RESUMO
Autophagy is a cellular process whereby cytoplasmic substrates are targeted for degradation in the lysosome via the membrane structures autophagosomes. This process is initiated by specific phosphoinositides, PtdIns3P and PtdIns5P, which play a key role in autophagy by recruiting effectors such as Atg18/WIPI2. Therefore, quantifying those lipids is important to better understand the assembly of the complex autophagic machinery. Herein, we describe in detail methods to quantify PtdIns3P and PtdIns5P by specific mass assays feasible in most laboratories.
Assuntos
Autofagia , Biologia Molecular/métodos , Fosfatos de Fosfatidilinositol/análise , Animais , Autofagia/fisiologia , Autorradiografia/métodos , Lipídeos/isolamento & purificação , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
We previously described a new form of recessive ataxia, Salih ataxia, in a large consanguineous Saudi Arabian family with three affected children carrying a new identified mutation in the KIAA0226 gene (c.2624delC; p.Ala875ValfsX146) coding for Rubicon. The pathogenicity of such mutation remains to be identified. Hence, we address the cellular impact of Rubicon p.Ala875ValfsX146 on endosomal/lysosomal machinery on cultured cells. We confirm that Rubicon colocalizes with the late endosome marker Rab7 and demonstrate that it also colocalizes with LampI at lysosomes. The Salih ataxia mutation leads to a diffuse cytosolic distribution and mislocalized protein from the late endosomes, indicating that deletion of the diacylglycerol binding-like motif in the mutant protein interferes with normal Rubicon subcellular localization and confirming the pathogenicity of the mutation.
Assuntos
Ataxia/genética , Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação/genética , Animais , Proteínas Relacionadas à Autofagia , Células Cultivadas , Chlorocebus aethiops , Diglicerídeos/metabolismo , Expressão Gênica/genética , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , Pele/citologia , Transfecção , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
In the peripheral nervous system, utrophin and the short dystrophin isoform (Dp116) are co-localized at the outermost layer of the myelin sheath of nerve fibers; together with the dystroglycan complex. Dp116 is associated with multiple glycoproteins, i.e. sarcoglycans, and alpha- and beta-dystroglycan, which anchor the cytoplasmic protein subcomplex to the extracellular basal lamina. In peripheral nerve, matrix metalloproteinase activity disrupts the dystroglycan complex by cleaving the extracellular domain of beta-dystroglycan. Metalloproteinase creates a 30 kDa fragment of beta-dystroglycan, leading to a disruption of the link between the extracellular matrix and the cell membrane. Here we asked if the processing of the beta-dystroglycan could influence the anchorage of Dp116 and/or utrophin in normal and mdx Schwann cell membrane. We showed that metalloproteinase-9 was more activated in mdx nerve than in wild-type ones. This activation leads to an accumulation of the 30 kDa beta-dystroglycan isoform and has an impact on the anchorage of Dp116 and utrophin isoforms in mdx Schwann cells membrane. Our results showed that Dp116 had greater affinity to the full length form of beta-dystroglycan than the 30 kDa form. Moreover, we showed for the first time that the short isoform of utrophin (Up71) was over-expressed in mdx Schwann cells compared with wild-type. In addition, this utrophin isoform (Up71) seems to have greater affinity to the 30 kDa beta-dystroglycan which could explain the increased stabilization of this 30 kDa form at the membrane compartment. Our results highlight the potential participation of the short utrophin isoform and the cleaved form of beta-dystroglycan in mdx Schwann cell membrane architecture. We proposed that these two proteins could be implicated in Schwann cell proliferation in response to a microenvironment stress such as mediated by accumulating macrophages in mdx mouse muscle inflammation sites.
