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[Cancer Research 63, 7975-7978, November 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Potent Inhibitor of N-Myristoylation

A Novel Molecular Target For Cancer

Anuraag Shrivastav1, Mohammed K. Pasha1, Ponniah Selvakumar1, Sweta Gowda1, Douglas J. H. Olson3, Andrew R. S. Ross3, Jonathan R. Dimmock2 and Rajendra K. Sharma1

1 Department of Pathology and Saskatoon Cancer Center, College of Medicine
2 College of Pharmacy and Nutrition, University of Saskatchewan,
3 Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the NH2 terminus of the glycine residue of a number of important proteins that have diverse biological functions and thus have been proposed as potential targets for chemotherapeutic drug design. Earlier, we demonstrated that NMT is more active in colonic epithelial neoplasms than in corresponding normal-appearing colonic tissue. Furthermore, an increased expression of NMT was also observed in gallbladder carcinoma. In the present study, we report a novel protein inhibitor of NMT. This protein caused a potent concentration-dependent inhibition of human NMT with half-maximal inhibition at 4.5 ± 0.35 nM. This study will serve as a template for further investigations in the area of protein myristoylation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
N-myristoylation is a cotranslational modification of proteins (1) in which myristate (a 14-carbon fatty acid) is covalently attached to the NH2 terminus of various cellular proteins, viral proteins, and oncoproteins (2, 3, 4) . Cellular myristoylated proteins have diverse biological functions in signal transduction and oncogenesis. Examples include the catalytic subunit of cAMP4 -dependent protein kinase, various tyrosine kinases (pp60src, pp60yes, pp56lck, pp59fyn/syn, and c-Abl), the ß-subunit of calcineurin, the myristoylated alanine-rich C kinase substrate, and the {alpha}-subunit of several guanine nucleotide-binding proteins and ADP ribosylation factors (2, 3, 4) . The importance of myristoylation of proteins in virus assembly and structure has been extensively investigated (5) . In addition, several reports have indicated that myristoylation may also occur posttranslationally (6 , 7) .

The field of protein myristoylation is still in its infancy, and relatively little is known about the role it may play in oncogenesis. The enzyme that catalyzes protein myristoylation, NMT, is a ubiquitously distributed eukaryotic enzyme. We have reported for the first time in rat model that NMT is more active in colonic epithelial neoplasms than in the corresponding normal-appearing colonic tissue and that an increase in NMT activity appears at an early stage in colonic carcinogenesis (8) . Increased NMT activity was also observed in human colonic tumors and was predominantly cytosolic (8) . Furthermore, colorectal tumors showed increased immunohistochemical staining for NMT compared with normal mucosa (9) . In addition, gallbladder carcinoma displayed strong cytoplasmic positivity for NMT with an increased intensity in the invasive component, whereas normal gallbladder mucosa showed weak to negative cytoplasmic staining (10) . These findings have significant implications with regard to the prognosis of cancer and the design of chemotherapeutic drugs.

Work in our laboratory is focused on understanding the regulators of NMT activity by discovering novel potent inhibitors and activators. A variety of compounds have been reported to inhibit NMT activity (2 , 3 , 11 , 12) . During the search for inhibitors of NMT, we discovered that enolase is a potent inhibitor of the N-myristoylation reaction in vitro. Enolase is a glycolytic enzyme that catalyzes the reversible removal of a water molecule from 2-phosphoglycerate to yield phosphoenolpyruvate. A {gamma}-{gamma} isoform of enolase called neuron-specific enolase is a putative marker of small cell lung carcinoma (13) . In this paper, for the first time, we report that enolase, a ubiquitous enzyme, is an inhibitor of NMT, which is one of the therapeutic targets for the treatment of cancer. Furthermore, we observe that full inhibitory activity remained in the clear supernatant after boiling enolase for 2 min in a boiling water bath.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
[9,10-3H]Myristic acid (39.3 Ci/mmol) was purchased from Perkin-Elmer. Pseudomonas acyl-CoA synthetase, rabbit muscle enolase as a crystalline suspension in 2.8 M (NH4)2SO4 containing 50 mM imidazole and 1 mM MgSO4, trypsin, and general laboratory reagents of analytical grade were obtained from Sigma (Toronto, Ontario, Canada). PVDF membrane and powdered milk were purchased from Bio-Rad Laboratories (Mississauga, Ontario, Canada). Monoclonal antibody to NMT-1 and horseradish peroxidase-conjugated goat antimouse antibody were obtained from BD Biosciences (Mississauga, Ontario, Canada). Chemiluminescence Reagent Plus was obtained from NEN Life Science Products. Peptide substrate based on the NH2-terminal ends of cAMP-dependent protein kinase (GNAAAAKKRR), pp60src (GSSKSKPKR), myristoylated alanine-rich C kinase substrate (GAQFSKTARR), and the M2 gene segment of reovirus type 3 (GNASSIKKK) were synthesized by the Alberta Peptide Institute (Edmonton, Alberta, Canada). Recombinant hNMT was purified as described previously (14) .

