The Molecular Basis of Serpin Function and Disfunction

描述（由申请人提供）：丝氨酸蛋白酶抑制剂类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。丝氨酸蛋白酶抑制剂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。丝氨酸蛋白酶抑制剂功能的核心是其独特的抑制机制，该机制涉及从亚稳态形式到稳定形式的大的构象变化，并且该机制对丝氨酸蛋白酶抑制剂施加了不寻常的要求，即它们折叠成亚稳态状态。折叠，构象变化和功能是密不可分的丝氨酸蛋白酶抑制剂，了解这种联系是必不可少的了解正常的丝氨酸蛋白酶抑制剂的功能，以及与各种丝氨酸蛋白酶抑制剂突变相关的病理。已知错误折叠是许多丝氨酸蛋白酶抑制剂相关疾病的基础，包括血栓形成和肺气肿。人类最常见的病理性丝氨酸蛋白酶抑制剂突变，α-1抗胰蛋白酶的Z变体，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抗胰蛋白酶和其他丝氨酸蛋白酶抑制剂的错误折叠形式对于设计有效的治疗剂是必不可少的。拟议的工作将采用氢交换和质谱的独特能力，提供以前无法访问的信息的构象动力学的丝氨酸蛋白酶抑制剂在溶液中和折叠途径的正常和病理丝氨酸蛋白酶抑制剂。我们的具体目标是：1）使用氢交换/质谱法测量野生型α-1抗胰蛋白酶和已知具有受损活性的许多突变体中构象柔性的分布。2)通过使用氢交换/质谱法测定α-1抗胰蛋白酶的折叠途径，以监测在重折叠期间整个α-1抗胰蛋白酶分子的天然结构的获得。3)通过氢交换/质谱法确定α-1抗胰蛋白酶的致病性Z变体的折叠途径。重要性：α-1抗胰蛋白酶缺乏症是一种严重的遗传性疾病，估计美国有10万人患有严重的形式。这种疾病可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.