hnRNPH2 as an Inhibitor of Chicken MDA5-Mediated Type I Interferon Response: Analysis Using Chicken MDA5-Host Interactome.

RIG-I and MDA5 are two key pattern recognition receptors that sense the invasion of RNA viruses and initiate type I interferon (IFN) response. Although these receptors are generally conserved in vertebrates, RIG-I is absent in chickens, whereas MDA5 is present. Chicken MDA5 (chMDA5) plays a pivotal role in sensing the invasion of RNA viruses into cells. However, unlike mammalian MDA5, where there are in-depth and extensive studies, regulation of the chMDA5-mediated signaling pathway remains unexplored. In this study, we performed a pulldown assay and mass spectrometry analysis to identify chicken proteins that could interact with the N terminal of chMDA5 (chMDA5-N) that contained two CARDs responsible for binding of the well-known downstream adaptor MAVS. We found that 337 host proteins could potentially interact with chMDA5-N, which were integrated to build a chMDA5-N-host association network and analyzed by KEGG pathway and Gene Ontology annotation. Results of our analysis revealed that diverse cellular processes, such as RNA binding and transport and protein translation, ribosome, chaperones, and proteasomes are critical cellular factors regulating the chMDA5-mediated signaling pathway. We cloned 64 chicken genes to investigate their effects on chMDA5-mediated chicken IFN-ß production and confirmed the association of chicken DDX5, HSPA8, HSP79, IFIT5, PRDX1, and hnRNPH2 with chMDA5-N. In particular, we found that chicken hnRNPH2 impairs the association between chMDA5-N and MAVS and thus acts as a check on the chMDA5-mediated signaling pathway. To our knowledge, this study is the first to analyze the chicken MDA5-host interactome, which provides fundamental but significant insights to further explore the mechanism of chicken MDA5 signaling regulation in detail.

Identification of Hnrph3 as an autoantigen for acute anterior uveitis.

Acute anterior uveitis (AAU) is the most common form of autoimmune uveitis in the eye with few known autoantigens. Identification of autoantigens will improve our understanding of the molecular mechanisms and capability for disease diagnosis. Phage display is a powerful technology for autoantigen identification. However, because of uncontrollable reading frames, phage display with conventional cDNA libraries identifies high percentage of non-open reading frames (non-ORFs) with minimal implications for autoantigen identification. We recently developed ORF phage display technology with minimal reading frame problem. Herein we used ORF phage display to identify 18 patient-specific clones, including 16 ORFs encoding endogenous proteins as candidate autoantigens for AAU. One of the identified antigens was heterogeneous nuclear ribonucleoprotein H3 (Hnrph3) that was further characterized for AAU relevance and independently verified by Western blot. These results demonstrate that ORF phage display is a valuable approach for identification of unknown autoantigens.

Heterogeneous nuclear ribonucleoprotein h1, a novel nuclear autoantigen.

BACKGROUND: Serum samples from patients with autoimmune connective tissue diseases that show a finely speckled antinuclear antibody (ANA) on indirect immune-fluorescence often have antibodies against unknown nuclear target antigens. To search for such autoantigens we applied a proteomic approach using sera from patients with a high ANA titer (>or=640) and finely speckled fluorescence but in whom no antibodies to extractable nuclear antigens (ENA) could be identified. METHODS: Using an immunoproteomics approach we identified heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) as a novel nuclear target of autoantibody response. RESULTS: Recombinant rat hnRNP H1 reacted in Western blot analyses with 48% of 93 sera from patients with primary Sjögren syndrome and with 5.2% of 153 sera from patients with other connective tissue diseases (diseased controls). For comparison, the diagnostic sensitivity and specificity of anti-Sjögren syndrome A (SSA) antibodies for primary Sjögren syndrome in the same patient cohort were 88.2% and 76.3%, respectively. Interestingly, 5 of 11 primary Sjögren syndrome patients with no anti-SSA or anti-SSB antibodies had anti-hnRNP H1 antibodies. Anti-hnRNP H1 antibodies were preabsorbed by hnRNP H1, as demonstrated by indirect immunofluorescence. In an evaluation of the presence of anti-hnRNP H1 antibodies in 188 consecutive samples submitted to the clinical laboratory with positive ANA (titer >or=160), anti-hnRNP H1 antibodies were found in 3 of 7 (2 primary and 5 secondary) Sjögren syndrome patients and in 8.3% of the diseased controls. CONCLUSIONS: HnRNP H1 is a newly discovered autoantigen that could become an additional diagnostic marker.

