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P4–052: Accumulation of sphingolipids increases secretion of the amyloid β–peptide by stabilization of the β–amyloid precursor protein
Author(s) -
Tamboli Irfan Y.,
Hampel Heike,
Sandhoff Konrad,
Walter Jochen
Publication year - 2006
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2006.05.1790
Subject(s) - sphingolipid , amyloid precursor protein , endocytic cycle , microbiology and biotechnology , endosome , sphingomyelin , secretion , biochemistry , amyloid precursor protein secretase , p3 peptide , chemistry , neurodegeneration , amyloid (mycology) , biology , endocytosis , alzheimer's disease , cholesterol , intracellular , medicine , receptor , inorganic chemistry , disease
mechanisms of enzyme-substrate interaction and the substrate requirements for -secretase-dependent cleavage remain unclear. It is possible that substrate sequences distant from the cleavage sites are required for these actions. Objective(s): To elucidate the structural requirements for -secretase recognition and cleavage of APP. Methods: We constructed a fusion protein of the Gal4 DNA-binding/VP16 transactivation (GVP) domain with the C99 form of APP, which is an immediate substrate for -secretase in vivo. Upon transfection into HEK293 cells, the -seceretase-dependent cleavage of this hybrid protein can be quantified with A ELISAs and a luciferase reporter assay measuring APP intracellular domain (AICD) generation. Using this assay platform, we then performed mutagenesis studies to examine the functional role of APP luminal juxtamembrane domain (LJD) in substrate recognition and/or cleavage by -secretase. Results: C99-GVP is a robust -secretase substrate, as shown by the aforementioned assays. Major deletion of the LJD resulted in dramatic inhibition of its cleavage by -secretase. In addition, replacement of the LJD of APP with corresponding regions from other known type I membrane proteins, such as Notch, amyloid precursor-like protein 2 (APLP2) and sterol regulatory element-binding protein (SREBP), also caused moderate to severe reduction in cleavage efficiency. Conclusions: Taken together, our results suggest an important role of the luminal juxtamembrane domain of APP in substrate recognition and/or cleavage by -secretase.