Multiomics analysis identifies oxidative phosphorylation as a cancer vulnerability arising from myristoylation inhibition.

BACKGROUND: In humans, two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, catalyze myristate transfer to proteins to facilitate membrane targeting and signaling. We investigated the expression of NMTs in numerous cancers and found that NMT2 levels are dysregulated by epigenetic suppression, particularly so in hematologic malignancies. This suggests that pharmacological inhibition of the remaining NMT1 could allow for the selective killing of these cells, sparing normal cells with both NMTs. METHODS AND RESULTS: Transcriptomic analysis of 1200 NMT inhibitor (NMTI)-treated cancer cell lines revealed that NMTI sensitivity relates not only to NMT2 loss or NMT1 dependency, but also correlates with a myristoylation inhibition sensitivity signature comprising 54 genes (MISS-54) enriched in hematologic cancers as well as testis, brain, lung, ovary, and colon cancers. Because non-myristoylated proteins are degraded by a glycine-specific N-degron, differential proteomics revealed the major impact of abrogating NMT1 genetically using CRISPR/Cas9 in cancer cells was surprisingly to reduce mitochondrial respiratory complex I proteins rather than cell signaling proteins, some of which were also reduced, albeit to a lesser extent. Cancer cell treatments with the first-in-class NMTI PCLX-001 (zelenirstat), which is undergoing human phase 1/2a trials in advanced lymphoma and solid tumors, recapitulated these effects. The most downregulated myristoylated mitochondrial protein was NDUFAF4, a complex I assembly factor. Knockout of NDUFAF4 or in vitro cell treatment with zelenirstat resulted in loss of complex I, oxidative phosphorylation and respiration, which impacted metabolomes. CONCLUSIONS: Targeting of both, oxidative phosphorylation and cell signaling partly explains the lethal effects of zelenirstat in select cancer types. While the prognostic value of the sensitivity score MISS-54 remains to be validated in patients, our findings continue to warrant the clinical development of zelenirstat as cancer treatment.

Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

Machine learning reveals serum myristic acid, palmitic acid and heptanoylcarnitine as biomarkers of coronary artery disease risk in patients with type 2 diabetes mellitus.

BACKGROUND: Coronary heart disease (CHD) is the most important complication of type 2 diabetes mellitus (T2DM) and the leading cause of death. Identifying the risk of CHD in T2DM patients is important for early clinical intervention. METHODS: A total of 213 participants, including 81 healthy controls (HCs), 69 T2DM patients and 63 T2DM patients complicated with CHD were recruited in this study. Serum metabolomics were conducted by using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Demographic information and clinical laboratory test results were also collected. RESULTS: Metabolic phenotypes were significantly altered among HC, T2DM and T2DM-CHD. Acylcarnitines were the most disturbed metabolites between T2DM patients and HCs. Lower levels of bile acids and higher levels of fatty acids in serum were closely associated with CHD risk in T2DM patients. Artificial neural network model was constructed for the discrimination of T2DM and T2DM complicated with CHD based on myristic acid, palmitic acid and heptanoylcarnitine, with accuracy larger than 0.95 in both training set and testing set. CONCLUSION: Altogether, these findings suggest that myristic acid, palmitic acid and heptanoylcarnitine have a good prospect for the warning of CHD complications in T2DM patients, and are superior to traditional lipid, blood glucose and blood pressure indicators.

Dibutyl phthalate degradation by Paenarthrobacter ureafaciens PB10 through downstream product myristic acid and its bioremediation potential in contaminated soil.

