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J. Biol. Chem., Vol. 283, Issue 33, 22529-22540, August 15, 2008

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Direct and Potent Regulation of -Secretase by Its Lipid Microenvironment^*

Pamela Osenkowski, Wenjuan Ye, Rong Wang, Michael S. Wolfe¹, and Dennis J. Selkoe²

From the Center for Neurologic Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115 and the Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York 10029

-Secretase is an unusual and ubiquitous aspartyl protease with an intramembrane catalytic site that cleaves many type-I integral membrane proteins, most notably APP and Notch. Several reports suggest that cleavage of APP to produce the Aβ peptide is regulated in part by lipids. As -secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate -activity. To determine the direct contribution of the lipid microenvironment to -secretase activity, we purified the human protease from overexpressing mammalian cells, reconstituted it in vesicles of varying lipid composition, and examined the effects of individual phospholipids, sphingolipids, cholesterol, and complex lipid mixtures on substrate cleavage. A conventional -activity assay was modified to include a detergent-removal step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased -secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids and sphingolipids robustly increased the cleavage of APP- and Notch-like substrates in a dose-dependent manner. Reconstitution of -secretase in complex lipid mixtures revealed that a lipid raft-like composition supported the highest level of activity compared with other membrane compositions. Taken together, these results demonstrate that membrane lipid composition is a direct and potent modulator of -secretase and that cholesterol, in particular, plays a major regulatory role.

Received for publication, March 10, 2008 , and in revised form, May 12, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants AG00222-15 (to P. O.), AG15379 from the NIA (to D. J. S. and M. S. W.), and AG17574 and NS41355 (to M. S. W.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence may be addressed. Tel.: 617-525-5511; Fax: 617-525-5305; E-mail: mwolfe{at}rics.bwh.harvard.edu. ² To whom correspondence may be addressed. Tel.: 617-525-5200; Fax: 617-525-5306; E-mail: dselkoe{at}rics.bwh.harvard.edu.

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