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1.
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
2.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200497

RESUMO

Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFß and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFß/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFß/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFß/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Insuficiência Cardíaca/patologia , Miofibroblastos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Estudos de Casos e Controles , Células Cultivadas , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Masculino , Miofibroblastos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Ativação Transcricional
3.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200573

RESUMO

In recent years, cyclic guanosine 3',5'-cyclic monophosphate (cGMP) and guanylyl cyclases (GCs), which catalyze the formation of cGMP, were implicated in a growing number of plant processes, including plant growth and development and the responses to various stresses. To identify novel GCs in plants, an amino acid sequence of a catalytic motif with a conserved core was designed through bioinformatic analysis. In this report, we describe the performed analyses and consider the changes caused by the introduced modification within the GC catalytic motif, which eventually led to the description of a plasma membrane receptor of peptide signaling molecules-BdPepR2 in Brachypodium distachyon. Both in vitro GC activity studies and structural and docking analyses demonstrated that the protein could act as a GC and contains a highly conserved 14-aa GC catalytic center. However, we observed that in the case of BdPepR2, this catalytic center is altered where a methionine instead of the conserved lysine or arginine residues at position 14 of the motif, conferring higher catalytic activity than arginine and alanine, as confirmed through mutagenesis studies. This leads us to propose the expansion of the GC motif to cater for the identification of GCs in monocots. Additionally, we show that BdPepR2 also has in vitro kinase activity, which is modulated by cGMP.


Assuntos
Brachypodium/enzimologia , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Mutação , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Guanilato Ciclase/química , Guanilato Ciclase/genética , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica , Conformação Proteica , Homologia de Sequência , Transdução de Sinais
4.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201004

RESUMO

Cadmium is a carcinogen that can induce ER stress, DNA damage, oxidative stress and cell death. The yeast mitogen-activated protein kinase (MAPK) signalling pathways paly crucial roles in response to various stresses. Here, we demonstrate that the unfolded protein response (UPR) pathway, the high osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway are all essential for yeast cells to defend against the cadmium-induced toxicity, including the elevated ROS and cell death levels induced by cadmium. We show that the UPR pathway is required for the cadmium-induced phosphorylation of HOG_MAPK Hog1 but not for CWI_MAPK Slt2, while Slt2 but not Hog1 is required for the activation of the UPR pathway through the transcription factors of Swi6 and Rlm1. Moreover, deletion of HAC1 and IRE1 could promote the nuclear accumulation of Hog1, and increase the cytosolic and bud neck localisation of Slt2, indicating crucial roles of Hog1 and Slt2 in regulating the cellular process in the absence of UPR pathway. Altogether, our findings highlight the significance of these two MAPK pathways of HOG and CWI and their interrelationship with the UPR pathway in responding to cadmium-induced toxicity in budding yeast.


Assuntos
Cádmio/toxicidade , Parede Celular/química , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glicerol/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Parede Celular/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Concentração Osmolar , Fosforilação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais
5.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34201918

RESUMO

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) plays a critical role in the pathogenesis of inflammatory bowel diseases (IBD). Mice lacking PTPN2 in dendritic cells (DCs) develop skin and liver inflammation by the age of 22 weeks due to a generalized loss of tolerance leading to uncontrolled immune responses. The effect of DC-specific PTPN2 loss on intestinal health, however, is unknown. The aim of this study was to investigate the DC-specific role of PTPN2 in the intestine during colitis development. PTPN2fl/flxCD11cCre mice were subjected to acute and chronic DSS colitis as well as T cell transfer colitis. Lamina propria immune cell populations were analyzed using flow cytometry. DC-specific PTPN2 deletion promoted infiltration of B and T lymphocytes, macrophages, and DCs into the lamina propria of unchallenged mice and elevated Th1 abundance during acute DSS colitis, suggesting an important role for PTPN2 in DCs in maintaining intestinal immune cell homeostasis. Surprisingly, those immune cell alterations did not translate into increased colitis susceptibility in acute and chronic DSS-induced colitis or T cell transfer colitis models. However, macrophage depletion by clodronate caused enhanced colitis severity in mice with a DC-specific loss of PTPN2. Loss of PTPN2 in DCs affects the composition of lamina propria lymphocytes, resulting in increased infiltration of innate and adaptive immune cells. However, this did not result in an elevated colitis phenotype, likely because increased infiltration of macrophages in the intestine upon loss of PTPN2 loss in DCs can compensate for the inflammatory effect of PTPN2-deficient DCs.


