title: Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes.
abstract: Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clinical autoimmunity. Previous studies from our laboratory support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are associated with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90β/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90β]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response experiments reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled molecular dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context.
authors.affiliation: Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213;
authors.forename: Timothy S
authors.identifier: 0000-0003-0105-3844
authors.initials: TS
authors.lastname: Travers
authors.affiliation: Division of Rheumatology, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136;
authors.forename: Lisa
authors.initials: L
authors.lastname: Harlow
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115;
authors.forename: Ivan O
authors.identifier: 0000-0003-4622-618X
authors.initials: IO
authors.lastname: Rosas
authors.affiliation: Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Bernadette R
authors.initials: BR
authors.lastname: Gochuico
authors.affiliation: Division of Rheumatology, Department of Medicine, University of Nebraska Medical Center, Omaha, NE 68198; and.
authors.forename: Ted R
authors.identifier: 0000-0002-0897-2272
authors.initials: TR
authors.lastname: Mikuls
authors.affiliation: Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, FL 33136.
authors.forename: Sanjoy K
authors.identifier: 0000-0003-3759-647X
authors.initials: SK
authors.lastname: Bhattacharya
authors.affiliation: Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; CCamacho@pitt.edu DAscherman@med.miami.edu.
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Camacho
authors.affiliation: Division of Rheumatology, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136; CCamacho@pitt.edu DAscherman@med.miami.edu.
authors.forename: Dana P
authors.identifier: 0000-0002-1387-0860
authors.initials: DP
authors.lastname: Ascherman
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001323:Autoantibodies; D002956:Citrulline / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000235:genetics; D013058:Mass Spectrometry; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000378:metabolism; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D000076342:Protein-Arginine Deiminases
chemical_list: D001323:Autoantibodies; D000939:Epitopes; D018841:HSP90 Heat-Shock Proteins; D010455:Peptides; D020033:Protein Isoforms; D002956:Citrulline; D001120:Arginine; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases