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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205064

RESUMO

Cortactin is a well-known regulatory protein of the host actin cytoskeleton and represents an attractive target of microbial pathogens like Helicobacter pylori. H. pylori manipulates cortactin's phosphorylation status by type-IV secretion-dependent injection of its virulence protein CagA. Multiple host tyrosine kinases, like FAK, Src, and Abl, are activated during infection, but the pathway(s) involved is (are) not yet fully established. Among them, Src and Abl target CagA and stimulate tyrosine phosphorylation of the latter at its EPIYA-motifs. To investigate the role of cortactin in more detail, we generated a CRISPR/Cas9 knockout of cortactin in AGS gastric epithelial cells. Surprisingly, we found that FAK, Src, and Abl kinase activities were dramatically downregulated associated with widely diminished CagA phosphorylation in cortactin knockout cells compared to the parental control. Together, we report here a yet unrecognized cortactin-dependent signaling pathway involving FAK, Src, and Abl activation, and controlling efficient phosphorylation of injected CagA during infection. Thus, the cortactin status could serve as a potential new biomarker of gastric cancer development.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Quinase 1 de Adesão Focal/genética , Infecções por Helicobacter/genética , Helicobacter pylori/genética , Proteínas Oncogênicas v-abl/genética , Regulação Bacteriana da Expressão Gênica/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Fosforilação/genética , Quinases da Família src/genética
2.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205123

RESUMO

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRK1A, 1B, 2-4) and cdc2-like kinases (CLK1-4) belong to the CMGC group of serine/threonine kinases. These protein kinases are involved in multiple cellular functions, including intracellular signaling, mRNA splicing, chromatin transcription, DNA damage repair, cell survival, cell cycle control, differentiation, homocysteine/methionine/folate regulation, body temperature regulation, endocytosis, neuronal development, synaptic plasticity, etc. Abnormal expression and/or activity of some of these kinases, DYRK1A in particular, is seen in many human nervous system diseases, such as cognitive deficits associated with Down syndrome, Alzheimer's disease and related diseases, tauopathies, dementia, Pick's disease, Parkinson's disease and other neurodegenerative diseases, Phelan-McDermid syndrome, autism, and CDKL5 deficiency disorder. DYRKs and CLKs are also involved in diabetes, abnormal folate/methionine metabolism, osteoarthritis, several solid cancers (glioblastoma, breast, and pancreatic cancers) and leukemias (acute lymphoblastic leukemia, acute megakaryoblastic leukemia), viral infections (influenza, HIV-1, HCMV, HCV, CMV, HPV), as well as infections caused by unicellular parasites (Leishmania, Trypanosoma, Plasmodium). This variety of pathological implications calls for (1) a better understanding of the regulations and substrates of DYRKs and CLKs and (2) the development of potent and selective inhibitors of these kinases and their evaluation as therapeutic drugs. This article briefly reviews the current knowledge about DYRK/CLK kinases and their implications in human disease.


Assuntos
Doença de Alzheimer/genética , Quinases relacionadas a CDC2 e CDC28/genética , Síndrome de Down/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Doença de Alzheimer/tratamento farmacológico , Diferenciação Celular/genética , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/genética , Síndrome de Down/tratamento farmacológico , Humanos , Fosforilação/genética , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos
3.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205883

