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Transformation of NOTCH3 protein in cerebral small vessel disease

NOTCH3蛋白在脑小血管疾病中的转化

基本信息

批准号：
10047287
负责人：
Michael M Wang
金额：
--
依托单位：
VETERANS HEALTH ADMINISTRATION
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-10-01 至 2021-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10047287
关键词：
Affect Aging American Animal Model Antibodies Arterial Disorder Arteries Binding Binding Proteins Biochemical Biochemistry Blood Vessels Brain Brain Diseases Brain Pathology Cell Culture Techniques Cells Cellular Stress Cellular biology Cerebral small vessel disease Cerebrum Cessation of life Cysteine Cytopathology Data Defect Dementia Detergents Development Disease Disulfides Event Family Functional disorder Generations Genetic Transcription Goals Health Priorities Heat shock proteins Human Inherited Light Location Maps Mediating Microvascular Dysfunction Modeling Molecular Molecular Biology Molecular Conformation Monoclonal Antibodies Mus Mutation NOTCH3 gene Oxidation-Reduction Pathogenesis Pathologic Pathology Pathway interactions Play Population Progress Review Group Property Protein Conformation Protein Secretion Proteins Reporting Risk Factors Role Series Site-Directed Mutagenesis Smooth Muscle Smooth Muscle Myocytes Stroke Structure Subcortical Infarctions Subcortical Leukoencephalopathy Techniques Testing Transcriptional Activation United States National Institutes of Health Vascular Dementia Vascular Diseases Vascular Smooth Muscle White Matter Disease age related cerebral artery conformational alteration disease-causing mutation disulfide bond experimental study human tissue in vivo in vivo evaluation mouse model mutant protein activation response stressor stroke risk tissue resource virtual

项目摘要

ABSTRACT Cerebral small vessel disease (SVD) affects over half of Americans over 65 and is a major risk factor for stroke and dementia. Although the pathological changes in blood vessels in SVD have been described for decades, the precise molecular defects are unclear. CADASIL is the most common inherited form of SVD and is caused by mutations in NOTCH3. We investigate the pathogenesis of CADASIL as a potential window into uncovering targetable mechanisms of vascular degeneration of the brain. Molecular mapping of affected families has provided important clues to the molecular pathways leading to arterial degeneration in CADASIL. The SVD-causing mutations in NOTCH3 either create or delete one cysteine residue, supporting the hypothesis that disulfide dependent conformational alterations in NOTCH3 trigger pathology of brain small vessels. In new preliminary data, we use a set of new NOTCH3 antibodies, including monoclonal probes, that bind to conformations of NOTCH3 that are strongly expressed in pathologically affected CADASIL arteries. These antibodies specifically bind to reduced NOTCH3 protein; other denaturants fail to induce the CADASIL conformational change. Site-directed mutagenesis of NOTCH3 demonstrates that the CADASIL conformation of NOTCH3 is generated after mutation of multiple cysteines, leading us to hypothesize that the unique NOTCH3 protein expressed in CADASIL results from reduction of more than one disulfide bond. We call this protein Multiple Reduced Cysteine NOTCH3 (mrc-N3). The experiments of this proposal aim to characterize mrc-N3. We propose to test the following hypotheses (Figure 1): (1) mrc-N3 is generated from reduction of at least two disulfide bonds and genesis of mrc-N3 is facilitate by NOTCH3 binding proteins from the vessel wall; (2) mrc-N3 activates transcription of disease related proteins and triggers smooth muscle cell stress pathways; (3) mrc-N3 functions in vivo to cause vascular dysfunction and pathology. These Aims will be tested using biochemical techniques incorporating purified NOTCH3 proteins, cell culture using primary human cerebral smooth muscle, pathological analysis of unique tissue resources available to our lab, and in vivo testing of mrc-N3 function in genetically modified mice. Successful execution of these studies will shed new light on key molecular mechanisms of small vessel disease.

摘要脑小血管疾病（SVD）影响超过一半的65岁以上的美国人，是一个主要的风险中风和痴呆的危险因素虽然SVD的血管病理改变具有一定的局限性，虽然已经描述了几十年，但精确的分子缺陷尚不清楚。CADASIL是世界上 SVD的常见遗传形式，由NOTCH 3突变引起。我们调查的 CADASIL的发病机制是揭示CADASIL靶向机制的潜在窗口大脑血管变性。受影响家庭的分子图谱提供了导致CADASIL动脉变性的分子途径的重要线索。的在NOTCH 3中引起SVD的突变要么产生要么缺失一个半胱氨酸残基，支持假设NOTCH 3中二硫键依赖性构象改变触发了脑内小血管在新的初步数据中，我们使用了一组新的NOTCH 3抗体，包括单克隆探针，其与NOTCH 3的构象结合，所述构象强烈地表达于病理受累的CADASIL动脉。这些抗体特异性结合减少的NOTCH 3蛋白;其他变性剂不能诱导CADASIL构象变化。 NOTCH 3的定点突变表明，NOTCH 3的CADASIL构象 NOTCH 3是在多个半胱氨酸突变后产生的，这使我们假设， CADASIL中表达的独特的NOTCH 3蛋白是由一个以上的二硫键我们将这种蛋白质称为多重还原半胱氨酸NOTCH 3（mrc-N3）。的该建议的实验旨在表征mrc-N3。我们建议测试以下内容假设（图1）：（1）mrc-N3是由至少两个二硫键的还原产生的 mrc-N3的形成受血管壁NOTCH 3结合蛋白的促进;（2）mrc-N3 激活疾病相关蛋白的转录并触发平滑肌细胞应激（3）mrc-N3在体内起作用以引起血管功能障碍和病理学。这些将使用生物化学技术结合纯化的NOTCH 3蛋白、细胞使用原代人脑平滑肌进行培养，对独特组织进行病理分析我们实验室可用的资源，以及转基因小鼠中mrc-N3功能的体内测试。小鼠这些研究的成功实施将为关键的分子机制提供新的线索小血管疾病。