MyD88 Palmitoylation mediates inflammatory responses in macrophages

Abstract

Recently, increasingly many proteins are known to be palmitoylated. However, the event of palmitoylatioMyn and its function in TLR signaling have not been previously studied. Here, we demonstrate that myeloid differentiation primary response protein (MyD88) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. There are 9 cysteine residues in MyD88 and all of them are highly conserved in closely related species, suggesting the importance of cysteine residue-specific post-translational modifications. MyD88 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 113 and 274 (C113, 274) were primarily palmitoylated. Palmitoylation of MyD88 was remarkably decreased in mutants where all 9 cysteines were mutated to either alanine or serine (CA or CS, respectively) and also where only C113, 274 were mutated to either alanine or serine (C113, 274A or C113, 274S, respectively). We found that the level of MyD88 palmitoylation correlated with the level of inflammatory responses in HEK293T cells, suggesting the role of palmitoylation in mediating P-p38 and NF-kB signaling. Indeed, both P-p38 and NF-kB activation were significantly inhibited in HEK293T cells stably expressing CA MyD88-GyrB compared to those in WT MyD88-GyrB. We demonstrated that the activation of P-p38 and NF-kB was specific to MyD88-dependent TLR4 signaling by the usage of MyD88-GyrB construct, which only self-dimerizes and being recruited to TLR4 subcellular domain upon coumermycin A1 treatment, excluding the possibility that P-p38 and NF-kB activation is affected by other signaling pathways. We further observed that MyD88 is potentially palmitoylated by DHHC6, a palmitoyl-transferase (PAT) enzyme containing aspartate-histidine-histidine-cysteine (DHHC) motif. A real time quantitative RT-PCR and immunoprecipitation analysis demonstrated that DHHC6 is highly expressed in macrophages and interacts strongly with MyD88 in vitro. Furthermore, acyl-biotin exchange assay showed that shDHHC6-transfected raw264.7 cells failed to palmitoylate endogenous MyD88 in the absence of DHHC6. These findings suggest that MyD88 palmitoylation is important in mediating inflammatory responses in macrophages.