RESUMO

Aneurysmal subarachnoid hemorrhage (SAH) is a devastating emergent event associated with high mortality and morbidity. Survivors usually experience functional neurological sequelae caused by vasospasm-related delayed ischemia. In this study, male Sprague-Dawley rats were randomly assigned to five groups: sham (non-SAH) group, SAH group, and three groups with SAH treated with different doses of valproic acid (VPA) (10, 20, 40 mg/kg, once-daily, for 7 days). The severity of vasospasm was determined by the ratio of cross-sectional areas to intima-media thickness of the basilar arteries (BA) on the seventh day after SAH. The BA showed decreased expression of phospho-Akt proteins. The dentate gyrus showed increased expression of cleaved caspase-3 and Bax proteins and decreased expression of Bcl-2, phospho-ERK 1/2, phospho-Akt and acetyl-histone H3 proteins. The incidence of SAH-induced vasospasm was significantly lower in the SAH group treated with VPA 40 mg/kg (p < 0.001). Moreover, all groups treated with VPA showed reversal of the above-mentioned protein expression in BA and the dentate gyrus. Treatment with VPA upregulated histone H3 acetylation and conferred anti-vasospastic and neuro-protective effects by enhancing Akt and/or ERK phosphorylation. This study demonstrated that VPA could alleviate delayed cerebral vasospasm induced neuro-apoptosis after SAH.


Assuntos
Neurônios/efeitos dos fármacos , Hemorragia Subaracnóidea/tratamento farmacológico , Ácido Valproico/farmacologia , Vasoespasmo Intracraniano/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neurônios/metabolismo , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Hemorragia Subaracnóidea/genética , Hemorragia Subaracnóidea/patologia , Vasoespasmo Intracraniano/genética , Vasoespasmo Intracraniano/patologia , Proteína X Associada a bcl-2/genética
4.
Nat Commun ; 12(1): 4230, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244494

RESUMO

Extracellular matrix protein-1 (ECM1) promotes tumorigenesis in multiple organs but the mechanisms associated to ECM1 isoform subtypes have yet to be clarified. We report in this study that the secretory ECM1a isoform induces tumorigenesis through the GPR motif binding to integrin αXß2 and the activation of AKT/FAK/Rho/cytoskeleton signaling. The ATP binding cassette subfamily G member 1 (ABCG1) transduces the ECM1a-integrin αXß2 interactive signaling to facilitate the phosphorylation of AKT/FAK/Rho/cytoskeletal molecules and to confer cancer cell cisplatin resistance through up-regulation of the CD326-mediated cell stemness. On the contrary, the non-secretory ECM1b isoform binds myosin and blocks its phosphorylation, impairing cytoskeleton-mediated signaling and tumorigenesis. Moreover, ECM1a induces the expression of the heterogeneous nuclear ribonucleoprotein L like (hnRNPLL) protein to favor the alternative mRNA splicing generating ECM1a. ECM1a, αXß2, ABCG1 and hnRNPLL higher expression associates with poor survival, while ECM1b higher expression associates with good survival. These results highlight ECM1a, integrin αXß2, hnRNPLL and ABCG1 as potential targets for treating cancers associated with ECM1-activated signaling.


Assuntos
Processamento Alternativo , Carcinoma Epitelial do Ovário/genética , Proteínas da Matriz Extracelular/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Neoplasias Ovarianas/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Carcinoma Epitelial do Ovário/mortalidade , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/terapia , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas da Matriz Extracelular/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/metabolismo , Estimativa de Kaplan-Meier , Camundongos , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Ovário/patologia , Ovário/cirurgia , Fosforilação/genética , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA-Seq , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
PLoS One ; 16(6): e0253089, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1282298

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , COVID-19/imunologia , Núcleo Celular/imunologia , Fator Regulador 3 de Interferon/imunologia , Proteínas de Ligação a RNA/imunologia , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Proteínas não Estruturais Virais/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , COVID-19/genética , Núcleo Celular/genética , Células HeLa , Humanos , Fator Regulador 3 de Interferon/genética , NF-kappa B/genética , NF-kappa B/imunologia , Fosforilação/genética , Fosforilação/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas de Ligação a RNA/genética , SARS-CoV-2/genética , Transdução de Sinais/genética , Proteínas não Estruturais Virais/genética
6.
PLoS One ; 16(6): e0253089, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34166398

