RESUMO
A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.
Assuntos
Complexo CD3/metabolismo , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Complexo CD3/química , Proteína Tirosina Quinase CSK/metabolismo , Linhagem Celular , Citocinas/metabolismo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Sobrevida , Vanadatos/farmacologiaRESUMO
Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.
Assuntos
Matriz Extracelular , Adesões Focais , Quinases da Família src , Adesões Focais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Quinases da Família src/metabolismo , Quinases da Família src/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Transdução de Sinais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Metaloproteinases da Matriz/metabolismoRESUMO
Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.
Assuntos
Reabsorção Óssea , Podossomos , Humanos , Osteoclastos/metabolismo , Podossomos/metabolismo , Actinas/metabolismo , Diferenciação Celular/fisiologia , Reabsorção Óssea/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Colágeno/metabolismo , Oligopeptídeos/metabolismoRESUMO
The function of Sprouty2 (Spry2) in T cells is unknown. Using 2 different (inducible and T cell-targeted) knockout mouse strains, we found that Spry2 positively regulated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling by modulating the activity of LCK. Spry2-/- CD4+ T cells were unable to activate LCK, proliferate, differentiate into T helper cells, or produce cytokines. Spry2 deficiency abrogated type 2 inflammation and airway hyperreactivity in a murine model of asthma. Spry2 expression was higher in blood and airway CD4+ T cells from patients with asthma, and Spry2 knockdown impaired human T cell proliferation and cytokine production. Spry2 deficiency up-regulated the lipid raft protein caveolin-1, enhanced its interaction with CSK, and increased CSK interaction with LCK, culminating in augmented inhibitory phosphorylation of LCK. Knockdown of CSK or dislodgment of caveolin-1-bound CSK restored ERK1/2 activation in Spry2-/- T cells, suggesting an essential role for Spry2 in LCK activation and T cell function.
Assuntos
Asma/fisiopatologia , Proteína Tirosina Quinase CSK/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas de Membrana/metabolismo , Adulto , Animais , Asma/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologiaRESUMO
In this work, readily achievable synthetic pathways were utilized for construction of a library of N/S analogues based on the pyrazolopyrimidine scaffold with terminal alkyl or aryl fragments. Subsequently, we evaluated the anticancer effects of these novel analogs against the proliferation of various cancer cell lines, including breast, colon, and liver lines. The results were striking, most of the tested molecules exhibited strong and selective cytotoxic activity against the MDA-MB-231 cancer cell line; IC50 1.13 µM. Structure-activity relationship (SAR) analysis revealed that N-substituted derivatives generally enhanced the cytotoxic effect, particularly with aliphatic side chains that facilitated favorable target interactions. We also investigated apoptosis, DNA fragmentation, invasion assay, and anti-migration effects, and discussed their underlying molecular mechanisms for the most active compound 7c. We demonstrated that 7c N-propyl analogue could inhibit MDA-MB-231 TNBC cell proliferation by inducing apoptosis through the regulation of vital proteins, namely c-Src, p53, and Bax. In addition, our results also revealed the potential of these compounds against tumor metastasis by downregulating the invasion and migration modes. Moreover, the in vitro inhibitory effect of active analogs against c-Src kinase was studied and proved that might be the main cause of their antiproliferative effect. Overall, these compelling results point towards the therapeutic potential of these derivatives, particularly those with N-substitution as promising candidates for the treatment of TNBC type of breast cancer.
