beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide.
Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.
Paganetti
Paolo
P
Institute for Research in Biomedicine, CH-6500 Bellinzona, Switzerland.
Calanca
Verena
V
Galli
Carmela
C
Stefani
Muriel
M
Molinari
Maurizio
M
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000096:biosynthesis; D016564:Amyloid beta-Protein Precursor / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001667:Binding, Competitive; D015153:Blotting, Western; D002457:Cell Extracts; D002460:Cell Line; D017077:Culture Media, Conditioned / Q000032:analysis; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D013379:Substrate Specificity; D014162:Transfection
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D017077:Culture Media, Conditioned; D010446:Peptide Fragments; D010455:Peptides; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human; C509280:BACE1 protein, human