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1.
Mol Cell ; 84(1): 17-19, 2024 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-38181757

RESUMEN

Ebner et al.1 discovered a nutrient-dependent molecular feedback circuit that employs mTORC1, lipid kinases, and phosphatases to generate phosphatidylinositol-3-phosphate [PI(3)P] or phosphatidylinositol-4-phosphate [PI(4)P] in a mutually exclusive manner on lysosomes, which respectively convert lysosomes into organelles that support anabolism or catabolism.


Asunto(s)
Crisis de Identidad , Fosfatidilinositoles , Lisosomas , Diana Mecanicista del Complejo 1 de la Rapamicina/genética
2.
Proc Natl Acad Sci U S A ; 121(23): e2217971121, 2024 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-38805272

RESUMEN

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.


Asunto(s)
Fusión Celular , Mioblastos , Fosfatos de Fosfatidilinositol , Podosomas , Animales , Fosfatos de Fosfatidilinositol/metabolismo , Ratones , Mioblastos/metabolismo , Mioblastos/citología , Podosomas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Desarrollo de Músculos/fisiología
3.
Acta Neuropathol ; 144(3): 537-563, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35844027

RESUMEN

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention.


Asunto(s)
Miopatías Estructurales Congénitas , Pez Cebra , Animales , Modelos Animales de Enfermedad , Epigénesis Genética , Ratones , Músculo Esquelético/metabolismo , Miopatías Estructurales Congénitas/tratamiento farmacológico , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Ácido Valproico/metabolismo , Ácido Valproico/farmacología , Pez Cebra/metabolismo
4.
Methods Mol Biol ; 2692: 121-137, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37365465

RESUMEN

Cells such as macrophages and neutrophils can internalize a diverse set of particulate matter, illustrated by bacteria and apoptotic bodies through the process of phagocytosis. These particles are sequestered into phagosomes, which then fuse with early and late endosomes and ultimately with lysosomes to mature into phagolysosomes, through a process known as phagosome maturation. Ultimately, after particle degradation, phagosomes then fragment to reform lysosomes through phagosome resolution. As phagosomes change, they acquire and divest proteins that are associated with the various stages of phagosome maturation and resolution. These changes can be assessed at the single-phagosome level by using immunofluorescence methods. Typically, we use indirect immunofluorescence methods that rely on primary antibodies against specific molecular markers that track phagosome maturation. Commonly, progression of phagosomes into phagolysosomes can be determined by staining cells for Lysosomal-Associated Membrane Protein I (LAMP1) and measuring the fluorescence intensity of LAMP1 around each phagosome by microscopy or flow cytometry. However, this method can be used to detect any molecular marker for which there are compatible antibodies for immunofluorescence.


Asunto(s)
Fagocitosis , Fagosomas , Fagosomas/metabolismo , Macrófagos/metabolismo , Lisosomas/metabolismo , Técnica del Anticuerpo Fluorescente , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo
5.
Methods Mol Biol ; 2251: 177-184, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33481239

RESUMEN

Following their generation by lipid kinases and phosphatases, phosphoinositides regulate important biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, and gene expression through the interaction of their phosphorylated inositol head group with a variety of protein domains such as PH, PX, and FYVE. Therefore, it is important to determine the specificity of phosphoinositides toward effector proteins to understand their impact on cellular physiology. Several methods have been developed to identify and characterize phosphoinositide effectors, and liposomes-based methods are preferred because the phosphoinositides are incorporated in a membrane, the composition of which can mimic cellular membranes. In this report, we describe the experimental setup for liposome flotation assay and a recently developed method called protein-lipid interaction by fluorescence (PLIF) for the characterization of phosphoinositide-binding specificities of proteins.


Asunto(s)
Liposomas/análisis , Fosfatidilinositoles/análisis , Mapeo de Interacción de Proteínas/métodos , Membrana Celular/metabolismo , Humanos , Liposomas/metabolismo , Fosfatidilinositoles/metabolismo , Fosforilación , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Proteínas/química , Transducción de Señal/fisiología
6.
Curr Protoc Protein Sci ; 89: 19.31.1-19.31.10, 2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28762494

RESUMEN

Phosphoinositides are key signaling and regulatory phospholipids that mediate important pathophysiological processes. This is achieved through the interaction of their phosphorylated inositol head group with a wide range of protein domains. Therefore, being able to determine the phosphoinositide specificity for effector protein is essential to the understanding of its cellular function. This unit describes a novel method named Protein-Lipid Interaction by Fluorescence, or PLIF. PLIF is a fast, reliable and high throughput assay that allows determination of the phosphoinositide specificity of proteins, simultaneously providing relative affinities. In addition, PLIF is suitable for screening inhibitors of protein- phosphoinositide interaction, allowing identification of potential pharmacological compounds. © 2017 by John Wiley & Sons, Inc.


Asunto(s)
Fosfatidilinositoles/química , Proteínas/química , Liposomas , Fosforilación , Unión Proteica , Espectrometría de Fluorescencia
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