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

总体目标：本研究项目将对自噬导致细胞存活和细胞生长的调控提供新的见解。背景：自噬是一种“自我消化”过程，其特征是双膜结构，自噬蛋白LC3-I在自噬体上转化为脂化的LC3-II形式。自噬体随后与溶酶体融合，产生维持细胞止血的营养物质。自噬是在饥饿、代谢应激（包括细胞增殖）、缺氧和氧化应激（如运动和海拔变化）等生理应激引起的过程中诱导的。根据不同的情况，自噬可以促进细胞存活、细胞死亡和/或细胞生长。这在人类细胞中是如何被调节的还不是很清楚。近年来，LC3结合蛋白在调节自噬功能中的重要作用已被描述。我们发现Bcl-2 BH3家族成员BNIP3在缺氧条件下上调，并通过与LC3的结合参与自噬。此外，我们发现一个新的基因TSSC4与LC3结合，阻止自噬。综上所述，了解LC3结合蛋白如何在LC3结合蛋白的背景下调节自噬，将有助于深入了解自噬介导的细胞存活和细胞生长。我们的短期目标是评估LC3结合蛋白如何调节自噬（Aim 1），以及生长因子如何通过LC3结合蛋白调节自噬（Aim 2）。我的长期目标是了解这两种LC3结合蛋白如何调节自噬途径，并发现这两种LC3结合蛋白调节细胞稳态的新的下游靶点。目的1：了解LC3结合蛋白如何调控自噬。在这个目的中，我们将确定在正常生长或生理应激条件下，细胞中TSSC4或BNIP3的过表达或低表达是否会改变自噬。我们将进一步确定缺乏LC3结合区的TSSC4或BNIP3是否会阻止自噬。最后，我们将确定LC3与TSSC4或BNIP3的结合是否会影响正常或应激条件下的细胞存活和细胞生长。所有的实验将由一名硕士生进行。目的2：确定生长因子是否通过TSSC4和BNIP3调控自噬。我们将确定在正常或应激条件下细胞自噬的生长因子调节。TSSC4或BNIP3与LC3的结合量也将在生长因子刺激后确定。最后，TSSC4或BNIP3在LIF中过表达、敲低或突变，确定生长因子处理对自噬、细胞存活和生长的影响。所有实验将由博士生进行。影响：这项基础研究将通过了解自噬在正常细胞稳态中所起的作用来提高我们的知识，并使细胞生物学研究人员受益。

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.