Differential Protein Citrullination in Human ER– and ER+ Tumor and Adjacent Healthy Breast Tissue

Post-translational modification of arginine to citrulline
is catalyzed
by members of the peptidylarginine deiminase (PAD) family. Dysregulation
of this catalysis is a significant driver of the pathogenesis of numerous
inflammatory diseases, including cancer. However, dysregulation of
PAD activity has not been examined in breast cancer with respect to
hormone receptor status. In this study, we measured PAD enzyme levels
using Western blotting and investigated protein citrullination using
a mass spectrometry-based proteomics approach in primary estrogen
receptor negative (ER−) or positive (ER+) breast tumor and
matched adjacent normal tissue. Our findings reveal 72 and 41 citrullinated
proteins in ER– tumor and adjacent healthy tissue, respectively,
where 20 of these proteins are common between the two groups. We detected
64 and 49 citrullinated proteins in ER+ tumor and adjacent healthy
tissue, respectively, where 32 proteins are common. Interestingly,
upon comparison of ER– and ER+ tumor tissue, only 32 citrullinated
proteins are shared between the two and the rest are unique to the
tumor’s receptor status. Using the STRING database for protein–protein
interaction network analysis, these proteins are involved in protein-folding
events (i.e., heat shock proteins) in ER– samples and blood-clotting
events (i.e., fibulin) in ER+ samples. Constituents of the extracellular
matrix structure (i.e., collagen and fibrinogen) were found in both.
Herein, we establish evidence that supports the role of this unique
post-translational modification in breast cancer biology. Finally,
to aid drug discovery against citrullination, we developed a liquid
chromatography–ultraviolet method to measure PAD enzymatic
activity and optimized glucagon-like peptide II to quantitatively
measure the ability of PADs to citrullinate its substrate.