeability participates in the progression of many pathological states, such as diabetic retinopathy, ischemic stroke, chronic bowel disease, and inflammatory disease. Here, using drug repositioning, we discovered that primaquine diphosphate (PD), previously known as an antimalarial drug, was a potential blocker of vascular leakage. PD inhibited the linear pattern of vascular endothelial growth factors (VEGF)-induced disruption at the cell boundaries, blocked the formation of VEGF-induced actin stress fibers, and stabilized the cortactin actin rings in endothelial cells. PD significantly reduced leakage in the Miles assay and mouse model of streptozotocin (STZ)-induced diabetic retinopathy. Targeted prediction programs and deubiquitinating enzyme activity assays identified a potential mechanism of action for PD and demonstrated that this operates via ubiquitin specific protease 1 (USP1). USP1 inhibition demonstrated a conserved barrier function by inhibiting VEGF-induced leakage in endothelial permeability assays. Taken together, these findings suggest that PD could be used as a novel drug for vascular leakage by maintaining endothelial integrity.

Background: Hepatocellular carcinoma (HCC) remains a medical challenge due to its high proliferation and metastasis. Although deubiquitinating enzymes (DUBs) play a key role in regulating protein degradation, their pathological roles in HCC have not been fully elucidated. Methods: By using biomass spectrometry, co-immunoprecipitation, western blotting and immunofluorescence assays, we identify ribosomal protein S16 (RPS16) as a key substrate of ubiquitin-specific peptidase 1 (USP1). The role of USP1-RPS16 axis in the progression of HCC was evaluated in cell cultures, in xenograft mouse models, and in clinical observations. Results: We show that USP1 interacts with RPS16. The depletion of USP1 increases the level of K48-linked ubiquitinated-RPS16, leading to proteasome-dependent RPS16 degradation. In contrast, overexpression of USP1-WT instead of USP1-C90A (DUB inactivation mutant) reduces the level of K48-linked ubiquitinated RPS16, thereby stabilizing RPS16. Consequently, USP1 depletion mimics RPS16 deficiency with respect to the inhibition of growth and metastasis, whereas transfection-enforced re-expression of RPS16 restores oncogenic-like activity in USP1-deficient HCC cells. Importantly, the high expression of USP1 and RPS16 in liver tissue is a prognostic factor for poor survival of HCC patients. Conclusions: These findings reveal a previously unrecognized role for the activation of USP1-RPS16 pathway in driving HCC, which may be further developed as a novel strategy for cancer treatment. [ABSTRACT FROM AUTHOR]

Endothelial barrier integrity is important for vascular homeostasis, and hyperpermeability participates in the progression of many pathological states, such as diabetic retinopathy, ischemic stroke, chronic bowel disease, and inflammatory disease. Here, using drug repositioning, we discovered that primaquine diphosphate (PD), previously known as an antimalarial drug, was a potential blocker of vascular leakage. PD inhibited the linear pattern of vascular endothelial growth factors (VEGF)-induced disruption at the cell boundaries, blocked the formation of VEGF-induced actin stress fibers, and stabilized the cortactin actin rings in endothelial cells. PD significantly reduced leakage in the Miles assay and mouse model of streptozotocin (STZ)-induced diabetic retinopathy. Targeted prediction programs and deubiquitinating enzyme activity assays identified a potential mechanism of action for PD and demonstrated that this operates via ubiquitin specific protease 1 (USP1). USP1 inhibition demonstrated a conserved barrier function by inhibiting VEGF-induced leakage in endothelial permeability assays. Taken together, these findings suggest that PD could be used as a novel drug for vascular leakage by maintaining endothelial integrity. [ABSTRACT FROM AUTHOR]

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro . Importantly, we found that USP1 interacts with nonstructural protein 1β (Nsp1β) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1β protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense.IMPORTANCEDeubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1β (Nsp1β) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1β, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1β, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response., Competing Interests: The authors declare no conflict of interest.

Ewing sarcoma is a cancer of bone and soft tissue in children and young adults that is driven by the EWS-ETS fusion transcription factor, most commonly EWS-FLI1. We previously reported that Ewing sarcoma harbors two populations of cells, the CD133 high population displaying higher growth rate and the CD133 low population displaying chemotherapy resistance. We now find that the ubiquitin-specific protease 1 (USP1) is a transcriptional target of the EWS-FLI1 fusion oncoprotein, expressed at high and low levels in the CD133 high and the CD133 low populations, respectively, and determines chemo-sensitivity. We also find that USP1 inhibits cdc42, increases EWS-FLI1 transcriptional output, and simulates Ewing sarcoma growth. We show that chemo-sensitization by USP1 is independent of cdc42. A pharmacological inhibitor of USP1 was able to activate cdc42 and inhibit Ewing sarcoma growth. These results uncover critical roles for USP1 in Ewing sarcoma, which regulates growth and chemo-sensitivity via distinct mechanisms., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare.

The prognosis of hepatocellular carcinoma (HCC) is closely associated with the occurrence of distant metastases, which is likely due to circulating tumor cells (CTCs). However, the low number of CTCs is the main obstacle limiting research of the mechanism of CTC metastasis. Here, We evaluated the role of ubiquitin-specific protease 1 (USP1) in promoting CTC survival during blood-borne metastases. We observed that USP1 was frequently upregulated in CTCs and correlated with metastasis and a reduced overall survival rate of patients. Additionally, genetic knockout of USP1 the survival rate of CTCs. Further analyses showed that USP1 mediates oncogenic activity by deubiquitinating and stabilizing transducin β-like 1 X-linked receptor 1 (TBLR1), which plays essential roles in regulating Wnt signaling. These results demonstrated that USP1 may act as an essential factor in promoting the survival of CTCs and suggest that inhibition of USP1 is a potential strategy for HCC treatment.

Mutations and altered expression of deubiquitinating enzymes (DUBs) have been found associated with many human diseases including cancers. In this study, Ubiquitin specific protease 1 (USP1) expression was found significantly increased in some colorectal cancers (CRC). The elevated USP1 level was associated with short overall survival of patients and with advanced stages of cancers. In cultured CRC cells, knockdown of USP1 induced growth arrest at G2/M of cell cycle and reduced the expression of anti-apoptotic proteins Bcl-2 and Mcl-1. Its knockdown also led to reduction of DNA-repair related substrates FANCD2 and ID1. Further investigations found that small molecular inhibitor of USP1 ML323 sensitized CRC cells to DNA-targeting chemotherapeutics, including doxorubicin, TOPI/II inhibitors, and PARP inhibitor, but not to 5-Fu. These results indicate that USP1 plays a critical in colorectal cancer cell survival and is a promising target for anti-colorectal cancer chemotherapy. Targeting USP1 may represent an effective strategy to regulate the DNA-repairing system.

The histone demethylase lysine‐specific demethylase 4A (KDM4A) is reported to be overexpressed and plays a vital in multiple cancers through controlling gene expression by epigenetic regulation of H3K9 or H3K36 methylation marks. However, the biological role and mechanism of KDM4A in prostate cancer (PC) remain unclear. Herein, we reported KDM4A expression was upregulation in phosphatase and tensin homolog knockout mouse prostate tissue. Depletion of KDM4A in PC cells inhibited their proliferation and survival in vivo and vitro. Further studies reveal that USP1 is a deubiquitinase that regulates KDM4A K48‐linked deubiquitin and stability. Interestingly, we found c‐Myc was a key downstream effector of the USP1‐KDM4A/androgen receptor axis in driving PC cell proliferation. Notably, upregulation of KDM4A expression with high USP1 expression was observed in most prostate tumors and inhibition of USP1 promotes PC cells response to therapeutic agent enzalutamide. Our studies propose USP1 could be an anticancer therapeutic target in PC. [ABSTRACT FROM AUTHOR]

PI3K‐Akt‐FoxO‐mTOR signaling is the central pathway controlling growth and metabolism in all cells. Ubiquitination of the protein kinase Akt prior to its phosphorylation is required for PI3K‐Akt activity. Here, we found that the deubiquitinating (DUB) enzyme USP1 removes K63‐linked polyubiquitin chains on Akt to restrict PI3K‐Akt‐FoxO signaling in mouse muscle during prolonged starvation. DUB screening platform identified USP1 as a direct DUB for Akt, and USP1 depletion in mouse muscle increased Akt ubiquitination, PI3K‐Akt‐FoxO signaling, and glucose uptake during fasting. Co‐immunoprecipitation and mass spectrometry identified disabled homolog‐2 (Dab2), the tuberous sclerosis complex TSC1/TSC2, and PHLPP1 as USP1 bound proteins. During starvation, Dab2 is essential for Akt recruitment to USP1‐TSC1‐PHLPP1 complex, and for PI3K‐Akt‐FoxO inhibition. Surprisingly, USP1 limits TSC1 levels to sustain mTOR‐mediated basal protein synthesis rates and maintain its own protein levels. We propose that Dab2 recruits Akt to USP1‐TSC1‐PHLPP1 complex to efficiently terminate the transmission of growth signals when cellular energy level is low. Synopsis: The deubiquitinating enzyme USP1 restricts PI3K‐Akt‐FoxO signaling in muscle atrophy by removing K63‐linked polyubiquitin chains on Akt. During fasting, Dab2 promotes the recruitment of Akt to USP1, which together with the phosphatase PHLPP1 inhibits Akt, thereby suppressing PI3K‐Akt signaling when cellular energy level is low. USP1 directly deubiquitinates and inhibits Akt to sustain PI3K‐Akt‐FoxO signaling low in the muscle during prolonged starvation (low insulin condition).During fasting, Dab2 promotes the recruitment of Akt to USP1, which also binds the tuberous sclerosis complex TSC1/TSC2, and the phosphatase PHLPP1.USP1‐mediated deubiquitination of Akt reduces Akt phosphorylation at T308 in fasting, but loss of Akt phosphorylation at S473 requires PHLPP1.To maintain its own protein levels high, USP1 limits TSC1 levels to sustain mTOR‐mediated basal protein synthesis rates in fasting. [ABSTRACT FROM AUTHOR]

Mutations and altered expression of deubiquitinating enzymes (DUBs) have been found associated with many human diseases including cancers. In this study, Ubiquitin specific protease 1 (USP1) expression was found significantly increased in some colorectal cancers (CRC). The elevated USP1 level was associated with short overall survival of patients and with advanced stages of cancers. In cultured CRC cells, knockdown of USP1 induced growth arrest at G2/M of cell cycle and reduced the expression of anti-apoptotic proteins Bcl-2 and Mcl-1. Its knockdown also led to reduction of DNA-repair related substrates FANCD2 and ID1. Further investigations found that small molecular inhibitor of USP1 ML323 sensitized CRC cells to DNA-targeting chemotherapeutics, including doxorubicin, TOPI/II inhibitors, and PARP inhibitor, but not to 5-Fu. These results indicate that USP1 plays a critical in colorectal cancer cell survival and is a promising target for anti-colorectal cancer chemotherapy. Targeting USP1 may represent an effective strategy to regulate the DNA-repairing system. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 belongs to the subgenus Sarbecovirus , which universally encodes the accessory protein ORF6. SARS-CoV-2 ORF6 is an antagonist of the interferon (IFN)-mediated antiviral response and plays an important role in viral infections. However, the mechanism by which the host counteracts the function of ORF6 to restrict viral replication remains unclear. In this study, we found that most ORF6 proteins encoded by sarbecoviruses could be ubiquitinated and subsequently degraded via the proteasome pathway. Through extensive screening, we identified that the deubiquitinase USP1, which effectively and broadly deubiquitinates sarbecovirus ORF6 proteins, stabilizes ORF6 proteins, resulting in enhanced viral replication. Therefore, ubiquitination and deubiquitination of ORF6 are important for antagonizing IFN-mediated antiviral signaling and influencing the virulence of SARS-CoV-2. These findings highlight an essential molecular mechanism and may provide a novel target for therapeutic interventions against viral infections.IMPORTANCEThe ORF6 proteins encoded by sarbecoviruses are essential for effective viral replication and infection and are important targets for developing effective intervention strategies. In this study, we confirmed that sarbecovirus ORF6 proteins are important antagonists of the host immune response and identified the regulatory mechanisms of ubiquitination and deubiquitination of most sarbecovirus ORF6 proteins. Moreover, we revealed that DUB USP1 prevents the proteasomal degradation of all ORF6 proteins, thereby promoting the virulence of SARS-CoV-2. Thus, impeding ORF6 function is helpful for attenuating the virulence of sarbecoviruses. Therefore, our findings provide a deeper understanding of the molecular mechanisms underlying sarbecovirus infections and offer potential new therapeutic targets for the prevention and treatment of these infections., Competing Interests: The authors declare no conflict of interest.

