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Mutants of alpha‐1‐antitrypsin cause the protein to self‐associate and form ordered aggregates ('polymers') that are retained within hepatocytes, resulting in a predisposition to the development of liver disease. The associated reduction in secretion, and for some mutants, impairment of function, leads to a failure to protect lung tissue against proteases released during the inflammatory response and an increased risk of emphysema. We report here a novel deficiency mutation (Gly192Cys), that we name the Sydney variant, identified in a patient in heterozygosity with the Z allele (Glu342Lys). Cellular analysis revealed that the novel variant was mostly retained as insoluble polymers within the endoplasmic reticulum. The basis for this behaviour was investigated using biophysical and structural techniques. The variant showed a 40% reduction in inhibitory activity and a reduced stability as assessed by thermal unfolding experiments. Polymerisation involves adoption of an aggregation‐prone intermediate and paradoxically the energy barrier for transition to this state was increased by 16% for the Gly192Cys variant with respect to the wild‐type protein. However, with activation to the intermediate state, polymerisation occurred at a 3.8‐fold faster rate overall. X‐ray crystallography provided two crystal structures of the Gly192Cys variant, revealing perturbation within the 'breach' region with Cys192 in two different orientations: in one structure it faces towards the hydrophobic core while in the second it is solvent‐exposed. This orientational heterogeneity was confirmed by PEGylation. These data show the critical role of the torsional freedom imparted by Gly192 in inhibitory activity and stability against polymerisation. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate serine proteases in the central coagulation and immune pathways, representing 2–10% of circulating proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian proteolytic pathways. Through interacting with extracellular and intracellular serine and cysteine proteases, viral serpins provide a new class of highly active virus-derived coagulation-, immune-, and apoptosis-modulating drug candidates. Viral serpins have unique characteristics: (1) function at micrograms per kilogram doses; (2) selectivity in targeting sites of protease activation; (3) minimal side effects at active concentrations; and (4) the demonstrated capacity to be modified, or fine-tuned, for altered protease targeting. To date, the virus-derived serpin class of biologics has proven effective in a wide range of animal models and in one clinical trial in patients with unstable coronary disease. Here, we outline the known viral serpins and review prior studies with viral serpins, considering their potential for application as new sources for immune-, coagulation-, and apoptosis-modulating therapeutics. [ABSTRACT FROM AUTHOR]

The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site-directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early-onset dementia. Our findings indicate that N401 glycosylation reports lability of the C-terminal end of neuroserpin in its native state. This C-terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant-specific N-linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin. [ABSTRACT FROM AUTHOR]

Serpins are protease inhibitors whose most stable state is achieved upon transition of a central 5-stranded β-sheet to a 6-stranded form. Mutations, low pH, denaturants and elevated temperatures promote this transition, which can result in a growing polymer chain of inactive molecules. Different types of polymer are possible, but, experimentally only heat has been shown to generate polymers in vitro consistent with ex vivo pathological specimens. Many mutations that alter the rate of heatinduced polymerization have been described, but interpretation is problematic because discrimination is lacking between the effect of global changes in native stability and specific effects on structural mechanism. We show that the temperature midpoint (Tm) of thermal denaturation reflects the transition of α1- antitrypsin to the polymerization intermediate, and determine the relationship with fixed-temperature polymerization halftimes (t0.5) in the presence of stabilizing additives [TMAO (trimethylamine W-oxide), sucrose and sodium sulfate], point mutations and disulfide bonds. Combined with a retrospective analysis of 31 mutants characterized in the literature, the results of the present study show that global changes to native state stability are the predominant basis for the effects of mutations and osmolytes on heat-induced polymerization, summarized by the equation: ln(t0.5_mutant/t0.5wild-type) = 0.34 x ΔTm. It is deviations from this relationship that hold key information about the polymerization process. [ABSTRACT FROM AUTHOR]

The common severe Z mutation (E342K) of a1-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular 'snapshots', the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α1-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P14P12 and P4, but not P10P8 or P2P1, resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)- based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P14P12 region self-inserts early during activation, while the interaction between P6P4 and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P6P4, but not P14P12, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol-1. [ABSTRACT FROM AUTHOR]

Abstract: Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α1-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α1-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α1-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation. [Copyright &y& Elsevier]

Serpins utilize conformational change to inhibit target proteinases; the price paid for this conformational flexibility is that many undergo temperature-induced polymerization. Despite this thermolability, serpins are present in the genomes of thermophilic prokaryotes, and here we characterize the first such serpin, thermopin. Thermopin is a proteinase inhibitor and, in comparison with human α1-antitrypsin, possesses enhanced stability at 60°C. The 1.5 A˚ crystal structure reveals novel structural features in regions implicated in serpin folding and stability. Thermopin possesses a C-terminal “tail” that interacts with the top of the A β sheet and plays an important role in the folding/unfolding of the molecule. These data provide evidence as to how this unusual serpin has adapted to fold and function in a heated environment. [Copyright &y& Elsevier]