MOLECULAR CHARACTERISTICS AND CLINICAL ASPECTS OF ALPHA 1-ANTITRYPSIN DEFICIENCY

α-1-抗胰蛋白酶（AAT）的突变Z型负责更多的 超过95%的人患有AAT缺乏症，这是一种重要的遗传性疾病。 导致肺气肿和肝病。Z AAT基因编码一种蛋白质 一个氨基酸的改变会导致细胞内的错误折叠 由于分泌的ZAAT是一种功能性抗蛋白酶，我们假设， 阻断滞留的Z-AAT的催化作用可能会增加 Z AAT的细胞外，并允许部分恢复细胞外 抗蛋白酶保护蛋白质翻译抑制剂 环己酰亚胺和蛋白酶体功能的特异性抑制剂， lactacystin阻止ZAAT的细胞内降解。 此外，委员会认为， 这种降解抑制与部分恢复有关 囊泡运输的影响。在一个模型中观察到了这种效应 转染的CHO细胞系统以及人肺泡巨噬细胞 合成Z AAT。这项研究表明，改变 错误折叠蛋白质的细胞内命运可能是一种选择， 治疗与错误折叠相关的疾病，但可能 功能蛋白质 AAT在粗面内质网中的滞留 内质网（RER）似乎是阻止AAT的重要步骤 包括ZAAT在内的几种缺陷变体的分泌。保留的 Z AAT通过将反应位点环（RSL）插入到聚合物中而聚合。 另一个Z分子的部分打开的B片A。活套单 聚合可能在某些截短AAT的RER保留中起作用 proteins.基于对AAT和其他SERPIN突变体的分析， 有人提出，这些蛋白质的A片层可能充当“快门”， 这允许RSL插入到片材A中，导致结构 改变和增加蛋白质稳定性。我们假设 直接在B页A下面包装可使SERPIN快门打开 作为环片聚合的前奏，RER保留和加速 细胞内降解确定是否在A页下包装 影响RSL的构象、蛋白质稳定性和细胞内 半衰期，我们使用体外翻译的AAT和转染的CHO细胞 具有正常AAT或在B表A下终止的变体， 密码子373或376，远远超过RSL。我们的结果支持这一假设 增加的聚合可能与SERPIN快门打开有关， 以及将RSL插入到B片材A中， 快门被干扰了。这种聚合可能是一种重要的机理 负责细胞内滞留和加速降解， AAT的一些截短形式。 这些分子占中性粒细胞蛋白质重量的30%。他们 具有广泛的靶点，包括细菌、病毒、真菌和 人类上皮细胞重要的是，中性粒细胞的细胞毒性 防御素被α-1-抗胰蛋白酶消除。我们已经确定 平均而言，存在10倍以上的人中性粒细胞防御素， 在α-1-抗胰蛋白酶缺乏的上皮衬里液中， 轻度肺功能障碍的个体比正常人。 此外，我们已经确定，中性粒细胞防御素的量 与中性粒细胞弹性蛋白酶的量和 中性粒细胞最近，我们已经证明，防御素是细胞毒性的， 重要的是，刺激它们释放 强大的化学引诱剂LTB 4的量是未刺激的5倍 细胞

The mutant Z form of alpha-1-antitrypsin (AAT) is responsible for more than 95% of all individuals with AAT deficiency, an important inherited cause of emphysema and liver disease. The Z AAT gene encodes a protein with a single amino acid change which causes intracellular misfolding. Since secreted Z AAT is a functional antiprotease, we hypothesized that interrupting catabolism of retained Z AAT might increase transport of Z AAT out of cells and allow partial restoration of extracellular antiprotease protection. Both the protein translation inhibitor cycloheximide and the specific inhibitor of proteasome function, lactacystin, prevented intracellular degradation of Z AAT. Moreover, this inhibition of degradation was associated with partial restoration of vesicular transport of Z AAT. This effect was observed in a model system of transfected CHO cells as well as in human alveolar macrophages synthesizing Z AAT. This study demonstrates that altering the intracellular fate of a misfolded protein may be an option in the treatment of diseases associated with misfolded, but potentially functional proteins. Retention of AAT in the rough endoplasmic reticulum (RER) appears to be an important step in the arrest of AAT secretion for several deficiency variants including Z AAT. RER-retained Z AAT polymerizes via insertion of a reactive site loop (RSL) into the partially open b sheet A of another Z molecule. Loop-sheet polymerization may play a role in RER retention of some truncated AAT proteins. Based on the analysis of AAT and other SERPIN mutants, it has been proposed that sheet A of these proteins may serve as a "shutter", which allows the RSL to insert into sheet A, resulting in a structural change and increased protein stability. We hypothesized that disturbed packing directly under b sheet A may allow the SERPIN shutter to open as a prelude to loop-sheet polymerization, RER retention and accelerated intracellular degradation. To determine whether packing under sheet A affects conformation of the RSL, protein stability and intracellular half-life, we used AAT translated in vitro and in CHO cells transfected with either normal AAT or variants that terminate under b sheet A at codon 373 or 376, well beyond the RSL.Our results support the hypothesis that increased polymerization may correlate with SERPIN shutter opening and insertion of the RSL into b sheet A, when packing beneath the shutter is disturbed. This polymerization may be one important mechanism responsible for intracellular retention and accelerated degradation in some truncated forms of AAT.Neutrophil defensins are highly cytoxic molecules which compose 30% of the protein weight of neutrophils. They have a wide spectrum of targets which include bacteria, virus, fungi and human epithelial cells. Importantly, the cytotoxicity of neutrophil defensins is abolished by alpha-1-antitrypsin. We have determine that on the average, there is 10 fold more human neutrophil defensins present in the epithelial lining fluid of alpha-1-antitrypsin deficient individuals with mild pulmonary dysfunction than in normals. Furthermore, we have determined that the amount of neutrophil defensins correlates with the amount of neutrophil elastase and number of neutrophils. Recently, we have demonstrated that defensins are cytotoxic for alveolar macrophages and importantly, stimulate them to release the powerful chemoattractant LTB4 in amounts 5-fold those from unstimulated cells.