MOLECULAR CHARACTERISTICS AND CLINICAL ASPECTS OF ALPHA 1-ANTITRYPSIN DEFICIENCY

α1-抗胰蛋白酶缺乏症的分子特征和临床表现

• 批准号: 6162714
• 负责人: J MOSS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6162714
• 关键词: alpha 1 antitrypsin; alpha 1 antitrypsin deficiency; asthma; bronchodilators; clinical research; cycloheximide; enzyme induction /repression; human therapy evaluation; immunoglobulin E; metabolism disorder chemotherapy; molecular pathology; patient /disease registry; protein degradation; respiratory disorder chemotherapy; respiratory function

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.

alpha-1-抗替丁蛋白（AAT）的突变Z形式负责更多 所有患有AAT缺乏症的人中的95％，这是一个重要的继承 肺气肿和肝病的原因。 Z AAT基因编码蛋白质 带有单个氨基酸的变化，会导致细胞内错误折叠。 由于分泌的Z AAT是一种功能性抗蛋白酶，我们假设 中断保留的Z AAT的分解代谢可能会增加运输的运输 Z AAT从细胞中脱出，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定抑制剂， 乳酸，阻止了Z AAT的细胞内降解。 而且， 这种降解的抑制与部分恢复有关 Z AAT的囊泡运输。在模型中观察到了这种效果 转染的CHO细胞以及人类肺泡巨噬细胞的系统 合成z aat。这项研究表明，改变 错误折叠蛋白的细胞内命运可能是一种选择 治疗与错误折叠有关的疾病，但可能 功能蛋白。 在粗糙的内质中保留AAT 网状（RER）似乎是逮捕AAT的重要一步 包括Z AAT在内的几种缺陷变体的分泌。重新保留 z AAT聚合通过将反应性位点环（RSL）插入 另一个Z分子的部分打开B板A。循环表 聚合可能在保留某些截断的AAT中发挥作用 蛋白质。基于对AAT和其他Serpin突变体的分析，它具有 被提出，这些蛋白质的表A可以用作“快门”， 这使RSL可以插入A板A，从而导致结构 变化并增加蛋白质稳定性。我们假设这不安 直接在B纸A下包装可能允许Serpin快门打开 作为循环单位聚合，保留和加速的序幕 细胞内降解。确定在纸上打包是否包装 影响RSL，蛋白质稳定性和细胞内的构象 半衰期，我们在体外翻译了AAT，在转染的CHO细胞中翻译 用正常的AAT或在B板A处终止的变体 密码子373或376，远远超出了RSL。我们的结果支持该假设 增加的聚合可能与Serpin快门开放有关 在包装下，将RSL插入B板A 百叶窗受到干扰。这种聚合可能是一种重要的机制 负责细胞内保留和加速降解 AAT的某些截短形式。中性磷脂防御素是高度细胞毒性的 占中性粒细胞蛋白质重量的30％的分子。他们 具有广泛的靶标，包括细菌，病毒，真菌和 人上皮细胞。重要的是，中性粒细胞的细胞毒性 防御素被α-1-抗抗肽消除。我们已经确定 平均而言，存在更多的人类嗜中性粒细胞防御素 在α-1-抗抗素的上皮衬里中缺乏 轻度肺功能障碍的个体比正常人中的个体。 此外，我们已经确定中性粒细胞防御素的量 与中性粒细胞弹性酶的量和数量相关 中性粒细胞。最近，我们证明了防御素是细胞毒性的 对于肺泡巨噬细胞，重要的是，刺激它们释放 强大的趋化剂LTB4的数量为5倍未刺激的元素 细胞。