ANALYSIS OF ESCRT FUNCTION IN ENDOLYSOSOMAL TRAFFICKING

内溶酶体转运中 ESCRT 功能的分析

基本信息

批准号：
10447626
负责人：
Phyllis I Hanson
金额：
$ 39.59万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-01-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10447626
关键词：
ATP phosphohydrolase Acute Area Autophagocytosis Autophagosome Biogenesis Biology Biomedical Engineering Cell Death Cell division Cells Cellular Membrane Chemicals Complex Cytokinesis Cytoplasm Disease Endosomes Epithelial Cells Esters Excision Face Funding Grant HIV Budding Health Homeostasis Image Inflammasome Inflammation Inflammatory Response Invaded Kinetics Leucine Lipid Peroxidation Lysosomes Mediating Membrane Membrane Proteins Metabolic Modeling Molecular Neck Necrosis Nuclear Envelope Organelles Particulate Pathway interactions Phagocytes Phagosomes Play Process Proteins Resolution Role Silicon Dioxide Sorting - Cell Movement Sterility Stress System Testing Thinking Vesicle Viral Work Yeasts cytokine endosome membrane extracellular insight nanoscale pathogen peroxisome protein degradation recruit repaired response seal trafficking vesicular release

项目摘要

The endolysosomal network is the portal by which extracellular material enters the cell. As such, the membranes of the endosomes, phagosomes, and lysosomes that comprise this network face challenges from pathogens and other internalized materials as well as from metabolic and chemical stresses. Consequences of damage vary according to the specific compartment and degree of damage, but extensive lysosomal membrane permeabilization triggers cell death while limited disruption of endosomes and phagosomes by particulate material and pathogens leads to inflammasome activation and ensuing cytokine responses. A widely deployed strategy for removing damaged organelles involves the use of selective autophagy, referred to as lysophagy. Removal is, however, unnecessary if organelles are instead repaired. We recently discovered a new role for the ESCRT (endosomal sorting complex required for transport) machinery in responding to nano- scale disruptions in endolysosomal membranes and promoting their repair. In this proposal, we will build on this discovery and test the hypothesis that ESCRTs (and in particular ESCRT-III proteins) act as a dynamic membrane stabilizing system to protect vulnerable membranes across the endolysosomal network and beyond. Two aims will exploit and explore responses to two experimentally tractable and sterile endolysosomal disruptants that potently engage the ESCRT machinery. In Aim 1, we will determine how the ESCRT machinery recognizes and counteracts lysosomal membrane stress induced by L-leucyl-L-leucine methyl ester (LLOMe). This will involve characterizing the membrane stress responsible for engaging ESCRTs, defining the molecular pathway(s) involved and identifying “keystone” ESCRT-III proteins, delineating the molecular features required for repair, and identifying pathways that trigger this stabilizing response. In Aim 2, we will examine how ESCRTs respond to and repair silica induced membrane damage in epithelial and phagocytic cells. This will include testing a role for Fe2+-dependent lipid peroxidation in engaging ESCRTs, imaging the relative role and dynamics of ESCRT components on phagosomal membranes, and testing the hypothesis that ESCRTs limit endolysosomal damage in phagocytic cells and thereby dampen inflammation associated with the many things that transit through these pathways. The insights gained from this work will be applicable to understanding how ESCRTs sense and respond to a broad range of membrane stresses.

内溶酶体网络是细胞外物质进入细胞的门户。构成该网络的内体、吞噬体和溶酶体的膜面临着来自以下方面的挑战：病原体和其他内化物质以及代谢和化学应激的后果。根据具体损伤部位和损伤程度而有所不同，但广泛的溶酶体膜透化作用引发细胞死亡，同时有限地破坏内体和吞噬体颗粒物质和病原体导致炎症小体激活和随后的细胞因子反应。广泛采用的去除受损细胞器的策略涉及使用选择性自噬，称为然而，如果细胞器被修复，则无需去除。 ESCRT（运输所需的内体分选复合物）机器在响应纳米- 在此提案中，我们将在此基础上进一步扩大内溶酶体膜的破坏并促进其修复。这一发现并检验了 ESCRT（特别是 ESCRT-III 蛋白）作为动态蛋白的假设膜稳定系统可保护内溶酶体网络中脆弱的膜，除此之外，还有两个目标将利用和探索对两种实验上易处理且无菌的内溶酶体的反应。在目标 1 中，我们将确定 ESRT 机制如何发挥作用。机器识别并抵消 L-亮氨酰-L-亮氨酸甲酯诱导的溶酶体膜应激 (LLOMe)。这将涉及表征负责参与 ESRT 的膜应力，定义涉及并识别“关键”ESCRT-III 蛋白的分子途径，描绘分子在目标 2 中，我们将研究修复所需的特征，并确定触发这种稳定反应的途径。检查 ESCRT 如何响应和修复二氧化硅诱导的上皮细胞和吞噬细胞膜损伤这将包括测试 Fe2+ 依赖性脂质过氧化在参与 ESRT 中的作用，并对细胞进行成像。 ESCRT 成分在吞噬体膜上的相对作用和动态，并检验以下假设： ESCRT 限制吞噬细胞的内溶酶体损伤，从而抑制与吞噬细胞相关的炎症从这项工作中获得的见解将适用于通过这些途径进行的许多事情。了解 ESRT 如何感知和响应广泛的膜应力。