ZDHHC5 Mediated NOD1 Palmitoylation Is Critical For Bacteria Induced Innate Immunity

The innate mmune system is the first line of defense that detects dangerassociated molecular patterns byseveral families of pattern recognition receptors(PRRs)that extensively distributed on cell surface and cytosol.Currently PRRs include the type I transmembrane molecules of the Toll-like receptor(TLR)family as well as the cytosolic Nod-like receptors(NLRs)and Rig-I-like receptors(RLRs)have been identified.Upon activation,PRRs interact with downstream proteins to activate nuclear factor-kB(NF-kB)and mitogen-activated protein kinase(MAPK)pathways,then promote interlukins and cytokines release.The NOD-like receptors(NLRs)NOD1 and NOD2 have key roles in regulating innate immune responses,and they are cytosolic pattern-recognition receptors critical for detection of microbial products,a NOD2 variant(3020ins C frameshift mutation that leads to a truncated NOD2 protein)is implicated in the pathogenesis of Crohn Disease.NOD1/2 have been demonstrated to be associated with plasma membrane and bacterial containing endosomes and this association is well understood to have a key role in microbial products detection.However,little is known about NOD1/2 recruitment mechanisms.Lacking recognizable membrane-targeting domains,NOD1/2 have been suggested to be anchored to membranes indirectly,through cytoskeletal components or to endosomes by endosomal proteins such as SLC15A3.However,these models do not fully explain the rapid redistribution of NODs from plasmalemma to endosomal compartments in response to bacterial invasion.In this thesis,we mainly focus on mechanisim behind relationship between NOD1 localization and its function.Protein Lipidation is essential not only for membrane binding but also for the interaction with effectors and the regulation of signaling processes,thereby playing a key role in controlling protein localization and function.By small molecular inhibitors screen,we found NOD1 is potentially palmitoylated,and further confirmed by Acyl-Biotin Exchange assay combined with mutugenesis.In order to get more clue about dynamic regulation of NOD1,a new technique named Bio ID was performed to identify NOD1 interacting proteins in resting cells.Among the NOD1 high confidence proximity interactors was ZDHHC5,a ubiquitously expressed integral membrane protein of the DHHC palmitoyltransferase(PAT)family.Knockdown,knock off or catalytically inactive ZDHHC5 lead to NOD1 mislocalization.In combination with biochemistry experiments,our preliminary studies suggest that:(1)palmitoylation is required for NOD1 recruitment to plasma and bacterial containing endosomes;(2)Multiple cysteine residues were found in NOD1 are palmitoylated,contained 558,567,952 cysteine sites;(3)we identified ZDHHC5 as the putative NOD1 palmitoyl-transferase which regulates NOD1 activation;(4)Palmitoylation of NOD1 is critical for its role in initiating immune signaling pathway;(5)ZDHHC5 is constitutively localized to phagosomes and that its catalytic activity is required for NOD1 phagosomal recruitment and NOD1 dependent signaling.In additional,A myeloid cell lineage–specific conditional ZDHHC5 knockout(KO)animal model was generated by crossing mice carrying a floxed-ZDHHC5 allele(ZDHHC5fl/fl)with myelomonocytic cell–specific Lysozyme M(Lys M)–Cre Recombinase mice.By isolating bone marrowed macrophage,we obersaved plasma membrane and endosome membrane localization of endogenous NOD1 in WT mice,while endogenous NOD1 was predominantly cytosolic in ZDHHC5 KO m BMDMs and ZDHHC5 KO m BMDMs displayed an impaired NOD1 dependent activation of NF-kB and p38 MAPK signaling,and reduction of CXCL-1/IL-6 release in response to C12-i E-DAP or C12-i E-DAP coated beads.Based on these promising preliminary experimental results,our findings will provide a better understanding of NOD1 in innate immunity and facilitate advances in discovery of novel treatments for NOD-driven inflammatory diseases.