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1.
Nat Commun ; 12(1): 5212, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471133

RESUMO

The autophagic degradation of misfolded and ubiquitinated proteins is important for cellular homeostasis. In this process, which is governed by cargo receptors, ubiquitinated proteins are condensed into larger structures and subsequently become targets for the autophagy machinery. Here we employ in vitro reconstitution and cell biology to define the roles of the human cargo receptors p62/SQSTM1, NBR1 and TAX1BP1 in the selective autophagy of ubiquitinated substrates. We show that p62 is the major driver of ubiquitin condensate formation. NBR1 promotes condensate formation by equipping the p62-NBR1 heterooligomeric complex with a high-affinity UBA domain. Additionally, NBR1 recruits TAX1BP1 to the ubiquitin condensates formed by p62. While all three receptors interact with FIP200, TAX1BP1 is the main driver of FIP200 recruitment and thus the autophagic degradation of p62-ubiquitin condensates. In summary, our study defines the roles of all three receptors in the selective autophagy of ubiquitin condensates.


Assuntos
Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Domínios Proteicos , Proteínas de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo
2.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
4.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360761

RESUMO

Regulated/activated protein kinase (PRAK) plays a crucial role in modulating biological function. However, the role of PRAK in mediating cardiac dysfunction and metabolic disorders remains unclear. We examined the effects of deletion of PRAK on modulating cardiac function and insulin resistance in mice exposed to a high-fat diet (HFD). Wild-type and PRAK-/- mice at 8 weeks old were exposed to either chow food or HFD for a consecutive 16 weeks. Glucose tolerance tests and insulin tolerance tests were employed to assess insulin resistance. Echocardiography was employed to assess myocardial function. Western blot was used to determine the molecular signaling involved in phosphorylation of IRS-1, AMPKα, ERK-44/42, and irisin. Real time-PCR was used to assess the hypertrophic genes of the myocardium. Histological analysis was employed to assess the hypertrophic response, interstitial myocardial fibrosis, and apoptosis in the heart. Western blot was employed to determine cellular signaling pathway. HFD-induced metabolic stress is indicated by glucose intolerance and insulin intolerance. PRAK knockout aggravated insulin resistance, as indicated by glucose intolerance and insulin intolerance testing as compared with wild-type littermates. As compared with wild-type mice, hyperglycemia and hypercholesterolemia were manifested in PRAK-knockout mice following high-fat diet intervention. High-fat diet intervention displayed a decline in fractional shortening and ejection fraction. However, deletion of PRAK exacerbated the decline in cardiac function as compared with wild-type mice following HFD treatment. In addition, PRAK knockout mice enhanced the expression of myocardial hypertrophic genes including ANP, BNP, and ßMHC in HFD treatment, which was also associated with an increase in cardiomyocyte size and interstitial fibrosis. Western blot indicated that deletion of PRAK induces decreases in phosphorylation of IRS-1, AMPKα, and ERK44/42 as compared with wild-type controls. Our finding indicates that deletion of PRAK promoted myocardial dysfunction, cardiac remodeling, and metabolic disorders in response to HFD.


Assuntos
Cardiomegalia/enzimologia , Diabetes Mellitus Experimental/enzimologia , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miocárdio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Volume Sistólico , Remodelação Ventricular
5.
Medicine (Baltimore) ; 100(32): e26868, 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34397900

RESUMO

BACKGROUND: In recent years, related studies have revealed that tripartite motif-containing 59 (TRIM59) is related to the prognosis of lung cancer. However, these results have not been proved by any evidence. Therefore, this study evaluated the relationship between TRIM59 and the prognosis of lung cancer by carrying out meta-analysis. In addition, we explored the mechanism and related pathways of TRIM59 in lung cancer through bioinformatics analysis. METHODS: Comprehensive literature search was performed in China National Knowledge Infrastructure, Chinese Biomedical literature Database, Chinese Scientific and Journal Database, Wan Fang, Web of Science, PubMed, and EMBASE databases, and eligible studies were obtained based on the inclusion and exclusion criteria. The pooled hazard ratios and odds ratios were applied to assess the clinical value of TRIM59 expression for overall survival and clinicopathological features. Meanwhile, meta-analysis was conducted on the Stata 16.0. The mRNA expression level of TRIM59 in lung cancer was analyzed using Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA) database. Gene Set Enrichment Analysis (GSEA) was used to predict the signaling pathways that TRIM59 might be involved in. The correlation between the expression level of TRIM59 in lung cancer and the abundance of immune cell invasion was analyzed by TIMER database. The survival analysis was verified by Kaplan-Meier Plotter database. RESULTS: The results of this meta-analysis would be submitted to peer-reviewed journals for publication. CONCLUSION: In this study, the application of meta-analysis and bioinformatics analysis will provide evidence support for the study on the prognosis and mechanism of TRIM59 in lung cancer.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares , Proteínas com Motivo Tripartido , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biologia Computacional , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Metanálise como Assunto , Prognóstico , Projetos de Pesquisa , Análise de Sobrevida , Revisões Sistemáticas como Assunto , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo
6.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445562

RESUMO

Synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, has been implicated in cancer progression, and therapeutic resistance, as well as cancer stem cell (CSC)-like properties. Previously, we demonstrated that SCP3 promotes these aggressive phenotypes via hyperactivation of the AKT signaling pathway; however, the underlying mechanisms responsible for SCP3-induced AKT activation remain to be elucidated. In this study, we demonstrated that the EGF-EGFR axis is the primary route through which SCP3 acts to activate AKT signaling. SCP3 triggers the EGFR-AKT pathway through transcriptional activation of EGF. Notably, neutralization of secreted EGF by its specific monoclonal antibody reversed SCP3-mediated aggressive phenotypes with a concomitant reversal of EGFR-AKT activation. In an effort to elucidate the molecular mechanisms underlying SCP3-induced transcriptional activation of EGF, we identified Jun activation domain-binding protein 1 (JAB1) as a binding partner of SCP3 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that SCP3 induces EGF transcription through physical interaction with JAB1. Thus, our findings establish a firm molecular link among SCP3, EGFR, and AKT by identifying the novel roles of SCP3 in transcriptional regulation. We believe that these findings hold important implications for controlling SCP3high therapeutic-refractory cancer.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento Epidérmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Complexo do Signalossomo COP9/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Peptídeo Hidrolases/genética , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
7.
Nat Commun ; 12(1): 4229, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244477

RESUMO

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved.


Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular/fisiologia , Mecanotransdução Celular/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Fibroblastos , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Pinças Ópticas , Paxilina/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Respiração , Organismos Livres de Patógenos Específicos , Talina/genética , Talina/metabolismo
8.
Methods Mol Biol ; 2312: 159-168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228290

RESUMO

Controlling gene expression in mammalian cells constitutes one of the core principles of mammalian synthetic biology. Especially for cell-based therapies, inducers of gene expression which demonstrate the highest degree of safety and patient adherence are needed. In this chapter, I describe methods to implement caffeine-controlled gene expression systems into mammalian cells. Using an array of different implementation strategies, from reconstituting transcription factors to activating endogenous signaling pathways, allows for a wide range of sensitivity and capacity of the resulting caffeine-responsive gene switches.


Assuntos
Cafeína/farmacologia , Engenharia Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Genes de Troca , Biologia Sintética , Técnicas de Cultura de Células , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
9.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298987

RESUMO

Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients' muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3ß (GSK-3ß), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.


Assuntos
Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Distrofia Muscular do Cíngulo dos Membros/tratamento farmacológico , Proteínas do Tecido Nervoso/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Sítio Alostérico/efeitos dos fármacos , Antígeno CD56/análise , Calpaína/deficiência , Calpaína/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/química , Humanos , Hidrazinas/farmacologia , Hidrazinas/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/enzimologia , Proteínas do Tecido Nervoso/química , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Quinolonas/farmacologia , Quinolonas/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/fisiologia , Tiadiazóis/farmacologia , Tiadiazóis/uso terapêutico , Via de Sinalização Wnt/efeitos dos fármacos
10.
Cell Death Dis ; 12(7): 697, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257278

RESUMO

The tripartite motif-containing protein 21 (TRIM21) plays important roles in autophagy and innate immunity. Here, we found that HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), as an interferon-stimulated gene 15 (ISG15) E3 ligase, catalyzes the ISGylation of TRIM21 at the Lys260 and Lys279 residues. Moreover, IFN-ß also induces TRIM21 ISGylation at multiple lysine residues, thereby enhancing its E3 ligase activity for K63-linkage-specific ubiquitination and resulting in increased levels of TRIM21 and p62 K63-linked ubiquitination. The K63-linked ubiquitination of p62 at Lys7 prevents its self-oligomerization and targeting to the autophagosome. Taken together, our study suggests that the ISGylation of TRIM21 plays a vital role in regulating self-oligomerization and localization of p62 in the autophagy induced by IFN-ß.


