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bioRxiv · 10.1101/2020.06.06.137885

Site-Specific Incorporation of Citrulline into Proteins in Mammalian Cells

Mondal, S.·Wang, S.·Zheng, Y.·Sen, S.·Chatterjee, A.·Thompson, P.
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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 novel technology that enables the site-specific incorporation of citrulline (Cit) into proteins in mammalian cells. This approach exploits an E. coli-derived engineered 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 characterized the effect of incorporating Cit at two known autocitrullination sites in Protein Arginine Deiminase 4 (PAD4, R372 and R374) and showed that the R372Cit and R374Cit mutants are 181- and 9-fold less active than the wild-type enzyme. This powerful technology possesses immense potential to decipher the biology of citrullination.

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Mondal, S., Wang, S., Zheng, Y., Sen, S., Chatterjee, A., Thompson, P.. 2020-06-06. Site-Specific Incorporation of Citrulline into Proteins in Mammalian Cells. https://doi.org/10.1101/2020.06.06.137885

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