Transformation of NOTCH3 protein in cerebral small vessel disease

摘要 脑部小血管疾病(SVD)影响着超过一半的65岁以上的美国人，是一个主要的风险因素 中风和痴呆症的因素。尽管SVD中血管的病理改变 几十年来一直被描述，但确切的分子缺陷尚不清楚。CADASIL是世界上 常见的遗传性SVD，由NOTCH3突变引起。我们调查了 CADASIL的发病机制是揭示其靶向机制的潜在窗口 脑部血管变性。受影响家庭的分子图谱提供了 CADASIL中导致动脉变性的分子途径的重要线索。这个 NOTCH3中导致SVD的突变可以创建或删除一个半胱氨酸残基，支持 假设NOTCH3中二硫化物依赖的构象变化触发了 脑部小血管。在新的初步数据中，我们使用了一组新的NOTCH3抗体， 包括与NOTCH3构象强烈结合的单克隆探针 在病变的CADASIL动脉中表达。这些抗体特异性地结合到 NOTCH3蛋白减少；其他变性剂不能诱导CADASIL构象变化。 NOTCH3的定点突变表明CADASIL的构象 NOTCH3是在多个半胱氨酸突变后产生的，这让我们假设 在CADASIL中表达的独特的NOTCH3蛋白是一个以上还原的结果 二硫键。我们称这个蛋白为多重还原半胱氨酸NOTCH3(MRC-N3)。这个 本方案的实验目的是对MRC-N3进行表征。我们建议测试以下内容 假设(图1)：(1)MRC-N3由至少两个二硫键还原而成 MRC-N3通过管壁的NOTCH3结合蛋白促进其发生；(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在体内发挥作用，导致血管功能障碍和病理。这些 AIMS将使用生化技术进行测试，其中包括纯化的NOTCH3蛋白、细胞 原代人脑平滑肌培养，独特组织病理学分析 我们实验室可用的资源，以及对转基因MRC-N3功能的体内测试 老鼠。这些研究的成功将为关键的分子机制提供新的线索 小血管疾病。

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.