N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence

Myristoylation by the myristoyl-CoA:protein N-myristoyltransferase (NMT) is an important lipid anchor modification of eukaryotic and viral proteins. Automated prediction of N-terminal N-myristoylation from the substrate protein sequence alone is necessary for large-scale sequence annotation projects but it requires a low rate of false positive hits in addition to a sufficient sensitivity.Our previous analysis of substrate protein sequence variability, NMT sequences and 3D structures has revealed motif properties in addition to the known PROSITE motif that are utilized in a new predictor described here. The composite prediction function (with separate ad hoc parameterization (a) for queries from non-fungal eukaryotes and their viruses and (b) for sequences from fungal species) consists of terms evaluating amino acid type preferences at sequences positions close to the N terminus as well as terms penalizing deviations from the physical property pattern of amino acid side-chains encoded in multi-residue correlation within the motif sequence. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set as well as with kinetic data for model substrates. The sensitivity in recognizing documented NMT substrates is above 95 % for both taxon-specific versions. The corresponding rate of false positive prediction (for sequences with an N-terminal glycine residue) is close to 0.5 %; thus, the technique is applicable for large-scale automated sequence database annotation. The predictor is available as public WWW-server with the URL http://mendel.imp.univie.ac.at/myristate/. Additionally, we propose a version of the predictor that identifies a number of proteolytic protein processing sites at internal glycine residues and that evaluates possible N-terminal myristoylation of the protein fragments.A scan of public protein databases revealed new potential NMT targets for which the myristoyl modification may be of critical importance for biological function. Among others, the list includes kinases, phosphatases, proteasomal regulatory subunit 4, kinase interacting proteins KIP1/KIP2, protozoan flagellar proteins, homologues of mitochondrial translocase TOM40, of the neuronal calcium sensor NCS-1 and of the cytochrome c-type heme lyase CCHL. Analyses of complete eukaryote genomes indicate that about 0.5 % of all encoded proteins are apparent NMT substrates except for a higher fraction in Arabidopsis thaliana ( approximately 0.8 %).     

N-Methyl transferase of Streptomyces erythraeus that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics: amino acid sequence and its homology to cognate R-factor enzymes from pathogenic bacilli and cocci

The nucleotide sequence of a structural gene ermE for ribosomal RNA (rRNA) N6-amino adenine N-methyl transferase (NMT) of Streptomyces erythraeus, cloned by Thompson et al. [Gene 20 (1982) 51-62], has been determined. The NMT amino acid (aa) sequence deduced from the nucleotide sequence contains extensive homology to aa sequences of cognate NMTs specified by: (1) plasmid pE194 from Staphylococcus aureus, 30% G + C, ermC; (2) plasmid pAM77 from Streptococcus sanguis, 43% G + C; as well as to (3) a chromosomal determinant from Bacillus licheniformis 759, 46% G + C, ermD, cloned in a recombinant plasmid pBD90. These findings suggest that all four NMT structural genes could have evolved from a common progenitor sequence despite the wide range of % G + C of the erm genes reflecting their current respective hosts. Comparison of the four NMT sequences with respect to localized hydrophobicity averaged over a moving window of 11 aa indicates that the common features of localized hydrophobicity that characterize the C-terminal portion of the ermE and ermD proteins are distinguishable from a contrasting pattern of hydrophobicity that characterizes the ermC and pAM77-coded proteins.     

