Quality control in the secretory pathway

分泌途径的质量控制

基本信息

批准号：
9908930
负责人：
Rachel Starr Plumb
金额：
$ 6.53万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-11 至 2022-12-10
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9908930
关键词：
Address Autophagocytosis Bacteria Biological Assay Cell Separation Cells Cellular biology Client Cytosol DNA sequencing Detection Diabetes Mellitus Disease Endoplasmic Reticulum Enzymes Future Genetic Screening Golgi Apparatus Heat shock proteins Homeostasis Hydroxymethylglutaryl-CoA reductase Immunoglobulin Fragments In Vitro Infection Libraries Mammals Mediating Medical Membrane Membrane Proteins Methods Molecular Molecular Chaperones Mutate Neurodegenerative Disorders Organism Pathway interactions Peptide Library Physiological Polyubiquitination Process Proteins Proteome Quality Control Reagent Saccharomyces cerevisiae Signal Transduction Site Sterols Structure System Techniques Technology Work Yeasts biochemical tools biological adaptation to stress glycosylation human disease in vivo milligram misfolded protein multicatalytic endopeptidase complex nanobodies pathogen pathogenic virus prevent protein degradation protein folding protein misfolding proteostasis reconstitution screening secretory protein therapeutic target tool

项目摘要

Abstract The endoplasmic reticulum (ER) is a major site for protein folding and maturation within the cell, and a wide array of human diseases, including neurodegenerative diseases, familial protein folding disorders, and diabetes, are associated with disruptions to ER protein folding homeostasis. Endoplasmic reticulum associated degradation (ERAD) is a highly conserved pathway that functions to promote protein homeostasis by preventing misfolded protein accumulation. It is an integral part of the ER unfolded protein stress response. In addition to degradation of misfolded proteins, ERAD regulates the protein levels of ER-resident enzymes, such as the HMG-CoA reductase, the rate-limiting enzyme in sterol synthesis. Interestingly, the ERAD machinery is hijacked by viral pathogens during infection, meaning it is a potential therapeutic target. The process of ERAD involves transferring target protein substrates from the ER lumen or membrane to the cytosol for degradation. It can be divided into five distinct steps: substrate recognition, retro-translocation across the ER membrane, polyubiquitination, extraction from the membrane, and proteasomal degradation. While the molecular details of the later steps have become increasingly clear, how ERAD machinery recognizes misfolded or other substrate targets remains ambiguous. Previous work identified substrate glycosylation state as an important influencer of interactions with ERAD machinery. Nevertheless, glycosylation is dispensable for degradation, while substrate misfolding is not. The aims of this proposal seek to identify principles of substrate recognition by ERAD and other protein quality control machinery in the secretory pathway. Combining DNA sequencing technology and cell sorting techniques, we will generate and screen libraries of mutated or degron-fused non-ERAD substrates in S. cerevisiae to identify features that are recognized by ERAD machinery. We will then validate features through cell biology and in vitro reconstitution assays. Understanding the principles of substrate recognition by ERAD will illuminate the physiological ERAD targets, should allow us to predict additional targets in higher organisms, and understand exploitation of the system by certain pathogens.

抽象的内质网 (ER) 是细胞内蛋白质折叠和成熟的主要场所，广泛的人类疾病，包括神经退行性疾病、家族性蛋白质折叠障碍和糖尿病与 ER 蛋白折叠稳态的破坏有关。内质网相关降解（ERAD）是一种高度保守的途径，通过以下方式促进蛋白质稳态：防止错误折叠的蛋白质积累。它是内质网未折叠蛋白应激反应的一个组成部分。在除了降解错误折叠蛋白外，ERAD 还调节 ER 驻留酶的蛋白水平，例如作为 HMG-CoA 还原酶，甾醇合成中的限速酶。有趣的是，ERAD 机械是在感染过程中被病毒病原体劫持，这意味着它是一个潜在的治疗靶点。 ERAD 的过程涉及从 ER 腔或膜转移靶蛋白底物进入细胞质进行降解。它可以分为五个不同的步骤：底物识别、逆转录转位穿过内质网膜、多聚泛素化、膜提取和蛋白酶体降解。虽然后续步骤的分子细节已变得越来越清晰，但 ERAD 机制如何识别错误折叠或其他底物目标仍然不明确。先前的工作确定了底物糖基化状态是与 ERAD 机制相互作用的重要影响因素。尽管如此，糖基化对于降解来说是可有可无的，而底物错误折叠则不然。本提案的目的是寻求确定 ERAD 和其他蛋白质质量控制机制的底物识别原理分泌途径。结合DNA测序技术和细胞分选技术，我们将生成并筛选酿酒酵母中突变或降解决定子融合的非 ERAD 底物文库，以确定以下特征获得 ERAD 机械认可。然后我们将通过细胞生物学和体外重建来验证特征化验。了解 ERAD 识别底物的原理将阐明生理 ERAD 目标，应该使我们能够预测高等生物中的其他目标，并了解对这些目标的利用系统受到某些病原体的影响。