Examining Molecular Regulation of Unconventional Cytoplasmic Protein Secretion

检查非常规细胞质蛋白分泌的分子调节

• 批准号: 10448325
• 负责人: Prabhodh Sai Abbineni
• 金额: $ 10万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10448325
• 关键词: 1-Phosphatidylinositol 3-Kinase; Address; Adipocytes; Autophagocytosis; Autophagosome; Biochemical; Biological; Biological Assay; Biological Process; Biology; Brefeldin A; CRISPR screen; CRISPR/Cas technology; Cell membrane; Cells; Cytolysis; Cytoplasm; Cytoplasmic Protein; Data; Destinations; Disease; Endoplasmic Reticulum; Endosomes; Extracellular Space; Fluorescence Microscopy; Foundations; Genes; Goals; Golgi Apparatus; Growth Factor; Inflammation Mediators; Inflammatory Response; Interleukin-1 beta; Kidney; Lipids; Mass Spectrum Analysis; Membrane; Mentors; Methodology; Methods; Molecular; Molecular Genetics; Natural Immunity; Neurons; Organelles; PIK3CG gene; Pathogenicity; Pathway interactions; Peptide Signal Sequences; Pharmacology; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Process; Protein Secretion; Proteins; Proteomics; Regulation; Research; Research Personnel; Resistance; Role; Route; Secretory Vesicles; System; Testing; Training; Work; angiogenesis; base; blood vessel development; cell type; complement C2a; cytokine; drug development; experimental study; fatty acid-binding proteins; functional genomics; genome-wide; hepatoma cell; human disease; interest; monocyte; new therapeutic target; novel; programs; protein transport; response; stressor; targeted treatment; tool

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.

摘要/项目摘要 分泌的蛋白质起着各种关键的生物学作用，因此，蛋白质分泌的分子调节。 正在进行深入的调查。许多优雅的研究揭示了潜在的保守分子原理 经典的分泌途径。携带信号肽的蛋白质共翻译插入到内质中 网状(ER)，在那里它们被适当地折叠，运输到高尔基体，并包装成分泌物 用于输出到各种细胞内目的地的囊泡或与质膜融合以使蛋白质能够 分泌物进入细胞外空间。然而，在过去的二十年里，几种细胞质蛋白参与了 在先天免疫中，血管生成和神经元致病性已被证明是通过内质网高尔基体分泌的。 独立的、非常规的分泌路线。因此，鉴于非常规药物具有广泛的生物学效用 细胞质蛋白分泌(UCPs)，了解这一过程的分子调控是相当有意义的 利息。为此，我开发了一种基因组规模的CRISPR屏幕来识别UCPs的新调控因子。我的 初步工作已确定II类磷脂酰肌醇-3-激酶(PI3K-C2α)是一种分泌调节因子 白介素1β是一种非常规分泌的细胞因子，在炎症反应中发挥重要作用。 此外，我还开发了一种基于质谱学的方法，可以识别非常规的分泌物 以高通量、不偏不倚的方式检测蛋白质。我的提案将应用尖端的分子遗传学和 蛋白质组学工具，揭示在不同细胞类型中使用UCPs途径的分泌蛋白的广度(目的 1)，并鉴定PI3K-C2α在调节UCP中的作用(目标2)。此外，CRISPR筛查 将在AIM 2中使用的策略可能会确定UCP的新调节器。总之，这些研究将解决 我的中心假设是，UCPs在不同的细胞类型中工作，并受一个保守的分子调控 支持分泌功能多样、缺乏信号肽的蛋白质的机械。这项研究将 在我的主要导师和指导委员会的指导下进行，他们都是该领域的专家 分子遗传学、蛋白质运输、肌醇磷脂生物学和蛋白质组学。他们的技术，学术， 专业指导将使我成功地完成拟议的实验，并帮助我 向独立过渡。作为一名独立调查员，我的研究项目的长期目标将是 破译分泌途径适应内在和外在应激源的机制，以及 描述导致疾病状态的不适应反应的特征。