Assuntos
Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Proteína Tirosina Quinase CSK/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Quinases da Família src , Relação Estrutura-Atividade , Pirimidinas/química , Pirimidinas/farmacologia , Pirazóis/química , Pirazóis/farmacologiaRESUMO
New chromene derivatives were synthesized based on 4-(3,4-dimethoxy)-4H-chromene scaffold. All target compounds exhibited cytotoxic activity against HepG2 cells (IC50 = 2.40-141.22 µM). Chromens 5 and 9 showed superior cytotoxicity over staurosporine (IC50 = 18.27 µM) and vinblastine (IC50 = 5.20 µM). c-Src kinase inhibition assay of compounds 5 and 9 displayed the dominant c-Src inhibitory activity of 5 (IC50 = 0.184 µM) over 9 (IC50 = 0.288 µM). The safety of the most potent compound 5 against normal WI-38 cells was confirmed via its IC50 of 115.75 µM comparable with 5-FU (IC50 = 16.28 µM). Moreover, the promising chromene 5 displayed potent cytotoxicity against resistant HepG2 cells with IC50 of 26.03 µM comparable with 5-FU (IC50 = 42.68 µM). The most active chromene 5 arrested the HepG2 cell cycle at the S phase and induced a 29-fold increase in the total number of apoptotic cells indicating pre-G1 apoptosis. The ability of compound 5 to induce apoptosis was supported via elevation of caspase-3, caspase-7, caspase-9 and proapoptotic Bax protein levels in addition to downregulation of the antiapoptotic Bcl2 protein. Molecular docking studies of compound 5 showed good binding interaction pattern inside c-Src kinase enzyme active site.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Estrutura Molecular , Relação Estrutura-Atividade , Benzopiranos/química , Simulação de Acoplamento Molecular , Proteína Tirosina Quinase CSK/metabolismo , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Hepáticas/tratamento farmacológico , Pontos de Checagem do Ciclo Celular , Antineoplásicos/química , Apoptose , Fluoruracila/farmacologia , Desenho de FármacosRESUMO
Morphine diminishes pain, but its long-term use is compromised by tolerance and hyperalgesia. Studies implicate δ receptors, ß-arrestin2 and Src kinase in tolerance. We examined whether these proteins are also involved in morphine-induced hypersensitivity (MIH). A common pathway for tolerance and hypersensitivity may provide a single target to guide improved analgesic approaches. We examined mechanical sensitivity using automated von Frey in wild-type (WT) and transgenic male and female C57Bl/6 mice before and after hind paw inflammation by complete Freund's adjuvant (CFA). CFA-evoked hypersensitivity ceased on day 7 in WT but persisted for the 15-day testing period in µ-/- . Recovery was delayed until day 13 in δ-/- . We explored the expression of opioid genes in the spinal cord using quantitative RT-PCR. Restoration to basal sensitivity in WT occurred with increased δ expression. By contrast, κ expression was reduced, while µ remained unchanged. Daily morphine reduced hypersensitivity in WT on day 3 compared to controls; however, hypersensitivity recurred on day 9 and beyond. By contrast, WT had no recurrence of hypersensitivity in the absence of daily morphine. We used ß-arrestin2-/- , δ-/- and Src inhibition by dasatinib in WT to establish whether these approaches, which diminish tolerance, also attenuate MIH. While none of these approaches affected CFA-evoked inflammation or acute hypersensitivity, all caused sustained morphine anti-hypersensitivity, abolishing MIH. Like morphine tolerance, MIH in this model requires δ receptors, ß-arrestin2 and Src activity. Our findings suggest that MIH is caused by a tolerance-induced reduction in endogenous opioid signalling. KEY POINTS: Morphine is effective for treating severe acute pain, but tolerance and hypersensitivity often develop during its use in treating chronic pain. It is unclear whether these detrimental effects share similar mechanisms; if so, it might be possible to develop a single approach to minimise both phenomena. Mice deficient in µ receptors, δ receptors or ß-arrestin2 and wild type mice treated with the Src inhibitor dasatinib exhibit negligible morphine tolerance. We show that these same approaches also prevent the development of morphine-induced hypersensitivity during persistent inflammation. This knowledge identifies strategies, such as the use of Src inhibitors, which may mitigate tolerance and morphine induced hyperalgesia.
Assuntos
Hiperalgesia , Morfina , Camundongos , Masculino , Feminino , Animais , Morfina/efeitos adversos , Hiperalgesia/induzido quimicamente , Analgésicos Opioides/efeitos adversos , Receptores Opioides delta/metabolismo , beta-Arrestina 1/metabolismo , Dasatinibe , Dor , Proteína Tirosina Quinase CSK/metabolismo , Receptores Opioides mu/metabolismo , Camundongos Endogâmicos C57BL , InflamaçãoRESUMO
In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Fator de Transcrição AP-1 , Humanos , Proteína Tirosina Quinase CSK/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Inflamação/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resveratrol/farmacologia , Resveratrol/metabolismo , Quinases da Família src/metabolismo , Trombina/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
The Src family kinases (SFKs) Src, Lyn, and Fyn are essential for platelet activation and also involved in megakaryocyte (MK) development and platelet production. Platelet SFKs are inhibited by C-terminal Src kinase (Csk), which phosphorylates a conserved tyrosine in their C-terminal tail, and are activated by the receptor-type tyrosine phosphatase PTPRJ (CD148, DEP-1), which dephosphorylates the same residue. Deletion of Csk and PTPRJ in the MK lineage in mice results in increased SFK activity, but paradoxically hypoactive platelets resulting from negative feedback mechanisms, including upregulation of Csk homologous kinase (Chk) expression. Here, we investigate the role of Chk in platelets, functional redundancy with Csk, and the physiological consequences of ablating Chk, Csk, and PTPRJ in mice. Platelet count was normal in Chk knockout (KO) mice, reduced by 92% in Chk;Csk double KO (DKO) mice, and partially rescued in Chk;Csk;Ptprj triple KO (TKO) mice. Megakaryocyte numbers were significantly increased in both DKO and TKO mice. Phosphorylation of the inhibitory tyrosine of SFKs was almost completely abolished in DKO platelets, which was partially rescued in Src and Fyn in TKO platelets. This residual phosphorylation was abolished by Src inhibitors, revealing an unexpected mechanism in which SFKs autoinhibit their activity by phosphorylating their C-terminal tyrosine residues. We demonstrate that reduced inhibitory phosphorylation of SFKs leads to thrombocytopenia, with Csk being the dominant inhibitor in platelets and Chk having an auxiliary role. PTPRJ deletion in addition to Chk and Csk ameliorates the extent of thrombocytopenia, suggesting targeting it may have therapeutic benefits in such conditions.
Assuntos
Plaquetas/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Animais , Biomarcadores , Tempo de Sangramento , Proteína Tirosina Quinase CSK/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Modelos Biológicos , Fosforilação , Ativação Plaquetária , Contagem de Plaquetas , Testes de Função Plaquetária , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Glioblastomas (GBs) are the most aggressive and common primary malignant brain tumors. Steroid hormone progesterone (P4) and its neuroactive metabolites, such as allopregnanolone (3α-THP) are synthesized by neural, glial, and malignant GB cells. P4 promotes cellular proliferation, migration, and invasion of human GB cells at physiological concentrations. It has been reported that 3α-THP promotes GB cell proliferation. Here we investigated the effects of 3α-THP on GB cell migration and invasion, the participation of the enzymes involved in its metabolism (AKR1C1-4), and the role of the c-Src kinase in 3α-THP effects in GBs. 3α-THP 100 nM promoted migration and invasion of U251, U87, and LN229 human-derived GB cell lines. We observed that U251, LN229, and T98G cell lines exhibited a higher protein content of AKR1C1-4 than normal human astrocytes. AKR1C1-4 silencing did not modify 3α-THP effects on migration and invasion. 3α-THP activated c-Src protein at 10 min (U251 cells) and 15 min (U87 and LN229 cells). Interestingly, the pharmacological inhibition of c-Src decreases the promoting effects of 3α-THP on cell migration and invasion. Together, these data indicate that 3α-THP promotes GB migration and invasion through c-Src activation.
