Quality control in the secretory pathway

分泌途径的质量控制

• 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
• 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

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部位，A 各种各样的人类疾病，包括神经退行性疾病，家族性蛋白质折叠和 糖尿病与ER蛋白质折叠稳态的干扰有关。内质网 降解（ERAD）是一种高度保守的途径，可以通过 防止错误折叠的蛋白积累。它是ER展开的蛋白质应激反应的组成部分。在 除了降解错误折叠的蛋白质，ERAD调节ER居民酶的蛋白质水平，例如 作为HMG-COA还原酶，固醇合成中的速率限制酶。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 到细胞质降解。它可以分为五个不同的步骤：底物识别，恢复转换 跨越ER膜，多泛素化，从膜上提取和蛋白酶体降解。 虽然以后步骤的分子细节变得越来越清晰，但如何擦除机械 识别错误折叠或其他底物目标仍然模棱两可。以前的工作确定了底物 糖基化状态是与Erad机械相互作用的重要影响者。尽管如此， 糖基化是可降解的，而底物错误折叠却不是。该提案的目的 确定ERAD和其他蛋白质质量控​​制机制的底物识别原则 分泌道路。结合DNA测序技术和细胞分类技术，我们将生成和 酿酒酵母中的突变或Degron融合的非校园底物的屏幕库，以识别特征 由Erad Machinery认可。然后，我们将通过细胞生物学和体外重构来验证特征 测定。了解ERAD的底物识别原理将阐明生理ERAD 目标，应该使我们能够预测更高生物体的其他目标，并了解对 某些病原体的系统。