The Molecular Basis of Serpin Function and Disfunction

说明(申请人提供)：丝氨酸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。Serpins参与许多生理过程的调节，包括纤溶、补体激活、血管生成和炎症。蛇针功能的中心是它们独特的抑制机制，它涉及从亚稳态到稳定形态的大范围构象变化，这一机制对蛇施加了不寻常的要求，即它们折叠到亚稳态。蛇针的折叠、构象变化和功能是密不可分的，了解这种联系对于了解正常的蛇针功能以及与各种蛇针突变相关的病理机制是至关重要的。错误折叠被认为是许多蛇毒相关疾病的基础，包括血栓形成和肺气肿。人类最常见的病理性丝氨酸突变是α-1抗胰蛋白酶的Z变种，在主要产生α-1抗胰蛋白酶的肝细胞中错误折叠和聚合。对错误折叠的α-1抗胰蛋白酶和其他蛇类的详细了解对于设计有效的治疗药物是必不可少的。这项拟议的工作将利用氢交换和质谱分析的独特能力，提供以前无法获得的关于蛇在溶液中的构象动力学以及正常和病理蛇的折叠路径的信息。我们的具体目标是：1)利用氢交换/质谱仪测量野生型α-1抗胰蛋白酶和一些已知活性受损的突变体的构象灵活性的分布。2)利用氢交换/质谱仪监测α-1抗胰蛋白酶分子在折叠过程中天然结构的获得，确定α-1抗胰蛋白酶的折叠途径。3)氢交换/质谱法测定α-1抗胰蛋白酶致病Z变异体的折叠路径。意义：α-1抗胰蛋白酶缺乏症是一种严重的遗传性疾病，据估计，美国有10万人患有这种严重的疾病。这种疾病可能会导致肝硬变和肺气肿。α-1抗胰蛋白酶的聚合是这些症状的根本原因。确定易于聚合的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.