Assuntos
Proteína Tirosina Quinase CSK , Glioblastoma , Pregnanolona , Proteína Tirosina Quinase CSK/metabolismo , Proliferação de Células , Glioblastoma/metabolismo , Humanos , Pregnanolona/metabolismo , Pregnanolona/farmacologia , Proteínas Tirosina QuinasesRESUMO
Multiple observations implicate T-cell dysregulation as a central event in the pathogenesis of rheumatoid arthritis. Here, we investigated mechanisms for suppressing T-cell activation via the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1). To determine how LAIR-1 affects T-cell receptor (TCR) signaling, we compared 1) T cells from LAIR-1-sufficient and -deficient mice, 2) Jurkat cells expressing either LAIR-1 mutants or C-terminal Src kinase (CSK) mutants, and 3) T cells from mice that contain a CSK transgene susceptible to chemical inhibition. Our results indicated that LAIR-1 engagement by collagen or by complement C1q (C1Q, which contains a collagen-like domain) inhibits TCR signaling by decreasing the phosphorylation of key components in the canonical T-cell signaling pathway, including LCK proto-oncogene SRC family tyrosine kinase (LCK), LYN proto-oncogene SRC family tyrosine kinase (LYN), ζ chain of T-cell receptor-associated protein kinase 70 (ZAP-70), and three mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase 1/2, and p38). The intracellular region of LAIR-1 contains two immunoreceptor tyrosine-based inhibition motifs that are both phosphorylated by LAIR-1 activation, and immunoprecipitation experiments revealed that Tyr-251 in LAIR-1 binds CSK. Using CRISPR/Cas9-mediated genome editing, we demonstrate that CSK is essential for the LAIR-1-induced inhibition of the human TCR signal transduction. T cells from mice that expressed a PP1 analog-sensitive form of CSK (CskAS) corroborated these findings, and we also found that Tyr-251 is critical for LAIR-1's inhibitory function. We propose that LAIR-1 activation may be a strategy for controlling inflammation and may offer a potential therapeutic approach for managing autoimmune diseases.
Assuntos
Receptores Imunológicos/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Bovinos , Colágeno Tipo I/metabolismo , Humanos , Células Jurkat , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Fosfotirosina/metabolismo , Proto-Oncogene Mas , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
BACKGROUND: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. METHODS: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. RESULTS: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; P<0.0001). CONCLUSIONS: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/toxicidade , Fibrilação Atrial/induzido quimicamente , Função do Átrio Esquerdo/efeitos dos fármacos , Proteína Tirosina Quinase CSK/antagonistas & inibidores , Átrios do Coração/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Piperidinas/toxicidade , Inibidores de Proteínas Quinases/toxicidade , Potenciais de Ação/efeitos dos fármacos , Adenina/toxicidade , Tirosina Quinase da Agamaglobulinemia/deficiência , Tirosina Quinase da Agamaglobulinemia/genética , Animais , Fibrilação Atrial/enzimologia , Fibrilação Atrial/fisiopatologia , Proteína Tirosina Quinase CSK/genética , Proteína Tirosina Quinase CSK/metabolismo , Bases de Dados Genéticas , Átrios do Coração/enzimologia , Átrios do Coração/fisiopatologia , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Medição de Risco , Fatores de RiscoRESUMO
Lower doses of capsaicin (8-methyl-N-vanillyl-6-nonenamide) have the potential to serve as an anticancer drug, however, due to its pungency, irritant effect, poor water solubility and high distribution volume often linked to various off-target effects, its therapeutic use is limited. This study aimed to determine the biodistribution and anticancer efficacy of capsaicin loaded solid lipid nanoparticles (SLNs) in human hepatocellular carcinoma in vitro. In this study, SLNs of stearic acid loaded with capsaicin was formulated by the solvent evaporation-emulsification technique and were instantly characterized for their encapsulation efficiency, morphology, loading capacity, stability, particle size, charge and in vitro drug release profile. Synthesized SLNs were predominantly spherical, 80 nm diameter particles that proved to be biocompatible with good stability in aqueous conditions. In vivo biodistribution studies of the formulated SLNs showed that 48 h after injection in the lateral tail vein, up to 15% of the cells in the liver, 1.04% of the cells in the spleen, 3.05% of the cells in the kidneys, 3.76% of the cells in the heart, 1.31% of the cells in the lungs and 0% of the cells in the brain of rats were determined. Molecular docking studies against the identified targets in HepG2 cells showed that the capsaicin is able to bind Abelson tyrosine-protein kinase, c-Src kinase, p38 MAP kinase and VEGF-receptor. Molecular dynamic simulation showed that capsaicin-VEGF receptor complex is highly stable at 50 nano seconds. The IC50 of capsaicin loaded SLNs in HepG2 cells in vitro was 21.36 µg × ml-1. These findings suggest that capsaicin loaded SLNs are stable in circulation for a period up to 3 d, providing a controlled release of loaded capsaicin and enhanced anticancer activity.
