Transformation of NOTCH3 protein in cerebral 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 大脑血管退化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。

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.