We reported previously that the carbohydrate domain of the amyloid precursor protein is involved in amyloid precursor protein (APP)-APP interactions. Functional in vitro studies suggested that this interaction occurs through the collagen binding site of APP. The physiological significance remained unknown, because it is not understood whether and how APP dimerization occurs in vivo. Here we report that cellular APP exists as homodimers matching best with a two-site model. Consistent with our published crystallographic data, we show that a deletion of the entire sequence after the kunitz protease inhibitor domain did not abolish APP homodimerization, suggesting that two domains are critically involved but that neither is essential for homodimerization. Finally, we generated stabilized dimers by expressing mutant APP with a single cysteine in the ectodomain juxtamembrane region. Mutation of Lys 624 to cysteine produced ϳ6 -8-fold more A␤ than cells expressing normal APP. Our results suggest that amyloid A␤ production can in principle be positively regulated by dimerization in vivo. We suggest that dimerization could be a physiologically important mechanism for regulating the proposed signal activity of APP.

We reported previously that stabilized ␤-amyloid peptide dimers were derived from mutant amyloid precursor protein with a single cysteine in the ectodomain juxtamembrane position. In vivo studies revealed that two forms of SDS-stable A␤ homodimers exist, species ending at A␤40 and A␤42. The phenomenon of the transformation of the initially deposited 42-residue ␤-amyloid peptide into the amyloid fibrils of Alzheimer's disease plaques remains to be explained in physical terms, i.e. energetically and structurally. We therefore performed spectroscopic analyses revealing that engineered dimeric peptides ending at residue 42 displayed a much more pronounced ␤-structural transition than corresponding monomers. Specifically, the single chemically induced dimerization of A␤ peptides significantly increased the ␤-sheet content by a factor of 2. The Cterminal residues Ile-41 and Ala-42 of dimeric forms further increased the ␤-sheet content by roughly one-third. In contrast to A␤42, the ␤-sheet content of the ␣-and ␥-secretase-generated p3 fragments did not necessarily correlate with the tendency to form fibrils, although p3/17-42 had a pronounced thread forming character with fibril lengths of up to 2.5 m. Electron microscopic images show that forms of p3/17-42 generated smaller granular particles than forms ending at residue 40. We discuss these findings in terms of A␤1-42 dimers representing paranuclei, which self-aggregate into ribbonlike ordered fibrils by elongation. Based on A␤42 dimerspecific titers of a polyclonal antiserum we propose that the A␤ homodimer represents a nidus for plaque formation and a well defined novel therapeutic target.In the amyloidogenic processing of APP, 1 the A␤ peptide is produced, circulates extracellularly, and usually does not deposit as plaques (1, 2). In the secretory pathway, APP is cleaved C-terminal from A␤ residue 16, thus precluding the formation of full-length A␤1-40/42 but generating 3-kDa fragments, termed A␤17-40/42 or p3/40/42 (3, 4). Familial AD-linked mutations in presenilins 1 and 2 and APP can cause Alzheimer's disease (AD) by increasing the cellular production of A␤42 (5), thereby accelerating the polymerization of A␤42 and promoting cerebral accumulation of A␤ as an essential early event in AD pathogenesis (6, 7). Furthermore, peptides ending at A␤42, i.e. A␤1-42 and A␤17-42, appear to be a major constituent of the diffuse plaques seen initially in AD and Down syndrome, and they have been proposed to serve as a nidus for the aggregation of the more abundant A␤1-40 peptides (8, 9). The presence of mixtures of A␤ assemblies, ranging from monomers to insoluble amyloid fibrils, has made it difficult to ascribe amyloidogenicity or toxicity principally to one or another A␤ species. Naturally secreted oligomers of A␤ are believed to have a direct role in AD pathology, both because of their short and long term neurotoxic effects and their ability to give rise to fibrillar assemblies characterized by the adopted ␤-sheet structure (10 -12). Fibrillar A␤ has been observed to bind...

This paper presents an extended study on the ion effects of a series of biocompatible hydrated choline based ionic liquids (ILs) on lactate oxidase (LOx), an important enzyme in biosensing technology for the in vitro detection of lactic acid. Secondary structural analysis revealed changes in the protein conformation in hydrated ILs, while thermal unfolding/aggregation dynamics showed different profiles in the presence or absence of ILs. Moreover, LOx thermally denaturised at 90 1C showed residual activity in the presence of chloride and dihydrogen phosphate anions. Kinetic and lifetime studies were also performed, providing a better understanding of the ion effects of ILs on the biocatalytic activity of the enzyme.

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