Unraveling Cell Death via Top-Down/Bottom-Up Proteomics

通过自上而下/自下而上的蛋白质组学揭示细胞死亡

• 批准号: 7045979
• 负责人: CHENG S LEE
• 金额: $ 25.75万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7045979
• 关键词: Drosophilidae; arthropod genetics; autophagy; biomarker; cell death; cysteine endopeptidases; enzyme activity; high throughput technology; protein purification; protein quantitation /detection; proteolysis; proteomics

DESCRIPTION (provided by applicant): Apoptosis and autophagic cell death are the two most prominent forms of programmed cell death that occur during animal development. While much is known about the regulation of apoptosis, relatively little is known about the mechanisms underlying autophagic cell death, and what mechanism and biochemical changes distinguish these most prominent forms of physiological cell killing. Drosophila embryos and salivary glands are an ideal system to study apoptosis and autophagic programmed cell death. The mass of embryos and salivary gland cells enable rapid purification and separation from other tissues, providing one of the few higher animal genetic systems where molecular and biochemical approaches can be used to study a purified population of dying cells in the context of a developing organism. These attributes argue that studies of Drosophila apoptosis and autophagic cell death will lead to the identification of new cell death proteins, and provide an excellent system to study their function. The programmed cell death genetic pathway is extremely conserved between fruit flies and humans, indicating that studies of cell death in Drosophila will be directly relevant to higher organisms. Our research goal is to develop, optimize, and apply innovative proteome technology for the comprehensive analysis of protein profiles during programmed cell death. By using the fruit fly Drosophila melanogaster as a model system, these proteome studies will explore pathways and identify biomarkers associated with Caspase activation during cell death in developing animals. This challenge will be addressed through the development and application of a capillary-based and integrated top-down/bottom-up multidimensional separation platform capable of ultrahigh resolution of intact proteins, followed by in situ proteolytic digestion of size-resolved proteins, high throughput peptide separation, and ultrasensitive peptide/protein identification. The combined strength of Drosophila genetics and the proposed innovative bioanalytical technologies enables investigation into the regulation of programmed cell death at the genome-wide level including the expression of proteins on a global scale.

描述（由申请人提供）：细胞凋亡和自噬性细胞死亡是动物发育过程中发生的两种最突出的程序性细胞死亡形式。虽然对细胞凋亡的调控了解很多，但对自噬性细胞死亡的机制以及这些最突出的生理性细胞杀伤形式的机制和生化变化知之甚少。果蝇胚胎和唾液腺是研究细胞凋亡和自噬性程序性细胞死亡的理想系统。大量的胚胎和唾液腺细胞能够快速纯化并与其他组织分离，提供了为数不多的高等动物遗传系统之一，其中分子和生物化学方法可用于研究发育生物体中纯化的垂死细胞群体。这些特性表明，对果蝇细胞凋亡和自噬性细胞死亡的研究将导致新的细胞死亡蛋白的鉴定，并为研究其功能提供一个很好的系统。程序性细胞死亡遗传途径在果蝇和人类之间是非常保守的，这表明果蝇细胞死亡的研究将直接与高等生物有关。 我们的研究目标是开发，优化和应用创新的蛋白质组技术，用于程序性细胞死亡过程中蛋白质谱的综合分析。通过使用果蝇Drosophila melanogaster作为模型系统，这些蛋白质组研究将探索途径并确定发育中动物细胞死亡期间与Caspase激活相关的生物标志物。这一挑战将通过开发和应用一个基于毛细管的和集成的自上而下/自下而上的多维分离平台来解决，该平台能够对完整蛋白质进行快速分辨，然后对大小分辨的蛋白质进行原位蛋白水解消化，高通量肽分离和超灵敏肽/蛋白质鉴定。果蝇遗传学和拟议的创新生物分析技术的综合实力，使调查程序性细胞死亡的调控在全基因组水平，包括蛋白质的表达在全球范围内。