Dibutyl phthalate (DBP) is a widely used plasticizer to make plastic flexible and long-lasting. It is easily accessible in a broad spectrum of environments as a result of the rising level of plastic pollution. This compound is considered a top-priority toxicant and persistent organic pollutant by international environmental agencies for its endocrine disruptive and carcinogenic propensities. To mitigate the DBP in the soil, one DBP-degrading bacterial strain was isolated from a plastic-polluted landfill and identified as Paenarthrobacter ureafaciens PB10 by 16S rRNA gene sequence-based homology. The strain was found to develop a distinct transparent halo zone around grown colonies on an agar plate supplemented with DBP. The addition of yeast extract (100 mg/L) as a nutrient source accelerated cell biomass production and DBP degradation rate; however, the presence of glucose suppressed DBP degradation by the PB10 strain without affecting its ability to proliferate. The strain PB10 was efficient in eliminating DBP under various pH conditions (5.0-8.0). Maximum cell growth and degradation of 99.49% at 300 mg/L DBP were achieved in 72 h at the optimized mineral salt medium (MS) conditions of pH 7.0 and 32 °C. Despite that, when the concentration of DBP rose to 3000 mg/L, the DBP depletion rate was measured at 79.34% in 72 h. Some novel intermediate metabolites, like myristic acid, hexadecanoic acid, stearic acid, and the methyl derivative of 4-hydroxyphenyl acetate, along with monobutyl phthalate and phthalic acid, were detected in the downstream degradation process of DBP through GC-MS profiling. Furthermore, in synchronization with native soil microbes, this PB10 strain successfully removed a notable amount of DBP (up to 54.11%) from contaminated soil under microcosm study after 10 d. Thus, PB10 has effective DBP removal ability and is considered a potential candidate for bioremediation in DBP-contaminated sites.

Fabrication of myristic acid-potato starch complex nanostructures and assessment of their cytotoxic behavior.

BACKGROUND: Lipids and carbohydrates perform essential functions in foods. In recent decades, food scientists have studied the effects of carbohydrate-lipid interactions on the functional properties of food. However, the ways in which carbohydrate-lipid complex-derived materials affect the biological system are unknown. In this study, a myristic acid-potato starch complex was created using a simple cooking approach. The complex was employed as a precursor for the fabrication of myristic acid-potato starch complex-based nanostructured materials (MPS-NMs) through a liquid-liquid extraction approach. A study was conducted on the structural and cytotoxic features of the fabricated MPS-NMs. RESULTS: Transmission electron microscopy images confirmed the formation of spherical nanostructures, 3-60 nm in size. After 24 h exposure, the chloroform fraction-based and n-hexane fraction-based MPS-NMs increased cell death by ~90% and ~ 82%, respectively. Chloroform fraction-based MPS-NMs (CMPS-NMs) triggers apoptotic cell death in human mesenchymal stem cells (hMSCs). n-Hexane fraction-based MPS-NMs (HMPS-NMs) treated cells have red color-intact nuclei, attributed to necrotic cell death. The CMPS-NMs and HMPS-NMs significantly decreased the mitochondria membrane potential and increased the intracellular reactive oxygen species (ROS) levels. We observed significant downregulation in flavin-containing monooxygenase (FMO), Ataxia Telangiectasia Mutated (ATM), and uridine diphosphate glucuronosyltransferases (UGT) gene expression levels in the exposed cells of CMPS-NMs and HMPS- NMs. In addition, we found upregulation of glutathione-disulfide reductase (GSR) and glutathione S-transferase A4 (GSTA4) genes in CMPS-NMs, and HMPS-NMs exposure. CONCLUSION: The cooking process may lead to the formation of nanostructured material in food systems. Chloroform fraction-based MPS-NMs and HMPS-NMs may contribute to cell metabolic disorders. © 2023 Society of Chemical Industry.

TNF-α-induced Inhibition of Protein Myristoylation Via Binding Between NMT1 and Sorbs2 in Osteoblasts.

