Expression Of N-myristoyltransferase Research Articles

N-Myristoyltransferase: A Novel Therapeutic Target for Cancer, Inflammation and Radiation Response Modifications

N-Myristoyltransferase (NMT) is a well conserved monomeric protein that catalyzes myristoyl group transfer to various target proteins carrying consensus amino acid sequence motif GXXXS/T (where ‘X’ is any amino acid). NMT mediated myristoylation promotes protein-protein and protein-membrane interactions, leading to diverse signaling including cell proliferation, carcinogenesis, inflammation and immune responses. However, inter-networking linkage between NMT over-expression and protein myristoylation under oxidative stress coupled with inflammation and carcinogenesis is completely missing and needs further investigation. Various types of tumor and cancer tissue showed significant increase in NMT expression. Therefore, inhibition of NMT expression either by using chemical NMT inhibitors or gene silencing could be a promising approach in controlling carcinogenesis. Various scientific evidence suggests that NMT mediated myristoylation promotes membrane association of some cell cycle and cell proliferation regulatory proteins and thus may contribute to cell cycle regulation and cell survival homeostasis. Several NMT inhibitors have been evaluated and tested for their probable anticancer potential. Molecular linkage between oxidative stress and carcinogenesis has been established in several studies. In this review, the authors cover the role and therapeutic potential of NMT in cancer, inflammatory disease and radiation response modulation.

Targeting N-Myristoylation for Adult Acute Myeloid Leukemia Therapy

Protein N-myristoylation is the covalent attachment of myristic acid (C14:0) by an amide linkage to the NH2-terminal glycine residue of numerous eukaryotic and viral proteins, thereafter regulating their membrane targeting or enzymatic activity. A single myristoyl-CoA: protein N-myristoyltransferase (NMT) isoform catalyses the acylation reaction in yeast, insects, plants, and unicellular parasites. It is two separate isoforms however; NMT1 and NMT2, with predominantly overlapping substrates that perform this task in mammals. Myristoylated proteins that fail to undergo the modification are recognised by a dedicated glycine N-degron system and rapidly degraded. We recently identified a remarkable reduction of NMT2 expression in a wide variety of cancers, especially hematological cancers. As a consequence, we analysed if NMT1/NMT2 could be associated with AML progression. Data analysis from the TCGA transcriptome database showed that high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML, and high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. One oncogenic myristoylated target identified initially in B-cell lymphoma is the family of Src kinases (SFKs). Here, we demonstrate that PCLX-001 significantly reduces total and phosphorylated SFKs, which are essential for pro-survival signalling downstream frequently mutated c-Kit and FLT3 receptors in multiple AML cell lines. Moreover, inhibiting myristoylation led to ER stress induction and apoptosis in cell lines and patient blast samples at concentrations sparing most peripheral blood lymphocytes and monocytes from healthy individuals. We further validated the therapeutic potential of our lead myristoylation inhibitor ongoing clinical trial evaluation against lymphoma and solid tumours (PCLX-001) in a variety of AML xenograft models. PCLX-001 monotherapy had a dose-dependent tumoricidal effect in an ectopic sub-cutaneous MV-4-11 xenograft model, in two tail vein injected patient derived xenografts (PDXs) as well as in 3 out of 5 intra-femoral PDXs. Of note, five days of PCLX-001 treatment at the highest concentration was sufficient to obtain complete tumour eradication in our CDX model. In tail-vein injected PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow with minimal toxicity or long-term impact on body weight (Fig.1). Further analyses are required to characterize the cytogenetics, common mutations or NMTs expression profile that could explain the sensitivity of certain patient models compared to the others. An ongoing challenge in treatment of leukemia resides in leukemic stem cells (LSCs), a low-frequency sub-population functioning as progenitor cells. Without eradication of LSCs no patient can be cured. Relapse rates are high in AML and are often featuring additional mutations and drug resistance. Here, we demonstrate that LSC subpopulations (CD34+/CD38- and CD117+/ CD15-) in the OCI-AML-22 patient derived model are exquisitely sensitive to PCLX-001 treatment in vitro and began shrinking after 48 hours of treatment. In addition, the LSC subpopulation showed lower IC50 compared to the general cell population at 72 hours. Interestingly, we identified that functionally validated LSC+ patient subpopulations were expressing lower amount of NMT2 compared to LSC-. This observation could partially explain the sensitivity of the LSC subpopulation. In aggregate, our preclinical observations suggest that PCLX-001 is highly potent against AML cells in vitro and in vivo (both circulating and in the bone marrow), and, that LSCs are also more sensitive to PCLX-001 than non-leukemic stem cells. This further supports the initiation of PCLX-001evaluation in AML clinical trials scheduled to start in late 2022. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nuclear localization of endothelial nitric oxide synthase and nitric oxide production attenuates aphidicolin-induced endothelial cell death

Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays an essential role in maintenance of endothelial integrity including endothelial cell (EC) survival. Previously, we reported that aphidicolin increases NO production in bovine aortic ECs (BAECs). However, the role of aphidicolin-induced NO on EC viability and its molecular mechanism remain to be elucidated. Treatment with 20 μM aphidicolin for 24 h reduced BAEC viability by ~40%, which was accompanied by increased NO production, phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179), and eNOS protein expression. The aphidicolin-increased eNOS expression and p-eNOS-Ser1179 were not altered by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable and specific intracellular Ca2+ chelator. Co-treatment with 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), an NO scavenger, or Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor, exacerbated aphidicolin-stimulated BAEC death. Knockdown of eNOS gene expression using siRNA aggravated aphidicolin-induced BAEC death. However, exogenous NO donors including S-nitroso-l-glutathione (GSNO) or diethylenetriamine NONOate (DETA NO) had no effect on aphidicolin-decreased BAEC viability and aggravated BAEC viability at higher doses. Interestingly, aphidicolin accumulated eNOS protein in the active form, p-eNOS-Ser1179, in the nucleus. When cells were ectopically transfected with a wild-type (WT)-eNOS gene, aphidicolin induced significant localization of the protein product in the nucleus. Additionally, aphidicolin-elicited cell death was significantly reversed in WT-eNOS gene-transfected BAECs. Furthermore, overexpression of the eNOS gene containing nuclear localization signal (NLS) but not nuclear export signal (NES) significantly attenuated aphidicolin-induced BAEC death. When G2A-eNOS mutant lacking myristoylation at Gly2 was transfected, its intracellular distribution became diffuse and included the nucleus. Finally, expression of N-myristoyltransferase 2 (NMT2) but not NMT1 significantly decreased in aphidicolin-treated BAECs. Taken together, our results suggest that aphidicolin attenuates BAEC death in part by increasing nuclear eNOS localization and NO production.

Inhibition of N-myristoyltransferase1 affects dengue virus replication.

Dengue virus (DENV) causes dengue fever, a self‐limiting disease that could be fatal due to serious complications. No specific treatment is currently available and the preventative vaccine is only partially protective. To develop a potential drug target for dengue fever, we need to understand its biology and pathogenesis thoroughly. N‐myristoyltransferase (NMT) is an N‐terminal protein lipidation enzyme that catalyzes the covalent cotranslational attachment of fatty acids to the amino‐terminal glycine residue of a number of proteins, leading to the modulation of various signaling molecules. In this study, we investigated the interaction of dengue viral proteins with host NMT and its subsequent effect on DENV. Our bioinformatics, molecular docking, and far‐western blotting analyses demonstrated the interaction of viral envelope protein (E) with NMT. The gene expression of NMT was strongly elevated in a dependent manner during the viral replication phase in dendritic cells. Moreover, NMT gene silencing significantly inhibited DENV replication in dendritic cells. Further studies investigating the target cell types of other host factors are suggested.

A Tetracycline-Repressible Transactivator System to Study Essential Genes in Malaria Parasites

SummaryA major obstacle in analyzing gene function in apicomplexan parasites is the absence of a practical regulatable expression system. Here, we identified functional transcriptional activation domains within Apicomplexan AP2 (ApiAP2) family transcription factors. These ApiAP2 transactivation domains were validated in blood-, liver-, and mosquito-stage parasites and used to create a robust conditional expression system for stage-specific, tetracycline-dependent gene regulation in Toxoplasma gondii, Plasmodium berghei, and Plasmodium falciparum. To demonstrate the utility of this system, we created conditional knockdowns of two essential P. berghei genes: profilin (PRF), a protein implicated in parasite invasion, and N-myristoyltransferase (NMT), which catalyzes protein acylation. Tetracycline-induced repression of PRF and NMT expression resulted in a dramatic reduction in parasite viability. This efficient regulatable system will allow for the functional characterization of essential proteins that are found in these important parasites.

Expression and activity of N-myristoyltransferase in lung inflammation of cattle and its role in neutrophil apoptosis

N-myristoyltransferase (NMT) attaches a 14 carbon fatty acid, myristic acid, to the N-terminal glycine residue of proteins. NMT exists in two isoforms NMT1 and NMT2. Myristoylated proteins play critical roles in protein–protein interactions, cell signaling and oncogenesis. Although elevated expression of NMT1 has been described in colorectal carcinoma, its expression and roles in normal and inflamed lungs of the cattle are unknown. Therefore, we investigated the expression and activity of NMT in a bovine model of lung inflammation induced with Mannheimia hemolytica and in vitro in neutrophils and macrophages. Western blots revealed increased expression of NMT1 in lungs from infected animals compared to control animals. Total NMT activity was reduced in inflamed lungs compared to control animals (p < 0.05) along with increased expression of enolase, a putative inhibitor of NMT. NMT1 staining was observed in the septum, vascular endothelium and the epithelium in the lungs from control as well as infected calves. NMT1 expression was intense in neutrophils in the necrotic areas in the inflamed lungs. Immuno-electron microscopy localized NMT1 in cytoplasm and nuclei of endothelium, pulmonary intravascular macrophages and airway epithelium. Total NMT activity and NMT1 expression were increased in neutrophils and macrophages exposed to Escherichia coli LPS in vitro. NMT knockdown increased apoptosis in activated neutrophils. This is the first report demonstrating expression of NMT in normal and inflamed lungs and a novel role for NMT in regulation of neutrophil lifespan.

Myristoyl-CoA:protein N-myristoyltransferase depletion in trypanosomes causes avirulence and endocytic defects

The enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyses the co-translational covalent attachment of the fatty acid myristate to the N-terminus of target proteins. NMT is known to be essential for viability in Trypanosoma brucei and Leishmania major. Here we describe phenotypic analysis of T. brucei bloodstream form cells following knockdown of NMT expression by tetracycline-inducible RNA interference. Cell death occurs from 72h post-induction, with approximately 50% of cells displaying a defect in endocytic uptake by this time. The majority of these induced cells do not have an enlarged flagellar pocket typical of a block in endocytosis but vesicle accumulation around the flagellar pocket indicates a defect in vesicular progression following endocytic fusion. Induced parasites have a wild-type or slightly enlarged Golgi apparatus, unlike the phenotype of cells with reduced expression of a major N-myristoylated protein, ARL1. Critically we show that following NMT knockdown, T. brucei bloodstream form cells are unable to establish an infection in a mouse model, therefore providing further validation of this enzyme as a target for drug development.

N-myristoyltransferase: A potential novel diagnostic marker for colon cancer

BackgroundColon cancer is the second leading cause of cancer deaths in the western world. If detected early, colorectal cancer is one of the most treatable forms of cancer. Unfortunately, very few people are screened. N-myristoyltransferase (NMT) catalyzes myristoylation of various proteins including oncoproteins. We have demonstrated earlier the alteration of NMT activity during the progression of colorectal cancer and established NMT as a putative therapeutic target for cancer.MethodsPeripheral blood samples and bone marrow were collected from the colon cancer patients and azoxymethane induced colonic tumor rats and their controls respectively. NMT activity and expression was determined as reported earlier. Immunohistochemical studies were carried out using standard procedures.ResultsIn this study we demonstrate for the first time altered expression and localization of NMT in the peripheral blood and bone marrow in colon cancer patients. Immunohistochemical analysis revealed weak to negative staining for NMT in peripheral blood mononuclear cells (PBMC) of controls, whereas strong positivity was observed in PBMC colon cancer patients. In addition, we observed that NMT was localized mostly in the nuclei of the bone marrow (BM) mononuclear cells of the colon cancer patients, whereas NMT remained cytoplasmic in the control bone marrow specimens.ConclusionThe strikingly different NMT expression offers the basis of a potential adjunct investigative tool for screening or diagnosis of patients at risk for or suspected of having colon cancer. Furthermore, altered localization of NMT in BM of tumor bearing hosts may serve as an added investigative tool for the diagnostic purpose.

Expression of myristoyltransferase and its interacting proteins in epilepsy

N-Myristoylation is a co-translational, irreversible addition of a fatty acyl moiety to the amino terminus of many eukaryotic cellular proteins. This modification is catalyzed by N-myristoyltransferase (NMT) and is recognized to be a widespread and functionally important modification of proteins. The myristoylated Src family kinases are involved in various signaling cascades, including the N-methyl- d-aspartate receptor functions. We examined the expression of NMT and its interacting proteins to gain further insight into the mechanisms in epileptic fowl. Higher expression of NMT1 and NMT2 was observed in carrier and epileptic fowl whereas expression of heat shock cognate protein 70, an inhibitor of NMT, was lower. Furthermore, protein–protein interaction of NMT with m-calpain, caspase-3, and p53 was established. The interaction of NMT2 with caspase-3 and p53 was weak in epileptic fowl compared with normal chicks while the interaction of NMT1 with m-calpain was weak in epileptics. Understanding the regulation of NMT by specific inhibitors may help us to control the action of this enzyme on its specific substrates and may lead to improvements in the management of various neurological disorders like Alzheimer’s disease, ischemia, and epilepsy.

Regulation of &lt;I&gt;N&lt;/I&gt;-Myristoyltransferase by Novel Inhibitor Proteins

N-myristoylation ensures the proper function and intracellular trafficking of proteins. Many proteins involved in a wide variety of signaling, including cellular transformation and oncogenesis, are myristoylated. The myristoylation of proteins is catalyzed by the ubiquitously distributed eukaryotic enzyme N-myristoyltransferase (NMT). Previously, we reported that NMT activity is higher in colonic epithelial neoplasms than in normal-appearing colonic tissue and that the increase in NMT activity appears at an early stage in colonic carcinogenesis. Furthermore, we observed that NMT expression is elevated in colorectal and gallbladder carcinoma. In our laboratory, an endogenous NMT inhibitor protein (NIP71) was discovered from bovine brain that inhibited NMT activity in rat colonic tumors. Very recently we have demonstrated that the protein NIP71, which is a potential inhibitor of NMT, is homologous to heat-shock cognate protein (HSC70). In addition, we have discovered that enolase is a potent inhibitor of NMT. Further work may elucidate the role of HSC70 and/or enolase in the regulation of NMT, which may lead to the development of a gene-based therapy of colorectal cancer. The interaction of oncoproteomic and oncogenomic data sets through powerful bioinformatics will yield a comprehensive database of protein properties, which will serve as an invaluable tool for cancer researchers to understand the progress of tumorigenesis.

Expression of N-myristoyltransferase in Human Brain Tumors

N-myristoylation is a process of covalent irreversible protein modification that promotes association of proteins with membranes. Based on our previous findings of elevated N-myristoyltransferase (NMT) activity in colonic epithelial neoplasms that appears at an early stage in colonic carcinogenesis, together with elevated NMT expression in human colorectal and gallbladder carcinomas, we investigated NMT activity and protein expression of NMT1 and NMT2 in human brain tumors and documented elevated NMT activity and higher protein expressions. For the first time, we have demonstrated that NMT has the potential to be used as a marker of human brain tumors. However, further studies with larger number of patients are required to establish its role as a complementary diagnostic tool. This finding has significant implications for further understanding of biological mechanisms involved in tumorigenesis, as well as for diagnosis and therapy of human brain tumors.

Expression, localization, and correlation of N-myristoyltransferase and its inhibitor in bovine eye.

N-myristoyltransferase (NMT) is a ubiquitously distributed eukaryotic enzyme that catalyzes myristoylation of proteins. Very little is known about the process of myristoylation, particularly in the eye. In the present study, the distribution, expression, and correlation of NMT and its inhibitor (NMT inhibitor protein, NIP) in the bovine eye were investigated. Whole bovine eyes were either homogenized or regionally dissected to determine the activity and localization of NMT and NIP. Dissected tissues were homogenized, and Western blot analysis was performed using polyclonal anti-NMT and anti-NIP antibodies. The NMT activity was assayed using cAMP-dependent protein kinase or pp60(src) derived peptide as a substrate. Fresh samples were then prepared for immunohistochemical analysis and probed with polyclonal anti-NMT and anti-NIP antibodies. The total bovine eye cytosolic fraction displayed both NMT and NIP expression. NMT was present in all the regions of the eye at various levels of expression. The highest expression of NMT was in the cornea, whereas NIP was present in the retina, optic nerve, sclera, and choroid only. NIP expression was the highest in the optic nerve, sclera, and retina. NMT activity was observed in the cornea, iris, and retina after DEAE-Sepharose CL-6B column chromatography. The inhibitory activity of crude homogenate on recombinant human NMT activity was found to be greater for optic nerve and choroid. Immunohistochemistry results displayed similar findings. The varied expression of NMT in different regions of the eye reveals a regulatory relationship of NMT with NIP. These findings indicate that NMT and NIP are present in various regions of the eye and will lead to further understanding of visual signaling in ocular cells.

