No abstract

The mutation in the Z deficiency variant of ␣ 1 -antitrypsin perturbs the structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepatocytes to form inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The process of polymer formation has been investigated here by intrinsic tryptophan fluorescence, fluorescence polarization, circular dichroic spectra and extrinsic fluorescence with 8-anilino-1-naphthalenesulfonic acid and tetramethylrhodamine-5-iodoacetamide. These biophysical techniques have demonstrated that ␣ 1 -antitrypsin polymerization is a twostage process and have allowed the calculation of rates for both of these steps. The initial fast phase is unimolecular and likely to represent temperature-induced protein unfolding, while the slow phase is bimolecular and associated with loop-sheet interaction and polymer formation. The naturally occurring Z, S, and I variants and recombinant site-directed reactive loop and shutter domain mutants of ␣ 1 -antitrypsin were used to demonstrate the close association between protein stability and rate of ␣ 1 -antitrypsin polymerization. Taken together, these data allow us to propose a kinetic mechanism for ␣ 1 -antitrypsin polymer formation that involves the generation of an unstable intermediate, which can form polymers or generate latent protein.␣ 1 -Antitrypsin is a member of the serpin protein superfamily that encompasses a wide range of serine proteinase inhibitors involved in coagulation, inflammation, fibrinolysis, and the complement cascade (1-3). The members of the superfamily have a common tertiary structure based on a central ␤-sheet (sheet A), surrounded by two other sheets (B and C) and a mobile, inhibitory reactive center loop (Fig. 1). The mechanism of action of these proteins as proteinase inhibitors is most unusual in comparison to other serine protease inhibitors, as cleavage of the P 1 -P 1 Ј bond by non-target enzymes causes a large rearrangement of the serpin with the loop incorporated into the A ␤-sheet and the P 1 and P 1 Ј residues separated by over 60 Å (4 -6). The loop may be stably incorporated into the A ␤-sheet in the absence of cleavage by formation of the latent species (7-10). This is typically prepared by heating serpins for prolonged periods in the presence of stabilizing concentrations of sodium citrate (8, 10, 11). The description of this conformational change from x-ray crystal structures of the native (7,8,(12)(13)(14)(15), latent (7-9), and cleaved (4 -6, 16) forms of the serpins, combined with the fact that the cleaved and latent forms have a much higher stability compared with the native species (10,(17)(18)(19)(20), has led to the suggestion that the serpin fold is metastable (21, 22).In 1992, we showed that severe deficiency of the Z variant of ␣ 1 -antitrypsin (Glu-342 3 Lys) resulted from a conformational transition and a unique linkage between the reactive center loop of one molecule and ...

Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutations, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

The reactive site loop of the serpin family of serine proteinase inhibitors is flexible and can adopt a number of diverse conformations. A 2.9 A resolution structure of alpha 1-antitrypsin-the principal proteinase inhibitor in human plasma-shows the loop in a stable canonical conformation matching that found in all other families of serine proteinase inhibitors. This unexpected finding in the absence of loop insertion into the body of the molecule favours a two-stage mechanism of inhibition and provides a model for the heparin activation of antithrombin. The beta-pleated strand conformation of the loop also accounts for the polymerization of the serpins in disease and for their association with other beta-sheet structures, most notably the beta-amyloid of Alzheimer's disease.