ANALYSIS OF DROSOPHILA Hsp27 IN DEVELOPMENTALLY REGULATED APOPTOSIS

果蝇 Hsp27 在发育调控细胞凋亡中的分析

基本信息

批准号：
8063587
负责人：
JONATHAN S MINDEN
金额：
$ 7.46万
依托单位：
CARNEGIE-MELLON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-15 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8063587
关键词：
Acridine Orange Address Adult Affect Alzheimer&apos s Disease Animals Apoptosis Apoptotic Biochemical Biochemistry Biological Biological Assay Caspase Cell Death Cell Death Process Cell Death Signaling Process Cell Nucleus Cells Cellular biology Cessation of life Co-Immunoprecipitations Complement Complex Congenital Abnormality Data Defect Development Diabetes Mellitus Disease Dissection Drosophila genus Embryo Ensure Epithelial Cells Event Excision Eye Failure Foundations Genes Genetic Genetic Epistasis Genetic Suppression Growth HSPB1 gene Health Heart Heat shock proteins Heat-Shock Response Hemolymph Human Development In Vitro Laboratories Lead Mammalian Cell Mammals Messenger RNA Methods Modification Molecular Mutate Mutation Nature Nuclear Decay Organ Organism Pathway interactions Pattern Pattern Formation Phenotype Phosphorylation Play Post-Translational Protein Processing Process Protein Family Protein Isoforms Proteins Proteomics RNA Interference Regulation Research Research Personnel Resistance Role Scheme Screening procedure Signal Pathway Signal Transduction Small Interfering RNA Staining method Stains Stimulus System Tissues Vertebrates Wing base blastomere structure cancer cell cytochrome c genetic analysis in vitro Assay in vivo knock-down man member mimetics mutant null mutation overexpression protein folding research study response tumor

项目摘要

DESCRIPTION (Provided by Applicant): The long term objective of the proposed research is to understand the early protein changes that occur during developmentally regulated apoptosis in Drosophila embryos. Developmentally regulated cell death is a complex and dynamic process, with many input signals and many downstream cellular responses. These cell death inputs funnel through a highly conserved caspase activation cascade that leads to the systematic dismantling of the cell. Using a proteomics screening method developed in his laboratory, the investigator discovered that a specific isoform of the ubiquitous small heat shock protein, HSP27, increases in embryos with elevated levels of cell death. This HSP27 change, which is most likely a change in phosphorylation, occurs early in apoptosis, before any overt signs of nuclear decay. Removal of Hsp27 by mutation, or reduction by RNA interference, causes a dramatic loss of cell death in embryos and adult tissues, as evidenced by a lack of acridine orange staining of apoptotic nuclei in embryos and the rescue of defects caused by over-expression of pro-apoptotic genes in adult tissue. In mammalian systems, heat shock proteins, including Hsp27, have been shown to serve both pro- and anti-apoptotic functions. An important difference between Drosophila and mammalian apoptosis is that release of cytochrome c and the involvement of the Bcl-family of proteins, which does not appear to play as central a role in triggering cell death in Drosophila as in mammalian cells. Thus the cell death pathway in Drosophila appears to be more streamlined than in mammals. The investigators propose to take advantage of Drosophila's amenability to genetic, cell biological, and biochemical dissection to establish where in the cell death pathway HSP27 functions and how it is regulated. Moreover, analysis of cell death in Drosophila allows one to assess apoptosis in the context of an intact, developing organism. Three sets of aims are described to address the following questions: (1) How does Hsp27 function in embryonic cell death? Genetic rescue and replacement methods will be used to determine if phosphomimetic and non-phosphorylatable forms of Hsp27 are able to rescue the loss-of-cell-death phenotype of a null Hsp27 mutation. (2) How does Hsp27 function in the apoptotic pathway in eye and wing development? Dominant suppression assays and epistasis experiments will be used to determine where in the apoptotic pathway Hsp27 operates. (3) What is the molecular basis for Hsp27 function in cell death? Co-immunoprecipitation and protein-folding assays as well as proteomic analysis will be used to study the molecular mechanisms of Hsp27's role in apoptosis. These aims are intended to lay the foundation for studying Hsp27's role in Drosophila cell death. These studies will lead to a better understanding of the molecular events and factors required for this vitally important process. RELEVANCE: Human development, as well as continued health, depends on the removal of excess and defective cells by a cell death process called apoptosis. The molecular mechanisms of apoptosis are highly conserved from fruit flies to man. Failures in apoptosis during development lead to severe birth defects and death; while failures in cell death in adults lead to diseases such as cancer, where cells resist apoptosis, or diabetes and Alzheimer's disease, where cells die prematurely. In this application, the investigators intend to use genetics, cell biology, and biochemistry to study the role Hsp27, a conserved, ubiquitous protein, plays in fruit fly apoptosis.