BACKGROUND/AIM: Bone resolution due to tumor invasion often occurs on the surface of the jaw and is important for clinical prognosis. Although cytokines, such as TNF-α are known to impair osteoblasts, the underlying mechanism remains unclear. Protein myristoylation, a post-translational modification, plays an important role in the development of immune responses and cancerization of cells. A clear understanding of the mechanisms underlying this involvement will provide insights into molecular-targeted therapies. N-myristoyltransferase1 (NMT1), a specific enzyme involved in myristoylation, is expressed in cancer cells and in other normal cells, suggesting that changes in myristoylation may result from the regulation of NMT1 in cancer cells. MATERIALS AND METHODS: Using newly emerging state-of-the-art techniques such as the Click-it assay, RNA interference, mass spectrometry, immunoprecipitation, immunocytochemistry, and western blotting, the expression of myristoylated proteins and the role of TNF-α stimulation on NMT1 and Sorbs2 binding were evaluated in a murine osteoblastic cell line (MC3T3-E1). RESULTS: The expression of myristoylated proteins was detected; however, TNF-α stimulation resulted in their inhibition in MC3T3-E1 cells. The expression of NMT1 also increased. Immunoprecipitation and mass spectrometry identified Sorbs2 as a novel binding protein of NMT1, which upon TNF-α stimulation, inhibited myristoylation. CONCLUSION: The binding between NMT1 and Sorbs2 can regulate myristoylation, and NMT1 can be considered as a potential target molecule for tumor invasion.

GC-MS Fingerprinting Combined with Chemical Pattern-Recognition Analysis Reveals Novel Chemical Markers of the Medicinal Seahorse.

Seahorse is a valuable marine-animal drug widely used in traditional Chinese medicine (TCM), and which was first documented in the "Ben Cao Jing Ji Zhu" during the Liang Dynasty. Hippocampus kelloggi (HK) is the most common seahorse species in the medicinal material market and is one of the genuine sources of medicinal seahorse documented in the Chinese pharmacopeia. It is mainly cultivated in the Shandong, Fujian, and Guangxi Provinces in China. However, pseudo-HK, represented by Hippocampus ingens (HI) due to its similar appearance and traits, is often found in the market, compromising the safety and efficacy of clinical use. Currently, there is a lack of reliable methods for identifying these species based on their chemical composition. In this study, we employed, for the first time, a strategy combining gas chromatography-mass spectrometry (GC-MS) fingerprints and chemical patterns in order to identify HK and HI; it is also the first metabolomic study to date of HI as to chemical components. The obtained results revealed remarkable similarities in the chemical fingerprints, while significant differences were also observed. By employing hierarchical cluster analysis (HCA) and principal component analysis (PCA), based on the relative contents of their characteristic peaks, all 34 samples were successfully differentiated according to their species of origin, with samples from the same species forming distinct clusters. Moreover, nonadecanoic acid and behenic acid were exclusively detected in HK samples, further distinguishing them from HI samples. Additionally, the relative contents of lauric acid, tetradecanoic acid, pentadecanoic acid, n-hexadecanoic acid, palmitoleic acid, margaric acid, oleic acid, fenozan acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) exhibited significant differences between HK and HI (p < 0.0001), as determined by an unpaired t-test. Orthogonal partial least squares discriminant analysis (OPLS-DA) identified seven components (DHA, EPA, n-hexadecanoic acid, tetradecanoic acid, palmitoleic acid, octadecanoic acid, and margaric acid) with high discriminatory value (VIP value > 1). Thus, nonadecanoic acid, behenic acid, and these seven compounds can be utilized as chemical markers for distinguishing HK from HI. In conclusion, our study successfully developed a combined strategy of GC-MS fingerprinting and chemical pattern recognition for the identification of HK and HI, and we also discovered chemical markers that can directly differentiate between the two species. This study can provide a foundation for the authentication of Hippocampus and holds significant importance for the conservation of wild seahorse resources.

Protein N-myristoylation plays a critical role in the mitochondrial localization of human mitochondrial complex I accessory subunit NDUFB7.