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Summary Repair of interstrand crosslinks by the Fanconi anemia (FA) pathway requires both monoubiquitination and de-ubiquitination of the FANCI/FANCD2 (FANCI/D2) complex. In the standing model, the phosphorylation of six sites in the FANCI S/TQ cluster domain occurs upstream of, and promotes, FANCI/D2 monoubiquitination. We generated phospho-specific antibodies against three different S/TQ cluster sites (serines 556, 559, and 565) on human FANCI and found that, in contrast to the standing model, distinct FANCI sites were phosphorylated either predominantly upstream (ubiquitination independent; serine 556) or downstream (ubiquitination-linked; serines 559 and 565) of FANCI/D2 monoubiquitination. Ubiquitination-linked FANCI phosphorylation inhibited FANCD2 de-ubiquitination and bypassed the need to de-ubiquitinate FANCD2 to achieve effective interstrand crosslink repair. USP1 depletion suppressed ubiquitination-linked FANCI phosphorylation despite increasing FANCI/D2 monoubiquitination, providing an explanation of why FANCD2 de-ubiquitination is important for function of the FA pathway. Our work results in a refined model of how FANCI phosphorylation activates the FANCI/D2 complex. [ABSTRACT FROM AUTHOR]

The Fanconi anemia ( FA) pathway regulates DNA interstrand crosslink ( ICL) repair. A critical step in this pathway is mono-ubiquitination of FANCD2 ( FANCD2-Ub). Deubiquitinase USP1 removes ubiquitin from FANCD2 resulting in inactivation of the FA pathway. USP1 is autocleaved and subsequently degraded for its down-regulation. Here, we investigated the functional consequences of Usp1-autocleavage defect. Usp1-autocleavage mutant (Usp1 GG/ AA) corrected the level of Fancd2-Ub similar to Usp1 WT in Usp1−/− MEFs. However, Usp1 GG/ AA only partially rescued MMC sensitivity with defective Fancd2 foci formation and homologous recombination defect. Contrary to this, Usp1 GG/ AA was capable of recovering UV resistance of Usp1−/− MEFs to a similar extent with Usp1 WT. Taken together, our findings suggest that Usp1 regulation by autocleavage is critical for Usp1 to exert its function in ICL repair. [ABSTRACT FROM AUTHOR]

Over the past half-century, we have become increasingly aware of the ubiquity of DNA damage. Under the constant exposure to exogenous and endogenous genomic stress, cells must attempt to replicate damaged DNA. The encounter of replication forks with DNA lesions triggers several cellular responses, including the activation of translesion DNA synthesis (TLS), which largely depends upon specialized DNA polymerases with flexible active sites capable of accommodating bulky DNA lesions. A detrimental aspect of TLS is its intrinsic mutagenic nature, and thus the activity of the TLS polymerases must ideally be restricted to synthesis on damaged DNA templates. Despite their potential clinical importance in chemotherapy, TLS inhibitors have been difficult to identify since a direct assay designed to quantify genomic TLS events is still unavailable. Herein we discuss the methods that have been used to validate TLS inhibitors such as USP1, p21 and Spartan, highlighting research that has revealed their contribution to the control of DNA synthesis on damaged and undamaged templates. [ABSTRACT FROM AUTHOR]

