Abstract
Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H2O2 and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H2O2 at concentrations above 40 µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H2O2 at concentrations up to 1000 µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.
Highlights
Citrullination, i.e. conversion of peptidyl-arginine into peptidyl-citrulline, is a post-translational protein modification catalysed by peptidylarginine deiminase (PAD) in a calcium-dependent manner[1]
Using human fibrinogen coated on microtiter plates as substrate, we measured the catalytic activity of Recombinant human PAD2 (rhPAD2) or rhPAD4 in the presence of a reaction buffer containing 15 mM GSH and 5 mM calcium, previously shown to be optimal for PADs to be enzymatically active[11]
At concentrations above 40 mM, exogenously added H2O2 lowered the catalytic activity of rhPAD2 and rhPAD4 in a dosedependent manner, which did not differ between the two PAD isoforms (Figure 1A) Half-maximal PAD activity was observed at $130 mM H2O2, and virtually no activity was observed at concentrations above 600 mM
Summary
Citrullination, i.e. conversion of peptidyl-arginine into peptidyl-citrulline, is a post-translational protein modification catalysed by peptidylarginine deiminase (PAD) in a calcium-dependent manner[1]. The catalytic activity of PAD requires reducing conditions[8] These are usually obtained in vitro using the non-physiological reducing agent dithiothreitol (DTT). Intracellular ROS may promote intracellular histone H3 citrullination[23,24] and NETosis[23,25], which has led to the suggestion that ROS may activate PAD4 downstream[26]. Such studies have generally not concerned extracellular proteins, which account for a large part of citrullinated proteins in the synovium of RA patients[27,28]. The role of NOX2 in regulation of extracellular PAD activity is examined
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