The present study examined human N-myristoylated proteins that specifically localize to mitochondria among the 1,705 human genes listed in MitoProteome, a mitochondrial protein database. We herein employed a strategy utilizing cellular metabolic labeling with a bioorthogonal myristic acid analog in transfected COS-1 cells established in our previous studies. Four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were identified as N-myristoylated proteins that specifically localize to mitochondria. Among these proteins, DMAC1 and NDUFB7 play critical roles in the assembly of complex I of the mitochondrial respiratory chain. DMAC1 functions as an assembly factor, and NDUFB7 is an accessory subunit of complex I. An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on NDUFB7 was important for the mitochondrial localization of this protein. Furthermore, an analysis of the role of the CHCH domain in NDUFB7 using Cys to Ser mutants revealed that it was essential for the mitochondrial localization of NDUFB7. Therefore, the present results showed that NDUFB7, a vital component of human mitochondrial complex I, was N-myristoylated, and protein N-myrisotylation and the CHCH domain were both indispensable for the specific targeting and localization of NDUFB7 to mitochondria.

Application of Liquid-Liquid Extraction for N-terminal Myristoylation Proteomics.

Proteins can be modified by lipids in various ways, for example, by myristoylation, palmitoylation, farnesylation, and geranylgeranylation-these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable to in vivo systems. Here, we introduce a proteomic methodology for global analysis of endogenous protein N-terminal myristoylation sites that combines liquid-liquid extraction of hydrophobic lipidated peptides with liquid chromatography-tandem mass spectrometry using a gradient program of acetonitrile in the high concentration range. We applied this method to explore myristoylation sites in HeLa cells and identified a total of 75 protein N-terminal myristoylation sites, which is more than the number of high-confidence myristoylated proteins identified by myristic acid analog-based chemical proteomics. Isolation of myristoylated peptides from HeLa digests prepared with different proteases enabled the identification of different myristoylated sites, extending the coverage of N-myristoylome. Finally, we analyzed in vivo myristoylation sites in mouse tissues and found that the lipidation profile is tissue-specific. This simple method (not requiring chemical labeling or affinity purification) should be a promising tool for global profiling of protein N-terminal myristoylation.

Capric Acid and Myristic Acid Permeability Enhancers in Curved Liposome Membranes.

Liposomes are considered as advanced drug delivery systems for cancer treatment. A generation of pH-sensitive liposomes is being developed that use fatty acids (FAs) as a trigger for drug release in tumor tissues. However, FAs are also known to enhance permeability, and it is unclear whether FAs in liposomes may cause drug leakage or premature drug release. The passive permeability of the drug through the membrane of the liposome is thus a crucial factor for timely drug delivery. To investigate how the curvature and lipid composition of liposomes affect their passive permeability, coarse-grained molecular dynamics were performed. The permeability was determined with a counting method. Flat bilayers and three liposomes with varying diameters were studied, which had varying lipid compositions of dipalmitoylphosphatidylcholine, cholesterol, and deprotonated or neutral saturated FAs. The investigated permeants were water and two other small permeants, which have different free energy profiles (solubility) across the membrane. First, for the curvature effect, our results showed that curvature increases the water permeability by reducing the membrane thickness. The permeability increase for water is about a factor of 1.7 for the most curved membranes. However, a high curvature decreases permeability for permeants with free energy profiles that are a mix of wells and barriers in the headgroup region of the membrane. Importantly, the type of experimental setup is expected to play a dominant role in the permeability value, i.e., whether permeants are escaping or entering the liposomes. Second, for the composition effect, FAs decrease both the area per lipid (APL) and the membrane thickness, resulting in permeability increases of up to 55%. Cholesterol has a similar effect on the APL but has the opposite impact on membrane thickness and permeability. Therefore, FAs and cholesterol have opposing effects on permeability, with cholesterol's effect being slightly stronger in our simulated bilayers. As all permeability values were well within a factor of 2, and with liposomes usually being larger and less curved in experimental applications, it can be concluded that the passive drug release from a pH-sensitive liposome does not seem to be significantly affected by the presence of FAs.

