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The cross‐regulation of immunity and metabolism is currently a research hotspot in life sciences and immunology. Metabolic immunology plays an important role in cutting‐edge fields such as metabolic regulatory mechanisms in immune cell development and function, and metabolic targets and immune‐related disease pathways. Protein post‐translational modification (PTM) is a key epigenetic mechanism that regulates various biological processes and highlights metabolite functions. Currently, more than 400 PTM types have been identified to affect the functions of several proteins. Among these, metabolic PTMs, particularly various newly identified histone or non‐histone acylation modifications, can effectively regulate various functions, processes and diseases of the immune system, as well as immune‐related diseases. Thus, drugs aimed at targeted acylation modification can have substantial therapeutic potential in regulating immunity, indicating a new direction for further clinical translational research. This review summarises the characteristics and functions of seven novel lysine acylation modifications, including succinylation, S‐palmitoylation, lactylation, crotonylation, 2‐hydroxyisobutyrylation, β‐hydroxybutyrylation and malonylation, and their association with immunity, thereby providing valuable references for the diagnosis and treatment of immune disorders associated with new acylation modifications. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is a fundamental cellular catabolic process that delivers cytoplasmic components into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their contents are degraded. Autophagy recycles cytoplasmic components, including misfolded proteins, dysfunctional organelles and even microbial invaders, thereby playing an essential role in development, immunity and cell death. Autophagosome formation is the main step in autophagy, which is governed by a set of ATG (autophagy related) proteins. ATG16L1 interacts with ATG12–ATG5 conjugate to form an ATG12–ATG5-ATG16L1 complex. The complex acts as a ubiquitin-like E3 ligase that catalyzes the lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), which is crucial for autophagosome formation. In the present study, we found that ATG16L1 was subject to S-palmitoylation on cysteine 153, which was catalyzed by ZDHHC7 (zinc finger DHHC-type palmitoyltransferase 7). We observed that re-expressing ATG16L1 but not the S-palmitoylation-deficient mutant ATG16L1C153S rescued a defect in the lipidation of LC3 and the formation of autophagosomes in ATG16L1-KO (knockout) HeLa cells. Furthermore, increasing ATG16L1 S-palmitoylation by ZDHHC7 expression promoted the production of LC3-II, whereas reducing ATG16L1 S-palmitoylation by ZDHHC7 deletion inhibited the LC3 lipidation process and autophagosome formation. Mechanistically, the addition of a hydrophobic 16-carbon palmitoyl group on Cys153 residue of ATG16L1 enhances the formation of ATG16L1-WIPI2B complex and ATG16L1-RAB33B complex on phagophore, thereby facilitating the LC3 lipidation process and autophagosome formation. In conclusion, S-palmitoylation of ATG16L1 is essential for the lipidation process of LC3 and the formation of autophagosomes. Our research uncovers a new regulatory mechanism of ATG16L1 function in autophagy.Abbreviation: ABE: acyl-biotin exchange; ATG: autophagy related; Baf-A1: bafilomycin A1; 2-BP: 2-bromopalmitate; CCD: coiled‐coil domain; co-IP: co-immunoprecipitation; CQ: chloroquine; EBSS: Earle’s balanced salt solution; HAM: hydroxylamine; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NP-40: Nonidet P-40; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3K-C1: class III phosphatidylinositol 3-kinase complex I; PTM: post-translational modification; RAB33B: RAB33B, member RAS oncogene family; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscope; WD: tryptophan and aspartic acid; WIPI2B: WD repeat domain, phosphoinositide interacting 2B; WT: wild-type; ZDHHC: zinc finger DHHC-type. [ABSTRACT FROM AUTHOR]

Gasdermin D (GSDMD)-mediated pyroptotic cell death drives inflammatory cytokine release and downstream immune responses upon inflammasome activation, which play important roles in host defense and inflammatory disorders. Upon activation by proteases, the GSDMD N-terminal domain (NTD) undergoes oligomerization and membrane translocation in the presence of lipids to assemble pores. Despite intensive studies, the molecular events underlying the transition of GSDMD from an autoinhibited soluble form to an oligomeric pore form inserted into the membrane remain incompletely understood. Previous work characterized S-palmitoylation for gasdermins from bacteria, fungi, invertebrates, as well as mammalian gasdermin E (GSDME). Here, we report that a conserved residue Cys191 in human GSDMD was S-palmitoylated, which promoted GSDMD-mediated pyroptosis and cytokine release. Mutation of Cys191 or treatment with palmitoyltransferase inhibitors cyano-myracrylamide (CMA) or 2-bromopalmitate (2BP) suppressed GSDMD palmitoylation, its localization to the membrane and dampened pyroptosis or IL-1β secretion. Furthermore, Gsdmd-dependent inflammatory responses were alleviated by inhibition of palmitoylation in vivo. By contrast, coexpression of GSDMD with palmitoyltransferases enhanced pyroptotic cell death, while introduction of exogenous palmitoylation sequences fully restored pyroptotic activities to the C191A mutant, suggesting that palmitoylation-mediated membrane localization may be distinct from other molecular events such as GSDMD conformational change during pore assembly. Collectively, our study suggests that S-palmitoylation may be a shared regulatory mechanism for GSDMD and other gasdermins, which points to potential avenues for therapeutically targeting S-palmitoylation of gasdermins in inflammatory disorders. [ABSTRACT FROM AUTHOR]

Purpose: Kidney clear cell carcinoma (KIRC) has a poor prognosis, high morbidity and mortality rates, and high invasion and metastasis rate, and effective therapeutic targets are lacking. zDHHC3 has been implicated in various cancers, but its specific role in KIRC remains unclear. Methods: In this study, we performed a pan-cancer analysis, bioinformatics analysis, and cell experiment to detect the role of zDHHC3 in KIRC. Results: zDHHC3 was significantly down-regulated in KIRC, and that its high expression was associated with favorable patient outcomes. We identified 202 hub genes that were most relevant to high zDHHC3 expression and KIRC, and found that they were involved mainly in ion transport and renal cell carcinoma. Among these hub genes, SLC9A2 was identified as a downstream gene of zDHHC3. zDHHC3 suppression led to decreased expression and S-palmitoylation of SLC9A2, which further inhibited the apoptosis of Caki-2 cells. Conclusion: Our findings suggest that zDHHC3 plays an important role in KIRC, due partly to its regulation of SLC9A2 S-palmitoylation. The targeting of the zDHHC3–SLC9A2 axis may provide a new option for the clinical treatment of KIRC. [ABSTRACT FROM AUTHOR]

S-palmitoylation is a reversible posttranslational modification, and the palmitoylation reaction in human-derived cells is mediated by the zDHHC family, which is composed of S-acyltransferase enzymes that possess the DHHC (Asp-His-His-Cys) structural domain. zDHHC proteins form an autoacylation intermediate, which then attaches the fatty acid to cysteine a residue in the target protein. zDHHC proteins sublocalize in different neuronal structures and exert dif-ferential effects on neurons. In humans, many zDHHC proteins are closely related to human neu-rological disor-ders. This review focuses on a variety of neurological disorders, such as AD (Alz-heimer's disease), HD (Huntington's disease), SCZ (schizophrenia), XLID (X-linked intellectual disability), attention deficit hyperactivity disorder and glioma. In this paper, we will discuss and summarize the research progress regarding the role of zDHHC proteins in these neu-rological disorders. [ABSTRACT FROM AUTHOR]

