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.

阿尔茨海默病 (AD) 的一个显着特征是细胞凋亡导致神经元损失。细胞凋亡的特征是质膜出血、核浓缩和 DNA 断裂，并由天冬氨酸蛋白酶家族的激活引发。细胞凋亡的启动涉及通过蛋白水解将半胱天冬酶原连续激活至其活性形式。该家族的两个关键成员是 caspase-8（Caspase 的最顶端成员）和 caspase-3（通常被称为该家族的刽子手成员）。目前，检查细胞凋亡在神经退行性疾病中的作用的主要技术包括能够检测细胞中DNA片段的TUNEL或ISEL方法。然而，这些方法存在一些“陷阱”，即它们可能检测凋亡细胞和坏死细胞中的 DNA 链断裂。此外，DNA 链断裂会随着死后时间间隔的增加而增加，并且是一种晚期核事件，可以在多种情况下发生，而不会导致细胞凋亡。此外，观察到的关键形态特征，例如细胞体的圆化和表面出血，即使在没有细胞核的情况下也会发生，并且归因于半胱天冬酶对细胞骨架蛋白的蛋白水解切割。由于这些限制，需要更新且更特异的细胞凋亡探针来证实 TUNEL 实验提供的证据。由于半胱天冬酶是特异性的，在天冬氨酸残基后进行切割，这将产生抗原上不同的半胱天冬酶切割产物 (CCP)，因此代表切割定点抗体的理想靶标。利用这种方法，我们设计了一种针对 fodrin（一种神经元细胞骨架蛋白）CCP 的抗体，并显示这些产物在 AD 中广泛积累。在本提案中，我们建议 1) 进一步表征该抗体，以确定多种细胞凋亡途径是否导致共同效应 caspase 的激活； 2) 开发针对 caspase-8 活性片段的切割位点定向抗体，并使用细胞凋亡模型系统表征这些抗体； 3) 使用该抗体与 forbin CCP 抗体一起确定 caspase 激活和 CCP 积累与 AD 相关其他事件（包括 β-淀粉样蛋白沉积和神经原纤维缠结形成）之间的关系。