Toll-like receptor (TLR)/myeloid differentiation primary response protein (MYD88) signaling aggravates sepsis by impairing neutrophil migration to infection sites. However, the role of intracellular fatty acids in TLR/MYD88 signaling is unclear. Here, inhibition of fatty acid synthase by C75 improved neutrophil chemotaxis and increased the survival of mice with sepsis in cecal ligation puncture and lipopolysaccharide-induced septic shock models. C75 specifically blocked TLR/MYD88 signaling in neutrophils. Treatment with GSK2194069 that targets a different domain of fatty acid synthase, did not block TLR signaling or MYD88 palmitoylation. De novo fatty acid synthesis and CD36-mediated exogenous fatty acid incorporation contributed to MYD88 palmitoylation. The binding of IRAK4 to the MYD88 intermediate domain and downstream signal activation required MYD88 palmitoylation at cysteine 113. MYD88 was palmitoylated by ZDHHC6, and ZDHHC6 knockdown decreased MYD88 palmitoylation and TLR/MYD88 activation upon lipopolysaccharide stimulus. Thus, intracellular saturated fatty acid-dependent palmitoylation of MYD88 by ZDHHC6 is a therapeutic target of sepsis.
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Any Supplementary information, chemical compound information are available in the online version of the paper. The data that support the findings of this study are available from the corresponding author upon request.
Hotchkiss, R. S., Monneret, G. & Payen, D. Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy. Nat. Rev. Immunol. 13, 862–874 (2013).
Cohen, J. et al. Sepsis: a roadmap for future research. Lancet Infect. Dis. 15, 581–614 (2015).
Foxman, E. F., Campbell, J. J. & Butcher, E. C. Multistep navigation and the combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139, 1349–1360 (1997).
Phillipson, M. & Kubes, P. The neutrophil in vascular inflammation. Nat. Med. 17, 1381–1390 (2011).
Fessler, M. B., Rudel, L. L. & Brown, M. J. Toll-like receptor signaling links dietary fatty acids to the metabolic syndrome. Curr. Opin. Lipidol. 20, 379 (2009).
Huang, S. et al. Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways. J. Lipid Res. 53, 2002–2013 (2012).
Fritsche, K. L. The science of fatty acids and inflammation. Adv. Nutr. 6, 293S–301S (2015).
Lancaster, G. I. et al. Evidence that TLR4 is not a receptor for saturated fatty acids but mediates lipid-induced inflammation by reprogramming macrophage metabolism. Cell Metab. 27, 1096–1110 e1095 (2018).
Berod, L. et al. De novo fatty acid synthesis controls the fate between regulatory T and T helper 17 cells. Nat. Med. 20, 1327–1333 (2014).
Wei, X. et al. Fatty acid synthesis configures the plasma membrane for inflammation in diabetes. Nature 539, 294–298 (2016).
Salaun, C., Greaves, J. & Chamberlain, L. H. The intracellular dynamic of protein palmitoylation. J. Cell Biol. 191, 1229–1238 (2010).
Alves-Filho, J. C., de Freitas, A., Russo, M. & Cunha, F. Q. Toll-like receptor 4 signaling leads to neutrophil migration impairment in polymicrobial sepsis. Crit. Care Med. 34, 461–470 (2006).
Kuhajda, F. P. et al. Synthesis and antitumor activity of an inhibitor of fatty acid synthase. Proc. Natl Acad. Sci. USA 97, 3450–3454 (2000).
Duarte, D. B., Vasko, M. R. & Fehrenbacher, J. C. Models of inflammation: carrageenan air pouch. Curr. Protoc. Pharmacol. 72, 1–9 (2016).
Tamassia, N. et al. The MYD88-Independent pathway is not mobilized in human neutrophils stimulated via TLR4. J. Immunol. 178, 7344–7356 (2007).
Medzhitov, R. et al. MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Mol. Cell 2, 253–258 (1998).