Despite its prevalence and the severity of symptoms, little is known about the pathogenesis and etiology of adenomyosis. In our previous study, Scribble localization has been found to be partially translocated to cytoplasm; however, its regulatory mechanism is known. In consideration of the important role of supraphysiologic estrogen production in the endometrium in the development of adenomyosis, we analyzed the effect and mechanism of estrogen on Scribble localization in vivo and in vitro. Firstly, we found Scribble translocation from the basolateral membrane to the cytoplasm was easily to be seen in women and mice with adenomyosis (68% vs 27%, 60% vs 10% separately). After treatment with the S-palmitoylation inhibitor 2-bromopalmitate for 48H, cytoplasmic enrichment of Scribble and the reduced level of palm-Scribble was observed by immunofluorescence, Western blot, and acyl-biotin exchange palmitoylation assay. High estrogen exposure could not only induce partially cytoplasmic translocation of Scribble but also decrease the expression level of palm-Scribble, which can be recovered by estrogen receptor inhibitor ICI182,780. Based on following experiments, we found that estrogen regulated Scribble localization by APT through S-palmitoylation of Scribble protein. At last, IHC was performed to verify the expression of APT1 and APT2 in human clinical tissue specimens and found that they were all increased dramatically. Furthermore, positive correlations were found between APT1 or APT2 and aromatase P450. Therefore, our research may provide a new understanding of the pathogenesis of adenomyosis. [ABSTRACT FROM AUTHOR]

S‐palmitoylation is a reversible posttranslational lipid modification that targets cysteine residues of proteins and plays critical roles in regulating the biological processes of substrate proteins. The innate immune system serves as the first line of defense against pathogenic invaders and participates in the maintenance of tissue homeostasis. Emerging studies have uncovered the functions of S‐palmitoylation in modulating innate immune responses. In this review, we focus on the reversible palmitoylation of innate immune signaling proteins, with particular emphasis on its roles in the regulation of protein localization, protein stability, and protein–protein interactions. We also highlight the potential and challenge of developing therapies that target S‐palmitoylation or de‐palmitoylation for various diseases. [ABSTRACT FROM AUTHOR]

Protein posttranslational modification regulates synaptic protein stability, sorting and trafficking, and is involved in emotional disorders. Yet the molecular mechanisms regulating emotional disorders remain unelucidated. Here we report unknown roles of protein palmitoylation/nitrosylation crosstalk in regulating anxiety-like behaviors in rats. According to the percentages of open arm duration in the elevated plus maze test, the rats were divided into high-, intermediate- and low-anxiety groups. The palmitoylation and nitrosylation levels were detected by acyl-biotin exchange assay, and we found low palmitoylation and high nitrosylation levels in the basolateral amygdala (BLA) of high-anxiety rats. Furthermore, we observed that 2-bromopalmitate (2-BP), a palmitoylation inhibitor, induced anxiety-like behaviors, accompanied with decreased amplitude and frequency of mEPSCs and mIPSCs in the BLA. Additionally, we also found that inhibiting nNOS activity with 7-nitroindazole (7-NI) in the BLA caused anxiolytic effects and reduced the synaptic transmission. Interestingly, diazepam (DZP) rapidly elevated the protein palmitoylation level and attenuated the protein nitrosylation level in the BLA. Specifically, similar to DZP, the voluntary wheel running exerted DZP-like anxiolytic action, and induced high palmitoylation and low nitrosylation levels in the BLA. Lastly, blocking the protein palmitoylation with 2-BP induced an increase in protein nitrosylation level, and attenuating the nNOS activity by 7-NI elevated the protein palmitoylation level. Collectively, these results show a critical role of protein palmitoylation/nitrosylation crosstalk in orchestrating anxiety behavior in rats, and it may serve as a potential target for anxiolytic intervention.

The immune checkpoint molecules programmed cell death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) are one of the most promising targets for tumor immunotherapy. PD-L1 is overexpressed on the surface of tumor cells and inhibits T cell activation upon binding to PD⁃1 on the surface of T cells, resulting in tumor immune escape. The therapeutic strategy of targeting PD-1/ PD-L1 involves blocking this binding and restoring the tumor-killing effect of immune cells. However, in clinical settings, a relatively low proportion of cancer patients have responded well to PD-1/PD-L1 blockade, and clinical outcomes have reached a bottleneck and no substantial progress has been made. In recent years, PD-L1 post-translation modifications (PTMs) have gradually become a hot topic in the field of PD-L1 research, which will provide new insights to improve the efficacy of current anti-PD-1/PD-L1 therapies. Here, we summarized and discussed multiple PTMs of PD-L1, including glycosylation, ubiquitination, phosphorylation, acetylation and palmitoylation, with a major emphasis on mechanism-based therapeutic strategies (including relevant enzymes and targets that are already in clinical use and that may become drugs in the future). We also summarized the latest research progress of PTMs of PD-L1/PD-1 in regulating immunotherapy. The review provided novel strategies and directions for tumor immunotherapy research based on the PTMs of PD-L1/PD-1. [ABSTRACT FROM AUTHOR]

The neurotransmitter serotonin (5-HT) is transported back into serotonergic neurons by the serotonin transporter (SERT). SERT is a main target of antidepressants, and much effort has therefore focused on finding relationships between SERT and depression. However, it is not fully understood how SERT is regulated at the cellular level. Here, we report post-translational regulation of SERT by S -palmitoylation, in which palmitate is covalently attached to cysteine residues of proteins. Using AD293 cells (a human embryonic kidney 293-derived cell line with improved cell adherence) transiently transfected with FLAG-tagged human SERT, we observed S -palmitoylation of immature SERT containing high-mannose type N -glycans or no N -glycan, which is presumed to be localized in the early secretory pathway, such as the endoplasmic reticulum. Mutational analysis by alanine substitutions shows that S -palmitoylation of immature SERT occurs at least at Cys-147 and Cys-155, juxtamembrane cysteine residues within the first intracellular loop. Furthermore, mutation of Cys-147 reduced cellular uptake of a fluorescent SERT substrate that mimics 5-HT without decreasing SERT on the cell surface. On the other hand, combined mutation of Cys-147 and Cys-155 inhibited SERT surface expression and reduced the uptake of the 5-HT mimic. Thus, S -palmitoylation of Cys-147 and Cys-155 is important for both the cell surface expression and 5-HT uptake capacity of SERT. Given the importance of S -palmitoylation in brain homeostasis, further investigation of SERT S -palmitoylation could provide new insights into the treatment of depression. • The serotonin transporter (SERT) is post-translationally S -palmitoylated. • Most S -palmitoylated SERTs are immature based on their N -glycosylation state. • Immature SERT is S -palmitoylated at Cys-147 and Cys-155. • S -Palmitoylation promotes cell surface expression of SERT and serotonin uptake. [ABSTRACT FROM AUTHOR]

Gasdermins are effectors of pyroptosis downstream of diverse signaling pathways. Emerging evidence suggests that a number of post-translational modifications regulate the function of gasdermins in pyroptosis, a highly inflammatory form of cell death, and lytic or non-lytic secretion of intracellular contents. These include processing by different caspases and other proteases that may activate or suppress pyroptosis, ubiquitination by a bacterial E3 ligase that suppresses pyroptosis as an immune evasion mechanism, modifications at Cys residues in mammalian or microbial gasdermins that promote or inhibit pyroptosis, and potential phosphorylation that represses pyroptosis. Such diverse regulatory mechanisms by host and microbial proteases, ubiquitin ligases, acyltransferases, kinases and phosphatases may underlie the divergent physiological and pathological functions of gasdermins, and furnish opportunities for therapeutic targeting of gasdermins in infectious diseases and inflammatory disorders. [ABSTRACT FROM AUTHOR]

Neuronal ankyrin-B is an intracellular scaffolding protein that plays multiple roles in the axon. By contrast, relatively little is known about the function of ankyrin-B in dendrites, where ankyrin-B is also localized in mature neurons. Recently, we showed that ankyrin-B acts as a scaffold for the voltage-gated sodium channel, NaV1.2, in dendrites of neocortical pyramidal neurons. How ankyrin-B is itself targeted to the dendritic membrane is not well understood. Here, we report that ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic localization of NaV1.2. We identify the palmitoyl acyl transferase zDHHC17 as a key mediator of ankyrin-B palmitoylation in heterologous cells and in neurons. Additionally, we find that zDHHC17 regulates ankyrin-B protein levels independently of its S-acylation function through a conserved binding mechanism between the ANK repeat domain of zDHHC17 and the zDHHC ankyrin-repeat binding motif of ankyrin-B. We subsequently identify five cysteines in the N-terminal ankyrin repeat domain of ankyrin-B that are necessary for ankyrin-B palmitoylation. Mutation of these five cysteines to alanines not only abolishes ankyrin-B palmitoylation, but also prevents ankyrin-B from scaffolding NaV1.2 at dendritic membranes of neurons due to ankyrin-B's inability to localize properly at dendrites. Thus, we show palmitoylation is critical for localization and function of ankyrin-B at dendrites. Strikingly, loss of ankyrin-B palmitoylation does not affect ankyrin-B-mediated axonal cargo transport of synaptic vesicle synaptotagmin-1 in neurons. This is the first demonstration of S-palmitoylation of ankyrin-B as an underlying mechanism required for ankyrin-B localization and function in scaffolding NaV1.2 at dendrites. [ABSTRACT FROM AUTHOR]

Protein lipidation is a common post-translational modification of proteins that plays an important role in human physiology and pathology. One form of protein lipidation, S-palmitoylation, involves the addition of a 16-carbon fatty acid (palmitate) onto proteins. This reversible modification may affect the regulation of protein trafficking and stability in membranes. From multiple recent experimental studies, a picture emerges whereby protein S-palmitoylation is a ubiquitous yet discrete molecular switch enabling the expansion of protein functions and subcellular localization in minutes to hours. Neural tissue is particularly rich in proteins that are regulated by S-palmitoylation. A surge of novel methods of detection of protein lipidation at high resolution allowed us to get better insights into the roles of protein palmitoylation in brain physiology and pathophysiology. In this review, we specifically discuss experimental work devoted to understanding the impact of protein palmitoylation on functional changes in the excitatory and inhibitory synapses associated with neuronal activity and neuronal plasticity. The accumulated evidence also implies a crucial role of S-palmitoylation in learning and memory, and brain disorders associated with impaired cognitive functions. [ABSTRACT FROM AUTHOR]

Interferon-induced transmembrane proteins (IFITM1, 2 and 3) are important host antiviral defense factors. They are active against viruses like the influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV). In this review, we focus on IFITM3 S-palmitoylation, a reversible lipid modification, and describe its role in modulating IFITM3 antiviral activity. Our laboratory discovered S-palmitoylation of IFITMs using chemical proteomics and demonstrated the importance of highly conserved fatty acid-modified Cys residues in IFITM3 antiviral activity. Further studies showed that site-specific S-palmitoylation at Cys72 is important for IFITM3 trafficking to restricted viruses (IAV and EBOV) and membrane–sterol interactions. Thus, site-specific lipid modification of IFITM3 directly regulates its antiviral activity, cellular trafficking, and membrane-lipid interactions.

Neuronal ankyrin-B is an intracellular scaffolding protein that plays multiple roles in the axon. By contrast, relatively little is known about the function of ankyrin-B in dendrites, where ankyrin-B is also localized in mature neurons. Recently, we showed that ankyrin-B acts as a scaffold for the voltage-gated sodium channel, NaV1.2, in dendrites of neocortical pyramidal neurons. How ankyrin-B is itself targeted to the dendritic membrane is not well understood. Here, we report that ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic localization of NaV1.2. We identify the palmitoyl acyl transferase zDHHC17 as a key mediator of ankyrin-B palmitoylation in heterologous cells and in neurons. Additionally, we find that zDHHC17 regulates ankyrin-B protein levels independently of its S-acylation function through a conserved binding mechanism between the ANK repeat domain of zDHHC17 and the zDHHC ankyrin-repeat binding motif of ankyrin-B. We subsequently identify five cysteines in the N-terminal ankyrin repeat domain of ankyrin-B that are necessary for ankyrin-B palmitoylation. Mutation of these five cysteines to alanines not only abolishes ankyrin-B palmitoylation, but also prevents ankyrin-B from scaffolding NaV1.2 at dendritic membranes of neurons due to ankyrin-B’s inability to localize properly at dendrites. Thus, we show palmitoylation is critical for localization and function of ankyrin-B at dendrites. Strikingly, loss of ankyrin-B palmitoylation does not affect ankyrin-B-mediated axonal cargo transport of synaptic vesicle synaptotagmin-1 in neurons. This is the first demonstration of S-palmitoylation of ankyrin-B as an underlying mechanism required for ankyrin-B localization and function in scaffolding NaV1.2 at dendrites.

