Journal of Biotechnology & Biomaterials
Open Access

Abstract

Autophagy is an important catabolic program to response a variety of cellular stresses by forming a double membrane vesicle, autophagosome. Autophagy plays key roles in various cellular functions. Accordingly, dysregulation of autophagy is closely associated with diseases such as diabetes, neurodegenerative diseases, cardiomyopathy and cancer. In this sense, autophagy is emerging as an important therapeutic target for disease control. Among the autophagy machineries, PIK3C3/VPS34 complex functions as autophagy-triggering kinase to recruit the subsequent autophagy protein machineries on phagophore membrane. Accumulating evidence showing that inhibition of PIK3C3/VPS34 complex successfully inhibits autophagy makes the complex as an attractive target for developing autophagy inhibitors. However, one concern in PIK3C3/VPS34 complex is that many different PIK3C3/VPS34 complexes have distinct cellular functions. In this study, we have developed in vitro PIK3C3/VPS34 complex monitoring assay for autophagy inhibitor screening in a high-throughput assay format instead of targeting the catalytic activity of PIK3C3/VPS34 complex which shuts down all PIK3C3/VPS34 complexes. We performed in vitro reconstitution of an essential autophagy-promoting PIK3C3/VPS34 complex, Vps34-Beclin1-ATG14L complex in the microwell (96 well formats) plate and successfully monitored the complex formation in many different conditions. This PIK3C3/VPS34 complex protein assay would provide a reliable tool for the screening of autophagy-specific inhibitors.

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