Regulation of N-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.     

Potential role of N-myristoyltransferase in cancer

Colorectal cancer is the second leading cause of malignant death, and better preventive strategies are needed. The treatment of colonic cancer remains difficult because of the lack of effective chemotherapeutic agents; therefore it is important to continue to search for cellular functions that can be disrupted by chemotherapeutic drugs resulting in the inhibition of the development and progression of cancer. The current knowledge of the modification of proteins by myristoylation involving myristoyl-CoA: protein N-myristoyltransferase (NMT) is in its infancy. This process is involved in the pathogenesis of cancer. We have reported for the first time that NMT activity and protein expression were higher in human colorectal cancer, gallbladder carcinoma and brain tumors. In addition, an increase in NMT activity appeared at an early stage in colonic carcinogenesis. It is conceivable therefore that NMT can be used as a potential marker for the early detection of cancer. These observations lead to the possibility of developing NMT specific inhibitors, which may be therapeutically useful. We proposed that HSC70 and/or enolase could be used as an anticancer therapeutic target. This review summarized the status of NMT in cancer which has been carried in our laboratory.     

Expression of N-myristoyltransferase inhibitor protein and its relationship to c-Src levels in human colon cancer cell lines

Earlier, we have reported that N-myristoyltransferase (NMT) activity is higher in colonic epithelial neoplasms than in normal appearing colonic tissue and that increase in NMT activity appears at an early stage in colonic carcinogenesis [Magnuson, B., Raju, R. V. S., Moyana, T. N., and Sharma, R. K. (1995) J. Natl. Cancer Inst. 87, 1630-1635]. In this study, we demonstrate increased NMT mRNA in well-differentiated adenocarcinomas. NMT and c-Src mRNA levels were generally elevated in a subset of human colon cancer cell lines. Western blotting analysis employing N-myristoyltransferase inhibitory protein (NIP(71)) antibody demonstrated low levels of NIP(71) in high-expressing c-Src cell lines and high levels of NIP(71) in low-expressing c-Src cell lines. Interestingly, down regulation of c-Src by antisense expression in the HT-29 cell line resulted in increased expression of NIP(71), suggesting c-Src may negatively regulate NIP(71) expression. Furthermore, this is the first study demonstrating the expression of NIP(71) in human colon cancer cell lines and a possible relationship to colon carcinogenesis.     

A novel inhibitor protein of N-myristoyltransferase from Escherichia coli

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal of the glycine residue of various eukaryotic and viral proteins of diverse functions. Earlier, we have demonstrated that NMT activity is elevated in colon and gall bladder cancer. Attenuation of NMT activity may prove a novel therapeutic protocol for cancer. We report here a novel inhibitor protein of NMT being expressed in Escherichia coli cells containing the human NMT gene on increasing the incubation period from 5 to 24h. The inhibitor protein was purified by SP-Sepharose column chromatography, heat treatment, ammonium sulfate precipitation, and Superose 12 HR/30 FPLC column chromatography. The inhibitor protein had an apparent molecular mass of 10kDa by gel filtration. It inhibited human NMT in a concentration-dependent manner with 50% inhibition at 640+/-4.68nM. The inhibitor protein showed no direct interaction with myristoyl-CoA and demonstrated no demyristoylase or protease activity. Therefore, we conclude that the inhibitor protein acts directly on NMT.     

A new,robust, and nonradioactive approach for exploring N-myristoylation

Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, thus promoting protein-protein and protein-membrane interactions. NMT is essential for the viability of numerous human pathogens and is also up-regulated in several tumors. Here we describe a new, nonradioactive, ELISA-based method for measuring NMT activity. After the NMT-catalyzed reaction between a FLAG-tagged peptide and azido-dodecanoyl-CoA (analog of myristoyl-CoA), the resulting azido-dodecanoyl-peptide-FLAG was coupled to phosphine-biotin by Staudinger ligation, captured by plate-bound anti-FLAG antibodies and detected by streptavidin-peroxidase. The assay was validated with negative controls (including inhibitors), corroborated by HPLC analysis, and demonstrated to function with fresh or frozen tissues. Recombinant murine NMT1 and NMT2 were characterized using this new method. This versatile assay is applicable for exploring recombinant NMTs with regard to their activity, substrate specificity, and possible inhibitors as well as for measuring NMT-activity in tissues.     