S-acylation is a post-translational linkage of long chain fatty acids to cysteines, playing a key role in normal physiology and disease. In human cells, the reaction is catalyzed by a family of 23 membrane DHHC-acyltransferases (carrying an Asp-His-His-Cys catalytic motif) in two stages: (1) acyl-CoA-mediated autoacylation of the enzyme; and (2) further transfer of the acyl chain to a protein substrate. Despite the availability of a 3D-structure of human acyltransferase (hDHHC20), the molecular aspects of lipid selectivity of DHHC-acyltransferases remain unclear. In this paper, using molecular dynamics (MD) simulations, we studied membrane-bound hDHHC20 right before the acylation by C12-, C14-, C16-, C18-, and C20-CoA substrates. We found that: (1) regardless of the chain length, its terminal methyl group always reaches the "ceiling" of the enzyme's cavity; (2) only for C16, an optimal "reactivity" (assessed by a simple geometric criterion) permits the autoacylation; (3) in MD, some key interactions between an acyl-CoA and a protein differ from those in the reference crystal structure of the C16-CoA-hDHHS20 mutant complex (probably, because this structure corresponds to a non-native dimer). These features of specific recognition of full-size acyl-CoA substrates support our previous hypothesis of "geometric and physicochemical selectivity" derived for simplified acyl-CoA analogues. [ABSTRACT FROM AUTHOR]

ABSTRACT Interferon-inducible transmembrane (IFITM) proteins are small homologous proteins that are encoded by the interferon-stimulated genes (ISGs), which can be strongly induced by interferon (IFN) and provide resistance to invasion by a variety of viral pathogens. However, the exact molecular mechanisms underlying this function have remained elusive. The antiviral activity of IFITMs from different species depends on S-palmitoylation at conserved cysteine residues. However, specific enzymes involved in the dynamic palmitoylation cycle of IFITMs, especially depalmitoylase, have not yet been reported. Here, we demonstrate that α/-hydrolase domain-containing 16A (ABHD16A) is a depalmitoylase and a negative regulator of IFITM protein that can catalyze the depalmitoyl reaction of S-palmitoylated IFITM proteins, thereby decreasing their antiviral activities on RNA viruses. Using the acyl-PEGyl exchange gel shift (APEGS) assay, we identified ABHD16A proteins from humans, pigs, and mice that can directly participate in the palmitoylation/depalmitoylation cycles of IFITMs in the constructed abhd16a−/− cells and ABHD16A-overexpressing cells. Furthermore, we showed that ABHD16A functions as a regulator of subcellular localization of IFITM proteins and is related to the immune system. It is tempting to suggest that pharmacological intervention in IFITMs and ABHD16A can be achieved either through controlling their expression or regulating their activity, thereby providing a broad-spectrum therapeutic strategy for animal viral diseases. IMPORTANCE IFITM protein is the cells first line of antiviral defense that blocks early stages of viral replication; the underlying mechanism might be associated with the proper distribution in cells. The palmitoylation/depalmitoylation cycle can dynamically regulate protein localization, stability, and function. This work is the first one that found the critical enzyme that participates in the palmitoylation/depalmitoylation cycle of IFITM, and this type of palmitoyl loss may be an essential regulation mode for balancing the antiviral functions of the IFN pathway. These findings imply that the pharmacological intervention in IFITM and ABHD16A, either through controlling their expression or regulating their activities, could provide a broad-spectrum therapeutic strategy for animal viral diseases and complications linked to interferon elevation.

Diabetic neuropathy is regarded as one of the most debilitating outcomes of diabetes. It can affect both the peripheral and central nervous systems, leading to pain, decreased motility, cognitive decline, and dementia. S-palmitoylation is a reversible posttranslational lipid modification, and its dysregulation has been implicated in metabolic syndrome, cancers, neurological disorders, and infections. However, the role of S-palmitoylation in diabetic neuropathy remains unclear. Here we demonstrate a potential association between activating protein palmitoylation and diabetic neuropathy. We compared the proteomic data of lumbar dorsal root ganglia (DRG) of diabetes mice and palmitoylome profiling data of the HUVEC cell line. The mapping results identified peroxiredoxin-6 (PRDX6) as a novel target in diabetic neuropathy, whose biological mechanism was associated with S-palmitoylation. Bioinformatic prediction revealed that PRDX6 had two palmitoylation sites, Cys47 and Cys91. Immunofluorescence results indicated PRDX6 translocating between the cytoplasm and cell membrane. Protein function analysis proposed that increased palmitoylation could competitively inhibit the formation of disulfide-bond between Cys47 and Cys91 and change the spatial topology of PRDX6 protein. Cl-HCO3-anion exchanger 3 (AE3) was one of the AE family members, which was proved to express in DRG. AE3 activity evoked Cl-influx in neurons which was generally associated with increased excitability and susceptibility to pain. We demonstrated that the S-palmitoylation status of Cys47 could affect the interaction between PRDX6 and the C-terminal domain of AE3, thereby regulating the activity of AE3 anion exchanger enzyme in the nervous system. The results highlight a central role for PRDX6 palmitoylation in protection against diabetic neuropathy. [ABSTRACT FROM AUTHOR]

The reversible lipid modification, S-palmitoylation, plays regulatory roles in various physiological processes, e.g., neuronal plasticity and organs development; however, the roles of palmitoylation engaged in testis have yet remained unexplored. Here, we used combined approaches of palm-proteomics, informatics and quantitative PCR to systematically analyze the expression of key enzymes related to protein palmitoylation and identify proteome-wide palmitoylated proteins during the processes of spermatogenesis. Specifically, different timepoints were chosen to collect samples to cover the initiation of meiosis (postnatal, P12), the appearance of the first batch of sperm (P36) and fully fertile status (P60) in mouse. Interestingly, our results showed that only a few enzymes related to protein palmitoylation are highly expressed at later stages (from P36 to P60), rather than in the earlier phase of testis development (P12). To focus on the molecular event of spermatogenesis, we examined the palm-proteomics of testes in P36 and P60 mouse. In total, we identified 4,883 palmitoylated proteins, among which 3,310 proteins match the published palmitoyl-proteome datasets and 1,573 proteins were firstly identified as palmitoylated proteins in this study. Informatics analysis suggested that palmitoylation is involved in events of protein transport, metabolic process, protein folding and cell adhesion, etc. Importantly, further analysis revealed that several networks of palmitoylated proteins are closely associated with sperm morphology and motility. Together, our study laid a solid ground for understanding the roles of protein palmitoylation in spermatogenesis for future studies. [ABSTRACT FROM AUTHOR]

The ZDHHC13/ZDHHC17 subfamily belongs to the zinc finger DHHC-domain containing (ZDHHC) family, including ZDHHC13 and ZDHHC17. Recent studies have shown that the ZDHHC13/ZDHHC17 subfamily is involved in various pathological and physiological processes, including S-palmitoylation, Mg 2+ transport, and CALCOCO1-mediated Golgiphagy. Moreover, the ZDHHC13/ZDHHC17 subfamily plays a crucial role in the occurrence and development of many diseases, including Huntington disease (HD), osteoporosis, atopic dermatitis, diabetes, and cancer. In the present review, we describe the distribution, structure, and post-translational modifications (PTMs) of the ZDHHC13/ZDHHC17 subfamily. Moreover, we effectively summarize the biological functions and associated diseases of this subfamily. Given the pleiotropy of the ZDHHC13/ZDHHC17 subfamily, it is imperative to conduct further research on its members to comprehend the pertinent pathophysiological mechanisms and to devise tactics for managing and controlling various diseases., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of funds or interest. Conflicts of interest The authors declare that there is no conflict of funds or interest., (Copyright © 2024. Published by Elsevier Ltd.)

Protein S -palmitoylation is a reversible lipophilic posttranslational modification regulating a diverse number of signaling pathways. Within transmembrane proteins (TMPs), S -palmitoylation is implicated in conditions from inflammatory disorders to respiratory viral infections. Many small-scale experiments have observed S -palmitoylation at juxtamembrane Cys residues. However, most large-scale S -palmitoyl discovery efforts rely on trypsin-based proteomics within which hydrophobic juxtamembrane regions are likely underrepresented. Machine learning- by virtue of its freedom from experimental constraints - is particularly well suited to address this discovery gap surrounding TMP S -palmitoylation. Utilizing a UniProt-derived feature set, a gradient boosted machine learning tool (TopoPalmTree) was constructed and applied to a holdout dataset of viral S -palmitoylated proteins. Upon application to the mouse TMP proteome, 1591 putative S -palmitoyl sites (i.e. not listed in SwissPalm or UniProt) were identified. Two lung-expressed S -palmitoyl candidates (synaptobrevin Vamp5 and water channel Aquaporin-5) were experimentally assessed. Finally, TopoPalmTree was used for rational design of an S -palmitoyl site on KDEL-Receptor 2. This readily interpretable model aligns the innumerable small-scale experiments observing juxtamembrane S -palmitoylation into a proteomic tool for TMP S -palmitoyl discovery and design, thus facilitating future investigations of this important modification., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Gasdermin D (GSDMD)-mediated pyroptotic cell death drives inflammatory cytokine release and downstream immune responses upon inflammasome activation, which play important roles in host defense and inflammatory disorders. Upon activation by proteases, the GSDMD N-terminal domain (NTD) undergoes oligomerization and membrane translocation in the presence of lipids to assemble pores. Despite intensive studies, the molecular events underlying the transition of GSDMD from an autoinhibited soluble form to an oligomeric pore form inserted into the membrane remain incompletely understood. Previous work characterized S -palmitoylation for gasdermins from bacteria, fungi, invertebrates, as well as mammalian gasdermin E (GSDME). Here, we report that a conserved residue Cys191 in human GSDMD was S -palmitoylated, which promoted GSDMD-mediated pyroptosis and cytokine release. Mutation of Cys191 or treatment with palmitoyltransferase inhibitors cyano-myracrylamide (CMA) or 2-bromopalmitate (2BP) suppressed GSDMD palmitoylation, its localization to the membrane and dampened pyroptosis or IL-1β secretion. Furthermore, Gsdmd -dependent inflammatory responses were alleviated by inhibition of palmitoylation in vivo. By contrast, coexpression of GSDMD with palmitoyltransferases enhanced pyroptotic cell death, while introduction of exogenous palmitoylation sequences fully restored pyroptotic activities to the C191A mutant, suggesting that palmitoylation-mediated membrane localization may be distinct from other molecular events such as GSDMD conformational change during pore assembly. Collectively, our study suggests that S -palmitoylation may be a shared regulatory mechanism for GSDMD and other gasdermins, which points to potential avenues for therapeutically targeting S -palmitoylation of gasdermins in inflammatory disorders., Competing Interests: Competing interests statement:The authors declare no competing interest.

The interferon-induced transmembrane protein 3 (IFITM3), a small molecule transmembrane protein induced by interferon, is generally conserved in vertebrates, which can inhibit infection by a diverse range of pathogenic viruses such as influenza virus. However, the precise antiviral mechanisms of IFITM3 remain unclear. At least four post-translational modifications (PTMs) were found to modulate the antiviral effect of IFITM3. These include positive regulation provided by S-palmitoylation of cysteine and negative regulation provided by lysine ubiquitination, lysine methylation, and tyrosine phosphorylation. IFITM3 S-palmitoylation is an enzymatic addition of a 16-carbon fatty acid on the three cysteine residues within or adjacent to its two hydrophobic domains at positions 71, 72, and 105, that is essential for its proper targeting, stability, and function. As S-palmitoylation is the only PTM known to enhance the antiviral activity of IFITM3, enzymes that add this modification may play important roles in IFN-induced immune responses. This study mainly reviews the research progresses on the antiviral mechanism of IFITM3, the regulation mechanism of S-palmitoylation modification on its subcellular localization, stability, and function, and the enzymes that mediate the S-palmitoylation modification of IFITM3, which may help elucidate the mechanism by which this IFN effector restrict virus replication and thus aid in the design of therapeutics targeted at pathogenic viruses. [ABSTRACT FROM AUTHOR]

Inactivating mutations in the PPT1 gene encoding palmitoyl‐protein thioesterase‐1 (PPT1) underlie the CLN1 disease, a devastating neurodegenerative lysosomal storage disorder. The mechanism of pathogenesis underlying CLN1 disease has remained elusive. PPT1 is a lysosomal enzyme, which catalyzes the removal of palmitate from S‐palmitoylated proteins (constituents of ceroid lipofuscin) facilitating their degradation and clearance by lysosomal hydrolases. Thus, it has been proposed that Ppt1‐deficiency leads to lysosomal accumulation of ceroid lipofuscin leading to CLN1 disease. While S‐palmitoylation is catalyzed by palmitoyl acyltransferases (called ZDHHCs), palmitoyl‐protein thioesterases (PPTs) depalmitoylate these proteins. We sought to determine the mechanism by which Ppt1‐deficiency may impair lysosomal degradative function leading to infantile neuronal ceroid lipofuscinosis pathogenesis. Here, we report that in Ppt1−/− mice, which mimic CLN1 disease, low level of inositol 3‐phosphate receptor‐1 (IP3R1) that mediates Ca++ transport from the endoplasmic reticulum to the lysosome dysregulated lysosomal Ca++ homeostasis. Intriguingly, the transcription factor nuclear factor of activated T‐cells, cytoplasmic 4 (NFATC4), which regulates IP3R1‐expression, required S‐palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8, which catalyzed S‐palmitoylation of NFATC4. Notably, in Ppt1−/− mice, reduced ZDHHC4 and ZDHHC8 levels markedly lowered S‐palmitoylated NFATC4 (active) in the nucleus, which inhibited IP3R1‐expression, thereby dysregulating lysosomal Ca++ homeostasis. Consequently, Ca++‐dependent lysosomal enzyme activities were markedly suppressed. Impaired lysosomal degradative function impaired autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1‐overexpression in Ppt1−/− mouse fibroblasts ameliorated this defect. Our results reveal a previously unrecognized role of Ppt1 in regulating lysosomal Ca++ homeostasis and suggest that this defect contributes to pathogenesis of CLN1 disease. [ABSTRACT FROM AUTHOR]

The interferon-induced transmembrane protein 3 (IFITM3), a small molecule transmembrane protein induced by interferon, is generally conserved in vertebrates, which can inhibit infection by a diverse range of pathogenic viruses such as influenza virus. However, the precise antiviral mechanisms of IFITM3 remain unclear. At least four post-translational modifications (PTMs) were found to modulate the antiviral effect of IFITM3. These include positive regulation provided by S-palmitoylation of cysteine and negative regulation provided by lysine ubiquitination, lysine methylation, and tyrosine phosphorylation. IFITM3 S-palmitoylation is an enzymatic addition of a 16-carbon fatty acid on the three cysteine residues within or adjacent to its two hydrophobic domains at positions 71, 72, and 105, that is essential for its proper targeting, stability, and function. As S-palmitoylation is the only PTM known to enhance the antiviral activity of IFITM3, enzymes that add this modification may play important roles in IFN-induced immune responses. This study mainly reviews the research progresses on the antiviral mechanism of IFITM3, the regulation mechanism of S-palmitoylation modification on its subcellular localization, stability, and function, and the enzymes that mediate the S-palmitoylation modification of IFITM3, which may help elucidate the mechanism by which this IFN effector restrict virus replication and thus aid in the design of therapeutics targeted at pathogenic viruses.

