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.

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.

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.

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.

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.

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.

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.

Dual Role of ACBD6 in the Acylation Remodeling of Lipids and Proteins.

The transfer of acyl chains to proteins and lipids from acyl-CoA donor molecules is achieved by the actions of diverse enzymes and proteins, including the acyl-CoA binding domain-containing protein ACBD6. N-myristoyl-transferase (NMT) enzymes catalyze the covalent attachment of a 14-carbon acyl chain from the relatively rare myristoyl-CoA to the N-terminal glycine residue of myr-proteins. The interaction of the ankyrin-repeat domain of ACBD6 with NMT produces an active enzymatic complex for the use of myristoyl-CoA protected from competitive inhibition by acyl donor competitors. The absence of the ACBD6/NMT complex in ACBD6.KO cells increased the sensitivity of the cells to competitors and significantly reduced myristoylation of proteins. Protein palmitoylation was not altered in those cells. The specific defect in myristoyl-transferase activity of the ACBD6.KO cells provided further evidence of the essential functional role of the interaction of ACBD6 with the NMT enzymes. Acyl-CoAs bound to the acyl-CoA binding domain of ACBD6 are acyl donors for the lysophospholipid acyl-transferase enzymes (LPLAT), which acylate single acyl-chain lipids, such as the bioactive molecules LPA and LPC. Whereas the formation of acyl-CoAs was not altered in ACBD6.KO cells, lipid acylation processes were significantly reduced. The defect in PC formation from LPC by the LPCAT enzymes resulted in reduced lipid droplets content. The diversity of the processes affected by ACBD6 highlight its dual function as a carrier and a regulator of acyl-CoA dependent reactions. The unique role of ACBD6 represents an essential common feature of (acyl-CoA)-dependent modification pathways controlling the lipid and protein composition of human cell membranes.

Blocking AMPK ß1 myristoylation enhances AMPK activity and protects mice from high-fat diet-induced obesity and hepatic steatosis.

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis and a therapeutic target for metabolic diseases. Co/post-translational N-myristoylation of glycine-2 (Gly2) of the AMPK ß subunit has been suggested to regulate the distribution of the kinase between the cytosol and membranes through a "myristoyl switch" mechanism. However, the relevance of AMPK myristoylation for metabolic signaling in cells and in vivo is unclear. Here, we generated knockin mice with a Gly2-to-alanine point mutation of AMPKß1 (ß1-G2A). We demonstrate that non-myristoylated AMPKß1 has reduced stability but is associated with increased kinase activity and phosphorylation of the Thr172 activation site in the AMPK α subunit. Using proximity ligation assays, we show that loss of ß1 myristoylation impedes colocalization of the phosphatase PPM1A/B with AMPK in cells. Mice carrying the ß1-G2A mutation have improved metabolic health with reduced adiposity, hepatic lipid accumulation, and insulin resistance under conditions of high-fat diet-induced obesity.

Inhibition of vaccinia virus L1 N-myristoylation by the host N-myristoyltransferase inhibitor IMP-1088 generates non-infectious virions defective in cell entry.

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors.

SVMyr: A Web Server Detecting Co- and Post-translational Myristoylation in Proteins.

Myristoylation (MYR) is a protein modification where a myristoyl group is covalently attached to an exposed (N-terminal) glycine residue. Glycine myristoylation occurs during protein translation (co-translation) or after (post-translation). Myristoylated proteins have a role in signal transduction, apoptosis, and pathogen-mediated processes and their prediction can help in functionally annotating the fraction of proteins undergoing MYR in different proteomes. Here we present SVMyr, a web server allowing the detection of both co- and post-translational myristoylation sites, based on Support Vector Machines (SVM). The input encodes composition and physicochemical features of the octapeptides, known to act as substrates and to physically interact with N-myristoyltransferases (NMTs), the enzymes catalyzing the myristoylation reaction. The method, adopting a cross validation procedure, scores with values of Area Under the Curve (AUC) and Matthews Correlation Coefficient (MCC) of 0.92 and 0.61, respectively. When benchmarked on an independent dataset including experimentally detected 88 medium/high confidence co-translational myristoylation sites and 528 negative examples, SVMyr outperforms available methods, with AUC and MCC equal to 0.91 and 0.58, respectively. A unique feature of SVMyr is the ability to predict post-translational myristoylation sites by coupling the trained SVMs with the detection of caspase cleavage sites, identified by searching regular motifs matching upstream caspase cleavage sites, as reported in literature. Finally, SVMyr confirms 96% of the UniProt set of the electronically annotated myristoylated proteins (31,048) and identifies putative myristoylomes in eight different proteomes, highlighting also new putative NMT substrates. SVMyr is freely available through a user-friendly web server at https://busca.biocomp.unibo.it/lipipred.

