Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase.
Proteolytic processing plays crucial roles in physiological and pathophysiological cellular functions such as peptide generation, cell cycle, and apoptosis. We developed a novel biophysical bioluminescence resonance energy transfer (BRET) system between a secreted Vargula luciferase (Vluc) and an enhanced yellow fluorescent protein (EYFP) for visualization of cell biological processes. The bioluminescence spectrum of the fusion protein (Vluc-EYFP) is bimodal (lambdamax = 460 nm (Vluc) and 525nm (EYFP)), indicating that the excited-state energy of Vluc transfers to EYFP (in short, BRET). The BRET signal can be measured in the culture medium and pursue quantitative production of two neuropeptides, nocistatin (NST) and nociceptin/orphanin FQ (N/OFQ) in living cells. NST and N/OFQ are located in tandem on the same precursor, but NST exhibits antagonistic action against N/OFQ-induced central functions. Insertion of a portion of the NST-N/OFQ precursor (Glu-Gln-Lys-Gln-Leu-Gln-Lys-Arg-Phe-Gly-Gly-Phe-Tyr-Gly) in Vluc-EYFP makes the fusion protein cleavable at Lys-Arg in NG108-15 cells, and proprotein convertase 1 enhances this digestion. The change in BRET signals quantifies the processing of the fusion protein. Our novel intramolecular BRET system using a secreted luciferase is useful for investigating peptide processing in living cells.
Otsuji
Tomomi
T
Special Division for Human Life Technology, Cell Dynamics Research Group, National Institute of AIST, Ikeda 563-8577, Japan.
Okuda-Ashitaka
Emiko
E
Kojima
Satoshi
S
Akiyama
Hidefumi
H
Ito
Seiji
S
Ohmiya
Yoshihiro
Y
D001426:Bacterial Proteins; D008156:Luciferases; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D010452:Peptide Biosynthesis / Q000502:physiology*; D013053:Spectrophotometry; D013194:Staining and Labeling / Q000379:methods*
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D008156:Luciferases