Protein S-palmitoylation is a reversible post-translational lipidation in which palmitic acid (16:0) is added to protein cysteine residue by a covalent thioester bond. This modification plays an active role in membrane targeting of soluble proteins, protein–protein interaction, protein trafficking, and subcellular localization. Moreover, palmitoylation is related to different diseases, such as neurodegenerative pathologies, cancer, and developmental defects. The aim of this research is to provide a straightforward and sensitive procedure to detect protein palmitoylation based on Acyl Biotin Exchange (ABE) chemistry. Our protocol setup consists of co-immunoprecipitation of native proteins (i.e., CD63), followed by the direct detection of palmitoylation on proteins immobilized on polyvinylidene difluoride (PVDF) membranes. With respect to the conventional ABE-based protocol, we optimized and validated a rapid semi-quantitative assay that is shown to be significantly more sensitive and highly reproducible.

Protein lipidation is a common post-translational modification of proteins that plays an important role in human physiology and pathology. One form of protein lipidation, S-palmitoylation, involves the addition of a 16-carbon fatty acid (palmitate) onto proteins. This reversible modification may affect the regulation of protein trafficking and stability in membranes. From multiple recent experimental studies, a picture emerges whereby protein S-palmitoylation is a ubiquitous yet discrete molecular switch enabling the expansion of protein functions and subcellular localization in minutes to hours. Neural tissue is particularly rich in proteins that are regulated by S-palmitoylation. A surge of novel methods of detection of protein lipidation at high resolution allowed us to get better insights into the roles of protein palmitoylation in brain physiology and pathophysiology. In this review, we specifically discuss experimental work devoted to understanding the impact of protein palmitoylation on functional changes in the excitatory and inhibitory synapses associated with neuronal activity and neuronal plasticity. The accumulated evidence also implies a crucial role of S-palmitoylation in learning and memory, and brain disorders associated with impaired cognitive functions.

The spatiotemporal cellular distribution of lysosomes depends on active transport mainly driven by microtubule motors such as kinesins and dynein. Different protein complexes attach these molecularmotors to their vesicular cargo. TMEM55B (also known as PIP4P1), as an integral lysosomal membrane protein, is a component of such a complex that mediates the retrograde transport of lysosomes by establishing interactions with the cytosolic scaffold protein JIP4 (also known as SPAG9) and dynein-dynactin. Here, we show that TMEM55B and its paralog TMEM55A (PIP4P2) are S-palmitoylated proteins that are lipidated at multiple cysteine residues. Mutation of all cysteines in TMEM55B prevents S-palmitoylation and causes retention of the mutated protein in the Golgi. Consequently, non-palmitoylated TMEM55B is no longer able tomodulate lysosomal positioning and the perinuclear clustering of lysosomes. Additional mutagenesis of the dileucine-based lysosomal sorting motif in non-palmitoylated TMEM55B leads to partial missorting to the plasma membrane instead of retention in the Golgi, implicating a direct effect of Spalmitoylation on the adaptor protein-dependent sorting of TMEM55B. Our data suggest a critical role for S-palmitoylation in the trafficking of TMEM55B and TMEM55B-dependent lysosomal positioning. [ABSTRACT FROM AUTHOR]

Protein palmitoylation—a lipid modification in which one or more cysteine thiols on a substrate protein are modified to form a thioester with a palmitoyl group—is a significant post-translational biological process. This process regulates the trafficking, subcellular localization, and stability of different proteins in cells. Since palmitoylation participates in various biological processes, it is related to the occurrence and development of multiple diseases. It has been well evidenced that the proteins whose functions are palmitoylation-dependent or directly involved in key proteins' palmitoylation/depalmitoylation cycle may be a potential source of novel therapeutic drugs for the related diseases. Many researchers have reported palmitoylation of proteins, which are crucial for host-virus interactions during viral infection. Quite a few explorations have focused on figuring out whether targeting the acylation of viral or host proteins might be a strategy to combat viral diseases. All these remarkable achievements in protein palmitoylation have been made to technological advances. This paper gives an overview of protein palmitoylation modification during viral infection and the methods for palmitoylated protein detection. Future challenges and potential developments are proposed. [ABSTRACT FROM AUTHOR]

Protein palmitoylation—a lipid modification in which one or more cysteine thiols on a substrate protein are modified to form a thioester with a palmitoyl group—is a significant post-translational biological process. This process regulates the trafficking, subcellular localization, and stability of different proteins in cells. Since palmitoylation participates in various biological processes, it is related to the occurrence and development of multiple diseases. It has been well evidenced that the proteins whose functions are palmitoylation-dependent or directly involved in key proteins’ palmitoylation/depalmitoylation cycle may be a potential source of novel therapeutic drugs for the related diseases. Many researchers have reported palmitoylation of proteins, which are crucial for host-virus interactions during viral infection. Quite a few explorations have focused on figuring out whether targeting the acylation of viral or host proteins might be a strategy to combat viral diseases. All these remarkable achievements in protein palmitoylation have been made to technological advances. This paper gives an overview of protein palmitoylation modification during viral infection and the methods for palmitoylated protein detection. Future challenges and potential developments are proposed.

Protein S -acylation is a prevalent post-translational protein lipidation that is dynamically regulated by 'writer' protein S -acyltransferases and 'eraser' acylprotein thioesterases. The protein S -acyltransferases comprise 23 aspartate–histidine–histidine–cysteine (DHHC)–containing proteins, which transfer fatty acid acyl groups from acyl-coenzyme A onto protein substrates. DHHC proteins are increasingly recognized as critical regulators of S -acylation–mediated cellular processes and pathology. As our understanding of the importance and breadth of DHHC-mediated biology and pathology expands, so too does the need for chemical inhibitors of this class of proteins. In this review, we discuss the challenges and progress in DHHC inhibitor development, focusing on 2-bromopalmitate, the most commonly used inhibitor in the field, and N -cyanomethyl- N -myracrylamide, a new broad-spectrum DHHC inhibitor. We believe that current and ongoing advances in structure elucidation, mechanistic interrogation, and novel inhibitor design around DHHC proteins will spark innovative strategies to modulate these critical proteins in living systems. [ABSTRACT FROM AUTHOR]

The membrane protein claudin-3 (CLDN3) is critical for the formation and maintenance of tight junction and its high expression has been implicated in dictating malignant progression in various cancers. However, the post-translational modification of CLDN3 and its biological function remains poorly understood. Here, we report that CLDN3 is positively correlated with ovarian cancer progression both in vitro and in vivo. Of interest, CLDN3 undergoes S-palmitoylation on three juxtamembrane cysteine residues, which contribute to the accurate plasma membrane localization and protein stability of CLDN3. Moreover, the deprivation of S-palmitoylation in CLDN3 significantly abolishes its tumorigenic promotion effect in ovarian cancer cells. By utilizing the co-immunoprecipitation assay, we further identify ZDHHC12 as a CLDN3-targating palmitoyltransferase from 23 ZDHHC family proteins. Furthermore, the knockdown of ZDHHC12 also significantly inhibits CLDN3 accurate membrane localization, protein stability and ovarian cancer cells tumorigenesis. Thus, our work reveals S-palmitoylation as a novel regulatory mechanism that modulates CLDN3 function, which implies that targeting ZDHHC12-mediated CLDN3 S-palmitoylation might be a potential strategy for ovarian cancer therapy.

