title: Conserved residues within the putative active site of gamma-secretase differentially influence enzyme activity and inhibitor binding.
abstract: Gamma-secretase performs the final processing step in the generation of amyloid-beta (Abeta) peptides, which are believed to be causative for Alzheimer's disease. Presenilins (PS) are required for gamma-secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen-bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue gamma-secretase inhibitor. Here we report that replacement of either tyrosine residue alters gamma-secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Abeta42 peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.
authors.affiliation: Department of Molecular & Cellular Neuroscience, Merck Sharp & Dohme Research Laboratories, The Neuroscience Research Centre, Terlings Park, Harlow, Essex, UK.
authors.forename: Jonathan D J
authors.initials: JD
authors.lastname: Wrigley
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Nunn
authors.forename: Omar
authors.initials: O
authors.lastname: Nyabi
authors.forename: Earl E
authors.initials: EE
authors.lastname: Clarke
authors.forename: Peter
authors.initials: P
authors.lastname: Hunt
authors.forename: Alan
authors.initials: A
authors.lastname: Nadin
authors.forename: Bart
authors.initials: B
authors.lastname: De Strooper
authors.forename: Mark S
authors.initials: MS
authors.lastname: Shearman
authors.forename: Dirk
authors.initials: D
authors.lastname: Beher
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002474:Cell-Free System; D002793:Cholic Acids / Q000494:pharmacology; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence / Q000502:physiology*; D017077:Culture Media, Conditioned / Q000032:analysis; D003902:Detergents / Q000494:pharmacology; D004622:Embryo, Mammalian; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007668:Kidney; D016297:Mutagenesis, Site-Directed / Q000502:physiology*; D010446:Peptide Fragments; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D002793:Cholic Acids; D002868:Chromosomal Proteins, Non-Histone; D017077:Culture Media, Conditioned; D003902:Detergents; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); D001224:Aspartic Acid; D014443:Tyrosine; C048531:chapso; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human