Defining the mechanism of polymerization in the serpinopathies

Abstract

The serpinopathies result from the ordered polymerization of mutants of members of the serine proteinase inhibitor (serpin) superfamily. These polymers are retained within the cell of synthesis where they cause a toxic gain of function. The serpinopathies are exemplified by inclusions that form with the common severe Z mutant of α 1 -antitrypsin that are associated with liver cirrhosis. There is considerable controversy as to the pathway of serpin polymerization and the structure of pathogenic polymers that cause disease. We have used synthetic peptides, limited proteolysis, monoclonal antibodies, and ion mobility-mass spectrometry to characterize the polymerogenic intermediate and pathological polymers formed by Z α 1 -antitrypsin. Our data are best explained by a model in which polymers form through a single intermediate and with a reactive center loop-β-sheet A linkage. Our data are not compatible with the recent model in which polymers are linked by a β-hairpin of the reactive center loop and strand 5A. Understanding the structure of the serpin polymer is essential for rational drug design strategies that aim to block polymerization and so treat α 1 -antitrypsin deficiency and the serpinopathies.alpha-1-antitrypsin deficiency | protein folding | serpins | polymers | cirrhosis

Update on alpha-1 antitrypsin deficiency: New therapies

“…This antibody binds polymers formed by heating monomeric α 1 -antitrypsin, but not those formed by refolding from guanidine and urea [15]. Our NMR studies followed the polymerisation of Queen's (Lys154Asn) α 1 -antitrypsin under physiological conditions or in urea.…”

“…The application of heat to monomeric α 1 -antitrypsin recapitulated the 2C1 neo-epitope of polymers associated with disease [15].…”

“…The patent β-sheet A can then accept insertion of the reactive site loop motif. Sequential insertion of the loop of one α 1 -antitrypsin molecule into β-sheet A of a neighbour to form first a loop-sheet dimer, and then longer species linking more molecules, is the simplest model to explain the formation of elongated polymers [2,8,15] ( Fig. 1B(i)).…”

Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

“…While denaturant can induce polymerization at concentrations that favour population of an unfolding intermediate state (I denat ) [6,20], observations indicate that there are multiple pathways favoured depending on the manner in which polymers are formed, and as a result intermediate ensembles represented by I pol and I denat could be structurally distinct [19]. In support of this, it has recently been shown that polymers produced in the presence of denaturant lack an epitope that is expressed on polymer obtained from patient samples [18,21]. Given this diversity it is unsurprising that there are currently three main models for the terminal polymer that differ from one another in fundamental respects ( Figure 1B).…”

“…During the activation to intermediate, there is a change in far-UV circular dichroic signal, intrinsic tryptophan fluorescence, the interaction with ANS (8-anilinonaphthalene-1-sulfonic acid) or bis-ANS (4,4 -dianilino-1,1 -binaphthyl-5,5 -disulfonic acid) dyes [14,17], ion mobility mass spectrum collision crosssectional area [18] and NMR cross-peaks [19]. While denaturant can induce polymerization at concentrations that favour population of an unfolding intermediate state (I denat ) [6,20], observations indicate that there are multiple pathways favoured depending on the manner in which polymers are formed, and as a result intermediate ensembles represented by I pol and I denat could be structurally distinct [19].…”