Assuntos
Autofagossomos/enzimologia , Citocinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Ribonucleoproteínas/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitinas/metabolismo , Células A549 , Autofagossomos/efeitos dos fármacos , Autofagossomos/genética , Autofagia , Citocinas/genética , Células HEK293 , Humanos , Interferon beta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ribonucleoproteínas/genética , Proteína Sequestossoma-1/genética , Ubiquitinação , Ubiquitinas/genética
11.
Cell Death Dis ; 12(7): 699, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262020

RESUMO

Butylate hydroxyanisole (BHA) is a synthetic phenol that is widely utilized as a preservative by the food and cosmetic industries. The antioxidant properties of BHA are also frequently used by scientists to claim the implication of reactive oxygen species (ROS) in various cellular processes, including cell death. We report on the surprising finding that BHA functions as a direct inhibitor of RIPK1, a major signaling hub downstream of several immune receptors. Our in silico analysis predicts binding of 3-BHA, but not 2-BHA, to RIPK1 in an inactive DLG-out/Glu-out conformation, similar to the binding of the type III inhibitor Nec-1s to RIPK1. This predicted superior inhibitory capacity of 3-BHA over 2-BHA was confirmed in cells and using in vitro kinase assays. We demonstrate that the reported protective effect of BHA against tumor necrosis factor (TNF)-induced necroptotic death does not originate from ROS scavenging but instead from direct RIPK1 enzymatic inhibition, a finding that most probably extends to other reported effects of BHA. Accordingly, we show that BHA not only protects cells against RIPK1-mediated necroptosis but also against RIPK1 kinase-dependent apoptosis. We found that BHA treatment completely inhibits basal and induced RIPK1 enzymatic activity in cells, monitored at the level of TNFR1 complex I under apoptotic conditions or in the cytosol under necroptosis. Finally, we show that oral administration of BHA protects mice from RIPK1 kinase-dependent lethality caused by TNF injection, a model of systemic inflammatory response syndrome. In conclusion, our results demonstrate that BHA can no longer be used as a strict antioxidant and that new functions of RIPK1 may emerge from previously reported effects of BHA.


Assuntos
Apoptose/efeitos dos fármacos , Hidroxianisol Butilado/farmacologia , Fibroblastos/efeitos dos fármacos , Aditivos Alimentares/farmacologia , Necroptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Síndrome de Resposta Inflamatória Sistêmica/prevenção & controle , Animais , Antioxidantes/farmacologia , Modelos Animais de Doenças , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Ligação Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Síndrome de Resposta Inflamatória Sistêmica/enzimologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Fator de Necrose Tumoral alfa
12.
EMBO J ; 40(18): e108249, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34296442

RESUMO

SARS-CoV-2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS-CoV-2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS-CoV-2 viral proteins. Here, we show that the nucleocapsid of SARS-CoV-2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS-CoV-2-infected monocytes show enhanced cellular interleukin-1ß (IL-1ß) expression, but reduced IL-1ß secretion. While SARS-CoV-2 infection promotes activation of the NLRP3 inflammasome and caspase-1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS-CoV-2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase-1. These insights into how SARS-CoV-2 antagonizes cellular inflammatory responses may open new avenues for treating COVID-19 in the future.


Assuntos
COVID-19/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Nucleocapsídeo/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/fisiologia , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Caspase 1/imunologia , Caspase 1/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Camundongos , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Ligação a Fosfato/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células THP-1
13.
Cell Death Dis ; 12(7): 668, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215720

RESUMO

Endocrine therapy for prostate cancer (PCa) mainly inhibits androgen receptor (AR) signaling, due to increased androgen synthesis and AR changes, PCa evolved into castration-resistant prostate cancer (CRPC). The function of Family With Sequence Similarity 64 Member A (FAM64A) and its association with prostate cancer has not been reported. In our research, we first reported that FAM64A is up-regulated and positively associated with poor prognosis of patients with prostate cancer (PCa) by TCGA database and immunohistochemistry staining. Moreover, knockdown of FAM64A significantly suppressed the proliferation, migration, invasion, and cell cycle of PCa cells in vitro. Mechanistically, FAM64A expression was increased by dihydrotestosterone (DHT) through direct binding of AR to FAM64A promoter, and notably promoted the proliferation, migration, invasion, and cell cycle of androgen-dependent cell line of PCa. In addition, abnormal expression of FAM64A affects the immune and interferon signaling pathway of PCa cells. In conclusion, FAM64A was up-regulated by AR through directly binding to its specific promoter region to promote the development of PCa, and was associated with the immune mechanism and interferon signaling pathway, which provided a better understanding and a new potential for treating PCa.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Sítios de Ligação , Ciclo Celular , Movimento Celular , Proliferação de Células , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Humanos , Interferons/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas Nucleares/genética , Células PC-3 , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Transdução de Sinais , Regulação para Cima
14.
Nat Cell Biol ; 23(7): 758-770, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34226698

