ABSTRACT
Ubiquitin ligases play important roles in immune regulation. The human RNF114 (RING finger protein 114), an ubiquitin ligase, was recently reported to be involved in immune response to double-stranded RNA in disease pathogenesis. Here, we identified a RNF114 homolog in Chinese sturgeon (Acipenser sinensis) and investigated its potential role in immune response. The full-length cDNA of Chinese sturgeon RNF114 (csRNF114) contains an open reading frame (ORF) of 681 nucleotides coding a protein of 227 amino acids. csRNF114 shares the highest identity of 76% at amino acid level to other RNF114 homologs, clustering with bony fish RNF114s based on phylogenetic analysis. The main structural features of csRNF114, including a C3HC4 (Cys3-His-Cys4) RING domain, a C2HC (Cys2-His-Cys)-type zinc finger motif, a C2H2 (Cys2-His2)-type zinc finger motif, and a UIM (ubiquitin-interacting motif), take csRNF114 as an ubiquitin ligase. csRNF114 mRNA was widely expressed in various tissues and significantly up-regulated in poly(I:C)-treated Chinese sturgeon. Over-expression of csRNF114 in HEK293T cells significantly promoted both basal and poly(I:C)-induced activation of interferon regulatory transcription factor 3 (IRF3) and nuclear factor-κB (NF-κB) downstream retinoic acid inducible gene I (RIG-I) signaling pathway and expression of target genes type I interferon (IFN), which was nearly abolished by knockdown of RIG-I with specific human siRNA and by mutation of the C3HC4 RING domain (C28A/C31A) in csRNF114 as well. Furthermore, csRNF114 associated with ubiquitinated proteins in HEK293T cells, for which the C3HC4 RING domain was essential. These data suggested that an ubiquitin ligase RNF114 homolog with a potential role in antiviral response possibly through modulating RIG-I signaling pathway was cloned from Chinese sturgeon, which might contribute to our understanding of the immune biology of Chinese sturgeon.
Subject(s)
Fishes/genetics , Interferons/metabolism , RING Finger Domains/genetics , Signal Transduction/immunology , Ubiquitin-Protein Ligases/immunology , Analysis of Variance , Animals , Cloning, Molecular , Computational Biology , DNA Primers/genetics , Fishes/immunology , Gene Knockdown Techniques , Gene Library , HEK293 Cells , Humans , Immunoprecipitation , NF-kappa B/metabolism , Open Reading Frames/genetics , Phylogeny , Poly I-C , RING Finger Domains/immunology , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Ubiquitination of eukaryotic proteins regulates a broad range of cellular processes, including T cell activation and tolerance. We have previously demonstrated that GRAIL (gene related to anergy in lymphocytes), a transmembrane RING finger ubiquitin E3 ligase, initially described as induced during the induction of CD4 T cell anergy, is also expressed in resting CD4 T cells. In this study, we show that GRAIL can down-modulate the expression of CD83 (previously described as a cell surface marker for mature dendritic cells) on CD4 T cells. GRAIL-mediated down-modulation of CD83 is dependent on an intact GRAIL extracellular protease-associated domain and an enzymatically active cytosolic RING domain, and proceeds via the ubiquitin-dependent 26S proteosome pathway. Ubiquitin modification of lysine residues K168 and K183, but not K192, in the cytoplasmic domain of CD83 was shown to be necessary for GRAIL-mediated degradation of CD83. Reduced CD83 surface expression levels were seen both on anergized CD4 T cells and following GRAIL expression by retroviral transduction, whereas GRAIL knock-down by RNA interference in CD4 T cells resulted in elevated CD83 levels. Furthermore, CD83 expression on CD4 T cells contributes to T cell activation as a costimulatory molecule. This study supports the novel mechanism of ubiquitination by GRAIL, identifies CD83 as a substrate of GRAIL, and ascribes a role for CD83 in CD4 T cell activation.
Subject(s)
Antigens, CD/metabolism , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , Immunoglobulins/metabolism , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/metabolism , Membrane Proteins/physiology , Ubiquitin-Protein Ligases/physiology , Amino Acid Sequence , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , CD4-Positive T-Lymphocytes/metabolism , Cell Line , Clonal Anergy/immunology , Down-Regulation/immunology , Herpesvirus 1, Human/immunology , Humans , Immediate-Early Proteins/physiology , Immunoglobulins/biosynthesis , Immunoglobulins/genetics , Lymphocyte Activation/immunology , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Molecular Sequence Data , Peptide Hydrolases/metabolism , RING Finger Domains/immunology , Substrate Specificity/immunology , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , CD83 AntigenABSTRACT
Recently, novel E3 ubiquitin ligases that target MHC molecules for lysosomal degradation have been discovered by several groups. All these E3s are membrane-bound and possess a variant type RING domain, termed the RING-CH or RING variant (RINGv) domain. They belong to a new E3 family designated Modulator of Immune Recognition (MIR), based on the name of the first identified family members. The discovery of the MIR family has provided fresh insight into viral pathogenesis and immune regulation.