The modulation of choline phosphoglyceride metabolism in human colon cancer

Colorectal cancer has a high incidence of morbidity and mortality in the North American population. Elevated levels of plasmalogens have been reported in some neoplastic tissues including colon tumors, but the mechanism for this increase has not been defined. Since changes in plasmalogen level are usually associated with changes in the other phospholipid subclasses, a general increase in all phospholipid subclasses may also be found in colonic neoplasms. In this study, the levels of the major phospholipids, including their plasmalogen and diacylphospholipid subclasses, were found to be elevated in human malignant colonic tissues. Since phosphatidylcholine is the most prominent type of phospholipid found in both malignant and control tissues, the mechanism for its accumulation during malignancy was investigated. Decreases in phospholipase C and D activities were observed in tumor samples, but an enhancement of the CTP: phosphocholine cytidylyltransferase activity was also detected. Immunoblotting analysis revealed that the elevated cytidylyltransferase activity was caused by a three-fold increase in the level of enzyme protein during tumor development. Based on these enzyme studies, we conclude that the high level of phosphatidylcholine in colon tumors resulted from a decrease in its turnover and an increase in its expression.     

Mammalian myristoyl CoA: protein N-myristoyltransferase

Myristoyl CoA:Protein N-myristoyltransferase (NMT) is the enzyme which catalyses the covalent transfer of myristate from myristoyl CoA to the amino-terminal glycine residue of protein substrates. Although NMT is ubiquitous in eukaryotic cells, the enzyme levels and cellular distribution vary among tissues. In this article, we describe the properties of mammalian NMT(s) with reference to subcellular distribution, molecular weights, substrate specificity and the possible involvement of NMT in pathological processes. The cytosolic fraction of bovine brain contains multiple forms of NMT activity whereas bovine spleen contains only a single form. In bovine brain and spleen, the cytosol contained majority of NMT activity. In contrast, rabbit colon and rat liver NMT activity was predominantly particulate. Regional differences in NMT activity have been observed in both rabbit intestine and bovine brain. Results from our laboratory along with the existing knowledge, provide evidence for the existence of tissue specific isozymes of NMT.     

N-myristoylation regulates the SnRK1 pathway in Arabidopsis

Cotranslational and posttranslational modifications are increasingly recognized as important in the regulation of numerous essential cellular functions. N-myristoylation is a lipid modification ensuring the proper function and intracellular trafficking of proteins involved in many signaling pathways. Arabidopsis thaliana, like human, has two tightly regulated N-myristoyltransferase (NMT) genes, NMT1 and NMT2. Characterization of knockout mutants showed that NMT1 was strictly required for plant viability, whereas NMT2 accelerated flowering. NMT1 impairment induced extremely severe defects in the shoot apical meristem during embryonic development, causing growth arrest after germination. A transgenic plant line with an inducible NMT1 gene demonstrated that NMT1 expression had further effects at later stages. NMT2 did not compensate for NMT1 in the nmt1-1 mutant, but NMT2 overexpression resulted in shoot and root meristem abnormalities. Various data from complementation experiments in the nmt1-1 background, using either yeast or human NMTs, demonstrated a functional link between the developmental arrest of nmt1-1 mutants and the myristoylation state of an extremely small set of protein targets. We show here that protein N-myristoylation is systematically associated with shoot meristem development and that SnRK1 (for SNF1-related kinase) is one of its essential primary targets.     

Elevated Expression of the cdc25A Protein Phosphatase in Colon Cancer

The nuclear protein phosphatase cdc25A has been postulated to be a protooncogene. The total nuclear phosphotyrosyl protein phosphatase (PTP) activity and the expression of cdc25A were compared in normal and cancerous colon epithelial tissue. Nuclei derived from normal mucosal epithelium and tumors were analyzed for phosphotyrosyl protein phosphatase activity using the malachite green assay and a synthetic phosphotyrosyl peptide based on the sequence of cdc2, a known cdc25A phosphotyrosyl protein substrate. Tumorigenesis resulted in elevated nuclear PTP activity (343.0 ± 37.0% of normal epithelial PTP activity) in 52% (29 of 56) of colon tumors. In all cases elevated nuclear PTP activity correlated with an increase in the expression of cdc25A. The changes in PTP activity observed were not due to any increase in the rate of growth of the colonic mucosa as no corresponding changes occurred with PTP activity under conditions of rapid mucosal growth.     

A fluorescence-based assay for N-myristoyltransferase activity

N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.     

