title: The transmembrane domain region of nicastrin mediates direct interactions with APH-1 and the gamma-secretase complex.
abstract: Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the gamma-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other gamma-secretase components include presenilin-1 (PS1), APH-1, and PEN-2, all of which span the membrane multiple times. The mechanism by which NCT associates with the gamma-secretase complex and regulates its activity is unclear. To avoid the misfolding phenotype often associated with introducing deletions or mutations into heavily glycosylated and disulfide-bonded proteins such as NCT, we produced chimeras between human (hNCT) and Caenorhabditis elegans NCT (ceNCT). Although ceNCT did not associate with human gamma-secretase components, all of the ceNCT/hNCT chimeras interacted with gamma-secretase components from human, C. elegans, or both, indicating that they folded correctly. A region at the C-terminal end of hNCT, encompassing the last 50 residues of its ectodomain, the transmembrane domain, and the cytoplasmic domain was important for mediating interactions with human PS1, APH-1, and PEN-2. This finding is consistent with the fact that the bulk of the gamma-secretase complex proteins resides within the membrane, with relatively small extramembranous domains. Finally, hNCT associated with hAPH-1 in the absence of PS, consistent with NCT and APH-1 forming a subcomplex prior to association with PS1 and PEN-2 and indicating that the interactions between NCT with PS1 may be indirect or stabilized by the presence of APH-1.
authors.affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Vanessa A
authors.initials: VA
authors.lastname: Morais
authors.forename: Adam S
authors.initials: AS
authors.lastname: Crystal
authors.forename: Donald S
authors.initials: DS
authors.lastname: Pijak
authors.forename: Dan
authors.initials: D
authors.lastname: Carlin
authors.forename: Júlia
authors.initials: J
authors.lastname: Costa
authors.forename: Virginia M-Y
authors.initials: VM
authors.lastname: Lee
authors.forename: Robert W
authors.initials: RW
authors.lastname: Doms
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008051:Lipid Bilayers / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: C405066:APH-1 protein, C elegans; D029742:Caenorhabditis elegans Proteins; D018076:DNA, Complementary; D018398:Homeodomain Proteins; D008051:Lipid Bilayers; D008562:Membrane Glycoproteins; C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human