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , COVID-19/imunologia , Núcleo Celular/imunologia , Fator Regulador 3 de Interferon/imunologia , Proteínas de Ligação a RNA/imunologia , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Proteínas não Estruturais Virais/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , COVID-19/genética , Núcleo Celular/genética , Células HeLa , Humanos , Fator Regulador 3 de Interferon/genética , NF-kappa B/genética , NF-kappa B/imunologia , Fosforilação/genética , Fosforilação/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas de Ligação a RNA/genética , SARS-CoV-2/genética , Transdução de Sinais/genética , Proteínas não Estruturais Virais/genética
7.
Int J Mol Sci ; 22(11)2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067421

RESUMO

Base Excision Repair (BER) addresses base lesions and abasic sites induced by exogenous and endogenous stressors. X-ray cross complementing group 1 (XRCC1) functions as a scaffold protein in BER and single-strand break repair (SSBR), facilitating and coordinating repair through its interaction with a host of critical repair proteins. Alterations of XRCC1 protein and gene expression levels are observed in many cancers, including colorectal, ovarian, and breast cancer. While increases in the expression level of XRCC1 are reported, the transcription factors responsible for this up-regulation are not known. In this study, we identify the signal transducer and activator of transcription 3 (STAT3) as a novel regulator of XRCC1 through chromatin immunoprecipitation. Activation of STAT3 through phosphorylation at Y705 by cytokine (IL-6) signaling increases the expression of XRCC1 and the occupancy of STAT3 within the XRCC1 promoter. In triple negative breast cancer, the constitutive activation of STAT3 upregulates XRCC1 gene and protein expression levels. Increased expression of XRCC1 is associated with aggressiveness and resistance to DNA damaging chemotherapeutics. Thus, we propose that activated STAT3 regulates XRCC1 under stress and growth conditions, but constitutive activation in cancers results in dysregulation of XRCC1 and subsequently BER and SSBR.


Assuntos
Regiões Promotoras Genéticas/genética , Fator de Transcrição STAT3/genética , Neoplasias de Mama Triplo Negativas/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Linhagem Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Simples , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Interleucina-6/genética , Fosforilação/genética , Regulação para Cima/genética
8.
Mol Med Rep ; 23(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33760172

RESUMO

Sepsis is a life­threatening organ dysfunction caused by a dysregulated host response to infection, and is a leading cause of mortality worldwide. Myocardial dysfunction is associated with poor prognosis in patients with sepsis and contributes to a high risk of mortality. However, the pathophysiological mechanisms underlying sepsis­induced myocardial dysfunction are not completely understood. The aim of the present study was to investigate the role of toll­like receptor 4 (TLR4)/c­Jun N­terminal kinase (JNK) signaling in pro­inflammatory cytokine expression and cardiac dysfunction during lipopolysaccharide (LPS)­induced sepsis in mice. C57BL/6 mice were pretreated with TAK­242 or saline for 1 h and then subjected to LPS (12 mg/kg, intraperitoneal) treatment. Cardiac function was assessed using an echocardiogram. The morphological changes of the myocardium were examined by hematoxylin and eosin staining and transmission electron microscopy. The serum protein levels of cardiac troponin I (cTnI) and tumor necrosis factor­α (TNF­α) were determined by an enzyme­linked immunosorbent assay (ELISA). The TLR4 and JNK mRNA levels were analyzed via reverse transcription­quantitative PCR. TLR4, JNK and phosphorylated­JNK protein levels were measured by western blotting. In response to LPS, the activation of TLR4 and JNK in the myocardium was upregulated. There were significant increases in the serum levels of TNF­α and cTnI, as well as histopathological changes in the myocardium and suppressed cardiac function, following LPS stimulation. Inhibition of TLR4 activation using TAK­242 led to a decrease in the activation of JNK and reduced the protein expression of TNF­α in plasma, and alleviated histological myocardial injury and improved cardiac function during sepsis in mice. The present data suggested that the TLR4/JNK signaling pathway played a critical role in regulating the production of pro­inflammatory cytokines and myocardial dysfunction induced by LPS.


