Protein N-myristoylation Research Articles

IpaJ trims the fat

Shigella flexneri disrupts host protein N-myristoylation to interfere with Golgi-mediated cargo sorting and secretion.

Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ

Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein-protein and protein-membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.

High‐throughput profiling of N‐myristoylation substrate specificity across species including pathogens

One of the most critical modifications affecting the N-terminus of proteins is N-myristoylation. This irreversible modification affects the membrane-binding properties of crucial proteins involved in signal transduction cascades. This cotranslational modification, catalyzed by N-myristoyl transferase, occurs both in lower and higher eukaryotes and is a validated therapeutic target for several pathologies. However, this lipidation proves very difficult to be evidenced in vivo even with state-of-the-art proteomics approaches or bioinformatics tools. A large part of N-myristoylated proteins remains to be discovered and the rules of substrate specificity need to be established in each organism. Because the peptide substrate recognition occurs around the first eight residues, short peptides are used for modeling the reaction in vitro. Here, we provide a novel approach including a dedicated peptide array for high-throughput profiling protein N-myristoylation specificity. We show that myristoylation predictive tools need to be fine-tuned to organisms and that their poor accuracy should be significantly enhanced. This should lead to strongly improved knowledge of the number and function of myristoylated proteins occurring in any proteome.

Inhibition of Protein N-Myristoylation: A Therapeutic Protocol in Developing Anticancer Agents

N-myristoyltransferase (NMT) is an essential eukaryotic enzyme which catalyzes the transfer of the myristoyl group to the terminal glycine residue of a number of proteins including those involved in signal transduction and apoptotic pathways. Myristoylation is crucial for the cellular proliferation process and is required for the growth and development in a number of organisms including many human pathogens and viruses. Targeting the myristoylation process thus has emerged as a novel therapeutic strategy for anticancer drug design. The expression/activity of NMT is considerably elevated in a number of cancers originating in the colon, stomach, gallbladder, brain and breast and attenuation of NMT levels has been shown to induce apoptosis in cancerous cell lines and reduce tumor volume in murine xenograft models for cancer. A focus of current therapeutic interventions in novel cancer treatments is therefore directed at developing specific NMT inhibitors. The inhibition of the myristoyl lipidation process with respect to cancer drug development lies in the fact that many proteins involved in oncogenesis such as src and various kinases require myristoylation to perform their cellular functions. Inhibiting NMT functions to control malignancy is a novel approach in the area of anticancer drug design and there are rapidly expanding discoveries of synthetic NMT inhibitors as potential chemotherapeutic agents to be employed in the warfare against cancer. The current review focuses on developments of various chemical NMT inhibitors with potential roles as anticancer agents.

Performance improvement in protein N-myristoyl classification by BONSAI with insignificant indexing symbol.

Many N-myristoylated proteins play key roles in regulating cellular structure and function. In the previous study, we have applied the machine learning system BONSAI to predict patterns based on which positive and negative examples could be classified. Although BONSAI has helped establish 2 interesting rules regarding the requirements for N-myristoylation, the accuracy rates of these rules are not satisfactory. This paper suggests an enhancement of BONSAI by introducing an "insignificant indexing symbol" and demonstrates the efficiency of this enhancement by showing an improvement in the accuracy rates. We further examine the performance of this enhanced BONSAI by comparing the results of classification obtained the proposed method and an existing public method for the same sets of positive and negative examples.

Comparative Large Scale Characterization of Plant versus Mammal Proteins Reveals Similar and Idiosyncratic N-α-Acetylation Features

N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.

Protein N-Myristoylation Is Required for Cellular Morphological Changes Induced by Two Formin Family Proteins, FMNL2 and FMNL3

The subcellular localization of 13 recently identified N-myristoylated proteins and the effects of overexpression of these proteins on cellular morphology were examined with the aim of understanding the physiological roles of the protein N-myristoylation that occurs on these proteins. Immunofluorescence staining of HEK293T cells transfected with cDNAs coding for the proteins revealed that most of them were associated with the plasma membrane or the membranes of intracellular compartments, and did not affect cellular morphology. However, two proteins, formin-like2 (FMNL2) and formin-like3 (FMNL3), both of them are members of the formin family of proteins, were associated mainly with the plasma membrane and induced significant cellular morphological changes. Inhibition of protein N-myristoylation by replacement of Gly2 with Ala or by the use of N-myristoylation inhibitor significantly inhibited membrane localization and the induction of cellular morphological changes, indicating that protein N-myristoylation plays critical roles in the cellular morphological changes induced by FMNL2 and FMNL3.

