A quantitative and site-specific atlas of the citrullinome reveals widespread existence of citrullination and insights into PADI4 substrates

Alexandra S Rebak, Ivo A Hendriks, Jonas D Elsborg, Sara C Buch-Larsen, Claus H Nielsen, Lene Terslev, Rebecca Kirsch, Dres Damgaard, Nadezhda T Doncheva, Caroline Lennartsson, Martin Rykær, Lars J Jensen, Maria A Christophorou, Michael L Nielsen*

*Corresponding author for this work
1 Citation (Scopus)

Abstract

Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.

Original languageEnglish
JournalNature Structural and Molecular Biology
Volume31
Issue number6
Pages (from-to)977-995
Number of pages19
ISSN1545-9993
DOIs
Publication statusPublished - Jun 2024

Keywords

  • Arthritis, Rheumatoid/metabolism
  • Citrullination
  • Citrulline/metabolism
  • HL-60 Cells
  • Humans
  • Protein-Arginine Deiminase Type 4/metabolism
  • Protein-Arginine Deiminases/metabolism
  • Proteomics/methods
  • Substrate Specificity
  • Synovial Fluid/metabolism

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