Functional characterization of NF-kappaB inhibitor-like protein 1 (NFkappaBIL1), a candidate susceptibility gene for rheumatoid arthritis.
Several studies have implicated the NF-kappaB inhibitor-like protein 1 (NFkBIL1) gene located in the class III region of the major histocompatibility complex (MHC) as a possible susceptibility locus for rheumatoid arthritis (RA). Based on limited homology, it has been suggested to be a member of the inhibitor of NF-kappaB (IkappaB) family of proteins, but a role in mRNA processing has also been proposed. We have investigated the expression of NFkBIL1 in RA synovial tissue and characterized its function. Real-time PCR showed the two NFkBIL1 mRNA splice variants are expressed in a tissue-specific manner. Dual immunofluorescent staining of human RA synovium with polyclonal anti-NFkBIL1 antibodies and anti-CD68, anti-CD3 or anti-factor VIII showed that NFkBIL1 was expressed in the rheumatoid synovial lining and sub-lining layers and co-localized in CD68+ and CD3+, but not Factor VIII+ cells. Confocal microscopy of cultured synovial fibroblasts revealed expression in speckled nuclear and homogenous cytoplasmic distributions, suggesting shuttling between the cytoplasmic and nuclear compartments. Functional tests showed that NFkBIL1 isoforms were incapable of associating with NF-kappaB and did not inhibit it, thus disproving the hypothesis that NFkBIL1 functions as an IkappaB. Affinity purification of endogenous NFkBIL1 proteins and co-immunoprecipitation experiments showed that NFkBIL1 can associate with mRNA and with three protein partners, identified by mass spectrometry as leukophysin, translation elongation factor 1 alpha and CTP synthase I. These data support a potential role for NFkBL1 in the pathogenesis of RA and indicates that it may be involved in mRNA processing or the regulation of translation.
Greetham
Darren
D
Ellis
Charles D
CD
Mewar
Devesh
D
Fearon
Ursula
U
an Ultaigh
Sinead Nic
SN
Veale
Douglas J
DJ
Guesdon
François
F
Wilson
Anthony G
AG
D048868:Adaptor Proteins, Signal Transducing; D001172:Arthritis, Rheumatoid / Q000235:genetics* / Q000473:pathology; D001706:Biopsy; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002478:Cells, Cultured; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D005260:Female; D020022:Genetic Predisposition to Disease; D006367:HeLa Cells; D000949:Histocompatibility Antigens Class II / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008297:Male; D009363:Neoplasm Proteins / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D020033:Protein Isoforms / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology; D014018:Tissue Distribution
D048868:Adaptor Proteins, Signal Transducing; D000949:Histocompatibility Antigens Class II; C120111:NFKBIL1 protein, human; D009363:Neoplasm Proteins; D020648:Peptide Elongation Factor 1; D020033:Protein Isoforms; D012333:RNA, Messenger; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase