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An optimized Affinity Selection-Mass Spectrometry (AS-MS) workflow has been developed for the efficient identification of potent USP1 inhibitors. USP1 was immobilized on agarose beads, ensuring low small molecule retention, efficient protein capture, and protein stability. The binding affinity of 49 compounds to USP1 was evaluated using the optimized AS-MS method, calculating binding index (BI) values for each compound. Biochemical inhibition assays validated the AS-MS results, revealing a potential correlation between higher BI values and lower IC50 values. This optimized workflow enables rapid identification of high-quality USP1 inhibitor hits, facilitating structure-activity relationship studies and accelerating the discovery of potential cancer therapeutics.
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Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.
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ATPases Associadas a Diversas Atividades Celulares , Proteínas de Ligação a DNA , Antígeno Nuclear de Célula em Proliferação , Ubiquitina Tiolesterase , Peptidase 7 Específica de Ubiquitina , Ubiquitinação , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Ubiquitina/metabolismo , Dano ao DNA , Ligação Proteica , Proteases Específicas de UbiquitinaRESUMO
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.
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Doença de Hashimoto , Inflamassomos , Piroptose , Proteases Específicas de Ubiquitina , Animais , Inflamassomos/metabolismo , Camundongos , Doença de Hashimoto/metabolismo , Doença de Hashimoto/patologia , Humanos , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Progressão da Doença , Modelos Animais de Doenças , Glândula Tireoide/metabolismo , Glândula Tireoide/patologiaRESUMO
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.
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[This corrects the article DOI: 10.3389/fimmu.2022.871766.].
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USP1 has emerged as a novel and potential target for drug discovery in single therapeutic agents or combination with chemotherapy and molecular targeted therapy. In this study, based on the disclosed structure of ML323 and KSQ-4279, we designed and synthesized a series of pyrido[2,3-d]pyrimidin-7(8H)-one derivatives as potent USP1 inhibitors by cyclization strategy and the systematic structure-activity relationship exploration was conducted. The representative compounds 1k, 1m and 2d displayed excellent USP1/UAF inhibition and exhibited strong antiproliferation effect in NCI-H1299 cells. Further flow cytometry analysis revealed that they could arrest breast cancer cells MDA-MB-436 in the S phase. Inhibition mechanism study of compound 1m indicated these derivatives acted as reversible and noncompetitive USP1 inhibitors. Of note, the combination of compound 1m with PARP inhibitor olaparib generated enhanced cell killing in olaparib-resistant MDA-MB-436/OP cells, and compound 1m exhibited excellent oral pharmacokinetic properties in mice. Overall, our efforts may provide a reliable basis for the development of novel USP1 inhibitor as a single therapeutic agent and in combination with PARP inhibitors.
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Antineoplásicos , Proliferação de Células , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Pirimidinonas , Humanos , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Animais , Pirimidinonas/farmacologia , Pirimidinonas/química , Pirimidinonas/síntese química , Estrutura Molecular , Camundongos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/metabolismoRESUMO
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 CD133high population displaying higher growth rate and the CD133low 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 CD133high and the CD133low 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.
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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 1ß (Nsp1ß) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1ß 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 1ß (Nsp1ß) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1ß, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1ß, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.
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Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Feminino , Endopeptidases/genética , Peptídeo Hidrolases/metabolismo , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Suínos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
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.
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Relógios Circadianos , Animais , Camundongos , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Enzimas Desubiquitinantes , HumanosRESUMO
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.
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SARS-CoV-2 , Proteínas Virais , Viroses , Humanos , Enzimas Desubiquitinantes , Interferons/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
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.
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Ferroptose , Hipertensão Renal , Humanos , Hipertensão Renal/metabolismo , Hipertensão Renal/patologia , Estresse Oxidativo , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/farmacologiaRESUMO
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.
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Neoplasias , Proteases Específicas de Ubiquitina , Humanos , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Ubiquitinação , FosforilaçãoRESUMO
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.
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Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Receptores de Estrogênio/metabolismo , Inflamação/tratamento farmacológico , Receptores de Progesterona/metabolismo , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêuticoRESUMO
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.
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Consolidação da Fratura , Fraturas não Consolidadas , Osteogênese , Proteases Específicas de Ubiquitina , Animais , Camundongos , Ratos , Diferenciação Celular , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Proteases Específicas de Ubiquitina/metabolismo , Fraturas não Consolidadas/metabolismo , Fraturas não Consolidadas/patologiaRESUMO
RNA N6-methyladenosine (m6A) modification is involved in diverse biological processes. However, its role in spinal cord injury (SCI) is poorly understood. The m6A level increases in injured spinal cord, and METTL3, which is the core subunit of methyltransferase complex, is upregulated in reactive astrocytes and further stabilized by the USP1/UAF1 complex after SCI. The USP1/UAF1 complex specifically binds to and subsequently removes K48-linked ubiquitination of the METTL3 protein to maintain its stability after SCI. Moreover, conditional knockout of astrocytic METTL3 in both sexes of mice significantly suppressed reactive astrogliosis after SCI, thus resulting in widespread infiltration of inflammatory cells, aggravated neuronal loss, hampered axonal regeneration, and impaired functional recovery. Mechanistically, the YAP1 transcript was identified as a potential target of METTL3 in astrocytes. METTL3 could selectively methylate the 3'-UTR region of the YAP1 transcript, which subsequently maintains its stability in an IGF2BP2-dependent manner. In vivo, YAP1 overexpression by adeno-associated virus injection remarkably contributed to reactive astrogliosis and partly reversed the detrimental effects of METTL3 knockout on functional recovery after SCI. Furthermore, we found that the methyltransferase activity of METTL3 plays an essential role in reactive astrogliosis and motor repair, whereas METTL3 mutant without methyltransferase function failed to promote functional recovery after SCI. Our study reveals the previously unreported role of METTL3-mediated m6A modification in SCI and might provide a potential therapy for SCI.SIGNIFICANCE STATEMENT Spinal cord injury is a devastating trauma of the CNS involving motor and sensory impairments. However, epigenetic modification in spinal cord injury is still unclear. Here, we propose an m6A regulation effect of astrocytic METTL3 following spinal cord injury, and we further characterize its underlying mechanism, which might provide promising strategies for spinal cord injury treatment.
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Gliose , Traumatismos da Medula Espinal , Animais , Feminino , Masculino , Camundongos , Astrócitos/metabolismo , Gliose/metabolismo , Inflamação/metabolismo , Metiltransferases/metabolismo , Metiltransferases/farmacologia , RNA Mensageiro/metabolismo , Medula Espinal/metabolismoRESUMO
The balance between inflammatory T helper type 17 (Th17) and immunosuppressive regulatory T (Treg) cells is critical for maintaining immune homeostasis in the human body and is tightly regulated under healthy conditions. An increasing number of studies have reported that deubiquitinases (DUBs) play a vital role in regulating Th17- and Treg-cell differentiation. However, the biological functions of only a small fraction of DUBs in Th17- and Treg-cell differentiation are well defined. In this study, we identified ubiquitin-specific peptidase 1 (USP1) as a vital regulator of CD4+ T-cell differentiation. USP1 promoted Th17-cell differentiation but attenuated Treg-cell differentiation, thereby promoting the development of inflammatory diseases. Mechanistically, USP1 in CD4+ T cells enhanced the activity of RORγt but promoted the proteasomal degradation of Foxp3 through deubiquitination and stabilization of TAZ in vitro and in vivo. Notably, ML323, a specific inhibitor of the USP1/UAF1 deubiquitinase complex, inhibited Th17-cell differentiation and promoted Treg-cell differentiation in vitro and in vivo, indicating that ML323 might be a promising candidate for the treatment of diseases associated with an imbalance between Th17 and Treg cells. Our study highlights the critical role of USP1 in regulating adaptive immune responses and suggests that USP1 might be a drug target for the treatment of diseases associated with an imbalance between Th17 and Treg cells.
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Linfócitos T Reguladores , Células Th17 , Humanos , Diferenciação Celular , Fatores de Transcrição , Proteases Específicas de UbiquitinaRESUMO
Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUb D2). Here, we present a 4.1 Å cryo-EM structure of IUb D2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUb D2Ub , is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the IUb D2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2Ub -DNA complex. The IUb D2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2Ub -DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an IUb D2Ub -DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUb D2 complex.
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Anemia de Fanconi , Humanos , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Lisina/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Ubiquitinação , DNA/metabolismo , Dano ao DNA , Reparo do DNARESUMO
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 (H2O2) 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 H2O2 treatment. Overexpressing USP1 restored H2O2-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 H2O2. The involvement of USP1 and TRAF6 in NLRP3 inflammasome signaling suggests a future therapeutic potential to improve clinical symptoms in osteoporosis.
Assuntos
Piroptose , Fator 6 Associado a Receptor de TNF , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Inflamassomos , Peróxido de Hidrogênio/farmacologiaRESUMO
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.
RESUMO
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.