Activation of PAD4 in NET formation

Abstract

Peptidylarginine deiminases, or PADs, convert arginine residues to the non-ribosomally encoded amino acid citrulline in a variety of protein substrates. PAD4 is expressed in granulocytes and is essential for the formation of neutrophil extracellular traps (NETs) via PAD4-mediated histone citrullination. Citrullination of histones is thought to promote NET formation by inducing chromatin decondensation and facilitating the expulsion of chromosomal DNA that is coated with antimicrobial molecules. Numerous stimuli have been reported to lead to PAD4 activation and NET formation. However, how this signaling process proceeds and how PAD4 becomes activated in cells is largely unknown. Herein, we describe the various stimuli and signaling pathways that have been implicated in PAD4 activation and NET formation, including the role of reactive oxygen species generation. To provide a foundation for the above discussion, we first describe PAD4 structure and function, and how these studies led to the development of PAD-specific inhibitors. A comprehensive survey of the receptors and signaling pathways that regulate PAD4 activation will be important for our understanding of innate immunity, and the identification of signaling intermediates in PAD4 activation may also lead to the generation of pharmaceuticals to target NET-related pathogenesis.

Keywords: NET; PAD4; citrullination; deimination; neutrophil.

FIGURE 1

Conversion of arginine into citrulline.Peptidylarginine deiminase (PAD) enzymes convert an arginine residue into citrulline.

FIGURE 2

(A) PAD4 in complex with Histone H4 1–5 (SGRGK). PAD4 active site residue side chains are colored gray (D350, H471, D473, C645A) and residues that are involved in binding the H4 1–5 backbone and S1 are colored yellow (Q346, W347, R347). N-terminally acetylated H4 1–5 is shown in cyan with Arginine 3 bound in the PAD4 active site. Polar residue interactions between PAD4 and H4 1–5 are indicated by dashed lines. The mutation of the active site cysteine residue (C645) to an alanine (C645A) was necessary to achieve substrate binding in the crystal structure as described in Arita et al. (2006). Figure was created from the structure filed under PDB code 2dey. (B) Proposed catalytic mechanism for PAD4. (C) Haloacetamidine-based inhibitors targeting the PAD isozymes.

FIGURE 3

Model of PAD4 activation in NET formation. Pathways that activate NET formation are less defined than phagocytic pathways, but are known to require NADPH oxidase activity and the activation of PAD4 and subsequent histone citrullination. PAD enzymes are Ca²⁺-dependent. Since PAD4-mediated histone citrullination is abrogated by the NADPH inhibitor apocyanin (Neeli et al., 2009), we speculate that NADPH regulated ROS generation and increase Ca²⁺ levels may converge to activate PAD4 in neutrophils.