The Molecular Basis of Serpin Function and Disfunction

描述（由申请人提供）：serpin类蛋白酶抑制剂包括血液中的许多蛋白酶抑制剂，如抗凝血酶和α -1抗胰蛋白酶。蛇蛋白参与许多生理过程的调节，包括纤维蛋白溶解、补体激活、血管生成和炎症。蛇形蛋白功能的核心是其独特的抑制机制，该机制涉及从亚稳形式到稳定形式的巨大构象变化，而这种机制要求蛇形蛋白折叠到亚稳状态。折叠、构象变化和功能在蛇形蛋白中有着千丝万缕的联系，理解这种联系对于理解正常的蛇形蛋白功能以及与各种蛇形蛋白突变相关的病理是必不可少的。已知错误折叠是许多蛇形蛋白相关疾病的基础，包括血栓和肺气肿。人类最常见的病理性蛇形蛋白突变，α -1抗胰蛋白酶的Z变异，在主要产生α -1抗胰蛋白酶的肝细胞中错误折叠和聚合。详细了解α -1抗胰蛋白酶和其他蛇形蛋白的错误折叠形式对于设计有效的治疗剂是必不可少的。这项工作将利用氢交换和质谱的独特能力，提供以前无法获得的关于蛇形蛋白在溶液中的构象动力学以及正常和病理蛇形蛋白折叠途径的信息。我们的具体目标是：1)利用氢交换/质谱法测量野生型α -1抗胰蛋白酶和已知活性受损的一些突变体的构象灵活性分布。2)利用氢交换/质谱法监测α -1抗胰蛋白酶分子在重折叠过程中获得的天然结构，确定α -1抗胰蛋白酶的折叠途径。3)采用氢交换/质谱法确定α -1抗胰蛋白酶致病性Z变异的折叠途径。意义：α -1抗胰蛋白酶缺乏症是一种严重的遗传性疾病，在美国估计有10万人患有这种严重的疾病。这种疾病会导致肝硬化和肺气肿。α -1抗胰蛋白酶的聚合是这些症状的根本原因。确定易聚合中间体的结构是开发治疗剂的重要步骤。

DESCRIPTION (provided by applicant): The serpin class of protease inhibitors includes many of the protease inhibitors in blood, such as antithrombin and alpha-1 antitrypsin. Serpins are involved in the regulation of many physiological processes including fibrinolysis, complement activation, angiogenesis and inflammation. Central to serpin function is their unique inhibitory mechanism which involves a large conformational change from a metastable form to a stable form, and this mechanism imposes on serpins the unusual requirement that they fold to a metastable state. Folding, conformational change and function are inextricably linked in serpins, and understanding this linkage is essential for understanding normal serpin function as well as the pathologies associated with various serpin mutations. Misfolding is known to be the basis of many serpin-linked diseases including thrombosis and emphysema. The most common pathological serpin mutation in humans, the Z variant of alpha-1 antitrypsin, misfolds and polymerizes in hepatic cells where alpha-1 antitrypsin is primarily produced. Detailed knowledge of misfolded forms of alpha-1 antitrypsin and other serpins is essential for the design of effective therapeutic agents. The proposed work will employ the unique capabilities of hydrogen exchange and mass spectrometry to provide previously inaccessible information on the conformational dynamics of serpins in solution and the folding pathways of normal and pathological serpins. Our specific aims are: 1) Measure the distribution of conformational flexibility in wild type alpha-1 antitrypsin and a number of mutants known to have compromised activity using hydrogen exchange/mass spectrometry. 2) Determine the folding pathway of alpha-1 antitrypsin by using hydrogen exchange/mass spectrometry to monitor the acquisition of native structure throughout the alpha-1 antitrypsin molecule during refolding. 3) Determine the folding pathway of the pathogenic Z variant of alpha-1 antitrypsin by hydrogen exchange/ mass spectrometry. Significance: Alpha-1 antitrypsin deficiency is a serious genetic disorder, with an estimated 100,000 individuals in the U.S. suffering from the severe form. This disorder can lead to both liver cirrhosis and emphysema. Polymerization of alpha-1 antitrypsin is the underlying cause of these symptoms. Determining the structure of the polymerization prone intermediate is an important step in developing therapeutic agents.