Hyd ubiquitinates the NF-κB co-factor Akirin to operate an effective immune response in Drosophila

Author summary Ubiquitination has been recently identified in pathogenesis and progression of various diseases where inflammation is critical. NF-kappa B transcription factors are key actors in the transcriptional cascade leading to inflammation as they activate genes with pro- or anti-inflammatory activities. The similarity between the immune pathways in flies and mammals makes Drosophila melanogaster an excellent model to study the innate response. Accordingly, we decided to identify E3 ubiquitin-ligases involved in the regulation of NF-kappa B pathway, using Drosophila as a model system. A RNAi based screen in immortalized embryonic macrophage-like Drosophila cells points to the HECT-E3 ubiquitin ligase Hyd as a new regulator of the Immune-deficiency (IMD) NF-kappa B pathway, activated after Gram-negative immune challenge. More precisely, we showed that Hyd acts at the level of Akirin, an evolutionarily conserved player in the NF-kappa B pathway, required for the transcription of pro-inflammatory genes, but not for the NF-kappa B-dependent genes contributing to the down-regulation of inflammation. In addition, we could show that the human homologue of Hyd (UBR5) acts genetically at the level of human AKIRIN2, pointing to a unique dichotomy between Hyd/Akirin-dependent and -independent gene activation, allowing for the decoupling activation and resolution of inflammation. These results identified UBR5 as a putative target for anti-inflammatory compounds. The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor kappa B (NF-kappa B) signaling pathway, we undertook an in vitro RNAi screen targeting E3 ubiquitin ligases specifically and identified the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) as a new actor in the IMD pathway. Hyd mediated Lys(63) (K63)-linked polyubiquitination of the NF-kappa B cofactor Akirin was required for efficient binding of Akirin to the NF-kappa B transcription factor Relish. We showed that this Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-kappa B transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. We showed further that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-kappa B pathway for the activation of Interleukin 6 (IL6) transcription by IL-1 beta in cultured human cells. Our findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.