N-myristoyltransferase in the leukocytic development processes

The lipidic modification of proteins has recently been shown to be of immense importance, although many of the roles of these modifications remain as yet unidentified. One of such key modifications occurring on several proteins is the covalent addition of a 14-carbon long saturated fatty acid, a process termed myristoylation. Myristoylation can occur during both co-translational protein synthesis and posttranslationally, confers lipophilicity to protein molecules, and controls protein functions. The protein myristoylation process is catalyzed by the enzyme N-myristoyltransferase (NMT), which exists as two isoforms: NMT1 and NMT2. NMT1 is essential for growth and development, during which rapid cellular proliferation is required, in a variety of organisms. NMT1 is also reported to be elevated in many cancerous states, which also involve rapid cellular growth, albeit in an unwanted and uncontrolled manner. The delineation of myristoylation-dependent cellular functions is still in a state of infancy, and many of the roles of the myristoylated proteins remain to be established. The development of cells of the leukocytic lineage represents a phase of rapid growth and development, and we have observed that NMT1 plays a role in this process. The current review outlines the roles of NMT1 in the growth and differentiation of the cells of leukocytic origin. The described studies clearly demonstrate the roles of NMT1 in the regulation of the developmental processes of the leukocytes cells and provide a basis for further research with the aim of unraveling the roles of protein myristoylation in both cellular and physiological context.     

Requirement of N-myristoyltransferase 1 in the development of monocytic lineage

N-myristoyltransferase (NMT) exists in two isoforms, NMT1 and NMT2, that catalyze myristoylation of various proteins crucial in signal transduction, cellular transformation, and oncogenesis. We have recently demonstrated that NMT1 is essential for the early development of mouse embryo. In this report, we have demonstrated that an invariant consequence of NMT1 knock out is defective myelopoesis. Suppressed macrophage colony forming units were observed in M-CSF-stimulated bone marrow cells from heterozygous (+/-) Nmt1-deficient mice. Homozygous (-/-) Nmt1-deficient mouse embryonic stem cells resulted in drastic reduction of macrophages when stimulated to differentiate by M-CSF. Furthermore, to understand the requirement of NMT1 in the monocytic differentiation we investigated the role of NMT, pp60c-Src (NMT substrate) and heat shock cognate protein 70 (inhibitor of NMT), during PMA-induced differentiation of U937 cells. Src kinase activity and protein expression increased during the differentiation process along with regulation of NMT activity by hsc70. NMT1 knock down in PMA treated U937 cells showed defective monocytic differentiation. We report in this study novel observation that regulated total NMT activity and NMT1 is essential for proper monocytic differentiation of the mouse bone marrow cells.     

Expression of methionine aminopeptidase 2, N-myristoyltransferase, and N-myristoyltransferase inhibitor protein 71 in HT29

Protein myristoylation is a co-translational process, catalyzed by N-myristoyltransferase (NMT) that occurs after the initiating methionine is removed by methionine aminopeptidase (MetAP). The enzymes NMT and MetAP play a major role in the process of myristoylation of oncoproteins including the c-src family. In this study, we examined the levels of expression of MetAP2, NMT, and NMT inhibitor protein 71 (NIP71) in human colon cancer cell lines (HCCLs). We examined the influence of cell density on the expression of the above proteins in HT29 cells. Western blot analysis of MetAP2 and NMT demonstrated higher levels of protein expression in low density of HT29 while low expression in high density was observed. In addition, we observed that NIP71 and pp60(c-src) expressions were dependent on the cell density of HT29. This is the first study demonstrating the expression of MetAP2, NMT, pp60(c-src), and NIP71 in HCCLs.     

Immunocytochemical Characterization and Subcellular Localization of Human Myristoyl-CoA: ProteinN-Myristoyltransferase in HeLa Cells

Antisera have been raised to three synthetic peptides based on the sequence of human myristoyl-CoA:proteinN-myristoyl transferase (NMT) and to the purified enzyme following its expression inEscherichia coli.These antisera have been affinity purified and shown to react both with theE. coliexpressed human NMT, and specifically with a protein of molecular weight of 63 kDa in immunoblots of the human cell line HeLa. The affinity purified antibodies have also been used to localize NMT in methanol/acetone permeabilized HeLa cells by immunofluorescent staining. The immunofluorescence showed a diffuse staining pattern throughout the cell, suggesting that the enzyme is predominantly cytosolic. This was confirmed by determining the distribution of NMT activity in different subcellular fractions of HeLa cells. Over 90% of NMT enzymatic activity was released from cell lysates during either hypotonic or isotonic homogenization. However, a small amount of enzymatic activity remained associated with cell membranes, despite extensive washing, and this was confirmed by immunoblot analysis of these membranes for NMT. In comparison, over 99.5% of lactate dehydrogenase activity was released under the same conditions, which suggests that the NMT was genuinely associated with the cell membranes. The membrane-bound enzyme behaved like a peripheral membrane protein. Permeabilization of HeLa cells with 50 μMdigitonin resulted in the release of 90–93% of lactate dehydrogenase compared to 73–85% of NMT, again suggesting that the majority of the enzyme is cytosolic, but that some may be associated with cell membranes or organelles.     

