Abstract：
Mutants of alpha-1-antitrypsin cause the protein to self-associate and form ordered aggregates (\'polymers\') that are retained within hepatocytes, resulting in a predisposition to the development of liver disease. The associated reduction in secretion, and for some mutants, impairment of function, leads to a failure to protect lung tissue against proteases released during the inflammatory response and an increased risk of emphysema. We report here a novel deficiency mutation (Gly192Cys), that we name the Sydney variant, identified in a patient in heterozygosity with the Z allele (Glu342Lys). Cellular analysis revealed that the novel variant was mostly retained as insoluble polymers within the endoplasmic reticulum. The basis for this behaviour was investigated using biophysical and structural techniques. The variant showed a 40% reduction in inhibitory activity and a reduced stability as assessed by thermal unfolding experiments. Polymerisation involves adoption of an aggregation-prone intermediate and paradoxically the energy barrier for transition to this state was increased by 16% for the Gly192Cys variant with respect to the wild-type protein. However, with activation to the intermediate state, polymerisation occurred at a 3.8-fold faster rate overall. X-ray crystallography provided two crystal structures of the Gly192Cys variant, revealing perturbation within the \'breach\' region with Cys192 in two different orientations: in one structure it faces towards the hydrophobic core while in the second it is solvent-exposed. This orientational heterogeneity was confirmed by PEGylation. These data show the critical role of the torsional freedom imparted by Gly192 in inhibitory activity and stability against polymerisation.
摘要：
α-1-抗胰蛋白酶突变体导致蛋白质自缔合并形成有序聚集体（\'聚合物\'），保留在肝细胞内，导致肝脏疾病发展的倾向。相关的分泌减少，对一些变种人来说,功能受损，导致无法保护肺组织免受炎症反应过程中释放的蛋白酶的侵害，并增加肺气肿的风险。我们在这里报告了一个新的缺陷突变（Gly192Cys），我们命名为悉尼变种，在具有Z等位基因(Glu342Lys)的杂合性患者中鉴定。细胞分析显示，新变体大部分保留为内质网中的不溶性聚合物。使用生物物理和结构技术研究了这种行为的基础。如通过热解折叠实验所评估的,该变体显示出40%的抑制活性降低和降低的稳定性。聚合涉及采用易于聚集的中间体，矛盾的是，相对于野生型蛋白，Gly192Cys变体过渡到该状态的能障增加了16％。然而,激活到中间状态，聚合以整体快3.8倍的速率发生。X射线晶体学提供了Gly192Cys变体的两种晶体结构，揭示了Cys192在两个不同方向上的“破裂”区域内的扰动：在一个结构中，它面向疏水核，而在第二个结构中，它是溶剂暴露的。这种取向异质性通过聚乙二醇化得到证实。这些数据表明Gly192赋予的扭转自由在抑制活性和对聚合的稳定性中的关键作用。