NMT Assay.
NMT activity was assayed as described by King and Sharma (6) . Briefly, [3H]myristoyl-CoA was synthesized as described previously (15) , except where described below. The reaction mixture contained 40 mM Tris-HCl (pH 7.4), 0.1 mM EGTA, 10 mM MgCl2, 5 mM ATP, 1 mM Li-CoA, 1 µM [3H]myristic acid (7.5 µCi), and 0.3 unit/ml Pseudomonas acyl-CoA synthetase in a total volume of 200 µl. The reaction was carried out for 30 min at 30°C. The conversion to [3H]myristoyl-CoA was usually >95%. The assay mixture contained 40 mM Tris-HCl (pH 7.4), 0.5 mM EGTA, 0.45 mM 2-mercaptoethanol, 1% Triton X-100, peptide substrate (500 µM), and NMT in a total volume of 25 µl. The transferase reaction was initiated by the addition of freshly generated [3H]myristoyl-CoA and incubated at 30°C for 30 min. The reaction was terminated by spotting 15-µl aliquots of incubation mixture onto P81 phosphocellulose paper discs and drying under a stream of warm air. The P81 phosphocellulose paper discs were washed in two changes of 40 mM Tris-HCl (pH 7.3) for 60 min. The radioactivity was quantified in 7.5 ml of Beckman Ready Safe liquid scintillation mixture in a Beckman liquid scintillation counter. One unit of NMT activity was expressed as 1 pmol of myristoyl peptide formed/min.

NMT Inhibition Assay.
Both unboiled and boiled enolase were assayed by their inhibitory activity against standard hNMT. Purified hNMT (0.2 µg/assay) was assayed in the presence of cAMP-dependent protein kinase-derived peptide substrate and inhibitor protein. A control experiment was performed in the absence of enolase, and the hNMT activity was considered as 100%. All other conditions were as described above.

Western Blot Analysis.
Western blot analysis was performed essentially as described by Towbin et al. (16) . Samples were electrophoresed by SDS-PAGE and transferred to a PVDF membrane. Transblot PVDF membrane was incubated with blocking buffer (PBS-T plus 5% powdered milk) for 1 h at room temperature to block nonspecific binding. After washing, the blot was incubated overnight at 4°C with monoclonal antibody against NMT-1 (1:250 dilution in blocking buffer). After washing, the blot was incubated with horseradish peroxidase-conjugated goat antimouse secondary antibody (1:5000 dilution in blocking buffer), and the NMT band was detected using Chemiluminescence Reagent Plus and exposed to X-ray films.

Other Methods.
SDS-PAGE (10%) was performed according to the method of Laemmli (17) . Proteins were estimated by the Bradford method (18) . GraphPad Prism software was used to calculate 95% confidence intervals.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
When purified hNMT was incubated in the presence of various concentrations of enolase, NMT activity was inhibited in a concentration-dependent manner, with maximal inhibition at a concentration of 3.3 ± 0.5 µM and half-maximal inhibition at 0.52 ± 0.08 µM (Fig. 1A)Citation . The results suggest that hNMT can be inhibited by enolase or by a slight contamination in the enolase preparation.



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  		Fig. 1. Effect of enolase and boiled sample of enolase on hNMT activity. Purified hNMT (0.2 µg/assay) was assayed as described in "Materials and Methods" in the presence of cAMP-dependent protein kinase-derived peptide (GNAAAAKKRR) and various concentrations of the unboiled (A) and boiled (C) sample of enolase. Control experiment was performed in the absence of enolase, and the hNMT activity was considered as 100%. The boiled sample of enolase was also examined using three additional substrates, i.e., pp60src (GSSKSKPKR), myristoylated alanine-rich C kinase substrate (GAQFSKTARR), and the M2 gene segment of reovirus type 3 (GNASSIKKK), which led to results similar to those obtained with cAMP-dependent protein kinase substrate. Each value is the mean of two experiments, and each experiment was performed more than two times. All of the data are expressed as the mean ± 95% confidence intervals of four experiments. B, analysis of enolase by SDS-PAGE. Gel electrophoresis was carried out with an equal volume of enolase. Lane 1, unboiled sample; Lane 2, boiled sample. D, matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of the boiled sample of enolase. The boiled sample of enolase shows close to 30% amino acid similarity to ß-enolase (2-phospho-D-glycerate hydrolyase, skeletal muscle enolase) as indicated in bold type.

