Site-specific incorporation of citrulline into proteins in mammalian cells

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.

A Streamlined Data Analysis Pipeline for the Identification of Sites of Citrullination

“…Specifically, ionFinder unambiguously mapped 28 citrullinated residues on PAD1 (76% of arginine residues), 20 on PAD2 (61% of arginine residues), 19 on PAD3 (49% of arginine residues), and 21 on PAD4 (78% of arginine residues) (Figure ). We compared the PAD4 autocitrullination sites obtained from ionFinder to a previously published data set where sites of autocitrullination of PAD4 were evaluated in a time-dependent manner using tandem mass tag (TMT) labeling . The sites identified by ionFinder displayed significant overlap with the previously determined sites of PAD4 autocitrullination.…”

“…To evaluate the utility of ionFinder in a “real world” situation, we examined the autocitrullination of all four active PADs, i.e., PAD1–4. Note that extensive research has established that the PADs autocitrullinate in biological systems, and once modified, protein–protein interactions are impacted. − Notably, recombinant, purified PADs readily undergo autocitrullination under reducing conditions in the presence of Ca 2+ . As such, PAD1–4 were incubated with Ca 2+ (5 mM) and TCEP (1 mM) at 37 °C for 1 h to induce autocitrullination.…”

“…These qualities make this family of proteins prime targets for the PADs. Future work entails mechanistic investigation of these targets to understand the functional consequences of citrullination in vitro and in cells using our recently described approach to site-specifically incorporate citrulline into proteins …”

“…We compared the PAD4 autocitrullination sites obtained from ionFinder to a previously published data set where sites of autocitrullination of PAD4 were evaluated in a time-dependent manner using tandem mass tag (TMT) labeling. 36 The sites identified by ionFinder displayed significant overlap with the previously determined sites of PAD4 autocitrullination. Because these autocitrullination sites were obtained using purified, recombinant PADs, we cannot conclude that they all occur endogenously.…”

“…Future work entails mechanistic investigation of these targets to understand the functional consequences of citrullination in vitro and in cells using our recently described approach to site-specifically incorporate citrulline into proteins. 36 In summary, ionFinder and envoMatch automate the process of verifying the assignment of Cit annotations by standard database search algorithms, primarily through the automated assignment of diagnostic neutral loss species. The primary strength of ionFinder and envoMatch is the speed of analysis, which far exceeds the throughput that can be achieved by manual spectral matching.…”