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1.
J Med Chem ; 64(4): 1966-1988, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33593051

RESUMO

TYK2 mediates signaling of IL-23, IL-12, and Type I IFN-driven responses that are critical in immune-mediated diseases. Herein, we report the design, synthesis, and structure-activity relationships (SARs) of 3-(4-(2-((1H-indol-5-yl)amino)-5-fluoropyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile derivatives as selective TYK2 inhibitors. Among them, compound 14l exhibited acceptable TYK2 inhibition with an IC50 value of 9 nM, showed satisfactory selectivity characteristics over the other three homologous JAK kinases, and performed good functional potency in the JAK/STAT signaling pathway on lymphocyte lines and human whole blood. In liver microsomal assay studies, the clearance rate and half-life of 14l were 11.4 mL/min/g and 121.6 min, respectively. Furthermore, in a dextran sulfate sodium colitis model, 14l reduced the production of pro-inflammatory cytokines IL-6 and TNF-α and improved the inflammation symptoms of mucosal infiltration, thickening, and edema. Taken together, 14l was a selective TYK2 inhibitor and could be used to treat immune diseases deserving further investigation.


Assuntos
Fármacos Gastrointestinais/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , TYK2 Quinase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Colo/patologia , Estabilidade de Medicamentos , Fármacos Gastrointestinais/síntese química , Fármacos Gastrointestinais/metabolismo , Humanos , Doenças Inflamatórias Intestinais/patologia , Masculino , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Pirazóis/síntese química , Pirazóis/metabolismo , Pirimidinas/síntese química , Pirimidinas/metabolismo , Ratos Sprague-Dawley , Relação Estrutura-Atividade , TYK2 Quinase/metabolismo
2.
J Med Chem ; 64(1): 677-694, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33370104

RESUMO

A search for structurally diversified Tyk2 JH2 ligands from 6 (BMS-986165), a pyridazine carboxamide-derived Tyk2 JH2 ligand as a clinical Tyk2 inhibitor currently in late development for the treatment of psoriasis, began with a survey of six-membered heteroaryl groups in place of the N-methyl triazolyl moiety in 6. The X-ray co-crystal structure of an early lead (12) revealed a potential new binding pocket. Exploration of the new pocket resulted in two frontrunners for a clinical candidate. The potential hydrogen bonding interaction with Thr599 in the pocket was achieved with a tertiary amide moiety, confirmed by the X-ray co-crystal structure of 29. When the diversity search was extended to nicotinamides, a single fluorine atom addition was found to significantly enhance the permeability, which directly led to the discovery of 7 (BMS-986202) as a clinical Tyk2 inhibitor that binds to Tyk2 JH2. The preclinical studies of 7, including efficacy studies in mouse models of IL-23-driven acanthosis, anti-CD40-induced colitis, and spontaneous lupus, will also be presented.


Assuntos
Ciclopropanos/farmacologia , Descoberta de Drogas , Oxazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , TYK2 Quinase/antagonistas & inibidores , Animais , Catálise , Cristalografia por Raios X , Ciclopropanos/química , Humanos , Camundongos , Oxazóis/química , Ligação Proteica , Inibidores de Proteínas Quinases/química , Psoríase/tratamento farmacológico , Relação Estrutura-Atividade , TYK2 Quinase/metabolismo
3.
Dev Comp Immunol ; 102: 103474, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31437526

RESUMO

Tyrosine kinase 2 (TYK2), a member of Janus kinase family, has been identified as a crucial protein in signal transduction initiated by interferons or interleukins in mammals. However, the function of avian TYK2 in innate immune response remains largely unknown. In this study, the full-length duck TYK2 (duTYK2) cDNA was cloned for the first time, which encoded a putative protein of 1187 amino acid residues and showed the high sequence similarity with bald eagle, crested ibis, and white-tailed tropicbird TYK2s. The duTYK2 was widely expressed in all examined tissues of healthy ducks and showed diffuse cytoplasmic localization in duck embryo fibroblasts (DEFs). Overexpression of duTYK2 significantly enhanced ISRE promoter activity and induced the expression of viperin, PKR, 2',5'-OAS, Mx and ZAP in DEFs. The C-terminal kinase domain of duTYK2 is essential for duTYK2-mediated ISRE promoter activation. Furthermore, knockdown of duTYK2 dramatically decreased duck Tembusu virus (DTMUV)-, duck enteritis virus (DEV)-, poly(I:C)- or poly(dA:dT)-induced ISRE promoter activation. Additionally, duTYK2 expression exhibited antiviral activity against DTMUV. These results indicated that duTYK2 played a critical role in duck antiviral innate immunity.


Assuntos
Proteínas Aviárias/metabolismo , Patos/imunologia , TYK2 Quinase/metabolismo , Sequência de Aminoácidos , Animais , Antivirais/metabolismo , Proteínas Aviárias/química , Proteínas Aviárias/genética , Doenças das Aves/imunologia , Doenças das Aves/virologia , Linhagem Celular , Clonagem Molecular , Citoplasma/metabolismo , Patos/virologia , Flavivirus/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Interferons/metabolismo , Filogenia , Regiões Promotoras Genéticas , Alinhamento de Sequência , Transdução de Sinais/genética , Transdução de Sinais/imunologia , TYK2 Quinase/química , TYK2 Quinase/genética , Distribuição Tecidual
4.
FEBS Lett ; 594(5): 864-877, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31705658

RESUMO

Respirovirus C protein blocks the type I interferon (IFN)-stimulated activation of the JAK-STAT pathway. It has been reported that C protein inhibits IFN-α-stimulated tyrosine phosphorylation of STATs, but the underlying mechanism is poorly understood. Here, we show that the C protein of Sendai virus (SeV), a member of the Respirovirus genus, binds to the IFN receptor subunit IFN-α/ß receptor subunit (IFNAR)2 and inhibits IFN-α-stimulated tyrosine phosphorylation of the upstream receptor-associated kinases, JAK1 and TYK2. Analysis of various SeV C mutant (Cm) proteins demonstrates the importance of the inhibitory effect on receptor-associated kinase phosphorylation for blockade of JAK-STAT signaling. Furthermore, this inhibitory effect and the IFNAR2 binding capacity are observed for all the respirovirus C proteins examined. Our results suggest that respirovirus C protein inhibits activation of the receptor-associated kinases JAK1 and TYK2 possibly through interaction with IFNAR2.


Assuntos
Receptor de Interferon alfa e beta/metabolismo , Vírus Sendai/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Janus Quinase 1/metabolismo , Mutação , Fosforilação , Fatores de Transcrição STAT/metabolismo , Vírus Sendai/genética , TYK2 Quinase/metabolismo , Proteínas Virais/genética
5.
Proc Natl Acad Sci U S A ; 116(49): 24881-24891, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31754034

RESUMO

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.


Assuntos
Bortezomib/farmacologia , Processos Neoplásicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , TYK2 Quinase/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Linhagem Celular Tumoral , Feminino , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mieloma Múltiplo , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Neoplasias de Mama Triplo Negativas/patologia
6.
J Virol ; 93(23)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31511388

RESUMO

Equid herpesvirus 1 (EHV-1) is a viral pathogen of horse populations worldwide spread by the respiratory route and is known for causing outbreaks of neurologic syndromes and abortion storms. Previously, we demonstrated that an EHV-1 strain of the neuropathogenic genotype, T953, downregulates the beta interferon (IFN-ß) response in vitro in equine endothelial cells (EECs) at 12 h postinfection (hpi). In the present study, we explored the molecular correlates of this inhibition as clues toward an understanding of the mechanism. Data from our study revealed that EHV-1 infection of EECs significantly reduced both Toll-like receptor 3 (TLR3) and TLR4 mRNA expression at 6 hpi and 12 hpi. While EHV-1 was able to significantly reduce IRF9 mRNA at both 6 hpi and 12 hpi, the virus significantly reduced IFN regulatory factor 7 (IRF7) mRNA only at 12 hpi. EHV-1 did not alter the cellular level of Janus-activated kinase 1 (JAK1) at any time point. However, EHV-1 reduced the cellular level of expression of tyrosine kinase 2 (TYK2) at 12 hpi. Downstream of JAK1-TYK2 signaling, EHV-1 blocked the phosphorylation and activation of signal transducer and activator of transcription 2 (STAT2) when coincubated with exogenous IFN, at 12 hpi, although not at 3 or 6 hpi. Immunofluorescence staining revealed that the virus prevented the nuclear translocation of STAT2 molecules, confirming the virus-mediated inhibition of STAT2 activation. The pattern of suppression of phosphorylation of STAT2 by EHV-1 implicated viral late gene expression. These data help illuminate how EHV-1 strategically inhibits the host innate immune defense by limiting steps required for type I IFN sensitization and induction.IMPORTANCE To date, no commercial vaccine label has a claim to be fully protective against the diseases caused by equid herpesvirus 1 (EHV-1), especially the neurologic form. The interferon (IFN) system, of which type I IFN is of great importance, still remains a viable immunotherapeutic option against EHV-1 infection. The type I IFN system has been exploited successfully to treat other viral infections, such as chronic hepatitis B and C in humans. The current state of research on how EHV-1 interferes with the protective effect of type I IFN has indicated transient induction of type I IFN production followed by a rapid shutdown in vitro in equine endothelial cells (EECs). The significance of our study is the identification of certain steps in the type I IFN signaling pathway targeted for inhibition by EHV-1. Understanding this pathogen-host relationship is essential for the long-term goal of developing effective immunotherapy against EHV-1.


Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/virologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/metabolismo , Herpesvirus Equídeo 1/imunologia , Interferon Tipo I/metabolismo , Animais , Regulação da Expressão Gênica , Hepatite B Crônica , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Cavalos , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Janus Quinase 1/metabolismo , RNA Mensageiro/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , TYK2 Quinase/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
7.
Sci Rep ; 9(1): 12165, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434951

RESUMO

Type I interferon (IFN-I) pathway plays a central role in the systemic lupus erythematosus (SLE) pathogenesis. Recent data suggest that SLE is associated with variants in IFN-I genes, such as tyrosine kinase 2 (TYK2), which is crucial in anti-viral immunity. Here, five TYK2 single nucleotide polymorphisms (SNPs) were genotyped in 368 childhood-onset SLE Mexican patients and 516 sex-matched healthy controls. Allele frequencies were also estimated in four indigenous groups. SLE protection was associated with TYK2 risk infection variants affecting residually its catalytic domain, rs12720356 (OR = 0.308; p = 0.041) and rs34536443 (OR = 0.370; p = 0.034), but not with rs2304256, rs12720270, and rs280500. This association was replicated in a 506 adult-onset SLE patients sample (OR = 0.250; p = 0.005, and OR = 0.277; p = 0.008, respectively). The minor alleles of both associated SNPs had a lower frequency in Mestizos than in Spaniards and were absent or rare in indigenous, suggesting that the presence of these alleles in the Mexican Mestizo population was derived from the Spaniards. For the first time, we report genetic variants with a protective effect in childhood- and adult-onset SLE Mexican population. Our results suggest that the frequency of IFN-I alleles associated with SLE, may have been shaped in populations exposed to infectious diseases for long periods, and this could be an explanation why Native American ancestry is associated with a higher SLE prevalence and an earlier onset.


Assuntos
Lúpus Eritematoso Sistêmico/patologia , TYK2 Quinase/genética , Adulto , Alelos , Estudos de Casos e Controles , Domínio Catalítico , Criança , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Genótipo , Haplótipos , Humanos , Interferon Tipo I/genética , Desequilíbrio de Ligação , Lúpus Eritematoso Sistêmico/genética , Masculino , México , Razão de Chances , Polimorfismo de Nucleotídeo Único , Fatores de Risco , TYK2 Quinase/química , TYK2 Quinase/metabolismo
8.
Turk J Gastroenterol ; 30(8): 722-731, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31418417