描述（由申请人提供）：所提议研究的长期目标是了解果蝇胚胎发育调节细胞凋亡过程中发生的早期蛋白质变化。发育调节的细胞死亡是一个复杂且动态的过程，具有许多输入信号和许多下游细胞反应。这些细胞死亡输入通过高度保守的半胱天冬酶激活级联漏斗，导致细胞的系统性分解。研究人员利用其实验室开发的蛋白质组学筛选方法发现，普遍存在的小型热休克蛋白 HSP27 的一种特定异构体在胚胎中数量增加，细胞死亡水平升高。这种 HSP27 变化很可能是磷酸化的变化，发生在细胞凋亡早期，在任何明显的核衰变迹象之前。通过突变或 RNA 干扰减少 Hsp27 的去除，会导致胚胎和成体组织中细胞死亡的急剧减少，这可以通过胚胎中凋亡细胞核的吖啶橙染色的缺乏以及因过度表达 Hsp27 引起的缺陷的挽救来证明。成体组织中的促凋亡基因。在哺乳动物系统中，热休克蛋白（包括 Hsp27）已被证明具有促凋亡和抗凋亡功能。果蝇和哺乳动物细胞凋亡之间的一个重要区别是细胞色素 c 的释放和 Bcl 家族蛋白的参与，而 Bcl 家族蛋白在果蝇中似乎不像哺乳动物细胞那样在触发细胞死亡中发挥核心作用。因此，果蝇的细胞死亡途径似乎比哺乳动物更精简。研究人员建议利用果蝇对遗传、细胞生物学和生化解剖的适应性来确定 HSP27 在细胞死亡途径中的作用及其调控方式。此外，对果蝇细胞死亡的分析允许人们在完整的、发育中的生物体的背景下评估细胞凋亡。描述了三组目标来解决以下问题：（1）Hsp27 在胚胎细胞死亡中如何发挥作用？基因拯救和替代方法将用于确定 Hsp27 的磷酸化和非磷酸化形式是否能够拯救 Hsp27 无效突变的细胞死亡表型丧失。 (2) Hsp27在眼睛和翅膀发育的细胞凋亡途径中如何发挥作用？显性抑制测定和上位性实验将用于确定 Hsp27 在凋亡途径中的作用位置。 (3) Hsp27在细胞死亡中发挥作用的分子基础是什么？免疫共沉淀、蛋白质折叠测定以及蛋白质组分析将用于研究 Hsp27 在细胞凋亡中作用的分子机制。这些目标旨在为研究 Hsp27 在果蝇细胞死亡中的作用奠定基础。这些研究将有助于更好地理解这一至关重要的过程所需的分子事件和因素。相关性：人类发育以及持续健康取决于通过称为细胞凋亡的细胞死亡过程去除多余和有缺陷的细胞。细胞凋亡的分子机制从果蝇到人类都是高度保守的。发育过程中细胞凋亡失败会导致严重的出生缺陷和死亡；而成人细胞死亡失败会导致癌症（细胞抵抗细胞凋亡）、糖尿病和阿尔茨海默病（细胞过早死亡）等疾病。在此应用中，研究人员打算利用遗传学、细胞生物学和生物化学来研究 Hsp27（一种保守的、普遍存在的蛋白质）在果蝇细胞凋亡中的作用。