Amyloid precursor protein and Presenilin 1 interaction studied by FRET in human H4 cells.
The mayor pathologic hallmarks of Alzheimer's disease (AD) are senile plaque and neurofibrillary tangles. Senile plaque are primarily made up of deposits of amyloid-beta protein, a proteolytic product derived from the amyloid precursor protein (APP). APP is a transmembrane protein detected into the endoplasmic reticulum, in the Golgi apparatus, at the cell surface, recycled by endocytosis to endosomes, whose physiological function is unclear. Presenilins (PS), are a component of gamma-secretase complex that cleave alpha-CTFs (carboxy-terminal fragment), or beta-CTFs, leaving 40 or 42 amino acids amyloid-beta peptides and 58 or 56 amino acids intracellular domains (AICD). Where the amyloid-beta peptides is generated is not clear. The study of APP-PS interaction in specific cell compartments provides a good opportunity to light upon the molecular mechanisms regulating the activity of the "gamma-secretase complex," and where beta-amyloid is generated. In our study we used a biophysical assay of protein proximity: fluorescence resonance energy transfer (FRET), that can provide information about molecular interactions when two proteins are in the close proximity (<10 nm), to examine the subcellular localization and interaction between APP and PS1 in human neuroglioma cells (H4). Confocal microscopic analysis reveals extensive colocalization in different cells' compartment, and centrosomal or microtubule organizing center (MTOC) localization of APP and PS1, but not necessarily a close molecular interaction. We used FRET to determine if APP and PS1 interact at the cell centrosome. FRET data suggest a close interaction between APP and PS1 in subcellular compartments and at the centrosome of H4 cells. Using this approach we show that APP and PS1 are closely associated in the centrosomes of the H4 cell.
Nizzari
Mario
M
Pharmacology, Department of Oncology, Biology and Genetics, University of Genova, Genova, Italy.
Venezia
Valentina
V
Bianchini
Paolo
P
Caorsi
Valentina
V
Diaspro
Alberto
A
Repetto
Emanuela
E
Thellung
Stefano
S
Corsaro
Alessandro
A
Carlo
Pia
P
Schettini
Gennaro
G
Florio
Tullio
T
Russo
Claudio
C
D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis* / Q000737:chemistry; D045744:Cell Line, Tumor; D018385:Centrosome / Q000378:metabolism / Q000648:ultrastructure*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D053764:Presenilin-1 / Q000096:biosynthesis* / Q000737:chemistry; D011485:Protein Binding
D016564:Amyloid beta-Protein Precursor; D053764:Presenilin-1