ZDHHC18 negatively regulates cGAS‐mediated innate immunity through palmitoylation

Article19 April 2022free access Source DataTransparent process ZDHHC18 negatively regulates cGAS-mediated innate immunity through palmitoylation Chengrui Shi Chengrui Shi orcid.org/0000-0001-6869-8386 School of Pharmaceutical Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China Contribution: Resources, Data curation, Software, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Xikang Yang Xikang Yang orcid.org/0000-0002-3404-906X School of Pharmaceutical Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China Contribution: Data curation, Software, ​Investigation, Methodology, Writing - original draft Search for more papers by this author Ye Liu Ye Liu orcid.org/0000-0003-0841-9263 Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Contribution: Data curation, Software, Formal analysis, Validation Search for more papers by this author Hongpeng Li Hongpeng Li orcid.org/0000-0002-7121-7157 School of Pharmaceutical Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China School of Medicine, Tsinghua University, Beijing, China Contribution: Methodology Search for more papers by this author Huiying Chu Huiying Chu orcid.org/0000-0002-0018-6189 Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Contribution: Software Search for more papers by this author Guohui Li Corresponding Author Guohui Li [email protected] orcid.org/0000-0001-8223-705X Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Contribution: Supervision, Funding acquisition Search for more papers by this author Hang Yin Corresponding Author Hang Yin [email protected] orcid.org/0000-0002-9762-4818 School of Pharmaceutical Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Chengrui Shi Chengrui Shi orcid.org/0000-0001-6869-8386 School of Pharmaceutical Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China Contribution: Resources, Data curation, Software, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Xikang Yang Xikang Yang orcid.org/0000-0002-3404-906X School of Pharmaceutical Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China Contribution: Data curation, Software, ​Investigation, Methodology, Writing - original draft Search for more papers by this author Ye Liu Ye Liu orcid.org/0000-0003-0841-9263 Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Contribution: Data curation, Software, Formal analysis, Validation Search for more papers by this author Hongpeng Li Hongpeng Li orcid.org/0000-0002-7121-7157 School of Pharmaceutical Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China School of Medicine, Tsinghua University, Beijing, China Contribution: Methodology Search for more papers by this author Huiying Chu Huiying Chu orcid.org/0000-0002-0018-6189 Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Contribution: Software Search for more papers by this author Guohui Li Corresponding Author Guohui Li [email protected] orcid.org/0000-0001-8223-705X Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Contribution: Supervision, Funding acquisition Search for more papers by this author Hang Yin Corresponding Author Hang Yin [email protected] orcid.org/0000-0002-9762-4818 School of Pharmaceutical Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Chengrui Shi1,2,3,†, Xikang Yang1,2,3,†, Ye Liu4,†, Hongpeng Li1,2,3,5, Huiying Chu4, Guohui Li *,4 and Hang Yin *,1,2,3 1School of Pharmaceutical Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China 2Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China 3Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China 4Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 5School of Medicine, Tsinghua University, Beijing, China † These authors contributed equally to this work *Corresponding author: Tel: +81 0411 84379593; E-mail: [email protected] *Corresponding author: Tel: +81 010 62786005; E-mail: [email protected] The EMBO Journal (2022)41:e109272https://doi.org/10.15252/embj.2021109272 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Double-stranded DNA is recognized as a danger signal by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), triggering innate immune responses. Palmitoylation is an important post-translational modification (PTM) catalyzed by DHHC-palmitoyl transferases, which participate in the regulation of diverse biological processes. However, whether palmitoylation regulates cGAS function has not yet been explored. Here, we found that palmitoylation of cGAS at C474 restricted its enzymatic activity in the presence of double-stranded DNA. cGAS palmitoylation was catalyzed mainly by the palmitoyltransferase ZDHHC18 and double-stranded DNA promoted this modification. Mechanistically, palmitoylation of cGAS reduced the interaction between cGAS and double-stranded DNA, further inhibiting cGAS dimerization. Consistently, ZDHHC18 negatively regulated cGAS activation in human and mouse cell lines. In a more biologically relevant model system, Zdhhc18-deficient mice were found to be resistant to infection by DNA viruses, in agreement with the observation that ZDHHC18 negatively regulated cGAS mediated innate immune responses in human and mouse primary cells. In summary, the negative role of ZDHHC18-mediated cGAS palmitoylation may be a novel regulatory mechanism in the fine-tuning of innate immunity. Synopsis Detection of double-stranded DNA by cGAS triggers innate immune responses. ZDHHC18-mediated palmitoylation of cGAS sheds light on a novel posttranslational modification that leads to the fine-tuning of cGAS-mediated innate immune responses. cGAS is not palmitoylated under steady state conditions. In the presence of double-stranded DNA cGAS is palmitoylated at C474, which restricts the enzymatic activity. ZDHHC18 is the key palmitoyltransferase responsible for cGAS palmitoylation. Palmitoylation of cGAS impedes the interaction between cGAS and double-stranded DNA, further inhibiting cGAS dimerization. ZDHHC18 negatively regulates cGAS-mediated innate immune responses in human and mouse cell lines as well as in mice. Introduction As a marker of pathogen invasion or tissue damage, cytosolic DNA has long been known as a pathogen/danger-associated molecular pattern (P/DAMP) that triggers robust innate immune responses. Recently, cyclic GMP-AMP synthase (cGAS) was defined as a pattern-recognition receptor (PRR) that recognizes and binds to cytosolic double-stranded DNA (Sun et al, 2013). After binding, cGAS forms a 2:2 complex with DNA and catalyzes the transformation of ATP and GTP into 2′-3′-cGAMP. As a cytosolic second messenger, cGAMP binds to stimulator of interferon genes (STING, also known as TMEM173) and causes a 180° rotation of its carboxyl ligand-binding domain relative to its transmembrane domain, leading to STING activation (Burdette et al, 2011; Gao et al, 2013; Shang et al, 2019; Zhang et al, 2019). Activated STING is then trafficked from the endoplasmic reticulum (ER) to an ER-Golgi intermediate compartment (ERGIC) and then to the Golgi apparatus. Next, STING recruits and activates the kinase TANK-binding kinase 1 (TBK1), which in turn phosphorylates STING and IRF3 (Ishikawa et al, 2009; Saitoh et al, 2009; Dobbs et al, 2015). Phosphorylated dimeric IRF3 enters the nucleus and induces the expression of type I interferons (IFNs) (Chen et al, 2016). Based on the sensing of cytosolic DNA derived from both invaders and self-DNA, cGAS-mediated innate immune responses are involved in multiple biological and pathological processes. cGAS recognition of invading DNA exerts a nonredundant role in defending against infection by DNA viruses, including herpes simplex virus 1 (HSV-1), vaccinia virus (VACV), and retroviruses, such as human immunodeficiency virus 1 (HIV-1) (Yoh et al, 2015; Lahaye et al, 2018). On the other hand, in the presence of cytosolic self-DNA, including mitochondrial DNA (mtDNA) and chromatin exposed to the cytosol during mitosis, cGAS function must be well controlled to prevent overactivation. MtDNA released under specific stress is recognized by cGAS and induces the expression of type I IFNs and proinflammatory cytokines, and this process is related to multiple diseases (West et al, 2015; Aarreberg et al, 2019; Huang et al, 2020). In the nucleus, cGAS is tightly bound to nucleosomes, which is important for the restriction of its autoreactivity (Boyer et al, 2020; Kujirai et al, 2020; Michalski et al, 2020; Pathare et al, 2020; Zhao et al, 2020). Moreover, during mitosis, activation and autoreactivity of cGAS are prevented by hyperphosphorylation at its amino (N) terminus and chromatin tethering (Li et al, 2021). However, whether there are other mechanisms that control cGAS activation remains to be further understood. Post-translational modifications (PTMs) play critical roles in the regulation of protein functions. Palmitoylation is a post-translational lipid modification of proteins by which fatty acids, usually 16-carbon palmitate, are reversibly linked to cysteine residues via a liable thioester bond (Chamberlain & Shipston, 2015). Protein palmitoylation is catalyzed by a series of enzymes known as DHHC-palmitoyl transferases, which contain a signature DHHC motif (Jiang et al, 2018). Extensive studies have reported that this reversible, dynamic palmitoylation plays important roles in protein subcellular localization and signal transduction by altering their membrane association or conformational state (Baekkeskov & Kanaani, 2009; Zhang et al, 2016, 2020; Lu et al, 2019). For example, different palmitoylation profiles change the conformation of GPCRs (Chini & Parenti, 2009; Zheng et al, 2012). In the cGAS/STING signaling pathway, it has been reported that STING palmitoylation is important for TBK1 recruitment and activation to trigger the innate immune response (Mukai et al, 2016). However, whether cGAS is palmitoylated and whether that modification regulates cGAS function remain unclear. Here, we found that cGAS is palmitoylated at C474. This palmitoylation restricts cGAS activation in the presence of cytosolic DNA. Moreover, through cell-based screening, we identified that ZDHHC18 is a major acyltransferase catalyzing cGAS palmitoylation. DNA enhanced the interaction between ZDHHC18 and cGAS and promoted cGAS palmitoylation. cGAS palmitoylation causes a conformational change, subsequently inhibiting cGAS DNA binding and dimerization. Unlike other post-translational mechanisms of cGAS (e.g., ubiquitination and arginine methylation) (Wang et al, 2017a; Chen & Chen, 2019; Ma et al, 2021), the palmitoylation of cGAS unconventionally impairs its enzymatic activity. ZDHHC18 negatively regulates cGAS activation in human and mouse cell lines. Furthermore, Zdhhc18-deficient mice are resistant to infection by DNA viruses, in agreement with the fact that Zdhhc18-deficient macrophages upregulate antiviral gene expression under DNA stimulation. Taken together, our findings elucidate a new mechanism of cGAS regulation, which may imply a novel strategy for therapeutic biotechnology development. Results cGAS is palmitoylated at C474 Because palmitoylation is an important PTM involved in the regulation of protein function, we speculated that cGAS was modified by palmitoylation. To verify our hypothesis, we first detected cGAS palmitoylation using an acyl resin-assisted capture (acyl-RAC) assay in which the free cysteine thiol groups of overexpressed cGAS were irreversibly blocked by N-ethylmaleimide (NEM) and the acylated cysteines were then exposed to hydroxylamine; thus, the protein could undergo affinity capture by thiol-Sepharose beads (Appendix Fig S1A). As shown in Fig 1A, a cGAS band was present in the hydroxylamine addition group, indicating that cGAS was modified by palmitoylation. Given that 2-bromopalmitate (2-BP) reportedly effectively inhibits protein palmitoylation (Draper & Smith, 2009), we added 2-BP to the cells in acyl-RAC assays to confirm cGAS palmitoylation. The results showed that there was a dramatic reduction in the level of the band pulled down by thiol-Sepharose beads, indicating that cGAS acylation was indeed due to reversible palmitoylation. Figure 1. Palmitoylation of cGAS suppresses its activation A. Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. B. Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. C. Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264.7 cells. D, E. In vitro palmitoylation assay for the indicated proteins. NBD-Palm-CoA: (N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl] amino) palmitoyl-CoA. The recombinant cGAS protein used in the assay was indicated by Coomassie blue staining. F. LC-MS/MS analysis of palmitoylated peptides of cGAS. G. Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. H, I. THP-1 cells were treated with DMSO or 2-BP (50 μM) (H) or palmitic acid (100 μM) (I) for 12 h. Six hours after transfection with HT-DNA (2 μg/ml), cGAMP was extracted and quantified by cGAMP ELISA. J. THP-1 cells were treated with palmitic acid (0, 50, or 100 μM) for 12 h before a cGAMP bioassay. K–N. BMDMs were treated with palmitic acid (0, 100, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/ml) for 6 h before RT–qPCR analysis of Ifna4 (K), Ifnb1 (L), Cxcl10 (M), and Rantes (N) expression. O. L929 cells were treated with BSA and palmitic acid (100 μM) and transfected with HT-DNA (2 μg/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. Data information: 17-ODYA, 17-octadecanoic acid; 2-BP, 2-bromopalmitate; C, C-terminal domain; EtOH, ethanol; FL, full length; HAM, hydroxylamine; N, N-terminal domain; PA, palmitic acid. Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t-test; ns, not significant; **P < 0.005; ***P < 0.001 (H–N). Source data are available online for this figure. Source Data for Figure 1 [embj2021109272-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint cGAS contains three major domains: an N-terminal domain (1–160 aa), a nucleotidyltransferase domain (161–383 aa) and a Mab-21 domain (212–522 aa). The carboxy (C)-terminal domain (161–522 aa) is essential for enzymatic activity, while the N-terminal domain is important for the formation of phase condensates with DNA (Du & Chen, 2018). To determine which cGAS domain is important for its palmitoylation, we employed the two truncated forms of cGAS, with deletions of the N- and C-terminal domains, in acyl-RAC assays and found that the palmitoylated band disappeared for both the N- and C-terminal truncated cGAS mutants, indicating that only full-length cGAS can be palmitoylated (Fig 1B). Moreover, through a click chemistry assay, we found that the palmitoylation level of endogenous cGAS was elevated with the addition of herring testis DNA (HT-DNA), a long double-stranded DNA widely used to activate cGAS (Unterholzner, 2013; Fig 1C). To further study the palmitoylation properties of cGAS, we conducted a previously described biophysical palmitoylation assay (Rana et al, 2018) by adding nitrobenzoxadiazolyl (NBD)-linked palmitoyl-coenzyme A (CoA) into a solution containing purified recombinant cGAS and found that the NBD fluorescence signal increased over time (Fig 1D). This result suggested that cGAS can bind spontaneously to palmitoyl-CoA in vitro even in the absence of an acyltransferase. In addition, with the addition of HT-DNA, the NBD fluorescence signal decreased, indicating that HT-DNA decreased the binding ability of palmitoyl-CoA to cGAS. In line with this result, when using the C-terminal truncated cGAS mutant, which lacks the N-terminal region responsible for DNA binding, the NBD fluorescence signal did not change in the presence of HT-DNA, indicating that HT-DNA regulated cGAS binding to palmitoyl-CoA through the N terminus (Fig 1E). Moreover, to determine whether the downstream element, for example, STING, was important in cGAS palmitoylation, we overexpressed STING in acyl-RAC assays and found no dramatical change in cGAS palmitoylation (Appendix Fig S1B and C). Simultaneously, we detected the role of cGAMP by adding cGAMP into cells or using the cGAS C396S mutant, which failed to produce cGAMP, in acyl-RAC assays. The results showed that cGAS palmitoylation level showed little difference in these conditions, indicating that cGAS palmitoylation is not dependent on cGAMP or STING (Appendix Fig S1B and C). To further identify which cysteine residue on cGAS is modified by palmitoylation, we performed liquid chromatography-mass spectrometry analysis. As shown in Fig 1F, we identified C474 as a cGAS palmitoylation site with high confidence. To confirm the palmitoylation site identified from LC-MS/MS, we employed several systematically engineered mutants in acyl-RAC assays. We found that compared to wild-type (WT) cGAS, the C405S, C409S, and C474S mutants had drastically reduced palmitoylation signals (Fig 1G). Since sites C405 and C409 localized toward the inner side of the protein, these two sites were hardly palmitoylated structurally (Appendix Fig S1D–F). The reduced palmitoylation signal of C405S and C409S could be caused by a conformational change, further confirming that the palmitoylated residue of cGAS is C474, which is easily structurally modified (Appendix Fig S1D–F). Taken together, our data suggest that cGAS is palmitoylated at C474. Palmitoylation of cGAS restricts its enzymatic activity Next, we assessed whether cGAS function was regulated by palmitoylation. To do so, we measured the level of cGAMP, the enzymatic product of cGAS that is essential for downstream signal transduction, after adding 2-BP to THP-1 cells to block cGAS palmitoylation. As shown by the results of an enzyme-linked immunosorbent assay (ELISA), 2-BP addition increased the cGAMP production by cGAS stimulated by HT-DNA, indicating a negative role of palmitoylation on cGAS activation (Fig 1H). Given that palmitic acid reportedly promotes protein palmitoylation (Chamberlain & Shipston, 2015; Chen et al, 2017), we then focused on whether palmitic acid affected the activation of cGAS production of cGAMP. As shown by Fig 1I, addition of palmitic acid, which promoted cGAS palmitoylation, reduced the cGAMP production. Moreover, using a previously developed bioassay (Wu et al, 2013), we transfected THP-1 cells with HT-DNA to stimulate cGAMP production by cGAS. Cell lysates were digested with DNase and boiled to remove DNA and proteins, and the supernatant containing cGAMP was added to RAW264.7 cells with digitonin, followed by measurement of Ifn-β expression, which indirectly represents the cGAMP level. As shown in Fig 1J, the mRNA level of Ifn-β in RAW264.7 cells increased by approximately 60,000-fold with the addition of THP-1 cell lysates, indicating that cGAS was activated to produce a large amount of cGAMP. However, palmitic acid addition significantly reduced Ifn-β expression in a dose-dependent manner, suggesting that palmitic acid restricted cGAS activity and cGAMP production (Fig 1J). Next, we sought to clarify the regulatory effects of cGAS palmitoylation in primary cells by adding palmitic acid to mouse bone marrow-derived macrophages (BMDMs). After transfection with HT-DNA, the expression of antiviral genes, including Ifna, Ifnb, Rantes, and Cxcl10, in BMDMs was dramatically induced, while their expression was decreased in palmitic acid-treated cells compared to that in control cells, indicating that promoting cGAS palmitoylation negatively regulates the innate immune response (Fig 1K–N). The same results were obtained when we used mouse L929 cells (Appendix Fig S1G–I). Consistently, palmitic acid addition inhibited the cGAS/STING-mediated induction of IFN-β promoter expression in a reporter assay (Appendix Fig S1J). Since STING phosphorylation is an important event in cGAS/STING signal transduction, we assessed whether palmitic acid affected the phosphorylation level of STING. As shown in Fig 1O, STING was apparently phosphorylated 9 and 12 h after transfection of HT-DNA, whereas palmitic acid addition markedly slowed STING phosphorylation, confirming the negative role of palmitic acid in cGAS/STING signal transduction. Taken together, our findings suggest that palmitoylation suppresses cGAS activation. ZDHHC18 is a major acyltransferase for cGAS palmitoylation Protein palmitoylation is catalyzed by DHHC-palmitoyl transferases, which include 23 family members. To identify which palmitoyl transferase is important for cGAS palmitoylation, we first examined the relative expression of 23 ZDHHC protein S-acyl transferases in human HEK293T and THP-1 cells and found that several proteins, including ZDHHC3, ZDHHC5, ZDHHC6, ZDHHC7, ZDHHC9, ZDHHC12, ZDHHC13, ZDHHC16, ZDHHC17, and ZDHHC18, were highly expressed in both HEK293T and THP-1 cells (Appendix Fig S2A and B). To determine which one is important for cGAS palmitoylation, we adopted short heparin RNA (shRNA)-mediated knockdown followed by an acyl-RAC assay in HEK293T cells. The mRNA levels of the individual ZDHHC family proteins were downregulated after shRNA transfection (Appendix Fig S2C), among which ZDHHC18 knockdown significantly abrogated the cGAS palmitoylation signal, indicating that ZDHHC18 is important for cGAS palmitoylation (Fig 2A and B). Furthermore, to confirm the role of ZDHHC18 in cGAS palmitoylation, we co-overexpressed ZDHHC18 and cGAS in the acyl-RAC assay and observed an increase in the cGAS palmitoylation signal, indicating that ZDHHC18 promotes cGAS palmitoylation in mammalian cells (Fig 2C). Given that cGAS is palmitoylated at C474, we next sought to investigate whether ZDHHC18 affects cGAS function through this palmitoylation site. Interestingly, as shown in the results of an IFN-β promoter reporter assay, we found that the cGAS C474S mutant showed no difference from the WT control with respect to the induction of IFN-β promoter expression when coexpressed with STING in HEK293T cells. However, with ZDHHC18 expression, WT cGAS-induced IFN-β expression was dramatically reduced, whereas expression of the cGAS C474S mutant rescued the inhibitory effects of ZDHHC18 on WT cGAS, suggesting that ZDHHC18 specifically regulates cGAS at C474 (Fig 2D). Taken together, these results indicate that ZDHHC18 is a major acyltransferase mediating cGAS palmitoylation. Figure 2. ZDHHC18 is a major acyltransferase catalyzing cGAS palmitoylation A. Acyl-RAC assay of HEK293T cells transfected with Flag-cGAS 24 h after transfection with the indicated shRNA. B. Quantification of palmitoylation levels of Flag-cGAS in (A). C. Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. HAM: hydroxylamine. D. HEK293T cells were transfected with HA-cGAS (WT or C474S mutant) (200 ng), Flag-STING (200 ng), and Myc-ZDHHC18 (200 ng) expression plasmids for 24 h before luciferase reporter assays. E, F. Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids for 24 h. G. Schematic diagrams of ZDHHC18 and its truncation mutants. H. Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-cGAS and Flag-ZDHHC18 truncations or GFP for 24 h. I. Schematic diagrams of cGAS and its truncation mutants. J. Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-ZDHHC18 and Flag-cGAS truncates or GFP for 24 h. K. Immunofluorescence analysis of HA-cGAS (green) and Flag-ZDHHC18 (red) in HT-DNA-stimulated (or not) HeLa cells. Scale bars: 7 μm. L. Colocalization (merged volume of total cGAS signal) of cGAS and ZDHHC18 in (K). M, N. Comparison of the binding free energy (M) and binding energy decomposition of a per-residue to the binding affinity between ZDHHC18 and cGAS (N) in different complexes. Data information: FL, full length; IP, immunoprecipitation; NC, negative control; TM, transmembrane domain; WCL, whole-cell lysate. Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t-test; **P < 0.005; ***P < 0.001 (D and L). Source data are available online for this figure. Source Data for Figure 2 [embj2021109272-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint ZDHHC18 interacts with cGAS Given that ZDHHC18 promotes cGAS palmitoylation, we next investigated whether ZDHHC18 directly interacts with cGAS under physiological conditions. We conducted coimmunoprecipitation (co-IP) experiments to examine whether cGAS physically interacts with ZDHHC18 in cultured mammalian cells. The results showed that epitope tagged ZDHHC18 reciprocally coimmunoprecipitated with cGAS in transfected HEK293T cells (Fig 2E and F). Previous reports have shown that ZDHHC18 is a membrane-associated protein (De & Sadhukhan, 2018). To further clarify the subcellular localization of ZDHHC18, we performed cell staining of endogenous ZDHHC18 in HeLa cells. The results showed that endogenous ZDHHC18 showed a dispersed pattern in cells (Appendix Fig S2D). Moreover, we engineered two truncated constructs: an M truncation referring to the middle region consisting of the four predicted transmembrane domains and the linker region and a ΔM truncation with a deletion of the middle region (Fig 2G). We detected the contribution of these truncated regions to the subcellular localization of Z