Rapid ischemic tolerance: Ubiquitin-mediated structural reorganization

快速缺血耐受：泛素介导的结构重组

• 批准号: 7282685
• 负责人: ROBERT MELLER
• 金额: $ 16.93万
• 依托单位: LEGACY EMANUEL HOSPITAL AND HEALTH CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2008-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282685
• 关键词: Accounting; Actins; Acute; Address; Alanine; Biochemical; Brain; Brain Injuries; Cell Death; Co-Immunoprecipitations; Confocal Microscopy; Coupling; Cytoskeletal Proteins; Cytoskeleton; Data; Dendrites; Disruption; Down-Regulation; Event; Exposure to; Gene Expression; Genes; Glucose; Hour; Image; Ischemia; Ischemic Preconditioning; Mediating; Membrane Proteins; Microfilaments; Modeling; N-Methyl-D-Aspartate Receptors; Neuroprotective Agents; Organism; Oxygen; Phenotype; Phosphotransferases; Play; Postsynaptic Membrane; Process; Proteasome Inhibitor; Proteins; Proteomics; Research; Role; Stress; Stroke; Structure; Synapses; System; Testing; Therapeutic; Toxicant exposure; Translating; Ubiquitin; Vertebral column; acute stroke; base; brain cell; cell injury; deprivation; fascin; gene repression; in vitro Model; multicatalytic endopeptidase complex; neuroprotection; novel; postsynaptic; preconditioning; protein degradation; receptor function; research study; response; therapeutic target

DESCRIPTION (provided by applicant): Tolerance, the cellular response to mild stress, which protects against a toxic stress, is a conserved feature of many organisms. Most research has focused on long-term tolerance, which occurs 24-72 hours following preconditioning and is mediated by changes in gene expression. In contrast we have focused on short-term ischemic tolerance, which is mediated by rapid biochemical events and occurs 1 hour following the preconditioning. The identification of the mechanisms of short-term tolerance may identify novel rapid acting therapeutic targets to treat acute ischemic episodes (stroke). Our studies have suggested the ubiquitin-proteasome system, which regulates protein degradation, as being responsible for rapid ischemic tolerance. Using a proteomics screen we identified 2 candidate proteins, fascin and myristilated alanine-rich C kinase substrate (MARCKS), which are selectively ubiquitinated and degraded following preconditioning ischemia. The degradation of these candidate proteins results in the reorganization of the actin cytoskeleton, and may reduce NMDA receptor function, which is implicated in excitotoxic cell injury following ischemia. Our central hypothesis is the degradation of fascin and MARCKS following preconditioning ischemia, results in brief cytoskeleton re-arrangement and disruption of NMDA receptor function thereby protecting the brain from harmful ischemia. Our hypothesis will be tested using an in vitro model of ischemia. In Aim 1, Investigate the effect of preconditioning ischemia on cytoskeletal protein degradation by the ubiquitin-proteasome system, we will investigate the role of the ubiquitin-proteasome system on the rapid degradation of our two candidate proteins, fascin and MARCKS, following preconditioning ischemia. In the Aim 2, Investigate changes in postsynaptic structural reorganization in ischemic tolerance, we will study the effect of preconditioning ischemia on the reorganization of the postsynaptic membrane. Experiments will address the effect of preconditioning ischemia on the structure of dendrites. We will determine the relevance of actin reorganization on ischemic tolerance by using actin stabilizing/ destabilizing compounds. Using co- immunoprecipitation we will investigate actin-NMDA receptor interactions following preconditioning. Understanding how preconditioning remodels synaptic structure and function may help identify harmful vs. protective mechanisms induced by brief ischemia in the brain, and give a clearer understanding of brain cell death. We believe that the rapid and selective degradation of specific brain proteins induces a protective state and may reveal suitable targets for pharmacological therapeutics. Indeed, the long-term aim of these studies is to discover endogenous protective mechanisms that can be translated into effective rapid acting neuroprotective agents for stroke.

描述（由申请人提供）：耐受性，对轻度压力的细胞反应，可以防止有毒应激，是许多生物的保守特征。大多数研究都集中在长期耐受性上，这种耐受性发生在预处理后24-72小时，并由基因表达的变化介导。相比之下，我们专注于短期缺血性耐受性，这是由快速生化事件介导的，并在预处理后1小时发生。短期耐受性机制的鉴定可能会鉴定出新型的快速作用治疗靶标，以治疗急性缺血性发作（中风）。我们的研究表明，调节蛋白质降解的泛素 - 蛋白酶体系统是造成快速缺血性耐受性的原因。使用蛋白质组学筛选，我们确定了2种候选蛋白，即Fascin和肉豆蔻氨酸富含C激酶底物（MARCKS），它们在预处理缺血后有选择地泛素化并降解。这些候选蛋白的降解会导致肌动蛋白细胞骨架的重组，并可能降低NMDA受体功能，这与局部缺血后有关兴奋性细胞损伤。我们的中心假设是预处理缺血后Fascin和Marcks的降解，导致短暂的细胞骨架重新安排，并破坏NMDA受体功能，从而保护大脑免受有害缺血的影响。我们的假设将使用缺血的体外模型进行检验。在AIM 1中，研究预处理缺血对泛素 - 蛋白酶体系统的细胞骨架蛋白降解的影响，我们将研究泛素 - 凝激素系统对我们两种候选蛋白，fascin和Marcks的快速降解的泛素 - 蛋白酶体系统的作用。在目标2中，研究突触后结构重组中缺血性耐受性的变化，我们将研究预处理缺血对突触后膜重组的影响。实验将解决预处理缺血对树突结构的影响。我们将通过使用肌动蛋白稳定/不稳定化合物来确定肌动蛋白重组对缺血性耐受性的相关性。使用联合沉淀，我们将研究预处理后的肌动蛋白-NMDA受体相互作用。了解预处理的突触结构和功能如何有助于确定大脑中短暂缺血引起的有害和保护机制，并对脑细胞死亡有更清晰的了解。我们认为，特定脑蛋白的快速和选择性降解会诱导保护状态，并可能揭示适当的药理治疗靶标。实际上，这些研究的长期目的是发现内源性保护机制，可以将其转化为有效的快速作用神经保护剂的中风。