Increased myocardial N-myristoyltransferase activity in rotenone model of Parkinsonism.

There is widespread brain pathology in Parkinson's disease (PD), with the primary pathology in the substantia nigra. Oxidative stress is believed to play a role in cell death in PD. Rotenone is a mitochondrial toxin which can produce Parkinson syndrome (PS) in rats. Myristoyl-CoA:protein N-myristoyltransferase (NMT), which catalyzes the co-translational transfer of myristate from myristoyl-CoA to the amino-terminal glycine residue of selected polypeptides, is increased in the myocardium of ischemia-reperfusion rat model myocardium. Animals received rotoneone (n=10) or placebo vehicle (n=6) via Alzet osmotic pumps. Mean cardiac muscle NMT activity of placebo treated (control) rats was 0.608+/-0.366 units/mg protein. Rats with mild or no detectable PS features on rotenone showed slight (mean 0.853+/-0.192) but insignificantly increased activity. Rats that had moderately severe PS features had higher level of NMT activity (mean 1.223+/-0.057), which was borderline significant compared to controls (P=0.066). Rats with severe PS features had the highest NMT activity (1.353+/-0.128) which was significantly greater compared to controls (P=0.003) and to the rats that had equivocal or no motor slowing (P=0.005). Our data show cardiac metabolic dysfunction in a rotenone rat model of PS. The severity of this change correlates with the severity of motor manifestations. Further studies of NMT activity in human PD cases and patients with cardiomyopathy of unknown cause may provide valuable information in these disorders.

N-myristoyltransferase inhibitor protein is homologous to heat shock cognate protein 70.

Many of viral and eukaryotic proteins are required for signal transduction and regulatory functions which undergo a lipid modification by the enzyme N-myristoyltransferase (NMT). In this study, we demonstrated that heat shock cognate protein 70 (HSC70) is homologous to NMT inhibitor protein (NIP71), which was discovered in our laboratory, based on MALDI-TOF mass spectrometric analysis. The purified bovine cytosolic HSC70 and particulate NIP71 produced a dose-dependent inhibition of human NMT having half maximal inhibitions of 235 and 230 nM, respectively. Further, Western blot analysis revealed that the purified particulate NIP71 and cytosolic HSC70 cross-reacted with both anti-NIP71 and anti-HSC70 antibodies. The results we obtained imply that molecular chaperones could be involved in the regulation of NMT in normal and cancerous cells. Further studies directed to revealing the role of HSC70 in the regulation of NMT may lead to the development of gene based therapies of colon cancer.

Lipidic inhibitors of human N-myristoyltransferase.

This study was undertaken in order to identify compounds which inhibit the activity of human myristoyl-CoA:protein N-myristoyltransferase (hNMT). In particular, the structural features of such molecules which contribute to enzyme inhibition were investigated. Two groups of compounds, namely myristic acid and analogs 1-13 and derivatives of myristoyl-CoA 14-19 were evaluated. All compounds were examined using cAMP-dependent protein kinase derived peptide substrate. The IC(50) values were <1 micro M, between 1 and 100 micro M or >100 micro M in eight, four and seven compounds, respectively. Of the six myristoyl-CoA analogs, five had IC(50) values in the 0.06-0.59 micro M range. These molecules were examined using three additional substrates viz pp60src, MARCKS and M2 gene segment of reovirus type 3 which led to results similar to those obtained with the cAMP-dependent protein kinase substrate. On the other hand, evaluation of myristic acid and four related compounds revealed some differences in hNMT-inhibiting properties among the substrates. From the results obtained, the possible manner whereby potent inhibitors interact with the enzyme was formulated thus enabling the design of further analogs as candidate inhibitors of hNMT.

Enhanced activity of human N-myristoyltransferase by dimethyl sulfoxide and related solvents in the presence of serine/threonine-containing peptide substrates.

Human N-myristoyltransferase (hNMT) activity was found to be stimulated several-fold by DMSO and its analogues in the presence of serine-containing peptide substrates. DMSO caused a concentration-dependent 10-fold stimulation of hNMT activity in the presence of a pp60(src)-derived peptide substrate (Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys-Arg). However, the stimulation of hNMT activity was not observed by DMSO when a cyclic AMP (cAMP)-dependent protein kinase-derived Ser-free peptide substrate (Gly-Asn-Ala-Ala-Ala-Ala-Lys-Lys-Arg-Arg) was used. These findings suggested that the effect of DMSO is on the substrate rather than on the enzyme. When a MARCKS (myristoylated alanine-rich C-kinase substrate)-derived peptide substrate (Gly-Ala-Gln-Phe-Ser-Lys-Thr-Ala-Arg-Arg) and the M2 gene segment of the reovirus type 3 peptide substrate (Gly-Asn-Ala-Ser-Ser-Ile-Lys-Lys-Lys) were used, hNMT activity was increased by approximately 8.5- and 7-fold, respectively. Dimethyl sulfone (20%) increased hNMT activity between 2.5- and 3.5-fold in the presence of pp60(src), MARCKS, and M2 gene segment peptides. Dimethyl formamide (20%) increased the hNMT activity by 8.5-, 8.5-, 5.5- and 3.5-fold when pp60(src), MARCKS, M2, and cAMP-dependent protein kinase-derived peptide substrates were used, respectively. Acetone (20%) also increased the hNMT activity by 20-fold in the presence of the pp60(src) peptide substrate. Dimethyl ammonium chloride (20%) caused about 6.5- and 2.5-fold increases in the hNMT activity in the presence of the pp60(src) and cAMP-dependent protein kinase-derived peptide substrates, respectively. Infrared spectroscopy showed a decreased intensity in the band at 3500-3600cm(-1) when the infrared spectrum of the pp60(src)-derived peptide was determined in the presence of DMSO. These results suggest the involvement of hydrogen bonding between the heteroatoms of the organic molecules and the hydrogen atoms of the free hydroxyl groups of the serine/threonine-containing peptide substrates. Such interactions appear to enhance the activity of hNMT towards its serine-containing substrates.

Myristoyl-CoA:protein N-myristoyltransferase: a novel molecular approach for cancer therapy (Review).

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 in rats treated with azoxymethane that NMT activity was higher in colonic epithelial neoplasms than in normal colonic tissue and that an increase in NMT activity appeared at an early stage in colonic carcinogenesis. Increased NMT activity was also confirmed in human colonic tumors compared to normal tissue. Furthermore, colorectal tumors displayed increased immunohistochemical staining for NMT compared to normal mucosa in the cytoplasm. In addition, gallbladder carcinoma showed moderate to strong cytoplasmic positivity for NMT with increased intensity in the invasive component whereas the normal gallbladder mucosa showed weak to negative cytoplasmic staining for this enzyme. It is conceivable therefore that NMT can be used as a potential marker for the early detection of cancer. Of particular note is the very recent discovery of cytotoxic compounds in the laboratories of the authors which inhibit NMT and may offer a novel approach for the evolution of candidate antineoplastic agents which display greater potencies towards neoplasms than the corresponding normal tissues.

Localization and characterization of calcineurin in bovine eye.

PURPOSE: There are several aspects of the visual system that may be regulated by Ca2+- and calmodulin (CaM)-stimulated protein phosphatase. In the present study, the distribution and characterization of calcineurin (CaN) in bovine eye was determined. METHODS: Whole bovine eyes were either homogenized for purification or regionally dissected to determine CaN localization and activity. Dissected tissues were homogenized and Western blot analysis performed, using polyclonal anti-CaN antibodies, and assayed using p-nitrophenyl phosphate (PNPP) as a substrate to determine the dephosphorylation activity of CaN. Fresh samples were then prepared for immunohistochemistry and probed with polyclonal anti-CaN antibodies. RESULTS: CaN was found to be present in all eye tissues, although activity and protein expression varied. The highest levels of CaN activity and protein expression were found in the optic nerve, retina, and cornea. Immunohistochemical methods displayed similar results with additional staining of the optic nerve vasculature. Assays of purified CaN demonstrated that bovine eye CaN had regulatory properties similar to CaN isolated from other tissues. Probing eye tissues with CaN A isoform-specific antibodies demonstrated that eye tissues displayed variable distributions of the CaN Aalpha and CaN Abeta isoforms. CONCLUSIONS: The presence of CaN in the bovine eye provides a physiological pathway by which the phosphorylated state of proteins and intracellular Ca2+ concentrations can be coordinated. The authors propose that CaN is involved in the immunologic privilege of the cornea, retinal signal transduction, and the toxic effects of immunosuppressants on the eye. Further in vivo studies of CaN function are necessary to understand the contributions of CaN to ocular physiology.