Abstract
The serpin alpha1-antichymotrypsin is a major component of brain amyloid plaques in Alzheimer's disease. In vitro alpha1-antichymotrypsin interacts with the Alzheimer's amyloid peptide Abeta1-42 and stimulates both formation and disruption of neurotoxic Abeta1-42 fibrils in a concentration-dependent manner. We have constructed a new hybrid model of the complex between Abeta1-42 and alpha1-antichymotrypsin in which both amino and carboxyl sequences of Abeta1-42 insert into two different beta-sheets of alpha1-antichymotrypsin. We have tested this model and shown experimentally that full-length and amino-terminal segments of Abeta1-42 bind to alpha1-antichymotrypsin as predicted. We also show that Abeta1-42 forms both intra- and intermolecular SDS-stable complexes with alpha1-antichymotrypsin and that the binding of Abeta1-42 to alpha1-antichymotrypsin abolishes the inhibitory activity of the latter and its ability to form stable complex with chymotrypsin. The existence of both inter- as well as intramolecular complexes of Abeta1-42 explains the nonlinear concentration-dependent effects of alpha1-antichymotrypsin on Abeta1-42 fibril formation, which we have reinvestigated here over a broad range of Abeta1-42:alpha1-antichymotrypsin ratios. These data suggest a molecular basis for the distinction between amorphous and fibrillar Abeta1-42 in vivo. The reciprocal effects of Abeta1-42 and alpha1-antichymotrypsin could play a role in the etiology of Alzheimer's disease.
Highlights
␣1-antichymotrypsin (ACT)1 is a serpin serine proteinase inhibitor with specificity for cathepsin and chymotrypsin-like enzymes [1]
As in other serpins [12], the reactive site loop in the cleaved form of ACT is inserted into a preexisting, flexible -sheet [4], and this cleaved form is stabilized to denaturation relative to the uncleaved native form [2]
Preincubation of ACT with A2–9 (Fig. 1A, lane f) followed by incubation with A1–42 showed no formation of the higher molecular weight ACT1⁄7A1–42 band
Summary
Complexes of A with ACT (Recombinant)—A1–42 (Saveen, Denmark), A2–9 (Commonwealth Biotechnologies), and A1–11 (Bachem) were Ͼ98% pure. 125I-A1–42 and 125I-A1–11 were made by the method of Thorell and Johansson [24] using chloramine T, purified on a Sephadex PD-10 column (G25M), and stored in phosphate-buffered saline, pH 7.4, containing 0.02% sodium azide. An aliquot of lyophilized A1–42 equal to 100- or 200-fold molar excess over ACT was dissolved in sterile 0.015 M Tris, pH 7.4, 0.15 M NaCl and combined with ACT (29.7 M). This mixture was incubated under sterile conditions for various time periods from overnight to 3 weeks. Labeled A1–11 was mixed with unlabeled peptide to a final specific activity of approximately 2 mCi/mmol and incubated with ACT at a molar ratio of 200:1 (peptide:ACT) in phosphate-buffered saline for 24 and 48 h at room temperature. There are no constraints in this model for the structure of the strand s4A reactive loop strand and strand s1C of ACT and for residues 10 –27 of the bound A1–42 in the complex, both of which are depicted by a dotted line, as shown in Fig. 3, with unknown structure
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