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

LC3 结合蛋白在调节自噬介导的细胞存活和死亡中的作用。

• 批准号: RGPIN-2021-02905
• 负责人: Gibson, Spencer
• 金额: $ 3.06万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744193
• 关键词: Role; LC3; binding; proteins; regulating

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.

总体目标：这项研究计划将提供新的见解调控自噬导致细胞存活和细胞生长。背景资料：自噬是一种“自我消化”过程，其特征在于自噬蛋白LC 3-I在自噬体处的双膜结构和转化为脂化LC 3-II形式。然后自噬体将与溶酶体融合以产生维持细胞止血的营养物。自噬在饥饿、涉及细胞增殖的代谢应激、缺氧和由生理应激如运动和海拔变化引起的氧化应激期间诱导。根据上下文，自噬可能有助于细胞存活、细胞死亡和/或细胞生长。这在人类细胞中是如何调节的还不清楚。最近，LC 3结合蛋白在调节自噬功能中的重要性已被描述。我们发现Bcl-2 BH 3家族成员BNIP 3在缺氧条件下上调，并通过与LC 3结合促进自噬。此外，我们发现一个新的基因TSSC 4与LC 3结合，阻止自噬。总而言之，了解LC 3结合蛋白如何在LC 3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。提案目标：我们的短期目标是评估LC 3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LC 3结合蛋白调节自噬（目标2）。我的长期目标是了解这两种LC 3结合蛋白如何调节自噬途径，并发现这两种LC 3结合蛋白调节细胞稳态的新下游靶点。 目的1：研究LC 3结合蛋白对自噬的调控作用。 在这个目标中，我们将确定在正常生长或生理应激条件下，细胞中TSSC 4或BNIP 3的过表达或敲低是否会改变自噬。我们将进一步确定缺乏LC 3结合区的TSSC 4或BNIP 3是否会阻止自噬。最后，我们将确定LC 3与TSSC 4或BNIP 3的结合是否影响正常或应激条件下的细胞存活和细胞生长。所有实验将由一名硕士生进行。 目的2：确定生长因子是否通过TSSC 4和BNIP 3调节自噬。我们将确定在正常或应激条件下细胞中自噬的生长因子调节。TSSC 4或BNIP 3与LC 3的结合量也将在生长因子刺激后测定。最后，TSSC 4或BNIP 3将在LIF中过表达、敲低或突变，并且将确定生长因子处理对自噬、细胞存活和生长的影响。所有实验将由博士进行。学生.影响力：这项基础研究将通过了解自噬在正常细胞稳态中的作用来提高我们的知识并使细胞生物学研究人员受益。