Assuntos
Insuficiência Cardíaca/genética , Sistema de Sinalização das MAP Quinases/genética , Sepse/genética , Receptor 4 Toll-Like/genética , Animais , Apoptose/genética , Citocinas/genética , Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Humanos , Interleucina-6/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Lipopolissacarídeos/toxicidade , Camundongos , Miocárdio/patologia , NF-kappa B/genética , Fosforilação/genética , Sepse/induzido quimicamente , Sepse/complicações , Sepse/patologia , Troponina I/genética , Fator de Necrose Tumoral alfa/genética
9.
Medicine (Baltimore) ; 100(12): e25093, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33761671

RESUMO

ABSTRACT: Based on the Thompson classification of intervertebral discs (IVDs), we systematically analyzed gene expression differences between severely degenerated and mildly degenerated IVDs and explored the underlying molecular mechanisms using bioinformatics methods and multichip integration. We used multiomics analysis, includes mRNA microarray and methylation chips, to explore the genetic network and mechanisms of lumbar disc herniation (LDH). Subsequently, the Combat function of the R language SVA package was applied to eliminate heterogeneity between the gene expression data. And the protein-protein interaction (PPI) network, gene ontology (GO), and molecular pathways were used to constructs the mechanisms network. Consequently, we obtained 149 differentially expressed genes. Related molecular pathways are the following: ribosome activity, oxidative phosphorylation, extracellular matrix response. Besides, through PPI network analysis, genes with higher connectivity such as UBA52, RPLP0, RPL3, RPLP2, and RPL27 were also identified, suggesting that they play important regulatory roles in the complex network associated with LDH. Additionally, cg12556991 (RPL27) and cg06852319 (RPLP0) were found to be LDH-related candidate DNA methylation modification sites in the IVDs tissue of LDH patients. In conclusions, ribosome activity, oxidative phosphorylation, and extracellular matrix response may be potential molecular mechanisms underlying LDH, while hub genes involved in UBA52, RPLP0, RPL3, RPLP2, and RPL27, and candidate DNA methylation modification sites of cg12556991and cg06852319 are likely key regulators in the development of LDH.


Assuntos
Metilação de DNA/genética , Matriz Extracelular/genética , Deslocamento do Disco Intervertebral/genética , Fosforilação/genética , Proteínas Ribossômicas/genética , Biologia Computacional , Expressão Gênica/genética , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes/genética , Humanos , Vértebras Lombares/metabolismo , Análise em Microsséries , Mapas de Interação de Proteínas/genética , RNA Mensageiro/metabolismo
10.
Plant Sci ; 305: 110847, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33691973

RESUMO

The transcription factor ABA-INSENSITIVE(ABI)4 has diverse roles in regulating plant growth, including inhibiting germination and reserve mobilization in response to ABA and high salinity, inhibiting seedling growth in response to high sugars, inhibiting lateral root growth, and repressing light-induced gene expression. ABI4 activity is regulated at multiple levels, including gene expression, protein stability, and activation by phosphorylation. Although ABI4 can be phosphorylated at multiple residues by MAPKs, we found that S114 is the preferred site of MPK3. To examine the possible biological role of S114 phosphorylation, we transformed abi4-1 mutant plants with ABI4pro::ABI4 constructs encoding wild type (114S), phosphorylation-null (S114A) or phosphomimetic (S114E) forms of ABI4. Phosphorylation of S114 is necessary for the response to ABA, glucose, salt stress, and lateral root development, where the abi4 phenotype could be complemented by expressing ABI4 (114S) or ABI4 (S114E) but not ABI4 (S114A). Comparison of root transcriptomes in ABA-treated roots of abi4-1 mutant plants transformed with constructs encoding the different phosphorylation-forms of S114 of ABI4 revealed that 85 % of the ABI4-regulated genes whose expression pattern could be restored by expressing ABI4 (114S) are down-regulated by ABI4. Phosphorylation of S114 was required for regulation of 35 % of repressed genes, but only 17 % of induced genes. The genes whose repression requires the phosphorylation of S114 are mainly involved in embryo and seedling development, growth and differentiation, and regulation of gene expression.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Serina/genética , Serina/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação/genética , Fosforilação/fisiologia , Reguladores de Crescimento de Plantas/genética , Fatores de Transcrição/genética
11.
Molecules ; 26(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652602

