Inhibiting peptidylarginine deiminases (PAD1-4) by targeting a Ca2+ dependent allosteric binding site

Introduction

Protein citrullination is a post-translational modification catalyzed by the peptidylarginine deiminase (PAD) enzyme family in which arginine residues are converted to citrulline. There are five human PAD isozymes, including the catalytically active members PAD1-4 and PAD6, which is reported to be enzymatically inactive^{1, 2–3}. The PADs are calcium (Ca²⁺)-dependent enzymes with either four (PAD1), five (PAD3 and PAD4), or six (PAD2) Ca²⁺ ion binding sites (designated as Ca1-6) identified from protein crystal structure analysis^{4, 5, 6–7}. As a consequence, PAD functional activity is tightly controlled by local Ca²⁺ ion concentration. All the calcium binding sites are required to be occupied in order to induce a conformational change in the protein fold, leading to the holoenzyme with the active site cysteine (Cys645, PAD4) in the proper orientation for the enzymatic reaction⁸. The expression profile of the PAD family is broad and isozyme dependent, with localization to the epidermis and uterus (PAD1), hematopoietic cells (PAD2 and PAD4), central nervous system (PAD2), hair follicles (PAD3), and oocytes and embryos (PAD6)⁹. All of the PADs may be found in the cytoplasm of cells; however, PAD4 is additionally located in the nucleus¹⁰. PAD2 and PAD4 are also commonly observed in extracellular compartments as a consequence of release during certain cell death mechanisms¹¹.

Protein citrullination mediated by the PAD enzyme family has a profound impact on the electrostatics of substrate proteins since a positively charged arginine residue is replaced by a neutral citrulline. This change may have consequences on protein structure, protein-protein interactions, and protein stability, leading to altered signaling networks and cellular responses. The scope of known citrullination sites has recently been estimated at over 14,000, covering more than 4000 proteins¹². Elevated levels of citrullinated proteins or their corresponding autoantibodies have been associated with disorders including autoimmunity^{13, 14, 15–16}, neurodegenerative disease^17,18, and cancers¹⁹. Of particular note is the role that protein citrullination plays in neutrophils and its possible linkage to the progression of rheumatoid arthritis (RA). Neutrophils may undergo NETosis, a form of programmed cell death that releases neutrophil extracellular traps (NETs)²⁰. During this event, citrullinated proteins and PAD enzymes are released into...