RESUMO
This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.
Assuntos
Escherichia coli/genética , Microscopia Eletrônica/métodos , Coloração Negativa/métodos , Proteína Sequestossoma-1/ultraestrutura , Autofagia , Cromatografia de Afinidade/métodos , Expressão Gênica , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/isolamento & purificação , Solubilidade , Regulação para CimaRESUMO
Autophagy is a catabolic process to maintain intracellular homeostasis that degrades damaged proteins and organelles in mammalian cells. Podocytes are crucial for maintaining the normal function of the glomerular filtration barrier. In the present study, we aimed to investigate the high glucose-induced cell injury in human podocytes and the protective role of autophagy in this process. Here we show that the autophagy activity was decreased under the high glucose conditions and 72 h of high glucose exposure inhibited the cell viablity and aggravated cell injury. Moreover, autophagy upregulation by Sequestosome 1 (p62/SQSM1) knockdown ameliorated this cell injury and relieved insulin resistance. Collectively, the present study proposed a novel autophagy involved mechanism of high glucose-induced cell injury. The present study deepens our understanding of the role of autophagy in the pathogenesis of diabetic nephropathy and provided potential therapeutic strategy for diabetic nephropathy.
