Demonstration by FRET of BACE interaction with the amyloid precursor protein at the cell surface and in early endosomes.
Amyloid-beta peptide, which accumulates in senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by proteolytic processing. beta-secretase (Asp2), which cleaves APP at the N-terminus of amyloid-beta, has recently been identified to be the protease BACE. In the present study, we examined the subcellular localization of interactions between APP and BACE by using both double immunofluorescence and a fluorescence resonance energy transfer (FRET) approach. Cell surface APP and BACE, studied by using antibodies directed against their ectodomains in living H4 neuroglioma cells co-transfected with APP and BACE, showed exquisite co-localization and demonstrated a very close interaction by FRET analysis. The majority of cell surface APP and BACE were internalized after 15 minutes, but they remained strongly co-localized together in the early endosomal compartment, where FRET analysis demonstrated a continued close interaction. By contrast, at later timepoints, almost no co-localization or FRET was observed in lysosomal compartments. To determine whether the APP-BACE interaction on cell surface and endosomes contributed to amyloid-beta synthesis, we labeled cell surface APP and demonstrated detectable levels of labeled amyloid-beta within 30 minutes. APP-Swedish mutant protein enhanced amyloid-beta synthesis from cell surface APP, consistent with the observation that it is a better BACE substrate than wild-type APP. Taken together, these data confirm a close APP-BACE interaction in early endosomes, and highlight the cell surface as an additional potential site of APP-BACE interaction.
Kinoshita
Ayae
A
Alzheimer Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
Fukumoto
Hiroaki
H
Shah
Tejal
T
Whelan
Christa M
CM
Irizarry
Michael C
MC
Hyman
Bradley T
BT
D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis*; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases