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1.
Nat Commun ; 13(1): 1065, 2022 03 04.
Article in English | MEDLINE | ID: mdl-35246533

ABSTRACT

Naturally competent bacteria encode sophisticated protein machinery for the uptake and translocation of exogenous DNA into the cell. If this DNA is integrated into the bacterial genome, the bacterium is said to be naturally transformed. Most competent bacterial species utilise type IV pili for the initial DNA uptake step. These proteinaceous cell-surface structures are composed of thousands of pilus subunits (pilins), designated as major or minor according to their relative abundance in the pilus. Here, we show that the minor pilin FimT plays an important role in the natural transformation of Legionella pneumophila. We use NMR spectroscopy, in vitro DNA binding assays and in vivo transformation assays to understand the molecular basis of FimT's role in this process. FimT binds to DNA via an electropositive patch, rich in arginines, several of which are well-conserved and located in a conformationally flexible C-terminal tail. FimT orthologues from other Gammaproteobacteria share the ability to bind to DNA. Our results suggest that FimT plays an important role in DNA uptake in a wide range of competent species.


Subject(s)
Fimbriae Proteins , Legionella pneumophila , Bacterial Proteins/metabolism , DNA/metabolism , DNA, Bacterial/metabolism , Fimbriae Proteins/metabolism , Fimbriae, Bacterial/metabolism , Legionella pneumophila/genetics , Legionella pneumophila/metabolism , Transformation, Bacterial
2.
Chembiochem ; 21(13): 1861-1867, 2020 07 01.
Article in English | MEDLINE | ID: mdl-32011787

ABSTRACT

Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio-orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence-based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse-chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.


Subject(s)
Fluorescent Dyes/chemistry , Interleukins/metabolism , Amino Acids/chemistry , Amino Acids/metabolism , Amino Acyl-tRNA Synthetases/metabolism , HEK293 Cells , Half-Life , Humans , Interleukins/chemistry , Interleukins/genetics , Kinetics , Protein Folding , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism
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