RESUMO

Hepatitis B virus (HBV) is a circular, and partially double-stranded DNA virus. Upon infection, the viral genome is translocated into the cell nucleus, generating the covalently closed circular DNA (cccDNA) intermediate, and forming a mini chromosome. HBV HBx is a small protein displaying multiple roles in HBV-infected cells, and in different subcellular locations. In the nucleus, the HBx protein is required to initiate and maintain viral transcription from the viral mini chromosome. In contrast, HBx also functions in the cytoplasm, where it is able to alter multiple cellular functions such as mitochondria metabolism, apoptosis and signal transduction pathways. It has been reported that in cultured cells, at low expression levels, the HBx protein is localized in the nucleus, whereas at high expression levels, it accumulates in the cytoplasm. This dynamic subcellular distribution of HBx might be essential to exert its multiple roles during viral infection. However, the mechanism that regulates different subcellular localizations of the HBx protein is unknown. We have previously taken a bioinformatics approach to investigate whether HBx might be regulated via post-translational modification, and we have proposed that the multiple nucleocytoplasmic functions of HBx might be regulated by an evolutionarily conserved mechanism via phosphorylation. In the current study, phylogenetically conserved amino acids of HBx with a high potential of phosphorylation were targeted for site-directed mutagenesis. Two conserved serine (Ser25 and Ser41), and one conserved threonine (Thr81) amino acids were replaced by either alanine or aspartic acid residues to simulate an unphosphorylated or phosphorylated state, respectively. Human hepatoma cells were transfected with increasing amounts of the HBx DNA constructs, and the cells were analyzed by fluorescence microscopy. Together, our results show that the nucleocytoplasmic distribution of the HBx protein could be regulated by phosphorylation since some of the modified proteins were mainly confined to distinct subcellular compartments. Remarkably, both HBx Ser41A, and HBx Thr81D proteins were predominantly localized within the nuclear compartment throughout the different expression levels of HBx mutants.


Assuntos
Carcinoma Hepatocelular/genética , Hepatite B/genética , Neoplasias Hepáticas/genética , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias/genética , Sequência de Aminoácidos/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Sequência Conservada/genética , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Células Hep G2 , Hepatite B/patologia , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Fosforilação/genética , Filogenia
12.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669246

RESUMO

The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice (p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1's dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.


Assuntos
Envelhecimento/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Contração Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Debilidade Muscular/metabolismo , Transdução de Sinais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Junção Neuromuscular/metabolismo , Fosforilação/genética , Espécies Reativas de Oxigênio/metabolismo , Adulto Jovem
13.
Int J Mol Sci ; 22(4)2021 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-33668437

RESUMO

Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In Drosophila, the Warts (Wts) kinase, a component of the Hippo signalling pathway, plays an essential role in regulating transcription and growth by phosphorylating its substrate Yorkie (Yki). The phosphorylation of Yki critically influences its localisation and activity as a transcriptional coactivator. In this study, we identified the homeodomain-interacting protein kinase (Hipk) as another kinase that phosphorylates Yki and mapped several sites of Yki phosphorylated by Hipk, using in vitro analysis: Ser168, Ser169/Ser172 and Ser255. These sites might provide auxiliary input for Yki regulation in vivo, as transgenic flies with mutations in these show prominent phenotypes; Hipk, therefore, represents an additional upstream regulator of Yki that works in concert with Wts.


