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

总体目标：这项研究计划将对自噬调节导致细胞存活和细胞生长提供新的见解。背景：自噬是一种“自我消化”的过程，其特征是自噬蛋白Lc3-I在自噬小体上转化为脂化的Lc3-II形式。然后，自噬小体将与溶酶体融合，产生营养物质，以维持细胞止血。自噬是在饥饿、代谢应激、细胞增殖、缺氧和生理应激如运动和海拔变化引起的氧化应激过程中诱导的。根据上下文的不同，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控的还不是很清楚。最近，Lc3结合蛋白在调节自噬功能中的重要性已被描述。我们发现，只有BH3家族成员BNIP3在低氧条件下表达上调，并通过与LC3结合促进自噬。此外，我们还发现了一个新的基因TSSC4与LC3结合以防止自噬。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生存和细胞生长。提案目的：我们的短期目标是评估LC3结合蛋白如何调节自噬(目标1)以及生长因子如何通过这些LC3结合蛋白调节自噬(目标2)。我的长期目标是了解这两个Lc3结合蛋白是如何调节自噬途径的，并发现这两个Lc3结合蛋白调节细胞内稳态的新的下游靶点。目的1：研究LC3结合蛋白对自噬的调节作用。为此，我们将确定在正常生长或生理应激条件下，细胞中TSSC4或BNIP3的过度表达或敲除是否会改变自噬。我们将进一步确定缺少LC3结合区的TSSC4或BNIP3是否可以防止自噬。最后，我们将确定在正常或应激条件下，LC3与TSSC4或BNIP3的结合是否会影响细胞的存活和生长。所有实验都将由M.Sc进行。学生。目的：确定生长因子是否通过TSSC4和BNIP3调节自噬。我们将在正常或应激条件下确定生长因子对细胞自噬的调节。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将在LIF中过表达、敲除或突变，生长因子处理对自噬、细胞存活和生长的影响将被确定。所有实验将由一名博士生进行。影响：这项基础研究将提高我们的知识，并通过了解自噬在正常细胞稳态中所起的作用，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.