Serum, Urine, and Fecal Metabolome Alterations in the Gut Microbiota in Response to Lifestyle Interventions in Pediatric Obesity: A Non-Randomized Clinical Trial.

Pediatric obesity is associated with alterations in the gut microbiota and its metabolites. However, how they influence obesity and the effect of lifestyle interventions remains unknown.. In this non-randomized clinical trial, we analyzed metabolomes and microbial features to understand the associated metabolic pathways and the effect of lifestyle interventions on pediatric obesity. Anthropometric/biochemical data and fasting serum, urine, and fecal samples were collected at baseline and after an eight-week, weight-reduction lifestyle modification program. Post-intervention, children with obesity were classified into responder and non-responder groups based on changes in total body fat. At baseline, serum L-isoleucine and uric acid levels were significantly higher in children with obesity compared with those in normal-weight children and were positively correlated with obesogenic genera. Taurodeoxycholic and tauromuricholic α + ß acid levels decreased significantly with obesity and were negatively correlated with obesogenic genera. Branched-chain amino acid and purine metabolisms were distinguished metabolic pathways in the obese group. Post-intervention, urinary myristic acid levels decreased significantly in the responder group, showing a significant positive correlation with Bacteroides. Fatty acid biosynthesis decreased significantly in the responder group. Thus, lifestyle intervention with weight loss is associated with changes in fatty acid biosynthesis, and myristic acid is a possible therapeutic target for pediatric obesity.

Anti-inflammatory effects of myristic acid mediated by the NF-κB pathway in lipopolysaccharide-induced BV-2 microglial cells.

Parkinson's disease (PD) is a serious neurodegenerative disorder wherein changes in metabolites related to lipids, glutathione, and energy metabolism occur. Currently, metabolite changes in PD have been reported, yet their role in the prognosis of disease remains poorly understood. Functional metabolites can be used to diagnose diseases, especially PD, and can exert neuroprotective effects. This study used a PD animal model and a lipopolysaccharide (LPS)-mediated inflammatory response model (using the BV-2 mouse microglial cell line) to identify functional metabolites that can identify important metabolic disorders during PD, and comprehensively evaluated their profiles using a metabolomics-based approach. Our results showed that co-treatment with myristic acid and heptadecanoic acid downregulated the expression of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α in BV-2 cells. Additionally, myristic acid and 10 µM heptadecanoic acid significantly inhibited the LPS-induced inflammatory response through the nuclear factor-κB pathway in BV-2 microglial cells, which provides a potential approach for PD treatment. Myristic acid and heptadecanoic acid were the active metabolites found by active metabolomics technology, but at present, there is no research report about their function for PD treatment, and our findings offer a novel research strategy for PD diagnosis and treatment.

Non-Cardiotoxic Tetradecanoic Acid-2,4-Dinitrophenol Ester Nanomicelles in Microneedles Exert Potent Anti-Obesity Effect by Regulating Adipocyte Browning and Lipogenesis.

Sustained oral uncoupler 2,4-dinitrophenol (DNP) administration exerts prominent anti-obesity effects, but the adipose tissue off-target disadvantage leads to systemic adverse effects. A novel non-cardiotoxicity DNP delivery method using a biocompatible microneedles patch containing the amphiphilic tetradecanoic acid-DNP ester (TADNP) is described, which is synthesized via esterification on the phenolic hydroxyl of DNP. The TADNP is self-assembled as nanomicelles, which enhance the endocytosis rate of DNP by adipocytes and its permeation in isolated adipose tissues. The microenvironment of adipose tissues promotes the massive release of DNP and plasma and simulated gastrointestinal fluids. The microneedles-delivered TADNP nanomicelles (MN-TADNP) effectively deliver DNP in treated adipose tissues and reduce DNP content in off-target organs. Both oral and MN patch-delivered TADNP micelles effectively exert anti-obesity effects in a mouse model of high-fat diet-induced obesity; and noteworthily, MN-TADNP exhibit more satisfactory biosafety than oral administration. Here, a smart MN patch loaded with tetradecanoic acid-modified DNP is reported, which enhances its accumulation in adipose tissues and exerts an anti-obesity effect without causing any systemic toxicity.