Assuntos
Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Fosforilação/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética
14.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673213

RESUMO

Glioblastoma multiforme (GBM) is amongst the deadliest of human cancers, with a median survival rate of just over one year following diagnosis. Characterized by rapid proliferation and diffuse infiltration into the brain, GBM is notoriously difficult to treat, with tumor cells showing limited response to existing therapies and eventually developing resistance to these interventions. As such, there is intense interest in better understanding the molecular alterations in GBM to guide the development of more efficient targeted therapies. GBM tumors can be classified into several molecular subtypes which have distinct genetic signatures, and they show aberrant activation of numerous signal transduction pathways, particularly those connected to receptor tyrosine kinases (RTKs) which control glioma cell growth, survival, migration, invasion, and angiogenesis. There are also non-canonical modes of RTK signaling found in GBM, which involve G-protein-coupled receptors and calcium channels. This review uses The Cancer Genome Atlas (TCGA) GBM dataset in combination with a data-mining approach to summarize disease characteristics, with a focus on select molecular pathways that drive GBM pathogenesis. We also present a unique genomic survey of RTKs that are frequently altered in GBM subtypes, as well as catalog the GBM disease association scores for all RTKs. Lastly, we discuss current RTK targeted therapies and highlight emerging directions in GBM research.


Assuntos
Neoplasias Encefálicas/enzimologia , Proliferação de Células , Glioblastoma/enzimologia , Proteínas de Neoplasias/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Proteínas de Neoplasias/genética , Fosforilação/genética , Receptores Proteína Tirosina Quinases/genética
15.
BMC Cancer ; 21(1): 312, 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33761914

RESUMO

BACKGROUND: Forkhead transcription factors control cell growth in multiple cancer types. Foxd1 is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported. METHODS: Transcriptome data from the TCGA database was used to correlate FOXD1 expression with patient survival. FOXD1 was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of the FOXD1 pathway with cell cycle progression was investigated through correlation analysis using the TCGA database. RESULTS: FOXD1 expression level in ccRCC correlated inversely with patient survival. Knockout of FOXD1 in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-OFOXD1null. In vivo, xenografted 786-OFOXD1null showed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-OFOXD1null had an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-OFOXD1null, and increased DNA damage. Genes correlate with FOXD1 in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation. CONCLUSIONS: We show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression of FOXD1 and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.


Assuntos
Carcinoma de Células Renais/genética , Divisão Celular/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Técnicas de Inativação de Genes , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas de Transporte da Membrana Mitocondrial/genética , Fosforilação/genética , RNA-Seq , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Mol Biol Rep ; 48(2): 1439-1452, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33590416

RESUMO

Profilin-1 (PFN1) regulates actin polymerization and cytoskeletal growth. Despite the essential roles of PFN1 in cell integration, its subcellular function in keratinocyte has not been elucidated yet. Here we characterize the specific regulation of PFN1 in DNA damage response and repair machinery. PFN1 depletion accelerated DNA damage-mediated apoptosis exhibiting PTEN loss of function instigated by increased phosphorylated inactivation followed by high levels of AKT activation. PFN1 changed its predominant cytoplasmic localization to the nucleus upon DNA damage and subsequently restored the cytoplasmic compartment during the recovery time. Even though γH2AX was recruited at the sites of DNA double strand breaks in response to DNA damage, PFN1-deficient cells failed to recruit DNA repair factors, whereas control cells exhibited significant increases of these genes. Additionally, PFN1 depletion resulted in disruption of PTEN-AKT cascade upon DNA damage and CHK1-mediated cell cycle arrest was not recovered even after the recovery time exhibiting γH2AX accumulation. This might suggest PFN1 roles in regulating DNA damage response and repair machinery to protect cells from DNA damage. Future studies addressing the crosstalk and regulation of PTEN-related DNA damage sensing and repair pathway choice by PFN1 may further aid to identify new mechanistic insights for various DNA repair disorders.


