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.

Fig. 1

Overview of mass spectrometry results. a Correlation matrix of non-redundant protein hits for all patients and controls. Comparisons were performed using Fisher’s exact test with the Bonferroni correction. Black boxes indicate intra-group comparisons for each of the four clinically defined groups. Green controls; red RNA polymerase III (RNAP3); blue centromere protein (CENP); yellow topoisomerase I (TOP1). b-f Venn diagrams depict overlap in non-redundant peptide hits within and between groups. b healthy controls, c RNAP3, d CENP, e TOP1, and f overlap between groups

Fig. 2

Proteins differentially detected in systemic sclerosis (SSc). Semiquantitative enrichment of SSc-associated autoantibodies was determined using a binary assessment of autoantibody presence or absence in a sample. Preferential enrichment in SSc was defined as all proteins detected in >50 % of all patient samples at a frequency >1.5-fold relative to controls. a Heat map of proteins differentially detected in SSc. b Network analysis of differentially detected proteins. Community detection was performed using the GIANT global network; functional annotation was performed using gProfiler

Fig. 3

Validation of RNA processing bodies (PB)/stress granules (SG) as a target of the SSc autoimmune response. a HeLa cell lysates were immunoprecipitated using patient sera, resolved by SDS-PAGE, and probed with antibodies targeting known PB and SG proteins; HeLa whole cell lysate was used as a control. b Immunofluorescence was performed in U2OS cells treated with sodium (meta)arsenite to induce the formation of SG. Cells were then fixed with 4 % paraformaldehyde and permeabilized with 5 % normal horse serum and 0.1 % digitonin in Tris-buffered saline. Staining was performed with anti-eIF3b (SG marker), anti-SK1-Hedls (PB marker), and patient sera. Representative images depicting co-localization between patient sera and SG/PB markers are shown, with sites of co-localization circled in red. RNAP RNA polymerase, CENP centromere protein TOPI topoisomerase I