Hacker, H. et al. Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature 439, 204–207 (2006).
Into, T. et al. Regulation of MyD88-dependent signaling events by S-nitrosylation retards Toll-like receptor signal transduction and initiation of acute-phase immune responses. Mol. Cell. Biol. 28, 1338–1347 (2007).
Farrar, M. A., Alberol-Ila, J. & Perlmutter, R. M. Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization. Nature 383, 178–181 (1996).
Xie, Y. et al. GPS-Lipid: a robust tool for the prediction of multiple lipid modification sites. Sci. Rep. 6, 28249 (2016).
Dekker, F. J. et al. Small-molecule inhibition of APT1 affects ras localization and signaling. Nat. Chem. Biol. 6, 449–456 (2010).
Carroll, R. G. et al. An unexpected link between fatty acid synthase and cholesterol synthesis in proinflammatory macrophage activation. J. Biol. Chem. 293, 5509–5521 (2018).
Alexander, J. K. et al. Palmitoylation of nicotinic acetylcholine receptors. J. Mol. Neurosci. 40, 12–20 (2010).
Janssens, S., Burns, K., Vercammen, E., Tschopp, J. & Beyaert, R. MyD88S, a splice variant of MyD88, differentially modulates NF‐κB‐ and AP‐1‐dependent gene expression. FEBS Lett. 548, 103–107 (2003).
Burns, K. et al. Inhibition of interleukin 1 Receptor/Toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4. J. Exp. Med. 197, 263–268 (2003).
Avbelj, M., Horvat, S. & Jerala, R. The role of intermediary domain of MyD88 in cell activation and therapeutic inhibition of TLRs. J. Immunol. 187, 2394–2404 (2011).
Loiarro, M. et al. Identification of critical residues of the MyD88 death domain involved in the recruitment of downstream kinases. J. Biol. Chem. 284, 28093–28103 (2009).
Fukata, Y., Bredt, D. S. & Fukata, M. in The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology (eds Kittler, J. T. & Moss, S. J.) Chapter 5 (CRC Press, 2006).
Ohno, Y. et al. Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system. Mol. Biol. Cell 23, 4543–4551 (2012).
Fukata, Y. & Fukata, M. Protein palmitoylation in neuronal development and synaptic plasticity. Nat. Rev. Neurosci. 11, 161–175 (2010).
Fukata, Y., Iwanaga, T. & Fukata, M. Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells. Methods 40, 177–182 (2006).
Currie, E., Schulze, A., Zechner, R., Walther, T. C. & Farese, R. V. Jr. Cellular fatty acid metabolism and cancer. Cell Metab. 18, 153–161 (2013).
Zhu, X. et al. Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol. J. Lipid Res. 51, 3196–3206 (2010).
Tall, A. R. & Yvan-Charvet, L. Cholesterol, inflammation and innate immunity. Nat. Rev. Immunol. 15, 104–116 (2015).
Moon, J.-S. et al. UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis. J. Clin. Invest. 125, 665–680 (2015).
Ruysschaert, J.-M. & Lonez, C. Role of lipid microdomains in TLR-mediated signalling. Biochim. Biophys. Acta, Biomembr. 1848, 1860–1867 (2015).
Gorleku, O. A., Barns, A.-M., Prescott, G. R., Greaves, J. & Chamberlain, L. H. Endoplasmic reticulum localization of dhhc palmitoyltransferases mediated by lysine-based sorting signals. J. Biol. Chem. 286, 39573–39584 (2011).
Lakkaraju, A. K. K. et al. Palmitoylated calnexin is a key component of the ribosome–translocon complex. EMBO J. 31, 1823–1835 (2012).
Fredericks, G. J. et al. Stable expression and function of the inositol 1,4,5-triphosphate receptor requires palmitoylation by a DHHC6/selenoprotein K complex. Proc. Natl Acad. Sci. USA 111, 16478–16483 (2014).
