Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation

Abstract

Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation.

CXCR4 and MIF are required for neutrophil extracellular trap release triggered by Plasmodium-infected erythrocytes

“…The fundamental role for PAD4 in NETosis has been systematically addressed in a recent report [42]. Additionally, a recent report has shown that chromatin decondensation may procced in a PAD4-dependent, NE-independent mechanism [43], in…”

“…The fundamental role for PAD4 in NETosis has been systematically addressed in a recent report [ 42 ]. Additionally, a recent report has shown that chromatin decondensation may procced in a PAD4-dependent, NE-independent mechanism [ 43 ], in which other neutrophil proteases, like calpain, may function in concert with PAD4 to fully induce chromatin decondensation and nuclear lamina breakdown. We think that a similar mechanism may be operative in this model.…”

Role of HMGB1 in the Interplay between NETosis and Thrombosis in Ischemic Stroke: A Review

“…On the other hand, in case of the function of PAD4, PAD4 citrullinates HMGB1. PAD4 citrullinates HMGB1 at arginine residues localized in the inter-domain region between A and B boxes; this modification enables calpain to digest HMGB1 into two fragments, leading to efficient chromatin decondensation [ 129 ]. In the absence of PAD4, calpain alone did not induce chromatin decondensation, whereas the concerted action of calpain and PAD4 enhanced it, suggesting a novel mechanistic role of PAD4 in NET formation [ 129 ].…”

“…PAD4 citrullinates HMGB1 at arginine residues localized in the inter-domain region between A and B boxes; this modification enables calpain to digest HMGB1 into two fragments, leading to efficient chromatin decondensation [ 129 ]. In the absence of PAD4, calpain alone did not induce chromatin decondensation, whereas the concerted action of calpain and PAD4 enhanced it, suggesting a novel mechanistic role of PAD4 in NET formation [ 129 ]. As HMGB1 not only induce NETosis but is also a part of the extruded NETs [ 15 ], reciprocal regulation between HMGB1 and PAD4 may contribute to this aggravation cycle in cerebral ischemia.…”

Neutrophil Extracellular Traps: Signaling Properties and Disease Relevance

“…Moreover, citrullination has recently been shown to impact proteolysis: Tilvawala et al [45] demonstrated enhances proteolysis by serine proteases via PAD4-mediated inhibition of serine protease inhibitors. Moreover, proteolysis by calpain is enhanced by conformational changes induced by citrullination thus favoring nuclear lamina breakdown and chromatin decondensation [46].…”