Citrullination of Proteins as a Specific Response Mechanism in Plants

Abstract

Arginine deimination, also referred to as citrullination of proteins by L-arginine deiminases, is a post-translational modification affecting histone modifications, epigenetic transcriptional regulation, and proteolysis in animals but has not been reported in higher plants. Here we report, firstly, that Arabidopsis thaliana proteome contains proteins with a specific citrullination signature and that many of the citrullinated proteins have nucleotide-binding regulatory functions. Secondly, we show that changes in the citrullinome occur in response to cold stress, and thirdly, we identify an A. thaliana protein with peptidyl arginine deiminase activity that was shown to be calcium-dependent for many peptide substrates. Taken together, these findings establish this post-translational modification as a hitherto neglected component of cellular reprogramming during stress responses.

Keywords: arginine deiminase; citrullination; citrullinome; cold stress; post-translational modification.

FIGURE 1

(A) Enzymatic formation of a citrulline. Enzymatic hydrolysis of peptidyl-arginine to peptidyl-citrulline by peptidylarginine deiminase (PAD). (B) MS/MS spectra of a citrullinated Chromatin Remodeling 34 (ChR34). The MS/MS spectrum of Chromatin Remodeling 34 (At2g21450) contains citrullinated residues on positions 10 and 14. Indicated on the peptide sequence are the detected fragment ions, as indicated by the b- and y-ions and their relative intensities. The ion score of the peptide is 41. The insert shows a western blot of anti-citrulline immunoprecipitated proteins. The arrow shows the protein band identified as “Chromatin Remodeling 34.”

FIGURE 2

(A) Amino acid sequence, (B) surface, and (C) ribbon structure of At5g08170 (PDB ID: 3H7K). Amino acids appearing in the motif: W-X-R-D-[TS]-G-X(100,140)-H-[VIL]-D are highlighted in red in (A) the full-length At5g08170 amino acid sequence, and colored yellow in (B) the surface and (C) ribbon At5g08170 structures. The cysteines (C180 and C366) implicated in the catalytic activity of agmatine deiminase are highlighted in brownish red in (A) and orange in (C), the ribbon structures. Amino acids R93–G96 from the motif occupy a clear cavity that can spatially accommodate an R residue (B) and thus is assigned the catalytic center for arginine deiminase (highlighted in yellow). All analyses and images were created using UCSF Chimera (ver. 1.10.1). Chimera was developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311).