S-acylation is a post-translational linkage of long chain fatty acids to cysteines, playing a key role in normal physiology and disease. In human cells, the reaction is catalyzed by a family of 23 membrane DHHC-acyltransferases (carrying an Asp-His-His-Cys catalytic motif) in two stages: (1) acyl-CoA-mediated autoacylation of the enzyme; and (2) further transfer of the acyl chain to a protein substrate. Despite the availability of a 3D-structure of human acyltransferase (hDHHC20), the molecular aspects of lipid selectivity of DHHC-acyltransferases remain unclear. In this paper, using molecular dynamics (MD) simulations, we studied membrane-bound hDHHC20 right before the acylation by C12-, C14-, C16-, C18-, and C20-CoA substrates. We found that: (1) regardless of the chain length, its terminal methyl group always reaches the “ceiling” of the enzyme’s cavity; (2) only for C16, an optimal “reactivity” (assessed by a simple geometric criterion) permits the autoacylation; (3) in MD, some key interactions between an acyl-CoA and a protein differ from those in the reference crystal structure of the C16-CoA-hDHHS20 mutant complex (probably, because this structure corresponds to a non-native dimer). These features of specific recognition of full-size acyl-CoA substrates support our previous hypothesis of “geometric and physicochemical selectivity” derived for simplified acyl-CoA analogues.

S‐palmitoylation is a reversible posttranslational modification in which a 16‐carbon saturated fatty acid (generally palmitate) is attached to specific cysteine residues in polypeptides via thioester linkage. Dynamic S‐palmitoylation (palmitoylation‐depalmitoylation), like phosphorylation‐dephosphorylation, regulates the function of numerous proteins, especially in the brain. While a family of 23 palmitoyl‐acyl transferases (PATS), commonly known as ZDHHCs, catalyze S‐palmitoylation of proteins, the thioesterases, localized either in the cytoplasm (eg, APT1) or in the lysosome (eg, PPT1) mediate depalmitoylation. Previously, we reported that APT1 requires dynamic S‐palmitoylation for shuttling between the cytosol and the plasma membrane. APT1 depalmitoylated H‐Ras to regulate its signaling pathway that stimulates cell proliferation. Although we demonstrated that APT1 catalyzed its own depalmitoylation, the ZDHHC(s) that S‐palmitoylated APT1 had remained unidentified. We report here that ZDHHC5 and ZDHHC23 catalyze APT1 S‐palmitoylation. Intriguingly, lysosomal Ppt1‐deficiency in Cln1−/− mouse, a reliable animal model of INCL, markedly reduced ZDHHC5 and ZDHHC23 levels. Remarkably, in the brain of these mice decreased ZDHHC5 and ZDHHC23 levels suppressed membrane‐bound APT1, thereby, increasing plasma membrane‐localized H‐Ras, which activated its signaling pathway stimulating microglia proliferation. Increased inflammatory cytokines produced by microglia together with increased complement C1q level contributed to the transformation of astrocytes to neurotoxic A1 phenotype. Importantly, neuroinflammation was ameliorated by treatment of Cln1−/− mice with a PPT1‐mimetic small molecule, N‐tert(Butyl)hydroxylamine (NtBuHA). Our results revealed a novel pathway to neuropathology in an INCL mouse model and uncovered a previously unrecognized mechanism of the neuroprotective actions of NtBuHA and its potential as a drug target. [ABSTRACT FROM AUTHOR]

S-palmitoylation, catalyzed by a family of 23 zinc finger Asp-His-His-Cys (DHHC) domain-containing (ZDHHC) protein acyltransferases localized on the cell membrane. However, stemness genes modulated by ZDHHCs in lung adenocarcinoma (LUAD) remain to be defined. Previously, we have constructed a network of cancer stem cell genes, including INCENP, based on mRNA stemness indices (mRNAsi) of LUAD. INCENP has the function of a chromosomal passenger complex locating to centromeres, which is performed by the conserved region of its N-terminal domain. INCENP protein with a deletion of the first non-conserved 26 amino acid sequence failed to target centromeres. However, the exact function of the deleted sequence has not been elucidated. To identify novel cancer stem cell-relevant palmitoylated proteins and responsible ZDHHC enzymes in LUAD, we analyzed multi-omics data obtained from the database of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and the Human Protein Atlas (HPA). ZDHHC5 is distinguished from the ZDHHC family for being up-regulated in mRNA and protein levels and associated with malignant prognosis. ZDHHC5 was positively associated with INCENP, and the correlation score increased with LUAD stages. CSS-Palm results showed Cys15 was the S-palmitoylation site of INCENP. Interestingly, Cys15 locates in the 1–26 aa sequence of INCENP, and is a conserved site across species. As INCENP is a nuclear protein, we predicted that the nuclear localization signal of ZDHHC5 was specific to the importin αβ pathway, and the result of immunofluorescence proves that ZDHHC5 is located in the nucleoplasm, in addition to the plasma membrane. Therefore, our study indicates the S-palmitoylation of INCENP mediated by ZDHHC5 as a potential mechanism of S-palmitoylation to modulate CSCs in LUAD.

S-palmitoylation is a reversible posttranslational lipid modification of proteins. It controls protein activity, stability, trafficking and protein–protein interactions. Recent global profiling of immune cells and targeted analysis have identified many S-palmitoylated immunity-associated proteins. Here, we review S-palmitoylated immune receptors and effectors, and their dynamic regulation at cellular membranes to generate specific and balanced immune responses. We also highlight how this understanding can drive therapeutic advances to pharmacologically modulate immune responses.

The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and 3H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in Akt1 is important for proper its function, since Akt1-C344S was unable to support adipocyte cell differentiation in vitro. These results add an unexpected new layer to the already complex Akt molecular code, improving our understanding of cell decision-making mechanisms such as cell survival, differentiation and death.

Macrophages are present in all tissues within our body, where they promote tissue homeostasis by responding to microenvironmental triggers, not only through clearance of pathogens and apoptotic cells but also via trophic, regulatory, and repair functions. To accomplish these divergent functions, tremendous dynamic fine-tuning of their physiology is needed. Emerging evidence indicates that S-palmitoylation, a reversible post-translational modification that involves the linkage of the saturated fatty acid palmitate to protein cysteine residues, directs many aspects of macrophage physiology in health and disease. By controlling protein activity, stability, trafficking, and protein–protein interactions, studies identified a key role of S-palmitoylation in endocytosis, inflammatory signaling, chemotaxis, and lysosomal function. Here, we provide an in-depth overview of the impact of S-palmitoylation on these cellular processes in macrophages in health and disease. Findings discussed in this review highlight the therapeutic potential of modulators of S-palmitoylation in immunopathologies, ranging from infectious and chronic inflammatory disorders to metabolic conditions.

Calcium (Ca2+) regulates a multitude of cellular processes during fertilization and throughout adult life by acting as an intracellular messenger to control effector functions in excitable and non-excitable cells. Changes in intracellular Ca2+ levels are driven by the co-ordinated action of Ca2+ channels, pumps, and exchangers, and the resulting signals are shaped and decoded by Ca2+-binding proteins to drive rapid and long-term cellular processes ranging from neurotransmission and cardiac contraction to gene transcription and cell death. S-acylation, a lipid post-translational modification, is emerging as a critical regulator of several important Ca2+-handling proteins. S-acylation is a reversible and dynamic process involving the attachment of long-chain fatty acids (most commonly palmitate) to cysteine residues of target proteins by a family of 23 proteins acyltransferases (zDHHC, or PATs). S-acylation modifies the conformation of proteins and their interactions with membrane lipids, thereby impacting intra- and intermolecular interactions, protein stability, and subcellular localization. Disruptions of S-acylation can alter Ca2+ signalling and have been implicated in the development of pathologies such as heart disease, neurodegenerative disorders, and cancer. Here, we review the recent literature on the S-acylation of Ca2+ transport proteins of organelles and of the plasma membrane and highlight the molecular basis and functional consequence of their S-acylation as well as the therapeutic potential of targeting this regulation for diseases caused by alterations in cellular Ca2+ fluxes., (© 2024 The Author(s).)

Protein S-acylation is a reversible post-translational modification (PTM). It is present on diverse proteins and has important roles in regulating protein function. Aminolysis with hydroxylamine is widely used in the global identification of the PTM. However, the identification is indirect. Distinct criteria have been used for identification, and the false discovery rate has not been addressed. Here, we report a site-specific method for S-acylation identification based on tagging of S-acylation sites with iodoTMT0. Efforts to improve the performance of the method and confidence of identification are discussed, highlighting the importance of reducing contaminant peptides and keeping the recovery rate consistent between aliquots with or without hydroxylamine treatment. With very stringent criteria, presumptive S-acylation sites of 269, 684, 695, and 780 were identified from HK2 cells, HK11 cells, mouse brain, and mouse liver samples, respectively. Among them, the newly identified protein S-acylation sites are equivalent to 34% of human and 24% of mouse S-acylation sites reported previously. In addition, false-positive rates for S-acylation identification and S-acylation abundances were estimated. Significant differences in S-acylation abundance were found from different samples (from 0.08% in HK2 cells to 0.76% in mouse brain), and the false-positive rates were significantly higher for samples with a low abundance of S-acylation.

The ceroid lipofuscinosis neuronal 1 (CLN1) disease, formerly called infantile neuronal ceroid lipofuscinosis, is a fatal hereditary neurodegenerative lysosomal storage disorder. This disease is caused by loss-of-function mutations in the CLN1 gene, encoding palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes depalmitoylation of S-palmitoylated proteins for degradation and clearance by lysosomal hydrolases. Numerous proteins, especially in the brain, require dynamic S-palmitoylation (palmitoylation-depalmitoylation cycles) for endosomal trafficking to their destination. While 23 palmitoyl-acyl transferases in the mammalian genome catalyze S-palmitoylation, depalmitoylation is catalyzed by thioesterases such as PPT1. Despite these discoveries, the pathogenic mechanism of CLN1 disease has remained elusive. Here, we report that in the brain of Cln1 -/- mice, which mimic CLN1 disease, the mechanistic target of rapamycin complex-1 (mTORC1) kinase is hyperactivated. The activation of mTORC1 by nutrients requires its anchorage to lysosomal limiting membrane by Rag GTPases and Ragulator complex. These proteins form the lysosomal nutrient sensing scaffold to which mTORC1 must attach to activate. We found that in Cln1 -/- mice, two constituent proteins of the Ragulator complex (vacuolar (H + )-ATPase and Lamtor1) require dynamic S-palmitoylation for endosomal trafficking to the lysosomal limiting membrane. Intriguingly, Ppt1 deficiency in Cln1 -/- mice misrouted these proteins to the plasma membrane disrupting the lysosomal nutrient sensing scaffold. Despite this defect, mTORC1 was hyperactivated via the IGF1/PI3K/Akt-signaling pathway, which suppressed autophagy contributing to neuropathology. Importantly, pharmacological inhibition of PI3K/Akt suppressed mTORC1 activation, restored autophagy, and ameliorated neurodegeneration in Cln1 -/- mice. Our findings reveal a previously unrecognized role of Cln1/Ppt1 in regulating mTORC1 activation and suggest that IGF1/PI3K/Akt may be a targetable pathway for CLN1 disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

S-palmitoylation is a reversible posttranslational modification, and the palmitoylation reaction in human-derived cells is mediated by the zDHHC family, which is composed of S-acyltransferase enzymes that possess the DHHC (Asp-His-His-Cys) structural domain. zDHHC proteins form an autoacylation intermediate, which then attaches the fatty acid to cysteine a residue in the target protein. zDHHC proteins sublocalize in different neuronal structures and exert dif-ferential effects on neurons. In humans, many zDHHC proteins are closely related to human neu-rological disor-ders. This review focuses on a variety of neurological disorders, such as AD (Alz-heimer's disease), HD (Huntington's disease), SCZ (schizophrenia), XLID (X-linked intellectual disability), attention deficit hyperactivity disorder and glioma. In this paper, we will discuss and summarize the research progress regarding the role of zDHHC proteins in these neu-rological disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liao, Huang, Liu, Zhang, Shi, Li and Luo.)

Palmitic acid (C16:0) is the most abundant saturated fatty acid in animals serving as a substrate in synthesis and β-oxidation of other lipids, and in the modification of proteins called palmitoylation. The influence of dietary palmitic acid on protein S-palmitoylation remains largely unknown. In this study we performed high-throughput proteomic analyses of a membrane-enriched fraction of murine liver to examine the influence of a palm oil-rich diet (HPD) on S-palmitoylation of proteins. HPD feeding for 4 weeks led to an accumulation of C16:0 and C18:1 fatty acids in livers which disappeared after 12-week feeding, in contrast to an accumulation of C16:0 in peritoneal macrophages. Parallel proteomic studies revealed that HPD feeding induced a sequence of changes of the level and/or S-palmitoylation of diverse liver proteins involved in fatty acid, cholesterol and amino acid metabolism, hemostasis, and neutrophil degranulation. The HPD diet did not lead to liver damage, however, it caused progressing obesity, hypercholesterolemia and hyperglycemia. We conclude that the relatively mild negative impact of such diet on liver functioning can be attributed to a lower bioavailability of palm oil-derived C16:0 vs. that of C18:1 and the efficiency of mechanisms preventing liver injury, possibly including dynamic protein S-palmitoylation.