The Mechanistic Basis of Selective ER-Export of Misfolded Secretory Pathway Proteins

项目摘要/摘要 分泌途径中的蛋白质质量控制系统通常通过折叠或 破坏错误折叠的蛋白质。错误折叠的分泌途径蛋白的积累和聚集 包括跨膜蛋白和糖基磷脂酰肌醇锚定蛋白(GPI-APs)意味着 分泌途径蛋白质质量控制的崩溃，并经常与毁灭性的，不可治愈的， 以及致命的蛋白质错误折叠疾病。例子包括淀粉样前体蛋白(APP)和普恩蛋白 (PRP)其错误折叠分别与阿尔茨海默氏症和普恩病毒疾病有关。重置是一种新的 发现了蛋白质质量控制途径，可以处理包括人类疾病在内的各种错误折叠的GPI-AP PrP的突变体。我们的初步数据有力地表明，重置底物池扩大到选择 跨膜蛋白，包括一些错误折叠的APP突变体。在重置过程中，错误折叠的蛋白质 由内质网(ER)驻留的伴侣蛋白Calnexin释放，并由p24家族成员结合， TMP21，出口到高尔基群岛。错误折叠的蛋白质随后通过细胞表面到达 溶酶体被破坏的地方。RESET与更具特性的蛋白质质量控制形成对比 系统，内质网相关的降解和自噬，这阻止了错误折叠的蛋白质进入 分泌途径，并在内质网降解它们。一条选择性内质网输出途径的发现 错误折叠的蛋白质，重置，揭示了相关的发展的新的和未知的贡献者 错折的疾病。该项目的长期目标是充分了解重置的机制及其 在维持分泌途径中的蛋白质动态平衡方面的作用。这项提议的目的是推论 Calnexin和TMP21如何控制错误折叠蛋白的命运并确定其特异性的决定因素 这一途径通过表征多个重置底物来实现。我们的中心假设是，重置是受监管的 通过Calnexin、TMP21和相关的蛋白质质量控制因子来协调各种不同的出口，但 特定的、错误折叠的蛋白质离开内质网进行下游降解。我们将应用成像、生物物理、 生物化学和细胞生物学方法通过三个具体目标来检验这一假说(1)确定 CNX引导底物和调节RESET的机制。(2)通过以下方式确定机制 其中TMP21护送将底物重置出ER。(3)识别错误折叠带来的决定因素 引导它们进行重置的蛋白质。成功完成这些拟议的研究将提供关键的 对新发现的重置途径背后的机制的洞察，提供了必要的但 目前无法获得对指导的治疗策略的发展具有直接影响的见解 用于治疗和治愈各种蛋白质错误折叠疾病。

Project Summary/Abstract Protein quality control systems in the secretory pathway normally maintain protein homeostasis by refolding or destroying misfolded proteins. The accumulation and aggregation of misfolded secretory pathway proteins including transmembrane proteins and glycosylphosphatidylinositol-anchored proteins (GPI-APs) signify a breakdown in secretory pathway protein quality control, and are often associated with devastating, incurable, and fatal protein-misfolding diseases. Examples include amyloid precursor protein (APP) and prion protein (PrP) whose misfolding is associated with Alzheimer's and prion diseases, respectively. RESET is a newly discovered protein quality control pathway that handles diverse misfolded GPI-APs, including human disease mutants of PrP. Our preliminary data strongly suggests that the pool of RESET substrates extends to select transmembrane proteins, including some misfolding mutants of APP. During RESET, misfolded proteins are released by the endoplasmic reticulum (ER)-resident chaperone, calnexin, and bound by p24-family member, Tmp21, for export to the Golgi. The misfolded proteins subsequently transit the cell surface en route to lysosomes where they are destroyed. RESET contrasts with better-characterized protein quality control systems, ER associated degradation and autophagy, which precludes the entry of misfolded proteins into the secretory pathway and degrades them at the ER. The discovery of a selective ER-export pathway for misfolded proteins, RESET, reveals new and unexplored contributors to the development of associated misfolding diseases. The long-term goal of this project is to fully understand the mechanism of RESET and its role in maintaining protein homeostasis in the secretory pathway. The objective of this proposal is to deduce how calnexin and Tmp21 control the fate of misfolded proteins and to identify the determinants of specificity for this pathway by characterizing multiple RESET substrates. Our central hypothesis is that RESET is regulated by calnexin, Tmp21 and associated protein quality control factors that coordinate the export of diverse, but specific, misfolded proteins out of the ER for downstream degradation. We will apply imaging, biophysical, biochemical and cell biological approaches to test this hypothesis through three specific aims (1) Determine the mechanism by which CNX directs substrates to and regulates RESET. (2) Determine the mechanism by which Tmp21 escorts RESET substrates out of the ER. (3) Identify the determinants presented by misfolded proteins that route them for RESET. Successful completion of these proposed studies will provide critical insights into the mechanisms that underlie the newly discovered RESET pathway, providing essential but currently unavailable insights with direct implications for the development of therapeutic strategies directed towards the treatment and cures of diverse protein misfolding diseases.