RESUMO

The YAP/TAZ transcriptional programme is not only a well-established driver of cancer progression and metastasis but also an important stimulator of tissue regeneration. Here we identified Cerebral cavernous malformations 3 (CCM3) as a regulator of mechanical cue-driven YAP/TAZ signalling, controlling both tumour progression and stem cell differentiation. We demonstrate that CCM3 localizes to focal adhesion sites in cancer-associated fibroblasts, where it regulates mechanotransduction and YAP/TAZ activation. Mechanistically, CCM3 and focal adhesion kinase (FAK) mutually compete for binding to paxillin to fine-tune FAK/Src/paxillin-driven mechanotransduction and YAP/TAZ activation. In mouse models of breast cancer, specific loss of CCM3 in cancer-associated fibroblasts leads to exacerbated tissue remodelling and force transmission to the matrix, resulting in reciprocal YAP/TAZ activation in the neighbouring tumour cells and dissemination of metastasis to distant organs. Similarly, CCM3 regulates the differentiation of mesenchymal stromal/stem cells. In conclusion, CCM3 is a gatekeeper in focal adhesions that controls mechanotransduction and YAP/TAZ signalling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Adesões Focais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mecanotransdução Celular , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Comunicação Celular , Diferenciação Celular , Linhagem Celular Tumoral , Feminino , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/genética , Adesões Focais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Paxilina/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Estresse Mecânico , Fatores de Transcrição/genética , Quinases da Família src/metabolismo
15.
Mol Genet Genomics ; 296(5): 1135-1145, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34196769

RESUMO

Nik1 orthologs or group III hybrid histidine kinases (HHK3) represent a unique cytoplasmic osmosensor that act upstream of HOG/p38 MAPK pathway in fungi. It is an important molecular target for developing new antifungal agents against human pathogens. HHK3 orthologs contain a linear array of alternative HAMP and HAMP-like linker domains (poly-HAMP) in the N-terminal region. HAMP domains are quite common in prokaryotic histidine kinases where it mostly functions as signal transducer mediating conformational changes in the kinase domains. In contrast, poly-HAMP in HHK3 acts as a sensor and signal transducer to regulate histidine kinase activity. However, the mechanistic detail of this is poorly understood. Interestingly, recent studies indicate that the poly-HAMP-mediated regulation of the kinase activity varies among the orthologs. Hik1 is an important HHK3 ortholog from fungus Magnaporthe oryzae. In this paper, we aimed to decipher the role HAMP and HAMP-like linker domains in regulating the activity of Hik1p. We show that Hik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG/p38 MAPK pathway in Saccharomyces cerevisiae. Our data suggest a differential role of the HAMP domains in the functionality of Hik1p. Most interestingly, the deletion of individual domains in poly-HAMP resulted in distinct active forms of Hik1p and thereby indicating that the poly-HAMP domain, instead of acting as on-off switch, regulates the histidine kinase activity by transition through multiple conformational states.


Assuntos
Proteínas Fúngicas/metabolismo , Histidina Quinase/química , Histidina Quinase/metabolismo , Magnaporthe/enzimologia , Dioxóis/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Teste de Complementação Genética , Histidina Quinase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microrganismos Geneticamente Modificados , Mutação , Domínios Proteicos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pirróis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34269675

RESUMO

Rabies virus (RABV) infection can initiate the host immune defence response and induce an antiviral state characterized by the expression of interferon (IFN)-stimulated genes (ISGs), among which the family of genes of IFN-induced protein with tetratricopeptide repeats (Ifits) are prominent representatives. Herein, we demonstrated that the mRNA and protein levels of Ifit1, Ifit2 and Ifit3 were highly increased in cultured cells and mouse brains after RABV infection. Recombinant RABV expressing Ifit3, designated rRABV-Ifit3, displayed a lower pathogenicity than the parent RABV in C57BL/6 mice after intramuscular administration, and Ifit3-deficient mice exhibited higher susceptibility to RABV infection and higher mortality during RABV infection. Moreover, compared with their individual expressions, co-expression of Ifit2 and Ifit3 could more effectively inhibit RABV replication in vitro. These results indicate that murine Ifit3 plays an essential role in restricting the replication and reducing the pathogenicity of RABV. Ifit3 acts synergistically with Ifit2 to inhibit RABV replication, providing further insight into the function and complexity of the Ifit family.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Vírus da Raiva/fisiologia , Raiva/virologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Encéfalo/metabolismo , Encéfalo/virologia , Linhagem Celular , Feminino , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Raiva/imunologia , Vírus da Raiva/patogenicidade , Transcriptoma , Carga Viral , Replicação Viral
17.
Nat Commun ; 12(1): 4578, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321481

RESUMO

Mitochondria are transported along microtubules by opposing kinesin and dynein motors. Kinesin-1 and dynein-dynactin are linked to mitochondria by TRAK proteins, but it is unclear how TRAKs coordinate these motors. We used single-molecule imaging of cell lysates to show that TRAK2 robustly activates kinesin-1 for transport toward the microtubule plus-end. TRAK2 is also a novel dynein activating adaptor that utilizes a conserved coiled-coil motif to interact with dynein to promote motility toward the microtubule minus-end. However, dynein-mediated TRAK2 transport is minimal unless the dynein-binding protein LIS1 is present at a sufficient level. Using co-immunoprecipitation and co-localization experiments, we demonstrate that TRAK2 forms a complex containing both kinesin-1 and dynein-dynactin. These motors are functionally linked by TRAK2 as knockdown of either kinesin-1 or dynein-dynactin reduces the initiation of TRAK2 transport toward either microtubule end. We propose that TRAK2 coordinates kinesin-1 and dynein-dynactin as an interdependent motor complex, providing integrated control of opposing motors for the proper transport of mitochondria.


Assuntos
Dineínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesina/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase , Proteínas de Transporte/metabolismo , Complexo Dinactina/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Associadas aos Microtúbulos , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Transporte Proteico/fisiologia , Transcriptoma
18.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299370

RESUMO

Primary cilia are commonly found on most quiescent, terminally differentiated cells and play a major role in the regulation of the cell cycle, cell motility, sensing, and cell-cell communication. Alterations in ciliogenesis and cilia maintenance are causative of several human diseases, collectively known as ciliopathies. A key determinant of primary cilia is the histone deacetylase HDAC6, which regulates their length and resorption and whose distribution is regulated by the death inducer-obliterator 3 (Dido3). Here, we report that the atypical protein kinase Haspin is a key regulator of cilia dynamics. Cells defective in Haspin activity exhibit longer primary cilia and a strong delay in cilia resorption upon cell cycle reentry. We show that Haspin is active in quiescent cells, where it phosphorylates threonine 3 of histone H3, a known mitotic Haspin substrate. Forcing Dido3 detachment from the chromatin prevents Haspin inhibition from impacting cilia dynamics, suggesting that Haspin activity is required for the relocalization of Dido3-HDAC6 to the basal body. Exploiting the zebrafish model, we confirmed the physiological relevance of this mechanism. Our observations shed light on a novel player, Haspin, in the mechanisms that govern the determination of cilia length and the homeostasis of mature cilia.


Assuntos
Cílios/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Treonina/metabolismo , Animais , Ciclo Celular/fisiologia , Células Cultivadas , Cromatina/metabolismo , Células HEK293 , Humanos , Peixe-Zebra
19.
Nat Commun ; 12(1): 4541, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315898

RESUMO

Wntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 2.2 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and a lateral opening. Wnt3a interacts with WLS at multiple interfaces, with the lipid moiety on Wnt3a traversing a hydrophobic tunnel of WLS transmembrane domain and inserting into membrane. A ß-hairpin of Wnt3a containing the conserved palmitoleoylation site interacts with WLS extensively, which is crucial for WLS-mediated Wnt secretion. The flexibility of the Wnt3a loop/hairpin regions involved in the multiple binding sites indicates induced fit might happen when Wnts are bound to different binding partners. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling.


Assuntos
Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/ultraestrutura , Proteína Wnt3A/ultraestrutura , Receptores Frizzled/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Proteína Wnt3A/química , Proteína Wnt3A/metabolismo
20.
Nat Commun ; 12(1): 4288, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257283

RESUMO

The commonly mutated human KRAS oncogene encodes two distinct KRAS4A and KRAS4B proteins generated by differential splicing. We demonstrate here that coordinated regulation of both isoforms through control of splicing is essential for development of Kras mutant tumors. The minor KRAS4A isoform is enriched in cancer stem-like cells, where it responds to hypoxia, while the major KRAS4B is induced by ER stress. KRAS4A splicing is controlled by the DCAF15/RBM39 pathway, and deletion of KRAS4A or pharmacological inhibition of RBM39 using Indisulam leads to inhibition of cancer stem cells. Our data identify existing clinical drugs that target KRAS4A splicing, and suggest that levels of the minor KRAS4A isoform in human tumors can be a biomarker of sensitivity to some existing cancer therapeutics.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células A549 , Animais , Western Blotting , Proliferação de Células , Citometria de Fluxo , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas de Ligação a RNA/genética
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