Regulated N-Terminal Modification of Proteins Synthesized Using a Reconstituted Cell-Free Protein Synthesis System.

The N-terminal modification of nascent proteins, such as acetylation and myristoylation, is one of the most abundant post-translational modifications. To analyze the function of the modification, it is important to compare the modified and unmodified proteins under defined conditions. However, it is technically difficult to prepare unmodified proteins because cell-based systems contain endogenous modification systems. In this study, we developed a cell-free method to conduct N-terminal acetylation and myristoylation of nascent proteins in vitro using a reconstituted cell-free protein synthesis system (PURE system). Proteins synthesized using the PURE system were successfully acetylated or myristoylated in a single-cell-free mixture in the presence of modifying enzymes. Furthermore, we performed protein myristoylation in giant vesicles, which resulted in their partial localization to the membrane. Our PURE-system-based strategy is useful for the controlled synthesis of post-translationally modified proteins.

Small-angle x-ray scattering investigation of the integration of free fatty acids in polysorbate 20 micelles.

A critical quality attribute for liquid formulations is the absence of visible particles. Such particles may form upon polysorbate hydrolysis resulting in release of free fatty acids into solution followed by precipitation. Strategies to avoid this effect are of major interest for the pharmaceutical industry. In this context, we investigated the structural organization of polysorbate micelles alone and upon addition of the fatty acid myristic acid (MA) by small-angle x-ray scattering. Two complementary approaches using a model of polydisperse core-shell ellipsoidal micelles and an ensemble of quasiatomistic micelle structures gave consistent results well describing the experimental data. The small-angle x-ray scattering data reveal polydisperse mixtures of ellipsoidal micelles containing about 22-35 molecules per micelle. The addition of MA at concentrations up to 100 µg/mL reveals only marginal effects on the scattering data. At the same time, addition of high amounts of MA (>500 µg/mL) increases the average sizes of the micelles indicating that MA penetrates into the surfactant micelles. These results together with molecular modeling shed light on the polysorbate contribution to fatty acid solubilization preventing or delaying fatty acid particle formation.

Biochemical and structural analysis of N-myristoyltransferase mediated protein tagging.

N-terminal myristoylation is an essential eukaryotic modification crucial for cellular homeostasis in the context of many physiological processes. Myristoylation is a lipid modification resulting in a C14 saturated fatty acid addition. This modification is challenging to capture due to its hydrophobicity, low abundance of target substrates, and the recent discovery of unexpected NMT reactivity including myristoylation of lysine side chains and N-acetylation in addition to classical N-terminal Gly-myristoylation. This chapter details the high-end approaches developed to characterize the different features of N-myristoylation and its targets through in vitro and in vivo labeling.

Use of alkyne-tagged myristic acid to detect N-terminal myristoylation.

Protein N-terminal myristoylation is a lipidic modification typically occurring to the α-amino group of N-terminal glycine residues of proteins. It is catalyzed by the N-myristoyltransferase (NMT) enzyme family. Many studies in the past three decades have highlighted the importance of N-terminal glycine myristoylation as it affects protein localization, protein-protein interaction, and protein stability, thereby regulating multiple biological processes, including immune cell signaling, cancer progression, and infections. This book chapter will present protocols for using alkyne-tagged myristic acid to detect the N-myristoylation of targeted proteins in cell lines and compare global N-myristoylation levels. We then described a protocol of SILAC proteomics that compare the levels of N-myristoylation on a proteomic scale. These assays allow for the identification of potential NMT substrates and the development of novel NMT inhibitors.