Analysis of the compartmentalization of myristoyl-CoA:protein N-myristoyltransferase in Saccharomyces cerevisiae.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the cotranslational, covalent attachment of a rare fatty acid, myristic acid (C14:0), to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction as well as several viral proteins necessary for assembly-replication. NMT has become a target for both anti-viral and anti-fungal therapy. Analysis of purified Saccharomyces cerevisiae NMT plus yeast strains with conditional lethal nmt1 mutations have provided insights about how this process is regulated in vivo. We have now defined the location of NMT in two strains of S. cerevisiae to better understand the functional and spatial relationships between this enzyme and cellular systems that generate its acyl-CoA and peptide ligands. Western blot studies using an affinity purified antibody raised in rabbits against purified S. cerevisiae NMT indicate that the acyltransferase represents 0.06% of total cellular proteins in an exponentially growing haploid strain with a wild type NMT1 allele. Another strain containing a single, integrated copy of a GAL1/NMT1 fusion gene and a nmt1 null allele had 12-fold higher levels of NMT when grown on galactose-containing media. This increase in NMT production had no detectable effects on growth or cellular morphology. Cell fractionation studies, confocal fluorescence immunocytochemical analysis, and immunogold electron microscopic surveys of fixed, gelatin-embedded cryosections of both strains revealed that NMT is a cytosolic protein that is not associated with cellular membranes (including the endoplasmic reticulum and plasma membrane), the nucleus, mitochondria, Golgi apparatus, or vacuoles. This finding is discussed in light of what is known about the location and activities of enzymes involved in de novo fatty acid biosynthesis and in amino-terminal processing of nascent proteins.     

The Candida albicans myristoyl-CoA:protein N-myristoyltransferase gene. Isolation and expression in Saccharomyces cerevisiae and Escherichia coli.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) has recently been identified as a target for antiviral and antifungal therapy. Candida albicans is a dimorphic, asexual yeast that is a major cause of systemic fungal infections in immunosuppressed humans. Metabolic labeling studies indicate that C. albicans synthesizes one principal 20-kDa N-myristoyl-protein. The single copy C. albicans NMT gene (ca-NMT1) was isolated and encodes a 451-amino acid protein that has 55% identity with Saccharomyces cerevisiae NMT. C. albicans NMT1 is able to complement the lethal phenotype of S. cerevisiae nmt1 null mutants by directing efficient acylation of the approximately 12 endogenous N-myristoylproteins produced by S. cerevisiae. C. albicans NMT was produced in Escherichia coli, a prokaryote with no endogenous NMT activity. In vitro studies of purified E. coli-derived S. cerevisiae and C. albicans NMTs revealed species-specific differences in the kinetic properties of synthetic octapeptide substrates derived from known N-myristoylproteins. Together these data indicate that C. albicans and S. cerevisiae NMTs have similar yet distinct substrate specificities which may be of therapeutic significance.     

Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N- myristoylation

Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N-myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp.     

Molecular characterization of TRP1, a gene coding for tryptophan synthetase in the basidiomycete Coprinus cinereus

We utilized a cloned gene (TRP5) encoding tryptophan synthetase (TSase) from Saccharomyces cerevisiae to identify and clone the corresponding gene (TRP1) from the basidiomycete Coprinus cinereus. The primary nucleotide (nt) sequence of this gene was determined and compared to sequences from other filamentous fungi, as well as to other genes coding for TSase. A transformation assay was used to demonstrate that 321 nt, which do not include CAAT or TATAAA elements and precede the translation initiation codon, are sufficient for expression in a variety of chromosomal locations. The coding region (2584 nt) is interrupted at nine positions, and putative splicing signals (5'-GTRNGT...YAG-3') are present in each case. The predicted translation product contains 702 amino acids (aa) and is very similar to other TSases, except in the region of aa 257-296 that connects the alpha and beta functional domains. Both the number and the identity of the aa differ in this region between C. cinereus. S. cerevisiae, and Neurospora crassa. Comparison of exon boundaries in the C. cinereus sequence to the three-dimensional structure of Salmonella typhimurium TSase indicates that there is no simple correlation between exons and major functional domains in this protein.     

Genomic Organization and Complete Nucleotide Sequence of the HumanPWP2Gene on Chromosome 21

The humanPWP2gene is the human homologue of the yeast periodic tryptophan protein 2 (PWP2) gene and is a member of the gene family that contains tryptophan-aspartate (WD) repeats. Genomic sequencing revealed that the humanPWP2gene consists of 21 exons spanning approximately 24 kb and locates just between the two genes EHOC-1 and KNP-I and distal to aNotI site of LJ104 (D21S1460) on chromosome 21q22.3. Analysis of the 5′-flanking DNA sequence revealed that the upstream region of thePWP2gene is associated with a CpG island containing theNotI site of LJ104. SincePWP2is considered to be a candidate for genetic disorders mapped in the 21q22.3 region, the information including nucleotide sequence and genomic organization of thePWP2gene should be invaluable for the mutation analysis of the corresponding genetic disorders.     

