Role of LC3 binding proteins in regulating autophagy mediated cell survival and death.

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Overall objective: This research program will give novel insight into the regulation of autophagy leading to cell survival and cell growth. Background: Autophagy is a "self-digestion" process that is characterized by a double-membrane structure and conversion of autophagy protein LC3-I to a lipidated LC3-II form at the autophagosome. The autophagosome will then fuse with the lysosome to generate nutrients to maintain cellular hemostasis. Autophagy is induced during starvation, metabolic stress involving cell proliferation, hypoxia, and oxidative stress caused by physiological stresses such as exercise and changes in elevation. Depending on the context, autophagy could contribute to both cell survival, cell death and/or cell growth. How this is regulated in human cells is not well understood. Recently, the importance of LC3 binding protein in regulating autophagy function has been described. We found that Bcl-2 BH3 only family member BNIP3 is upregulated under hypoxia and contributes to autophagy through its binding to LC3. In addition, we discovered that a novel gene TSSC4 binds with LC3 preventing autophagy. Taken together, an understanding of how LC3 binding proteins regulates autophagy in the context of LC3 binding proteins will give insight into autophagy mediated cell survival and cell growth. Proposal Objective: Our short-term objective is to evaluate how LC3 binding proteins regulate autophagy (Aim 1) and how growth factors can regulate autophagy through these LC3 binding proteins (Aim 2). My long term goal is to understanding how these two LC3 binding proteins regulate the autophagy pathway and discover novel downstream targets for these two LC3 binding proteins that regulate cellular homeostasis. Aim 1: To determine how LC3 binding proteins regulation of autophagy. In this aim, we will determine whether autophagy is altered by TSSC4 or BNIP3 over expression or knockdown in cells under normal growth, or physiological stress conditions. We will further determine whether TSSC4 or BNIP3 lacking the LC3 binding region will prevent autophagy. Finally we will determine whether binding of LC3 to TSSC4 or BNIP3 affects cell survival and cell growth under normal or stress conditions. All experiments will be conducted by an M.Sc. student. Aim 2: To determine whether growth factors regulate autophagy through TSSC4 and BNIP3. We will determine the growth factor regulation of autophagy in cells under normal or stress conditions. The amount of binding of TSSC4 or BNIP3 to LC3 will be also determined after growth factor stimulation. Finally, TSSC4 or BNIP3 will be over-expressed, knockdown or mutated in the LIF and the effect of growth factor treatment on autophagy, cell survival and growth will be determined. All experiments will be conducted by a Ph.D. student. Impact: This fundamental research will improve our knowledge and benefit cell biology researchers by understanding the role autophagy plays in normal cellular homeostasis.