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(申请人提供)：耐受性，细胞对温和压力的反应，防止有毒压力，是许多生物体的保守特征。大多数研究都集中在长期耐受方面，这种耐受发生在预适应后24-72小时，并由基因表达的变化介导。相反，我们关注的是短期缺血耐受，这是由快速生化事件介导的，发生在预适应后1小时。短期耐受机制的确定可能确定治疗急性缺血性发作(卒中)的新的快速有效的治疗靶点。我们的研究表明，调节蛋白质降解的泛素-蛋白酶体系统与快速缺血耐受有关。利用蛋白质组学筛选，我们确定了两种候选蛋白，Fasin和富含肉豆蔻氨酸的C激酶底物(MARCKS)，它们在缺血预适应后选择性泛素化和降解。这些候选蛋白的降解导致肌动蛋白细胞骨架的重组，并可能降低NMDA受体的功能，这与缺血后的兴奋性细胞损伤有关。我们的中心假设是缺血预适应后，束蛋白和MARCKS的降解，导致短暂的细胞骨架重排和NMDA受体功能的破坏，从而保护大脑免受有害缺血的影响。我们的假设将使用体外缺血模型进行验证。目的1，通过泛素-蛋白酶体系统研究缺血预适应对细胞骨架蛋白降解的影响，探讨泛素-蛋白酶体系统在缺血预适应后两种候选蛋白Fasin和Marcks快速降解中的作用。目的2，探讨缺血耐受过程中突触后结构重组的变化，研究缺血预适应对突触后膜结构重组的影响。实验将解决预适应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与缺血耐受的相关性。利用免疫共沉淀法，我们将研究预适应后肌动蛋白-NMDA受体的相互作用。了解预适应如何重塑突触结构和功能，可能有助于识别脑内短暂缺血诱导的有害和保护机制，并对脑细胞死亡有更清晰的理解。我们认为，特定脑蛋白的快速和选择性降解诱导了一种保护状态，并可能揭示合适的药物治疗靶点。事实上，这些研究的长期目标是发现内源性保护机制，这些机制可以转化为有效的、快速起作用的中风神经保护剂。