Exploring protein lipidation with chemical biology.

Exploring protein lipidation with chemical biology.

Heterogeneous N-Terminal Acylation of Retinal Proteins Results from the Retina’s Unusual Lipid Metabolism

Protein N-myristoylation occurs by a covalent attachment of a C14:0 fatty acid to the N-terminal Gly residue. This reaction is catalyzed by a N-myristoyltransferase that uses myristoyl-coenzyme A as substrate. But proteins in the retina also undergo heterogeneous N-acylation with C14:2, C14:1, and C12:0 fatty acids. The basis and the role of this retina-specific phenomenon are poorly understood. We studied guanylate cyclase-activating protein 1 (GCAP1) as an example of retina-specific heterogeneously N-acylated protein. The types and the abundance of fatty acids bound to bovine retinal GCAP1 were C14:2, 37.0%; C14:0, 32.4%; C14:1, 22.3%; and C12:0, 8.3% as quantified by liquid chromatography coupled mass spectrometry. We also devised a method for N-acylating proteins in vitro and used it to modify GCAP1 with acyl moieties of different lengths. Analysis of these GCAPs both confirmed that N-terminal acylation of GCAP1 is critical for its high activity and proper Ca(2+)-dependent response and revealed comparable functionality for GCAP1 with acyl moieties of various lengths. We also tested the hypothesis that retinal heterogeneous N-acylation results from retinal enrichment of unusual N-myristoyltransferase substrates. Thus, acyl-coenzyme A esters were purified from both bovine retina and brain and analyzed by liquid chromatography coupled mass spectrometry. Substantial differences in acyl-coenzyme A profiles between the retina and brain were detected. Importantly, the ratios of uncommon N-acylation substrates--C14:2- and C14:1-coenyzme A to C14:0-coenzyme A--were higher in the retina than in the brain. Thus, our results suggest that heterogeneous N-acylation, responsible for expansion of retinal proteome, reflects the unique character of retinal lipid metabolism. Additionally, we propose a new hypothesis explaining the physiological relevance of elevated retinal ratios of C14:2- and C14:1-coenzyme A to C14:0-coenzyme A.

The consensus motif for N‐myristoylation of plant proteins in a wheat germ cell‐free translation system

Protein N-myristoylation plays key roles in various cellular functions in eukaryotic organisms. To clarify the relationship between the efficiency of protein N-myristoylation and the amino acid sequence of the substrate in plants, we have applied a wheat germ cell-free translation system with high protein productivity to examine the N-myristoylation of various wild-type and mutant forms of Arabidopsis thaliana proteins. Evaluation of the relationship between removal of the initiating Met and subsequent N-myristoylation revealed that constructs containing Pro at position 3 do not undergo N-myristoylation, primarily because of an inhibitory effect of this amino acid on elimination of the initiating Met by methionyl aminopeptidase. Our analysis of the consensus sequence for N-myristoylation in plants focused on the variability of amino acids at positions 3, 6 and 7 of the motif. We found that not only Ser at position 6 but also Lys at position 7 affects the selectivity for the amino acid at position 3. The results of our analyses allowed us to identify several A. thaliana proteins as substrates for N-myristoylation that had previously been predicted not to be candidates for such modification with a prediction program. We have thus shown that a wheat germ cell-free system is a useful tool for plant N-myristoylome analysis. This in vitro approach will facilitate comprehensive determination of N-myristoylated proteins in plants.

Strategy for comprehensive identification of human N‐myristoylated proteins using an insect cell‐free protein synthesis system

To establish a strategy for the comprehensive identification of human N-myristoylated proteins, the susceptibility of human cDNA clones to protein N-myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell-free protein synthesis system. One-hundred-and-forty-one cDNA clones with N-terminal Met-Gly motifs were selected as potential candidates from approximately 2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N-myristoylation was evaluated using fusion proteins, in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N-myristoylated. The metabolic labeling experiments both in an insect cell-free protein synthesis system and in the transfected COS-1 cells using full-length cDNA revealed that 27 out of 29 proteins were in fact N-myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N-myristoylated proteins that have not been reported previously to be N-myristoylated, indicating that this strategy is useful for the comprehensive identification of human N-myristoylated proteins from human cDNA resources.

Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli

BackgroundN-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogeneous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose.Methodology/Principal FindingsHere we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium.Conclusions/SignificanceThe single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.

N-myristoyltransferase inhibitors as new leads to treat sleeping sickness

African sleeping sickness or human African trypanosomiasis (HAT), caused by Trypanosoma brucei spp., is responsible for ~30,000 deaths each year. Available treatments for this neglected disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease, when the parasite has infected the central nervous system. Here, we report the validation of a molecular target and discovery of associated lead compounds with potential to address this unmet need. Inhibition of this target, T. brucei N-myristoyltransferase (TbNMT), leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have very promising pharmaceutical properties and represent an exciting opportunity to develop oral drugs to treat this devastating disease. Our studies validate TbNMT as a promising therapeutic target for HAT.

N-myristoylated proteins, key components in intracellular signal transduction systems enabling rapid and flexible cell responses

N-myristoylation, one of the co- or post-translational modifications of proteins, has so far been regarded as necessary for anchoring of proteins to membranes. Recently, we have revealed that Nα-myristoylation of several brain proteins unambiguously regulates certain protein–protein interactions that may affect signaling pathways in brain. Comparison of the amino acid sequences of myristoylated proteins including those in other organs suggests that this regulation is involved in signaling pathways not only in brain but also in other organs. Thus, it has been shown that myristoylated proteins in cells regulate the signal transduction between membranes and cytoplasmic fractions. An algorithm we have developed to identify myristoylated proteins in cells predicts the presence of hundreds of myristoylated proteins. Interestingly, a large portion of the myristoylated proteins thought to take part in signal transduction between membranes and cytoplasmic fractions are included in the predicted myristoylated proteins. If the proteins functionally regulated by myristoylation, a posttranslational protein modification, were understood as cross-talk points within the intracellular signal transduction system, known signaling pathways could thus be linked to each other, and a novel map of this intracellular network could be constructed. On the basis of our recent results, this review will highlight the multifunctional aspects of protein N-myristoylation in brain.

Mutation of SHOC2 promotes aberrant protein N-myristoylation and causes Noonan-like syndrome with loose anagen hair.

N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue.1,2 We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat-containing protein that positively modulates RAS-MAPK signal flow,3–6 underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair [OMIM 607721]7 shared the 4A>G missense change (Ser2Gly) in SHOC2 that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2S2G in vitro enhanced MAPK activation in a cell type-specific fashion. Induction of SHOC2S2G in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.

Molecular Recognition of the Palmitoylation Substrate Vac8 by Its Palmitoyltransferase Pfa3

Palmitoylation of the yeast vacuolar protein Vac8 is important for its role in membrane-mediated events such as vacuole fusion. It has been established both in vivo and in vitro that Vac8 is palmitoylated by the Asp-His-His-Cys (DHHC) protein Pfa3. However, the determinants of Vac8 critical for recognition by Pfa3 have yet to be elucidated. This is of particular importance because of the lack of a consensus sequence for palmitoylation. Here we show that Pfa3 was capable of palmitoylating each of the three N-terminal cysteines of Vac8 and that this reaction was most efficient when Vac8 is N-myristoylated. Additionally, when we analyzed the Src homology 4 (SH4) domain of Vac8 independent of the rest of the protein, palmitoylation by Pfa3 still occurred. However, the specificity of palmitoylation seen for the full-length protein was lost, and the SH4 domain was palmitoylated by all five of the yeast DHHC proteins tested. These data suggested that a region of the protein C-terminal to the SH4 domain was important for conferring specificity of palmitoylation. This was confirmed by use of a chimeric protein in which the SH4 domain of Vac8 was swapped for that of Meh1, another palmitoylated and N-myristoylated protein in yeast. In this case we saw specificity mimic that of wild type Vac8. Competition experiments revealed that the 11th armadillo repeat of Vac8 is an important element for recognition by Pfa3. This demonstrates that regions distant from the palmitoylated cysteines are important for recognition by DHHC proteins.