RESUMO

BACKGROUND/AIMS: We have previously identified a tight junction protein claudin-9 (CLDN9) as an upregulated gene in hepatocellular carcinoma (HCC) through an immunohistochemistry analysis. Here, we explore its function and clinical relevance in human HCC. MATERIALS AND METHODS: Stable transfection of the hepatocyte line HL7702 with CLDN9 was confirmed by the real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence. The impact of CLDN9 on the cell invasion and migration was assessed in vitro by a transwell assay and wound-healing experiment. Western blotting was used to determine the activation state of the Tyk2 (tyrosine kinase 2)/Stat3 (signal transducer and activator of transcription 3) pathway. Moreover, we used a Tyk2-RNAi assay to silence the expression of Tyk2 in CLDN9 expressing hepatocytes; subsequently, the impact of the Tyk2/Stat3 signaling pathway on the cell invasion and migration in vitro was assessed by a transwell assay and a wound-healing experiment. Furthermore, an immunohistochemistry method was utilized to explore the expression levels of CLDN9 and p-Stat3 in the HCC tissues and histologically non-neoplastic hepatic tissues. RESULTS: We confirmed that the expression of CLDN9 significantly enhanced the metastatic ability of hepatocytes in vitro, and the activation of the Stat3 pathway by Tyk2 was an important mechanism by which CLDN9 promoted hepatocyte aggressiveness in HCC. CONCLUSION: As an HCC proto-oncogene, CLDN9 affected the Stat3 signaling pathway via Tyk2 and ultimately enhanced the metastatic ability of hepatocytes.


Assuntos
Carcinoma Hepatocelular/genética , Claudinas/metabolismo , Neoplasias Hepáticas/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , TYK2 Quinase/metabolismo , Linhagem Celular Tumoral , Hepatócitos/metabolismo , Humanos , Metástase Neoplásica/genética
9.
Immunohorizons ; 3(5): 172-185, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31356171

RESUMO

Cytokine IL-17A (IL-17) acts on various cell types, including epidermal keratinocytes, and induces antimicrobial peptide and chemokine production to elicit antibacterial and antifungal defense responses. Excess IL-17 leads to inflammatory skin diseases such as psoriasis. The IκB family protein IκB-ζ mediates IL-17-induced responses. However, the mechanism controlling IκB-ζ expression in IL-17-stimulated cells remains elusive. In this study, we showed that JAK kinase TYK2 positively regulates IL-17-induced IκB-ζ expression. TYK2-deficient mice showed reduced inflammation and concomitant reduction of IκB-ζ mRNA compared with wild-type mice in imiquimod-induced skin inflammation. The analysis of the IκB-ζ promoter activity using human cell lines (HaCaT and HeLa) revealed that catalytic activity of TYK2 and its substrate transcription factor STAT3, but not IL-17, is required for IκB-ζ promoter activity. In contrast, IL-17-induced signaling, which did not activate STAT3, posttranscriptionally stabilized IκB-ζ mRNA via its 3'-untranslated region. IL-17 signaling protein ACT1 was required to counteract constitutive IκB-ζ mRNA degradation by RNase Regnase-1. These results suggested that transcriptional activation by TYK2-STAT3 pathway and mRNA stabilization by IL-17-mediated signals act separately from each other but complementarily to achieve IκB-ζ induction. Therefore, JAK/TYK2 inhibition might be of significance in regulation of IL-17-induced inflammatory reactions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Interleucina-17/metabolismo , Estabilidade de RNA , Fator de Transcrição STAT3/metabolismo , TYK2 Quinase/metabolismo , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Células HeLa , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Psoríase/induzido quimicamente , Psoríase/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , TYK2 Quinase/genética , Fatores de Transcrição/metabolismo
10.
Biomed Environ Sci ; 32(6): 406-418, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31262386

RESUMO

OBJECTIVE: Previous studies have indicated that the plasticizer di (2-ethylhexyl) phthalate (DEHP) affects lipid accumulation; however, its underlying mechanism remains unclear. We aim to clarify the effect of DEHP on lipid metabolism and the role of TYK2/STAT1 and autophagy. METHODS: In total, 160 Wistar rats were exposed to DEHP [0, 5, 50, 500 mg/(kg•d)] for 8 weeks. Lipid levels, as well as mRNA and protein levels of TYK2, STAT1, PPARγ, AOX, FAS, LPL, and LC3 were detected. RESULTS: The results indicate that DEHP exposure may lead to increased weight gain and altered serum lipids. We observed that DEHP exposure affected liver parenchyma and increased the volume or number of fat cells. In adipose tissue, decreased TYK2 and STAT1 promoted the expression of PPARγ and FAS. The mRNA and protein expression of LC3 in 50 and 500 mg/(kg•d) groups was increased significantly. In the liver, TYK2 and STAT1 increased compensatorily; however, the expression of FAS and AOX increased, while LPL expression decreased. Joint exposure to both a high-fat diet and DEHP led to complete disorder of lipid metabolism. CONCLUSION: It is suggested that DEHP induces lipid metabolism disorder by regulating TYK2/STAT1. Autophagy may play a potential role in this process as well. High-fat diet, in combination with DEHP exposure, may jointly have an effect on lipid metabolism disorder.


Assuntos
Autofagia/efeitos dos fármacos , Dietilexilftalato/toxicidade , Disruptores Endócrinos/toxicidade , Transtornos do Metabolismo dos Lipídeos/induzido quimicamente , Metabolismo dos Lipídeos/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos Wistar , Fator de Transcrição STAT1/metabolismo , TYK2 Quinase/metabolismo
11.
Mediators Inflamm ; 2019: 4143604, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31275057

RESUMO

A small group of only seven transcription factors known as STATs (signal transducer and activator of transcription) are considered to be canonical determinants of specific gene activation for a plethora of ligand/receptor systems. The activation of STATs involves a family of four tyrosine kinases called JAK kinases. JAK1 and JAK2 activate STAT1 in the cytoplasm at the heterodimeric gamma interferon (IFNγ) receptor, while JAK1 and TYK2 activate STAT1 and STAT2 at the type I IFN heterodimeric receptor. The same STATs and JAKs are also involved in signaling by functionally different cytokines, growth factors, and hormones. Related to this, IFNγ-activated STAT1 binds to the IFNγ-activated sequence (GAS) element, but so do other STATs that are not involved in IFNγ signaling. Activated JAKs such as JAK2 and TYK2 are also involved in the epigenetics of nucleosome unwrapping for exposure of DNA to transcription. Furthermore, activated JAKs and STATs appear to function coordinately for specific gene activation. These complex events have not been addressed in canonical STAT signaling. Additionally, the function of noncoding enhancer RNAs, including their role in enhancer/promoter interaction is not addressed in the canonical STAT signaling model. In this perspective, we show that JAK/STAT signaling, involving membrane receptors, is essentially a variation of cytoplasmic nuclear receptor signaling. Focusing on IFN signaling, we showed that ligand, IFN receptor, the JAKs, and the STATs all undergo endocytosis and ATP-dependent nuclear translocation to promoters of genes specifically activated by IFNs. We argue here that the vacuolar ATPase (V-ATPase) proton pump probably plays a key role in endosomal membrane crossing by IFNs for receptor cytoplasmic binding. Signaling of nuclear receptors such as those of estrogen and dihydrotestosterone provides templates for making sense of the specificity of gene activation by closely related cytokines, which has implications for lymphocyte phenotypes.


Assuntos
Esteroides/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Humanos , Interferons/metabolismo , Fatores de Transcrição STAT/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , TYK2 Quinase/metabolismo
12.
Cancer Med ; 8(11): 5232-5241, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31278855

RESUMO

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that arise most commonly in the setting of the Neurofibromatosis Type 1 (NF1) cancer predisposition syndrome. Despite aggressive multimodality therapy, outcomes are dismal and most patients die within 5 years of diagnosis. Prior genomic studies in our laboratory identified tyrosine kinase 2 (TYK2) as a frequently mutated gene in MPNST. Herein, we explored the function of TYK2 in MPNST pathogenesis. METHODS: Immunohistochemistry was utilized to examine expression of TYK2 in MPNSTs and other sarcomas. To establish a role for TYK2 in MPNST pathogenesis, murine and human TYK2 knockdown and knockout cells were established using shRNA and CRISPR/Cas9 systems, respectively. RESULTS: We have demonstrated that TYK2 was highly expressed in the majority of human MPNSTs examined. Additionally, we demonstrated that knockdown of Tyk2/TYK2 in murine and human MPNST cells significantly increased cell death in vitro. These effects were accompanied by a decrease in the levels of activated Stats and Bcl-2 as well as an increase in the levels of Cleaved Caspase-3. In addition, Tyk2-KD cells demonstrated impaired growth in subcutaneous and metastasis models in vivo. CONCLUSION: Taken together, these data illustrate the importance of TYK2 in MPNST pathogenesis and suggest that the TYK2 pathway may be a potential therapeutic target for these deadly cancers.


Assuntos
Biomarcadores Tumorais , Neoplasias da Bainha Neural/genética , Neoplasias da Bainha Neural/patologia , TYK2 Quinase/genética , Adulto , Idoso , Animais , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Suscetibilidade a Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Marcação de Genes , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias da Bainha Neural/metabolismo , Transdução de Sinais , TYK2 Quinase/metabolismo , Adulto Jovem
13.
Mol Cell Endocrinol ; 484: 52-58, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30660700

RESUMO

BACKGROUND: Mono-2-ethylhexyl phthalate (MEHP), an important metabolite of di (2-ethylhexyl) phthalate (DEHP), can induce lipid metabolic disorder. Previous studies have shown that MEHP promotes 3T3-L1 cell differentiation; however, the underlying mechanism is unclear. The present study was performed to investigate the effect of the TYK-2/STAT-3 pathway on lipid accumulation induced by MEHP. METHODS: A 3T3-L1 precursor adipocyte differentiation model was exposed to MEHP. 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin were used to establish the 3T3-L1 precursor adipocyte differentiation model. Then the model cells were exposed to MEHP for 8 d. The lipid droplet formation in 3T3-L1 cells was determined with Oil-Red-O staining, and isopropyl alcohol was used to extract the lipid droplets for quantification. Flow cytometry was used to detect the intracellular reactive oxygen species (ROS) and mitochondrial membrane potential. Quantitative real-time polymerase chain reaction (qPCR) was used to detect mRNA expression, and western blotting was used to detect the expression of proteins encoded by TYK-2/STAT-3 pathway genes and adipogenesis-related genes. RESULTS: MEHP treatment, compared with the control treatment, significantly promoted the differentiation of 3T3-L1 cells and increased the expression of STAT-3 mRNA and protein and P-STAT3 protein in the cells. In addition, MEHP down-regulated the phosphorylation of STAT-3 in mitochondria. MEHP was found to influence the mitochondrial membrane potential and intracellular ROS levels. CONCLUSION: MEHP may affect adipocyte differentiation and lead to lipid accumulation through the TYK-2/STAT-3 pathway.


Assuntos
Adipócitos/citologia , Dietilexilftalato/análogos & derivados , Metabolismo dos Lipídeos/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Diferenciação Celular , Sobrevivência Celular , Dexametasona/farmacologia , Dietilexilftalato/farmacologia , Insulina/farmacologia , Gotículas Lipídicas/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , TYK2 Quinase/metabolismo
14.
J Allergy Clin Immunol ; 143(6): 2131-2146, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30578870

RESUMO

BACKGROUND: The incidence of eosinophilic esophagitis (EoE) is greater in male than female subjects, and the underlying molecular basis for this sex bias remains unclear. OBJECTIVE: We sought to delineate the contribution of the sex hormone estrogen to the EoE phenotype and esophageal epithelial barrier function and remodeling. METHODS: We performed demographic and incidence analyses of EoE in male and female subjects from a single-center pediatric cohort. Estrogen-responsive gene expression analyses and estrogen receptor (ESR) immunofluorescence staining of esophageal biopsy specimens from patients with EoE and control subjects were performed. The effect of 17ß-estradiol (E2) on IL-13-induced signaling pathways, gene expression, and esophageal epithelial architecture and barrier function in a primary human esophageal keratinocyte cell (EPC2) culture system (EPC2-air-liquid interface) was examined. RESULTS: We observed a male predominance in patients with EoE. Analyses of RNA sequencing data sets revealed a significant dysregulation of the estrogen-responsive gene network and expression of ESR1 and ESR2 in esophageal biopsy specimens from patients with EoE compared with control subjects. IL-13 stimulation of EPC2-air-liquid interface cells led to altered cellular architecture with induced dilation of intercellular spaces and barrier dysfunction. Pretreatment of EPC2s with E2 prior to IL-13 exposure abrogated IL-13-induced architectural changes and esophageal barrier dysfunction. Mechanistically, E2-protective effects were dependent on ESR2 and associated with diminishing of IL-13-induced tyrosine kinase 2 and signal transducer and activator of transcription 6 phosphorylation and EoE-dysregulated gene expression. CONCLUSIONS: Estrogen-responsive genes are modified in patients with EoE compared with control subjects. E2 attenuated IL-13-induced architectural changes and esophageal epithelial barrier dysfunction through inhibition of the IL-13/tyrosine kinase 2/signal transducer and activator of transcription 6 pathway via ESR2-dependent process. Estrogen hormone signaling may protect against development of EoE in female subjects.


