N-terminal N-myristoylation of proteins: refinement of the sequence motif and its taxon-specific differences

N-terminal N-myristoylation is a lipid anchor modification of eukaryotic and viral proteins targeting them to membrane locations, thus changing the cellular function of modified proteins. Protein myristoylation is critical in many pathways; e.g. in signal transduction, apoptosis, or alternative extracellular protein export. The myristoyl-CoA:protein N-myristoyltransferase (NMT) recognizes the sequence motif of appropriate substrate proteins at the N terminus and attaches the lipid moiety to the absolutely required N-terminal glycine residue. Reliable recognition of capacity for N-terminal myristoylation from the substrate protein sequence alone is desirable for proteome-wide function annotation projects but the existing PROSITE motif is not practical, since it produces huge numbers of false positive and even some false negative predictions.As a first step towards a new prediction method, it is necessary to refine the sequence motif coding for N-terminal N-myristoylation. Relying on the in-depth study of the amino acid sequence variability of substrate proteins, on binding site analyses in X-ray structures or 3D homology models for NMTs from various taxa, and on consideration of biochemical data extracted from the scientific literature, we found indications that, at least within a complete substrate protein, the N-terminal 17 protein residues experience different types of variability restrictions. We identified three motif regions: region 1 (positions 1-6) fitting the binding pocket; region 2 (positions 7-10) interacting with the NMT's surface at the mouth of the catalytic cavity; and region 3 (positions 11-17) comprising a hydrophilic linker. Each region was characterized by physical requirements to single sequence positions or groups of positions regarding volume, polarity, backbone flexibility and other typical properties of amino acids (http://mendel.imp.univie.ac.at/myristate/). These specificity differences are confined partly to taxonomic ranges and are proposed for the design of NMT inhibitors in pathogenic fungal and protozoan systems including Aspergillus fumigatus, Leishmania major, Trypanosoma cruzi, Trypanosoma brucei, Giardia intestinalis, Entamoeba histolytica, Pneumocystis carinii, Strongyloides stercoralis and Schistosoma mansoni. An exhaustive search for NMT-homologues led to the discovery of two putative entomopoxviral NMTs.     

Prediction of potential GPI-modification sites in proprotein sequences.

Glycosylphosphatidylinositol (GPI) lipid anchoring is a common posttranslational modification known mainly from extracellular eukaryotic proteins. Attachment of the GPI moiety to the carboxyl terminus (omega-site) of the polypeptide follows after proteolytic cleavage of a C-terminal propeptide. For the first time, a new prediction technique locating potential GPI-modification sites in precursor sequences has been applied for large-scale protein sequence database searches. The composite prediction function (with separate parametrisation for metazoan and protozoan proteins) consists of terms evaluating both amino acid type preferences at sequence positions near a supposed omega-site as well as the concordance with general physical properties encoded in multi-residue correlation within the motif sequence. The latter terms are especially successful in rejecting non-appropriate sequences from consideration. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set of fully annotated sequences from the SWISS-PROT database as well as with a newly created database "big-Pi" (more than 300 GPI-motif mutations extracted from original literature sources). The accuracy of predicting the effect of mutations in the GPI sequence motif was above 83 %. Lists of potential precursor proteins which are non-annotated in SWISS-PROT and SPTrEMBL are presented on the WWW-page http://www.embl-heidelberg.de/beisenha/gpi/gpi_p rediction. html The algorithm has been implemented in the prototype software "big-Pi predictor" which may find application as a genome annotation and target selection tool.     

N-Terminal myristoylation predictions by ensembles of neural networks

N-terminal myristoylation is a post-translational modification that causes the addition of a myristate to a glycine in the N-terminal end of the amino acid chain. This work presents neural network (NN) models that learn to discriminate myristoylated and nonmyristoylated proteins. Ensembles of 25 NNs and decision trees were trained on 390 positive sequences and 327 negative sequences. Experiments showed that NN ensembles were more accurate than decision tree ensembles. Our NN predictor evaluated by the leave-one-out procedure, obtained a false positive error rate equal to 2.1%. That was better than the PROSITE pattern for myristoylation for which the false positive error rate was 22.3%. On a recent version of Swiss-Prot (41.2), the NN ensemble predicted 876 myristoylated proteins, while 1150 proteins were predicted by the PROSITE pattern for myristoylation. Finally, compared to the well-known NMT predictor, the NN predictor gave similar results. Our tool is available under http://www.expasy.org/tools/myristoylator/myristoylator.html.     

Glycosylphosphatidylinositol lipid anchoring of plant proteins. Sensitive prediction from sequence- and genome-wide studies for Arabidopsis and rice

Posttranslational glycosylphosphatidylinositol (GPI) lipid anchoring is common not only for animal and fungal but also for plant proteins. The attachment of the GPI moiety to the carboxyl-terminus after proteolytic cleavage of a C-terminal propeptide is performed by the transamidase complex. Its four known subunits also have obvious full-length orthologs in the Arabidopsis and rice (Oryza sativa) genomes; thus, the mechanism of substrate protein processing appears similar for all eukaryotes. A learning set of plant proteins (substrates for the transamidase complex) has been collected both from the literature and plant sequence databases. We find that the plant GPI lipid anchor motif differs in minor aspects from the animal signal (e.g. the plant hydrophobic tail region can contain a higher fraction of aromatic residues). We have developed the "big-Pi plant" program for prediction of compatibility of query protein C-termini with the plant GPI lipid anchor motif requirements. Validation tests show that the sensitivity for transamidase targets is approximately 94%, and the rate of false positive prediction is about 0.1%. Thus, the big-Pi predictor can be applied as unsupervised genome annotation and target selection tool. The program is also suited for the design of modified protein constructs to test their GPI lipid anchoring capacity. The big-Pi plant predictor Web server and lists of potential plant precursor proteins in Swiss-Prot, SPTrEMBL, Arabidopsis, and rice proteomes are available at http://mendel.imp.univie.ac.at/gpi/plants/gpi_plants.html. Arabidopsis and rice protein hits have been functionally classified. Several GPI lipid-anchored arabinogalactan-related proteins have been identified in rice.     

Prediction of lipid posttranslational modifications and localization signals from protein sequences: big-Pi,NMT and PTS1

Many posttranslational modifications (N-myristoylation or glycosylphosphatidylinositol (GPI) lipid anchoring) and localization signals (the peroxisomal targeting signal PTS1) are encoded in short, partly compositionally biased regions at the N- or C-terminus of the protein sequence. These sequence signals are not well defined in terms of amino acid type preferences but they have significant interpositional correlations. Although the number of verified protein examples is small, the quantification of several physical conditions necessary for productive protein binding with the enzyme complexes executing the respective transformations can lead to predictors that recognize the signals from the amino acid sequence of queries alone. Taxon-specific prediction functions are required due to the divergent evolution of the active complexes. The big-Pi tool for the prediction of the C-terminal signal for GPI lipid anchor attachment is available for metazoan, protozoan and plant sequences. The myristoyl transferase (NMT) predictor recognizes glycine N-myristoylation sites (at the N-terminus and for fragments after processing) of higher eukaryotes (including their viruses) and fungi. The PTS1 signal predictor finds proteins with a C-terminus appropriate for peroxisomal import (for metazoa and fungi). Guidelines for application of the three WWW-based predictors (http://mendel.imp.univie.ac.at/) and for the interpretation of their output are described.     

Phosphorylation of human N-myristoyltransferase by N-myristoylated SRC family tyrosine kinase members

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the amino terminal glycine residue of a number of important proteins especially the non-receptor tyrosine kinases whose activity is important for tumorigenesis. Human NMT was found to be phosphorylated by non-receptor tyrosine kinase family members of Lyn, Fyn and Lck and dephosphorylated by the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin. Deletion of 149 amino acids from the N-terminal end resulted in the absence of phosphorylation suggesting that the phosphorylation sites are located in the N-terminal end of NMT. Furthermore, a site-directed mutagenesis study indicated that substitution of tyrosine 100 with phenylalanine served NMT as a poor substrate for the Lyn kinase. A synthetic peptide corresponding to the amino-terminal region encompassing tyrosine 100 of NMT served as a good substrate for the Lyn and Fyn kinases. Our studies also indicated that NMT was found to interact with Lyn through its N-terminal end in a phosphorylation-dependent manner. This is the first study demonstrating the cross-talk between NMT and their myristoylated protein substrates in signaling pathways.     

Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase

Using synthetic octapeptides, we examined the amino-terminal sequence requirements for substrate recognition by myristoyl-CoA:protein N-myristoyl transferase (NMT). NMT is absolutely specific for peptides with amino-terminal Gly residues. Peptides with Asn, Gln, Ser, Val, or Leu penultimate to the amino-terminal Gly were substrates, whereas peptides with Asp, D-Asn, Phe, or Tyr at this position were not myristoylated. Peptides with aromatic residues at this position competitively inhibited myristoylation of substrates, introducing the possibility of developing specific in vivo inhibitors of NMT. Peptides having sequences which correspond to those of known N-myristoyl proteins, including p60src, appear to be recognized by a single enzyme, and yeast and murine NMT have identical substrate specificities. The catalytic selectivity of NMT for myristoyl transfer accounts for the remarkable acyl chain specificity of this enzyme.     

Molecular characterization of a gene encoding N-myristoyl transferase (NMT) from Triticum aestivum (bread wheat).

Myristoyl-CoA:protein N-myristoyl transferase (NMT; EC 2.3.1.97) acylates the Gly residue abutting the N-terminal Met with a myristic acid following the removal of the Met residue in certain eukaryotic proteins, and in some cases myristoylation is essential to cell growth and survival. We report the cloning of a full-length cDNA encoding NMT from Triticum aestivum (TaNMT). The cDNA included a predicted open reading frame of 1317 nucleotides, which encoded a predicted protein of 438 amino acids containing all of the residues that are important for NMT activity. The TaNMT amino acid and nucleotide sequences were compared with NMTs from 14 other species encompassing a wide array of taxonomic groups. Among the experimentally validated NMTs, TaNMT was most similar to that of Arabidopsis thaliana. Southern blot analysis of wheat genomic DNA showed that TaNMT is encoded by a single copy gene, with one copy per haploid genome. We expressed TaNMT in Escherichia coli cells and determined that the recombinant protein possessed NMT activity, catalyzing the N-myristoylation of peptides from known or putatively myristoylated proteins from plants and animals without a strong preference for the plant peptides. TaNMT is the second experimentally validated plant NMT sequence and the first from a monocotyledonous species.     

A critical assessment of the secondary structure alpha-helices and their termini in proteins

Secondary structure prediction from amino acid sequence is a key component of protein structure prediction, with current accuracy at approximately 75%. We analysed two state-of-the-art secondary structure prediction methods, PHD and JPRED, comparing predictions with secondary structure assigned by the algorithms DSSP and STRIDE. The specific focus of our study was alpha-helix N-termini, as empirical free energy scales are available for residue preferences at N-terminal positions. Although these prediction methods perform well in general at predicting the alpha-helical locations and length distributions in proteins, they perform less well at predicting the correct helical termini. For example, although most predicted alpha-helices overlap a real alpha-helix (with relatively few completely missed or extra predicted helices), only one-third of JPRED and PHD predictions correctly identify the N-terminus. Analysis of neighbouring N-terminal sequences to predicted helical N-termini shows that the correct N-terminus is often within one or two residues. More importantly, the true N-terminal motif is, on average, more favourable as judged by our experimentally measured free energies. This suggests a simple, but powerful, strategy to improve secondary structure prediction using empirically derived energies to adjust the predicted output to a more favourable N-terminal sequence.     

Human N-myristoyltransferases form stable complexes with lentiviral nef and other viral and cellular substrate proteins

Nef is a multifunctional virulence factor of primate lentiviruses that facilitates viral replication in the infected host. All known functions of Nef require that it be myristoylated at its N terminus. This reaction is catalyzed by N-myristoyltransferases (NMTs), which transfer myristate from myristoyl coenzyme A (myristoyl-CoA) to the N-terminal glycine of substrate proteins. Two NMT isoforms (NMT-1 and NMT-2) are expressed in mammalian cells. To provide a better mechanistic understanding of Nef function, we used biochemical and microsequencing techniques to isolate and identify Nef-associated proteins. Through these studies, NMT-1 was identified as an abundant Nef-associated protein. The Nef-NMT-1 complex is most likely a transient intermediate of the myristoylation reaction of Nef and is modulated by agents which affect the size of the myristoyl-CoA pool in the cell. We also examined two other proteins that bear an N-terminal myristoylation signal, human immunodeficiency virus type 1 Gag and Hck protein tyrosine kinase, and found that Gag bound preferentially the NMT-2 isoform, while Hck bound mostly to NMT-1. Recognition of different NMT isoforms by these viral and cellular substrate proteins suggests nonoverlapping roles for these enzymes in vivo and reveals a potential for the development of inhibitors that target the myristoylation of specific viral substrates more selectively.     

Identification of related proteins with weak sequence identity using secondary structure information

Molecular modeling of proteins is confronted with the problem of finding homologous proteins, especially when few identities remain after the process of molecular evolution. Using even the most recent methods based on sequence identity detection, structural relationships are still difficult to establish with high reliability. As protein structures are more conserved than sequences, we investigated the possibility of using protein secondary structure comparison (observed or predicted structures) to discriminate between related and unrelated proteins sequences in the range of 10%-30% sequence identity. Pairwise comparison of secondary structures have been measured using the structural overlap (Sov) parameter. In this article, we show that if the secondary structures likeness is >50%, most of the pairs are structurally related. Taking into account the secondary structures of proteins that have been detected by BLAST, FASTA, or SSEARCH in the noisy region (with high E: value), we show that distantly related protein sequences (even with <20% identity) can be still identified. This strategy can be used to identify three-dimensional templates in homology modeling by finding unexpected related proteins and to select proteins for experimental investigation in a structural genomic approach, as well as for genome annotation.     

Mechanisms of action of NIP71 on N-myristoyltransferase activity

N-Myristoyl-CoA:protein N-myristoyltransferase (NMT) is the enzyme that catalyses the transfer of myristate from myristoyl-CoA to the N-terminal glycine of protein substrates. NMT was highly purified from bovine brain by procedures involving sequential column chromatography on DEAE-Sepharose CL-6B, phosphocellulose, hydroxylapatite, and mono S and mono Q f.p.l.c.. The highly purified NMT (termed NMT·II) possessed high specific activity with peptide substrates derived from the N-terminal sequences of the cAMP-dependent protein kinase and pp60^src (29,800 and 47,600 pmol N-myristoylpeptide formed/min/mg, respectively), intermediate activity with a peptide based on the N-terminal sequence of a viral structural protein (l) (M2; 17,300 pmol N-myristoylpeptide formed/min/mg) and very low activity with a peptide derived from the N-terminal sequence ofmyristoylatedalanine-richC-kinasesubstrate (MARCKS; 1500 pmol myristoylpeptide formed/min/mg). An NMT protein inhibitor (NIP₇₁) isolated from the particulate fraction of bovine brain (King MJ and Sharma RK: Biochem J 291635-639, 1993) potently inhibited highly purified NMT activity (IC₅₀ 23.7 nM). A minor NMT activity (NMT·PU; 30% total NMT activity), which failed to bind to phosphocellulose, was insensitive to NIP₇₁ inhibition. Inhibition of NMT was observed to be via mixed inhibition with respect to both the myristoyl-CoA and peptide substrates with NIP₇₁ having an apparent higher affinity for NMT than the NMT·myristoyl·CoA complex. Inhibition by NIP₇₁ at subsaturating concentrations of myristolyl-CoA and peptide resulted in a sigmoidal pattern of inhibition indicating that bovine brain possesses a potent and delicate on/off switch to control NMT activity.Abbreviations NMT N-myristoyl-CoA:protein N-myristoyltransferase - NMT·I mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak I - NMT·II mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak II - NMT·III mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak III - NIP₇₁ 71 kDa heat-stable N-myristoyltransferase inhibitor protein     

Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis

The Arabidopsis genome contains approximately 200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR-encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.     

A novel representation of protein sequences for prediction of subcellular location using support vector machines

As the number of complete genomes rapidly increases, accurate methods to automatically predict the subcellular location of proteins are increasingly useful to help their functional annotation. In order to improve the predictive accuracy of the many prediction methods developed to date, a novel representation of protein sequences is proposed. This representation involves local compositions of amino acids and twin amino acids, and local frequencies of distance between successive (basic, hydrophobic, and other) amino acids. For calculating the local features, each sequence is split into three parts: N-terminal, middle, and C-terminal. The N-terminal part is further divided into four regions to consider ambiguity in the length and position of signal sequences. We tested this representation with support vector machines on two data sets extracted from the SWISS-PROT database. Through fivefold cross-validation tests, overall accuracies of more than 87% and 91% were obtained for eukaryotic and prokaryotic proteins, respectively. It is concluded that considering the respective features in the N-terminal, middle, and C-terminal parts is helpful to predict the subcellular location.     

A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans, Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe

The fungal transamidase complex that executes glycosylphosphatidylinositol (GPI) lipid anchoring of precursor proteins has overlapping but distinct sequence specificity compared with the animal system. Therefore, a taxon-specific prediction tool for the recognition of the C-terminal signal in fungal sequences is necessary. We have collected a learning set of fungal precursor protein sequences from the literature and fungal proteomes. Although the general four segment scheme of the recognition signal is maintained also in fungal precursors, there are taxon specificities in details. A fungal big-Pi predictor has been developed for the assessment of query sequence concordance with fungi-specific recognition signal requirements. The sensitivity of this predictor is close to 90%. The rate of false positive prediction is in the range of 0.1%. The fungal big-Pi tool successfully predicts the Gas1 mutation series described by C. Nuoffer and co-workers, and recognizes that the human PLAP C terminus is not a target for the fungal transamidase complex. Lists of potentially GPI lipid anchored proteins for five fungal proteomes have been generated and the hits have been functionally classified. The fungal big-Pi prediction WWW server as well as precursor lists are available at     

Bioverse: Functional,structural and contextual annotation of proteins and proteomes

Functional annotation is routinely performed for large-scale genomics projects and databases. Researchers working on more specific problems, for instance on an individual pathway or complex, also need to be able to quickly, completely and accurately annotate sequences. The Bioverse sequence annotation server (http://bioverse.compbio.washington.edu) provides a web-based interface to allow users to submit protein sequences to the Bioverse framework. Sequences are functionally and structurally annotated and potential contextual annotations are provided. Researchers can also submit candidate genomes for annotation of all proteins encoded by the genome (proteome).     

Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel

Electroblotting method employing a semidry blotting apparatus for the subsequent protein microsequence analysis (Hirano, 1987) was improved. This method is convenient and allows rapid and efficient transfer of the proteins from a polyacrylamide gel (1 mm thick) onto the Polybrene-coated glass-fiber sheet or polyvinylidene difluoride membrane filter in only 20 min. The electroblotted proteins could be sequenced directly with the gas-phase protein sequencer at a 20-pmole level. This method was applied to the sequence analysis of winged bean seed proteins. A portion of the crude extracts from only one-twentieth of a seed of the winged bean was separated by two-dimensional polyacrylamide gel electrophoresis and electroblotted, and the N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of about 60% of the blotted major proteins, including nine Kunitz trypsin inhibitor-like proteins with heterogeneity in the N-terminal sequences, a protein that has a homologous sequence to the leghaemoglobin, nitrogen-fixing root nodule-specific protein, and a soybean basic 7S globulin-like protein could be easily identified.     

Fragment peptide library for classification and functional prediction of proteins

From protein sequence comparison data found in the literature, a library was organized using peptide fragment sequences which are common to related proteins. Each of the fragments was then examined for its occurrence in all the protein superfamilies defined by the NBRF-PIR data base. We have selected those fragment peptides that appear exclusively in one or a few superfamilies, and thus made a library of fragment peptides that characterize specific superfamilies. Such characteristic peptides are, in general, five to seven residues long and contain unusually high proportions of glycine and cysteine. This collection is a useful resource for the classification and functional prediction of protein molecules.