Cancer cells have upregulated DNA repair mechanisms, enabling them survive DNA damage induced during repeated rapid cell divisions and targeted chemotherapeutic treatments. Cancer cell proliferation and survival targeting via inhibition of DNA repair pathways is currently a very promiscuous anti-tumor approach. The deubiquitinating enzyme, USP1 is known to promote DNA repair via complexing with UAF1. The USP1/UAF1 complex is responsible for regulating DNA break repair pathways such as trans-lesion synthesis pathway, Fanconi anemia pathway and homologous recombination. Thus, USP1/UAF1 inhibition poses as an efficient anti-cancer strategy. The recently made available high throughput screen data for anti USP1/UAF1 activity prompted us to compute bioactivity predictive models that could help in screening for potential USP1/UAF1 inhibitors having anti-cancer properties. The current study utilizes publicly available high throughput screen data set of chemical compounds evaluated for their potential USP1/UAF1 inhibitory effect. A machine learning approach was devised for generation of computational models that could predict for potential anti USP1/UAF1 biological activity of novel anticancer compounds. Additional efficacy of active compounds was screened by applying SMARTS filter to eliminate molecules with non-drug like features. The structural fragment analysis was further performed to explore structural properties of the molecules. We demonstrated that modern machine learning approaches could be efficiently employed in building predictive computational models and their predictive performance is statistically accurate. The structure fragment analysis revealed the structures that could play an important role in identification of USP1/UAF1 inhibitors. [ABSTRACT FROM AUTHOR]

Objectives: Deubiquitinase Ubiquitin Specific Protease 1 (USP1) is essential for bone formation, but how USP1 regulates bone formation in response to oxidative stress remains unclear. In this study, we aim to investigate the biological function of USP1 in osteoblastic MC3T3-E1 cells., Methods: Hydrogen peroxide (H 2 O 2 ) as an oxidative reagent was used to trigger osteoblastic MC3T3-E1 cellular damage. Flow cytometry was used to evaluate ROS production, apoptosis, and pyroptosis. Real-time PCR and western bolt assay were used to detect the mRNA and protein levels of USP1. Moreover, coimmunoprecipitation was used to validate the relationship between USP1 and TRAF6., Results: We demonstrated that USP1 was significantly decreased in MC3T3-E1 cells after H 2 O 2 treatment. Overexpressing USP1 restored H 2 O 2 -decreased alkaline phosphatase activity and reactive oxygen species production. USP1 overexpression inhibited cytokine release and NLP3 inflammasome activation, which was mediated by NF-κB. Overexpressing USP1 prevented NF-κB translocation. USP1 formed a complex with TRAF6, inhibiting TRAF6 ubiquitination., Conclusion: USP1 exhibits protective role in MC3T3-E1 cells by suppressing NF-κB-NLRP3 mediated pyroptosis in response to H 2 O 2 . The involvement of USP1 and TRAF6 in NLRP3 inflammasome signaling suggests a future therapeutic potential to improve clinical symptoms in osteoporosis., Competing Interests: The authors have no conflict of interest.

Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the bestcharacterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells. [ABSTRACT FROM AUTHOR]

Background: Ubiquitin specific protease 1 (USP1) tightly correlates with poor prognosis of multiple cancers. However, whether USP1 underlies ovarian cancer (OV) progression remains unclarified. Methods: First, GSEA strategy and WGCNA analysis were used to screen for anti-ovarian cancer drugs and furthern optimal module, respectively. In addition, functional enrichments of module genes were realized by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Kaplan-Meier was then employed to analyze the prognostic impact of USP1 expression on OV patients. Cell proliferation and cell cycle assays were used to confirm biological functions of USP1 in the final. Results: Through the forementioned methods, we obtained five candidate drugs against OV from 353 anticancer drugs, and proposed ML323 as a novel anti-OV drug. As our hypothesized, ML323 significantly inhibited the proliferation of OV cells. Combined with WGCNA and KEGG analysis, the turquoise module was related to ML323, together with cell cycle. USP1 was subsequently identified as a target of ML323 and according to the TCGA database, USP1 negatively correlated with prognosis in OV, and its reduction and ML323-treatment both inhibited the proliferation of OV cells, blocking the S phase of cell cycle in vitro . Conclusion: Taken together, ML323 exerts its inhibitory effect on the proliferation of OV cells by targeting USP1-regulated cell cycle, providing a therapeutical strategy and potential target against OV., (Copyright © 2022 Song, Jiang, Jiang, Lin and Liu.)

