Characterization of a ubiquitin-independent pathway for proteasomal degradation

蛋白酶体降解的不依赖于泛素的途径的表征

• 批准号: 10642425
• 负责人: elijah l mena
• 金额: $ 13.64万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642425
• 关键词: 26S proteasome; Advisory Committees; Aging; Alzheimer' s Disease; Award; Binding Sites; Biochemical; Biology; CRISPR/Cas technology; Cell Line; Cells; Chimeric Proteins; Committee Members; Complement; Cytidine Deaminase; Defect; Degradation Pathway; Development; Disease; Environment; Equipment; Fluorescence-Activated Cell Sorting; Fractionation; Future; Genes; Genetic; Genetic Screening; Genomics; Goals; Homeostasis; Hospitals; Huntington Disease; Huntington gene; Institution; Learning; Ligation; Mass Spectrum Analysis; Mentors; Mentorship; Methods; Mutagenesis; Mutation; Ornithine Decarboxylase; Parkinson Disease; Pathway interactions; Peptide Hydrolases; Peptides; Point Mutation; Postdoctoral Fellow; Probability; Process; Proteasome Inhibition; Proteins; Proteomics; Receptor Gene; Reporter; Reporting; Research; Research Personnel; Resources; Scientist; Signal Transduction; Structural Biologist; System; Techniques; Therapeutic; Training; Ubiquitin; Woman; Work; base editor; career; career development; enhanced green fluorescent protein; experimental study; inhibitor; insight; medical schools; misfolded protein; multicatalytic endopeptidase complex; mutant; oxidative damage; particle; protein degradation; proteostasis; receptor; skills

PROJECT SUMMARY/ABSTRACT The proteasome is the main intracellular protease and is responsible for degrading thousands of different proteins. The main signal for degradation by the proteasome is thought to be the post-translational attachment of ubiquitin molecules onto a substrate protein. While some proteasome substrates have been reported to be targeted to the proteasome without the use of ubiquitin, the extent and mechanism of ubiquitin-independent proteasomal degradation remain unclear. We have recently identified hundreds of peptides that, when fused to the C-terminus of enhanced green fluorescent protein (EGFP), are degraded by the proteasome in a ubiquitin- independent manner. Here, we propose to investigate the mechanism of this ubiquitin-independent degradation and determine the endogenous substrates of this pathway. First, we will determine if the EGFP fusion substrates are recognized by the proteasome directly, or if they are shuttled to the proteasome by an intervening factor (Aim 1). Next, we will characterize the interaction between the receptor and substrates using a combination of genetic and biochemical methods (Aim 2). Finally, we will identify endogenous substrates of the ubiquitin-independent pathway (Aim 3). Overall, these experiments will characterize in detail this newly-uncovered ubiquitin-independent proteasome pathway, which may have profound implications for our understanding of the proteasome and of protein homeostasis more generally. I will perform this research as a postdoctoral fellow under the mentorship of Dr. Stephen Elledge at Brigham and Women’s Hospital/Harvard Medical School (HMS) and into my future independent research career at a top U.S. institution. To accomplish my proposed aims, I will need to learn essential skills in the K99 period from Dr. Elledge pertaining to advanced genetic screening techniques. I have assembled a team of collaborators and advisors from around HMS (Dr. Alfred Goldberg, Dr. Wade Harper, Dr. Eric Fisher, Dr. Phil Cole, and Dr. Eugene Oh) who complement my skill set and will help me effectively accomplish my proposed projects and prepare for my future independent career. The local training environment in the Elledge lab and at HMS will support me with all the materials, equipment, and professional development opportunities necessary to become a world-class researcher. Receiving the K99/R00 Pathway to Independence Award would provide me with critical resources to realize my project and career goals.

项目概要/摘要 蛋白酶体是主要的细胞内蛋白酶，负责降解数千种不同的物质。 蛋白酶体降解的主要信号被认为是翻译后附着。 泛素分子附着在底物蛋白上，而一些蛋白酶体底物已被报道。 不使用泛素靶向蛋白酶体，不依赖泛素的程度和机制 我们最近发现了数百种肽，当它们融合时，其降解过程仍不清楚。 增强型绿色荧光蛋白 (EGFP) 的 C 末端被泛素中的蛋白酶体降解 在这里，我们建议研究这种不依赖于泛素的机制。 降解并确定该途径的内源底物。 首先，我们将确定 EGFP 融合底物是否被蛋白酶体直接识别，或者是否被蛋白酶体识别。 通过干预因子穿梭至蛋白酶体（目标 1）。接下来，我们将表征之间的相互作用。 最后，我们将结合遗传和生化方法来研究受体和底物（目标 2）。 总体而言，这些实验将鉴定泛素独立途径的内源性底物（目标 3）。 详细描述了这种新发现的不依赖于泛素的蛋白酶体途径，该途径可能具有 对我们更广泛地理解蛋白酶体和蛋白质稳态具有广泛的意义。 我将在布里格姆大学 Stephen Elledge 博士的指导下以博士后研究员的身份进行这项研究 和妇女医院/哈佛医学院 (HMS) 并进入我未来的独立研究生涯 为了实现我提出的目标，我需要在 K99 期间向 Dr. 学习基本技能。 我组建了一个与先进基因筛查技术相关的团队。 来自 HMS 各地的顾问（Alfred Goldberg 博士、Wade Harper 博士、Eric Fisher 博士、Phil Cole 博士和 Dr. Eugene Oh），他补充了我的技能，并将帮助我有效地完成我提出的项目和 Elledge 实验室和 HMS 的本地培训环境将为我未来的独立职业生涯做好准备。 支持我提供所有必要的材料、设备和专业发展机会 获得 K99/R00 独立之路奖将为我提供成为世界级研究员的机会。 拥有实现我的项目和职业目标的关键资源。