Blomenkamp Keith S, Teckman Jeffrey H
Department of Pediatrics, Saint Louis University, Saint Louis, MO, USA.
Department of Pediatrics, Saint Louis University School of Medicine, Saint Louis, MO, USA.
Methods Mol Biol. 2017;1639:227-234. doi: 10.1007/978-1-4939-7163-3_23.
Alpha-1 antitrypsin (a1AT) deficiency, in its classical form, is an autosomal recessive disease associated with an increased risk of liver disease in adults and children, and with lung disease in adults. The vast majority of liver disease is associated with homozygosity for the Z mutant allele, also called PiZZ. This homozygous allele synthesizes large quantities of a1AT mutant Z protein in the liver, but the mutant protein also folds improperly during biogenesis. As a result, approximately 85% of the molecules are retained within the hepatocytes instead of being appropriately secreted. The resulting low, or "deficient," serum level leaves the lungs vulnerable to inflammatory injury from uninhibited neutrophil proteases. Most of the mutant Z protein retained within hepatocytes is directed into intracellular proteolysis pathways, but some molecules remain in the endoplasmic reticulum for long periods of time and others adopt an unusual aggregated or "polymerized" conformation. It is thought that these intracellular polymers trigger a cascade of intracellular injury which can lead to end organ liver injury including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. It is widely accepted that the disease causing factor in mutant Z-alpha-1 antitrypsin deficiency (AATD-Z) is the toxic build-up of the mutant Z protein. Since misfolding of some but not all of the Z protein during its maturation leads to homopolymerization, an assay to assess the amount of normally folded ATZ and accumulated polymeric ATZ would be very useful. Here we describe a method to semiquantitatively assess these two fractions in a tissue or cell culture source.
α-1抗胰蛋白酶（a1AT）缺乏症的经典形式是一种常染色体隐性疾病，与成人和儿童患肝病的风险增加以及成人患肺病的风险增加有关。绝大多数肝病与Z突变等位基因的纯合性有关，也称为PiZZ。这种纯合等位基因在肝脏中合成大量的a1AT突变Z蛋白，但突变蛋白在生物合成过程中也会错误折叠。结果，大约85%的分子保留在肝细胞内，而不是被适当分泌。由此导致的低血清水平，即“缺乏”，使肺部容易受到未受抑制的中性粒细胞蛋白酶的炎症损伤。保留在肝细胞内的大多数突变Z蛋白被导向细胞内蛋白水解途径，但一些分子会在很长一段时间内留在内质网中，而其他分子则呈现出异常的聚集或“聚合”构象。人们认为，这些细胞内聚合物会引发一系列细胞内损伤，从而导致包括慢性肝炎、肝硬化和肝细胞癌在内的终末器官肝损伤。人们普遍认为，突变型Z-α-1抗胰蛋白酶缺乏症（AATD-Z）的致病因素是突变型Z蛋白的毒性积累。由于Z蛋白在成熟过程中部分而非全部错误折叠会导致同聚物形成，因此一种评估正常折叠的ATZ和积累的聚合ATZ量的检测方法将非常有用。在这里，我们描述了一种在组织或细胞培养源中半定量评估这两个部分的方法。
