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.

项目摘要/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不同 蛋白质。蛋白酶体降解的主要信号被认为是翻译后的附件 泛素分子的底物蛋白质。据报道某些蛋白酶体底物是 针对蛋白酶体的靶向不使用泛素，泛素无关的程度和机制 蛋白酶体降解尚不清楚。我们最近确定了数百个宠物，当融合到 增强的绿色荧光蛋白（EGFP）的C末端被泛素 - 蛋白质组降解 独立的方式。在这里，我们建议研究这种泛素独立的机制 降解并确定该途径的内源性底物。 首先，我们将确定EGFP融合基板是否直接识别出蛋白酶体，还是它们是否是 通过中间因素将蛋白酶体撞到蛋白酶体（AIM 1）。接下来，我们将表征 接收器和底物结合了遗传和生化方法（AIM 2）。最后，我们会的 识别泛素独立途径的内源性底物（AIM 3）。总体而言，这些实验将 详细描述了这种新的泛素蛋白独立于蛋白酶体途径，可能具有 对我们对蛋白酶体和蛋白质稳态的理解的深刻影响。 我将在Brigham的Stephen Elledge博士的心态下作为博士后研究员进行这项研究 以及妇女医院/哈佛医学院（HMS），进入我未来的独立研究职业 美国机构。为了实现我的拟议目标，我需要从Dr. Dr.的K99期间学习基本技能。 与先进的基因筛查技术有关的ELLEDGE。我组建了一个合作者团队， 来自HMS附近的顾问（Alfred Goldberg博士，Wade Harper博士，Eric Fisher博士，Phil Cole博士和博士 尤金OH）完成了我的技能，并将帮助我有效地完成我的拟议项目和 为我未来的独立职业做准备。 Eledge实验室和HMS的本地培训环境将 为我提供所需的所有材料，设备和专业发展机会 成为世界一流的研究员。获得K99/R00独立奖的奖励将为我提供 拥有重要的资源来实现我的项目和职业目标。