title: Identification of a tetratricopeptide repeat-like domain in the nicastrin subunit of γ-secretase using synthetic antibodies.
abstract: The γ-secretase complex, composed of presenilin, anterior-pharynx-defective 1, nicastrin, and presenilin enhancer 2, catalyzes the intramembranous processing of a wide variety of type I membrane proteins, including amyloid precursor protein (APP) and Notch. Earlier studies have revealed that nicastrin, a type I membrane-anchored glycoprotein, plays a role in γ-secretase assembly and trafficking and has been proposed to bind substrates. To gain more insights regarding nicastrin structure and function, we generated a conformation-specific synthetic antibody and used it as a molecular probe to map functional domains within nicastrin ectodomain. The antibody bound to a conformational epitope within a nicastrin segment encompassing residues 245-630 and inhibited the processing of APP and Notch substrates in in vitro γ-secretase activity assays, suggesting that a functional domain pertinent to γ-secretase activity resides within this region. Epitope mapping and database searches revealed the presence of a structured segment, located downstream of the previously identified DAP domain (DYIGS and peptidase; residues 261-502), that is homologous to a tetratricopeptide repeat (TPR) domain commonly involved in peptide recognition. Mutagenesis analyses within the predicted TPR-like domain showed that disruption of the signature helical structure resulted in the loss of γ-secretase activity but not the assembly of the γ-secretase and that Leu571 within the TPR-like domain plays an important role in mediating substrate binding. Taken together, these studies offer provocative insights pertaining to the structural basis for nicastrin function as a "substrate receptor" within the γ-secretase complex.
authors.affiliation: Department of Neurobiology, University of Chicago, Chicago, IL 60637, USA.
authors.forename: Xulun
authors.initials: X
authors.lastname: Zhang
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Hoey
authors.forename: Guoqing
authors.initials: G
authors.lastname: Lin
authors.forename: Akiko
authors.initials: A
authors.lastname: Koide
authors.forename: Brenda
authors.initials: B
authors.lastname: Leung
authors.forename: Kwangwook
authors.initials: K
authors.lastname: Ahn
authors.forename: Georgia
authors.initials: G
authors.lastname: Dolios
authors.forename: Marcin
authors.initials: M
authors.lastname: Paduch
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ikeuchi
authors.forename: Rong
authors.initials: R
authors.lastname: Wang
authors.forename: Yue-Ming
authors.initials: YM
authors.lastname: Li
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
authors.forename: Sangram S
authors.initials: SS
authors.lastname: Sisodia
mesh_terms: D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D015153:Blotting, Western; D002478:Cells, Cultured; D002942:Circular Dichroism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry
chemical_list: D016564:Amyloid beta-Protein Precursor; D000906:Antibodies; D000939:Epitopes; D008562:Membrane Glycoproteins; D009842:Oligopeptides; C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases