Zebrafish ZDHHC11 palmitoylates STING to potentiate antiviral immune response

Abstract

In mammals, the activation of stimulator of interferon gene (STING), a pivotal adaptor protein mediating type I interferon (IFN-I) induction upon DNA virus infection, is regulated by palmitoylation. This post-translational modification is catalyzed by zinc finger DHHC domain-containing palmitoyltransferases (ZDHHCs). However, the specific palmitoyltransferases accountable for mediating STING palmitoylation remain predominantly unknown. This study demonstrated that ZDHHC11 plays a critical role by modifying STING palmitoylation and subsequently enhancing the antiviral response in zebrafish. The expression of zebrafish ZDHHC11 was found to be upregulated upon stimulation with GCRV, SVCV, B-DNA, and Z-DNA in the tested tissues or cells. Consistently, the overexpression of zebrafish ZDHHC11 led to an increase in IFN-I expression, while its knockdown resulted in a suppression of IFN-I expression. Mechanistically, ZDHHC11 specifically bound to STING at the endoplasmic reticulum (ER) and then promoted STING palmitoylation via its palmitoyltransferase activity thereby activating the STING-IRF3-mediated IFN I response. In addition, we have demonstrated that ZDHHC11 is capable of inhibiting SVCV replication both in vitro and in vivo. In conclusion, our findings revealed a novel role for ZDHHC11 in regulation of antiviral response. This implies that ZDHHC11 serves as a valuable target for modulating STING signaling and for the development of antiviral therapies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-025-02643-9.

Introduction

Following the viral invasion of host cells, intracellular proteins frequently undergo post-translational modifications (PTMs), such as phosphorylation, ubiquitination, acetylation, ISGylation, and so on. These modifications are not mere “decorations”, but serve to regulate the protein activity states, including their localization, stability, intracellular transport and interactions.

Palmitoylation is a significant post-translational modification that plays a crucial role in regulating protein function. It is catalyzed by a family of enzymes known as palmitoyl acyltransferases (PATs). In mammals, twenty-four distinct DHHC proteins, designated DHHC1 through DHHC24, have been identified [1]. These proteins possess a Cys4 zinc finger metal-binding motif, which classifies them as ZDHHC (zinc-finger DHHC) proteins [2]. Furthermore, the conserved Asp-His-His-Cys (DHHC) motif within their active sites has resulted in the alternative designation of these enzymes as the DHHC family [3, 4]. Palmitoylation is required for antiviral immune response. Mammalian ZDHHC7-catalyzed palmitoylation of MAVS at Cys508 promotes RLR signaling [5]. ML349, a inhibitor for depalmitoylase APT2, enhances fish innate immunity [6]. Fish ZDHHC18 facilitates palmitoylation of NLRP3 and inflammatory response [7]. Therefore, palmitoyltransferase ZDHHC family serves as activators for innate immunity.

Palmitoyltransferase ZDHHC11 was initially identified as a potential marker in a whole-genome copy number analysis of bladder cancer patients. Its aberrant expression is strongly correlated with the development of multiple cancers [8]. Testis-specific heterogeneous nuclear ribonucleoproteins (hnRNPs) promote cancer cell proliferation by stabilizing ZDHHC11 mRNA [9]. Moreover, miR-150 promotes the proliferation of Burkitt lymphoma and diffuse large B-cell lymphoma by modulating the ZDHHC11/MYB/MYC complex [10]. Furthermore, ZDHHC11 promotes TRAF6 oligomerization and ligase activity, activating TAK1 and IKK complexes [11]. ZDHHC11 acts as a critical role in antiviral responses against both DNA and RNA viruses. For example, ZDHHC11-deficient cells show impaired interferon-I (IFN-I) production during infections with HSV-1 or Zika Virus viruses [12, 13]. Although ZDHHC11 is involved in regulating carcinogenesis and immune response, its precise functional mechanism still remains unclear.

STING, also known as MITA, ERIS, MPYS, or TMEM173, is a transmembrane protein that plays a crucial role in innate immunity. It is localized to the endoplasmic reticulum (ER), mitochondria, and mitochondrial-associated membranes. Upon sensing cytoplasmic DNA, cGAS initiates the innate immune response by synthesizing the second messenger cGAMP, which subsequently binds to STING. This binding promotes STING translocation from the ER to the Golgi apparatus, where it recruits the downstream kinase TBK1 and transcription factor IRF3. This cascade ultimately leads to the induction of type I interferons (IFN-I) [14].

However, STING activity is tightly regulated by post-translational modifications (PTMs). Among the frequently observed PTMs are ubiquitination, SUMOylation, phosphorylation and ISGylation [15–18]. Moreover, it is reported that STING can be modified by palmitoylation [19]. However, the precise regulatory mechanisms governing STING palmitoylation, as well as the specific molecular pathways through which palmitoylated STING activates innate immunity, require further elucidation.

In this study, we reported the identification of zinc finger DHHC domain-containing palmitoyltransferase 11 (ZDHHC11) as a positive regulator of innate immune signaling. Zebrafish ZDHHC11 promotes the induction of IFN-I by directly activating STING-mediated downstream pathway. Mechanically, ZDHHC11 catalyzed STING palmitoylation through its palmitoyltransferase activity. Our results reveal a novel regulatory mechanism of STING activity by ZDHHC11.

Results

ZDHHC11 expression is elevated in response to GCRV, SVCV, B-DNA and Z-DNA stimulation

Phylogenetic tree showed that ZDHHC11 is conserved among fish, reptiles and mammals (Fig. S1), the species name and Genebank ID were shown in Table S1. To investigate the role of ZDHHC11 in the innate immune response, we assessed its expression following viral stimulation using qRT-PCR. As shown in Fig. 1A, ZDHHC11 expression was significantly elevated in zebrafish kidney at 12 h post-GCRV stimulation. Similarly, SVCV induced ZDHHC11 expression in all tested tissues. The highest levels of ZDHHC11 were observed at 48 h in the liver (Fig. 1A).

Fig. 1.

Zebrafish ZDHHC11 is induced by the stimulation with GCRV, SVCV, B-DNA and Z-DNA. A and B In vivo ZDHHC11 expression. Zebrafish were injected intraperitoneally with 100 µl of either 10^–7 TCID₅₀ GCRV, 10^–7 TCID₅₀ SVCV, or 10 µM B-DNA/Z-DNA. ZDHHC11 mRNA levels were measured at the indicated time points (6, 12, 24, 48 and 72 h post-injection) in heart, kidney and liver (n = 6 zebrafish per group). In vitro ZDHHC11 expression. CIK cells and ZFIN cells were infected with 10^–7 TCID50 GCRV and SVCV, respectively. ZFIN cells were transfected with 10 µM B-DNA or Z-DNA. ZDHHC11 mRNA levels were detected at the indicated time points (6, 12, 24, 48 and 72 h post-treatment). Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. Significant differences (p < 0.05) are indicated by different letters. Data represent one representative experiment performed with three biological replicates (mean ± SD) or representative data from at least three independent experiments

Stimulation with extracellular B-form DNA (B-DNA) robustly induced the expression of ZDHHC11 compared to other stimulants across all tested tissues, with the strongest effect observed at 24 h in the liver; stimulation with Z-DNA also increased ZDHHC11 levels in several tissues, including heart, kidney and liver (Fig. 1B). ZDHHC11 expression was also detected in other zebrafish tissues, suggesting that its can be widely induced in different tissues after viral stimulation (Fig. S2). These findings suggested that DNA pathogens (B-DNA) elicit a greater induction of ZDHHC11 expression than RNA pathogens (GCRV or SVCV). So, we used B-DNA as an agonist for ZDHHC11-mediated pathway in subsequent experiment.

Furthermore, ZDHHC11 expression was also detected in cell lines following viral stimulation. As illustrated in each last panel of Fig. 1A and B, its expression initially increased and subsequently decreased with prolonged stimulation. Although this expression profile in cell lines differs from that observed in tissues, all tested stimulants still induced ZDHHC11 expression to varying degrees.

Zebrafish ZDHHC11 positively regulates IFN-I response

It is important to elucidate the role of ZDHHC11 in the regulation of the IFN-I response. ZDHHC11 demonstrated a dose-dependent increase in IFN-I expression at both the transcriptional and translational levels, correlating with increasing ZDHHC11 transfection concentrations (Fig. 2A-C). Moreover, dual-luciferase reporter assays showed that overexpression of ZDHHC11 augmented the transcriptional activity of ISRE and NF-κB (Fig. 2D and E).

Fig. 2.

ZDHHC11 is a positive regulator for IFN-I response. A-E ZFIN cells were transfected with different concentrations of ZDHHC11 plasmid (0.5, 1, 1.5, and 2 µg). An empty plasmid transfection (2 µg) served as the control. After 24 h, cells were stimulated with 10 µl of 10 µM B-DNA for 12 h. The mRNA levels, promoter activity and protein levels of IFN-I were measured. Additionally, promoter activities of ISRE and NF-κB were assessed. F–H In a separate experiment, ZFIN cells were transfected with negative control siRNA (N.C), siRNA-173, siRNA-502, and siRNA-833. After 24 h, cells were stimulated with 10 µl of 10 µM B-DNA for 12 h. The mRNA levels of ZDHHC11 and IFN-I, as well as IFN-I promoter activity, were measured. I Western blot analysis was performed to detect IFN-I, p-IRF3, p-IRF7, and p-TBK1. Statistical significance was determined using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. Differences with p < 0.05 are indicated by different letters. Data represent one representative experiment performed in triplicate, with at least three independent experiments (mean ± SD). Western blot band intensities were quantified using Image J software

To further elucidate the positive role of ZDHHC11 in regulating IFN-I expression, a knockdown assay was performed. Knockdown efficiency of ZDHHC11 was confirmed at the mRNA level (Fig. 2F). Both qRT-PCR and dual-luciferase reporter assays revealed that ZDHHC11 knockdown attenuated IFN-I expression (Fig. 2G and H). In line with these observations, knockdown of ZDHHC11 led to decreased protein levels of IFN-I, p-IRF3, and p-TBK1, while showing no significant impact on p-IRF7 (Fig. 2I). These findings collectively establish ZDHHC11 as an activator of the IFN-I response.

ZDHHC11 specifically interacts with STING and activates STING-dependent IFN-I expression

To further validate the substrate of ZDHHC11, cellular co-IP, co-localization, and BIFC assays were conducted. Co-IP results indicated a specific interaction between ZDHHC11 and STING, while no interaction was observed with IRF3, IRF7, or TBK1 (Fig. 3A). Similarly, co-localization and BIFC assays demonstrated that the ZDHHC11-STING interaction is formed within the cytoplasm (Fig. 3B and C). Additionally, a subcellular localization assay revealed that both ZDHHC11 and STING are localized to the endoplasmic reticulum (ER) (Fig. 3D). Consequently, ZDHHC11 interacts with STING on the ER following B-DNAstimulation.

Fig. 3.

ZDHHC11 specifically binds to STING and synergistically activates IFN-I response. A ZFIN cells were co-transfected with ZDHHC11-Flag and IRF3-GFP (or IRF7-GFP, STING-GFP, TBK1-GFP). After 24 h, cells were stimulated with B-DNA for 12 h. Co-IP assays were performed to identify substrates of ZDHHC11. B ZFIN cells were transfected with indicated plasmids: bFOS-VC155 and bJun-VN155; ZDHHC11-VC155 and STING-VN155; bFOS-VC155 (deltazip) and bJun-VN155 (152L); STING-VN155; ZDHHC11-VC155. After 24 h, cells were stimulated with B-DNA for 12 h. BiFC assays were used to analyze the interaction between ZDHHC11 and STING. C ZFIN cells were co-transfected with ZDHHC11-Flag and STING-GFP. After 24 h, cells were stimulated with B-DNA for 12 h. Co-localization assays were conducted to examine the interaction of ZDHHC11 and STING. D ZFIN cells were co-transfected with ZDHHC11-GFP (or STING-GFP) and pDsRed2-ER plasmids. After 24 h, cells were stimulated with B-DNA for 12 h. Subcellular localization of ZDHHC11 or STING was imaged. E–H One group of ZFIN cells was co-transfected with pcDNA3.1-ZDHHC11 and pcDNA3.1-IRF3 (or IRF7, STING, TBK1). A second group was co-transfected with siRNA-ZDHHC11 and the same plasmids. After 24 h, cells were stimulated with B-DNA for 12 h. qRT-PCR and Western blot analyses were performed to assess IFN-I expression. Statistical significance was determined using Student’s t-test (*p < 0.05, **p < 0.01). I-L ZFIN cells were transfected with IRF3-GFP. After 12 h, cells were transfected with indicated plasmids. Subcellular localization, Nuclear and cytoplasmic protein extraction, Native-PAGE gel electrophoresis, Co-IP assays were performed to analyze the nuclear translocation, dimerization and phosphorylation levels of IRF3, respectively. M ZFIN cells were co-transfected with STING-Flag and IRF3-GFP plasmids, then the cells were transfected with ZDHHC11-pcDNA3.1 or siRNA-ZDHHC11. Co-IP experiments were performed to verify the interaction between STING and IRF3. N ZFIN cells were co-transfected with IRF3-Flag plasmids and siRNA-STING, then the cells were transfected with ZDHHC11-pcDNA3.1 or siRNA-ZDHHC11. IRF3 phosphorylation was detected. Data are representative of one experiment performed in triplicate, with at least three independent repeats (mean ± SD). Western blot band intensities were quantified using Image J software

Subsequently, ZDHHC11 enhanced IFN-I expression induced by IRF3, IRF7, and TBK1, with a particularly strong effect on STING-induced IFN-I expression (Fig. 3E). Conversely, knockdown of ZDHHC11 restrained the induction by IRF3, STING, and TBK1, while showing minimal influence on IRF7-induced IFN-I expression (Fig. 3F). This same conclusion was supported by Western blot (Fig. 3G and H).

In addition, we investigated whether ZDHHC11 regulates IRF3 activity. Subcellular localization assay indicated that both ZDHHC11 and STING promoted the nuclear translocation of IRF3, leading to increased IRF3 dimerization and phosphorylation (Fig. 3I-L). Then, IP assays demonstrated that ZDHHC11 enhanced the interaction between IRF3 and STING (Fig. 3M). Furthermore, the increase of IRF3 phosphorylation level induced by ZDHHC11 can be blocked by knockdown of STING (Fig. 3N). The knockdown efficiency of STING was confirmed in Fig. S3. Therefore, ZDHHC11 targets STING to activate IRF3-induced IFN-I expression.

Transmembrane domains (TMs) are required for the activities of ZDHHC11 and STING

Transmembrane domains are known to be critical for precise protein localization, which in turn can dictate their function. We observed that zebrafish ZDHHC11 and STING possess multiple transmembrane domains. Consequently, we generated several transmembrane (TM)-deficient mutants of ZDHHC11 and STING (Fig. 4A). Subcellular localization assays revealed that ZDHHC11 mutants (TM0, TM1, or TM2) lost their cytoplasmic localization and exhibited reduced activity in IFN-I induction (Fig. 4B), as demonstrated by qRT-PCR and Western blot (Fig. 4C-D). Furthermore, we mapped the region of ZDHHC11 that interacts with STING. Our results indicated that only the ZDHHC11 mutants (TM3 and TM4) were capable of interacting with STING, suggesting that having at least three transmembrane (TM) domains is essential for ZDHHC11 to effectively bind STING (Fig. 4E).

Fig. 4.

Transmembrane domain is essential for the role of ZDHHC11 and STING. A The transmembrane (TM) mutants of ZDHHC11 (ZDHHC11-TM0, ZDHHC11-TM1, ZDHHC11-TM2, ZDHHC11-TM3, and ZDHHC11-TM4) and STING (STING-TM0, STING-TM1, STING-TM2, and STING-TM3) were generated. B-D ZFIN cells were separately transfected with the indicated ZDHHC11-TM mutants. Subcellular localization was analyzed to determine the localization characteristics of these mutants. qRT-PCR and Western blot analyses were performed to detect IFN-I expression. E ZFIN cells were co-transfected with STING-Flag and the ZDHHC11-TM mutants. After 24 h, cells were stimulated with B-DNA for 12 h. Co-IP assays were performed to map the interaction region between ZDHHC11 and STING. F–H ZFIN cells were separately transfected with the STING-TM mutants. After 24 h, cells were stimulated with B-DNA for 12 h. Subcellular localization of these mutants was determined, and IFN-I expression was measured using qRT-PCR and Western blot. I ZFIN cells were transfected with STING at a concentration gradient, and IFN-I expression was assessed using qRT-PCR. Statistical significance was determined using the Kruskal–Wallis (K-W) test followed by Dunn's multiple comparisons test. Differences with p < 0.05 are indicated by different letters. Data represent one representative experiment performed in triplicate with at least three independent repeats (mean ± SD). Western blot band intensities were quantified using Image J software

Quantitative RT-PCR results indicated that STING promoted IFN-I expression (Fig. 4F). Furthermore, we investigated the function of STING's transmembrane (TM) domains. Subcellular localization assays showed that STING mutants (TM0, TM1, or TM2) lost their cytoplasmic localization (Fig. 4G). This loss resulted in reduced STING activity in IFN-I induction, as confirmed by qRT-PCR and Western blot (Fig. 4H and I). Collectively, these results suggested that a sufficient number of TM domains in ZDHHC11 or STING is essential for their proper cytoplasmic localization and functional activity in regulating the IFN-I response.

ZDHHC11 facilitates palmitoylation of STING through DHHC domain

Although above findings demonstrated that ZDHHC11 interacts with STING within the three TM regions, whether the enzyme-active domain (DHHC) is responsible for the interactions is still unknown. To address this, we constructed a mutant lacking the DHHC domain (Fig. 5A). Co-immunoprecipitation (Co-IP) assays showed that this DHHC mutant failed to interact with STING, strongly suggesting that the DHHC domain is indeed responsible for this interaction (Fig. 5B). To further investigate the role of palmitoylation in STING-mediated IFN-I response, we employed 2-bp, an inhibitor of palmitoylation. qRT-PCR, Western blot, and dual-luciferase reporter assays revealed that treatment with 2-bp progressively inhibited B-DNA-induced IFN-I expression in a dose-dependent manner (Fig. 5C-5D). Furthermore, B-DNA treatment induced STING palmitoylation compared to PBS-treated control cells (Fig. 5E). Subsequently, overexpression of ZDHHC11 enhanced STING palmitoylation, and cells expressing the ΔDHHC mutant exhibited reduced STING palmitoylation compared to those overexpressing full-length ZDHHC11 (Fig. 5F and G). Collectively, these data demonstrate that ZDHHC11 promotes STING palmitoylation via its DHHC domain.

Fig. 5.

