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

Wenjuan Ye,Dennis J. Selkoe,Michael S. Wolfe,Matthew J. LaVoie,Beth L. Ostaszewski,Patrick C. Fraering

doi:10.1074/jbc.m501368200

Wenjuan Ye, Dennis J. Selkoe + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m501368200

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Abstract

gamma-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin composed of its N- and 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 gamma-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Abeta, 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 gamma-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified gamma-secretase preparations; the labeling was blocked by ATP itself and APP-selective gamma-secretase inhibitors. We concluded that a nucleotide-binding site exists within gamma-secretase, and certain compounds that bind to this site can specifically modulate the generation of Abeta while sparing Notch. Drugs targeting the gamma-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.

Highlights

Alzheimer disease is characterized by the progressive accumulation of amyloid ␤-protein (A␤)3 in brain regions subserving memory and cognition [1]
Sequential proteolytic cleavages of the amyloid ␤-protein precursor (APP) by the ␤- and ␥-secretases generate the amyloid ␤-protein (A␤) [1]. ␤-Secretase is a single membrane-spanning aspartyl protease expressed at high levels in neurons [2]. ␥-Secretase is 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 amyloid ␤-protein (A␤) precursor (APP) and the Notch receptors
Because Netzer et al [7] showed that Gleevec and inhibitor 2 inhibited APP but not Notch cleavage in their systems, we investigated the effects of selected protein-tyrosine kinase inhibitors on A␤ production and on Notch substrate cleavage using isolated, purified ␥-secretase

Summary

Introduction

Alzheimer disease is characterized by the progressive accumulation of amyloid ␤-protein (A␤)3 in brain regions subserving memory and cognition [1]. Gleevec Itself Is Not a Direct ␥-Secretase Inhibitor; a Gleevec Extract Inhibits the Generation of A␤ by Purified ␥-Secretase Without Affecting the Cleavage of a Notch-based Recombinant Substrate—Because ATP activated the purified ␥-secretase complex (Fig. 1, A and B), we used this preparation to examine the effects of a Gleevec extract (prepared from capsules and characterized as described in detail under “Materials and Methods”) on the cleavage efficiency of C100FLAG substrate.

Results

Conclusion