Bioinformatics of Differentially Expressed Genes in Phorbol 12-Myristate 13-Acetate-Induced Megakaryocytic Differentiation of K562 Cells by Microarray Analysis.

Megakaryocytes are large hematopoietic cells present in the bone marrow cavity, comprising less than 0.1% of all bone marrow cells. Despite their small number, megakaryocytes play important roles in blood coagulation, inflammatory responses, and platelet production. However, little is known about changes in gene expression during megakaryocyte maturation. Here we identified the genes whose expression was changed during K562 leukemia cell differentiation into megakaryocytes using an Affymetrix GeneChip microarray to determine the multifunctionality of megakaryocytes. K562 cells were differentiated into mature megakaryocytes by treatment for 7 days with phorbol 12-myristate 13-acetate, and a microarray was performed using RNA obtained from both types of cells. The expression of 44,629 genes was compared between K562 cells and mature megakaryocytes, and 954 differentially expressed genes (DEGs) were selected based on a p-value < 0.05 and a fold change >2. The DEGs was further functionally classified using five major megakaryocyte function-associated clusters­inflammatory response, angiogenesis, cell migration, extracellular matrix, and secretion. Furthermore, interaction analysis based on the STRING database was used to generate interactions between the proteins translated from the DEGs. This study provides information on the bioinformatics of the DEGs in mature megakaryocytes after K562 cell differentiation.

Reduced DMPC and PMPC in lung surfactant promote SARS-CoV-2 infection in obesity.

OBJECTIVE: Obesity is an established risk factor for higher SARS-CoV-2 viral loads, severe COVID-19 pneumonia requiring hospitalization, and worse outcomes. However, the underlying mechanisms for the increased risk are not well understood. SARS-CoV-2 is a respiratory virus with the primary route of entry through the lungs, where the Spike protein of SARS-CoV-2 binds to the ACE2 receptor on pneumocytes. Lung surfactant produced by type II pneumocytes plays a major role in respiratory defense against infections. Surfactant predominantly contains lipids, especially phosphatidylcholines (PC), and obesity is characterized by aberrant lipid metabolism. We hypothesized that altered lipid composition in lung surfactant in obesity may promote SARS-CoV-2 infection, leading to severe COVID-19 disease. METHODS: Lipidomic analysis of lung tissue and bronchoalveolar lavage fluid (BALF) was performed using LC-MS/MS. The effects of PCs on SARS-CoV-2 pseudovirus infection were studied in HEK293T cells with ACE2 overexpression and in Vero-E6 cells with endogenous ACE2 expression. For the cell-cell fusion assay, HEK293T-ACE2 and HEK293T expressing SARS-CoV-2 Spike/eGFP were used as the target and effector cells, respectively. RESULTS: Lipidomic analysis revealed that myristic acid-containing dimyristoyl-PC (DMPC) and palmitoylmyristoyl-PC (PMPC) were reduced in lung tissue and BALF from high fat diet-induced obese mice. DMPC and PMPC markedly inhibited wild type and D614G mutant SARS-CoV-2 infection in HEK293T-ACE2 and Vero-E6 cells. Feeding obese mice with trimyristin, the triglycerides of myristic acid, increased DMPC and PMPC levels in lung surfactant. Lipid extract from BALF of trimyristin-treated obese mice mitigated the elevated wild type and D614G mutant SARS-CoV-2 infection. The inhibitory effects of DMPC and PMPC on SARS-CoV-2 infection were reversed by cholesterol. CONCLUSIONS: The reduced DMPC and PMPC in lung surfactant may promote SARS-CoV-2 infection. Increasing DMPC and PMPC in lung surfactant could be an innovative strategy for preventing and treating severe COVID-19 disease in obesity.

Phorbol 12-myristate 13-acetate stimulation under hypoxia induces nuclear swelling with DNA outflow but not extracellular trap formation of neutrophils.

Neutrophils stand sentinel over infection and possess diverse antimicrobial weapons, including neutrophil extracellular traps (NETs). NETs are composed of web-like extracellular DNA decorated with antimicrobial substances and can trap and eliminate invading microorganisms. Although phorbol 12-myristate 13-acetate (PMA) is a potent NET inducer, previous studies have demonstrated that not all neutrophils exhibit NET formation even if stimulated by PMA at high concentrations. This study first showed that some neutrophils stimulated by PMA displayed a swollen nucleus but not NET formation and that hypoxic environments suppressed the NET release. Next, characterization of PMA-stimulated neutrophils with a swollen nucleus was accomplished by differentiating between suicidal-type NETosis and apoptosis. Furthermore, the significance of the phenomenon was examined using formalin-fixed, paraffin-embedded human lung disease tissues with and without pneumonia. As a result, histone H3 citrullination, DNA outflow, propidium iodide labeling, resistance to DNase I, and suspended actin rearrangement were characteristics of PMA-stimulated neutrophils with a swollen nucleus distinct from neutrophils that underwent either suicidal-type NETosis or apoptosis. Neutrophils stimulated by PMA under hypoxic conditions secreted matrix metalloproteinase-9 cytotoxic to human lung-derived fibroblasts. Further, deposition of neutrophil-derived citrullinated histone H3+ chromatin substances in pulmonary lesions was greater in patients with pneumonia than in patients without pneumonia and positively correlated with hypoxia-inducible factor-1α expression. The collective findings suggested that neutrophils activated under hypoxic conditions could be putative modulators of hypoxia-related disease manifestations.

Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: The myristoylated pre-S1 peptide (Myr47) synthesized to mimic pre-S1 domain (2-48) in large (L) surface protein of hepatitis B virus (HBV) prevents HBV infection to hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP). We previously demonstrated that yeast-derived nanoparticles containing L protein (bio-nanocapsules: BNCs) bind scavenger receptor class B type 1 (SR-B1). In this study, we examined the binding of Mry47 to SR-B1. (2) Methods: The binding and endocytosis of fluorescence-labeled Myr47 to SR-B1 (and its mutants)-green fluorescence protein (GFP) fusion proteins expressed in HEK293T cells were analyzed using flow cytometry and laser scanning microscopy (LSM). Various ligand-binding properties were compared between SR-B1-GFP and NTCP-GFP. Furthermore, the binding of biotinylated Myr47 to SR-B1-GFP expressed on HEK293T cells was analyzed via pull-down assays using a crosslinker and streptavidin-conjugated beads. (3) Conclusions: SR-B1 bound not only Myr47 but also its myristoylated analog and BNCs, but failed to bind a peptide without myristoylation. However, NTCP only bound Myr47 among the ligands tested. Studies using SR-B1 mutants suggested that both BNCs and Myr47 bind to similar sites of SR-B1. Crosslinking studies indicated that Myr47 binds preferentially SR-B1 multimer than monomer in both HEK293T and HepG2 cells.

Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus.

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial interaction between fungi and land plants and promotes global phosphate cycling in terrestrial ecosystems. AM fungi are recognised as obligate symbionts that require root colonisation to complete a life cycle involving the production of propagules, asexual spores. Recently, it has been shown that Rhizophagus irregularis can produce infection-competent secondary spores asymbiotically by adding a fatty acid, palmitoleic acid. Furthermore, asymbiotic growth can be supported using myristate as a carbon and energy source for their asymbiotic growth to increase fungal biomass. However, the spore production and the ability of these spores to colonise host roots were still limited compared to the co-culture of the fungus with plant roots. Here we show that a combination of two plant hormones, strigolactone and jasmonate, induces the production of a large number of infection-competent spores in asymbiotic cultures of Rhizophagus clarus HR1 in the presence of myristate and organic nitrogen. Inoculation of asymbiotically-generated spores promoted the growth of host plants, as observed for spores produced by symbiotic culture system. Our findings provide a foundation for the elucidation of hormonal control of the fungal life cycle and the development of inoculum production schemes.

A metabolic labeling protocol to enrich myristoylated proteins from Caenorhabditis elegans.

Myristoylation is a type of lipidation with important functions. Owing to the lack of high-quality antibodies against myristoylation, developing alternative methods for profiling myristoylated proteins is important. Here, we provide a protocol for metabolic labeling using click chemistry to profile myristoylated proteins in C. elegans. Our approach improves the signal/noise ratio by covalently linking the myristoylated proteins to the beads. This protocol provides a highly specific and reproducible way for enriching myristoylated proteins, which could be modified to analyze other types of lipidations. For complete details on the use and execution of this protocol, please refer to Tang et al. (2021).

Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion.

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of "pseudoschizonts," which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.