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

这项研究将促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有机体的细胞免受饥饿、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导致作物产量以及动物和人类健康的改善。除了它的科学目标，这个项目还将通过在东密歇根大学提供一个研究丰富的学习环境，帮助创造一支多元化的、具有技术素养的劳动力队伍。东密歇根大学是一所以本科生为主的机构，服务于不同种族和社会经济背景的学生群体。将资助12名本科生和2名硕士研究生各进行1-2年的尖端生物化学研究。此外，该项目将为参加研究型高级生物化学实验室课程的约24名本科生提供为期一学期的指导研究体验。这两项活动将为学生提供成为下一代科学、技术和工程专业人员所需的实践和智力技能。这个项目使用酵母遗传学来促进我们对自噬小体形成的理解，自噬小体是一种包裹细胞货物并将其运送到液泡(在后生动物中，是溶酶体)进行降解的双膜囊。尽管之前对酿酒酵母的研究已经确定了这一过程所需的蛋白质，但关于它们如何发挥作用进行这一过程仍有许多有待了解的地方。其中一个中心角色是Atg8连接系统，它将小的泛素样蛋白Atg8连接到脂质磷脂酰乙醇胺上，从而定义了形成自噬膜的过程。先前的研究表明，细胞中Atg8的数量决定了自噬小体变得多大，但不决定产生的数量。然而，ATG7催化了导致Atg8结合的第一步，决定了自噬小体的大小和数量。该项目将研究该途径中的其他蛋白质，以解决这一难题，并确定该途径的哪个分支影响自噬体数。该项目还将研究Atg11的分子细节，Atg11是一种通过与核心自噬蛋白结合并排列来组织自噬货物周围的自噬小体形成的蛋白质。对Atg11的一个区域进行突变分析，初步数据表明该区域对与其关键伙伴之一的结合至关重要，这将证明哪些特定残基对这种相互作用是必不可少的。最后，将开发一种改进的数学模拟，以便从透射电子显微镜切片中更准确地估计自噬小体的大小和数量。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.