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

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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.

OriginalsprogEngelsk
TidsskriftNature Structural and Molecular Biology
ISSN1545-9993
DOI
StatusE-pub ahead of print - 2024

Bibliografisk note

Funding Information:
The work carried out in this study was in part supported by the Novo Nordisk Foundation Center for Protein Research, the Novo Nordisk Foundation (grant agreement numbers NNF14CC0001 and NNF13OC0006477), The Danish Council of Independent Research (grant agreement numbers 4002-00051, 4183-00322A and 8020-00220B) and The Danish Cancer Society (grant agreement R146-A9159-16-S2). The proteomics technology was part of a project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement EPIC-XS-823839. We thank members of the NNF-CPR Mass Spectrometry Platform for instrument support and technical assistance. M.A.C was supported by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society (grant no. 105642/A/14/Z) and by funding from the UK Biotechnology and Biological Sciences Research Council (BBS/E/B/000C0421). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Publisher Copyright:
© The Author(s) 2024.

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