Reid, D. W. & Nicchitta, C. V. Diversity and selectivity in mRNA translation on the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. 16, 221–231 (2015).
Fiorentino, M. et al. Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of beta-catenin in prostate cancer. Lab. Invest. 88, 1340–1348 (2008).
Wei, X. et al. De novo lipogenesis maintains vascular homeostasis through endothelial nitric-oxide synthase (eNOS) palmitoylation. J. Biol. Chem. 286, 2933–2945 (2011).
Wei, X. et al. Fatty acid synthase modulates intestinal barrier function through palmitoylation of mucin 2. Cell Host Microbe 11, 140–152 (2012).
Coleman, R. A., Rao, P., Fogelsong, R. J. & Bardes, E. 2-Bromopalmitoyl-CoA and 2-bromopalmitate: promiscuous inhibitors of membrane-bound enzymes. Biochim. Biophys. Acta, Lipids Lipid Metab. 1125, 203–209 (1992).
Zheng, B., Zhu, S. & Wu, X. Clickable analogue of cerulenin as chemical probe to explore protein palmitoylation. ACS Chem. Biol. 10, 115–121 (2015).
Thupari, J. N., Landree, L. E., Ronnett, G. V. & Kuhajda, F. P. C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity. Proc. Natl Acad. Sci. USA 99, 9498–9502 (2002).
Jang, H. D., Yoon, K., Shin, Y. J., Kim, J. & Lee, S. Y. PIAS3 suppresses NF-kappaB-mediated transcription by interacting with the p65/RelA subunit. J .Biol. Chem. 279, 24873–24880 (2004).
Fujii, S. et al. Nr0b1 is a negative regulator of Zscan4c in mouse embryonic stem cells. Sci. Rep. 5, 9146 (2015).
Shin, J. et al. Aurkb/PP1-mediated resetting of Oct4 during the cell cycle determines the identity of embryonic stem cells. eLife 5, e10877 (2016).
Yap, M. C. et al. Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry. J. Lipid Res. 51, 1566–1580 (2010).
Charron, G. et al. Robust fluorescent detection of protein fatty-acylation with chemical reporters. J. Am. Chem. Soc. 131, 4967–4975 (2009).
Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).
Hwang, H. et al. In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation. Anal. Bioanal. Chem. 406, 7999–8011 (2014).
Sun, N. et al. Quantitative proteome and transcriptome analysis of the archaeon thermoplasma acidophilum cultured under aerobic and anaerobic conditions. J. Proteome Res. 9, 4839–4850 (2010).
Millius, A. & Weiner, O. D. Chemotaxis in neutrophil-like HL-60 cells. Methods Mol. Biol. 571, 167–177 (2009).
Soderberg, O. et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995–1000 (2006).
Rittirsch, D., Huber-Lang, M. S., Flierl, M. A. & Ward, P. A. Immunodesign of experimental sepsis by cecal ligation and puncture. Nat. Protoc. 4, 31–36 (2009).
We thank M.A. Farrar (Univ. of Minnesota) for the pKS-GyrB construct; M. Fukata (National Institute for Physiological Science and National Institutes of Natural Sciences) for the 24 pEF-Bos-zDHHC-HA constructs. This study was supported by grants from the Korea Health Technology R&D Project ‘Strategic Center of Cell and Bio Therapy’ (grant no. HI17C2085; H.-S.K.) and ‘Korea Research-Driven Hospital’ (grant no. HI14C1277; H.-S.K.) through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Korea and from the National Research Foundation of Korea funded by the Korea Government (grant no. 2018R1C1B5086482; S.E.L.).
The authors declare no competing interests.
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Kim, YC., Lee, S.E., Kim, S.K. et al. Toll-like receptor mediated inflammation requires FASN-dependent MYD88 palmitoylation. Nat Chem Biol 15, 907–916 (2019). https://doi.org/10.1038/s41589-019-0344-0
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