The role of N-myristoyltransferase 1 in tumour development.

N-myristoyltransferase 1 (NMT1) is an indispensable eukaryotic enzyme that catalyses the transfer of myristoyl groups to the amino acid terminal residues of numerous proteins. This catalytic process is required for the growth and development of many eukaryotes and viruses. Elevated expression and activity of NMT1 is observed to varying degrees in a variety of tumour types (e.g. colon, lung and breast tumours). Furthermore, an elevated level of NMT1 in tumours is associated with poor survival. Therefore, a relationship exists between NMT1 and tumours. In this review, we discuss the underlying mechanisms by which NMT1 is associated with tumour development from the perspective of oncogene signalling, involvement in cellular metabolism, and endoplasmic reticulum stress. Several NMT inhibitors used in cancer treatment are introduced. The review will provide some directions for future research.Key MessagesElevated expression and activity of NMT1 is observed to varying degrees in a variety of tumour types which creates the possibility of targeting NMT1 in tumours.NMT1-mediated myristoylation plays a pivotal role in cancer cell metabolism and may be particularly relevant to cancer metastasis and drug resistance. These insights can be used to direct potential therapeutic avenues for NMT1 inhibitors.

Myristic acid as a checkpoint to regulate STING-dependent autophagy and interferon responses by promoting N-myristoylation.

Stimulator of interferon gene (STING)-triggered autophagy is crucial for the host to eliminate invading pathogens and serves as a self-limiting mechanism of STING-induced interferon (IFN) responses. Thus, the mechanisms that ensure the beneficial effects of STING activation are of particular importance. Herein, we show that myristic acid, a type of long-chain saturated fatty acid (SFA), specifically attenuates cGAS-STING-induced IFN responses in macrophages, while enhancing STING-dependent autophagy. Myristic acid inhibits HSV-1 infection-induced innate antiviral immune responses and promotes HSV-1 replication in mice in vivo. Mechanistically, myristic acid enhances N-myristoylation of ARF1, a master regulator that controls STING membrane trafficking. Consequently, myristic acid facilitates STING activation-triggered autophagy degradation of the STING complex. Thus, our work identifies myristic acid as a metabolic checkpoint that contributes to immune homeostasis by balancing STING-dependent autophagy and IFN responses. This suggests that myristic acid and N-myristoylation are promising targets for the treatment of diseases caused by aberrant STING activation.

Blockade of NMT1 enzymatic activity inhibits N-myristoylation of VILIP3 protein and suppresses liver cancer progression.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Identification of the underlying mechanism of HCC progression and exploration of new therapeutic drugs are urgently needed. Here, a compound library consisting of 419 FDA-approved drugs was taken to screen potential anticancer drugs. A series of functional assays showed that desloratadine, an antiallergic drug, can repress proliferation in HCC cell lines, cell-derived xenograft (CDX), patient-derived organoid (PDO) and patient-derived xenograft (PDX) models. N-myristoyl transferase 1 (NMT1) was identified as a target protein of desloratadine by drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR) assays. Upregulation of NMT1 expression enhanced but NMT1 knockdown suppressed tumor growth in vitro and in vivo. Metabolic labeling and mass spectrometry analyses revealed that Visinin-like protein 3 (VILIP3) was a new substrate of NMT1 in protein N-myristoylation modification, and high NMT1 or VILIP3 expression was associated with advanced stages and poor survival in HCC. Mechanistically, desloratadine binds to Asn-246 in NMT1 and inhibits its enzymatic activity, disrupting the NMT1-mediated myristoylation of the VILIP3 protein and subsequent NFκB/Bcl-2 signaling. Conclusively, this study demonstrates that desloratadine may be a novel anticancer drug and that NMT1-mediated myristoylation contributes to HCC progression and is a potential biomarker and therapeutic target in HCC.