Molecular analysis of intramembrane proteolysis by combinatorial reconstitution and chemical genetics of membrane protein complex.

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Several lines of evidence suggest that aggregation and deposition of amyloid-β peptide (Aβ) are involved in the pathogenesis of Alzheimer's dipease, that is the most common cause of dementia with aging. Thus, γ-secretase, that is a pivotal enzyme in generating Aβ, is a plausible therapeutic target for AD. However, as γ-secretase is an atypical protease and endoproteolyzes a scissile bond within the hydrophobic lipid bilayer, the mode of action whereby γ-secretase cleaves its substrate still remains unknown. To elucidate the molecular mechanism of γ-secretase mediated cleavage for the development of AD therapeutics, we have extensively analyzed recombinant γ-secretase complex reconstituted by mammalian and insect ?cell expression systems. Moreover, through world-wide collaborations, we have investigated γ-secretase by structural and chemical biological approaches. Finally, we have proposed the "catalytic pore" model for intramembrane proteolysis by γ-secretage. We found that the intramembrane-cleaving reaction takes place within this pore, and can be regulated by the pharmacological and genetic modulation of the catalytic pore structure. Moreover, we analyzed the physiological function of γ-secreta se to prove that the functional modulation of γ-secretase activity is plausible therapeutics for AD. To this end, we characterized the stmcture of γ-secretape complex by single particle analysis. Further extensive efforts for the understanding the molecular mechanism of γ-secretase may contribute to the development of promising therapeutics using safe bioavailable compounds for AD.