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

错误折叠分泌途径蛋白选择性 ER 输出的机制基础

• 批准号: 10006833
• 负责人: Prasanna Satpute-Krishnan
• 金额: $ 31.08万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-03 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10006833
• 关键词: Address; Alzheimer' s Disease; Amyloid beta-Protein Precursor; Autophagocytosis; Binding; Biochemical; Biological; Biological Assay; Biophysics; Calnexin; Cell surface; Cells; Cysteine; Data; Development; Disease; Dissociation; Disulfides; Dithiothreitol; Drug Targeting; Endopeptidase K; Endoplasmic Reticulum; Estrogen receptor positive; Family; Family member; Fluorescence Recovery After Photobleaching; GPI Membrane Anchors; Glycoproteins; Goals; Golgi Apparatus; Heat shock proteins; Image; Integral Membrane Protein; Knowledge; Link; Lipids; Lysosomes; Mediating; Modification; Molecular; Molecular Chaperones; Monitor; Mutation; Pathogenesis; Pathway interactions; Physiological; PrP; Prion Diseases; Protein Family; Proteins; Quality Control; Resolution; Role; Route; Signal Transduction; Solubility; Specificity; System; Testing; Thapsigargin; Travel; Viral; cytokine; deletion analysis; disulfide bond; endoplasmic reticulum stress; glycosylation; human disease; insight; misfolded protein; mutant; prevent; protein misfolding; proteostasis; stressor; therapeutic development; trafficking

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.

项目总结/摘要 分泌途径中的蛋白质质量控制系统通常通过重折叠或重结晶来维持蛋白质稳态。 破坏错误折叠的蛋白质错误折叠的分泌途径蛋白的聚集和聚集 包括跨膜蛋白和糖基磷脂酰肌醇锚定蛋白（GPI-AP）， 分泌途径蛋白质质量控制的破坏，并且通常与破坏性的，不可治愈的， 和致命的蛋白质错误折叠疾病。例子包括淀粉样前体蛋白（APP）和朊病毒蛋白 (PrP)其错误折叠分别与阿尔茨海默氏症和朊病毒疾病有关。是一个新的 发现了蛋白质质量控制途径，处理各种错误折叠的GPI-AP，包括人类疾病 PrP突变体。我们的初步数据有力地表明，该池的底物延伸到选择 跨膜蛋白，包括APP的一些错误折叠突变体。 由内质网（ER）驻留的伴侣蛋白钙连接蛋白释放，并由p24家族成员结合， Tmp 21，用于输出到高尔基体。错误折叠的蛋白质随后通过细胞表面， 溶酶体在那里被破坏。与更好表征的蛋白质质量控制相比， 系统，ER相关的降解和自噬，这阻止了错误折叠的蛋白质进入细胞内。 分泌途径并在ER降解它们。一种选择性ER输出途径的发现 错误折叠的蛋白质，ESTA，揭示了新的和未探索的贡献者的发展相关的 错误折叠疾病本项目的长期目标是充分了解生物学行为的机制， 在分泌途径中维持蛋白质稳态的作用。本提案的目的是推断 钙连接蛋白和Tmp 21如何控制错误折叠蛋白的命运，并确定特异性的决定因素， 这一途径通过表征多种底物。我们的中心假设是， 通过钙连接蛋白、Tmp 21和相关蛋白质质量控制因子协调多种，但 特异性错误折叠的蛋白质从ER中排出用于下游降解。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测试这一假设：（1）确定 CNX引导底物并调节底物的机制。(2)通过以下方式确定机制 其Tmp 21将底物护送出ER。(3)确定错误折叠的决定因素 这些蛋白质将它们运送到大肠杆菌中。成功完成这些拟议的研究将提供关键的 深入了解新发现的重置途径的机制，提供必要但 目前无法获得的见解与直接影响的治疗策略的发展， 用于治疗和治愈各种蛋白质错误折叠疾病。