The molecular basis of polymer accumulation in alpha-1-antitrypsin deficiency (360G-Wellcome-211980_Z_18_Z)

α1-Antitrypsin, a circulating protein inhibitor of neutrophil elastase, is normally secreted by hepatocytes; the most common pathological ('Z') mutant of this protein results in the formation of an ordered aggregate (‘polymer’) that largely accumulates within the cell instead. The result is the formation of insoluble protein deposits within the liver, with a consequent toxic-gain-of- function phenotype (neonatal hepatitis and cirrhosis) and loss-of-function phenotype within the lung (early-onset emphysema, due to a protease- antiprotease imbalance). Recent data suggests that there is a partition between soluble and insoluble polymer populations within the cell. The overall goal of this study is to determine, at a molecular level, the characteristics of these polymer forms, to address the question: is there evidence for a 'decision point' that determines whether a polymer will accumulate in the insoluble fraction and thereby contribute to the liver polymer burden? To achieve this, biochemical and biophysical techniques - including SEC-MALS, immunoassays, native PAGE, and concanavalin A affinity - will be applied to α1-antitrypsin polymers (i) induced artificially in vitro, (ii) extracted from a mammalian model cell line, and (iii) previously extracted from a tissue sample. In addition, FRET and filter-trap assays will be used to study the kinetic mechanism in vitro.