Alzheimer's disease is the most fatal neurodegenerative disorder wherein the process of amyloid-beta (Abeta) amyloidogenesis appears causative. Here, we present the 3D structure of the fibrils comprising Abeta(1-42), which was obtained by using hydrogen-bonding constraints from quenched hydrogen/deuterium-exchange NMR, side-chain packing constraints from pairwise mutagenesis studies, and parallel, in-register beta-sheet arrangement from previous solid-state NMR studies. Although residues 1-17 are disordered, residues 18-42 form a beta-strand-turn-beta-strand motif that contains two intermolecular, parallel, in-register beta-sheets that are formed by residues 18-26 (beta1) and 31-42 (beta2). At least two molecules of Abeta(1-42) are required to achieve the repeating structure of a protofilament. Intermolecular side-chain contacts are formed between the odd-numbered residues of strand beta1 of the nth molecule and the even-numbered residues of strand beta2 of the (n - 1)th molecule. This interaction pattern leads to partially unpaired beta-strands at the fibrillar ends, which explains the sequence selectivity, the cooperativity, and the apparent unidirectionality of Abeta fibril growth. It also provides a structural basis for fibrillization inhibitors.

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NMR studies of amyloid b-peptides (Ab) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer's disease-related conformational polymorphism of Ab. In the aqueous medium, neither of the polypeptides Ab ox or Ab(1-42) ox (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of Ab(1-40) ox and Ab ox are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in Ab (1 -42) ox when compared to Ab(1 -40) ox . The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that Ab (1 -42) ox in aqueous solution has much higher plaquecompetence than Ab(1 -40) ox .Keywords: Alzheimer's disease; aqueous solution; NMR; polypeptide; aggregation.Alzheimer's disease (AD) is associated with the progressive accumulation of amyloid deposits in the brain and is identified by extracellular neuritic plaques and neurofibrillary tangles [1]. The major components of the plaques are polypeptides with 39 -43 amino-acid residues, which are commonly referred to as 'amyloid b-peptides' (Ab). Once it is cleaved from the much larger amyloid precursor protein (APP), which is a glycoprotein containing a single hydrophobic transmembrane region, Ab has a high propensity for the formation of b-sheet-containing aggregates [2]. Production of Ab and its aggregation and deposition appear to play a causative role in the onset of AD, and genetic factors or brain injuries may accelerate this process [3,4].Ab occurs as a heterogeneous ensemble of variant polypeptides in body fluids as well as in supernatants of cell cultures and brain amyloid deposits [5 -7]. Heterogeneity occurs at both chain ends as well as in nonterminal sequence positions. For example, a substantial proportion of the peptides in brain deposits contain methionine in position 35 as sulfoxide (Ab ox ) [6]. The heterogeneity at the C-terminus is of particular importance. About 90% of the molecules end at position 40 [Ab(x240)] and 10% at position 42 [Ab(x242)]. The extension by the dipeptide segment Ile41-Ala42 results in dramatically increased aggregation propensity when compared with the corresponding Ab(x240) [8,9]. The importance of this effect lies in the fact that some individuals with inherited early onset AD bear a mutation which increases the proportion of Ab(x242) [10][11][12].In this paper we investigate whether the well documented, widely different competence for fibril formation of Ab ox and Ab(1-42) ox [13] might be related to different conformational properties of the monomeric forms of the two polypeptides. NMR spectroscopy in aqueous solution was used to study the two polypeptides Ab ox and Ab(1 -42) ox , which were both generated in a dedicated procedure to yield monomeric species that could be dissolved i...

The full-length and ectodomain forms of ␤-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the ␤-secretase site, a critical step in the Alzheimer's disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2 and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM2DAEFR, K m ‫؍‬ 7 M, K cat ‫؍‬ 0.002 s ؊1 ; Swedish mutant, SEVNL2DAEFR, K m ‫؍‬ 9 M, K cat ‫؍‬ 0.02 s ؊1 ). A new substrate (VVEVDA2AVTP, K m ‫؍‬ 1 M, K cat ‫؍‬ 0.004) was identified by serendipity.Alzheimer's disease is characterized by the extracellular deposition of insoluble amyloid plaques. The main component of amyloid plaques is the 39 -43-amino acid ␤-amyloid peptide (A␤), 1 which derives from a larger protein precursor (amyloid precursor protein, APP). A␤ is excised from APP by the sequential action of two proteases known, respectively, as ␤-secretase, which cuts amino-terminal to A␤, and ␥-secretase, which cleaves at the carboxyl terminus. Several reports appeared recently describing the cloning and characterization of ␤-secretase (1-5). This protein, designated Asp-2, BACE, or memapsin 2, according to the laboratory in which it was discovered, is a novel transmembrane aspartic protease that cleaves APP at the ␤-secretase site. BACE possesses all the characteristics expected for ␤-secretase in terms of substrate preference, pH optimum for activity, tissue distribution, and subcellular localization. In addition, two recent reports indicate that A␤ levels in the brains of BACE knockout mice are reduced by more than 90% compared with control mice (6, 7). In addition to cleaving APP at the ␤-secretase site, BACE cuts APP further downstream within the amyloid region (between Tyr-10 and Glu-11 of A␤), generating a truncated form of A␤ that is probably still amyloidogenic (3,8). Parallel to the discovery of BACE, a second, homologous transmembrane aspartic protease termed Asp-1, BACE2, memapsin 1, or down region aspartic protease was reported (4, 5, 9, 10). Preliminary analysis of BACE2 indicated that it can also function as a ␤-secretase in vitro (8,9).In seeking to develop a disease-modifying therapy for Alzheimer's Disease, BACE presents itself as an ideal drug target. It belongs to a well understood class of protease where inhibitors...