title: Development of in vitro PIK3C3/VPS34 complex protein assay for autophagy-specific inhibitor screening.
abstract: Autophagy is an important catabolic program to respond to 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 an autophagy-triggering kinase to recruit the subsequent autophagy protein machineries on the phagophore membrane. Accumulating evidence showing that inhibition of PIK3C3/VPS34 complex successfully inhibits autophagy makes the complex an attractive target for developing autophagy inhibitors. However, one concern about PIK3C3/VPS34 complex is that many different PIK3C3/VPS34 complexes have distinct cellular functions. In this study, we have developed an in vitro PIK3C3/VPS34 complex monitoring assay for autophagy inhibitor screening in a high-throughput assay format instead of targeting the catalytic activity of the 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 a microwell plate (96-well format) 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.
authors: Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 130-701, Republic of Korea.
authors: Tae-Mi
authors: TM
authors: Kim
authors: Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 130-701, Republic of Korea.
authors: Jong-Hyuk
authors: JH
authors: Baek
authors: Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 130-701, Republic of Korea; Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Kyung Hee University, Seoul 130-701, Republic of Korea.
authors: Jeong Hee
authors: JH
authors: Kim
authors: Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 130-701, Republic of Korea; Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea. Electronic address: msohok@khu.ac.kr.
authors: Myung Sook
authors: MS
authors: Oh
authors: Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Kyung Hee University, Seoul 130-701, Republic of Korea. Electronic address: jok011@khu.ac.kr.
authors: Joungmok
authors: J
authors: Kim
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D002478:Cells, Cultured; D058546:Class III Phosphatidylinositol 3-Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D047428:Protein Kinase Inhibitors; D058546:Class III Phosphatidylinositol 3-Kinases
keywords: ATG14L; Autophagy-specific inhibitor screening method; In vitro PIK3C3/VPS34 complex protein assay