Identification of immunodominant autoantigens in rat autoimmune orchitis.

Infection and inflammation of the genital tract are amongst the leading causes of male infertility. Experimental autoimmune orchitis (EAO) in the rat serves as a model for the investigation of inflammatory testicular impairment. In this study, experiments were conducted to identify the molecules that are responsible for eliciting the autoimmune attack on the testis. EAO was induced in in-bred Wistar rats by active immunization with testis homogenates (EAO group I). Development of disease was observed using histological techniques and a new non-invasive three-dimensional (3D) imaging technology for in vivo monitoring, termed flat-panel volumetric computed tomography (fpvCT). Examination of control and EAO testes demonstrated the superior image quality of high-resolution fpvCT. A proteomics approach using 2D SDS-PAGE and immunoblotting analysis with EAO sera identified 12 spots. Seven were subsequently identified by mass spectrometry as heat shock proteins 60 (Hsp60) and 70 (Hsp70), disulphide isomerase ER-60, alpha-1-anti-trypsin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), sperm outer dense fibre major protein 2 (ODF-2), and phosphoglycerate kinase 1. Hsp70, ODF-2, hnRNP H1, and ER-60 were identified by all EAO sera studied. To test the capacity of the identified proteins to elicit testicular autoimmune disease, recombinant proteins were used either individually or in combination to immunize rats (EAO group II). In all groups, the incidence of EAO was 25%. Inflammatory-type (ED1+) and resident (ED2+) macrophages, lymphocytes (CD45RA+), and dendritic cells (Ox-62+) were strongly increased in EAO group II animals, comparable to the testes of EAO I rats. Pre-immunization with a low dose of recombinant Hsp 70, hnRNP H1 or ODF-2 before induction of EAO with testis homogenate significantly delayed the onset of EAO but could not prevent disease. The identification of testicular autoantigens will allow a better understanding of disease pathogenesis and could provide a basis for the development of novel therapies for inflammation-based male infertility.

Heterogeneous nuclear ribonucleoproteins F and H/H' show differential expression in normal and selected cancer tissues.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H/H', containing the quasi-RNA recognition motif (qRRM) domains, are implicated in several steps of pre-mRNA processing and in cellular differentiation. We have compared a set of tissues and found striking differences in their levels of expression as well as in the nuclear versus the cytoplasmic distribution. Generally, hnRNP F is broadly expressed in many tissues with extremely strong expression in the prostate gland while hnRNP H/H' shows a more restricted degree of expression with low expression in some tissues, for example, liver, exocrine acini of the pancreas, thyroid gland and heart. At the cellular level, hnRNP F is, with few exceptions, predominantly expressed in the cytoplasm while hnRNP H/H' is more abundant in the nuclei. A quite pronounced heterogeneous expression pattern is seen in the proximal tubules of the kidney where hnRNP F is present at moderate cytoplasmic levels while hnRNP H/H' is undetectable, whereas both proteins are more evenly expressed in distal tubules and collecting ducts. Generally, tumor tissues reveal a broad expression of hnRNP F in the nuclei as well as in the cytoplasm while hnRNP H/H' is expressed at higher levels in the nuclei than in the cytoplasm. Up-regulation of hnRNP H/H' is found in a few tissues that normally express low cytoplasmic levels of hnRNP H/H', for example, adenocarcinoma of the pancreas, hepatocellular carcinoma and gastric carcinoma. hnRNP F is down-regulated in hepatocellular carcinoma and up-regulated in gastric carcinoma. The present study indicates the important potential role of this subset of hnRNPs on the gene expression in many tissues.