Characterisation of the polymerisation properties of at-antitrypsin and its pathological Z variant
Creators: name: Levina, Vitalina
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Protease inhibitors of the serpin family are characterised by a metastable native fold which is necessary for their function. Under the influence of aberrant physical and chemical conditions or in the presence of destabilising mutations, this metastability in conformation often leads to misfolding which gives rise to thermodynamically more stable long-chain polymers. Such serpin polymer depositions found in tissue are pathological and they lead to a range of diseases, called serpinopathies. At the start of this project, the most popular models for serpin polymerisation are s4A-Reactive Centre Loop (RCL) swap, s4A-RCL/s5A swap and C-terminal swap. The current study tested these models using a proline scan method, where P9-P3 residues of RCL in UJ.antitrypsin (a1-AT) was mutated singly, in combination or together in order to map the molecular interactions of the RCL in polymerisation. It was hypothesized that if RCL becomes a ~-strand within a ~-sheet of the polymer then the rate of polymerisation would be affected. Indeed, it was found that, proline mutations at all positions, except for P5, have a significantly reduced rate of heat-induced polymerisation. Further kinetics analysis showed that proline mutations affect only the aggregation step, which is the transition state between polymerogenic intermediate and polymerisation. The ability of a1-A T to polymerise despite the proline mutations together with a shift of the "folding core" domains between the monomeric and polymeric conformations observed in this study suggests an extensive domain interchange and molecular repacking during polymerisation. Together, it provides evidence supporting the s4A-RCL/s5A or Cterminal exchange models instead of the s4A-RCL swap (also known as loop-sheet polymerisation) model. Further studies on the proline mutants also revealed the molecular details of the serpin mechanism. Notably, characterisation of the functional (inhibitory) properties of the proline mutants of a1-AT elucidated roles of the P9-P3 residues and the results correlated well with previous understanding on the functional role assigned to the individual residues of the RCL, showing that the residue at P4 is important for complex stabilisation especially during the formation of Michaelis complexes. The residue at P3 plays an important role in substrate specificity, as the Ut-AT mutant with Pro at P3 remains inhibitory against human neutrophil elastase (HNE) but is a substrate of chymotrypsin. In addition, the current study suggests that the hydrophobic pocket around P6 residue plays an important role in the progress of the RCL insertion into ~-sheet A during inhibition. Prior to the current studies, the purification of the monomeric Z a1-AT was shown to be problematic due to its high propensity to polymerise. One of the focuses of this study was to establish the protocol for the expression and purification of the most common pathological a1-AT variant, Z a1-AT, in monomeric conformation and it was successfully developed. The polymerisation analysis of Z a1-AT showed that similar to wild type UJAT, Z a1-AT polymerises via three phases. The first fast phase of polymerisation is the formation of the polymerogenic intermediate, M*. The intermediate formation is followed by the oligomerisation step, and finally by a slow phase of longer polymer formation. No concentration dependency of the Z a1-AT polymerisation was detected under the study conditions. The investigations of the interaction of the extracellular heat shock protein, clusterin, with Z a1-AT showed that under the thermal stress conditions, clusterin does not inhibit the conformational change of Z a1-AT leading to the formation of polymerogenic intermediate, M*. However, it does inhibit the formation of the large insoluble Z a1-AT aggregates. Clusterin, therefore, could be an important physiologically relevant inhibitor of the Z a1-AT polymerisation in the circulation as well as at functional sites, such as the lungs or in alveori fluids. The data presented in this thesis suggest that a1-AT undergoes a more extensive domain interchange during polymerisation supporting the s4A-RCL/s5A or C-terminal exchange models. Importantly, it was shown that Clusterin can be used as a potential therapeutics for the prevention of the build up of toxic polymers of Z a 1-AT in the circulation and at the sites of function.