Assuntos
Colite/etiologia , Colite/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/deficiência , Animais , Colite/patologia , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Fator de Transcrição STAT1/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia
6.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202125

RESUMO

Alzheimer's disease is a neurodegenerative disorder associated with age, and is characterized by pathological markers such as amyloid-beta plaques and neurofibrillary tangles. Symptoms of AD include cognitive impairments, anxiety and depression. It has also been shown that individuals with AD have impaired neurotransmission, which may result from the accumulation of amyloid plaques and neurofibrillary tangles. Preclinical studies showed that melatonin, a monoaminergic neurotransmitter released from the pineal gland, is able to ameliorate AD pathologies and restore cognitive impairments. Theoretically, inhibition of the pathological progression of AD by melatonin treatment should also restore the impaired neurotransmission. This review aims to explore the impact of AD on neurotransmission, and whether and how melatonin can enhance neurotransmission via improving AD pathology.


Assuntos
Doença de Alzheimer/metabolismo , Melatonina/metabolismo , Neurotransmissores/metabolismo , Transmissão Sináptica , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Monoaminas Biogênicas/metabolismo , Suscetibilidade a Doenças , Humanos , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Fosforilação , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Proteínas tau/metabolismo
7.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199748

RESUMO

BACKGROUND: Psoriasis, a chronic inflammatory disease affecting 2-3% of the population, is characterised by epidermal hyperplasia, a sustained pro-inflammatory immune response and is primarily a T-cell driven disease. Previous work determined that Connexin26 is upregulated in psoriatic tissue. This study extends these findings. METHODS: Biopsies spanning psoriatic plaque (PP) and non-involved tissue (PN) were compared to normal controls (NN). RNA was isolated and subject to real-time PCR to determine gene expression profiles, including GJB2/CX26, GJB6/CX30 and GJA1/CX43. Protein expression was assessed by immunohistochemistry. Keratinocytes and fibroblasts were isolated and used in 3D organotypic models. The pro-inflammatory status of fibroblasts and 3D cultures was assessed via ELISA and RnD cytokine arrays in the presence or absence of the connexin channel blocker Gap27. RESULTS: Connexin26 expression is dramatically enhanced at both transcriptional and translational level in PP and PN tissue compared to NN (>100x). In contrast, CX43 gene expression is not affected, but the protein is post-translationally modified and accumulates in psoriatic tissue. Fibroblasts isolated from psoriatic patients had a higher inflammatory index than normal fibroblasts and drove normal keratinocytes to adopt a "psoriatic phenotype" in a 3D-organotypic model. Exposure of normal fibroblasts to the pro-inflammatory mediator peptidoglycan, isolated from Staphylococcus aureus enhanced cytokine release, an event protected by Gap27. CONCLUSION: dysregulation of the connexin26:43 expression profile in psoriatic tissue contributes to an imbalance of cellular events. Inhibition of connexin signalling reduces pro-inflammatory events and may hold therapeutic benefit.


Assuntos
Conexinas/genética , Regulação da Expressão Gênica , Psoríase/genética , Adulto , Idoso , Biópsia , Conexinas/metabolismo , Conexinas/farmacologia , Epiderme/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT , Humanos , Mediadores da Inflamação , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Pessoa de Meia-Idade , Modelos Biológicos , Oligopeptídeos/farmacologia , Peptidoglicano/isolamento & purificação , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Psoríase/patologia , Staphylococcus aureus/fisiologia , Adulto Jovem
8.
Biochemistry (Mosc) ; 86(6): 737-745, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34225596

RESUMO

The review summarizes the results of studies on the cellular and molecular mechanisms mediating the impact of stress on the pathogenesis of neurodegenerative brain pathologies (Alzheimer's disease, Parkinson's disease, etc.) and presents current information on the role of stress in the hyperphosphorylation of tau protein, aggregation of beta-amyloid, and hyperactivation of the hypothalamic-pituitary-adrenal axis involved in the hyperproduction of factors that contribute to the pathogenetic role of stress in neurodegeneration. The data on the participation of microglia in the effects of stress on the pathogenesis of neurodegenerative diseases are presented.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Doenças Neurodegenerativas/metabolismo , Estresse Psicológico , Proteínas tau/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Demência/etiologia , Demência/metabolismo , Demência/fisiopatologia , Humanos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/fisiopatologia , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Fosforilação , Agregação Patológica de Proteínas , Processamento de Proteína Pós-Traducional
9.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199160

RESUMO

Acadesine (ACA), a pharmacological activator of AMP-activated protein kinase (AMPK), showed a promising beneficial effect in a mouse model of colitis, indicating this drug as an alternative tool to manage IBDs. However, ACA displays some pharmacodynamic limitations precluding its therapeutical applications. Our study was aimed at evaluating the in vitro and in vivo effects of FA-5 (a novel direct AMPK activator synthesized in our laboratories) in an experimental model of colitis in rats. A set of experiments evaluated the ability of FA5 to activate AMPK and to compare the efficacy of FA5 with ACA in an experimental model of colitis. The effects of FA-5, ACA, or dexamethasone were tested in rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis to assess systemic and tissue inflammatory parameters. In in vitro experiments, FA5 induced phosphorylation, and thus the activation, of AMPK, contextually to the activation of SIRT-1. In vivo, FA5 counteracted the increase in spleen weight, improved the colon length, ameliorated macroscopic damage score, and reduced TNF and MDA tissue levels in DNBS-treated rats. Of note, FA-5 displayed an increased anti-inflammatory efficacy as compared with ACA. The novel AMPK activator FA-5 displays an improved anti-inflammatory efficacy representing a promising pharmacological tool against bowel inflammation.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Benzofuranos/uso terapêutico , Desenvolvimento de Medicamentos , Ativadores de Enzimas/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Animais , Benzofuranos/farmacologia , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Colo/efeitos dos fármacos , Colo/patologia , Dinitrofluorbenzeno/análogos & derivados , Eletroforese em Gel Bidimensional , Ontologia Genética , Doenças Inflamatórias Intestinais/patologia , Interleucina-10/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
10.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204432

RESUMO

Several transmembrane mucins have demonstrated that they contribute intracellularly to induce fibrotic processes. The extracellular domain of MUC16 is considered as a biomarker for disease progression and death in IPF patients. However, there is no evidence regarding the signalling capabilities of MUC16 that contribute to IPF development. Here, we demonstrate that MUC16 was overexpressed in the lung tissue of IPF patients (n = 20) compared with healthy subjects (n = 17) and localised in fibroblasts and hyperplastic alveolar type II cells. Repression of MUC16 expression by siRNA-MUC16 transfection inhibited the TGF-ß1-induced fibrotic processes such as mesenchymal/ myofibroblast transformations of alveolar type II A549 cells and lung fibroblasts, as well as fibroblast proliferation. SiRNA-MUC16 transfection also decreased the TGF-ß1-induced SMAD3 phosphorylation, thus inhibiting the Smad Binding Element activation. Immunoprecipitation assays and confocal immunofluorescence showed the formation of a protein complex between MUC16/p-SMAD3 in the cell membrane after TGF-ß1 stimulation. This study shows that MUC16 is overexpressed in IPF and collaborates with the TGF-ß1 canonical pathway to induce fibrotic processes. Therefore, direct or indirect targeting of MUC16 could be a potential drug target for human IPF.


Assuntos
Antígeno Ca-125/genética , Expressão Gênica , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/metabolismo , Proteínas de Membrana/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Idoso , Biomarcadores , Antígeno Ca-125/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Proliferação de Células , Suscetibilidade a Doenças , Feminino , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/diagnóstico , Imuno-Histoquímica , Pulmão/metabolismo , Pulmão/patologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Miofibroblastos/metabolismo , Fosforilação , Testes de Função Respiratória
11.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204449

RESUMO

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including ß-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR-/- mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR-/- mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.


Assuntos
Proteínas de Arcabouço Homer/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Osteoblastos/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Cálcio/metabolismo , Linhagem da Célula , Sobrevivência Celular , Células Cultivadas , Feminino , Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Detecção de Cálcio/genética
12.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204506

RESUMO

Ergosta-7, 9 (11), 22-trien-3ß-ol (EK100) was isolated from Cordyceps militaris, which has been used as a traditional anti-inflammatory medicine. EK100 has been reported to attenuate inflammatory diseases, but its anti-inflammatory mechanism is still unclear. We were the first to investigate the effect of EK100 on the Toll-like receptor 4 (TLR4)/nuclear factor of the κ light chain enhancer of B cells (NF-κB) signaling in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells and the green fluorescent protein (GFP)-labeled NF-κB reporter gene of Drosophila. EK100 suppressed the release of the cytokine and attenuated the mRNA and protein expression of pro-inflammatory mediators. EK100 inhibited the inhibitor kappa B (IκB)/NF-κB signaling pathway. EK100 also inhibited phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (Akt) signal transduction. Moreover, EK100 interfered with LPS docking to the LPS-binding protein (LBP), transferred to the cluster of differentiation 14 (CD14), and bonded to TLR4/myeloid differentiation-2 (MD-2) co-receptors. Compared with the TLR4 antagonist, resatorvid (CLI-095), and dexamethasone (Dexa), EK100 suppressed the TLR4/AKT signaling pathway. In addition, we also confirmed that EK100 attenuated the GFP-labeled NF-κB reporter gene expression in Drosophila. In summary, EK100 might alter LPS docking to LBP, CD14, and TLR4/MD-2 co-receptors, and then it suppresses the TLR4/NF-κB inflammatory pathway in LPS-stimulated RAW264.7 cells and Drosophila.


Assuntos
Anti-Inflamatórios/farmacologia , Drosophila/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , Anti-Inflamatórios/química , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Receptores de Lipopolissacarídeos/química , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Antígeno 96 de Linfócito/química , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Ligação Proteica , Relação Estrutura-Atividade , Receptor 4 Toll-Like/química
13.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203978

RESUMO

Alzheimer's disease (AD) is the major cause of dementia, and affected individuals suffer from severe cognitive, mental, and functional impairment. Histologically, AD brains are basically characterized by the presence of amyloid plaques and neurofibrillary tangles. Previous reports demonstrated that protein kinase CK1δ influences the metabolism of amyloid precursor protein (APP) by inducing the generation of amyloid-ß (Aß), finally contributing to the formation of amyloid plaques and neuronal cell death. We therefore considered CK1δ as a promising therapeutic target and suggested an innovative strategy for the treatment of AD based on peptide therapeutics specifically modulating the interaction between CK1δ and APP. Initially, CK1δ-derived peptides manipulating the interactions between CK1δ and APP695 were identified by interaction and phosphorylation analysis in vitro. Selected peptides subsequently proved their potential to penetrate cells without inducing cytotoxic effects. Finally, for at least two of the tested CK1δ-derived peptides, a reduction in Aß levels and amyloid plaque formation could be successfully demonstrated in a complex cell culture model for AD. Consequently, the presented results provide new insights into the interactions of CK1δ and APP695 while also serving as a promising starting point for further development of novel and highly innovative pharmacological tools for the treatment of AD.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Caseína Quinase Idelta/metabolismo , Peptídeos/metabolismo , Precursor de Proteína beta-Amiloide/química , Caseína Quinase Idelta/química , Morte Celular , Sobrevivência Celular , Humanos , Simulação de Acoplamento Molecular , Neurônios/metabolismo , Fosforilação , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Ligação Proteica
14.
Science ; 373(6550)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210851

RESUMO

Synthetic biological networks comprising fast, reversible reactions could enable engineering of new cellular behaviors that are not possible with slower regulation. Here, we created a bistable toggle switch in Saccharomyces cerevisiae using a cross-repression topology comprising 11 protein-protein phosphorylation elements. The toggle is ultrasensitive, can be induced to switch states in seconds, and exhibits long-term bistability. Motivated by our toggle's architecture and size, we developed a computational framework to search endogenous protein pathways for other large and similar bistable networks. Our framework helped us to identify and experimentally verify five formerly unreported endogenous networks that exhibit bistability. Building synthetic protein-protein networks will enable bioengineers to design fast sensing and processing systems, allow sophisticated regulation of cellular processes, and aid discovery of endogenous networks with particular functions.


