We have used the yeast two-hybrid system to isolate cDNAs encoding proteins that specifically interact with the 42-aa ␤-amyloid peptide (A␤), a major constituent of senile plaques in Alzheimer's disease. The carboxy terminus of ␣ 2 -macroglobulin (␣2M), a proteinase inhibitor released in response to inf lammatory stimuli, was identified as a strong and specific interactor of A␤, utilizing this system. Direct evidence for this interaction was obtained by co-immunoprecipitation of ␣2M with A␤ from the yeast cell, and by formation of SDS-resistant A␤ complexes in polyacrylamide gels by using synthetic A␤ and purified ␣2M. The association of A␤ with ␣2M and various purified amyloid binding proteins was assessed by employing a method measuring proteinprotein interactions in liquid phase. The dissociation constant by this technique for the ␣2M-A␤ association using labeled purified proteins was measured (K d ‫؍‬ 350 nM). Electron microscopy showed that a 1:8 ratio of ␣2M to A␤ prevented fibril formation in solution; the same ratio to A␤ of another acute phase protein, ␣ 1 -antichymotrypsin, was not active in preventing fibril formation in vitro. These results were corroborated by data obtained from an in vitro aggregation assay employing Thiof lavine T. The interaction of ␣2M with A␤ suggests new pathway(s) for the clearance of the soluble amyloid peptide.Senile plaques in the brain and cerebral blood vessels of patients with Alzheimer's disease are composed primarily of the aggregated form of A␤ (1, 2). The A␤ peptide is derived post-translationally by proteolytic activity from a larger amyloid precursor protein (3-10). The mechanism for A␤ clearance or for its deposition is not known. Two proteinase inhibitors, ␣ 2 -macroglobulin (␣2M) and ␣ 1 -antichymotrypsin (␣1ACT), have been identified as being associated with senile plaques (11,12,13). ␣2M is capable of binding to and blocking the proteolytic activity of most proteinases before rapid clearance of these ␣2M -proteinase complexes by the low density lipoprotein receptor-related protein (LRP). Internalization and degradation of ␣1ACT-proteinase complexes are mediated by the serpin-enzyme complex receptor. Significantly increased levels of both ␣2M and ␣1ACT are often found in localized areas of inflammation (14,15,16). The full range of biological activities of ␣2M and ␣1ACT still remains to be defined.In an effort to identify proteins that interact in vivo with A␤ and therefore might play a role in its clearance or deposition, we screened a HeLa library using the yeast two-hybrid system (17-21). One of the proteins determined to have a strong and specific interaction with A␤ was ␣2M. To examine the possible role of this interaction in neurotoxic amyloid fibril formation, we investigated the following: (i) the in vivo binding of A␤ to ␣2M in the yeast cell; (ii) the in vitro binding affinity of A␤ to ␣2M compared with that of A␤ to other amyloid-binding proteins; and (iii) the effect of ␣2M, ␣1ACT, and apolipoprotein (apo) J on fibril formation. EXPERIMENTA...

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