Development of Site-Directed Caspase-Cleavage Antibodies

定点 Caspase 切割抗体的开发

• 批准号: 6331329
• 负责人: TROY T ROHN
• 金额: $ 5.63万
• 依托单位: BOISE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6331329
• 关键词: Alzheimer's disease; DNA damage; abzyme; apoptosis; chemical cleavage; cysteine endopeptidases; enzyme activity; immunologic substance development /preparation; laboratory rat; molecular pathology; molecular site; neurotoxicology; protein degradation; spectrin

A prominent feature of Alzheimer's disease (AD) is loss of neurons by apoptotic cell death. Apoptosis is characterized by plasma membrane bleeding, nuclear condensation, and DNA fragmentation and is initiated by the activation of capspases, a family of aspartate proteases. The initiation of apoptosis involves the sequential activation of procaspases to their active form by proteolysis. Two key members of this family are caspase-8, the most apical member of the caspase, and caspase-3 which is commonly referred to as the executioner member of this family. Presently, the major technique to examine the role of apoptosis in neurodegenerative diseases consist of TUNEL or ISEL methods that are able to detect DNA fragmentation in cells. However, there are several 'pitfalls' associated with these methods, namely that they may detect DNA strand breaks in both apoptotic and necrotic cells. In addition, DNA strand breaks can be increase with postmortem interval and are a late stage nuclear event that can occur in a variety of situations without apoptosis. Furthermore, key morphological features observed such as rounding up of the cell body and surface bleeding occur even in the absence of nuclei and have been attributed to proteolytic cleavage of cytoskeletal proteins by caspases. Due to these limitations, newer and more specific probes for apoptosis are necessary to confirm evidence provided by TUNEL experiments. Because caspases are specific, cleaving after aspartic residues, this will generate caspase cleavage products (CCPs) that are antigenically distinct and therefore, represent desirable targets for cleavage site-directed antibodies. Using this approach, we designed an antibody to CCPs of fodrin, a neuronal cytoskeleton protein, and showed widespread accumulation of these products in AD. In the present proposal we propose to 1) further characterize this antibody to determine whether multiple pathways of apoptosis lead to the activation of a common effector caspase; 2) develop cleavage site-directed antibodies against the active fragments of caspase-8 and characterize these antibodies using model systems of apoptosis; 3) use this antibody together with the forbin CCP antibody to determine the relationship between caspase activation and accumulation of CCPs with other events associated with AD including beta-amyloid deposition and neurofibrillary tangle formation.

阿尔茨海默氏病（AD）的重要特征是凋亡细胞死亡丧失神经元。凋亡的特征是质膜出血，核凝结和DNA碎片化，并由天冬氨酸蛋白酶家族的Capspases激活引发。凋亡的起始涉及procassass通过蛋白水解的顺序激活其活性形式。这个家族的两个主要成员是caspase-8，是caspase的最高成员，caspase-3通常称为该家族的执行者成员。目前，检查凋亡在神经退行性疾病中的作用的主要技术由能够检测细胞中DNA片段化的TUNEL或ISEL方法组成。但是，这些方法有几种“陷阱”，即它们可以检测到凋亡和坏死细胞中的DNA链断裂。此外，DNA链断裂可以随着死后间隔而增加，并且是晚期核事件，可以在各种情况下发生而不会凋亡。此外，观察到的关键形态特征，例如在细胞体的四舍五入和表面出血，即使在没有核的情况下也会发生，并且已归因于胱天蛋白酶细胞骨架蛋白的蛋白水解裂解。由于这些局限性，需要对凋亡的更新和更具体的探针确认Tunel实验提供的证据。由于胱天蛋白酶是特异性的，在天冬氨酸残基后切割，因此这将产生caspase裂解产物（CCP），它们在抗原上截然不同，因此代表了切割位点定向抗体的理想靶标。使用这种方法，我们设计了一种与神经元细胞骨架蛋白CCP的抗体，并在AD中显示了这些产物的广泛积累。在本提案中，我们提出的是1）进一步表征该抗体，以确定多种凋亡途径是否导致共同效应子caspase的激活； 2）开发针对caspase-8的活性片段的切割位置定向抗体，并使用凋亡模型系统表征这些抗体； 3）使用该抗体与福生CCP抗体一起确定caspase激活与CCP的积累与与AD有关的其他事件之间的关系，包括β-淀粉样蛋白沉积和神经纤维纤维缠结的形成。