Genetically Encodable Bacterial Flavin Transferase for Fluorogenic Protein Modification in Mammalian Cells.

Abstract:

:A bacterial flavin transferase (ApbE) was recently employed for flavin mononucleotide (FMN) modification on the Na+-translocating NADH:quinone oxidoreductase C (NqrC) protein in the pathogenic Gram-negative bacterium Vibrio cholerae. We employed this unique post-translational modification in mammalian cells and found that the FMN transfer reaction robustly occurred when NqrC and ApbE were genetically targeted in the cytosol of live mammalian cells. Moreover, NqrC expression in the endoplasmic reticulum (NqrC-ER) induced the retro-translocation of NqrC to the cytosol, leading to the proteasome-mediated ER-associated degradation of NqrC, which is considered to be an innate immunological response toward the bacterial protein. This unexpected cellular process of NqrC-ER could be exploited for the construction of an in cellulo proteasome inhibitor screening system, and our proposed approach yielded substantially improved results compared to a previous method. In addition, a truncated version of RnfG (half-RnfG) was found to be potentially useful as a genetically encoded tag for monitoring protein-protein interactions in a specific compartment, even in the ER, in a live cell according to its fluorogenic post-translational modification via ApbE. This new genetically encoded system in mammalian cells should serve as a valuable tool for anticancer drug screening and other applications in molecular and synthetic biology.

journal_name

ACS Synth Biol

journal_title

ACS synthetic biology

authors

Kang MG,Park J,Balboni G,Lim MH,Lee C,Rhee HW

doi

10.1021/acssynbio.6b00284

subject

Has Abstract

pub_date

2017-04-21 00:00:00

pages

667-677

issue

4

issn

2161-5063

journal_volume

6

pub_type

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