Involvement of ubiquitin system on the hepatitis B virus X protein (HBx)-mediated transactivation activity

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We have identified an RING-IBR-RING (RBR) family ubiquitin ligase, HOIL-1L, as a predominant intracellular isoform of HOIL-1 (heme-oxidized IRP2 ligase-1). HOIL-1L forms a high molecular mass (〜600 kDa) complex with HOIP (HOIL-1L-interacting protein) in vivo. HOIP also belongs to RBR family, and the HOIL-1L/HOIP complex exhibits ubiquitin polymerization activity in vitro. To examine the physiological function of HOIL-1L/HOIP, we first screened the effect of HOIL-1L/HOIP on transcriptional activity, since HOIL-1 has shown to bind protein kinase C and hepatitis B virus X protein, and both of them affect transcription. Then, we found by luciferase reporter assay that combined expression of HOIL-1L and HOIP, but not by single expression of respective ligase, specifically induced NF-KB activation. Indeed, co-expression of HOIL-1L/HOIP caused intranuclear translocation of p65 and p50, and enhanced phosphorylation of IKBa. Using various domain-deleted mutants, we identified that the RING domains of HOIP played a crucial role on the NF-KB activation. Notably, a dysfunctional RING-finger mutant of HOIP inhibited TNF-a-and TAK1-mediated NF-KB activation, but had no effect on NIK-mediated NF-KB activation. These results indicate that HOIL-1L/HOIP activates canonical NF-KB pathway through the ubiquitin ligase activity of HOIP. The ligase complex may have a pivotal role on the inflammation, anti-apoptosis, and immune system through the regulation of NF-KB pathway.