title: Stress granules and RNA processing bodies are novel autoantibody targets in systemic sclerosis.
abstract: BACKGROUND
  Autoantibody profiles represent important patient stratification markers in systemic sclerosis (SSc). Here, we performed serum-immunoprecipitations with patient antibodies followed by mass spectrometry (LC-MS/MS) to obtain an unbiased view of all possible autoantibody targets and their associated molecular complexes recognized by SSc.
  
  
  METHODS
  HeLa whole cell lysates were immunoprecipitated (IP) using sera of patients with SSc clinically positive for autoantibodies against RNA polymerase III (RNAP3), topoisomerase 1 (TOP1), and centromere proteins (CENP). IP eluates were then analyzed by LC-MS/MS to identify novel proteins and complexes targeted in SSc. Target proteins were examined using a functional interaction network to identify major macromolecular complexes, with direct targets validated by IP-Western blots and immunofluorescence.
  
  
  RESULTS
  A wide range of peptides were detected across patients in each clinical autoantibody group. Each group contained peptides representing a broad spectrum of proteins in large macromolecular complexes, with significant overlap between groups. Network analyses revealed significant enrichment for proteins in RNA processing bodies (PB) and cytosolic stress granules (SG) across all SSc subtypes, which were confirmed by both Western blot and immunofluorescence.
  
  
  CONCLUSIONS
  While strong reactivity was observed against major SSc autoantigens, such as RNAP3 and TOP1, there was overlap between groups with widespread reactivity seen against multiple proteins. Identification of PB and SG as major targets of the humoral immune response represents a novel SSc autoantigen and suggests a model in which a combination of chronic and acute cellular stresses result in aberrant cell death, leading to autoantibody generation directed against macromolecular nucleic acid-protein complexes.
authors.affiliation: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. michael.e.johnson@dartmouth.edu.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Johnson
authors.affiliation: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. andrew.v.grassetti.gr@dartmouth.edu.
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Grassetti
authors.affiliation: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. jaclyn.n.taroni.gr@dartmouth.edu.
authors.forename: Jaclyn N
authors.initials: JN
authors.lastname: Taroni
authors.affiliation: Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, MA, USA. smlyons@partners.org.
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Lyons
authors.affiliation: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. dkschwep@uw.edu.
authors.forename: Devin
authors.initials: D
authors.lastname: Schweppe
authors.affiliation: Department of Rheumatology, Hospital for Special Surgery, New York, NY, USA. gordonj@hss.edu.
authors.forename: Jessica K
authors.initials: JK
authors.lastname: Gordon
authors.affiliation: Department of Rheumatology, Hospital for Special Surgery, New York, NY, USA. spierar@hss.edu.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Spiera
authors.affiliation: Boston University School of Medicine, Boston, MA, USA. rlafyatis@gmail.com.
authors.forename: Robert
authors.initials: R
authors.lastname: Lafyatis
authors.affiliation: Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, MA, USA. panderson@rics.bwh.harvard.edu.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Anderson
authors.affiliation: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. scott.a.gerber@dartmouth.edu.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Gerber
authors.affiliation: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. michael.l.whitfield@dartmouth.edu.|Dartmouth Medical School, Hinman Box 7400, Hanover, NH, 03755, USA. michael.l.whitfield@dartmouth.edu.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Whitfield
mesh_terms: D000328:Adult; D000368:Aged; D001323:Autoantibodies / Q000032:analysis* / Q000276:immunology*; D001324:Autoantigens / Q000032:analysis* / Q000276:immunology*; D015415:Biomarkers / Q000032:analysis; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008875:Middle Aged; D012323:RNA Processing, Post-Transcriptional / Q000276:immunology*; D012595:Scleroderma, Systemic / Q000276:immunology* / Q000473:pathology; D055815:Young Adult
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D015415:Biomarkers