Navegando por Palavras-chave "NAADP"
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- ItemSomente MetadadadosEstudo da autofagia mediada por TFEB: desvendando um papel para os receptores Two-Pore Channels(Universidade Federal de São Paulo (UNIFESP), 2020-12-18) Guarache, Gabriel Cicolin [UNIFESP]; Pereira, Gustavo Jose Da Silva [UNIFESP]; Universidade Federal de São PauloAutophagy is a lysosomal catabolic process that degrades and recycles intracellular components in response to cellular stress. One of important regulator of this process is the transcription factor EB (TFEB), which is translocated to the nucleus through the lysosomal Ca2+ release and further activates the transcription machinery related to lysosomal biogenesis and autophagy genes. Recent studies have demonstrated that the Ca2+ signals from the endolysosomal system mediated by the newly second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) via Two-Pore Channels (TPCs) activation induces autophagy. Since TFEB activation and consequent autophagy induction by TPCs signaling are dependent on lysosomal Ca2+, this present study investigated the possible participation of the TFEB on TPCs-mediated autophagy in HeLa cell line. For this purpose, firstly, was verified that autophagy was induced by NAADP-AM [100 nM], a membrane permeant analog of NAADP, accompanied by decrease in TFEB cytosolic levels during 4 hours. In HeLaGFP-TFEB cells, NAADP-AM [100 nM] for 1 hour induced TFEB nuclear translocation, whereas Ned-19 [10 μM], a NAADP non-competitive antagonist, inhibited autophagy, increased TFEB cytosolic levels and decreased nuclear/cytosolic TFEB ratio. To evaluate the possible participation of TPCs on TFEB translocation, cells overexpressing Myc-TPC1 or Myc-TPC2 were treated with NAADP-AM [100 nM] for 1 hour. As result, TPC1-overexpressing cells treated with NAADP-AM [100 nM] as well as the TPC2-overexpressing cells were able to translocate TFEB to the nucleus. Furthermore, TFEB translocation induced by starvation was potentiated by TPCs overexpression, especially in TPC2-overexpressing cells. Taken together, these results indicated that TFEB subcellular localization can be modulated by Ca2+/TPCs cell signaling in response to NAADP and starvation. The comprehension of the underlying mechanisms associated to TFEB-mediated autophagy is important for potential therapeutic targets in several diseases.
- ItemAcesso aberto (Open Access)Estudo da sinalização de Ca2+ na modulação e agregação da proteína huntingtina mutante em modelo celular da doença de Huntington(Universidade Federal de São Paulo, 2022-05-23) Pereira, Cássia Arruda Souza [UNIFESP]; Smaili, Soraya Soubhi [UNIFESP]; Pereira, Gustavo José da Silva [UNIFESP]; Erustes, Adolfo Garcia [UNIFESP]; http://lattes.cnpq.br/7125107888186769; http://lattes.cnpq.br/4111992747501625; http://lattes.cnpq.br/6368730022418127; http://lattes.cnpq.br/9272838242229426A doença de Huntington (DH) é uma doença neurodegenerativa causada por uma mutação no gene responsável pela codificação da proteína huntingtina (Htt), que resulta na formação da huntingtina mutante (mHtt). Essa proteína mutante possui um ganho de função tóxica, visto que seus fragmentos podem formar agregados proteicos. A autofagia consiste em um mecanismo de degradação intracelular, que pode ter um efeito neuroprotetor em doenças neurodegenerativas, inclusive na DH. E como o NAADP (ácido nicotínico adenina dinucleotídeo fosfato), que é um potente mobilizador de Ca2+ de organelas ácidas, se mostrou capaz de induzir a autofagia, estudos têm relacionado o seu envolvimento com a autofagia disfuncional em doenças neurodegenerativas, porém pouco se sabe sobre o papel dessa via de sinalização na DH. Portanto, o objetivo desse trabalho foi investigar o efeito da sinalização Ca2+ mediada pelo NAADP nos níveis da mHtt, na formação de agregados proteicos e na modulação da autofagia, em um modelo celular da DH. Nossos resultados mostraram que o NAADP-AM promoveu um aumento do número de agregados de mHtt e que esse efeito foi dependente de Ca2+, visto que o tratamento com BAPTA-AM (quelante de Ca2+) ou Ned-19 (antagonista dos receptores de NAADP) reverteu esse efeito. Estudos de localização intracelular, mostraram que a mHtt apresenta colocalização com os receptores do NAADP (TPC1 e TPC2), indicando uma possível interação entre essas proteínas. Nos estudos de modulação da autofagia, o NAADP-AM não alterou a expressão de proteínas marcadoras autofágicas (LC3-II e p62), o que indica que o NAADP não é capaz modula a autofagia em células contendo a mHtt. Em conjunto, nossos resultados indicam que há um envolvimento da sinalização de Ca2+ mediada pelo NAADP na agregação da mHtt, que pode estar relacionada com uma possível interação da mHtt com os receptores do NAADP. A ausência da estimulação autofágica pelo NAADP também pode contribuir para uma maior agregação da mHtt. Logo, a desregulação dessa via pode contribuir com o aumento da toxicidade da mHtt e consequentemente para a neurodegeneração.
