Post-translational modifications such as ubiquitination play a key role in regulation of inflammatory nuclear factor-κB (NF-κB) signalling. The Drosophila IκB kinase γ (IKKγ) Kenny is a central regulator of the Drosophila Imd pathway responsible for activation of the NF-κB Relish. We found the Drosophila E3 ligase and HOIL-1L interacting protein (HOIP) orthologue linear ubiquitin E3 ligase (LUBEL) to catalyse formation of M1-linked linear ubiquitin (M1-Ub) chains in flies in a signal-dependent manner upon bacterial infection. Upon activation of the Imd pathway, LUBEL modifies Kenny with M1-Ub chains. Interestingly, the LUBEL-mediated M1-Ub chains seem to be targeted both directly to Kenny and to K63-linked ubiquitin chains conjugated to Kenny by DIAP2. This suggests that DIAP2 and LUBEL work together to promote Kenny-mediated activation of Relish. We found LUBEL-mediated M1-Ub chain formation to be required for flies to survive oral infection with Gram-negative bacteria, for activation of Relish-mediated expression of antimicrobial peptide genes and for pathogen clearance during oral infection. Interestingly, LUBEL is not required for mounting an immune response against systemic infection, as Relish-mediated antimicrobial peptide genes can be expressed in the absence of LUBEL during septic injury. Finally, transgenic induction of LUBEL-mediated M1-Ub drives expression of antimicrobial peptide genes and hyperplasia in the midgut in the absence of infection. This suggests that M1-Ub chains are important for Imd signalling and immune responses in the intestinal epithelia, and that enhanced M1-Ub chain formation is able to drive chronic intestinal inflammation in flies.

Methionine 1 (M1)-linked ubiquitination plays a key role in the regulation of inflammatory nuclear factor-jB (NF-jB) signalling and is important for clearance of pathogen infection in Drosophila melanogaster. M1-linked ubiquitin (M1-Ub) chains are assembled by the linear ubiquitin E3 ligase (LUBEL) in flies. Here, we have studied the role of LUBEL in sterile inflammation induced by different types of cellular stresses. We have found that the LUBEL catalyses formation of M1-Ub chains in response to hypoxic, oxidative and mechanical stress conditions. LUBEL is shown to be important for flies to survive low oxygen conditions and paraquat-induced oxidative stress. This protective action seems to be driven by stress-induced activation of the NF-jB transcription factor Relish via the immune deficiency (Imd) pathway. In addition to LUBEL, the intracellular mediators of Relish activation, including the transforming growth factor activating kinase 1 (Tak1), Drosophila inhibitor of apoptosis (IAP) Diap2, the IjB kinase c (IKKc) Kenny and the initiator caspase Death-related ced-3/Nedd2-like protein (Dredd), but not the membrane receptor peptidoglycan recognition protein (PGRP)-LC, are shown to be required for sterile inflammatory response and survival. Finally, we showed that the stress-induced upregulation of M1-Ub chains in response to hypoxia, oxidative and mechanical stress is also induced in mammalian cells and protects from stress-induced cell death. Taken together, our results suggest that M1-Ub chains are important for NF-jB signalling in inflammation induced by stress conditions often observed in chronic inflammatory diseases and cancer.Abbreviations AMP, antimicrobial peptide; daGal4, daughterless-Gal4; DAMP, danger-associated molecular pattern; Diap, Drosophila inhibitor of apoptosis; Dif, Dorsal-related immunity factor; Dredd, death-related ced-3/Nedd2-like protein; DUB, deubiquitinating enzyme; Fadd, fas-associated death domain; HIF, hypoxia-inducible factor; HOIL-1L, heme-oxidised iron-responsive element-binding protein 2 ubiquitin ligase-1L; HOIP, HOIL-1interacting protein; IKK, IjB kinase; Imd, immune deficiency; LDD, linear ubiquitin chain-determining domain; LUBAC, linear ubiquitin chain assembly complex; LUBEL, linear ubiquitin E3 ligase; NF-jB, nuclear factor j-light-chain enhancer of activated B cells; NZF, npl4 zinc finger; PAMP, pathogen-associated molecular pattern; PGRP, peptidoglycan recognition protein; PIM, PUB-interacting motif; PNG, peptide N-glycanase; PRR, pattern-recognition receptor; PUB, UBA or UBX-containing protein; RBR, RING in between RING; RING, really interesting new gene; SAMP, stress-associated molecular pattern; SHARPIN, SH3 and multiple ankyrin repeat domains protein (SHANK)-associated RBCK1 homology (RH) domain-interacting protein; Sima, similar; Tab2, transforming growth factor-b-activated kinase 1 binding protein 2; Tak1, transforming growth factor activating kinase; TGF, transforming growth factor; TNF, tumour necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; TUB...

Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.