Dimerization mechanisms of two procaspase subfamilies

两个 procaspase 亚家族的二聚化机制

• 批准号: 6613276
• 负责人: ALLAN CLAY CLARK
• 金额: $ 24.51万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6613276
• 关键词: active sites; apoptosis; binding sites; chemical cleavage; clinical research; cysteine endopeptidases; dimer; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate complex; inflammation; molecular assembly /self assembly; molecular chaperones; protein engineering; zymogens

DESCRIPTION (provided by applicant): The mechanisms that regulate the activation of procaspases play central roles in the regulation of apoptosis and inflammation. It has been shown that formation of a procaspase dimer is a critical event in maturation. For example, procaspase-1 is thought to be a monomer until interactions in the caspase recruitment domain (CARD) drive dimerization of the protease domains. The scaffold is sufficient to allow autolytic processing. In contrast, we have shown that procaspase-3 is a stable dimer, even though it does not contain a CARD. This suggests different folding and regulatory mechanisms for the activation of procaspases-1 and -3. We hypothesize that differences in the dimer interfaces are the key to whether the protein is a monomer or dimer. In addition, we show that dimerization and enzymatic activity are linked. Based on our protein engineering studies, we hypothesize that the gains in protein stability and enzyme activity are linked via a network of amino acids that extends from the dimer interface to the two active sites. We suggest that procaspases act as molecular machines in which side chain movements in the dimer interface affect the movements in the active site, allowing the substrate-binding pocket to form. We will approach this problem by addressing three key questions in the following specific aims. 1. Do procaspases-1 and -3 fold and assemble via similar mechanisms? Established biophysical methods will be employed to determine the oligomeric properties of procaspase-1 in order to test the current paradigm. 2. How is dimerization linked to active site formation? Using protein engineering techniques, we will examine the apparent linkage of amino acids at four positions near the dimer interface and active sites that affect proper insertion of the active site loops. 3. How does the pro-domain function in folding and assembly? Evidence is presented that the pro-peptide functions as an intramolecular chaperone. Molecular biological and biophysical studies will be employed to determine the precise mechanism of action. This work has the potential to affect therapeutic strategies for a number of autoimmune diseases, leart disease, and cancers because apoptosis is a common factor to these diseases. Learning to electively manipulate the level of apoptosis may well lead to therapeutic strategies for these diseases.

描述（由申请人提供）： 调节procaspase激活的机制在调节细胞凋亡和炎症中起着核心作用。已经表明，procaspase二聚体的形成是成熟的关键事件。例如，直到蛋白酶域的caspase募集域（CARD）驱动二聚体中的相互作用之前，procaspase-1被认为是单体。脚手架足以允许自溶处理。相比之下，我们已经表明，procaspase-3是一个稳定的二聚体，即使它不包含卡。这暗示了用于激活procaspase -1和-3的不同折叠和调节机制。我们假设二聚体界面的差异是蛋白质是单体还是二聚体的关键。此外，我们表明二聚化和酶活性是连接的。基于我们的蛋白质工程研究，我们假设蛋白质稳定性和酶活性的增长是通过从二聚体界面延伸到两个活性位点的氨基酸网络连接的。我们建议procaspase充当分子机器，其中二聚体界面中的侧链移动会影响活性位点的运动，从而使底物结合口袋形成。我们将通过以下特定目标解决三个关键问题来解决这个问题。 1。procaspase -1和-3折叠是否通过相似的机制组装？将采用已建立的生物物理方法来确定procaspase-1的寡聚特性，以测试当前的范式。 2。如何将二聚化链接到主动站点形成？使用蛋白质工程技术，我们将检查氨基酸在二聚体界面附近的四个位置和活动位点附近的氨基酸的明显连接，这些位置会影响活动现场环的适当插入。 3。亲域如何在折叠和组装中起作用？有证据表明，促肽作为分子内伴侣的作用。将采用分子生物学和生物物理研究来确定确切的作用机理。这项工作有可能影响多种自身免疫性疾病，学习疾病和癌症的治疗策略，因为细胞凋亡是这些疾病的常见因素。学习选举操纵凋亡水平可能会导致这些疾病的治疗策略。