Aph-1 associates directly with full-length and C-terminal fragments of gamma-secretase substrates.
Gamma-secretase is a ubiquitous, multiprotein enzyme composed of presenilin, nicastrin, Aph-1, and Pen-2. It mediates the intramembrane proteolysis of many type 1 proteins, plays an essential role in numerous signaling pathways, and helps drive the pathogenesis of Alzheimer disease by excising the hydrophobic, aggregation-prone amyloid beta-peptide from the beta-amyloid precursor protein. A central unresolved question is how its many substrates bind and enter the gamma-secretase complex. Here, we provide evidence that both the beta-amyloid precursor protein holoprotein and its C-terminal fragments, the immediate substrates of gamma-secretase, can associate with Aph-1 at overexpressed as well as endogenous protein levels. This association was observed using bi-directional co-immunoprecipitation in multiple systems and detergent conditions, and an beta-amyloid precursor protein-Aph-1 complex was specifically isolated following in situ cross-linking in living cells. In addition, another endogenous canonical gamma-substrate, Jagged, showed association of both its full-length and C-terminal fragment forms with Aph-1. We were also able to demonstrate that this interaction with substrates was conserved across the multiple isoforms of Aph-1 (beta, alphaS, and alphaL), as they were all able to bind beta-amyloid precursor protein with similar affinity. Finally, two highly conserved intramembrane histidines (His-171 and His-197) within Aph-1, which were recently shown to be important for gamma-secretase activity, are required for efficient binding of substrates. Taken together, our data suggest a dominant role for Aph-1 in interacting with gamma-secretase substrates prior to their processing by the proteolytic complex.
Chen
Allen C
AC
Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Guo
Lucie Y
LY
Ostaszewski
Beth L
BL
Selkoe
Dennis J
DJ
LaVoie
Matthew J
MJ
D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry*; D016229:Amyloid beta-Peptides / Q000737:chemistry; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D010450:Endopeptidases; D006639:Histidine / Q000737:chemistry; D006801:Humans; D016086:Lentivirus / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D010447:Peptide Hydrolases; D053763:Presenilins / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; D053763:Presenilins; D006639:Histidine; C475154:APH1A protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases