MOLECULAR CHARACTERISTICS AND CLINICAL ASPECTS OF ALPHA 1-ANTITRYPSIN DEFICIENCY

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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.