Cellular Consequences, Citrullination Substrates, and Antigenicity of Targeting PAD4 to Cell Surfaces

Protein arginine deiminase-4 (PAD4) catalyzes hydrolysis of arginine to citrulline in proteins that promote widespread cellular changes that can induce innate immunity and promote cancer. Hyperactivity of PAD4 leads to a form of cell death called NETosis, releasing PAD4 to the extracellular space to promote various autoimmune diseases through the generation of anticitrulline protein antibodies (ACPAs). Little is known about the specific citrullinated substrates that lead to autoimmunity, but there is growing evidence that PAD4 is localized to the cell surface in response to inflammation. Here, we characterize the cellular consequences of exogenous PAD4, showing that it induces morphological changes that increase cell migration, a hallmark of cancer. We then devised a robust proteomics approach to identify PAD4 substrates. We identified ∼ 3000 citrullinated peptides from 1300 proteins upon exogenous addition of PAD4 both inside and outside of cells. This extracellular set can be further augmented by targeting PAD4 to cancer cells using a HER2 binding protein conjugate. Finally, we studied how citrullinated cells can induce a humoral response in vivo to produce ACPAs. We believe these studies further our understanding of cellular consequences of extracellular PAD4 and identify new PAD4 substrates that are potential neoepitopes for ACPA generation.