- ItemAcesso aberto (Open Access)Glutamate induces autophagy via the two-pore channels in neural cells(Impact Journals Llc, 2017) Pereira, Gustavo José da Silva [UNIFESP]; Antonioli, Manuela; Hirata, Hanako [UNIFESP]; Ureshino, Rodrigo Portes [UNIFESP]; Nascimento, Aline Rosa [UNIFESP]; Bincoletto, Claudia [UNIFESP]; Vescovo, Tiziana; Piacentini, Mauro; Fimia, Gian Maria; Smaili, Soraya Soubhi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)NAADP (nicotinic acid adenine dinucleotide phosphate) has been proposed as a second messenger for glutamate in neuronal and glial cells via the activation of the lysosomal Ca2+ channels TPC1 and TPC2. However, the activities of glutamate that are mediated by NAADP remain unclear. In this study, we evaluated the effect of glutamate on autophagy in astrocytes at physiological, non-toxic concentration. We found that glutamate induces autophagy at similar extent as NAADP. By contrast, the NAADP antagonist NED-19 or SiRNA-mediated inhibition of TPC1/2 decreases autophagy induced by glutamate, confirming a role for NAADP in this pathway. The involvement of TPC1/2 in glutamate-induced autophagy was also confirmed in SHSY5Y neuroblastoma cells. Finally, we show that glutamate leads to a NAADP-dependent activation of AMPK, which is required for autophagy induction, while mTOR activity is not affected by this treatment. Taken together, our results indicate that glutamate stimulates autophagy via NAADP/TPC/AMPK axis, providing new insights of how Ca2+ signalling glutamate-mediated can control the cell metabolism in the central nervous system.
- ItemSomente MetadadadosNAADP-sensitive two-pore channels are present and functional in gastric smooth muscle cells(Churchill Livingstone, 2014-08-01) Pereira, Gustavo José da Silva [UNIFESP]; Hirata, Hanako [UNIFESP]; Garcez-do-Carmo, Lucia [UNIFESP]; Stilhano, Roberta Sessa [UNIFESP]; Ureshino, Rodrigo Portes [UNIFESP]; Medaglia, Natalia de Castro [UNIFESP]; Han, Sang Won [UNIFESP]; Churchill, Grant; Bincoletto, Claudia [UNIFESP]; Patel, Sandip; Smaili, Soraya Soubhi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Oxford; UCLNicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as an important modulator of Ca2+ release from the endo-lysosomal system in a variety of cells by a new and ubiquitous class of endo-lysosomal ion channels known as the two-pore channels (TPCs). However, the role of TPCs in NAADP action in smooth muscle is not known. in the present work, we investigated the effects of NAADP in gastric smooth muscle cells and its ability to release Ca2+ by TPCs. We show that Ca2+ signals mediated by NAADP were inhibited by disrupting Ca2+ handling by either acidic organelles (using bafilomycin A1) or the Endoplasmic Reticulum (using thapsigargin, ryanodine or 2-APB). Transcripts for endogenous TPC1 and TPC2 were readily detected and recombinant TPCs localized to the endosomes and/or lysosomes. Overexpression of wild-type TPCs but not pore mutants enhanced NAADP-mediated cytosolic Ca2+ signals. Desensitizing the NAADP pathway inhibited Ca2+-responses to extracellular stimulation with carbachol but not ATP. Taken together, these results indicate that NAADP likely induces Ca2+ release from the endolysosomal system through TPCs which is subsequently amplified via the ER in an agonist-specific manner. Thus, we suggest a second messenger role for NAADP in smooth muscle cells. (C) 2014 Elsevier B.V. All rights reserved.