Dual Bioorthogonal Labeling of the Amyloid-ß Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products.
J Alzheimers Dis
; 72(2): 537-548, 2019.
Article
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| MEDLINE
| ID: mdl-31609694
The amyloid-ß protein precursor (AßPP) is critical in the pathophysiology of Alzheimer's disease (AD), since two-step proteolytic processing of AßPP generates the neurotoxic amyloid-ß peptide (Aß). We developed a dual fluorescence labeling system to study the exact subcellular location of γ-secretase cleavage of AßPP. The C-terminal tail of AßPP was fluorescently labeled using a SNAP-tag, while the Aß region of AßPP was fluorescently tagged with a dye at a genetically-encoded noncanonical amino acid (ncAA). The ncAA was introduced at specific positions in AßPP using a genetic code expansion strategy and afterwards, the reactive side-chain of the ncAA was coupled to the dye using a bioorthogonal labeling chemistry. In proof-of-concept experiments, HEK293T cells were transfected with plasmids containing engineered AßPP harboring an amber mutation and an amber codon suppression system with an evolved tRNA synthetase/tRNA pair and grown in the presence of a lysine-derived ncAA. Processing of the AßPP variants was validated with ELISA and immunoblotting, and seven AßPP mutants that showed similar cleavage pattern as wild-type AßPP were identified. The AßPP mutant was fluorescently labeled with 6-methyl-tetrazine-BDP-FL and TMR-Star at the ncAA and SNAP-tag, respectively. Using this approach, AßPP was fluorescently labeled at two sites in living cells with minimal background to allow monitoring of Aß and C-terminal cleavage products simultaneously. The method described provides a powerful tool to label Aß with minimal perturbations of its processing, thus enabling studies of the trafficking of the cleavage products of AßPP.
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Base de datos:
MEDLINE
Asunto principal:
Precursor de Proteína beta-Amiloide
Límite:
Humans
Idioma:
En
Revista:
J Alzheimers Dis
Asunto de la revista:
GERIATRIA
/
NEUROLOGIA
Año:
2019
Tipo del documento:
Article
País de afiliación:
Suecia