title: A synthetic antibody fragment targeting nicastrin affects assembly and trafficking of γ-secretase.
abstract: The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid β (Aβ) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid β peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch.
authors.affiliation: From the Departments of Neurobiology and.
authors.forename: Xulun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637.
authors.forename: Robert
authors.initials: R
authors.lastname: Hoey
authors.affiliation: Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637.
authors.forename: Akiko
authors.initials: A
authors.lastname: Koide
authors.affiliation: the Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Georgia
authors.initials: G
authors.lastname: Dolios
authors.affiliation: Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637.
authors.forename: Marcin
authors.initials: M
authors.lastname: Paduch
authors.affiliation: the Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla, California 92093, and.
authors.forename: Phuong
authors.initials: P
authors.lastname: Nguyen
authors.affiliation: Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, New York 10065.
authors.forename: Xianzhong
authors.initials: X
authors.lastname: Wu
authors.affiliation: Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, New York 10065.
authors.forename: Yueming
authors.initials: Y
authors.lastname: Li
authors.affiliation: the Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla, California 92093, and.
authors.forename: Steven L
authors.initials: SL
authors.lastname: Wagner
authors.affiliation: the Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Rong
authors.initials: R
authors.lastname: Wang
authors.affiliation: Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637.
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
authors.affiliation: From the Departments of Neurobiology and ssisodia@bsd.uchicago.edu.
authors.forename: Sangram S
authors.initials: SS
authors.lastname: Sisodia
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D059748:Proteolysis; D051880:Receptors, Notch / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D008562:Membrane Glycoproteins; D051880:Receptors, Notch; D057127:Single-Chain Antibodies; C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases
keywords: Alzheimer Disease; Amyloid Precursor Protein (APP); Cancer; Notch Receptor; Synthetic Antibody; γ-Secretase