Binding of Amyloid  Protein to the 20 S Proteasome* (Received for publication, May 17, 1996, and in revised form, September 23, 1996) Luisa Gregori‡, James F. Hainfeld§, Martha N. Simon§, and Dmitry Goldgaber From the Department of Psychiatry and Behavioral Science, School of Medicine, State University of New York, Stony Brook, New York 11794 and the §Biology Department, Brookhaven National Laboratory, Upton, New York 11973 Neurodegenerative disorders of aging are character- ized by the intraneuronal accumulation of ubiquitin conjugates into tangles and inclusions. Ubiquitin conju- gates are degraded by cellular particles known as pro- teasomes. We have previously shown that amyloid  pro- tein (A) inhibits proteasomal activity and thereby blocks ubiquitin conjugate degradation. In the present studies, we found that A binds the 20 S proteasome and forms a proteasome-A complex. The complex was de- tected by Western blot with anti-A antibodies. Using a 1.4 nm Nanogold-labeled A, we visualized protea- some-A complexes by scanning transmission electron microscopy (STEM). Analysis of the side-on oriented proteasome-A complexes revealed a single gold parti- cle, corresponding to one gold-labeled A, in the middle portion of the proteasome. On end-on views of protea- some-A complexes, gold was detected within the area delimited by the proteasome circular projection. Both STEM views are consistent with A localization inside the proteasome along the peptide channel. Direct inter- action of A with the inner catalytic compartment of the proteasome may explain the generation of ubiquitin- containing lesions in Alzheimer’s disease and other neu- rodegenerative disorders. In addition, detection of Nanogold-labeled peptide inside the 20 S eukaryotic proteasome suggests that conformational constraints for protein degradation in eukaryotic proteasomes are different from those in archaebacteria proteasomes. The ubiquitin/proteasome protein degradation pathway is one of the two major cellular proteolytic systems. In this path- way, damaged and abnormal proteins are targeted for degra- dation by the covalent attachment of several ubiquitin moieties (ubiquitination) to form ubiquitin conjugates. Ubiquitinated proteins are rapidly degraded by the 26 S proteasome, the proteolytic component of the ubiquitin degradation system (1– 3). Several findings link the ubiquitin/proteasome pathway to some pathological manifestations of neurodegenerative disor- ders such as Alzheimer’s disease (AD), 1 Lewy bodies disease, and to a lesser extent normal aging of the brain, as well as pathologies of non-central nervous system-related diseases such as inclusion body myositis (IBM). In these disorders, high levels of ubiquitin and ubiquitin conjugates are detected in abnormal intracellular inclusions and cytoskeletal-derived fibrils (4 – 6). In AD, microtubule associated protein  is ubiq- uitinated but is not degraded. Ubiquitinated  is found in paired helical filaments forming neurofibrillary tangles (NFT) (7). Development of NFT has been linked directly to neuronal degeneration in AD (8). Other, yet unidentified, proteins also accumulate into intracellular inclusions. These observations suggest that the ubiquitin/proteasome proteolytic pathway may be involved in the formation of ubiquitinated intracellular lesions. Proteasomes are abundant nonlysosomal multicatalytic pro- teinases involved in a variety of cell functions (1, 9 –12). Pro- teasomes are localized both in the cytoplasm and in the nucleus of cells (10). In addition, proteasomes have been detected asso- ciated with plasma and internal membranes (10). The 20 S proteasome is the catalytic core of the 26 S proteasome (9 –11). An increasing body of evidence points to a role of proteasomes in the accumulation of ubiquitinated proteins and ubiquitin- containing lesions in neurodegenerative disorders. Antibodies to proteasomes decorate some Lewy bodies and NFT (13). Cul- tured cells exposed to proteasome inhibitors accumulate ubiq- uitin conjugates (14, 15) and in at least one neuronal cell line these ubiquitin conjugates accumulated into intracellular AD- like inclusion bodies. 2 In addition, we found that in vitro amy- loid  protein (A) blocked the degradation of ubiquitinated proteins by inhibiting the proteasome activity (16). A is a 39 – 42-amino acid peptide, derived from the proteolytic cleav- age of the membrane-bound amyloid  protein precursor, which has been implicated in AD (17). Intracellular A has been detected in cultured cells (18, 19) and associated with NFT in AD (20, 21) and with cytoplasmic tubulofilaments of vacuolated muscle fibers in IBM (5). We showed that A selectively inhib- its the chymotrypsin-like activity in the proteasome (16). Among the proteasome’s different proteolytic activities, the chymotrypsin-like activity has been linked directly to the deg- radation of ubiquitin conjugates. Defects in the yeast protea- some subunits bearing this catalytic specificity cause accumu- lation of ubiquitin conjugates and reduced protein degradation rates (22). Selective inhibition of the proteasome chymotrypsin- like activity by A is consistent with a critical function of the proteasome-peptide interaction in the accumulation of ubiq- uitin conjugates in AD. Furthermore, a recent finding that A, used as the bait in a yeast two-hybrid system, binds two dis- tinct yeast proteasome subunits, supports our results on the ability of A to interact with the proteasome (23). In this work, we characterized the 20 S proteasome-A com- plex and established the spatial localization of A in the com- plex. The results provide evidence for a molecular mechanism of A interaction with the proteasome, which may be involved in the accumulation of ubiquitin conjugates in neurodegenera- tive disorders and other age-related diseases. * This work was supported by The Alzheimer’s Association through Grant ZEN-91-017 (to D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: Dept. of Psychiatry and Behavioral Science, Health Science Center T-10, School of Medi- cine, State University of New York, Stony Brook, NY 11794-8101. Tel.: 516-444-3426; Fax: 516-444-7534. 1 The abbreviations used are: AD, Alzheimer’s disease; IBM, inclu- sion body myositis; A, amyloid  protein; NFT, neurofibrillary tangles; STEM, scanning transmission electron microscopy; PVDF, polyvinyli- dene difluoride; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine. 2 M. Figueiredo-Pereira, personal communication. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 1, Issue of January 3, pp. 58 –62, 1997 Printed in U.S.A. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 58 by guest on March 19, 2016 http://www.jbc.org/ Downloaded from