Mutations and altered expression of deubiquitinating enzymes (DUBs) profoundly influence tumor progression. Ubiquitin-specific protease 1 (USP1) is a well-characterized human DUB reportedly overexpressed in and associated with maintaining the mesenchymal stem cell status of osteosarcoma (OS); however, the potential mechanisms of USP1 in OS remain poorly understood. In this study, we identified that USP1 directly interacts with Transcriptional Co-Activator With PDZ-Binding Motif (TAZ) in OS cell lines, and with mechanistic analysis indicating that the anti-OS effects of USP1 inhibition could be partially attributed to TAZ instability, with its reduced nuclear accumulation responsible for a subsequent decrease in the expression of downstream genes associated with the Hippo signaling pathway. Moreover, pharmacological inhibition USP1 by ML323 presented the similar effects on Hippo signaling pathway and suppressed OS growth and metastasis both in vitro and in vivo . Taken together, our results revealed a novel molecular mechanism underlying the function of USP1 in OS and a potential role of ML323 as a therapeutic strategy for the clinical treatment of OS., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Hyperactivation of phosphatidylinositol 3-kinase/Akt signaling is commonly associated with human tumors including lung cancers. PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), which terminates Akt signaling by directly dephosphorylating and inactivating Akt, has been identified as a tumor suppressor. The protein level of PHLPP1 is regulated by E3 ligase beta-TRCP, however, the deubiquitinase for PHLPP1 is still not known. Methods: The mRNA levels of USP1 and PHLPP1 in lung cancer cells and tissues were determined by real-time PCR. The half-life of PHLPP1 was detected by CHX assay. The interaction between USP1 and PHLPP1 was examined by immunoprecipitation and GST pull-down assay. Results: Both USP1 and PHLPP1 are low expressed in lung cancer cells and tissues and silencing of USP1 by RNA interference significantly decreased the half-life of PHLPP1, which in turn amplified Akt1 phosphorylation. Our data identified a novel USP1-PHLPP1-Akt signaling axis, and decreased USP1 level in lung cancer cells may play an important role in lung cancer progress. [ABSTRACT FROM AUTHOR]

Abstract: Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation plays a crucial role in maintaining genomic stability during DNA replication. DNA damage stalling the DNA replication fork induces PCNA ubiquitylation that activates DNA damage bypass to prevent the collapse of DNA replication forks that could potentially produce double-strand breaks and chromosomal rearrangements. PCNA ubiquitylation dictates the mode of bypass depending on the level of ubiquitylation; monoubiquitylation and polyubiquitylation activate error-prone translesion synthesis and error-free template switching, respectively. Due to the error-prone nature of DNA damage bypass, PCNA ubiquitylation needs to be tightly regulated. Here, we review the molecular mechanisms to remove ubiquitin from PCNA including the emerging role of USP1 and ELG1 in this fascinating process. [Copyright &y& Elsevier]

Nasopharyngeal carcinoma (NPC) development involves many genetic alterations. This study profiled differentially expressed microRNAs (DE-miRNAs) and selected miR-375 for further study. DE-miRNAs were screened using online databases and subjected to various analyzes. miR-375 mimics with negative control (NC) cDNA, and a ubiquitin-specific protease 1 (USP1) as well as a NC group were transfected into NPC cells for analysis by quantitative PCR, western blotting, wound healing, Transwell, flow cytometry, cell counting kit-8 (CCK-8), and luciferase gene reporter assays. Among these DE-miRNAs, miR-375 was downregulated and miR-21 was upregulated in NPC cells. Bioinformatical analysis identified USP1 as a potential target gene of miR-375. Increased USP1 expression was associated with poor survival of head and neck cancer patients. The luciferase assay confirmed miR-375 binding to the USP1 3'-untranslated region (UTR), while the transfection experiment confirmed miR-375 expression reduced USP1 expression. USP1 overexpression reversed the anti-tumor activity of miR-375 in NPC cells as determined by tumor cell migration, invasion, apoptosis, and viability assays. In addition, USP1 overexpression activated phosphoinositide 3-kinase (PI3K) signaling, whereas a selective PI3K inhibitor (S2739) could reverse the effects of USP1 on NPC cells in vitro. miR-375 and miR-21 are both related to NPC and miR-375 can target USP1. Further experiments revealed that up-regulated miR-375 expression led to USP1 down-regulation, and miR-375 overexpression suppressed PI3K/Akt signaling and inhibited NPC cell migration and invasion, but promoted NPC cell apoptosis. [ABSTRACT FROM AUTHOR]