Assuntos
Bioengenharia , Mapas de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas de Saccharomyces cerevisiae/genética
16.
Se Pu ; 39(4): 384-390, 2021 Apr 08.
Artigo em Chinês | MEDLINE | ID: mdl-34227758

RESUMO

Protein phosphorylation is an important type of post-translational protein modification. In Western Blot experiment, the assay of phosphoproteins need special phospho antibodies, which are expensive, difficult to preserve, poorly reproducible. To this end, the immobilized metal ion affinity luminescent silica nanoparticles for instead of phospho antibodies were prepared. A layer of polymer was created on the surface of the silica nanoparticles via co-polymerization to protect the nanoparticles and to functionalize them with the immobilized metal ion affinity property to specifically label the phosphorylated proteins in Western Blot assays. The affinity luminescent silica nanoparticles were prepared with the following procedure. First, the sol-gel precursor fluorescein isothiocyanate-3-aminopropyltriethoxysilane (FITC-APTES) with the fluorescent moiety was prepared by modifying APTES with FITC. The luminescent silica nanoparticles (FITC@SiO2) were synthesized using the Stöber synthesis method in a reversed microemulsion. Briefly, 123.2 mL of cyclohexane, 25.6 mL of n-hexanol, and 5.44 mL of deionized water were ultrasonically mixed, and then 28.3 g of Triton X-100 were added and the mixture was magnetically stirred for 15 min to form a clear and transparent microemulsion system. Within 10 min, 0.8 mL of FITC-APTES precursor, 1.6 mL of tetraethoxysilane (TEOS), and 0.96 mL of concentrated ammonia (25%-27%, mass fraction) were added to the microemulsion, and the mixture was stirred at 24 ℃ for 24 h. After the reaction, the microemulsion system was destroyed by adding 200 mL of ethanol. The resulting FITC@SiO2 luminescent silica nanoparticles were centrifuged, and washed three times with ethanol. After dryness, the FITC@SiO2 nanoparticles were modified with methacryloxy-propyltrimethoxysilane (MPS) to introduce the double bonds for further modification. The functional monomer nitrilotriacetic acid (NTA) and glycidyl methacrylate (GMA) were copolymerized on the surface of the nanoparticles to convert FITC@SiO2-MPS to FITC@SiO2-MPS-GMA-NTA. The polymer coating of the silica nanoparticles was not only able to protect the silica from hydrolysis, but also to introduce the functional groups of nitrilotriacetic acid, which can chelate with metal ions. Elemental analysis demonstrated that the NTA groups had been bonded to the surface of the nanoparticles via copolymerization. The polymerization did not affect the morphology and fluorescence properties of the nanoparticles. The FITC@SiO2-MPS-GMA-NTA nanoparticles were activated with three different metal ions Zr4+, Fe3+, and Ti4+, for the enrichment of phosphorylated peptides derived form α-casein tryptic digestion. HPLC-MS analysis indicated that the FITC@SiO2-MPS-GMA-NTA-Ti 4+ nanoparticles are the best for the enrichment of phosphorylated peptides. The FITC@SiO2-MPS-GMA-NTA-Ti4+ nanoparticles were used for labelling the phosphorylated proteins in Western Blot experiment. The electrophoretic band of α-casein could be clearly labeled with the FITC@SiO2-MPS-GMA-NTA-Ti 4+ nanoparticles, while the bovine albumin band could not be labelled. This indicates that the luminescent FITC@SiO2-MPS-GMA-NTA-Ti4+nanoparticles can be used to label the phosphorylated proteins in Western Blot experiments.


Assuntos
Western Blotting , Nanopartículas , Proteínas Proto-Oncogênicas A-raf/química , Dióxido de Silício , Animais , Bovinos , Íons , Fosforilação , Soroalbumina Bovina , Titânio
17.
Nat Commun ; 12(1): 4284, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257281

RESUMO

The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.


