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.

项目总结/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 proteins.蛋白酶体降解的主要信号被认为是翻译后附着 将泛素分子连接到底物蛋白上。虽然已经报道了一些蛋白酶体底物是 针对蛋白酶体不使用泛素，泛素非依赖性的程度和机制 蛋白酶体降解仍不清楚。我们最近已经鉴定出数百种肽，当与 增强型绿色荧光蛋白（EGFP）的C-末端，被蛋白酶体降解， 独立的方式。在这里，我们建议研究这种不依赖于泛素的机制， 降解和确定该途径的内源性底物。 首先，我们将确定EGFP融合底物是否被蛋白酶体直接识别，或者它们是否被蛋白酶体识别。 通过干预因子穿梭于蛋白酶体（Aim 1）。接下来，我们将描述 受体和底物使用遗传和生物化学方法的组合（目的2）。最后我们将 确定内源性底物的泛素非依赖性途径（目的3）。总的来说，这些实验将 详细描述这种新发现的不依赖于泛素的蛋白酶体途径， 这对我们理解蛋白酶体和蛋白质稳态有着深远的意义。 我将在布里格姆大学斯蒂芬·埃利奇博士的指导下作为博士后研究员进行这项研究 和妇女医院/哈佛医学院（HMS），并进入我未来的独立研究生涯的顶部 美国机构。为了实现我提出的目标，我需要在K99期间从博士那里学习基本技能。 关于先进的基因筛选技术。我召集了一个合作小组， 来自HMS的顾问（Alfred Goldberg博士、Wade哈珀博士、Eric Fisher博士、Phil科尔博士和Dr. 尤金哦）谁补充我的技能，并将帮助我有效地完成我提出的项目， 为我未来的独立事业做准备。Elledge实验室和HMS的本地培训环境将 为我提供所有必要的材料、设备和专业发展机会， 成为世界级的研究员。获得K99/R 00独立之路奖将为我提供 关键资源来实现我的项目和职业目标。