Assuntos
Antineoplásicos/farmacologia , Capsaicina/farmacologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Proteína Tirosina Quinase CSK/metabolismo , Capsaicina/síntese química , Capsaicina/farmacocinética , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Concentração Inibidora 50 , Lipídeos , Neoplasias Hepáticas/tratamento farmacológico , Modelos Moleculares , Simulação de Dinâmica Molecular , Nanopartículas , Tamanho da Partícula , Proteínas Proto-Oncogênicas c-abl/metabolismo , Ratos , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Solubilidade , Distribuição Tecidual , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Full T cell activation depends on stimulation of the TCR in conjunction with a costimulatory receptor. The involvement of costimulatory molecules is potent, and a mechanistic understanding of how downstream signaling is regulated is required to fully understand T cell responsiveness. In this study, a proteomic approach was taken to identify the interactomes of the coreceptors CD2 and CD28. These coreceptors are both positive regulators of T cell activation, but CD28 less potently induces TCR-proximal signaling. C-terminal Src kinase (CSK), a negative regulator of TCR signaling, was identified as a specific and direct interactor only of activated CD28. CSK is recruited to CD28 upon T cell activation, and the in vitro kinase activity of CSK is enhanced in the presence of phosphorylated CD28. Interruption of the CSK/CD28 interaction prior to TCR/CD28 costimulation induces a signaling response which mimics the more potent CD2-induced TCR-proximal pathway activation. Thus, CD28 functions as a novel adaptor protein for CSK, and CSK regulates signaling downstream of CD28.
Assuntos
Antígenos CD28/imunologia , Proteína Tirosina Quinase CSK/imunologia , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Antígenos CD28/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Células Cultivadas , Humanos , Linfócitos T/metabolismoRESUMO
Angelica sinensis (AS; Dang Gui), a traditional Chinese herb, has for centuries been used for the treatment of bone diseases, including osteoporosis and osteonecrosis. However, the effective ingredient and underlying mechanisms remain elusive. Here, we identified guaiacol as the active component of AS by two-dimensional cell membrane chromatography/C18 column/time-of-flight mass spectrometry (2D CMC/C18 column/TOFMS). Guaiacol suppressed osteoclastogenesis and osteoclast function in bone marrow monocytes (BMMCs) and RAW264.7 cells in vitro in a dose-dependent manner. Co-immunoprecipitation indicated that guaiacol blocked RANK-TRAF6 association and RANK-C-Src association. Moreover, guaiacol prevented phosphorylation of p65, p50, IκB (NF-κB pathway), ERK, JNK, c-fos, p38 (MAPK pathway) and Akt (AKT pathway), and reduced the expression levels of Cathepsin K, CTR, MMP-9 and TRAP. Guaiacol also suppressed the expression of nuclear factor of activated T-cells cytoplasmic 1(NFATc1) and the RANKL-induced Ca2+ oscillation. In vivo, it ameliorated ovariectomy-induced bone loss by suppressing excessive osteoclastogenesis. Taken together, our findings suggest that guaiacol inhibits RANKL-induced osteoclastogenesis by blocking the interactions of RANK with TRAF6 and C-Src, and by suppressing the NF-κB, MAPK and AKT signalling pathways. Therefore, this compound shows therapeutic potential for osteoclastogenesis-related bone diseases, including postmenopausal osteoporosis.
