Characterizing Stress-dependent Secretory Protein Mistargeting

项目摘要 分泌途径必须折叠和运输三分之一的蛋白质组，同时处理蛋白质组。 出色的客户端负载动态范围。质量控制机制防止蛋白质稳态， 分泌途径被非天然状态的蛋白质淹没。一个这样 机制是抢先质量控制（preQC），其中ER应激抑制ER易位， 新生蛋白质这个过程保护内质网不被新生蛋白质淹没 在错误折叠的蛋白质应激期间，与多种疾病相关的情况包括糖尿病， 神经退行性疾病然而，这种保护导致分泌性蛋白质在细胞中积累， 细胞溶质，其可以是蛋白毒性的并且导致细胞死亡和功能障碍。由于 方法的局限性，底物，原因，机制和preQC的后果是 大部分未知。因此，尽管预期preQC的生理相关性是显著的， 并没有得到很好的理解。我们建议应用我们最近开发的分泌蛋白测定法 错误地对preQC进行系统表征。我们将确定哪些应力会导致预QC 在来自不同人类谱系的分泌细胞中的一系列蛋白质底物。我们将确定 什么因素控制着聚集和聚集之间的错配分泌蛋白的分类？ 降解机制因为我们的检测是在活细胞中进行的，我们将确定 信号通路介导ER应激对preQC的诱导。最后，我们将把我们的分析扩展到 允许蛋白质组范围内定量基础和应激依赖性ER错误启动。这 延伸将使我们能够确定决定蛋白质对preQC敏感性的因素。的 拟议的研究结果将是细胞如何重塑的系统表征 应激反应中的内质网转位。

PROJECT SUMMARY The secretory pathway must fold and traffic one third of the proteome, while handling an exceptional dynamic range of client load. Quality control mechanisms prevent proteostasis in the secretory pathway from being overwhelmed by proteins in non-native states. One such mechanism is preemptive quality control (preQC), wherein ER stress inhibits ER translocation of nascent proteins. This process protects the ER from being overwhelmed with nascent protein during misfolded protein stress, a situation associated with diverse disease included diabetes and neurodegenerative diseases. However, this protection leads to secretory protein accumulating in the cytosol, which can be proteotoxic and lead to cell death and dysfunction. Due to methodological limitations, the substrates, causes, mechanism, and consequences of preQC are largely unknown. Hence, the physiological relevance of preQC, while expected to be significant, is not well understood. We propose to apply our recently developed assay for secretory protein mistargeting to systematically characterize preQC. We will identify which stresses induce preQC for a series of protein substrates in secretory cells from diverse human lineages. We will identify what factors govern triaging of mistargeted secretory proteins between aggregation and degradation mechanisms. Because our assay is performed in living cells, we will determine which signaling pathways mediate induction of preQC by ER stress. Finally, we will extend our assay to allow proteome-wide quantification of both basal and stress-dependent ER mistargeting. This extension will allow us to identify the factors that govern susceptibility of proteins to preQC. The outcome of the proposed research will be systematic characterization of how the cell remodels ER translocation in response to stress.