γ-Secretase Substrate Selectivity Can Be Modulated Directly via Interaction with a Nucleotide-binding Site*,s

γ-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid β-protein (Aβ) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of γsecretase: heterodimeric presenilin composed of its Nand C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified γ-secretase to selectively block cleavage of APPbut not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Aβ, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified γ-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified γ-secretase preparations; the labeling was blocked by ATP itself and APP-selective γ-secretase inhibitors. We concluded that a nucleotide-binding site exists within γ-secretase, and certain compounds that bind to this site can specifically modulate the generation of Aβ while sparing Notch. Drugs targeting the γ-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease. Alzheimer disease is characterized by the progressive accumulation of amyloid β-protein (Aβ)3 in brain regions subserving memory and cognition (1). Sequential proteolytic cleavages of the amyloid β-protein precursor (APP) by the βand γ-secretases generate the amyloid βprotein (Aβ) (1). β-Secretase is a single membrane-spanning aspartyl pro-tease expressed at high levels in neurons (2). γ-Secretase is also an aspartyl protease but with an unprecedented intramembranous catalytic site (3,4) that is required for the cleavage of a wide range of type I membrane proteins that include APP and the Notch receptors (for a review see Ref. 5). We recently reported a specific and reproducible procedure for the high grade purification of active human γ-secretase and characterized various factors that affect its activity in vitro (6). In further investigating the properties of the purified enzyme, we have observed that ATP can activate purified γ-secretase in vitro by up to 2-fold. This observation is in agreement with the recent *This work was supported by Swiss National Science Foundation Grant 81FR-68842 (to P. C. F.), NIA Grant AG15379 from the National Institutes of Health, a Pioneer award from the Alzheimer Association (to D. J. S.), and by National Institutes of Health Grants AG17574, AG15379, and NS41355 (to M. S. W.). sThe on-line version of this article (available at http://www.jbc.org) contains Table S1. 1 To whom correspondence may be addressed: Center for Neurologic Diseases, Harvard Institute of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5200; Fax: 617-525-5252; E-mail: [email protected].. 2 To whom correspondence may be addressed. E-mail: [email protected].. 3The abbreviations used are: Aβ, amyloid β-protein; AD, Alzheimer disease; APP, amyloid β-protein precursor; AICD, APP intracellular domain; CHO, Chinese hamster ovary; CTF, C-terminal fragment; ELISA, enzyme-linked immunosorbent assay; GST, glutathione Stransferase; NCT, nicastrin; NTF, N-terminal fragment; PC, Phosphatidylcholine; PE, Phosphatidylethanolamine; PS, presenilin; CHAPSO, 3-[(3-cholamido-propyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; ATPγS, adenosine 5′-O-(thiotriphosphate); BN, blue native; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. NIH Public Access