Gene Families Encoding 11S Globulin and 2S Albumin Isoforms of Jelly Fig (Ficus awkeotsang) Achenes

Seed storage proteins of plants commonly comprise several groups of multiple isoforms encoded by gene families. From about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes, gene families encoding precursor polypeptides of two storage protein classes, including six 11S globulin isoforms and two 2S albumin isoforms, were identified. Complete sequences encoding the precursor polypeptides of these eight storage proteins were obtained by sequencing the pertinent EST clones that contained full-length cDNA fragments. Matrix-assisted laser desorption/ionization mass spectrometry analysis confirmed the presence of these storage protein isoforms in the extract of jelly fig achenes resolved in SDS–PAGE. The amino acid compositions of the deduced storage proteins indicated that achene proteins in jelly fig are nutritive, for both isoforms of 2S albumin are sulfur-rich, and one of them is also rich in tryptophan.     

BRCA1蛋白的序列及空间结构的分析

查询人的BRCA1蛋白的氨基酸序列,利用生物信息学的方法进行相似性搜索,获得一系列BRCA1蛋白的氨基酸序列。选择了其中的11条序列,对BRCA1蛋白进行了多重序列分析和进化分析,对BRCA1蛋白的BRCT结构域进行三维同源模型的构建与比较分析。分析结果表明:BRCA1中某些特定部位的氨基酸序列高度保守;确定氨基酸的保守位点并联合进化分析可对基因错义突变的致病性做初步地猜测;相近物种来源的BRCA1具有较近的亲缘关系,而且具有极其相似的三维空间结构。这些为研究BRCA1蛋白的结构与功能关系提供指导意义。     

Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.     

Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli

A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous alpha subunits of the signal-transducing, heterotrimeric G proteins. Exogenous [3H]myristate was incorporated into rat Gi alpha 1 and rat Go alpha but not into bovine Gs alpha or human Gz alpha. Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity. Metabolic labeling studies with 6-, 11-, or 13-[3H]oxatetradecanoic acid indicated that they were incorporated into rat Gi alpha 1 and Go alpha with an efficiency that could be correlated with their accumulation into E. coli and their interactions with purified NMT in vitro. Octapeptides derived from the NH2-terminal sequences of these four G alpha polypeptides were tested as substrates for purified S. cerevisiae NMT. None were bound by the enzyme. Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat Go alpha produced active substrates. These results imply that productive interactions between NMT and G alpha protein substrates in vivo require structural features that are not fully represented within their NH2-terminal 8 residues.     

Conservation and dispersion of sequence and function in fungal TRK potassium transporters: focus on Candida albicans

TRK proteins – essential potassium (K⁺) transporters in fungi and bacteria, as well as in plants – are generally absent from animal cells, which makes them potential targets for selective drug action. Indeed, in the human pathogen Candida albicans , the single TRK isoform (CaTrk1p) has recently been demonstrated to be required for activity of histidine-rich salivary antimicrobial peptides (histatins). Background for a detailed molecular investigation of TRK-protein design and function is provided here in sequence analysis and quantitative functional comparison of CaTrk1p with its better-known homologues from Saccharomyces cerevisiae . Among C. albicans strains (ATCC 10261, SC5314, WO-1), the DNA sequence is essentially devoid of single nucleotide polymorphisms in regions coding for evolutionarily conserved segments of the protein, meaning the four intramembranal [membrane–pore–membrane (MPM)] segments thought to be involved directly with the conduction of K⁺ ions. Among 48 fungal (ascomycete) TRK homologues now described by complete sequences, clades (but not the detailed order within clades) appear conserved for all four MPM segments, independently assessed. The primary function of TRK proteins, 'active' transport of K⁺ ions, is quantitatively conserved between C. albicans and S. cerevisiae . However, the secondary function, chloride efflux channeling, is present but poorly conserved between the two species, being highly variant with respect to activation velocity, amplitude, flickering (channel-like) behavior, pH dependence, and inhibitor sensitivity.     

