The transition metal ion copper(II) has a critical role in chronic neurologic diseases. The amyloid precursor protein (APP) of Alzheimer's disease or a synthetic peptide representing its copper-binding site reduced bound copper(II) to copper(I). This copper ion-mediated redox reaction led to disulfide bond formation in APP, which indicated that free sulfhydryl groups of APP were involved. Neither superoxide nor hydrogen peroxide had an effect on the kinetics of copper(II) reduction. The reduction of copper(II) to copper(I) by APP involves an electron-transfer reaction and could enhance the production of hydroxyl radicals, which could then attack nearby sites. Thus, copper-mediated toxicity may contribute to neurodegeneration in Alzheimer's disease.

Progressive cerebral amyloid beta-protein (A beta) deposition is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). Elevated levels of A beta(42) peptide formation have been linked to early-onset familial AD-causing gene mutations in the amyloid beta-protein precursor (A beta PP) and the presenilins. Sequential cleavage of A beta PP by the beta- and gamma-secretases generates the N- and C-termini of the A beta peptide, making both the beta- and gamma-secretase enzymes potential therapeutic targets for AD. The identity of the A beta PP gamma-secretase and the mechanism by which the C-termini of A beta are formed remain uncertain, although it has been suggested that the presenilins themselves are novel intramembrane-cleaving gamma-secretases of the aspartyl protease class [Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517]. In this study we report the identification of L-685,458 as a structurally novel inhibitor of A beta PP gamma-secretase activity, with a similar potency for inhibition of A beta(42) and A beta(40) peptides. This compound contains an hydroxyethylene dipeptide isostere which suggests that it could function as a transition state analogue mimic of an aspartyl protease. The preferred stereochemistry of the hydroxyethylene dipeptide isostere was found to be the opposite to that required for inhibition of the HIV-1 aspartyl protease, a factor which may contribute to the observed specificity of this compound. Specific and potent inhibitors of A beta PP gamma-secretase activity such as L-685,458 will enable important advances toward the identification and elucidation of the mechanism of action of this enigmatic protease.

Resveratrol is a plant polyphenol capable of exerting beneficial metabolic effects which are thought to be mediated in large by the activation of the NAD + -dependent protein deacetylase SIRT1. Although resveratrol has been claimed to be a bona fide SIRT1 activator using a peptide substrate (Fluor de Lys-SIRT1 peptide substrate), recent reports indicate that this finding might be an experimental artifact and need to be clarified. Here, we show that: (i) the Fluor de Lys-SIRT1 peptide is an artificial SIRT1 substrate because in the absence of the covalently linked fluorophore the peptide itself is not a substrate of the enzyme, (ii) resveratrol does not activate SIRT1 in vitro in the presence of either a p53-derived peptide substrate or acetylated PGC1a isolated from cells, and (iii) although SIRT1 deacetylates PGC-1a in both in vitro and cell-based assays, resveratrol did not activate SIRT1 under these conditions. Based on these observations, we conclude that the pharmacological effects of resveratrol in various models are unlikely to be mediated by a direct enhancement of the catalytic activity of the SIRT1 enzyme. In consequence, our data challenge the overall utility of resveratrol as a pharmacological tool to directly activate SIRT1. Key words: Fluor de Lys, resveratrol, SIRT1Abbreviations: AMPK, AMP-activated protein kinase; HPLC, high performance liquid chromatography; NAM, nicotinamide; PGC-1a, peroxisome proliferator-activated receptor-c coactivator-1a; Sir2, silence information regulator 2; SIRTs, sirtuins. Sirtuins constitute the unique family of NAD + -dependent protein deacetylases. Silent information regulator 2 (Sir2) is a sirtuin in budding yeast Saccharomyces cerevisiae and its activity mediates lifespan extension induced by calorie restriction (1). The mammalian Sir2 ortholog, SIRT1, is also induced by calorie restriction and promotes cell survival (2), triggers lipolysis and loss of fat (3), and controls glucose homeostasis (4). The biological effects of SIRT1 are mediated by its ability to deacetylate several important transcriptional factors such as Peroxisome proliferator-activated receptor-c coactivator 1alpha (PGC-1a), p53, and FOXO proteins and consequently regulate their activities (5). PGC-1a, which is activated upon deacetylation by SIRT1, plays an important role in the regulation of mitochondrial function and fatty acid oxidation (4). In contrast, p53 is inactivated once deacetylated by SIRT1 (5), suggesting that SIRT1 may protect cells from apoptosis under conditions of nutrient restriction. Taken together, these findings demonstrate that SIRT1 activity stimulates energy metabolism, improves mitochondrial function and promotes cell survival. Therefore, pharmacological activation of SIRT1 in vivo may provide a new avenue to maintain metabolic homeostasis.Molecular screening of SIRT1 activators led to the identification of plant polyphenols as SIRT1 activators, among which is resveratrol (6). The notion that resveratrol is a SIRT1 activator is consistent with two later pharmacological ...

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.

Previously it has been shown that the extracellular domain of transmembrane BA4 amyloid precursor protein (APP) includes binding sites for zinc(I1) and for molecules of the extracellular matrix such as collagen, laminin and the heparin sulfate chains of proteoglycans (HSPGs). Here we report that APP also binds copper ions. A copper type I1 binding site was located within residues 135-I 55 of the cysteine-rich domain of APP,,, which is present in all eight APP splice isoforms known so far. The two essential histidines in the type II copper binding site of APP are conserved in the related protein APLP2. Copper(I1) binding is shown to inhibit homophilic APP binding. The identification of a copper(I1) binding site in APP suggests that APP and APLP2 may be involved in electron transfer and radical reactions.