RESUMO
Endocytic internalization of extracellular proteins plays roles in signaling, nutrient uptake, immunity, and extracellular protein quality control. However, there are few protocols for analyzing the lysosomal degradation of extracellular protein. Here, we purified secreted proteins fused with pH-sensitive GFP and acid- and protease-resistant RFP from mammalian cells and describe an internalization assay for mammalian cells. This protocol enables quantification of cellular uptake and lysosomal degradation of protein-of-interest (POI) via cell biological and biochemical analyses. For full details on the use and execution of this protocol, please refer to Itakura et al. (2020).
Assuntos
Citometria de Fluxo/métodos , Immunoblotting/métodos , Lisossomos , Proteínas , Anticorpos Monoclonais , Endocitose/fisiologia , Células HEK293 , Humanos , Proteínas Luminescentes , Lisossomos/química , Lisossomos/metabolismo , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Proteínas RecombinantesRESUMO
The accumulation of aberrant proteins leads to various neurodegenerative disorders. Mammalian cells contain several intracellular protein degradation systems, including autophagy and proteasomal systems, that selectively remove aberrant intracellular proteins. Although mammals contain not only intracellular but also extracellular proteins, the mechanism underlying the quality control of aberrant extracellular proteins is poorly understood. Here, using a novel quantitative fluorescence assay and genome-wide CRISPR screening, we identified the receptor-mediated degradation pathway by which misfolded extracellular proteins are selectively captured by the extracellular chaperone Clusterin and undergo endocytosis via the cell surface heparan sulfate (HS) receptor. Biochemical analyses revealed that positively charged residues on Clusterin electrostatically interact with negatively charged HS. Furthermore, the Clusterin-HS pathway facilitates the degradation of amyloid ß peptide and diverse leaked cytosolic proteins in extracellular space. Our results identify a novel protein quality control system for preserving extracellular proteostasis and highlight its role in preventing diseases associated with aberrant extracellular proteins.