Autocitrullination of PAD4 does not alter its enzymatic activity: In vitro and in silico studies

Xiaosong Liu; Kanin Wichapong; Sebastiaan Lamers; Chris P M Reutelingsperger; Gerry A F Nicolaes

doi:10.1016/j.biocel.2021.105938

Autocitrullination of PAD4 does not alter its enzymatic activity: In vitro and in silico studies

Int J Biochem Cell Biol. 2021 May:134:105938. doi: 10.1016/j.biocel.2021.105938. Epub 2021 Jan 30.

Authors

Xiaosong Liu¹, Kanin Wichapong¹, Sebastiaan Lamers¹, Chris P M Reutelingsperger¹, Gerry A F Nicolaes²

Affiliations

¹ Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6200 MD, Maastricht, the Netherlands.
² Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6200 MD, Maastricht, the Netherlands. Electronic address: g.nicolaes@maastrichtuniversity.nl.

PMID: 33529715
DOI: 10.1016/j.biocel.2021.105938

Abstract

Background: Protein arginine deiminase 4 (PAD4) is an enzyme capable of converting arginine (positively charged residue) into citrulline (neutral residue). PAD4 is a promiscuous enzyme since it citrullinates various substrates, including small peptides, large proteins and itself. The effect of autocitrullination on PAD4 activity remains controversial and inconclusive. We hypothesized that PAD4 autocitrullination may influence the activity of PAD4 by indirectly altering its binding to substrate.

Methods: We employed mass spectrometry analysis to study the process of autocitrullination. The kinetics of citrullination of PAD4 and citrullinated PAD4 (citPAD4) towards substrates of different sizes (0.17-15.4 kDa), i.e. free arginine, a peptidyl substrate, and histone H3, were studied by colorimetric assay and Western blotting. Molecular dynamics (MD) simulations were performed to investigate structural dynamic and binding properties of PAD4/citPAD4 in the absence and presence of substrates.

Results: We observed that 23/27 arginine residues in PAD4 (85 %) can be citrullinated, including R372, R374 and R639 located near the substrate binding pocket. PAD4 and citPAD4 expressed comparable enzymatic activities towards different substrates. In agreement with experimental results, MD simulations indicated that autocitrullination does not change the shape of the substrate binding pocket and PAD4/citPAD4 exhibited comparable binding free energy with a H3-derived peptidyl substrate (6-TARKS-10).

Conclusion: While the effect of autocitrullination on PAD4 activity thus far remained unclear and controversial, here we have demonstrated that autocitrullination does not affect the activity of PAD4. Thus, the regulation of PAD4 activity is probably not controlled by autocitrullination but likely by other mechanisms that need further investigation.

Keywords: Autocitrullination; Citrullination; Histone H3; PAD4; PAD4 activity; Protein-protein interactions (PPIs).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Arginine / metabolism
Arthritis, Rheumatoid / enzymology
Arthritis, Rheumatoid / metabolism*
Arthritis, Rheumatoid / pathology
Citrullination
Citrulline / metabolism*
Computer Simulation
Enzyme Assays / methods
Humans
In Vitro Techniques
Mass Spectrometry / methods
Molecular Dynamics Simulation
Protein-Arginine Deiminase Type 4 / chemistry
Protein-Arginine Deiminase Type 4 / metabolism*
Recombinant Proteins / chemistry
Recombinant Proteins / isolation & purification
Recombinant Proteins / metabolism

Substances

Recombinant Proteins
Citrulline
Arginine
PADI4 protein, human
Protein-Arginine Deiminase Type 4