Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms

The N-terminal protein processing pathway is an essential mechanism found in all organisms. However, it is widely believed that deformylase, a key enzyme involved in this process in bacteria, does not exist in eukaryotes, thus making it a target for antibacterial agents such as actinonin. In an attempt to define this process in higher eukaryotes we have used Arabidopsis thaliana as a model organism. Two deformylase cDNAs, the first identified in any eukaryotic system, and six distinct methionine aminopeptidase cDNAs were cloned. The corresponding proteins were characterized in vivo and in vitro. Methionine aminopeptidases were found in the cytoplasm and in the organelles, while deformylases were localized in the organelles only. Our work shows that higher plants have a much more complex machinery for methionine removal than previously suspected. We were also able to identify deformylase homologues from several animals and clone the corresponding cDNA from human cells. Our data provide the first evidence that lower and higher eukaryotes, as well as bacteria, share a similar N-terminal protein processing machinery, indicating universality of this system.     

An unusual peptide deformylase features in the human mitochondrial N-terminal methionine excision pathway

Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated.     

Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis

Arabidopsis (ecotype Columbia-0) genes, AtDEF1 and AtDEF2, represent eukaryotic homologs of the essential prokaryotic gene encoding peptide deformylase. Both deduced proteins contain three conserved protein motifs found in the active site of all eubacterial peptide deformylases, and N-terminal extensions identifiable as chloroplast-targeting sequences. Radiolabeled full-length AtDEF1 was imported and processed by isolated pea (Pisum sativum L. Laxton's Progress No. 9) chloroplasts and AtDEF1 and 2 were immunologically detected in Arabidopsis leaf and chloroplast stromal protein extracts. The partial cDNAs encoding the processed forms of Arabidopsis peptide deformylase 1 and 2 (pAtDEF1 and 2, respectively) were expressed in Escherichia coli and purified using C-terminal hexahistidyl tags. Both recombinant Arabidopsis peptide deformylases had peptide deformylase activity with unique kinetic parameters that differed from those reported for the E. coli enzyme. Actinonin, a specific peptide deformylase inhibitor, was effective in vitro against Arabidopsis peptide deformylase 1 and 2 activity, respectively. Exposure of several plant species including Arabidopsis to actinonin resulted in chlorosis and severe reductions in plant growth and development. The results suggest an essential role for peptide deformylase in protein processing in all plant plastids.     

Peptide aldehyde inhibitors of bacterial peptide deformylases.

Bacterial peptide deformylases (PDF, EC 3.5.1.27) are metalloenzymes that cleave the N-formyl groups from N-blocked methionine polypeptides. Peptide aldehydes containing a methional or norleucinal inhibited recombinant peptide deformylase from gram-negative Escherichia coli and gram-positive Bacillus subtilis. The most potent inhibitor was calpeptin, N-CBZ-Leu-norleucinal, which was a competitive inhibitor of the zinc-containing metalloenzymes, E. coli and B. subtilis PDF with Ki values of 26.0 and 55.6 microM, respectively. Cobalt-substituted E. coli and B. subtilis deformylases were also inhibited by these aldehydes with Ki values for calpeptin of 9.5 and 12.4 microM, respectively. Distinct spectral changes were observed upon binding of calpeptin to the Co(II)-deformylases, consistent with the noncovalent binding of the inhibitor rather than the formation of a covalent complex. In contrast, the chelator 1,10-phenanthroline caused the time-dependent inhibition of B. subtilis Co(II)-PDF activity with the loss of the active site metal. The fact that calpeptin was nearly equipotent against deformylases from both gram-negative and gram-positive bacterial sources lends further support to the idea that a single deformylase inhibitor might have broad-spectrum antibacterial activity.     

Control of protein life-span by N-terminal methionine excision

Peptide deformylases (PDFs) have been discovered recently in eukaryotic genomes, and it appears that N-terminal methionine excision (NME) is a conserved pathway in all compartments where protein synthesis occurs. This work aimed at uncovering the function(s) of NME in a whole proteome, using the chloroplast-encoded proteins of both Arabidopsis thaliana and Chlamydomonas reinhardtii as model systems. Disruption of PDF1B in A.thaliana led to an albino phenotype, and an extreme sensitivity to the PDF- specific inhibitor actinonin. In contrast, a knockout line for PDF1A exhibited no apparent phenotype. Photosystem II activity in C.reinhardtii cells was substantially reduced by the presence of actinonin. Pulse-chase experiments revealed that PDF inhibition leads to destabilization of a crucial subset of chloroplast-encoded photosystem II components in C. reinhardtii. The same proteins were destabilized in pdf1b. Site-directed substitutions altering NME of the most sensitive target, subunit D2, resulted in similar effects. Thus, plastid NME is a critical mechanism specifically influencing the life-span of photosystem II polypeptides. A general role of NME in modulating the half-life of key subsets of proteins is suggested.     