 
A common, although not universal, feature of activators and/or inhibitors is their stability to heat. To test this possibility, the enolase sample from rabbit muscles was boiled for 2 min in a boiling water bath, during which time >99% of the proteins were precipitated (Fig. 1B)Citation . The inhibitory effect of the clear supernatant from the boiled sample on hNMT was then examined and compared with unboiled sample of enolase. Fig. 1CCitation shows that after heating for 2 min in a boiling water bath, the clear supernatant of enolase caused a potent concentration-dependent inhibition of hNMT. Half-maximal inhibition was seen at 4.5 ± 0.35 nM, suggesting that hNMT can be inhibited by a boiled sample of enolase. To test whether the inhibitor factor is a contaminant protein in enolase, we further investigated the nature of inhibitor. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of a tryptic digest of the boiled sample was carried out, and the resulting mass fingerprint was compared with a protein sequence database. Fig. 1DCitation shows that the percentage match of the boiled sample with that of enolase was close to 30%. To further verify that the inhibitor is a protein molecule, the boiled sample of enolase was preincubated with trypsin for 30 min at 30°C. The inhibitor was proved to be a protein because its activity was readily destroyed by trypsin (Table 1)Citation .


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  		Table 1 Effect of trypsin on the inhibitory activity of boiled enolase sample

Boiled enolase sample (4.8 µg) was preincubated in the presence of 25 mM potassium phosphate buffer (pH 7.0) with and without trypsin (48 µg) at 30°C for 30 min in a total volume of 150 µl. The reaction was terminated by boiling the incubation mixture for 2 min and by addition of the soybean trypsin inhibitor (96 µg) and leupeptin (240 µg). Inhibitory activity was assayed using 10 ng/assay of boiled enolase sample and trypsin-treated boiled enolase sample as described in "Materials and Methods." Control experiment was performed in the absence of boiled enolase sample. Results are expressed as units per assay or percentage activity (in parentheses).

 
To test whether the inhibitory activity comigrates with the identified protein, the sample of enolase was applied on a Superpose 12 HR/30 FPLC gel filtration column and analyzed for NMT inhibition and protein concentration. Fig. 2Citation shows that inhibitory activity and identified protein comigrated on the FPLC gel filtration, suggesting that inhibitory activity resides in the enolase protein.



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  		Fig. 2. The Superose 12 HR/30 gel filtration FPLC column (Pharmacia, LKB Biotechnology, Inc.) pre-equilibrated with 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 M NaCl, 0.1 mM EGTA, 10 mM 2-mercaptoethanol, and 0.5 mM MgSO4·7H2O was calibrated with calcineurin (Mr 80,000), BSA (Mr 67,000), calmodulin (Mr 40,000), and myoglobin (Mr 17,800) as indicated by arrows. Enolase sample (500 µg) was applied to the calibrated column, and fractions of 500 µl were collected. Fractions were assayed for the inhibition of NMT and protein determination as described in "Materials and Methods."

 
To investigate the interaction between the inhibitory protein and myristoyl-CoA, Sephadex G-25 gel filtration column chromatography was carried out where the inhibitor protein and myristoyl-CoA had significantly different elution volumes. A mixture of inhibitor protein with myristoyl-CoA was incubated at 30°C for 10 min and chromatographed on a Sephadex G-25 column that had been pre-equilibrated with buffer [50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EGTA and 10 mM 2-mercaptoethanol]. Fig. 3ACitation shows that the inhibitor protein was eluted in the void volume, whereas myristoyl-CoA was eluted in the total volume, suggesting that the inhibitor protein does not bind to myristoyl-CoA. In a separate experiment, a mixture of hNMT and myristoyl-CoA was chromatographed on a Sephadex G-25 column under conditions identical to those described above. Fig. 3BCitation shows that some myristoyl-CoA was eluted in the void volume along with hNMT. When a mixture of myristoyl-CoA, the inhibitor protein, and hNMT was chromatographed, the elution profile was similar to that of myristoyl-CoA and hNMT alone (Fig. 3B)Citation , i.e., there was no increase or loss of myristoyl-CoA associated with the protein peak (Fig. 3C)Citation . These results suggest that the inhibitor protein does not bind to myristoyl-CoA, nor does it prevent the binding of myristoyl-CoA to hNMT. Furthermore, the binding of the inhibitor protein to substrate is unlikely because the concentration of peptide substrate is very high in the reaction mixture (high micromolar concentration versus low nanomolar concentration).



