FRET probes for analysis of serpin-mediated apoptosis in focal brain ischemia

FRET 探针用于分析局灶性脑缺血中丝氨酸蛋白酶抑制剂介导的细胞凋亡

• 批准号: 9037073
• 负责人: VLADIMIR V DIDENKO
• 金额: $ 30.81万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9037073
• 关键词: Alzheimer' s Disease; Apoptosis; Apoptotic; Biochemistry; Brain Ischemia; Caspase; Cell Culture Techniques; Cell Death; Cell Nucleus; Cells; Cellular biology; Cerebral Ischemia; Cessation of life; Characteristics; Clinical Research; Color; DNA; Deoxyribonucleases; Detection; Development; Diagnostic; Disease; Enzymes; Evaluation; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Future; Goals; Health; Hypoxia; Imagery; In Situ; Investigation; Ischemia; Ischemic Brain Injury; Label; Life; Light; Malignant Neoplasms; Mediating; Medical; Metabolic stress; Methods; Modeling; Molecular; Nuclear Localization Signal; Outcome; Pathology; Pathway interactions; Pattern; Peptide Hydrolases; Process; Proteins; Rattus; Regulation; Research; Role; Serine Protease; Serpins; Signal Transduction; Site; Stroke; System; Technology; Therapeutic Intervention; Tissues; Work; base; endonuclease; member; molecular pathology; new technology; nuclease; programs; relating to nervous system; tool

DESCRIPTION (provided by applicant): FRET probes for analysis of serpin-mediated apoptosis in focal brain ischemia. In light of the profound significance of apoptosis research, it s essential to have probes which can discriminate between different apoptotic pathways. The serine protease- dependent pathway is one of the most recently identified apoptotic programs, which still lacks specific tools for its visualization in situ. Serpin 1B is a key member of this pathway and is an important effector of apoptotic death in neural tissue. In metabolic stress it transforms into an active endonuclease. This unique transition occurs in hypoxia and simultaneously exposes an active DNase site and a nuclear localization signal. The newly formed DNA degrading enzyme promptly moves into the nucleus causing cell death. In spite of the high importance of this non-caspase apoptosis mechanism, there are currently no methods to selectively label it in tissue sections and in live cell cultures. In this project we will develp such methods and will apply them to study serpin-mediated apoptosis in focal brain ischemia. Specific aims: 1. To develop the new FRET-based approach for selective labeling of serpin-mediated apoptosis in tissue sections. The approach will simultaneously co-detect serpin 1B-derived endonuclease protein and characteristic DNA breaks which it produces. 2. To develop the new FRET-based approach for selective labeling of serpin-mediated apoptosis in live cell cultures. The FRET probes will become fluorescent only after they detect a specific marker of serpin-mediated pathway. 3. To apply the newly developed molecular tools to study focal cerebral ischemia. To evaluate the role of serpin-mediated apoptosis in focal brain ischemia in the rat model; to investigate its initiation mechanisms, dynamics, and patterns of distribution. The project will introduce enabling technologies for apoptosis research in general, and for studies in ischemia in particular. Their first systematic application to focal brain ischemia will provide information useful for the future clinical and research investigations of stroke, and for the development of effective therapeutic interventions.

描述（由申请人提供）：用于分析局灶性脑缺血中丝氨酸蛋白酶抑制剂介导的细胞凋亡的FRET探针。鉴于细胞凋亡研究的深远意义，有必要拥有能够区分不同凋亡途径的探针。丝氨酸蛋白酶依赖性途径是最近鉴定的凋亡程序之一，其仍然缺乏用于其原位可视化的特定工具。丝氨酸蛋白酶抑制剂1B是该通路的关键成员，并且是神经组织中凋亡性死亡的重要效应物。在代谢应激中，它转化为活性核酸内切酶。这种独特的转变发生在缺氧状态，同时暴露了一个活性DNA酶位点和一个核定位信号。新形成的DNA降解酶迅速进入细胞核，导致细胞死亡。尽管这种非半胱天冬酶凋亡机制非常重要，但目前还没有方法在组织切片和活细胞培养物中选择性地标记它。在本项目中，我们将发展这种方法，并将其应用于研究局灶性脑缺血中丝氨酸蛋白酶抑制剂介导的细胞凋亡。具体目标：1.建立一种基于FRET的选择性标记组织切片中丝氨酸蛋白酶抑制剂介导的细胞凋亡的新方法。该方法将同时共同检测丝氨酸蛋白酶抑制剂1B衍生的核酸内切酶蛋白和其产生的特征性DNA断裂。2.建立一种基于FRET的选择性标记丝氨酸蛋白酶抑制剂介导的活细胞凋亡的新方法。FRET探针只有在检测到丝氨酸蛋白酶抑制剂介导途径的特异性标记物后才会发出荧光。3.应用新发展的分子生物学工具研究局灶性脑缺血。评价丝氨酸蛋白酶抑制剂介导的细胞凋亡在大鼠局灶性脑缺血模型中的作用，研究其启动机制、动力学和分布模式。该项目将介绍一般的凋亡研究，特别是缺血研究的使能技术。他们的首次系统应用于局灶性脑缺血将提供有用的信息，为未来的临床和研究调查中风，有效的治疗干预措施的发展。