Increased expression of N‐myristoyltransferase in gallbladder carcinomas

BACKGROUND Activated Src, which has intrinsic protein tyrosine kinase activity, has been found in human solid tumors such as colorectal and breast carcinomas. The Src gene encodes a cytoplasmic tyrosine kinase p60src, which attaches to the inner surface of the membrane after N-terminal myristoylation and is implicated in transduction of signals to the nucleus. N-myristoyltransferase (NMT) catalyzes the biochemical modification process called N-myristoylation. To investigate whether, through Src, NMT contributes to the pathogenesis of gallbladder carcinoma, the authors investigated expression of NMT and p53 in in situ and invasive carcinomas. METHODS One hundred cases of documented gallbladder carcinoma were reviewed, and 30 cases were selected randomly to evaluate expression of NMT and p53 by immunohistochemistry in both in situ and in invasive tumor components. RESULTS Eighteen cases (60%) of gallbladder carcinoma showed moderate to strong cytoplasmic positivity for NMT with increased intensity in the invasive component, and 12 cases (40%) were negative. The in situ component revealed mild to moderate cytoplasmic staining in 20 cases (67%), whereas the normal gallbladder mucosa showed weak to negative cytoplasmic staining. Moderate to strong p53 staining was observed in 17 in situ cases (63%) and 24 invasive cases (80%). The in situ staining patterns of p53 were unrelated to the clinical outcome of the tumor. However, moderate to strong staining of the invasive component as observed in 15 cases (50%) was associated with a mean survival of 8.8 months. Amplification of intron-8 in normal gallbladder mucosa and invasive carcinoma were similar in intensity, suggesting the absence of NMT gene amplification in these tumors. CONCLUSIONS The increased expression of NMT in these tumors could be due to transcriptional activation. Tumors with increased expression of NMT and p53 were associated with poor clinical outcomes as evidenced by their mean survival times. NMT is likely to play a pathogenic role in gallbladder carcinoma. Cancer 2000;88:1992–9. © 2000 American Cancer Society.

Increased expression of N-myristoyltransferase in gallbladder carcinomas

Activated Src, which has intrinsic protein tyrosine kinase activity, has been found in human solid tumors such as colorectal and breast carcinomas. The Src gene encodes a cytoplasmic tyrosine kinase p60src, which attaches to the inner surface of the membrane after N-terminal myristoylation and is implicated in transduction of signals to the nucleus. N-myristoyltransferase (NMT) catalyzes the biochemical modification process called N-myristoylation. To investigate whether, through Src, NMT contributes to the pathogenesis of gallbladder carcinoma, the authors investigated expression of NMT and p53 in in situ and invasive carcinomas. One hundred cases of documented gallbladder carcinoma were reviewed, and 30 cases were selected randomly to evaluate expression of NMT and p53 by immunohistochemistry in both in situ and in invasive tumor components. Eighteen cases (60%) of gallbladder carcinoma showed moderate to strong cytoplasmic positivity for NMT with increased intensity in the invasive component, and 12 cases (40%) were negative. The in situ component revealed mild to moderate cytoplasmic staining in 20 cases (67%), whereas the normal gallbladder mucosa showed weak to negative cytoplasmic staining. Moderate to strong p53 staining was observed in 17 in situ cases (63%) and 24 invasive cases (80%). The in situ staining patterns of p53 were unrelated to the clinical outcome of the tumor. However, moderate to strong staining of the invasive component as observed in 15 cases (50%) was associated with a mean survival of 8.8 months. Amplification of intron-8 in normal gallbladder mucosa and invasive carcinoma were similar in intensity, suggesting the absence of NMT gene amplification in these tumors. The increased expression of NMT in these tumors could be due to transcriptional activation. Tumors with increased expression of NMT and p53 were associated with poor clinical outcomes as evidenced by their mean survival times. NMT is likely to play a pathogenic role in gallbladder carcinoma.

Expression of N-myristoyl transferase is developmentally regulated in mammary epithelial cells.

Expression of N-myristoyl transferase is developmentally regulated in mammary epithelial cells.