Abstract
Citrullination is a post-translational modification (PTM) of arginine that is crucial for several physiological processes, including gene regulation and neutrophil extracellular trap formation. Despite recent advances, studies of protein citrullination remain challenging due to the difficulty of accessing proteins homogeneously citrullinated at a specific site. Herein, we report a technology that enables the site-specific incorporation of citrulline (Cit) into proteins in mammalian cells. This approach exploits an engineered E. coli-derived leucyl tRNA synthetase-tRNA pair that incorporates a photocaged-citrulline (SM60) into proteins in response to a nonsense codon. Subsequently, SM60 is readily converted to Cit with light in vitro and in living cells. To demonstrate the utility of the method, we biochemically characterize the effect of incorporating Cit at two known autocitrullination sites in Protein Arginine Deiminase 4 (PAD4, R372 and R374) and show that the R372Cit and R374Cit mutants are 181- and 9-fold less active than the wild-type enzyme. This technology possesses the potential to decipher the biology of citrullination.
Highlights
Citrullination is a post-translational modification (PTM) of arginine that is crucial for several physiological processes, including gene regulation and neutrophil extracellular trap formation
SM60 was synthesized over two steps from L-ornithine, and was characterized by 1H, 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) (Supplementary Figs. 1 and 2a)
Using liquid chromatography-multiple sclerosis (MS) (LC-MS), we found that SM60 can be quantitatively converted to Cit in phosphate-buffered saline (PBS) supplemented with dithiothreitol (DTT) using 365 nm UV radiation for 5 min (Fig. 1d and Supplementary Fig. 3)
Summary
Citrullination is a post-translational modification (PTM) of arginine that is crucial for several physiological processes, including gene regulation and neutrophil extracellular trap formation. To demonstrate the utility of the method, we biochemically characterize the effect of incorporating Cit at two known autocitrullination sites in Protein Arginine Deiminase 4 (PAD4, R372 and R374) and show that the R372Cit and R374Cit mutants are 181- and 9-fold less active than the wild-type enzyme This technology possesses the potential to decipher the biology of citrullination. The in vitro translation systems or post-translational mutagenesis approaches that have been used to incorporate Cit are limited by their cumbersome nature, the need for specialized equipment, and for the latter approach, the need to incorporate a dehydroalanine at the site of modification, which is itself challenging and generates a mixture of D- and L-stereoisomers[18,19] These strategies preclude the expression of site- citrullinated proteins in living cells, and are ineffective for interpreting the downstream implications of this PTM. Genetically encoding Cit using this technology has remained elusive so far
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