Tetrahydrobiopterin Recycling, a Key Determinant of Endothelial Nitric-oxide Synthase-dependent Signaling Pathways in Cultured Vascular Endothelial Cells

Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated "knockdown" GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.

2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro

Pharmacologic approaches to studying palmitoylation are limited by the lack of specific inhibitors. Recently, screens have revealed five chemical classes of small molecules that inhibit cellular processes associated with palmitoylation (Ducker, C. E., L. K. Griffel, R. A. Smith, S. N. Keller, Y. Zhuang, Z. Xia, J. D. Diller, and C. D. Smith. 2006. Discovery and characterization of inhibitors of human palmitoyl acyltransferases. Mol. Cancer Ther. 5: 1647-1659). Compounds that selectively inhibited palmitoylation of N-myristoylated vs. farnesylated peptides were identified in assays of palmitoyltransferase activity using cell membranes. Palmitoylation is catalyzed by a family of enzymes that share a conserved DHHC (Asp-His-His-Cys) cysteine-rich domain. In this study, we evaluated the ability of these inhibitors to reduce DHHC-mediated palmitoylation using purified enzymes and protein substrates. Human DHHC2 and yeast Pfa3 were assayed with their respective N-myristoylated substrates, Lck and Vac8. Human DHHC9/GCP16 and yeast Erf2/Erf4 were tested using farnesylated Ras proteins. Surprisingly, all four enzymes showed a similar profile of inhibition. Only one of the novel compounds, 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one [Compound V (CV)], and 2-bromopalmitate (2BP) inhibited the palmitoyltransferase activity of all DHHC proteins tested. Hence, the reported potency and selectivity of these compounds were not recapitulated with purified enzymes and their cognate lipidated substrates. Further characterization revealed both compounds blocked DHHC enzyme autoacylation and displayed slow, time-dependent inhibition but differed with respect to reversibility. Inhibition of palmitoyltransferase activity by CV was reversible, whereas 2BP inhibition was irreversible.

Biochemical Characterization of Bovine Brain Myristoyl‐CoA:Protein N‐Myristoyltransferase Type 2

Protein N-myristoylation is a lipidic modification which refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral, and fungal proteins. In this paper, we have cloned the gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) from Bos tarus brain. The open reading frame codes for a 410-amino-acid protein and overexpressed in Escherichia coli. Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities. The metal ion Ca2+ had stimulatory effects on NMT2 activity while Mn2+ and Zn2+ inhibited the enzyme activity. In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect. Biochemical characterization suggested that both forms of NMT have unique characteristics. Further analysis towards functional role NMT2 will lead the development of therapeutic target for the progression of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases.

Expanded impact of protein N-myristoylation in plants

N-MYR controls the function of the plant protein complex SnRK1, described as one of the most important plant regulatory protein in stress and energy signalling. In plant cells, N-MYR is involved in a significantly higher number of metabolic pathways than in yeast or human. Some N-myristoylated protein families are solely encountered in plant cells. This lipid modification could be involved in the control of the redox imbalances originating from different stresses in plants. This prevalence of N-MYR in such proteins is unique to the plant kingdom. We hypothesize that this expansion of the mechanism in plants improves the control of the damages induced by environmental changes.Addendum to: Pierre M, Traverso JA, Boisson B, Domenichini S, Bouchez D, Giglione C, Meinnel T. N-myristoylation regulates the SnRK1 pathway in Arabidopsis. Plant Cell 2007; 19:2804-21.