An improved method and cost effective strategy for soluble expression and purification of human N-myristoyltransferase 1 in E. coli

N-myristoyltransferase (NMT) is an indispensible enzyme, which exists as two isoforms (NMT1 and NMT2) in humans and has proven roles in development of cancerous states. It is thus a target for novel anti-cancer drug design, but understanding of the biochemical and functional differences of these isozymes is not fully deciphered. A soluble expression under the T7 promoter for human NMT1 was achieved in E. coli BL21 (DE3) cells, devoid of any isopropyl β-d-1-thiogalactopyranoside-based induction. The identity of expressed protein was confirmed by matrix-assisted laser desorption ionization mass spectrometry peptide-fingerprint analysis and a two-step purification protocol yielded homogeneous enzyme. The intact mass of the purified protein was verified by electrospray ionization mass spectrometry and found to be in agreement with the theoretical mass (48.141 vs. 48.140 kDa). The fluorescence spectrophotometric analyses of the ligand binding and enzyme activity demonstrated that the recombinant form is functional. The yield of purified protein was ~8–10 mg/L culture (batch to batch variation) with a specific activity value of 18,500 ± 513 U/mg of protein under the experimental conditions used. The final verification of the myristoylation was demonstrated by mass spectrometry analysis of reaction product. The described approach could be readily adapted for production of human NMT1, with high yields of pure enzyme preparations, which should aid in downstream applications involving inhibitor design and structure–function studies of NMT’s.     

Downregulation of FIP200 induces apoptosis of glioblastoma cells and microvascular endothelial cells by enhancing Pyk2 activity

The expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies of glioblastoma tumors, we detected FIP200 in the tumor cells, tumor-associated endothelial cells, and occasional glial cells. Human glioblastoma tumor cell lines and immortalized human astrocytes cultured in complete media also expressed FIP200 as did primary human brain microvessel endothelial cells (MvEC), which proliferate in culture and resemble reactive endothelial cells. Downregulation of endogenous expression of FIP200 using small interfering RNA resulted in induction of apoptosis in the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC. It has been shown by other investigators using cells from other tissues that FIP200 can interact directly with, and inhibit, proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK). In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK. Coimmunoprecipitation and colocalization studies indicated that the endogenous FIP200 was largely associated with Pyk2, rather than FAK, in the glioblastoma tumor cells and brain MvEC. Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells. In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors.     

Expression of human N-myristoyltransferase inEscherichia coli. Comparison with N-myristoyltransferases expressed in different tissues

Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins.Escherichia coli transformed with human NMT expression construct produced high levels of N-myristoyltransferase. Using the combination of ammonium sulfate precipitation, chromatography on SP-Sepharose fast flow and fast protein liquid chromatography on Mono-S, the enzyme was purified more than 100 fold with 40% yield. The hNMT fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-polyacrylamide gel electrophoresis. Upon cleavage by the Enterokinase [(Asp)₄-Lys], the hNMT exhibited an apparent molecular mass of 49 kDa without loss of catalytic activity. The hNMT activity could be greatly activated severalfold with the use of Tris, SDS, ethanol and acetonitrile. The catalytic activity of hNMT was potently inhibited in a concentration dependent manner by NIP₇₁₁ a bovine brain NMT inhibitory protein with a half maximal inhibition of 31.0 nM. TheE. coli expressed hNMT was homogeneous and showed enzyme activity.Abbreviations NMT N-myristoyl CoA:protein N-myristoyltransferase - NIP₇₁ 71 kDa heat stable membrane bound N-myristoyltransferase inhibitor protein - hNMT human NMT - DTNB N-5,5dithiobis (2-nitrobenzoic acid) - FPLC fast protein liquid chromatography - IPTG isopropyl -D-thiogalactopyranoside - cDNA complementarydeoxyribonucleic acid - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride