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1.
Nat Commun ; 12(1): 4852, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381028

RESUMO

Oncogenic activation of KRAS and its surrogates is essential for tumour cell proliferation and survival, as well as for the development of protumourigenic microenvironments. Here, we show that the deubiquitinase USP12 is commonly downregulated in the KrasG12D-driven mouse lung tumour and human non-small cell lung cancer owing to the activation of AKT-mTOR signalling. Downregulation of USP12 promotes lung tumour growth and fosters an immunosuppressive microenvironment with increased macrophage recruitment, hypervascularization, and reduced T cell activation. Mechanistically, USP12 downregulation creates a tumour-promoting secretome resulting from insufficient PPM1B deubiquitination that causes NF-κB hyperactivation in tumour cells. Furthermore, USP12 inhibition desensitizes mouse lung tumour cells to anti-PD-1 immunotherapy. Thus, our findings propose a critical component downstream of the oncogenic signalling pathways in the modulation of tumour-immune cell interactions and tumour response to immune checkpoint blockade therapy.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente Tumoral/imunologia , Ubiquitina Tiolesterase/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Quimiocinas/metabolismo , Regulação para Baixo , Humanos , Tolerância Imunológica , Imunoterapia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Proteína Fosfatase 2C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores
2.
FASEB J ; 35(9): e21870, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34436790

RESUMO

COVID-19 is often characterized by dysregulated inflammatory and immune responses. It has been shown that the Traditional Chinese Medicine formulation Qing-Fei-Pai-Du decoction (QFPDD) is effective in the treatment of the disease, especially for patients in the early stage. Our network pharmacology analyses indicated that many inflammation and immune-related molecules were the targets of the active components of QFPDD, which propelled us to examine the effects of the decoction on inflammation. We found in the present study that QFPDD effectively alleviated dextran sulfate sodium-induced intestinal inflammation in mice. It inhibited the production of pro-inflammatory cytokines IL-6 and TNFα, and promoted the expression of anti-inflammatory cytokine IL-10 by macrophagic cells. Further investigations found that QFPDD and one of its active components wogonoside markedly reduced LPS-stimulated phosphorylation of transcription factor ATF2, an important regulator of multiple cytokines expression. Our data revealed that both QFPDD and wogonoside decreased the half-life of ATF2 and promoted its proteasomal degradation. Of note, QFPDD and wogonoside down-regulated deubiquitinating enzyme USP14 along with inducing ATF2 degradation. Inhibition of USP14 with the small molecular inhibitor IU1 also led to the decrease of ATF2 in the cells, indicating that QFPDD and wogonoside may act through regulating USP14 to promote ATF2 degradation. To further assess the importance of ubiquitination in regulating ATF2, we generated mice that were intestinal-specific KLHL5 deficiency, a CUL3-interacting protein participating in substrate recognition of E3s. In these mice, QFPDD mitigated inflammatory reaction in the spleen, but not intestinal inflammation, suggesting CUL3-KLHL5 may function as an E3 for ATF2 degradation.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Regulação para Baixo/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Flavanonas/farmacologia , Glucosídeos/farmacologia , Inflamação/tratamento farmacológico , Proteólise/efeitos dos fármacos , Ubiquitina Tiolesterase/deficiência , Animais , Linhagem Celular , Colite/induzido quimicamente , Colite/tratamento farmacológico , Proteínas Culina/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana/farmacologia , Sulfato de Dextrana/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Flavanonas/uso terapêutico , Glucosídeos/uso terapêutico , Inflamação/induzido quimicamente , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirróis/farmacologia , Pirrolidinas/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitinação
3.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207520

RESUMO

The 26S proteasome is the principal protease for regulated intracellular proteolysis. This multi-subunit complex is also pivotal for clearance of harmful proteins that are produced throughout the lifetime of eukaryotes. Recent structural and kinetic studies have revealed a multitude of conformational states of the proteasome in substrate-free and substrate-engaged forms. These conformational transitions demonstrate that proteasome is a highly dynamic machinery during substrate processing that can be also controlled by a number of proteasome-associated factors. Essentially, three distinct family of deubiquitinases-USP14, RPN11, and UCH37-are associated with the 19S regulatory particle of human proteasome. USP14 and UCH37 are capable of editing ubiquitin conjugates during the process of their dynamic engagement into the proteasome prior to the catalytic commitment. In contrast, RPN11-mediated deubiquitination is directly coupled to substrate degradation by sensing the proteasome's conformational switch into the commitment steps. Therefore, proteasome-bound deubiquitinases are likely to tailor the degradation events in accordance with substrate processing steps and for dynamic proteolysis outcomes. Recent chemical screening efforts have yielded highly selective small-molecule inhibitors for targeting proteasomal deubiquitinases, such as USP14 and RPN11. USP14 inhibitors, IU1 and its progeny, were found to promote the degradation of a subset of substrates probably by overriding USP14-imposed checkpoint on the proteasome. On the other hand, capzimin, a RPN11 inhibitor, stabilized the proteasome substrates and showed the anti-proliferative effects on cancer cells. It is highly conceivable that these specific inhibitors will aid to dissect the role of each deubiquitinase on the proteasome. Moreover, customized targeting of proteasome-associated deubiquitinases may also provide versatile therapeutic strategies for induced or repressed protein degradation depending on proteolytic demand and cellular context.


Assuntos
Inibidores Enzimáticos , Proteínas de Neoplasias , Neoplasias , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Pirróis , Pirrolidinas , Ubiquitina Tiolesterase , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Pirróis/química , Pirróis/uso terapêutico , Pirrolidinas/química , Pirrolidinas/uso terapêutico , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/metabolismo
4.
Aging (Albany NY) ; 13(11): 14999-15012, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34081623

RESUMO

The ubiquitin-specific protease 8 (USP8) is a prototypic multidomain deubiquitinating enzyme with pleiotropic functions. We investigated the role of USP8 in hepatocellular carcinoma (HCC) by analyzing expression patterns of USP8 in HCC patients, and evaluating its functions and underlying signaling. Among 20 HCC patients investigated, we found that USP8 protein upregulation was a common phenomenon (17 out of 20) in HCC compared to normal liver tissue. Furthermore, the upregulation of USP8 was not associated with any clinicopathology. USP8 inhibition via genetic and pharmacological approaches resulted in growth inhibition and apoptosis induction in both sensitive and doxorubicin-resistant HCC cells. Of note, USP8 inhibition significantly enhanced doxorubicin or sorafenib's efficacy in HCC cells and mouse models. We further found that USP8 inhibition decreased levels of multiple receptor tyrosine kinases (RTKs) by ~90%, such as epidermal growth factor receptor (EGFR) and c-Met. Consistently, the downstream signaling regulated by RTKs was disrupted in HCC cells after USP8 inhibition, as shown by the decreased p-Akt, p-STAT3 and p-Raf. Our findings demonstrate that USP8 is a novel therapeutic target in HCC. Inhibiting USP8 has potential to overcome current drug resistance, particularly on HCC patients with high USP8 expression.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Receptores Proteína Tirosina Quinases/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação para Baixo/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos SCID , Transdução de Sinais , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Regulação para Cima/genética
5.
Eur J Pharmacol ; 900: 174045, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-33745956

RESUMO

The ubiquitin-proteasome system (UPS) plays an essential role in cellular homeostasis and myocardial function. Ubiquitin carboxy-terminal hydrolase 1 (UCHL1) is involved in cardiac remodeling, but its underlying mechanisms are largely unknown. Here, we observed that the UCHL1 was significantly up-regulated in angiotensin II-infused heart and primary cardiac fibroblast (CF). Systemic administration of the UCHL1 inhibitor LDN57444 significantly ameliorated cardiac fibrosis and improved cardiac function induced by angiotensin II. Also, LDN57444 inhibited CF cell proliferation as well as attenuated collagen I, and CTGF gene expression in the presence of Ang II. Mechanistically, UCHL1 promotes angiotensin II-induced fibrotic responses by way of activating nuclear factor kappa B (NF-κB) signaling. Moreover, suppression of the NF-κB pathway interfered with UCHL1 overexpression-mediated fibrotic responses. Besides, the chromatin immunoprecipitation assay demonstrated that NF-κB can bind to the UCHL1 promoter and trigger its transcription in cardiac fibroblasts. These findings suggest that UCHL1 positively regulates cardiac fibrosis by modulating NF-κB signaling pathway and identify UCHL1 could be a new treatment strategy for cardiac fibrosis.


Assuntos
Fibroblastos/efeitos dos fármacos , Miocárdio/patologia , NF-kappa B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina Tiolesterase/antagonistas & inibidores , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/antagonistas & inibidores , Colágeno Tipo I/biossíntese , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Fibrose/prevenção & controle , Camundongos , Ratos , Ratos Sprague-Dawley
6.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668938

RESUMO

The deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK. Structure-activity relationship studies identified modifications to improve activity versus the target and a full cellular characterization was carried out for the first time with this scaffold. The studies produced a new inhibitor, 34, with an IC50 value of 7.7 µM against UCHL1 and no observable activity versus the closest related DUB UCHL3. The molecule was also capable of selectively inhibiting UCHL1 in cells and did not demonstrate any discernible off-target toxicity. Finally, the molecule was used for initial probe studies to assess the role of UCHL1 role in proliferation of myeloma cells and migration behavior in small cell lung cancer cells making 34 a new tool to be used in the biological evaluation of UCHL1.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteases/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-Atividade , Ubiquitina Tiolesterase/metabolismo
7.
Mar Drugs ; 19(2)2021 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-33572615

RESUMO

In 2019, streptoglutarimide H (SGH) was characterized as a new glutarimide from the secondary metabolites produced by a marine-derived actinomycete Streptomyces sp. ZZ741 and shown to have in vitro antiglioma activity. However, the antiproliferative activity and potential mechanism of SGH against lung cancer cells have not yet been characterized. This study demonstrated that SGH significantly inhibited the proliferation of different lung cancer cells. In terms of mechanism of action, SGH downregulated cell cycle- and nucleotide synthesis-related proteins to block cell cycle at G0/G1 phase, reduced the expression levels of glycolytic metabolic enzymes to inhibit glycolysis, and downregulated the important cancer transcription factor c-Myc and the therapeutic target deubiquitinase USP28. Potent anticancer activity and multiple mechanisms indicated SGH to be a novel antitumor compound against lung cancer cells.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Streptomyces/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Ubiquitina Tiolesterase/antagonistas & inibidores
8.
Int J Biol Macromol ; 176: 490-497, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33582217

RESUMO

Disulfiram is a promising repurposed drug that, combining with radiation and chemotherapy, exhibits effective anticancer activities in several preclinical models. The cellular metabolites of disulfiram have been established, however, the intracellular targets of disulfiram remain largely unexplored. We have previously reported that disulfiram suppresses the coronaviral papain-like proteases through attacking their zinc-finger domains, suggesting an inhibitory function potentially on other proteases with similar catalytic structures. Ubiquitin-specific proteases (USPs) share a highly-conserved zinc-finger subdomain that structurally similar to the papain-like proteases and are attractive anticancer targets as upregulated USPs levels are found in a variety of tumors. Here, we report that disulfiram functions as a competitive inhibitor for both USP2 and USP21, two tumor-related deubiquitinases. In addition, we also observed a synergistic inhibition of USP2 and USP21 by disulfiram and 6-Thioguanine (6TG), a clinical drug for acute myeloid leukemia. Kinetic analyses revealed that both drugs exhibited a slow-binding mechanism, moderate inhibitory parameters, and a synergistically inhibitory effect on USP2 and USP21, suggesting the potential combinatory use of these two drugs for USPs-related tumors. Taken together, our study provides biochemical evidence for repurposing disulfiram and 6TG as a combinatory treatment in clinical applications.


Assuntos
Dissulfiram/química , Inibidores Enzimáticos/química , Tioguanina/química , Ubiquitina Tiolesterase , Dissulfiram/agonistas , Sinergismo Farmacológico , Humanos , Tioguanina/agonistas , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/química
9.
Arch Biochem Biophys ; 701: 108811, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33600786

RESUMO

USP8 is a deubiquitinating enzyme in the family of ubiquitin-specific proteases (USPs) which can remove ubiquitin from the substrate and protect the substrate from degradation. The upregulated or mutated USP8 becomes hyperactivated and stabilizes numerous oncogenes or proto-oncogenes leading to cancer progression and survival by activating multiple signaling pathways. Moreover, USP8 inhibition is also important to overcome anticancer drug-resistant. This review is the first study to find, combine, analyze, and represent the multiple oncogenic signaling pathways with their downstream and upstream regulation activated or enhanced by USP8, which will help the researchers to find any therapeutic strategy for drug discovery by inhibiting or suppressing the multi-targeted USP8.


Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Endopeptidases , Complexos Endossomais de Distribuição Requeridos para Transporte , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina Tiolesterase , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo
10.
J Cell Biol ; 220(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33507233

RESUMO

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.


Assuntos
Ribossomos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Anticorpos/metabolismo , Biocatálise , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Estabilidade Proteica , Reprodutibilidade dos Testes , Ribonucleoproteínas/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores
11.
Am J Physiol Lung Cell Mol Physiol ; 320(4): L497-L507, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33438509

RESUMO

Increasing evidence suggests an important role for deubiquitinating enzymes (DUBs) in modulating a variety of biological functions and diseases. We previously identified the upregulation of the DUB ubiquitin carboxyl terminal hydrolase 1 (UCHL1) in murine ventilator-induced lung injury (VILI). However, the role of UCHL1 in modulating vascular permeability, a cardinal feature of acute lung injury (ALI) in general, remains unclear. We investigated the role of UCHL1 in pulmonary endothelial cell (EC) barrier function in vitro and in vivo and examined the effects of UCHL1 on VE-cadherin and claudin-5 regulation, important adherens and tight junctional components, respectively. Measurements of transendothelial electrical resistance confirmed decreased barrier enhancement induced by hepatocyte growth factor (HGF) and increased thrombin-induced permeability in both UCHL1-silenced ECs and in ECs pretreated with LDN-57444 (LDN), a pharmacological UCHL1 inhibitor. In addition, UCHL1 knockdown (siRNA) was associated with decreased expression of VE-cadherin and claudin-5, whereas silencing of the transcription factor FoxO1 restored claudin-5 levels. Finally, UCHL1 inhibition in vivo via LDN was associated with increased VILI in a murine model. These findings support a prominent functional role of UCHL1 in regulating lung vascular permeability via alterations in adherens and tight junctions and implicate UCHL1 as an important mediator of ALI.


Assuntos
Permeabilidade Capilar , Endotélio Vascular/patologia , Ubiquitina Tiolesterase/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Animais , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Técnicas In Vitro , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oximas/farmacologia , Transdução de Sinais , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Ubiquitinação , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo
12.
J Cell Mol Med ; 25(2): 1001-1011, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33314748

RESUMO

Cardiac fibrosis, featuring abnormally elevated extracellular matrix accumulation, decreases tissue compliance, impairs cardiac function and accelerates heart failure. Mounting evidence suggests that the ubiquitin proteasome pathway is involved in cardiac fibrosis. In the present study, ubiquitin-specific protease 2 (USP2) was identified as a novel therapeutic target in cardiac fibrosis. Indeed, USP2 expression was increased in angiotensin II-induced primary cardiac fibroblasts (CFs) from neonatal rats. In addition, USP2 inhibition suppressed CFs proliferation, collagen synthesis and cell cycle progression. Furthermore, USP2 interacted with ß-catenin, thereby regulating its deubiquitination and stabilization in CFs. To sum up, these findings revealed that USP2 has a therapeutic potential for the treatment of cardiac fibrosis.


Assuntos
Angiotensina II/farmacologia , Ciclina D1/metabolismo , Fibroblastos/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/citologia , Ubiquitina Tiolesterase/metabolismo , Regulação para Cima , beta Catenina/metabolismo , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular/metabolismo , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo III/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/efeitos dos fármacos , Proteínas Musculares/antagonistas & inibidores , Estabilidade Proteica/efeitos dos fármacos , Ratos Sprague-Dawley , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Regulação para Cima/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos
13.
Bioorg Med Chem ; 30: 115931, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33341501

RESUMO

The ubiquitin-proteasome system (UPS) plays an important role in maintaining protein homeostasis by degrading intracellular proteins. In the proteasome, poly-ubiquitinated proteins are deubiquitinated by three deubiquitinases (DUBs) associated with 19S regulatory particle before degradation via 20S core particle. Ubiquitin carboxyl-terminal hydrolase L5 (UCHL5) is one of three proteasome-associated DUBs that control the fate of ubiquitinated substrates implicated in cancer survival and progression. In this study, we have performed virtual screening of an FDA approved drug library with UCHL5 and discovered tiaprofenic acid (TA) as a potential binder. With molecular docking analysis and in-vitro DUB assay, we have designed, synthesized, and evaluated a series of TA derivatives for inhibition of UCHL5 activity. We demonstrate that one TA derivative, TAB2, acts as an inhibitor of UCHL5.


Assuntos
Inibidores Enzimáticos/farmacologia , Propionatos/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Propionatos/síntese química , Propionatos/química , Relação Estrutura-Atividade , Ubiquitina Tiolesterase/metabolismo
14.
Nature ; 588(7838): 479-484, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33177714

RESUMO

Cholesterol is an essential lipid and its synthesis is nutritionally and energetically costly1,2. In mammals, cholesterol biosynthesis increases after feeding and is inhibited under fasting conditions3. However, the regulatory mechanisms of cholesterol biosynthesis at the fasting-feeding transition remain poorly understood. Here we show that the deubiquitylase ubiquitin-specific peptidase 20 (USP20) stabilizes HMG-CoA reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthetic pathway, in the feeding state. The post-prandial increase in insulin and glucose concentration stimulates mTORC1 to phosphorylate USP20 at S132 and S134; USP20 is recruited to the HMGCR complex and antagonizes its degradation. The feeding-induced stabilization of HMGCR is abolished in mice with liver-specific Usp20 deletion and in USP20(S132A/S134A) knock-in mice. Genetic deletion or pharmacological inhibition of USP20 markedly decreases diet-induced body weight gain, reduces lipid levels in the serum and liver, improves insulin sensitivity and increases energy expenditure. These metabolic changes are reversed by expression of the constitutively stable HMGCR(K248R). This study reveals an unexpected regulatory axis from mTORC1 to HMGCR via USP20 phosphorylation and suggests that inhibitors of USP20 could be used to lower cholesterol levels to treat metabolic diseases including hyperlipidaemia, liver steatosis, obesity and diabetes.


Assuntos
Colesterol/biossíntese , Ingestão de Alimentos/fisiologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Linhagem Celular , Glucose/metabolismo , Humanos , Insulina/metabolismo , Fígado/metabolismo , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Metabolismo/genética , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fosfosserina/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/deficiência , Ubiquitinação , Ganho de Peso
15.
Biosci Rep ; 40(10)2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33030206

RESUMO

Oocyte maturation is a prerequisite for successful fertilization and embryo development. Incomplete oocyte maturation can result in infertility. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) has been found to be implicated in oocyte maturation and embryo development. However, the cellular and molecular mechanisms of UCH-L1 underlying oocyte maturation have not been fully elucidated. In the present study, we observed that the introduction of UCH-L1 inhibitor LDN-57444 suppressed first polar body extrusion during mouse oocyte maturation. The inhibition of UCH-L1 by LDN-57444 led to the notable increase in reactive oxygen species (ROS) level, conspicuous reduction in glutathione (GSH) content and mitochondrial membrane potential (MMP), and blockade of spindle body formation. As a conclusion, UCH-L1 inhibitor LDN-57444 suppressed mouse oocyte maturation by improving oxidative stress, attenuating mitochondrial function, curbing spindle body formation and down-regulating extracellular signal-related kinases (ERK1/2) expression, providing a deep insight into the cellular and molecular basis of UCH-L1 during mouse oocyte maturation.


Assuntos
Indóis/administração & dosagem , Oócitos/crescimento & desenvolvimento , Oximas/administração & dosagem , Ubiquitina Tiolesterase/metabolismo , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Feminino , Glutationa/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Animais , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores
16.
J Am Chem Soc ; 142(39): 16825-16841, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32886496

RESUMO

Many reagents have emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement, allowing investigations of the function of individual enzymes in their native cellular context. Here we report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and an in vivo animal model. The probe labels active ubiquitin carboxy-terminal hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and Parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1 to 2% of the total brain protein. UCHL1 variants have been linked with neurodegenerative disorders Parkinson's and Alzheimer's diseases. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 activity or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1-specific activity tools exist. We show that the reagents reported here are specific to UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our cell-penetrable probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 in an activity-dependent manner. Its use is demonstrated by the fluorescent labeling of active UCHL1 both in vitro and in live cells. We furthermore show that this probe can selectively and spatiotemporally report UCHL1 activity during the development of zebrafish embryos. Our results indicate that our probe has potential applications as a diagnostic tool for diseases with perturbed UCHL1 activity.


Assuntos
Corantes Fluorescentes/química , Bibliotecas de Moléculas Pequenas/química , Ubiquitina Tiolesterase/análise , Ubiquitina Tiolesterase/metabolismo , Proteínas de Peixe-Zebra/análise , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Sobrevivência Celular , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Células HEK293 , Humanos , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Proteínas de Peixe-Zebra/antagonistas & inibidores
17.
J Biol Chem ; 295(49): 16630-16642, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-32967969

RESUMO

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein-coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule-binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Endossomos/metabolismo , Receptores de Glucagon/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismo , Linhagem Celular , Regulação para Baixo , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Glucagon/farmacologia , Humanos , Monensin/farmacologia , Mutagênese , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Glucagon/agonistas , Receptores de Glucagon/genética , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/efeitos dos fármacos , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
18.
Comput Biol Chem ; 89: 107376, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32979815

RESUMO

Human ubiquitin carboxyl-terminal hydrolase-2 (USP2) inhibitors, such as thiopurine analogs, have been reported to inhibit SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb-palm-fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated in-silico efforts. After an extensive virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC50 value against Jurkat (9.67 µM) and MOTL-4 cells (11.8 µM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Antivirais/química , COVID-19/virologia , Linhagem Celular Tumoral , Proteases 3C de Coronavírus/metabolismo , Inibidores de Protease de Coronavírus/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Células Jurkat , Modelos Moleculares , Estrutura Molecular , Ubiquitina Tiolesterase/metabolismo
19.
J Am Chem Soc ; 142(28): 12020-12026, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32579346

RESUMO

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme that is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells. We further demonstrate that potent and selective UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting the potential of UCHL1 as a potential therapeutic target in fibrotic diseases.


Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Inibidores Enzimáticos/química , Células HeLa , Humanos , Estrutura Molecular , Ubiquitina Tiolesterase/metabolismo
20.
Bioorg Chem ; 101: 103962, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32480171

RESUMO

USP8, one member of deubiquitinating enzymes (DUBs) families, maintains the ubiquitination level of EGFR and regulates the downstream signaling pathways. The deregulation of USP8 has been implicated in many human diseases, especially in cancer. Therefore, USP8 has been identified as a promising target for drug design. Herein, via high throughput screening based on Ubiquitin-rhodamine-110 (Ubiquitin-Rho-110) fluorometric activity assay, we discovered a novel inhibitor DC-U43. By structure optimization, DC-U43-10 reached a half-maximal inhibitory concentration (IC50) value of 2.6 ± 1.1 µM and exhibited 10-fold selectivity against USP7. The binding between DC-U43-10 and USP8 was validated by surface plasmon resonance (SPR) assay with a KD value of 10.5 ± 3.7 µM. It also inhibited the colony formation of H1975 cells. Hence, DC-U43-10 represents a kind of USP8 inhibitors with novel scaffold and has broad prospects for being a probe for USP8-related academic and clinical research.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Ubiquitina Tiolesterase/antagonistas & inibidores , Endopeptidases , Humanos , Simulação de Acoplamento Molecular , Transdução de Sinais , Espectrometria de Fluorescência/métodos , Ressonância de Plasmônio de Superfície , Ubiquitinação
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