Characterization of the five replication factor C genes of Saccharomyces cerevisiae.

Replication factor C (RFC) is a five-subunit DNA polymerase accessory protein that functions as a structure-specific, DNA-dependent ATPase. The ATPase function of RFC is activated by proliferating cell nuclear antigen. RFC was originally purified from human cells on the basis of its requirement for simian virus 40 DNA replication in vitro. A functionally homologous protein complex from Saccharomyces cerevisiae, called ScRFC, has been identified. Here we report the cloning, by either peptide sequencing or by sequence similarity to the human cDNAs, of the S. cerevisiae genes RFC1, RFC2, RFC3, RFC4, and RFC5. The amino acid sequences are highly similar to the sequences of the homologous human RFC 140-, 37-, 36-, 40-, and 38-kDa subunits, respectively, and also show amino acid sequence similarity to functionally homologous proteins from Escherichia coli and the phage T4 replication apparatus. All five subunits show conserved regions characteristic of ATP/GTP-binding proteins and also have a significant degree of similarity among each other. We have identified eight segments of conserved amino acid sequences that define a family of related proteins. Despite their high degree of sequence similarity, all five RFC genes are essential for cell proliferation in S. cerevisiae. RFC1 is identical to CDC44, a gene identified as a cell division cycle gene encoding a protein involved in DNA metabolism. CDC44/RFC1 is known to interact genetically with the gene encoding proliferating cell nuclear antigen, confirming previous biochemical evidence of their functional interaction in DNA replication.     

肺形侧耳变温结实相关基因Ppcsl-1的克隆及功能预测

CSL（CBF1/RBP-Jκ/suppressor of hairless/LAG-1）转录因子家族在真菌发育和细胞分化过程中扮演重要角色。前期研究已构建了肺形侧耳变温结实相关消减杂交文库,并从中筛选到一个代表csl基因部分序列的EST。通过TAIL PCR（thermal asymmetric interlaced PCR）技术克隆了该基因（Pleurotus pulmonarius csl-1,简写为 Ppcsl-1）,并利用RACE（rapid-amplification of cDNA ends）技术获得该基因的cDNA全长。Ppcsl-1 cDNA全长2 991bp,编码一个996个氨基酸组成的蛋白（命名为PpCSL-1）。进化分析显示在担子菌CSL中,PpCSL-1与糙皮侧耳Pleurotus ostreatus CSL（PoCSL）亲缘关系最近。荧光定量检测结果表明Ppcsl-1在菌丝经过5℃ 12h冷处理之后的表达量最高,表明其有可能被冷刺激诱导表达并在开启子实体形成的过程中起重要作用。     

Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeast-phase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall beta-glucan levels by about 50%. The purified C. albicans beta-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified beta-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate beta-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of beta-1,3-glucan synthase.     

The C1orf9 gene encodes a putative transmembrane member of a novel protein family

Here we report the characterization of a human mRNA encoding a novel protein denoted C1orf9 (chromosome 1 open reading frame 9). The cDNA sequence, derived from a testis cDNA library, contains 5700 bp which encodes an open reading frame of 1254 amino acids. The deduced protein contains a putative N-terminal signal peptide and one putative transmembrane region, indicating membrane localization. No significant homology was found with known characterized proteins. However, a 150 amino acid region has significant homology to deduced protein sequences from other organisms, including Caenorhabditis elegans (43% identity), Saccharomyces cerevisiae (47% identity), Schizosaccharomyces pombe (48% identity), and two proteins from Arabidopsis thaliana (42% and 40% identity), suggesting a novel family of conserved domains. The C1orf9 gene was assigned to chromosome 1q24. The gene spans approximately 78.7 kb and is organized into at least 24 exons. Expression analysis revealed a single C1orf9 mRNA species of approximately 6.0 kb with a predominant expression in pancreas and testis, and only low levels of expression in other tissues examined.