Implication of Novel Biochemical Property of -Amyloid 1 Danek Elbaum,* , † ,2 Maria Brzyska,* Andrzej Bacia,* and Daniel L. Alkon† *Laboratory of Biophysical Methods, Nencki Institute of Experimental Biology, Warsaw, Poland; and †Laboratory of Adaptive Systems, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Room B-312, Building 36, Bethesda, Maryland 20892 Received December 6, 1999 Alzheimer disease (AD) is a heterogeneous disorder with a variety of molecular pathologies converging pre- dominantly on abnormal amyloid deposition particu- larly in the brain. -Amyloid aggregation into senile plaques is one of the pathological hallmarks of AD. -Amyloid is generated by a proteolytic cleavage of a large membrane protein, amyloid precursor protein (APP). We have observed a new property of -amyloid. The amyloid 1-42  fragment, when aggregated, pos- sesses proteolytic and esterase-like activity, in vitro. Three independent methods were used to test the new property of -amyloid. While esterase activity involves imidazole catalysis, proteolytic activity is consistent with participation of a serine peptidase triad: catalytic Ser, His and Glu (or Asp). Although the amino acid triad is a necessary requirement for the protease reactivity, it is not sufficient since the secondary structure of the protein significantly contributes to the proteolytic activ- ity. The ability of -amyloid to cleave peptide or ester bonds could be thus responsible for either inactivation of other proteins and/or APP proteolysis itself. This property may be responsible for early pathogenesis of AD since there is emerging evidence that non-plaque amy- loid is elevated in Alzheimer patients. © 2000 Academic Press -Amyloid peptides are proteolytic fragments 39 to 44 amino-acid derived from amyloid precursor protein (APP), a transmembrane glycoprotein that is coded by chromosome 21 and expressed in most mammalian cells (review 1). Amyloid fragments form the core of Alzheimer disease (AD) senile plaques and are impli- cated as a major determinant of the pathology (2, 3). In addition to amyloid deposits, these proteolytic frag- ments have been detected in plasma and cerebrospinal fluid (4, 5). APP can be cleaved by multiple proteases, amyloidogenic and nonamyloidogenic, to generate sev- eral secreted and membrane bound derivatives. Al- though the enzymes catalyzing the APP proteolytic cleavage have not been identified, the heterogeneity in N- and C-termini in soluble and insoluble species of amyloidogenic fragments imply a nonspecific nature of the reaction (6). The precise mechanism of -amyloid cell toxicity has not been clearly defined. Several or- ganelles have been implicated as sites of proteolysis of putative -secretase: endosome, lysosome, endoplasmic reticulum, and the trans Golgi network (7–9). In addition, free radical generation by the peptide has been suggested to occur by an oxygen-dependent mechanism that could disrupt cell integrity and even- tually cause cell death (10). Hydrogen peroxide-mediated amyloid protein toxicity was demonstrated using primary central nervous system cells and clonal cell lines (11). This observation was fur- ther supported by demonstration that the Fenton reac- tion facilitates amyloid toxicity of the clonal B 12 cells (12). The methionine residue has been implicated, but is not required, for amyloid induced toxicity in rat hip- pocampal cells (13). Although several nontoxic proteins and peptides generated spin trapping products, they showed no neurotoxicity (10). Amyloid induced plasma membrane lipid peroxidation, glutamate uptake, disrup- tion of intracellular calcium homeostasis, impairment of metabolism by inhibition of glucose uptake, uncoupling of a G-protein linked receptor, impairment of ATPases have all been observed to contribute to amyloid induced cellu- lar injury (rev: 14). These and other observations support the hypothesis that -amyloid formation is critical in initiating cellular dysfunction. Such dysfunction may arise from -amyloid’s ability to generate free radicals as well as from other properties that contribute to -amyloid neurotoxicity (15). Inhibition of amyloid production and deposition, as well as enhancement of the aggregate proteolytic deg- radation are logical targets for reduction of -amyloid cytotoxicity. Thus, inhibition of its release has been suggested to be a prime therapeutic goal. The broad 1 This work was supported in part by the Polish Committee for Scientific Research, Project No 4PO5A 114 12. 2 To whom correspondence should be addressed at Nencki Insti- tute of Experimental Biology, PAN, Laboratory of Biophysical Meth- ods, ul. Pasteura 3, Warsaw, Poland. Fax: 48-22-822-53-42. E-mail: elbaum@nencki.gov.pl. Biochemical and Biophysical Research Communications 267, 733–738 (2000) doi:10.1006/bbrc.1999.2024, available online at http://www.idealibrary.com on 733 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.