Requirement for small side chain residues within the GxGD-motif of presenilin for gamma-secretase substrate cleavage.
gamma-Secretase is a pivotal intramembrane-cleaving protease complex and important drug target for Alzheimer's disease. The protease not only releases small peptides, such as the amyloid-beta peptide, which drives Alzheimer's disease pathogenesis, but also intracellular domains, which can have critical functions in nuclear signaling. Unlike typical aspartyl proteases, gamma-secretase contains a non-classical GxGD active site motif in its catalytic subunit presenilin (PS) 1 or PS2. It is not known whether both glycines are of similar functional relevance and why the glycine residues are invariant elements of the motif. Here we identify the N-terminal glycine of the GxGD motif in PS1, G382, as a critical residue of the active site domain of gamma-secretase. Substitution of G382 by a number of different amino acids abrogated gamma-secretase activity. Only the smallest possible G382A substitution allowed substantial gamma-secretase activity. Depending on the substrate, however, the presence of G382 could become even an absolute functional requirement of gamma-secretase. Very similar results were obtained for the C-terminal glycine residue (G384) of the GxGD motif. Our data thus identify a requirement for small side chain residues in the active site domain of gamma-secretase and suggest that the glycines of the GxGD motif could be evolutionary conserved to allow cleavage of all possible gamma-secretase substrates, including those, which are highly sensitive to minimal alteration of the PS active site domain. These findings broaden our understanding of gamma-secretase substrate recognition and cleavage, which may prove crucial for therapeutic targeting of the enzyme.
Pérez-Revuelta
Blanca Isabel
BI
Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE) and Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, München, Germany.
Fukumori
Akio
A
Lammich
Sven
S
Yamasaki
Aya
A
Haass
Christian
C
Steiner
Harald
H
D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D005998:Glycine / Q000235:genetics*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D053764:Presenilin-1 / Q000737:chemistry* / Q000235:genetics; D053766:Presenilin-2 / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014162:Transfection / Q000379:methods
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D018960:Hyaluronan Receptors; D053764:Presenilin-1; D053766:Presenilin-2; D053829:Amyloid Precursor Protein Secretases; D000409:Alanine; D005998:Glycine