Global Analysis of Proteolysis in Apoptosis

细胞凋亡中蛋白水解的整体分析

• 批准号: 8054743
• 负责人: JAMES A WELLS
• 金额: $ 28.77万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8054743
• 关键词: Amines; Apoptosis; Apoptotic; Area; Aspartate; Biological Assay; Biological Process; Biology; Caspase; Cell Death; Cell physiology; Cells; Cleaved cell; Data; Databases; Development; Disease; Drug Delivery Systems; Engineering; Enzymes; Esters; Event; Feedback; Fractionation; Goals; Harvest; Homeostasis; Hour; Human; In Vitro; Induction of Apoptosis; Jurkat Cells; Label; Life; Link; Logic; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Methods; Neurodegenerative Disorders; Pathway interactions; Peptide Hydrolases; Peptides; Process; Protease Gene; Protein Analysis; Proteins; Proteolysis; Proteomics; Protocols documentation; Reporting; Research; Research Personnel; Sampling; Sea; Signal Transduction; Site; Small Interfering RNA; Stroke; Supporting Cell; Testing; Time; Western Blotting; Work; cancer cell; cancer therapy; inhibitor/antagonist; knock-down; liquid chromatography mass spectrometry; programs; research study; subtiligase; therapeutic target

DESCRIPTION (provided by applicant): The long-term goal of the proposed work is to elucidate the process leading to programmed cell death (apoptosis) by applying a new and general method we have developed for global proteomic profiling of proteolysis ("degradomics"). Our hypothesis is that proteases, and in particular caspases, act like demolition experts to target key control points to disrupt cellular homeostasis. Finding these points of attack, identifying the proteases involved, and determining the sequence of events are all fundamental to understanding cell death. Apoptosis is a key cellular process that regulates diseases such as cancer, stroke and neurodegenerative diseases among others. By discovering the critical regulatory points in apoptosis, new and important drug targets for these diseases may be identified. Apoptosis is dominated by proteolytic events that are primarily mediated by caspases, aspartate specific proteases. However, the protein substrates they cleave and the cascades they ignite are only partially understood. This proposal applies a new and general method that allows one to selectively label and enrich for a-amino peptides, the product of proteolysis. Tagged products can be readily identified and sequenced by LC/MS/MS. Preliminary studies in Jurkat cells induced to apoptose have already identified 68 asparate cleavage sites of which two-thirds are new substrates. Data suggest the total set of caspase substrates may be up to 3 to 6-times larger than the entire data base of reported caspase substrates across all cells. A combination of cellular ("forward") and in vitro ("reverse") degradomics experiments are proposed. These experiments will provide the most comprehensive map to date of proteins cleaved and proteases responsible for intrinsic and extrinsic apoptosis pathways. There are over a 1000 different human protease genes. The degradomics approaches developed here for caspases will be general and applicable to mapping other protease signaling events. Proteases are involved in most areas of biology and mediate many diseases. It is known that prosurvival proteins are cleaved during apoptosis and many cancer treatments involve compounds that selectively induce apoptosis through inhibiting prosurvival proteins in cancer cells. Thus, in addition to mapping the process of apoptosis and demonstrating a general degradomics method, the proposed work may identify new drug targets for the development of cancer therapies.

描述（由申请人提供）：拟议工作的长期目标是通过应用我们为蛋白质水解的全局蛋白质组学分析（“降解组学”）开发的新的通用方法来阐明导致程序性细胞死亡（细胞凋亡）的过程。我们的假设是，蛋白酶，特别是半胱天冬酶，就像破坏专家一样，针对关键控制点来破坏细胞稳态。找到这些攻击点、识别所涉及的蛋白酶以及确定事件的顺序都是理解细胞死亡的基础。细胞凋亡是调节癌症、中风和神经退行性疾病等疾病的关键细胞过程。通过发现细胞凋亡的关键调节点，可以确定这些疾病的新的重要药物靶点。 细胞凋亡主要由蛋白水解事件主导，蛋白水解事件主要由半胱天冬酶、天冬氨酸特异性蛋白酶介导。然而，它们裂解的蛋白质底物和它们引发的级联反应仅被部分了解。该提案应用了一种新的通用方法，允许人们选择性地标记和富集蛋白质水解产物α-氨基肽。标记的产品可以通过 LC/MS/MS 轻松识别和测序。对诱导凋亡的 Jurkat 细胞的初步研究已经确定了 68 个天冬氨酸切割位点，其中三分之二是新底物。数据表明，Caspase 底物的总集可能比所有细胞中报道的 Caspase 底物的整个数据库大 3 至 6 倍。提出了细胞（“正向”）和体外（“反向”）降解组学实验的组合。这些实验将提供迄今为止最全面的负责内在和外在细胞凋亡途径的蛋白质和蛋白酶的图谱。有超过 1000 种不同的人类蛋白酶基因。这里开发的半胱天冬酶降解组学方法将是通用的，适用于绘制其他蛋白酶信号事件。 蛋白酶涉及生物学的大部分领域并介导许多疾病。众所周知，促存活蛋白在细胞凋亡过程中被裂解，并且许多癌症治疗涉及通过抑制癌细胞中的促存活蛋白来选择性诱导细胞凋亡的化合物。因此，除了绘制细胞凋亡过程并展示通用降解组学方法之外，拟议的工作还可以确定用于开发癌症疗法的新药物靶点。