A Genetic Code Expansion‐Derived Molecular Beacon for the Detection of Intracellular Amyloid‐β Peptide Generation

Abstract

Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often‐small size and intricate post‐translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue‐specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid‐β peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.

Minimal genetically encoded tags for fluorescent protein labeling in living neurons

“…As a proof-of-principle, they showed incorporation of O -methyltyrosine in a reporter GFP TAG protein in neurons. In the meantime, other types of UAAs were applied in further studies of neuronal proteins, such as irreversible or reversible light-induced control of NMDA- and AMPA-type glutamate receptors with photoresponsive UAAs 49 – 52 , fluorescent labeling of NMDA receptors 53 and the Shaker B voltage‐dependent potassium channel 54 , as well as the development of reporters for amyloid precursor protein trafficking and processing 55 , 56 . However, all of these studies were performed using standard cell lines, and the number of studies showing UAA incorporation in neurons has remained limited.…”

Minimal genetically encoded tags for fluorescent protein labeling in living neurons

“…As a proof-of-principle, they showed incorporation of O-methyltyrosine in a reporter GFP TAG protein in neurons. In the meantime, other types of UAAs were applied in further studies of neuronal proteins, such as irreversible or reversible light-induced control of NMDA-and AMPA-type glutamate receptors with photoresponsive UAAs [34][35][36][37] , fluorescent labeling of NMDA receptors 38 and the Shaker B voltage-dependent potassium channel 39 , as well as the development of reporters for amyloid precursor protein trafficking and processing 40,41 . However, all of these studies were performed using standard cell lines, and the number of studies showing UAA incorporation in neurons has remained limited.…”

Molecular probes for cellular imaging of post-translational proteoforms

“…159,160 (b) Genetic code expansion-mediated labeling of intracellular amyloid-b peptides. 162 The amyloid-bcontaining segment within APP is site-specifically labeled with an organic fluorophore (red circle) via genetic code expansion. The fluorescence signal from this label is suppressed prior to proteolysis by an intramolecular quencher (dark grey circle) appended to APP via enzyme-mediated labeling.…”

“…5b ). 162 Here, a fluorophore was embedded in the peptide-containing segment of the pro-protein via genetic code expansion, while a fluorescence quencher was attached to another site within the pro-protein. Fluorescence was suppressed via distance-dependent FRET-based quenching while the pro-protein was intact, but elicited when the pro-protein was proteolyzed and the processed peptide liberated.…”