ABSTRACT
Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins. Additionally, C- and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid beta-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the gamma-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of gamma-secretase activity.
Subject(s)
Endopeptidases/metabolism , Gene Expression Regulation, Enzymologic , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Adenoviridae/genetics , Alleles , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , Cell Membrane/metabolism , Culture Media, Conditioned/pharmacology , DNA, Complementary/metabolism , Fibroblasts/metabolism , Genes, Reporter , Genotype , Green Fluorescent Proteins , Heterozygote , Humans , Luciferases/metabolism , Luminescent Proteins/metabolism , Membrane Glycoproteins/physiology , Membrane Proteins/physiology , Mice , Mice, Knockout , Peptides/chemistry , Phenotype , Presenilin-1 , Presenilin-2 , Protein Structure, Tertiary , RNA, Messenger/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Time FactorsABSTRACT
Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.