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  		Fig. 3. Investigation of the interaction between the myristoyl-CoA and boiled sample of enolase by gel filtration. A, [3H]myristoyl-CoA (0.27 µM) was incubated at 30°C for 10 min with enolase (50 µg) and chromatographed in 100 µl of total volume on a Sephadex G-25 column (16 x 0.8 cm) that had been pre-equilibrated in 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EGTA and 10 mM 2-mercaptoethanol, and fractions of 0.5 ml were collected. B and C, [3H]myristoyl-CoA (0.27 µM) was incubated with hNMT (B; 6 µg) or [3H]myristoyl-CoA was incubated with hNMT and enolase (C) as described above. Solid arrows and broken arrows indicate the position of enolase and free [3H]myristoyl-CoA, respectively. Radiolabeled myristoyl-CoA ({blacksquare}) and protein ({square}) are shown.

 
To investigate whether the inhibitor protein possesses any protease activity, two approaches were used. In the first of these, hNMT was incubated with the inhibitor protein for 30 min at 30°C, and SDS-PAGE was carried out with 10% acrylamide gel and transferred to a PVDF membrane for Western blot analysis (Fig. 4)Citation . The intensity of the hNMT polypeptide (Mr 50,000) band was similar to the control hNMT, and no proteolytic fragments of the Mr 50,000 polypeptide band of hNMT were observed when hNMT was incubated with boiled sample of enolase. This suggests that the inhibitor protein did not contain protease activity. In the second approach, the inhibitor protein was added 10 min after initiation of the transferase reaction, which gave the same degree of inhibition as that observed for inhibitor protein added before the initiation of the transferase reaction (Fig. 5)Citation . This experiment also argues against the inhibitor protein exerting its effect by a demyristoylation of the radiolabeled myristoyl-CoA peptide. If the inhibitor protein possessed any demyristoylase activity, a time-dependent loss of radioactivity recovered in the assay would be observed in Fig. 4Citation . Therefore, we conclude that the inhibitor protein may act directly on NMT.



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  		Fig. 4. Determination of protease activity against hNMT in boiled sample of enolase. Purified hNMT (6 µg) was incubated either with or without boiled sample of enolase (2 µg) at 30°C for 30 min. Incubation mixtures were subjected to SDS-PAGE and transblotted onto a PVDF membrane as described in "Materials and Methods." Lane 1, boiled enolase sample; Lane 2, boiled enolase and hNMT; Lane 3, hNMT.

 


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  		Fig. 5. Time-dependent inhibition of hNMT by boiled sample of enolase. Purified hNMT (3.6 µg) was incubated in the presence of cAMP-dependent protein kinase-derived peptide in a final volume of 450 µl. Transferase reactions were initiated by addition of 0.27 µM [3H]myristoyl-CoA as described in "Materials and Methods." Boiled sample of enolase was added to the incubation mixture at 0 min ({blacksquare}) or 10 min ({blacktriangleup}). In the control experiment ({blacktriangledown}), 25 mM potassium phosphate buffer (pH 7.0) was added. Aliquots of 15 µl from the incubation mixtures were assayed. Results are expressed in pmol of myristoyl peptide formed as a function of time (mean ± 95% confidence intervals of three independent experiments).

 
In conclusion, we have shown that the novel protein inhibitor potently inhibited hNMT, suggesting that this novel inhibitor may be involved in the regulation of NMT activity. Once the cDNA of the novel inhibitor is available, it would greatly facilitate the manipulation of this novel NMT inhibitor by generating mutations in the DNA using oligonucleotide-directed mutagenesis and deletion analysis. This will help to define the key regions within the protein as a whole and to assign the specific functions for NMT in cancer progression.


   FOOTNOTES
 
Grant support: Canadian Institutes of Health Research, Canada, Grant MOP-36484.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Rajendra K. Sharma, Research Unit, Saskatoon Cancer Center, 20 Campus Drive, Saskatoon, Saskatchewan, S7N 4H4 Canada. Phone: (306) 966-7733; Fax: (306) 655-2635; E-mail: rsharma{at}scf.sk.ca

4 The abbreviations used are: cAMP, cyclic AMP; hNMT, human N-myristoyltransferase; PVDF, polyvinylidene difluoride; FPLC, fast protein liquid chromatography; NMT, N-myristoyltransferase. Back

Received 6/10/03. Revised 9/ 3/03. Accepted 9/ 5/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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