Identification And Functional Characterization Of Two Autophagic Gene, Barkor And Keap1

Autophagy is a highly conserved and well regulated eukaryotic intracellular degradation pathway. During autophagy a portion of cytoplasm materials such as protein aggregates, damaged organelles, invaded bacterial and virus as well as cellular debris are transported to lysosome for degradation through a unique double membrane vesicle called autophagosome. Due to its varies types of substrates, autophagy contributes to cell survival during starvation, elimination of protein mutants, maintaining cellular homeostasis, autophagy also functions as another type of programmed cell death in certain situation. Autophagy has strong connections with many human diseases such as neuron degeneration diseases, diabetes and cancer et al.The process of autophagy depends on the coordination of a group of proteins. By genetic screening in yeast, more than 30 genes has been identified that function in autophagy. They are named AuTophGy (ATG) genes. And this number is keeping increasing. Among these ATG genes, those which are essential for autophagosome formation are called core molecules. Phosphotidylinositol-3 kinase (PI3K) plays a fundamental role; it's responsible of producing PI3P in the cell and it functions at the early step during autophagosome formation and determines the activities of other ATG genes. PI3K complex is comprised by Vps34 and Vps15 in yeast, they further forms a complex with autophagic protein ATG6. In yeast, PI3K is critical for both autophagy and vacuole protein sorting pathway. Two proteins, Vps38 and ATG 14 interact with ATG6 and determine the function of PI3K in VPS and autophagy respectively.The mammalian homolog of PI3K proteins are hVPS34 and p150, they form a complex called class III PI3K or PI3KC3. PI3KC3 also interacts with the mammalian homolog of ATG6-Beclinl. The PI3KC3-Beclin1 complex functions at both autophagy and endocytosis in mammalian cells. We are proposing three major discoveries in this paper: 1, By protein purification, we identified a novel gene, Barkor (Beclin 1-associated autophagy-related key regulator) and characterized its fuction in autophagy.1.1 By immunoprecipitation and in vitro binding assay, we approved that Barkor directly interacts with Beclin1 and recruits PI3KC3 complex to autophagosome for its function;1.2 Barkor could also regulate PI3KC3 kinase activity;1.3 Barkor is a key gene in autophagy pathway, inhibiting the expression level of Barkor by shRNA blocked autophagy, while overexpression Barkor elevated autophagy activity.2, By analyzing protein sequence and structure of Barkor, we found a novel functional domain, BATS (Barkor Autophagosome Targeting Sequence), which is the first autophagosome specific binding protein sequence.2.1 After testing the autophagosome targeting ability of differet Barkor fragments, we eventually approved BATS is comprised with the very C terminal 80 amino acids of Barkor;2.2 By structure analysis, we found BATS contains an amphiphathic helix. BATS directly interacts with autophagosome membrane through its amphiphathic helix. Mutant with three hydrophobic amino acids or lacking extreme C terminal 10 amino acids lost their ability to interact with autophagosome;2.3 By in vitro liposome binding assay, we approved BATS directly binds with membrane.3, By purifying LC3 protein complex, we found another autophagic gene-Keapl.3.1 By immune co-precipitation we showed Keap1 stays in same complex with LC3 and p62, the interaction between Keapl and p62 is induced by oxdative stress;3.2 The expression level of Keapl during autophagy indicated that Keapl is not a substrate of autophagy;3.3 The accumulation of ubiquitinated protein aggregates in Keapl knock out cells showed Keapl regulates autophagic clearance of ubiquitinated proteins; 3.4 The difference in resisting ubiquitinated protein aggregates between Keap 1+/+ and Keap 1-/-indicated Keap1 is important in cellular defense to ubiquitinated protein aggregates.