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
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.
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