title: 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.
authors.forename: Myeong-Gyun
authors.initials: MG
authors.lastname: Kang
authors.forename: Jumi
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Life and Environmental Sciences, Pharmaceutical, Pharmacological and Nutraceutical Sciences Unit, University of Cagliari , I-09124 Cagliari, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Balboni
authors.forename: Mi Hee
authors.identifier: 0000-0003-3377-4996
authors.initials: MH
authors.lastname: Lim
authors.forename: Changwook
authors.initials: C
authors.lastname: Lee
authors.forename: Hyun-Woo
authors.identifier: 0000-0002-3817-3455
authors.initials: HW
authors.lastname: Rhee
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000069286:Bortezomib / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D060746:Endoplasmic Reticulum-Associated Degradation / Q000187:drug effects; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007976:Leupeptins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D018613:Microscopy, Confocal; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D020543:Proteome / Q000037:antagonists & inhibitors / Q000378:metabolism; D011808:Quinone Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014166:Transferases / Q000235:genetics / Q000378:metabolism*; D014734:Vibrio cholerae / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D007976:Leupeptins; D020543:Proteome; D011994:Recombinant Proteins; D000069286:Bortezomib; C084142:sodium-translocating NADH-quinone reductase; D011808:Quinone Reductases; D014166:Transferases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: ER-associated degradation; flavin mononucleotide modification; flavin transferase; proteasome inhibitor screening; protein−protein interaction; subcellular compartment