Alpha-1-antitrypsin (AT) deficiency and the serpinopathies: pathobiology and new therapeutic strategies

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25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)使用生物物理分析和冷冻电子显微镜确定病理聚合物的结构。我们将定义从人体组织中分离的病理性聚合物的结构，并利用这些信息制定策略来阻断导致抗胰蛋白酶缺乏和丝状病的异常蛋白-蛋白连接。（ii）建立细胞对细胞内丝氨酸聚合物的反应。我们将使用表达聚合物的细胞成像来定义聚合物形成对细胞功能的影响。我们还将确定细胞功能障碍是否可以通过阻断聚合物形成的小分子来逆转，从而确定是否是聚合物本身导致了细胞功能障碍和疾病。（iii）确定多细胞背景下的疾病机制，并利用抗胰蛋白酶缺乏症的蠕虫（秀丽隐杆线虫）模型确定新的治疗靶点。我们已经开发了一种新的蠕虫（秀丽隐杆线虫）模型，其中Z抗胰蛋白酶作为聚合物保留，与运动功能受损，体薄结构和发育迟缓有关。我们将评估小分子和聚合物阻断抗体对细胞内聚合和蠕虫运动的影响，剖析细胞间抗胰蛋白酶聚合物在细胞内发出毒性信号的机制，并对能够降低聚合物在蠕虫体内毒性的新型药物进行化学筛选。这些将作为设计用于人类的新疗法的基础。（iv）确定体内抗胰蛋白酶聚合物的动态及其作为疾病生物标志物的效用。我们将研究肝和肺移植中抗胰蛋白酶缺乏的个体，以评估肺和循环中抗胰蛋白酶聚合物的变化。我们还将评估循环聚合物作为预测和诊断抗胰蛋白酶缺乏相关肝病的第一个特异性生物标志物。(v)开发体内Z型抗胰蛋白酶聚合物成像诊断技术。我们将使用细胞穿透单克隆抗体或小分子特异性聚合物成像细胞内聚合物在疾病的小鼠模型。我们的长期目标是将其用于人类，这样我们就可以评估聚合物负担和肝脏疾病之间是否存在相关性，以及这种新的成像测试是否有助于加速人类药物的开发。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 25 years of work by our group and has 5 interlinking projects within a programme of work. We propose to:(i) define the structure of the pathological polymer using biophysical analysis and cryo-electron microscopy. We will define the structure of the pathological polymer isolated from human tissues and use this information to develop strategies to block the abnormal protein-protein linkage that underlies antitrypsin deficiency and the serpinopathies.(ii) establish the cellular response to intracellular serpin polymers. We will use the imaging of cells that express polymers to define the effect of polymer formation on cell function. We will also establish whether cell dysfunction can be reversed by small molecules that block polymer formation and so determine whether it is the polymers themselves that cause cell dysfunction and disease.(iii) define disease mechanism in a multicellular context and identify new therapeutic targets using a worm (C. elegans) model of antitrypsin deficiency. We have developed a novel worm (C. elegans) model in which Z antitrypsin is retained as polymers in association with impaired motility, thin body structure and delayed development. We will assess the effect of small molecules and polymer blocking antibodies on intracellular polymerisation and the motility of the worm, dissect the mechanism by which intercellular antitrypsin polymers signal toxicity within the cell and undertake a chemical screen for novel agents that can reduce the toxicity of polymers in the worm. These will be used as the basis to design new therapies for use in man.(iv) define the dynamics of antitrypsin polymers in vivo and their utility as a biomarker of disease. We will study individuals with antitrypsin deficiency undergoing liver and lung transplantation to evaluate the changes in antitrypsin polymers within the lung and circulation. We will also evaluate circulating polymers as the first biomarker that is specific for predicting and diagnosising antitrypsin deficiency related liver disease.(v) develop a diagnostic technology for imaging Z antitrypsin polymers in vivo. We will use cell penetrating monoclonal antibodies or small molecules that are specific for polymers to image intracellular polymers in mouse models of disease. Our longer term aim is to develop this for use in man so we can assess whether there is a correlation between polymer burden and liver disease and if this new imaging test will be useful 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.