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: 10867561
• 负责人: Prabhodh Sai Abbineni
• 金额: $ 24.9万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 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 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10867561
• 关键词: Adipocytes; Autophagocytosis; Autophagosome; Binding; 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; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Process; Protein Biosynthesis; Protein Secretion; Proteins; Proteomics; Regulation; Research; Research Personnel; Resistance; Role; Route; Secretory Vesicles; System; Testing; Training; Work; angiogenesis; 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; pharmacologic; 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 分泌的蛋白质发挥了多种关键生物学作用，因此，蛋白质分泌的分子调节 经过深入研究。许多优雅的研究表明，构成了构成的分子原理 古典秘书途径。轴承信号肽的蛋白质在同时插入内质中 网状（ER）正确折叠，运输到高尔基体并包装成分泌物 出口到各种细胞内目的地或与质膜融合的工厂以实现蛋白质 分泌到细胞外空间。但是，在过去的二十年中，涉及几种细胞质蛋白 在先天免疫中，血管生成和神经元的致病性已被证明是通过er-golgi分泌的 独立的，非常规的分泌路线。考虑到非常规的广泛生物学效用 细胞质蛋白质分泌（UCP），了解该过程的分子调节是考虑的 兴趣。为此，我开发了一个基因组规模的CRISPR屏幕来识别UCP的新调节剂。我的 初步工作已将II类磷脂酰肌醇-3-激酶（PI3K-C2α）确定为分泌的调节剂 白介素-1β（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，我已经开发了一种基于质谱的方法，该方法可以识别不常见的分泌 蛋白质以高通量，公正的方式。我的建议将应用最先进的分子遗传和 蛋白质组学工具可揭示在潜水细胞类型中使用UCPS途径的分泌蛋白的广度（AIM 1），并表征PI3K-C2α在调节UCP中的作用（AIM 2）。此外，CRISPR筛选 AIM 2将使用的策略可以识别UCP的新型调节剂。这些研究将共同​​解决 我的中心假设是UCP在潜水细胞类型中起作用，并由保守的分子调节 支持功能潜水，信号肽缺乏蛋白质的机械。这项研究会 在我的主要导师和心理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， 专业的指导将使我能够成功完成拟议的实验，并帮助我 过渡到独立。作为独立研究者，我的研究计划的长期目标将是 破译秘密途径适应固有和外在应激源的机制，以及 表征导致疾病状态的不良适应反应。