title: Immature nicastrin stabilizes APH-1 independent of PEN-2 and presenilin: identification of nicastrin mutants that selectively interact with APH-1.
abstract: Gamma-secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx-defective-1 (APH-1) and presenilin enhancer-2 (PEN-2). The complex mediates the intramembraneous proteolysis of beta-secretase cleaved beta-amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease-associated amyloid beta-peptide (Abeta). In order to dissect functionally important domains of Nct required for gamma-secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with significantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH-1 and PEN-2 expression as well as by monitoring the accumulation of the APP C-terminal fragments, the immediate substrates of gamma-secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH-1 but not of PEN-2 or PS and thus failed to restore gamma-secretase activity. Interestingly, these immature Nct variants selectively bound to APH-1, suggesting a stable Nct/APH-1 interaction independent of PS and PEN-2. Consistent with this finding, expression of APH-1 remained largely unaffected in the PS double knock-out and immature Nct co-immunoprecipitated with APH-1 in the absence of PS and PEN-2. Taken together, our findings suggest that immature Nct can stably interact with APH-1 to form a potential scaffold for binding of PS and PEN-2. Moreover, binding of the latter two complex partners critically depends on the integrity of the Nct ectodomain.
authors.affiliation: Adolf-Butenandt Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Ludwig-Maximilians University, 80336 Munich, Germany.
authors.forename: Keiro
authors.initials: K
authors.lastname: Shirotani
authors.forename: Dieter
authors.initials: D
authors.lastname: Edbauer
authors.forename: Marcus
authors.initials: M
authors.lastname: Kostka
authors.forename: Harald
authors.initials: H
authors.lastname: Steiner
authors.forename: Christian
authors.initials: C
authors.lastname: Haass
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism*; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D010450:Endopeptidases; D006801:Humans; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases; D011233:Precipitin Tests; D053764:Presenilin-1; D053766:Presenilin-2; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D014162:Transfection
chemical_list: D016564:Amyloid beta-Protein Precursor; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C508853:PSEN1 protein, human; C508861:PSEN2 protein, human; C469349:PSENEN protein, human; D053764:Presenilin-1; D053766:Presenilin-2; D020033:Protein Isoforms; C413604:nicastrin protein; C475154:APH1A protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases