Structural and cellular basis of alpha-1-antitrypsin (AT) deficiency and the serpinopathies

抗胰蛋白酶在血液中以高浓度存在，其主要作用是保护肺部免受炎症引起的组织损伤。肝细胞（肝细胞）通常释放单个抗胰蛋白酶分子进入循环。抗胰蛋白酶缺乏症的结果时，一个人继承了两个基因的抗胰蛋白酶蛋白质的微小变化。在大约4%的北欧人中发现的Z变体是严重抗胰蛋白酶缺乏症的最常见原因。Z变体导致抗胰蛋白酶形成长链的连接分子（称为“聚合物”），这些分子被困在肝细胞内。聚合物的积聚会损害细胞，增加患肝硬化和肝癌的机会。循环中适当形成的蛋白质的减少意味着肺部不能很好地保护免受炎症的影响，因此个体会发展为肺气肿。我们已经证明，类似的过程发生在抗胰蛋白酶所属的蛋白质家族的其他成员的突变体中。这些包括神经丝氨酸蛋白酶抑制剂的突变体在大脑中聚合而导致痴呆。我们把所有的病症都归为一类疾病，我们称之为“蛇症”。该应用程序建立在我们小组30年的工作基础上，在工作方案中有5个相互关联的项目。我们建议：i）通过冷冻电子显微镜以原子分辨率定义从Z抗胰蛋白酶纯合子肝脏中分离的病理聚合物中的连接。我们将确定从组织中分离的病理聚合物的结构，从而为阻断抗胰蛋白酶缺乏症的异常蛋白质-蛋白质连接的治疗策略提供新的机会。(ii)确定聚合机制的一般性：由快门结构域突变体（Siiyama和Mmalton抗胰蛋白酶）和引起FENIB的神经丝氨酸蛋白酶抑制剂突变体引起的病理性聚合物的结构。我们将定义致病聚合物的结构，这些聚合物是由于分子不同部分的点突变而形成的。(iii)使用NMR表征丝氨酸蛋白酶抑制剂聚合途径上的中间体和指纹，并确定病理聚合物的结构。这项工作将定义蛋白质的中间体，之前形成的抗胰蛋白酶和神经丝氨酸蛋白酶抑制剂聚合物，并提供了一个了解的区域的抗胰蛋白酶聚合物的结构，没有观察到在冷冻电子显微镜，因为它们是太移动的。它将支持目标（v）中确定的聚合物阻滞剂和显像剂的开发和优化。(iv)在细胞环境中原位观察Z抗胰蛋白酶聚合物。我们将使用一种新的高分辨率技术，低温聚焦离子束（FIB）铣削，提供前所未有的洞察Z抗胰蛋白酶聚合物引起的细胞内的变化。(v)使用病理聚合物作为抗胰蛋白酶缺乏症的生物标志物和诊断工具。我们将跟踪一组患有抗胰蛋白酶缺乏症的儿童，以证实我们的初步观察，即循环抗胰蛋白酶聚合物是肝脏疾病的生物标志物。如果得到证实，这将使我们能够招募最高风险的个体进行临床试验。此外，我们将使用NMR来鉴定特异性结合抗胰蛋白酶聚合物的小分子，以开发一种允许非侵入性测量肝内抗胰蛋白酶聚合物/内含物的测定方法。这将使我们能够解决两个关键问题：（i）相关肝内聚合物负荷与肝脏疾病的严重程度和（ii）使用这种成像技术，以加速药物开发的人。总之，这项工作将增加我们的抗胰蛋白酶缺乏症和丝氨酸蛋白酶抑制剂病的机制的理解，并允许开发新的治疗方法。

Antitrypsin is found at high concentrations in the bloodstream, where its main role is to protect the lungs against tissue damage from inflammation. Liver cells (hepatocytes) normally release individual molecules of antitrypsin into the circulation. Antitrypsin deficiency results when an individual inherits two genes with small changes in the antitrypsin protein. The Z variant, found in about 4% of people of North European decent, is the most common cause of severe antitrypsin deficiency. The Z variant causes antitrypsin to form long chains of linked molecules (called "polymers") that are trapped inside liver cells. The build-up of polymers damages the cell and increases the chance of developing liver cirrhosis and liver cancer. The reduced amount of properly formed protein in the circulation means the lungs are not as well protected against inflammation and so individuals develop emphysema. We have shown that a similar process occurs in mutants of other members of the protein family to which antitrypsin belongs. These include polymerisation of mutants of neuroserpin in the brain to cause dementia. We have grouped all the conditions together as a single class of disease that we have called 'the serpinopathies'. The application builds on 30 years of work by our group and has 5 interlinking projects within a programme of work. We propose to:i) define of the linkage in pathological polymers isolated from the livers of Z antitrypsin homozygotes at atomic resolution by cryo-electron microscopy. We will define the structure of the pathological polymer isolated from tissues and so provide new opportunities for therapeutic strategies to block the abnormal protein-protein linkage that underlies antitrypsin deficiency.(ii) determine the generality of the polymerization mechanism: structure of the pathological polymers caused by shutter domain mutants (Siiyama and Mmalton antitrypsin) and mutants of neuroserpin that cause FENIB. We will define the structure of disease causing polymers that form as a consequence of point mutations in different parts of the molecule from the 'Z mutation'. (iii) use NMR to characterize intermediates on serpin polymerisation pathways and fingerprint and define the structure of the pathological polymer. This work will define protein intermediates that precede the formation of antitrypsin and neuroserpin polymers and provide an understanding of regions of the antitrypsin polymer structure that are not observed within cryo-electron microscopy as they are too mobile. It will support the development and optimization of polymer blockers and the imaging agents identified in aim (v). (iv) visualise Z antitrypsin polymers in situ within the cellular environment. We will use a new high resolution technique, cryo-focused ion beam (FIB) milling, to provide unprecedented insight into the changes induced by Z antitrypsin polymers within the cell. (v) use the pathological polymers as a biomarker and diagnostic tool for antitrypsin deficiency. We will follow a cohort of children with antitrypsin deficiency to confirm our initial observation that circulating antitrypsin polymers are a biomarker of liver disease. If confirmed this will allow us to recruit the most high risk individuals to clinical trials. Moreover, we will use NMR to identify small molecules that specifically bind to antitrypsin polymers to develop an assay that allows non-invasive measurement of intra-hepatic antitrypsin polymers/inclusions. This will allow us to address two key issues: (i) correlating intrahepatic polymer load with the severity of liver disease and (ii) the use of this imaging technique to accelerate drug development in man.Taken together this work will increase our understanding of mechanism of antitrypsin deficiency and the serpinopathies and allow the development of new approaches to treatment.