title: Glu-333 of nicastrin directly participates in gamma-secretase activity.
abstract: gamma-Secretase is a proteolytic membrane complex that processes a variety of substrates including the amyloid precursor protein and the Notch receptor. Earlier we showed that one of the components of this complex, nicastrin (NCT), functions as a receptor for gamma-secretase substrates. A recent report challenged this, arguing instead that the Glu-333 residue of NCT predicted to participate in substrate recognition only participates in gamma-secretase complex maturation and not in activity per se. Here, we present evidence that Glu-333 directly participates in gamma-secretase activity. By normalizing to the active pool of gamma-secretase with two separate methods, we establish that gamma-secretase complexes containing NCT-E333A are indeed deficient in intrinsic activity. We also demonstrate that the NCT-E333A mutant is deficient in its binding to substrates. Moreover, we find that the cleavage of substrates by gamma-secretase activity requires a free N-terminal amine but no minimal length of the extracellular N-terminal stub. Taken together, these studies provide further evidence supporting the role of NCT in substrate recognition. Finally, because gamma-secretase cleaves itself during its maturation and because NCT-E333A also shows defects in gamma-secretase complex maturation, we present a model whereby Glu-333 can serve a dual role via similar mechanisms in the recruitment of both Type 1 membrane proteins for activity and the presenilin intracellular loop during complex maturation.
authors.affiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Dries
authors.forename: Sanjiv
authors.initials: S
authors.lastname: Shah
authors.forename: Yu-Hong
authors.initials: YH
authors.lastname: Han
authors.forename: Cong
authors.initials: C
authors.lastname: Yu
authors.forename: Sophie
authors.initials: S
authors.lastname: Yu
authors.forename: Mark S
authors.initials: MS
authors.lastname: Shearman
authors.forename: Gang
authors.initials: G
authors.lastname: Yu
mesh_terms: D019943:Amino Acid Substitution; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D011485:Protein Binding / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D018411:Spodoptera; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D008562:Membrane Glycoproteins; C413604:nicastrin protein; D018698:Glutamic Acid; D053829:Amyloid Precursor Protein Secretases