Assuntos
Proteína Tirosina Quinase CSK/metabolismo , Guaiacol/farmacologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Adipogenia , Animais , Células da Medula Óssea/citologia , Reabsorção Óssea , Proliferação de Células , Feminino , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , Microtomografia por Raio-XRESUMO
MicroRNAs (miRNAs) fine-tune cellular signaling by regulating expression of signaling proteins, and aberrant expression of miRNAs is observed in many cancers. The tyrosine kinase c-Src is upregulated in various human cancers, but the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. In previous investigations of miRNA-mediated control of c-Src-related oncogenic pathways, we identified miRNAs that were downregulated in association with c-Src transformation and uncovered the signaling networks by predicting their target genes, which might act cooperatively to control tumor progression. Here, to further elucidate the process of cell transformation driven by c-Src, we analyzed the expression profiles of miRNAs in a doxycycline-inducible Src expression system. We found that miRNA (miR)-129-1-3p was downregulated in the early phase of c-Src-induced cell transformation, and that reexpression of miR-129-1-3p disrupted c-Src-induced cell transformation. In addition, miR-129-1-3p downregulation was tightly associated with tumor progression in human colon cancer cells/tissues. Expression of miR-129-1-3p in human colon cancer cells caused morphological changes and suppressed tumor growth, cell adhesion, and invasion. We also identified c-Src and its critical substrate Fer, and c-Yes, a member of the Src family of kinases, as novel targets of miR-129-1-3p. Furthermore, we found that miR-129-1-3p-mediated regulation of c-Src/Fer and c-Yes is important for controlling cell adhesion and invasion. Downregulation of miR-129-1-3p by early activation of c-Src increases expression of these target genes and synergistically promotes c-Src-related oncogenic signaling. Thus, c-Src-miR-129-1-3p circuits serve as critical triggers for tumor progression in many human cancers that harbor upregulation of c-Src.
Assuntos
Proteína Tirosina Quinase CSK/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/metabolismo , Regulação para Baixo , MicroRNAs/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Animais , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Neoplasias do Colo/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Camundongos , Transplante de NeoplasiasRESUMO
Upon detection of viral DNA, the cytoplasmic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) utilizes GTP and ATP as substrates to synthesize the second messenger molecule 2'3'cyclic GMP-AMP (cGAMP), which binds to the ER-associated adaptor protein MITA/STING to signal innate antiviral response to DNA virus. How the cGAS-MITA pathways are post-translationally regulated is not fully understood. In this study, we identified the tyrosine kinase CSK as a positive regulator of cGAS-MITA mediated innate antiviral response. CSK-deficiency inhibits DNA virus-triggered induction of downstream antiviral effector genes. Following DNA virus infection, CSK phosphorylates MITA at Y240 and Y245, which is important for its activation. These results suggest that CSK plays a role in modulating innate immune response to DNA virus.
