Saudi Cultural Missions Theses & Dissertations
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Item Restricted The molecular basis of pathological alpha-1-antitrypsin polymerisation(University College London, 2025) Aldobiyan, Ibrahim Fahad I; Lomas, David A; Irving, James A; Orlova, Elena VAlpha-1-antitrypsin deficiency (AATD) is a rare protein misfolding disease, primarily afflicting Northern European and Iberian populations. The deficiency is due to mutations in the gene encoding alpha-1-antitrypsin which promote its misfolding and subsequent aggregation into polymeric chains within hepatocytes. This results in both gain-of- toxicity and loss-of-function phenotypes, with accumulating polymers inducing cellular damage to hepatocytes, causing liver cirrhosis in the long term. The marked reduction in circulating antitrypsin levels leads to early-onset emphysema. AATD has been studied for over half a century and the mechanism defining the polymerisation pathway in molecular detail remains elusive. Several theories have been proposed, with varying degrees of experimental support, on artificially inducedantitrypsin polymers. We propose that understanding the nature of the intermolecular linkage between the monomeric subunits of ex vivo liver-derived antitrypsin polymers is fundamental to developing therapeutics that inhibit or reverse polymerization. Such therapeutics are necessary as currently, liver transplantation is the only means of curing the condition. The principal aim of this study is to use cryo-electron microscopy (cryo-EM) with single- particle reconstruction techniques to establish the structural changes that lead to polymer formation, using material obtained from human explant tissue. Polymers are technically challenging targets due to their flexibility and heterogeneity. Decorating antitrypsin liver polymers with Fab fragments of known epitope restricted their flexibility and provided valuable orientational information, assisting particle alignment. Despite the challenging sample preparation, the structure of the monomeric subunit of alpha-1-antitrypsin has successfully been reconstructed to 4.2 Å resolution. This reconstruction, in the context of other experiments, allows conclusions to be drawn regarding the polymerisation mechanism, and its implications for the polymerisation of other members of the serpin family. It has been established that an early step in polymerisation involves a monomeric activated state, M*, with perturbations in a region known as the breach. A secondary aim of this project is to better understand the role of the breach region of AAT in the polymerisation pathway. Site-directed mutagenesis was performed on numerous sites within and around the breach. A potent small molecule polymerisation inhibitor was used to probe the effect of these mutations on the ability of antitrypsin to transition to the intermediate M* conformation and polymerise.10 0