title: Evidence That the "Lid" Domain of Nicastrin Is Not Essential for Regulating γ-Secretase Activity.
abstract: Understanding of the structure of the γ-secretase complex consisting of presenilin (PS), anterior pharynx-defective 1 (APH-1), nicastrin (NCT), and presenilin enhancer 2 (PEN-2) is of significant therapeutic interest for the design of γ-secretase modulators for Alzheimer disease. The structure of γ-secretase revealed by cryo-EM approaches suggested a substrate binding mechanism for NCT, a bilobar structure that involved rotation of the two lobes around a central pivot and opening of a "lid" region that facilitates substrate recruitment. To validate this proposal, we expressed NCT that lacks the lid entirely, or a variety of NCT variants that harbor mutations at highly conserved residues in the lid region inNCT-deficient cells, and then assessed their impact on γ-secretase assembly, activity, and stability. In addition, we assessed the impact of mutating a critical residue proposed to be a pivot around which the two lobes of NCT rotate. Our results show that neither the mutations on the lid tested here nor the entire lid deletion has any significant impact on γ-secretase assembly, activity, and stability, and that NCT with the mutation of the proposed pivot rescues γ-secretase activity inNCT-deficient cells in a manner indistinguishable from WT NCT. These findings indicate that the NCT lid is not an essential element necessary for γ-secretase assembly, activity, and stability, and that rotation of the two lobes appears not to be a prerequisite for substrate binding and γ-secretase function.
authors.affiliation: From the Department of Neurobiology, The University of Chicago, Chicago, Illinois 60637 and.
authors.forename: Xulun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: From the Department of Neurobiology, The University of Chicago, Chicago, Illinois 60637 and.
authors.forename: Eric
authors.initials: E
authors.lastname: Sullivan
authors.affiliation: From the Department of Neurobiology, The University of Chicago, Chicago, Illinois 60637 and.
authors.forename: Maggie
authors.initials: M
authors.lastname: Scimeca
authors.affiliation: the Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065.
authors.forename: Xianzhong
authors.initials: X
authors.lastname: Wu
authors.affiliation: the Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065.
authors.forename: Yue-Ming
authors.initials: YM
authors.lastname: Li
authors.affiliation: From the Department of Neurobiology, The University of Chicago, Chicago, Illinois 60637 and ssisodia@bsd.uchicago.edu.
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*; D000818:Animals; D002460:Cell Line; D010450:Endopeptidases; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D053763:Presenilins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D015398:Signal Transduction; D013329:Structure-Activity Relationship
chemical_list: D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C469349:PSENEN protein, human; D053763:Presenilins; C413604:nicastrin protein; C475154:APH1A protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases
keywords: Alzheimer disease; Notch protein; amyloid precursor protein (APP); gamma-secretase; lid; mutagenesis; nicastrin; substrate binding