Protein ubiquitination based neuroprotection for stroke

基于蛋白质泛素化的中风神经保护

• 批准号: 6862336
• 负责人: ROBERT MELLER
• 金额: $ 17.92万
• 依托单位: LEGACY EMANUEL HOSPITAL AND HEALTH CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-15 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6862336
• 关键词: cell death; cerebral ischemia /hypoxia; immunoprecipitation; ischemic preconditioning; laboratory rat; mass spectrometry; neuroprotectants; newborn animals; posttranslational modifications; tissue /cell culture; ubiquitin

Tolerance, the cellular response to mild stress, which protects against a toxic stress, is a conserved feature of many organisms. Classic tolerance is gene-mediated, protein synthesis-dependent and requires at least 24 hours to occur. In contrast, rapid ischemic tolerance occurs within 1 hour of the preconditioning ischemia and is not dependent on the synthesis of new proteins. As such understanding the endogenous mechanism of neuroprotection which are involved in rapid ischemic tolerance, may identify novel fast acting therapeutic strategies to treat acute stroke. Our preliminary data suggests that cell death promoting proteins are rapidly down regulated, following preconditioning, which may account for the neuroprotective effects observed in rapid ischemic tolerance. Our central hypothesis is that brief ischemia stimulates the generation of rapid ischemic tolerance by acute alterations in the levels of cell death promoting proteins by the process of ubiquitination, which targets the proteins for degradation by the proteasome. The specific aims are:-1. We will test our hypothesis using a candidate cell death promoting protein, Bim. Experiments are designed to investigate Bim ubiquitination and degradation in the 1 hour period following ischemic preconditioning. 2. We will further test our hypothesis broadly with immunoprecipitation and mass spectrographic analysis to identify novel proteins that are ubiquitinated following preconditioning ischemia. Experiments will then further validate this data, and identify potential new targets for regulating cell death following ischemia. These studies will support our hypothesis that protein down regulation is a powerful endogenous neuroprotective mechanism in the brain. We will identify the molecular mechanisms that favor the degradation of pro-apoptotic proteins, which may offer new acute therapeutic targets for the treatment of stroke and other neurological insults, which would not be available via gene modulation strategies based on classic tolerance, requiring over 24h to exert a neuroprotective effect.

耐受性是细胞对轻微应激的反应，可以防止有毒应激，是许多生物体的保守特征。经典耐受是基因介导的、蛋白质合成依赖性的，并且需要至少 24 小时才能发生。相比之下，快速缺血耐受发生在预处理缺血后 1 小时内，并且不依赖于新蛋白质的合成。因此，了解参与快速缺血耐受的神经保护的内源性机制，可以确定治疗急性中风的新型快速作用治疗策略。我们的初步数据表明，预处理后细胞死亡促进蛋白迅速下调，这可能解释了在快速缺血耐受中观察到的神经保护作用。我们的中心假设是，短暂的缺血通过泛素化过程导致细胞死亡促进蛋白水平的急剧改变，从而刺激快速缺血耐受的产生，泛素化的目标是被蛋白酶体降解的蛋白质。具体目标是：-1。我们将使用候选细胞死亡促进蛋白 Bim 来检验我们的假设。实验旨在研究缺血后 1 小时内的 Bim 泛素化和降解 预处理。 2. 我们将通过免疫沉淀和质谱分析进一步广泛检验我们的假设，以鉴定在预处理缺血后泛素化的新型蛋白质。然后实验将进一步验证这些数据，并确定调节缺血后细胞死亡的潜在新靶点。这些研究将支持我们的假设，即蛋白质下调是大脑中强大的内源性神经保护机制。我们将确定有利于促凋亡蛋白降解的分子机制，这可能为治疗细胞凋亡提供新的急性治疗靶点。 中风和其他神经损伤，通过基于经典耐受性的基因调节策略无法实现，需要超过 24 小时才能发挥神经保护作用。