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

抗胰蛋白酶在血液中浓度很高，其主要作用是保护肺部免受炎症造成的组织损伤。肝细胞（肝细胞）通常释放单个抗胰蛋白酶分子进入循环。当个体遗传了两个抗胰蛋白酶蛋白变化很小的基因时，就会导致抗胰蛋白酶缺乏症。在大约4%的北欧人身上发现的Z型变异是导致严重的抗胰蛋白酶缺乏症的最常见原因。Z型变异导致抗胰蛋白酶形成长链分子（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。血液循环中正常形成的蛋白质数量减少意味着肺部不能很好地抵御炎症，因此个体会患上肺气肿。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。其中包括大脑中神经丝氨酸突变体的聚合，从而导致痴呆。我们把所有的情况归类为一类疾病，我们称之为“蛇形病”。该应用程序建立在我们小组30年的工作基础上，并在工作计划中有5个相互关联的项目。我们建议：i)用冷冻电镜在原子分辨率下定义从Z抗胰蛋白酶纯合子肝脏分离的病理聚合物中的连锁。我们将定义从组织中分离出来的病理性聚合物的结构，从而为治疗策略提供新的机会，以阻断导致抗胰蛋白酶缺乏症的异常蛋白-蛋白连接。（ii）确定聚合机制的普遍性：由shutter domain突变（Siiyama和Mmalton抗胰蛋白酶）和引起FENIB的neuroserpin突变引起的病理性聚合物的结构。我们将定义导致疾病的聚合物的结构，这些聚合物是由于分子不同部分的点突变而形成的，与“Z突变”不同。（iii）利用核磁共振表征丝氨酸丝氨酸聚合途径的中间体和指纹图谱，并确定病理聚合物的结构。这项工作将定义在抗胰蛋白酶和神经丝氨酸聚合物形成之前的蛋白质中间体，并提供对抗胰蛋白酶聚合物结构区域的理解，这些区域在低温电子显微镜下无法观察到，因为它们太过移动。它将支持聚合物阻滞剂和目标(v)中确定的显像剂的开发和优化。（iv）在细胞环境中原位可视化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，为细胞内Z抗胰蛋白酶聚合物引起的变化提供前所未有的见解。(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。我们将跟踪一组抗胰蛋白酶缺乏的儿童，以证实我们最初的观察，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，我们就可以招募高危人群进行临床试验。此外，我们将使用核磁共振来识别特异性结合抗胰蛋白酶聚合物的小分子，以开发一种允许无创测量肝内抗胰蛋白酶聚合物/包涵体的检测方法。这将使我们能够解决两个关键问题：(i)肝内聚合物负荷与肝脏疾病严重程度的相关性；（ii）使用这种成像技术加速人类药物开发。总之，这项工作将增加我们对抗胰蛋白酶缺乏症和丝状病变机制的理解，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.