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

基本信息

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
项目类别：
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&apos 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

项目摘要

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SERPIN介导的凋亡的FRET探针。鉴于凋亡研究的深刻意义，必须进行探针，以区分不同的凋亡途径。丝氨酸蛋白酶依赖性途径是最近确定的凋亡程序之一，它仍然缺乏特定的原位可视化工具。 Serpin 1b是该途径的关键成员，是神经组织中凋亡死亡的重要效应因子。在代谢压力下，它转化为活性内切清除酶。这种独特的过渡发生在缺氧中，同时暴露了活跃的DNase位点和核定位信号。新形成的DNA降解酶迅速移入导致细胞死亡的细胞核。尽管这种非asp酶凋亡机制具有很高的重要性，但目前尚无在组织切片和活细胞培养物中选择性地标记它的方法。在这个项目中，我们将开除这种方法，并将其应用于研究局灶性脑缺血的SERPIN介导的凋亡。具体目的：1。开发新的基于FRET的方法来选择性标记组织切片中SERPIN介导的凋亡。该方法将同时共同检测SERPIN 1B衍生的核酸内切酶蛋白和特征性DNA断裂。 2。开发新的基于FRET的方法来选择性标记SERPIN介导的活细胞培养物中的细胞凋亡。只有在检测到Serpin介导的途径的特定标记后，FRET探针才会变成荧光。 3。应用新开发的分子工具来研究局灶性脑缺血。评估SERPIN介导的凋亡在大鼠模型中局灶性脑缺血中的作用；研究其启动机制，动力学和分布模式。该项目将介绍一般凋亡研究的能力技术，尤其是缺血研究。他们在局灶性脑部缺血上的首次系统应用将为未来的中风临床和研究研究以及开发有效的治疗干预措施提供有用的信息。