Elucidating the function of mammalian autophagy receptors in selective autophagy

阐明哺乳动物自噬受体在选择性自噬中的功能

• 批准号: 10091469
• 负责人: Christopher J Shoemaker
• 金额: $ 24.9万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091469
• 关键词: Advisory Committees; Affect; Aging; Alleles; Autophagocytosis; Award; Biochemical; Biological Assay; CRISPR interference; CRISPR screen; CRISPR/Cas technology; Cell Survival; Cells; Complex; Cues; Data; Disease; Encapsulated; Faculty; Goals; Grant; Health; Human; Institution; Leadership; Lysosomes; Malignant Neoplasms; Mammalian Cell; Mammals; Mass Spectrum Analysis; Mediating; Membrane; Mentors; Methods; Molecular; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Organelles; Pathway interactions; Phase; Phosphorylation; Phosphotransferases; Process; Protein Kinase; Publishing; Receptor Activation; Regulation; Reporter; Research; Role; Starvation; Stress; System; Technical Expertise; Therapeutic; Training Support; Vesicle; Work; Writing; Yeasts; career; chemical genetics; frontotemporal lobar dementia-amyotrophic lateral sclerosis; human disease; man; novel; programs; protein aggregation; protein degradation; public health relevance; receptor; receptor function; recruit; response; screening; skills; tenure track; therapeutic candidate; therapeutic target; tissue culture; tool

Project Summary / Abstract The long-term goal of this project is to elucidate the molecular mechanism by which autophagy receptors effect selective autophagy in both normal and disease conditions. Autophagy is a process of non-selective cytoplasmic degradation that is required for cell survival during periods of starvation or stress (termed bulk autophagy), as well as for the selective degradation of protein aggregates, damaged organelles, and other large targets that otherwise accumulate and cause disease (selective autophagy). In nearly all cases, the induction of autophagy requires the activation of a dedicated kinase, Atg1, that is conserved from yeast (Atg1) to man (ULK1 and ULK2). Neither how ULK1/2 are activated, nor how phosphorylation of ULK1/2 substrates enables autophagy induction is well understood. The goal of this proposal is to take an autophagy-receptor- centric approach to understanding selective autophagy in mammalian systems. Specifically, in Aim 1, I will biochemically characterize the role of autophagy receptors in ULK1/2 activation. I will then determine the effect of receptor mutations associated with human disease. In Aim 2, I will use CRISPR/Cas9 technology to screen for novel modulators of selective autophagy in mammalian cells, including screens for suppressors of disease- associated alleles. These studies will identify potential therapeutic targets for autophagy-associated diseases. In Aim 3, I will use a novel cell free assay for ULK1/2 activity to identify substrates of these autophagy kinases and determine the mechanism by which ULK1/2 activation by receptors drives selective autophagy. Collectively, my combined approaches will reveal conserved principles of autophagy induction and, ultimately, inform our understanding of autophagy dysregulation in various disease states. During the early stage of this award, I will gain valuable technical skills, including in mammalian tissue culture, CRISPR screening and mass spectrometry, that will enable me to develop a unique research program. Under the mentoring of my formal advisory committee, I will develop important soft skills, such as grant writing, presentation skills and lab leadership. This combination of training, support and career mentoring will be instrumental in my transition to independence as a tenure-track faculty at a leading institution.

项目摘要/摘要 这个项目的长期目标是阐明自噬受体作用的分子机制。 在正常和疾病条件下的选择性自噬。自噬是一个非选择性的过程 在饥饿或应激期间细胞生存所需的细胞质降解(称为块状 自噬)，以及选择性降解蛋白质聚集体、受损细胞器和其他 以其他方式积累并导致疾病的大靶点(选择性自噬)。在几乎所有的情况下， 自噬的诱导需要从酵母中保守的一种专用的激酶Atg1的激活(Atg1)。 对人(ULK1和ULK2)。无论ULK1/2是如何激活的，也不是ULK1/2底物的磷酸化 使自噬能够诱导是众所周知的。这项提议的目标是利用自噬受体-- 了解哺乳动物系统中选择性自噬的中心方法。具体地说，在目标1中，我将 生化表征自噬受体在ULK1/2激活中的作用。然后我会确定效果 与人类疾病相关的受体突变。在Aim 2中，我将使用CRISPR/Cas9技术进行筛选 对于哺乳动物细胞中选择性自噬的新调节器，包括疾病抑制因子的筛选- 关联的等位基因。这些研究将确定自噬相关疾病的潜在治疗靶点。 在目标3中，我将使用一种新的无细胞检测ULK1/2活性的方法来鉴定这些自噬蛋白激酶的底物 并确定受体激活ULK1/2驱动选择性自噬的机制。 总的来说，我的组合方法将揭示自噬诱导的保守原则，并最终， 告诉我们对不同疾病状态下自噬失调的理解。在这个早期阶段， 获奖后，我将获得宝贵的技术技能，包括哺乳动物组织培养、CRISPR筛查和质量 这将使我能够制定一个独特的研究计划。在我毕业典礼的指导下 作为顾问委员会，我将发展重要的软技能，如拨款撰写、演示技能和实验室 领导力。这种培训、支持和职业指导的结合将有助于我过渡到 作为一家领先机构的终身教职员工，独立性。