Assuntos
Esofagite Eosinofílica/tratamento farmacológico , Esôfago/imunologia , Estradiol/uso terapêutico , Mucosa Intestinal/fisiologia , Queratinócitos/fisiologia , Fatores Sexuais , Adolescente , Adulto , Células Cultivadas , Criança , Pré-Escolar , Esofagite Eosinofílica/epidemiologia , Esôfago/efeitos dos fármacos , Feminino , Humanos , Incidência , Interleucina-13/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Masculino , Cultura Primária de Células , Receptores Estrogênicos/metabolismo , Fator de Transcrição STAT6/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , TYK2 Quinase/metabolismo , Adulto Jovem
15.
Cytokine ; 111: 434-444, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29934048

RESUMO

Thermogenic fat is a promising target for new therapies in diabetes and obesity. Understanding how thermogenic fat develops is important to develop rational strategies to treat obesity. Previously, we have shown that Tyk2 and STAT3, part of the JAK-STAT pathway, are necessary for proper development of classical brown fat. Using primary preadipocytes isolated from newborn mice we demonstrate that STAT3 is required for differentiation and robust expression of Uncoupling Protein 1 (UCP1). We also confirm that STAT3 is necessary during the early induction stage of differentiation and is dispensable during the later terminal differentiation stage. The inability of STAT3-/- preadipocytes to differentiate can be rescued using Wnt ligand secretion inhibitors when applied during the induction stage. Through chemical inhibition and RNAi, we show that it is the canonical ß-catenin pathway that is responsible for the block in differentiation; inhibition or knockdown of ß-catenin can fully rescue adipogenesis and UCP1 expression in the STAT3-/- adipocytes. During the induction stage, Wnts 1, 3a, and 10b have increased expression in the STAT3-/- adipocytes, potentially explaining the increased levels and activity of ß-catenin. Our results for the first time point towards an interaction between the JAK/STAT pathway and the Wnt/ß-catenin pathway during the early stages of in-vitro adipogenesis.


Assuntos
Adipogenia/fisiologia , Tecido Adiposo Marrom/metabolismo , Fator Regulador Miogênico 5/metabolismo , Fator de Transcrição STAT3/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Adipócitos/metabolismo , Animais , Diferenciação Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , TYK2 Quinase/metabolismo , Proteína Desacopladora 1/metabolismo
16.
Methods Mol Biol ; 1794: 269-278, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29855964

RESUMO

KISS (KInase Substrate Sensor) is a recently developed two-hybrid technology that allows in situ analysis of protein-protein interactions in intact mammalian cells. In this method, which is derived from MAPPIT (mammalian protein-protein interaction trap), the bait protein is coupled to the kinase domain of TYK2, while the prey protein is fused to a fragment of the gp130 cytokine receptor chain. Bait and prey interaction leads to phosphorylation of the gp130 anchor by TYK2, followed by recruitment and activation of STAT3, resulting in transcription of a STAT3-dependent reporter system. This approach enables the identification of interactions between proteins, including transmembrane and cytosolic proteins, and their modulation in response to physiological or pharmacological challenges. Here, we describe a detailed step-by-step protocol for the detection of an interaction between two proteins of interest using KISS.


Assuntos
Receptor gp130 de Citocina/metabolismo , Mapeamento de Interação de Proteínas/métodos , Fator de Transcrição STAT3/metabolismo , TYK2 Quinase/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Transdução de Sinais
17.
Crit Care ; 22(1): 44, 2018 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-29477144

RESUMO

Pulmonary inflammation and vascular leakage are hallmarks of acute respiratory distress syndrome (ARDS), a life-threatening condition, for which there is no specific pharmacologic treatment.Recent literature suggests that leaky vessels in pulmonary infection and ARDS may be mediated through dysregulation of a non-redundant endothelial control pathway, the Tie2 receptor and its ligands, the angiopoietins.This Viewpoint summarizes results from cell-based experiments, animal models and clinical studies underlining the potential of Tie2 targeted interventions in reducing infection-mediated pulmonary hyperpermeability.


Assuntos
Angiopoietina-2/análise , Angiopoietina-2/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , /tratamento farmacológico , Angiopoietina-2/sangue , Biomarcadores/análise , Biomarcadores/sangue , Técnicas de Apoio para a Decisão , Humanos , Receptor TIE-2/efeitos dos fármacos , Receptor TIE-2/metabolismo , Sepse/tratamento farmacológico , Sepse/prevenção & controle , TYK2 Quinase/metabolismo
18.
Sci Rep ; 7(1): 15919, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29162862

RESUMO

STAT3 is a pleiotropic transcription factor involved in homeostatic and host defense processes in the human body. It is activated by numerous cytokines and growth factors and generates a series of cellular effects. Of the STAT-mediated signal transduction pathways, STAT3 transcriptional control is best understood. Jak kinase dependent activation of STAT3 relies on Y705 phosphorylation triggering a conformational switch that is stabilized by intermolecular interactions between SH2 domains and the pY705 motif. We here show that a second tyrosine phosphorylation within the SH2 domain at position Y640, induced by Tyk2, negatively controls STAT3 activity. The Y640F mutation leads to stabilization of activated STAT3 homodimers, accelerated nuclear translocation and superior transcriptional activity following IL-6 and LIF stimulation. Moreover, it unlocks type I IFN-dependent STAT3 signalling in cells that are normally refractory to STAT3 transcriptional activation.


Assuntos
Fosfotirosina/metabolismo , Fator de Transcrição STAT3/metabolismo , TYK2 Quinase/metabolismo , Transcrição Genética , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Interferon-alfa/farmacologia , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , TYK2 Quinase/química , Transcrição Genética/efeitos dos fármacos
19.
Hum Mol Genet ; 26(21): 4301-4313, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28973304

RESUMO

Psoriasis is a common inflammatory skin disorder for which multiple genetic susceptibility loci have been identified, but few resolved to specific functional variants. In this study, we sought to identify common and rare psoriasis-associated gene-centric variation. Using exome arrays we genotyped four independent cohorts, totalling 11 861 psoriasis cases and 28 610 controls, aggregating the dataset through statistical meta-analysis. Single variant analysis detected a previously unreported risk locus at TNFSF15 (rs6478108; P = 1.50 × 10-8, OR = 1.10), and association of common protein-altering variants at 11 loci previously implicated in psoriasis susceptibility. We validate previous reports of protective low-frequency protein-altering variants within IFIH1 (encoding an innate antiviral receptor) and TYK2 (encoding a Janus kinase), in each case establishing a further series of protective rare variants (minor allele frequency < 0.01) via gene-wide aggregation testing (IFIH1: pburden = 2.53 × 10-7, OR = 0.707; TYK2: pburden = 6.17 × 10-4, OR = 0.744). Both genes play significant roles in type I interferon (IFN) production and signalling. Several of the protective rare and low-frequency variants in IFIH1 and TYK2 disrupt conserved protein domains, highlighting potential mechanisms through which their effect may be exerted.


Assuntos
Psoríase/genética , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Alelos , Estudos de Casos e Controles , Estudos de Coortes , Exoma , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Masculino , Polimorfismo de Nucleotídeo Único/genética , Psoríase/fisiopatologia , Fatores de Risco , TYK2 Quinase/genética , TYK2 Quinase/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Sequenciamento Completo do Exoma
20.
J Phys Chem B ; 121(34): 8142-8148, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28759991

RESUMO

We present here the use of QM/MM LIE (linear interaction energy) based binding free energy calculations that greatly improve the precision and accuracy of predicting experimental binding affinities, in comparison to most current binding free energy methodologies, while maintaining reasonable computational times. Calculations are done for four sets of ligand-protein complexes, chosen on the basis of diversity of protein types and availability of experimental data, totaling 140 ligands binding to therapeutic protein targets BACE1, TYK2, HSP90, and PERK. This method allows calculations for a diverse set of ligands and multiple protein targets without the need for parametrization or extra calculations. The accuracy achieved with this method can be used to guide small molecule computational drug design programs.


Assuntos
Secretases da Proteína Precursora do Amiloide/química , Proteínas de Choque Térmico HSP90/química , Ligantes , TYK2 Quinase/química , eIF-2 Quinase/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Sítios de Ligação , Desenho de Fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Modelos Moleculares , Ligação Proteica , Teoria Quântica , TYK2 Quinase/metabolismo , Termodinâmica , eIF-2 Quinase/metabolismo
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