Quality control in the secretory pathway

摘要 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，以及 糖尿病与ER蛋白折叠稳态的破坏有关。内质网相关 蛋白质降解（ERAD）是一种高度保守的途径，其功能是通过以下途径促进蛋白质稳态： 防止错误折叠的蛋白质积累。它是ER未折叠蛋白应激反应的组成部分。在 除了降解错误折叠的蛋白质外，ERAD还调节ER驻留酶的蛋白质水平， 作为HMG-CoA还原酶，是甾醇合成的限速酶。有趣的是，ERAD机器是 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 到细胞质中降解。它可以分为五个不同的步骤：底物识别，反向转位， 穿过ER膜、聚遍在蛋白化、从膜中提取和蛋白酶体降解。 虽然后来步骤的分子细节已经变得越来越清楚，ERAD机器如何 识别错误折叠或其他衬底目标仍然不明确。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。然而，尽管如此， 糖基化是降解的主要原因，而底物错误折叠则不是。本提案的目的是 确定ERAD和其他蛋白质质量控制机器在蛋白质中的底物识别原理， 分泌途径结合DNA测序技术和细胞分选技术， 筛选S. cerevisiae来鉴定 由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功能 测定。了解ERAD识别底物的原理将有助于阐明生理ERAD 目标，应该使我们能够预测高等生物中的其他目标，并了解利用这些目标。 系统由某些病原体。

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.