ZDHHC11 promotes STING palmitoylation rely on DHHC domain. A A mutant lacking the DHHC domain was constructed. B ZFIN cells were co-transfected with STING-Flag and ZDHHC11-GFP or ZDHHC11-ΔDHHC-GFP. After 24 h, cells were stimulated with B-DNA for 12 h. Co-IP assays were performed to analyze their interaction. C and D ZFIN cells were treated with 2-bp at a concentration gradient (0 µM, 50 µM, 100 µM, 150 µM, and 200 µM). The 0 µM group represents cells treated with the same volume of culture medium. Cells in the "mock" group were untreated. After 24 h, cells were stimulated with B-DNA for 12 h. Expression of IFN-I was assessed using qRT-PCR, Western blot and dual-luciferase assays. E ZFIN cells were transfected with STING-Flag. After 24 h, cells were stimulated with B-DNA, or co-treated with B-DNA and 2-bp. F ZFIN cells were co-transfected with STING-Flag and ZDHHC11-GFP or GFP-basic. After 24 h, one group of cells was treated with 2-bp. G ZFIN cells were co-transfected with STING-Flag and ZDHHC11-GFP, GFP-basic, or ZDHHC11-ΔDHHC-GFP. After 24 h, cells were stimulated with B-DNA for 12 h. ABE assays were used to analyze STING palmitoylation. Statistical significance was determined using the Kruskal–Wallis (K-W) test followed by Dunn's multiple comparisons test. Significant differences (p < 0.05) are indicated by different letters. Data are representative of one experiment performed in triplicate with at least three independent repeats (mean ± SD). Western blot band intensities were quantified using Image J software

ZDHHC11 protects ZFIN cells from SVCV infection

To investigate the role of ZDHHC11 in viral infection, we performed crystal violet staining and cell viability assays in ZFIN cells. Compared to the control (pcDNA3.1-basic), overexpression of ZDHHC11 reduced cytopathic effects (CPEs) and improved cell viability following SVCV infection (Fig. 6A-B). The expression of SVCV N and P genes were decreased after transfection of ZDHHC11 (Fig. 6C-D). Conversely, ZDHHC11 knockdown enhanced CPEs and significantly decreased cell viability in the presence of SVCV (Fig. 6E-F). ZDHHC11 knockdown also promoted SVCV replication (Fig. 6G-H). Therefore, ZDHHC11 is essential for host cell defense against viral infection.

Fig. 6.

ZDHHC11 inhibits SVCV replication in ZFIN cells. A and B ZFIN cells were transfected with ZDHHC11-pcDNA3.1 or pcDNA3.1-basic at concentrations of 0.5, 1.0, 1.5, and 2 µg. After 24 h, cells were infected with SVCV; non-infected cells served as a mock. Crystal violet staining and cell viability assays were performed to assess cytopathic effects (CPEs). C and D ZFIN cells were transfected with ZDHHC11-pcDNA3.1 or pcDNA3.1-basic. After 24 h, cells were infected with SVCV. qRT-PCR and Western blot analyses were conducted to measure the expression levels of SVCV N and P genes. E and F ZFIN cells were transfected with either negative control siRNA (N.C) or siRNAs targeting siRNA-173, siRNA-502, or siRNA-833. Following transfection, cells were subjected to crystal violet staining and cell viability assays to evaluate cytopathic effects. G and H qRT-PCR and Western blot analyses were used to assess SVCV N and P gene expression in the knockdown experiments. Statistical differences between groups were analyzed using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). Data shown are representative of one experiment performed in triplicate, with at least three independent repeats (mean ± SD). Western blot band intensities were quantified using Image J software

ZDHHC11 is a critical regulator for antiviral response in vivo

To further investigate the antiviral function of ZDHHC11, we separately overexpressed and knocked down ZDHHC11 in zebrafish embryos. In the ZDHHC11 overexpression experiment, we transcribed ZDHHC11 mRNA in vitro and injected it into embryos. We first confirmed ZDHHC11 overexpression efficiency using qRT-PCR and Western blot. The results demonstrated that both ZDHHC11 mRNA and protein levels were significantly upregulated following ZDHHC11 mRNA injection (Fig. 7A). Subsequent viral challenge experiments revealed that the survival rate of larvae was significantly higher in the ZDHHC11-overexpressing group compared to the control group (Fig. 7B-C). Crystal violet staining indicated that the virulence of SVCV in the sample injected with ZDHHC11 mRNA was weaker than that in the sample injected with scramble sequence (Fig. 7D). The levels of ZDHHC11-downstream proteins, including phosphorylated TBK1 (p-TBK1) and IRF3 (p-IRF3), as well as IFN-I, were increased by ZDHHC11 overexpression, irrespective of SVCV stimulation (Fig. 7E). Similarly, qRT-PCR analysis revealed that ZDHHC11 injection enhanced the expression of antiviral genes, including IFN-I, Mxa, Mxb and Mxc (Fig. 7F).

Fig. 7.

Overexpression of ZDHHC11 in zebrafish increases antiviral response. Zebrafish embryos at the one-cell stage were injected with synthesized mRNA encoding ZDHHC11 or a scramble sequence (control). A Overexpression effects were confirmed via qRT-PCR and Western blot. B and C At 72 h post-injection, zebrafish larvae were infected with SVCV in water, and morphology and survival rates were assessed. D Viral homogenate solutions were prepared from infected larvae. Each well of ZFIN cells was infected with 100 µl of the homogenate solution for 72 h. Crystal violet staining was performed to assess SVCV virulence from each group's homogenate. E and F Western blot and qRT-PCR were used to analyze the expression of antiviral genes. Significance was determined using the Kruskal–Wallis (K-W) test followed by Dunn's multiple comparisons test. Differences with p < 0.05 are indicated by different letters. Data are representative of one experiment performed in triplicate with at least three independent repeats (mean ± SD). Western blot band intensities were quantified using Image J software

In the ZDHHC11 knockdown experiment, embryos were injected with either morpholino oligonucleotides targeting ZDHHC11 (Mo-ZDHHC11) or a standard control morpholino (Mo-STD) and subsequently stimulated with SVCV. We then assessed the morphology and survival rates. The results demonstrated that ZDHHC11 knockdown reduced larval survival following SVCV infection (Fig. 8A-B). Crystal violet staining showed that the virulence of SVCV in the sample injected with Mo-ZDHHC11 was stronger than that in the sample injected with Mo-STD (Fig. 8C). Knockdown efficiency was confirmed by analyzing ZDHHC11 protein levels. In ZDHHC11-knockdown larvae, the levels of downstream proteins, including phosphorylated TBK1 (p-TBK1), IRF3 (p-IRF3), and IFN-I, were decreased, irrespective of SVCV stimulation (Fig. 8D). Furthermore, the expression of antiviral genes, such as IFN-I, Mxa, Mxb and Mxc, was reduced following ZDHHC11 knockdown (Fig. 8E). These in vivo experiments collectively demonstrated that zebrafish ZDHHC11 plays a positive role in the antiviral response.