Assuntos
Distúrbios no Reparo do DNA/genética , Reparo do DNA/genética , Histonas/genética , Profilinas/genética , Actinas/genética , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Quinase 1 do Ponto de Checagem/genética , Citoplasma/genética , Citoesqueleto/genética , Dano ao DNA/genética , Distúrbios no Reparo do DNA/patologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Fosforilação/genética
17.
J Mol Biol ; 433(7): 166874, 2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33556406

RESUMO

The CREB-Regulated Transcriptional Coactivators (CRTCs) regulate the transcription of CREB target genes and have important functions in many biological processes. At the basal state, they are phosphorylated at multiple residues, which promotes their association with 14-3-3 that sequesters them in the cytoplasm. Upon dephosphorylation, they translocate into the nuclei and associate with CREB to activate the target gene transcription. Although three conserved serine residues in CRTCs have been implicated in their phosphorylation regulation, whether and how they mediate interactions with 14-3-3 is unclear. Here, we provide direct evidence that these residues and flanking regions interact with 14-3-3 and the structural basis of the interaction. Our study also identified a novel salt bridge in CRTC1 with an important function in binding 14-3-3, expanding the understanding of the interaction between 14-3-3 and its ligands.


Assuntos
Proteínas 14-3-3/genética , Proteína de Ligação a CREB/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Fatores de Transcrição/genética , Núcleo Celular/genética , Citoplasma/genética , Células HEK293 , Humanos , Fosforilação/genética , Ligação Proteica/genética , Transcrição Genética/genética , Ativação Transcricional/genética
18.
Molecules ; 26(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33573096

RESUMO

Phosphorylation represents one of the most important modifications of amino acids, peptides, and proteins. By modifying the latter, it is useful in improving the functional properties of foods. Although all these substances are broadly annotated in internet databases, there is no unified code for their annotation. The present publication aims to describe a simple code for the annotation of phosphopeptide sequences. The proposed code describes the location of phosphate residues in amino acid side chains (including new rules of atom numbering in amino acids) and the diversity of phosphate residues (e.g., di- and triphosphate residues and phosphate amidation). This article also includes translating the proposed biological code into SMILES, being the most commonly used chemical code. Finally, it discusses possible errors associated with applying the proposed code and in the resulting SMILES representations of phosphopeptides. The proposed code can be extended to describe other modifications in the future.


Assuntos
Sequência de Aminoácidos/genética , Aminoácidos/genética , Anotação de Sequência Molecular , Proteínas/genética , Aminoácidos/classificação , Código Genético/genética , Humanos , Fosfopeptídeos/classificação , Fosfopeptídeos/genética , Fosforilação/genética , Proteínas/classificação
19.
Nat Commun ; 12(1): 1285, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627652

RESUMO

The host defence peptide cathelicidin (LL-37 in humans, mCRAMP in mice) is released from neutrophils by de-granulation, NETosis and necrotic death; it has potent anti-pathogen activity as well as being a broad immunomodulator. Here we report that cathelicidin is a powerful Th17 potentiator which enhances aryl hydrocarbon receptor (AHR) and RORγt expression, in a TGF-ß1-dependent manner. In the presence of TGF-ß1, cathelicidin enhanced SMAD2/3 and STAT3 phosphorylation, and profoundly suppressed IL-2 and T-bet, directing T cells away from Th1 and into a Th17 phenotype. Strikingly, Th17, but not Th1, cells were protected from apoptosis by cathelicidin. We show that cathelicidin is released by neutrophils in mouse lymph nodes and that cathelicidin-deficient mice display suppressed Th17 responses during inflammation, but not at steady state. We propose that the neutrophil cathelicidin is required for maximal Th17 differentiation, and that this is one method by which early neutrophilia directs subsequent adaptive immune responses.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Células Th17/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Células Th1/citologia , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th17/citologia , Células Th17/efeitos dos fármacos
20.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33419940

RESUMO

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.


Assuntos
Proteínas de Arabidopsis/genética , Cromatina/genética , Fosfoproteínas/genética , Imunidade Vegetal/genética , Motivos AT-Hook/genética , Motivos AT-Hook/imunologia , Arabidopsis/genética , Arabidopsis/imunologia , Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/genética , Padrões Moleculares Associados a Patógenos/imunologia , Padrões Moleculares Associados a Patógenos/metabolismo , Fosfoproteínas/imunologia , Fosforilação/genética
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