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.

Autism spectrum disorder (ASD) is a developmental disorder of the nervous system characterized by a deficiency in interpersonal communication skills, a pathologic tendency for repetitive behaviors, and highly restrictive interests. The spectrum is a gradient-based construct used to categorize the widely varying degrees of ASD phenotypes, and has been linked to a genetic etiology in 25% of cases. Prior studies have revealed that 30% of ASD patients exhibit hyperserotonemia, or severely elevated whole blood serotonin (5HT), implicating the serotonergic system in the pathogenesis of ASD. Likewise, escitalopram, a selective-serotonin reuptake inhibitor (SSRI), has been demonstrated to effectively improve core ASD symptoms potentially by modulating abnormal brain activation in ASD patients. Molecular studies have uncovered proband patients with rare mutations in the serotonin transporter (SERT) that manifest enhanced surface expression and 5HT transport capacity, suggesting that abnormal enhancement of SERT function may be involved in the pathogenesis of ASD. Here, we reveal that palmitoylation is enhanced in the ASD SERT F465L and L550V coding variants, and confirm prior reports of enhanced kinetic activity and surface expression of F465L. Furthermore, treatment of F465L with the irreversible palmitoyl acyl-transferase inhibitor, 2-bromopalmitate (2BP), or escitalopram, rectified enhanced F465L palmitoylation, surface expression, and transport capacity to basal WT levels. Overall, our results implicate disordered SERT palmitoylation in the pathogenic mechanism of ASD, with basal recovery of these processes following escitalopram treatment providing insight into its molecular utility as an ASD therapeutic.

Nicotinamide adenine dinucleotide phosphate (NADPH) is a vital electron donor essential for macromolecular biosynthesis and protection against oxidative stress. Although NADPH is compartmentalized within the cytosol and mitochondria, the specific functions of mitochondrial NADPH remain largely unexplored. Here we demonstrate that NAD+ kinase 2 (NADK2), the principal enzyme responsible for mitochondrial NADPH production, is critical for maintaining protein lipoylation, a conserved lipid modification necessary for the optimal activity of multiple mitochondrial enzyme complexes, including the pyruvate dehydrogenase complex. The mitochondrial fatty acid synthesis (mtFAS) pathway utilizes NADPH for generating protein-bound acyl groups, including lipoic acid. By developing a mass-spectrometry-based method to assess mammalian mtFAS, we reveal that NADK2 is crucial for mtFAS activity. NADK2 deficiency impairs mtFAS-associated processes, leading to reduced cellular respiration and mitochondrial translation. Our findings support a model in which mitochondrial NADPH fuels the mtFAS pathway, thereby sustaining protein lipoylation and mitochondrial oxidative metabolism.

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.