Fig. 8.

Knockdown of ZDHHC11 in zebrafish decreases antiviral response. Zebrafish embryos at the one-cell stage were injected with either ZDHHC11-Mo or STD-Mo morpholino. Larvae were then infected with SVCV in water. A and B Morphology and survival rates were assessed at 72 h post-infection. C Crystal violet staining was performed to evaluate SVCV virulence in the larval homogenates. D Western blot analyses were conducted to determine both the knockdown efficiency of ZDHHC11 and the protein expression of select antiviral genes. E qRT-PCR was performed to measure the mRNA levels of antiviral genes. Statistical significance was determined using the Kruskal–Wallis (K-W) test followed by Dunn's multiple comparisons test. Differences with p < 0.05 are indicated by different letters. Data are representative of one experiment performed in triplicate with at least three independent repeats (mean ± SD). Western blot band intensities were quantified using Image J software

Discussion

STING plays a critical role as an antiviral factor against viral infections in both mammals and fish. Our previous studies showed that grass carp STING modulates the DNA receptor-induced innate immune response, specifically influencing the expression of genes like ISG15, Mre11A, and cGAS [20–22]. Consequently, STING acts as a key molecular switch for immune responses in fish. However, the mechanisms regulating fish STING activity are still largely unknown. Palmitoylation, a post-translational modification, can also enhance antiviral responses by modulating the modification of pathway proteins involved in the antiviral process [23]. In this study, we identified a zebrafish palmitoyltransferase, ZDHHC11, which positively modulates the STING-mediated antiviral innate immune response through palmitoylation.

Phylogenetic analysis indicated that ZDHHC11 exhibits a high degree of homology among mammals, fish and reptiles (Fig. S1), suggesting that its functional role may be conserved across these species. In mammals, ZDHHC11 is known to be involved in DNA-viral innate immune responses and positively regulates type I interferon (IFN-I) expression [11]. Similarly, our results showed that zebrafish ZDHHC11 can be induced by cytoplasmic DNA pathogens (B-DNA and Z-DNA). Furthermore, zebrafish ZDHHC11 was markedly upregulated following infection with RNA viruses such as GCRV and SVCV (Fig. 1 and Fig. S2). Recently, mammalian ZDHHC11 has also been reported as a critical protein in defending against Zika virus (ZIKV), and RNA-enveloped virus [13]. These findings suggested that ZDHHC11 may play a key role in the host response to both RNA and DNA virus infections.

Subsequently, we found that overexpression of ZDHHC11 activated the promoters of ISRE, IFN-I and NF-κB, and increased IFN-I expression. Conversely, ZDHHC11 knockdown reduced IFN-I levels and decreased phosphorylation of IRF3 and TBK1, with no apparent effect on IRF7 phosphorylation (Fig. 2). Consistent with this, studies in mice have shown that ZDHHC11 enhances ISRE and NF-κB promoter activities, thereby activating IRF3-dependent IFN-β expression [12]. These findings suggested that fish ZDHHC11 plays a critical role in regulating IRF3-dependent innate antiviral immune responses, similar to its function in mammals.

Next, we investigated the substrates of zebrafish ZDHHC11 during viral infection. As shown in Fig. 3A, ZDHHC11 specifically interacted with STING, but not with IRF3, IRF7, or TBK1. Bimolecular fluorescence complementation (BIFC) and co-localization assays confirmed this interaction at the endoplasmic reticulum (ER) (Fig. 3B-D). Furthermore, ZDHHC11 synergistically enhanced IFN-I expression mediated by IRF3, STING, and TBK1, but did not affect IRF7-mediated IFN-I expression (Fig. 3E-H). These data suggested that ZDHHC11 directly targets the STING-IRF3 axis to activate IFN-I expression. Subsequently, overexpression of both ZDHHC11 and STING increased IRF3 activity, as evidenced by nuclear translocation, phosphorylation and dimerization (Fig. 3I-L). The activated form of IRF3 is known to involve nuclear translocation, phosphorylation, and dimerization [24]. Consistent with our findings, mammalian STING stimulates the phosphorylation of IRF3 by the kinase TBK1 in the cytosolic DNA signaling pathway [25]. In this study, zebrafish ZDHHC11 targeted STING to activate IRF3 (Fig. 3M and N). Collectively, our data suggested the presence of a novel signaling axis, ZDHHC11-STING-IRF3, in the antiviral immune pathway in fish.

The transmembrane domain (TM) is critical for the subcellular localization and function of many transmembrane proteins. For example, MYCT1 protein relies on its second transmembrane domain to determine its cytoplasmic localization and function [26]. Similarly, the transmembrane domain of the rice small protein OsS1Fa1 is responsible for its subcellular localization and drought tolerance [27]. Zebrafish ZDHHC11 was predicted to contain five TM domains. Our results indicate that ZDHHC11 can localize in the cytoplasm when it contains at least three TM domains, and this configuration enables ZDHHC11 to interact with STING and activate the IFN-I response (Fig. 4A-E). Additionally, STING was found to contain four TM domains; only STING molecules with more than three TM domains can maintain proper cytoplasmic localization and effectively activate the IFN-I response (Fig. 4F-I). Similarly, human STING oligomerization and cytoplasmic localization also rely on its TM domains [28]. Therefore, TM domains are essential for the function of both ZDHHC11 and STING. The mechanism by which TM domains influence the activity of these proteins warrants further investigation in our future studies.

To further investigate whether ZDHHC11 regulates the STING-IRF3 signaling pathway through its palmitoyltransferase activity, we constructed a mutant lacking the DHHC domain (ΔDHHC). The entire DHHC domain is located between amino acids 124–278 of full-length ZDHHC11 (within the third transmembrane domain, TM3) (Fig. 5A). This DHHC domain is responsible for palmitoyltransferase activity, a crucial and highly conserved motif within the palmitoyl acyltransferase family. It is characterized by a zinc finger-like metal-binding site containing four cysteine residues, followed by a complex region rich in cysteine and histidine residues. Disrupting the DHHC domain through mutation in these enzymes typically leads to a loss of their palmitoyltransferase activity [29, 30]. In this study, the ΔDHHC mutant was unable to interact with STING (Fig. 5B), suggesting that the DHHC domain is a key interaction region. This finding supported the results presented in Fig. 4E, which suggested that the DHHC domain is critical for the interaction with STING.

The palmitoylation inhibitor 2-bp reduced IFN-I expression and impaired B-DNA–induced palmitoylation of STING (Fig. 5C-E). These results suggested that palmitoylation is essential for the STING-mediated IFN-I response. To further confirm the role of ZDHHC11 in regulating STING palmitoylation, we found that ZDHHC11 increased the level of STING palmitoylation. Conversely, treatment with 2-bp or transfection of the ΔDHHC mutant inhibited ZDHHC11-induced STING palmitoylation (Fig. 5F-G). These findings demonstrated that ZDHHC11 catalyzes the palmitoylation of STING.

Activation of the STING-dependent pathway is crucial for the antiviral response. DDX41 modulates the levels of double-stranded DNA (dsDNA), thereby promoting cGAS-STING activation and subsequently triggering the antiviral response [31]. Our previous work also demonstrated that grass carp DDX41 activates the STING-IRF7 axis and reduces GCRV infection [32]. Furthermore, ISGylation promotes STING activation and inhibits SARS-CoV-2 replication [18]. Based on these findings, we hypothesized that palmitoylation enhances STING activation. To investigate the antiviral role of ZDHHC11-induced palmitoylation, we overexpressed or knocked down ZDHHC11 in ZFIN cells. The results indicated that ZDHHC11 exhibited antiviral activity against SVCV (Fig. 6). Similar to other palmitoyltransferases, ZDHHC7 enhances the antiviral response by catalyzing the palmitoylation of MAVS during IAV infection [5]. Conversely, ZDHHC20 promotes IFITM3 palmitoylation, increasing cellular susceptibility to influenza virus infection [33]. Collectively, these results suggested that zebrafish ZDHHC11 is essential for host cells to defend against SVCV infection.

Zebrafish are a widely used model for studying the function of antiviral genes. To further validate the antiviral role of ZDHHC11 in vivo, we performed morpholino-mediated gene knockdown experiments in zebrafish embryos. This method allows for sustained gene silencing for up to 5 days [34]. Knockdown of ZDHHC11 led to a decreased survival rate and reduced expression of antiviral genes, while concomitantly increasing SVCV replication in zebrafish larvae (Fig. 8). Conversely, overexpression of ZDHHC11 in zebrafish embryos enhanced antiviral capacity (Fig. 7). These in vivo findings are consistent with our in vitro results.

In summary, our study demonstrates that zebrafish ZDHHC11 is upregulated by stimulation with GCRV, SVCV, B-DNA, and Z-DNA. Subsequently, ZDHHC11 promotes STING palmitoylation, activates the STING-IRF3-dependent IFN-I response, and ultimately enhances the host's antiviral innate immunity (Fig. 9). These findings represent the first elucidation of this mechanism, revealing a novel regulatory pathway for STING palmitoylation and identifying a potential target for developing antiviral therapies in fish. Based on current data, we plan to further explore the role of palmitoylation modification in other adaptor in antiviral signaling pathway.

Fig. 9.

A model of the regulatory mechanism by which zebrafish ZDHHC11 catalyzes STING palmitoylation to activate antiviral innate immunity. During viral infection, zebrafish ZDHHC11 is activated by viral RNA or DNA, subsequently interacting with STING at the endoplasmic reticulum (ER). This interaction facilitates the recruitment of TBK1, which phosphorylates IRF3, ultimately triggering the expression of IFN-I. The zebrafish ZDHHC11, a palmitoyltransferase, enhances STING palmitoylation through its DHHC domain

Materials and methods

Zebrafish and cell lines

Zebrafish of the AB strain, originally obtained from the China Zebrafish Resource Center (CZRC), were maintained in a recirculating aquaculture system (Esen, China) under controlled conditions. These conditions included a constant temperature of 28 °C ± 0.5 °C, a 14-h light/10-h dark photoperiod, and feeding twice daily with newly hatched brine shrimp. Optimal water quality was ensured by maintaining conductivity between 500 and 550 µs/cm and pH levels between 7.0 and 7.4. All experimental procedures involving zebrafish were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals approved by the Institutional Animal Care and Use Committee (IACUC) at Nanchang University (Issue No. NCUAUU-2020–563).

Ctenopharyngodon idella kidney (CIK) cells were cultured at 28 °C in Medium 199, supplemented with 10% fetal calf serum (FCS). Zebrafish caudal fin (ZFIN) cells and Epithelioma papulosum cyprini (EPC) cells were kindly provided by Professor Jun Zou (Shanghai Ocean University) and Professor Yibing Zhang (Institute of Hydrobiology, Chinese Academy of Sciences), respectively. ZFIN cells were grown at 28 °C in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FCS under a 5% CO₂ atmosphere. EPC cells were maintained at 28 °C in Medium 199 supplemented with 10% FCS and 5% CO₂, with the 5% CO₂ atmosphere.

Virus and viral analogues

Grass carp reovirus (GCRV) strain 873 was propagated in CIK cells, while Spring Viremia of Carp Virus (SVCV) was propagated in EPC cells until cytopathic effects (CPE) were evident. After repeated freeze–thaw cycles, virus-containing solutions were collected. Viral titers were determined using the 50% tissue culture infective dose (TCID₅₀) assay. For the infection of adult zebrafish, 100 µl of GCRV or SVCV (titer of 10^–7 TCID₅₀) was injected per fish. For cell infection, 10 µl of GCRV or SVCV (10^–7 TCID₅₀) was added to each well of a 6-well plate. For zebrafish larvae infection, 1 ml of GCRV 873 (10^–7 TCID₅₀) was added to 10 ml of culture medium.

Viral nucleotide analogs, B-DNA and Z-DNA, were synthesized by Sangon Biotech (Shanghai, China), with their sequences detailed in Table S2. For in vivo experiments, 100 µl of B-DNA or Z-DNA (10 µM) was injected into zebrafish. For in vitro experiments, 10 µM of B-DNA or Z-DNA was transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen, USA). Viral infection experiments were conducted at 6, 12, 24, 48 and 72 h post-infection (hpi). A control group (0 h) received an equivalent volume of PBS.

Plasmids construction and transfection

The full-length open reading frames (ORFs) of zebrafish ZDHHC11 and STING were separately cloned into pCMV-Flag, pEGFP-C1, and pcDNA3.1 vectors. Transmembrane domain mutants (TM0, TM1, TM2, TM3, TM4) of ZDHHC11 and STING, along with a ΔDHHC domain construct of ZDHHC11, were each constructed into the pEGFP-C1 vector. The pGL3-IFN-I promoter reporter plasmid was prepared in our previous study [35]. Additionally, the pISRE-TA-luc and pRL-TK plasmids were purchased from Beyotime (China). The primers used for the construction of these plasmids are listed in Table S2.

Cells were transfected with 1 µg of B-DNA or Z-DNA oligonucleotides using 2 µl of Lipofectamine 2000 transfection reagent (Invitrogen, USA). In addition, cells were transfected with recombinant plasmids employing the Lipo8000™ transfection reagent (Beyotime, China) at a DNA (µg) to reagent (µl) ratio of 1:1.6. Finally, cells were transfected with 1 µg of specific small interfering RNA (siRNA) using 3 µl of Hiperfect transfection reagent (Qiagen, Germany). All transfection protocols adhered to the manufacturers' instructions.

Quantitative Real-time PCR (qRT-PCR) analysis

Total RNA was extracted from tissues or cells using a Total RNA Extraction Kit (Tiangen, China). cDNA synthesis was performed using a Quantscript RT Kit (TaKaRa, Japan). qRT-PCR was conducted to quantify the mRNA levels of ZDHHC11, IFN-I, Mxa, Mxb and Mxc, utilizing TB Green PCR Master Mix (TaKaRa, Japan). The detailed qRT-PCR reaction setup has been described in our previous study [36]. Relative gene expression levels were calculated using the 2^−ΔΔCT method. β-actin served as the reference gene for qRT-PCR analysis. The primers used for qRT-PCR are listed in Table S2.

Dual-luciferase reporter system

ZFIN cells were seeded in 6-well plates and co-transfected with the following plasmids: 0.25 µg of pGL3-IFN-I-promoter plasmid (or pISRE-TA-luc or pNF-κB-TA-luc plasmid), 1 µg of the ZDHHC11-pcDNA3.1 plasmid, and 0.025 µg of the pRL-TK Renilla luciferase control plasmid. Transfection was performed using Lipo8000™ reagent. At 24 h post-transfection, cells in each well were lysed with 100 µl of 1 × passive lysis buffer (Promega, USA). Promoter activities of the reporter plasmids were measured using the Dual-luciferase® Reporter Assay System (Promega, USA) on a Luminoskan Ascent luminometer (Thermo, USA).

Knockdown of ZDHHC11 in cells and zebrafish embryos

Small interfering RNAs (siRNAs) specifically targeting ZDHHC11 and a negative control (N.C) were synthesized by GenePharma (China). ZFIN cells were transfected with these siRNAs or N.C at a concentration of 1 µg per well using Hiperfect transfection reagent. After 24 h post-transfection, total RNA and protein were extracted separately from the cells. qRT-PCR and Western blot were performed to examine the mRNA and protein levels of ZDHHC11, respectively. The specific siRNA and N.C sequences used in this study are provided in Table S2.

Morpholino (Mo)-mediated knockdown of ZDHHC11 was performed in zebrafish embryos. Morpholino oligonucleotides targeting the ATG of ZDHHC11 (Mo-ZDHHC11) and a standard control morpholino (Mo-STD) were designed and synthesized by Gene Tools LLC (USA). Either 4 ng of Mo-ZDHHC11 or 8 ng of Mo-STD was injected into one-cell stage embryos. At least 100 embryos were injected per group. Knockdown efficiency was verified by detecting the protein level of ZDHHC11 in embryos at 48 h post-fertilization (hpf). Sequences of Mo-ZDHHC11 and Mo-STD are provided in Table S2.

Antibodies and Western blot assay

Mouse anti-Flag and mouse anti-GFP antibodies were purchased from Sigma (USA) and Abmart (China), respectively. Rabbit polyclonal anti-IFN-I antisera was prepared in our laboratory [37]. Rabbit anti-ZDHHC11, anti-IRF3, anti-phospho-IRF3, anti-TBK1, and anti-phospho-TBK1 antibodies were obtained from Beyotime (China). Rabbit anti-IRF7 antibody was purchased from HUA Bio (China). Alexa Fluor 594-labeled goat anti-mouse antibody, goat anti-rabbit HRP antibody, and goat anti-mouse HRP antibody were purchased from ZSGB-Bio (China). The product codes of all commercial antibodies are listed in Table S3.

Cells were lysed in NP-40 lysis buffer (supplemented with protease inhibitors) on ice for 30 min to extract total protein. After centrifugation, the supernatant was collected and denatured by boiling in SDS-loading buffer for 10 min. The protein samples were then separated by 10% SDS-PAGE and transferred to nitrocellulose (NC) membranes. Membranes were washed and blocked before being incubated with the indicated primary antibodies overnight. Following washing, the membranes were incubated with anti-mouse or anti-rabbit HRP-conjugated secondary antibodies. Protein bands were visualized using a luminescent analyzer (CLINX, China). The intensity of each band was quantified using Image J software.

Nuclear and cytoplasmic protein extraction

ZFIN cells were separately transfected with 2 μg each of pcDNA3.1-basic, ZDHHC11-pcDNA3.1, STING-pcDNA3.1. At 24 h post-transfection, these cells were stimulated with B-DNA for an additional 12 h, then washed with PBS and collected in 1.5 ml microcentrifuge tubes. Nucleus-Mass Extraction Kit (Beyotime, China) was used to fractionate nuclear and cytoplasmic proteins from ZFIN cells. Cytoplasmic protein extraction lysis buffer was added, and the samples were vortexed vigorously at the highest speed for 15 s. The cytoplasmic protein fraction was isolated after centrifugation. Nuclear protein extraction was then performed by adding lysis buffer, followed by vortexing the samples for 30 s. The samples were subsequently placed in an ice bath for 2 min, vortexed again for 30 s, and this cycle was repeated for a total duration of 30 min. The nuclear protein fraction was collected after centrifugation. The concentrations of the protein were determined using the Enhanced BCA Protein Assay Kit (Beyotime, China).

Subcellular localization and co-localization analysis

ZFIN cells were seeded in confocal dishes and then transfected with the indicated GFP-tagged plasmids (2 μg). After 24 h of transfection, the cells were analyzed for subcellular localization. Subsequently, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, stained with DAPI for 20 min, and finally imaged using a laser confocal microscope (Leica, Germany).

ZFIN cells were seeded in confocal dishes and divided into two groups for distinct experimental procedures. For the first group, cells were co-transfected with STING-GFP (1 μg) and ZDHHC11-Flag (1 μg) plasmids. After 24 h of transfection, the cells were washed, fixed, permeabilized and blocked. Subsequently, they were incubated with a mouse anti-Flag antibody overnight at 4 °C. Following this, the cells were washed again and incubated with an Alexa Fluor 594-labeled goat anti-mouse antibody for 2 h at room temperature. For the second group, cells were co-transfected with pDsRed2-ER (1 μg) and either ZDHHC11-GFP (1 μg) or STING-GFP (1 μg). After 24 h of transfection, these cells were washed and fixed. Finally, for both groups, the cells were stained with DAPI and imaged using a laser confocal microscope (Leica, Germany).

Co-immunoprecipitation (co-IP) and bimolecular fluorescence complementation (BIFC) assays

For co-IP assay, ZFIN cells were seeded in 10-cm diameter dishes and co-transfected with a total of 5 μg of Flag- or GFP-recombinant plasmids. After 24 h, the cells were stimulated with SVCV for 12 h. Subsequently, the cells were lysed with NP40 lysis buffer (containing a 10% protease inhibitor cocktail) on ice for 30 min. The cell lysate was collected after centrifugation, and its protein concentration was determined using an Enhanced BCA Protein Assay Kit (Beyotime, China). The clarified supernatant was then incubated with agarose beads conjugated to GFP, Flag, or IgG antibodies (AlpaLife, China) overnight at 4 °C. The beads were washed with co-IP buffer and denatured in 2 × SDS loading buffer at 95 °C for 10 min. Finally, protein interactions were analyzed by Western blot.

For BIFC assay, ZFIN cells were seeded in confocal dishes and co-transfected with ZDHHC11-VC155 and STING-VN155 plasmids. Negative controls included cells transfected with bFos-VC155(deltazip) and bJun-VN155 (152L), STING-VN155, or ZDHHC11-VC155. The positive control involved transfecting cells with bFOS-VC155 and bJun-VN155. Cells were imaged at 24 h post-transfection using a fluorescence microscope (Nikon, Japan) at a 488 nm excitation wavelength.

Native PAGE electrophoresis

ZFIN cells were seeded in 6-well plates and transfected with the indicated plasmids. Twenty-four hours post-transfection, cells were stimulated with SVCV for 12 h. Cells were then harvested by sonication. The resulting lysates were diluted in 5 × loading buffer (without SDS) and subjected to electrophoresis on a 6% acrylamide gel (without SDS). The gel was run in Tris–glycine buffer (25 mM Tris–HCl, pH 8.4, 192 mM glycine, 0.5% deoxycholate) for 1 h at 35 mA on ice prior to immunoblotting.

Palmitoylation assay

ZFIN cells were seeded in 10-cm diameter dishes and transfected with STING-Flag (3 μg). 2-bromohexadecanoic acid (2-bp), a palmitoylation inhibitor, served as a negative control. At 48 h post-transfection, cells were washed with PBS and lysed in ABE lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 1% NP40, 10% glycerol, 50 mM NEM) for 1.5 h. The lysate was then incubated with Flag antibody-conjugated agarose beads for 3 h at 4 °C. Next, the beads were sequentially incubated with 1 M hydroxylamine and 5 µM Biotin-BMCC. After washing with ABE lysis buffer, beads were denatured in 2 × SDS loading buffer by heating at 95 °C for 10 min. Finally, samples were analyzed by Western blot, using Streptavidin-HRP and anti-Flag antibodies (Beyotime, China).

Crystal violet staining and cell viability analysis

For crystal violet staining experiments, ZFIN cells were separately seeded in 24-well plates. ZDHHC11 expression was manipulated via overexpression or knockdown. At 24 h post-transfection, cells were infected with SVCV. At 48 h post-infection, cells in 24-well plates were washed with PBS, fixed with 4% paraformaldehyde, and stained with 1% crystal violet solution (Sangon, China). Cytopathic effects (CPEs) were visualized and imaged. Simultaneously, cells in 24-well plates were treated with 350 μl of CCK8 reagent (Yeasen Biotechnology, China). After 1 h of incubation at 37 °C, absorbance at 450 nm was measured for each well using a microplate reader (MD, USA).

In vitro transcription and microinjection

The open reading frame (ORF) of ZDHHC11 was amplified and cloned into the pGEM®-T Easy plasmid. The recombinant plasmid was transcribed into mRNA in vitro using the Ambion™ MAXIscript™ T7 Transcription kit (Thermo Fisher Scientific, USA). One-cell stage zebrafish embryos were microinjected with 8 ng of the mRNA. A scrambled nucleotide sequence was used as a control. At least 100 embryos were injected per group. Expression efficiency was confirmed by qRT-PCR and Western blot analysis.

Statistical analysis

Data analysis and statistical significance were determined using GraphPad Prism 8.0 or IBM SPSS Statistics 27 software. Comparisons between two treatment groups were performed using Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001). For data that did not meet the assumptions of normality, the Kruskal–Wallis test was employed. Significant differences were marked with different letters (p < 0.05), and the same letter indicates no significant difference. Image J software was used to quantify Western blot band intensity (gray values). All data are presented as mean ± standard deviation (SD). The data represent the results of three independent experiments, each performed with three technical replicates.

Authors’ contributions

Tingting Yu: Conduct individual experiments and palmitoylation experiments, Process results. Meifeng Li: Investigation, Data curation. Hongying Zhang: Construction of plasmids. Jihuan Hu: Carry out some experiments. Quanling Zhang: Data curation. Miaomiao Li: Formal analysis. Xuefeng Liu: Formal analysis. Zhixin Fu: Data curation. Chengyu Hu: Writing – review & editing, Supervision, Funding acquisition, Formal analysis, Conceptualization. Xiaowen Xu: Writing – review & editing, Original draft, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Funding

This work was supported by Natural Science Foundation of JiangXi province (20242BAB20316 and 20243BCE51118) and National Natural Science Foundation of China (32160872).

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

No. NCUAUU-2020–563.

Consent for publication

All authors read and approved the final manuscript.

Competing interests

The authors declare no competing interests.