Assuntos
Transtorno do Espectro Autista/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transtorno do Espectro Autista/genética , Citosol/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Humanos , Microcefalia/genética , Microcefalia/metabolismo , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética
18.
Nat Commun ; 12(1): 4166, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230483

RESUMO

Classically considered a by-product of anaerobic metabolism, lactate is now viewed as a fundamental fuel for oxidative phosphorylation in mitochondria, and preferred over glucose by many tissues. Lactate is also a signaling molecule of increasing medical relevance. Lactate levels in the blood can increase in both normal and pathophysiological conditions (e.g., hypoxia, physical exercise, or sepsis), however the manner by which these changes are sensed and induce adaptive responses is unknown. Here we show that the carotid body (CB) is essential for lactate homeostasis and that CB glomus cells, the main oxygen sensing arterial chemoreceptors, are also lactate sensors. Lactate is transported into glomus cells, leading to a rapid increase in the cytosolic NADH/NAD+ ratio. This in turn activates membrane cation channels, leading to cell depolarization, action potential firing, and Ca2+ influx. Lactate also decreases intracellular pH and increases mitochondrial reactive oxygen species production, which further activates glomus cells. Lactate and hypoxia, although sensed by separate mechanisms, share the same final signaling pathway and jointly activate glomus cells to potentiate compensatory cardiorespiratory reflexes.


Assuntos
Artérias/metabolismo , Células Quimiorreceptoras/metabolismo , Ácido Láctico/metabolismo , Animais , Corpo Carotídeo/metabolismo , Homeostase , Concentração de Íons de Hidrogênio , Hipóxia/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Reflexo , Transdução de Sinais
19.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208643

RESUMO

Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.


Assuntos
Plaquetas/fisiologia , Sistemas Microeletromecânicos/métodos , Testes de Função Plaquetária/métodos , Algoritmos , Técnicas Biossensoriais , Plaquetas/ultraestrutura , Ensaio de Imunoadsorção Enzimática , Sistemas Microeletromecânicos/instrumentação , Modelos Teóricos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Testes de Função Plaquetária/instrumentação
20.
FASEB J ; 35(8): e21771, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34275172

RESUMO

Impaired mitochondrial fusion, due in part to decreased mitofusin 2 (Mfn2) expression, contributes to unrestricted cell proliferation and apoptosis-resistance in hyperproliferative diseases like pulmonary arterial hypertension (PAH) and non-small cell lung cancer (NSCLC). We hypothesized that Mfn2 levels are reduced due to increased proteasomal degradation of Mfn2 triggered by its phosphorylation at serine 442 (S442) and investigated the potential kinase mediators. Mfn2 expression was decreased and Mfn2 S442 phosphorylation was increased in pulmonary artery smooth muscle cells from PAH patients and in NSCLC cells. Mfn2 phosphorylation was mediated by PINK1 and protein kinase A (PKA), although only PINK1 expression was increased in these diseases. We designed a S442 phosphorylation deficient Mfn2 construct (PD-Mfn2) and a S442 constitutively phosphorylated Mfn2 construct (CP-Mfn2). The effects of these modified Mfn2 constructs on Mfn2 expression and biological function were compared with those of the wildtype Mfn2 construct (WT-Mfn2). WT-Mfn2 increased Mfn2 expression and mitochondrial fusion in both PAH and NSCLC cells resulting in increased apoptosis and decreased cell proliferation. Compared to WT-Mfn2, PD-Mfn2 caused greater Mfn2 expression, suppression of proliferation, apoptosis induction, and cell cycle arrest. Conversely, CP-Mfn2 caused only a small increase in Mfn2 expression and did not restore mitochondrial fusion, inhibit cell proliferation, or induce apoptosis. Silencing PINK1 or PKA, or proteasome blockade using MG132, increased Mfn2 expression, enhanced mitochondrial fusion and induced apoptosis. In a xenotransplantation NSCLC model, PD-Mfn2 gene therapy caused greater tumor regression than did therapy with WT-Mfn2. Mfn2 deficiency in PAH and NSCLC reflects proteasomal degradation triggered by Mfn2-S442 phosphorylation by PINK1 and/or PKA. Inhibiting Mfn2 phosphorylation has potential therapeutic benefit in PAH and lung cancer.


Assuntos
Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Hipertensão Pulmonar/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/metabolismo , Proteólise , Células A549 , Animais , GTP Fosfo-Hidrolases/genética , Humanos , Hipertensão Pulmonar/genética , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas de Neoplasias/genética , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Quinases/genética
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