RUI: Tools and Approaches for Investigating the Basic Mechanisms of Autophagy

RUI：研究自噬基本机制的工具和方法

• 批准号: 2243163
• 负责人: Steven Backues
• 金额: $ 41.44万
• 依托单位: Eastern Michigan University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-15 至 2026-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2243163&HistoricalAwards=false
• 关键词: RUI; Tools; Approaches; Investigating; Basic

This research will advance our basic understanding of autophagy, a cellular clean-up and recycling system that helps to protect cells of many organisms – including humans – from stresses such as starvation, pathogens, and the accumulation of cellular debris. Results of this research could lead to improvements in crop yields as well as animal and human health. In addition to its scientific goals, this project will help create a diverse and technically literate workforce by providing a research-rich learning environment at Eastern Michigan University, a primarily undergraduate institution that serves a racially and socioeconomically diverse population of students. Twelve undergraduate and two master’s students will be supported to perform cutting-edge biochemical research for 1-2 years each. In addition, the project will help support semester-long, guided research experiences for ~24 undergraduate students taking a research-based senior-level biochemistry laboratory course. These two activities will provide students with the practical and intellectual skills needed to become the next generation of science, technology, and engineering professionals. This project uses yeast genetics to advance our understanding of the formation of the autophagosome, a double-membraned vesicle that envelops cellular cargo and delivers it to the vacuole (or, in metazoans, the lysosome) for degradation. Although previous research in Saccharomyces cerevisiae has established the proteins necessary for this process, much remains to be learned about how they function to carry it out. One central player is the Atg8 conjugation system, which attaches the small ubiquitin-like protein Atg8 to the lipid phosphatidylethanolamine, thus defining the forming autophagic membrane. Previous research has shown that the amount of Atg8 in a cell determines how large autophagosomes become, but not the number created. However, Atg7, which catalyzes the first step in the pathway leading to Atg8 conjugation, determines both the size and the number of autophagosomes. This project will investigate additional proteins in this pathway to resolve this conundrum and determine which branch of the pathway affects autophagosome number. The project will also investigate the molecular details of Atg11, a protein that organizes autophagosome formation around autophagic cargo by binding to and arranging core autophagy proteins. Mutational analysis of a region of Atg11 that preliminary data has shown to be crucial for binding to one of its key partners will demonstrate which specific residues are essential for this interaction. Finally, an improved mathematical simulation to allow more accurate estimation autophagosome size and number from transmission electron microscopy sections will be developed.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

这项研究将推进我们对自噬的基本理解，自噬是一种细胞清理和回收系统，有助于保护包括人类在内的许多生物体的细胞免受饥饿，病原体和细胞碎片积累等压力的影响。这项研究的结果可能会导致作物产量以及动物和人类健康的改善。除了其科学目标，该项目将通过在东密歇根大学提供一个研究丰富的学习环境，帮助创建一个多元化和技术素养的劳动力，东密歇根大学是一个主要的本科院校，为种族和社会经济多样化的学生群体提供服务。将支持1 - 2名本科生和2名硕士生进行1-2年的前沿生物化学研究。此外，该项目将帮助支持长达一个学期的指导研究经验，为24名本科生采取了以研究为基础的高级生物化学实验室课程。这两项活动将为学生提供成为下一代科学，技术和工程专业人士所需的实践和智力技能。该项目使用酵母遗传学来推进我们对自噬体形成的理解，自噬体是一种双膜囊泡，它包裹细胞货物并将其运送到液泡（或后生动物中的溶酶体）进行降解。虽然先前在酿酒酵母中的研究已经确定了这一过程所必需的蛋白质，但关于它们如何发挥作用来实现这一过程，还有很多东西有待了解。其中一个重要的参与者是Atg 8缀合系统，它将小的泛素样蛋白Atg 8连接到脂质磷脂酰乙醇胺上，从而形成自噬膜。先前的研究表明，细胞中Atg 8的数量决定了自噬体的大小，但不是产生的数量。然而，催化导致Atg 8缀合的途径中的第一步的Atg 7决定了自噬体的大小和数量。这个项目将研究在这个途径中的其他蛋白质来解决这个难题，并确定该途径的哪个分支影响自噬体的数量。该项目还将研究Atg 11的分子细节，Atg 11是一种通过结合和排列核心自噬蛋白来组织自噬体形成的蛋白质。初步数据显示，Atg 11的一个区域对于与其关键伙伴之一的结合至关重要，该区域的突变分析将证明哪些特定残基对这种相互作用至关重要。最后，一个改进的数学模拟，使更准确地估计自噬体的大小和数量从透射电子显微镜切片将developed.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。