Examining Molecular Regulation of Unconventional Cytoplasmic Protein Secretion

摘要/项目摘要 分泌的蛋白质具有多种重要的生物学作用，因此，蛋白质分泌的分子调节 被深入调查。许多优雅的研究揭示了保守的分子原理， 经典分泌途径携带信号肽的蛋白质协同插入内质网， 内质网（ER），在那里它们被正确折叠，运输到高尔基体，并包装成分泌细胞。 用于输出到各种细胞内目的地或与质膜融合以使蛋白质 分泌到细胞外空间。然而，在过去的二十年里，几种细胞质蛋白参与了 在先天性免疫中，血管生成和神经元致病性已显示通过ER-高尔基体分泌 独立的非常规分泌途径因此，考虑到非常规的生物学效用， 细胞质蛋白分泌（UCPS），了解这一过程的分子调控是相当重要的 兴趣为此，我开发了一种基因组规模的CRISPR筛选，以识别UCPS的新型调节因子。我 初步工作已经确定了II类磷脂酰肌醇-3-激酶（PI 3 K-C2 α）作为分泌的调节剂 白细胞介素-1 β是一种非常规分泌的细胞因子，在炎症反应中起主要作用。 此外，我还开发了一种基于质谱的方法，可以识别非常规分泌的 以高通量、无偏见的方式来检测蛋白质。我的建议将应用尖端的分子遗传学和 蛋白质组学工具揭示了在不同细胞类型中使用UCPS途径的分泌蛋白的广度（Aim 目的2：探讨PI 3 K-C2 α在UCPS调节中的作用。此外，CRISPR筛选 将用于目标2的策略可以识别UCPS的新调节剂。这些研究将共同解决 我的中心假设是UCPS在不同的细胞类型中起作用，并受一种保守的分子调控， 支持功能多样的、缺乏信号肽的蛋白质的分泌的机制。这项研究将 在我的主要导师和指导委员会的指导下进行，他们是该领域的专家 分子遗传学、蛋白质运输、磷酸肌醇生物学和蛋白质组学。他们的技术，学术， 和专业的指导将使我成功地完成拟议的实验，并帮助我 过渡到独立。作为一名独立的研究者，我的研究计划的长期目标将是 破译分泌途径适应内在和外在压力的机制， 描述导致疾病状态的适应不良反应。

Abstract/Project Summary Secreted proteins serve a variety of critical biological roles and thus, the molecular regulation of protein secretion is intensively investigated. Many elegant studies have revealed conserved molecular principles underlying the classical secretory pathway. Proteins bearing signal peptides are co-translationally inserted into the endoplasmic reticulum (ER) where they are properly folded, transported to the Golgi apparatus, and packaged into secretory vesicles for export to various intracellular destinations or fusion with the plasma membrane to enable protein secretion into the extracellular space. However, over the past two decades, several cytoplasmic proteins involved in innate immunity, angiogenesis, and neuronal pathogenicity have been shown to be secreted via ER-Golgi independent, unconventional secretory routes. Thus, given the broad biological utility of unconventional cytoplasmic protein secretion (UCPS), understanding the molecular regulation of the process is of considerable interest. To this end, I developed a genome-scale CRISPR screen to identify novel regulators of UCPS. My preliminary work has identified a class II phosphatidylinositol-3-kinase (PI3K-C2α) as a regulator of the secretion of interleukin-1β, an unconventionally secreted cytokine that plays a major role in the inflammatory response. Furthermore, I have developed a mass-spectrometry based method that can identify unconventionally secreted proteins in a high-throughput, unbiased manner. My proposal will apply cutting-edge molecular genetic and proteomic tools to uncover the breadth of secreted proteins that use UCPS pathways in diverse cell types (Aim 1), and to characterize the role of PI3K-C2α in modulating UCPS (Aim 2). Furthermore, the CRISPR screening strategy that will be used in Aim 2 may identify novel regulators of UCPS. Together, these studies will address my central hypothesis that UCPS operates in diverse cell types, and is regulated by a conserved molecular machinery that supports the secretion of functionally diverse, signal-peptide lacking proteins. This research will be conducted under the guidance of my primary mentor and mentoring committee, who are experts in the fields of molecular genetics, protein trafficking, phosphoinositide biology, and proteomics. Their technical, academic, and professional guidance will allow me to successfully complete the proposed experiments, and help me transition to independence. As an independent investigator, the long-term goal of my research program will be to decipher the mechanisms by which the secretory pathway adapts to intrinsic and extrinsic stressors, and characterize the maladaptive responses that contribute to disease states.