Research article

Changes in γ-secretase activity and specificity caused by the introduction of consensus aspartyl protease active motif in Presenilin 1

Donald B Carter¹ , Edwige Dunn¹ , Adele M Pauley² , Denise D McKinley¹ , Timothy J Fleck¹ , Brenda R Ellerbrook³ , Nancy C Stratman³ , Xiangdong Zhou⁴ , Carol S Himes¹ , Jeffrey S Nye¹ , Alfredo Tomasselli^2,5 and Riqiang Yan^1,4

¹  Global Research and Development, Pfizer Inc. Kalamazoo, MI 49001, USA

²  Global Research and Development, Pfizer Inc. St Louis, MO 63017, USA

³  Global Research and Development, Pfizer Inc. Groton, CT 06340, USA

⁴  Department of Neurosciences, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA

⁵  St Louis Laboratories Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA

author email corresponding author email

Molecular Neurodegeneration 2008, 3:6doi:10.1186/1750-1326-3-6

Published:	12 May 2008

Abstract

Presenilin (PS1 or PS2) is an essential component of the active γ-secretase complex that liberates the Aβ peptides from amyloid precursor protein (APP). PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the γ-secretase activity in the presence or absence of γ-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Aβ₄₂ production compared to the wild type (wt), and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the γ-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Aβ₃₈ and Aβ₄₂, but to reduce levels of Aβ₃₉, showing a pattern of Aβ formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the γ-site without interfering with Notch processing.