Assuntos
Proteína Tirosina Quinase CSK/metabolismo , Vírus de DNA/imunologia , Imunidade Inata , Proteínas de Membrana/metabolismo , Animais , Proteína Tirosina Quinase CSK/deficiência , Linhagem Celular , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , FosforilaçãoRESUMO
Loss of E-cadherin elicits epithelial-mesenchymal transition (EMT). While both the Src family of membrane-associated non-receptor tyrosine kinases (SFKs) and Slit2 binding to Roundabout 1 (Robo1) have been shown to induce E-cadherin repression and EMT, whether these two signaling pathways are mechanistically coupled remains unknown in epithelial cells. Here we found that Slit2 and Robo1 overexpression activated Src kinases for tyrosine phosphorylation, degradation of E-cadherin and induction of EMT. Specific blockade of Slit2 binding to Robo1 inactivated Src, prevented E-cadherin phosphorylation and EMT induction. Biochemically, the cytoplasmic CC3 motif of Robo1 (CC3) bound directly to the SH2 and 3 domains of c-Src and the cytoplasmic domains of E-cadherin. Slit2 induced Robo1 association with endogenous c-Src and E-cadherin, whereas ectopic expression of CC3 dissociated this protein complex in colorectal epithelial cells. These results indicate that Slit2 not only induces Robo1 binding to Src, but also recruits Src to E-cadherin for tyrosine phosphorylation of E-cadherin, leading to E-cadherin degradation and EMT induction in colorectal epithelial cells.
Assuntos
Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Humanos , Fosforilação , Mapas de Interação de Proteínas , Proteínas RoundaboutRESUMO
Cell adhesion molecules act as tumor suppressors primarily by cell attachment activity, but additional mechanisms modifying signal transduction are suggested in some cases. Cell adhesion molecule 1 (CADM1), a membrane-spanning immunoglobulin superfamily, mediates intercellular adhesion by trans-homophilic interaction and acts as a tumor suppressor. Here, we investigated CADM1-associated proteins comprehensively using proteomic analysis of immune-precipitates of CADM1 by mass spectrometry and identified a transmembrane adaptor protein, Csk-binding protein (Cbp), known to suppress Src-mediated transformation, as a binding partner of CADM1. CADM1 localizes to detergent-resistant membrane fractions and co-immunoprecipitated with Cbp and c-Src. Suppression of CADM1 expression using siRNA reduces the amount of co-immunoprecipitated c-Src with Cbp and activates c-Src in colon cancer cells expressing both CADM1 and Cbp. On the other hand, co-replacement of CADM1 and Cbp in colon cancer cells lacking CADM1 and Cbp expression suppresses c-Src activation, wound healing and tumorigenicity in nude mice. Furthermore, expression of Cbp and CADM1 was lost in 55% and 83% of human colon cancer, respectively, preferentially in tumors with larger size and/or lymph node metastasis. CADM1 would act as a colon tumor suppressor by intervening oncogenic c-Src signaling through binding with Cbp besides its authentic cell adhesion activity.
Assuntos
Proteína Tirosina Quinase CSK/metabolismo , Carcinogênese/metabolismo , Molécula 1 de Adesão Celular/metabolismo , Neoplasias do Colo/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Animais , Ativação Enzimática , Feminino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Metal binding to sites engineered in proteins can provide an increase in their stability and facilitate new functions. Besides the sites introduced in purpose, sometimes they are present accidentally as a consequence of the expression system used to produce the protein. This happens with the copper- and nickel-binding (ATCUN) motif generated by the amino-terminal residues Gly-Ser-His. This ATCUN motif is fortuitously present in many proteins, but how it affects the structural and biophysical characterization of the proteins has not been studied. In this work, we have compared the structure and biophysical properties of a small modular domain, the SH3 domain of the c-Src tyrosine kinase, cloned with and without an ATCUN motif at the N terminus. At pH 7.0, the SH3 domain with the ATCUN motif binds nickel with a binding constant Ka = 28.0 ± 3.0 mM-1. The formation of the nickel complex increases the thermal and chemical stability of the SH3 domain. A comparison of the crystal structures of the SH3 domain with and without the ATCUN motif shows that the binding of nickel does not affect the overall structure of the SH3 domain. In all crystal structures analyzed, residues Gly-Ser-His in complex with Ni2+ show a square planar geometry. The CD visible spectrum of the nickel complex shows that this geometry is also present in the solution. Therefore, our results not only show that the ATCUN motif might influence the biophysical properties of the protein, but also points to an advantageous stabilization of the protein with potential biotechnological applications.