Peptidylarginine deiminase type 6 in rheumatoid arthritis pathogenesis

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PROJECT SUMMARY/ABSTRACT Citrullinated proteins are major targets of the immune system in rheumatoid arthritis (RA), with recent evidence suggesting that mechanisms that enhance citrullination are associated with the worst disease outcome. Protein citrullination is the enzymatic conversion of arginine residues to citrulline and is mediated by the family of calcium- dependent peptidylarginine deiminase (PAD) enzymes. Five PAD members have been found in humans, PAD1- 4, and the catalytically inactive PAD6. While several mechanisms have been linked to abnormal citrullination in RA, including hyperactivation of PADs (particularly PAD4) during neutrophil death and the production of autoantibodies that enhance PAD4 activity, regulatory pathways that control citrullination are poorly understood. Since PADs require supraphysiologic concentrations of calcium for activity in vitro, it is suspected that PAD activation in vivo requires additional factors that modulate the enzyme’s sensitivity for calcium. Such co-factors, however, have not been identified. Our preliminary data demonstrate that PAD6, which has no citrullinating activity, and 3 novel PAD6 splicing variants described for the first time in this proposal, act as co-factors that decrease the calcium requirement of PAD4 for catalysis. Moreover, we found that all PAD6 variants are abundant in neutrophils, which are considered a major source of citrullinated proteins in the RA joint. Taken together, we hypothesize that PAD6 and its variants may be responsible for modulating the magnitude of PAD4 activity both in physiologic and pathologic conditions. We will examine this hypothesis directly in the human model in three specific aims. In Aim 1, we will define how PAD6 variants regulate PAD4 activity with the goal of identifying the biochemical mechanisms by which PAD6 variants decrease the calcium requirement of PAD4. Aim 2 will define the expression and function of PAD6 and its splicing variants at the cellular level. Aim 3 will study the expression of PAD6 isoforms in RA synovial fluid to address their relationship with the abnormal production of citrullinated proteins in RA. Together, these studies will define novel interacting mechanisms of disease amplification relevant to sustain the production of citrullinated proteins in the RA joints. Furthermore, these studies may provide evidence for targeting PAD6 as a novel therapeutic strategy to modulate PAD4 activity, which is highly significant for diseases in which citrullination has been pathogenically implicated.