Protein Misfolding and the Serpinopathies

The serpins are the largest superfamily of protease inhibitors. They are found in almost all branches of life including viruses, prokaryotes and eukaryotes. They inhibit their target protease by a unique mechanism that involves a large conformational transition and the translocation of the enzyme from the upper to the lower pole of the protein. This complex mechanism, and the involvement of serpins in important biological regulatory processes, makes them prone to mutation-related diseases. For example the polymerization of mutant α₁-antitrypsin leads to the accumulation of ordered polymers within the endoplasmic reticulum of hepatocytes in association with cirrhosis. An identical process in the neuron specific serpin, neuroserpin, results in the accumulation of polymers in neurons and the dementia FENIB. In both cases there is a clear correlation between the molecular instability, the rate of polymer formation and the severity of disease. A similar process underlies the hepatic retention and plasma deficiency of antithrombin, C1 inhibitor, α₁-antichymotrypsin and heparin co-factor II. The common mechanism of polymerization has allowed us to group these conditions together as a novel class of disease, the serpinopathies.Key Words: serpins, α1-antitrypsin, neuroserpin, polymerization, dementia, conformational disease, serpinopathiesSerpins (or serine protease inhibitors) are the largest family of protease inhibitors. They have been found in all major branches of life including viruses, prokaryotes and eukaryotes.^1–3 Despite their name there is increasing evidence that serpins can also inhibit other classes of proteases as demonstrated by the viral serpin CrmA and recently by a plant serpin, serpin1.^4,5 They can even play a non-inhibitory role in events as diverse as blood pressure regulation (angiotensinogen), chromatin condensation (MENT), tumor progression (maspin), protein folding (hsp47) and hormone transport (cortisol and thyroxine binding globulin).⁶One of the most important roles of serpins is the regulation of enzymes involved in proteolytic cascades. Among these serpins are α₁-antitrypsin, α₁-antichymotrypsin, C1 inhibitor, antithrombin and plasminogen activator inhibitor-1, which play an important role in the control of proteases involved in the inflammatory, complement, coagulation and fibrinolytic pathways, respectively.^1,3 The serpin superfamily is characterised by more than 30% homology with the archetypal serpin α₁-antitrypsin and conservation of tertiary structure.^7,8 Serpins adopt a metastable conformation composed in most cases of 9 α-helices, three β-sheet (A to C) and an exposed mobile reactive centre loop (RCL). This flexible RCL typically contains 20 residues that act as a pseudo substrate for the target protease (Fig. 1A).^9–15 After formation of a Michaelis complex^16,17 the enzyme cleaves the P1-P1′ bond of the serpin, releasing the P1'' residue and forming an ester bond between the protease and the serpin.^18,19 This is then followed by a dramatic conformational transition from a stressed to relaxed conformation with the enzyme being pulled from the upper to the lower pole of the serpin and the insertion of the reactive loop as an extra strand in β-sheet A.^20–25 As a consequence of this conformational change the thermal stability of the serpin is greatly enhanced. Whereas a typical serpin in its native state exhibits a midpoint of thermal denaturation of around 50–60°C, a cleaved serpin with its RCL fully incorporated into β-sheet A denatures at temperatures >120°C.^9,26,27 Another consequence is the inactivation of the enzyme, stabilised at the acyl-intermediate and unable to proceed further to deacylation of the complex.^24,28 This serpin-protease complex then binds to members of the lipoprotein receptor family and is cleared from the circulation.^29–31Open in a separate windowFigure 1Inhibition of neutrophil elastase by α₁-antitrypsin and the structural basis of polymerization. (A) After docking (left) the neutrophil elastase (grey) is inactivated by movement from the upper to the lower pole of the protein (right). This is associated with the insertion of the RCL (red) as an extra strand into β-sheet A (green). (B) The structure of α₁-antitrypsin is centred on β-sheet A (green) and the mobile reactive centre loop (red). Polymer formation results from the Z variant of α₁-antitrypsin (Glu342Lys at P17; indicated by arrow) or mutations in the shutter domain (blue circle) that open β-sheet A to favour partial loop insertion and the formation of an unstable intermediate (M*). The patent β-sheet A then accepts the loop of another molecule to form a dimer (D), which then extends into polymers (P). The individual molecules of α₁-antitrypsin within the polymer, although identical, are coloured red, yellow and blue for clarity. Figure reproduced with permission from Lomas et al.⁹⁷Despite the evolutionary advantage conferred upon serpins by the remarkable mobility of the native state, their complexity is also their weak point.^19,32 Mutations affecting the serpins can lead to a variety of diseases, resulting from either a gain or loss of function.^6,19 For example mutations can cause aberrant conformational transitions that result in the retention of the serpin within the cell of synthesis. This will lead to either protein overload and death of the cell in which the serpin is synthesised, or disease as a consequence of the resulting plasma deficiency. Such a mechanism underlies diseases as diverse as cirrhosis, thrombosis, angio-oedema, emphysema and dementia. We review here the common mechanism underlying these diseases that we have grouped together as the serpinopathies.^33–35 The aggregation and accumulation of conformationally destabilized proteins is an important feature of many neurodegenerative diseases, including Alzheimer''s and Parkinson''s disease and the spongiform encephalopathies. Indeed we have used the serpinopathies as a paradigm for these other ‘conformational diseases’.³⁶