ANALYSIS OF DROSOPHILA Hsp27 IN DEVELOPMENTALLY REGULATED APOPTOSIS

描述(由申请人提供)：拟议研究的长期目标是了解果蝇胚胎在发育调节的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而动态的过程，有许多输入信号和许多下游的细胞反应。这些细胞死亡输入漏斗通过高度保守的caspase激活级联导致细胞的系统分解。利用实验室开发的蛋白质组学筛选方法，这位研究人员发现，在细胞死亡水平升高的胚胎中，普遍存在的小热休克蛋白的一种特定亚型HSP27会增加。这种HSP27的变化，很可能是磷酸化的变化，发生在细胞凋亡的早期，在任何明显的核衰变迹象之前。通过突变或通过RNA干扰减少Hsp27会导致胚胎和成人组织中细胞死亡的戏剧性损失，这体现在胚胎中缺乏吖啶橙染色的凋亡核，以及由于成人组织中促凋亡基因的过度表达而导致的缺陷的修复。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的功能。果蝇和哺乳动物细胞凋亡的一个重要区别是细胞色素c的释放和Bcl族蛋白的参与，而Bcl族蛋白似乎不像哺乳动物细胞那样在引发果蝇细胞死亡的过程中发挥核心作用。因此，果蝇的细胞死亡途径似乎比哺乳动物的更流畅。 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哪里发挥作用以及它是如何调节的。此外，对果蝇细胞死亡的分析使人们能够在一个完整的、发育中的有机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？基因挽救和替换方法将被用来确定仿磷酸型和非磷酸化形式的Hsp27是否能够挽救Hsp27缺失突变的细胞死亡表型。(2)Hsp27在眼和翅膀发育中的细胞凋亡途径中起什么作用？显性抑制分析和上位性实验将被用来确定Hsp27在凋亡途径中的作用。(3)Hsp27在细胞死亡中发挥作用的分子基础是什么？免疫共沉淀和蛋白质折叠分析以及蛋白质组学分析将用于研究HSP27‘S在细胞凋亡中作用的分子机制。为进一步研究HSP27基因S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极其重要的过程所需的分子事件和因素。 相关性：人类的发展以及持续的健康依赖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。从果蝇到人，细胞凋亡的分子机制是高度保守的。发育过程中细胞凋亡的失败会导致严重的出生缺陷和死亡；而成年人细胞死亡的失败会导致癌症或糖尿病和阿尔茨海默氏症等疾病，后者的细胞抵抗凋亡，而糖尿病和阿尔茨海默氏症的细胞过早死亡。在这一应用中，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，在果蝇细胞凋亡中所起的作用。

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.