CD36, a vital fatty acid translocase, has been reported to participate in multiple physiological functions through palmitoylation mediated by zinc finger Asp-His-His-Cys-type palmitoyltransferases (DHHCs). This study aimed to investigate the possible involvement of DHHC-mediated CD36 palmitoylation in high-fat diet (HFD)-induced impairment of pubertal mammary gland development and explore the underlying mechanisms involved. Palmitic acid (PA)-treated HC11 cells were used as the in vitro high-fat model, and the cell proliferation was examined by 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay. The palmitoylation of CD36 was determined by the acyl-biotin exchange (ABE) method. The expression of CD36, proliferative genes, and signaling molecules was detected by immunoblotting. The cellular localization of CD36 was determined by immunofluorescence. The bindings of CD36 with zinc finger DHHC-type palmitoyltransferases 9 (DHHC9) or Fyn/Lyn were detected by co-immunoprecipitation (Co-IP). The palmitoylation inhibitor 2-bromopalmitate (2BP), DHHC9 knockdown, and point mutation of CD36 cysteine residues were applied to construct a CD36 palmitoylation deficiency model in vitro to investigate the effects of CD36 palmitoylation on HC11 proliferation. In vivo, the pubertal mice were treated with HFD and/or 2BP. Mammary gland morphology was determined by whole mount staining, and the underlying mechanisms were verified by the methods used in the in vitro system. In vitro, the palmitoylation inhibitor 2BP eliminated PA-inhibited HC11 proliferation and inhibited CD36 palmitoylation and localization on the plasma membrane. Meanwhile, the binding of DHHC9 and CD36 in PA-treated HC11 cells was repressed by 2BP. In addition, both knockdown of DHHC9 and point mutation of CD36 cysteine residues suppressed the membrane palmitoylation and localization of CD36 and stimulated the proliferation of PA-treated HC11 cells. Furthermore, in PA-treated HC11 cells, the inhibition of CD36 palmitoylation, the knockdown of DHHC9, and the mutation of CD36 cysteine residues resulted in decreased formation of the CD36/Fyn/Lyn complex. Correspondingly, the downstream c-jun n-terminal kinase 1 (JNK1) pathway was inhibited, and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway was activated. Moreover, inhibition of the JNK pathway with SP600125 promoted the proliferation of PA-treated HC11 cells via activation of the ERK1/2 pathway. In vivo, the palmitoylation inhibitor 2BP ameliorated HFD-induced impairment of mammary gland development in pubertal female mice, which was associated with a decrease in DHHC9-mediated CD36 palmitoylation in the plasma membrane, a reduction in the CD36/Fyn/Lyn complex, inhibition of the JNK1 pathway, and activation of the ERK1/2 pathway. This study revealed that inhibition of DHHC9-mediated CD36 palmitoylation mitigated HFD-induced impairment of pubertal mammary gland development via the JNK1-ERK1/2 pathway.

Palmitoylation, a reversible post-translational modification, regulates a wide range of cellular processes by modulating protein localization, stability, and interactions. The zinc finger Asp-His-His-Cys (zDHHC) family of palmitoyltransferases is central to this process, yet their functions in immune cell death of lower vertebrates, remain poorly understood. Here, we investigate the role of zDHHC8 in modulating immune responses and necroptosis during infection in large yellow croaker (LczDHHC8). We demonstrate that LczDHHC8 is significantly upregulated in monocyte/macrophages (MO/MΦ) during Pseudomonas plecoglossicida infection and plays a key role in promoting pro-inflammatory cytokine production and M1 macrophage polarization. Through acyl-biotin exchange (ABE) assays and liquid chromatography-mass spectrometry (LC-MS/MS), we show that LczDHHC8 suppresses the palmitoylation of necrosome components, including receptor-interacting protein kinases 1 (RIPK1), receptor-interacting protein kinases 3 (RIPK3), and mixed lineage kinase domain-like protein (MLKL), thereby influencing necroptosis of MO/MΦ. LczDHHC8 deficiency exacerbates necroptosis, evidenced by increased phosphorylation of necroptosis-related proteins and enhanced cell death. Our findings suggest that LczDHHC8 may negatively regulates necroptosis through facilitating the palmitoylation of necrosome components, thereby influencing MO/MΦ function by modulating immune responses and cell survival during infection. This study provides novel insights into the immune regulatory roles of palmitoylation in teleosts and emphasizes the importance of LczDHHC8 in the modulation of necroptosis, which may have broader implications for understanding host-pathogen interactions and inflammatory responses in vertebrates.

Monocyte-to-macrophage differentiation and subsequent foam cell formation are key processes that contribute to plaque build-up during the progression of atherosclerotic lesions. Palmitoylation enzymes are known to play pivotal roles in the development and progression of inflammatory diseases. However, their specific impact on atherosclerosis development remains unclear. In this study, we discovered that the knockout of zDHHC1 in THP-1 cells, as well as Zdhhc1 in mice, markedly reduces the uptake of oxidized low-density lipoprotein (ox-LDL) by macrophages, thereby inhibiting foam cell formation. Moreover, the absence of Zdhhc1 in ApoE-/- mice significantly suppresses atherosclerotic plaque formation. Mass spectrometry coupled with bioinformatic analysis revealed an enrichment of the PI3K-Akt-mTOR signaling pathway. Consistent with this, we observed that knockout of zDHHC1 significantly decreases the palmitoylation levels of p110α, a crucial subunit of PI3K. Notably, the deletion of Zdhhc1 facilitates the nuclear translocation of p110α in macrophages, leading to a significant reduction in the downstream phosphorylation of Akt at Ser473 and mTOR at Ser2448. This cascade results in a decreased number of macrophages within plaques and ultimately mitigates the severity of atherosclerosis. These findings unveil a novel role for zDHHC1 in regulating foam cell formation and the progression of atherosclerosis, suggesting it as a promising target for clinical intervention in atherosclerosis therapy.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Cleavage-deficient GSDMD (D275A) is also palmitoylated after inflammasome stimulation or treatment with ROS activators and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. ZDHHC5 and ZDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated at their N termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that functions as a general switch for the activation of this pore-forming family.

Autophagy is a lysosome-dependent degradation pathway essential for cellular homeostasis, which decreases with age. However, it is unclear how aging induces autophagy decline. Here we show the role of protein S-palmitoylation in autophagy. We identify the palmitoyl acyltransferase DHHC5 as a regulator of autophagy by mediating the palmitoylation of beclin 1, which in turn promotes the formation of ATG14L-containing class III phosphatidylinositol-3-kinase complex I and its lipid kinase activity by promoting the hydrophobic interactions between beclin 1 and adapter proteins ATG14L and VPS15. In aging brains of human and nonhuman primate, the levels of DHHC5 exhibit a marked decrease in expression. We show that DHHC5 deficiency in neurons leads to reduced cellular protein homeostasis in two established murine models of Alzheimer's disease, which exaggerates neurodegeneration in an autophagy-dependent manner. These findings identify reduction of DHHC5-mediated beclin 1 S-palmitoylation as an underlying mechanism by which aging induces autophagy decline.