Specificity of cotranslational amino-terminal processing of proteins in yeast

Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.     

Distinctive features of the two classes of eukaryotic peptide deformylases.

Peptide deformylases (PDFs) are essential enzymes of the N-terminal protein processing pathway of eubacteria. The recent discovery of two types of PDFs in higher plants, PDF1A and PDF1B, and the detection of PDF1A in humans, have raised questions concerning the importance of deformylation in eukaryotes. Here, we have characterized fully in vitro and compared the properties of the two classes of eukaryotic PDFs, PDF1A and PDF1B, using the PDFs from Arabidopsis thaliana and Lycopersicon esculentum. We have shown that the PDFs of a given class (1A or 1B) all display similar features, independently of their origin. We also observed similar specificity of all plant PDFs for natural substrate peptides, but identified a number of biochemical differences between the two classes (1A or 1B). The main difference lies at the level of the bound cofactor, iron for PDF1B-like bacterial PDFs, and zinc for PDF1A. The nature of the metal cation has important consequences concerning the relative sensitivity to oxygen of the two plant PDFs. Investigation of the specificity of these enzymes with unusual substrates revealed additional differences between the two types of PDFs, enabling us to identify specific inhibitors with a lower affinity against PDF1As. However, the two plant PDFs were inhibited equally strongly in vitro by actinonin, an antibiotic that specifically acts on bacterial PDFs. Uptake of actinonin by A. thaliana seedlings was used to investigate the function of PDFs in the plant. Because it induces an albino phenotype, we conclude that deformylation is likely to play an essential role in the chloroplast.     

The crystal structures of four peptide deformylases bound to the antibiotic actinonin reveal two distinct types: a platform for the structure-based design of antibacterial agents

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.     

Extent of N-terminal modifications in cytosolic proteins from eukaryotes

Most proteins in all organisms undergo crucial N-terminal modifications involving N-terminal methionine excision, N-alpha-acetylation or N-myristoylation (N-Myr), or S-palmitoylation. We investigated the occurrence of these poorly annotated but essential modifications in proteomes, focusing on eukaryotes. Experimental data for the N-terminal sequences of animal, fungi, and archaeal proteins, were used to build dedicated predictive modules in a new software. In vitro N-Myr experiments were performed with both plant and animal N-myristoyltransferases, for accurate prediction of the modification. N-terminal modifications from the fully sequenced genome of Arabidopsis thaliana were determined by MS. We identified 105 new modified protein N-termini, which were used to check the accuracy of predictive data. An accuracy of more than 95% was achieved, demonstrating (i) overall conservation of the specificity of the modification machinery in higher eukaryotes and (ii) robustness of the prediction tool. Predictions were made for various proteomes. Proteins that had undergone both N-terminal methionine (Met) cleavage and N-acetylation were found to be strongly overrepresented among the most abundant proteins, in contrast to those retaining their genuine unblocked Met. Here we propose that the nature of the second residue of an ORF is a key marker of the abundance of the mature protein in eukaryotes.     

Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguis ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.     

Expression of Escherichia coli methionyl-tRNA formyltransferase in Saccharomyces cerevisiae leads to formylation of the cytoplasmic initiator tRNA and possibly to initiation of protein synthesis with formylmethionine

Protein synthesis in eukaryotic cytoplasm and in archaebacteria is initiated with methionine, whereas, that in eubacteria and in eukaryotic organelles, such as mitochondria and chloroplasts, is initiated with formylmethionine. In view of this clear distinction, we have investigated whether protein synthesis in the eukaryotic cytoplasm can be initiated with formylmethionine, and, if so, what the consequences are to the cell. For this purpose, we have expressed in an inducible manner the Escherichia coli methionyl-tRNA formyltransferase (MTF) in the cytoplasm of the yeast Saccharomyces cerevisiae. Expression of active MTF, but not of an inactive mutant, leads to formylation of methionine attached to the yeast cytoplasmic initiator tRNA to the extent of about 70%. As a consequence, the yeast strain grows slowly. Coexpression of the E. coli polypeptide deformylase (DEF), which removes the formyl group from the N-terminal formylmethionine in a polypeptide, rescues the slow-growth phenotype, whereas, coexpression of an inactive mutant of DEF does not. These results suggest that the cytoplasmic protein-synthesizing system of yeast, like that of eubacteria, can at least to some extent utilize formylated initiator Met-tRNA to initiate protein synthesis and that initiation of proteins with formylmethionine leads to the slow-growth phenotype. Removal of the formyl group in these proteins by DEF would explain the rescue of the slow-growth phenotype.     

Protein alpha-N-acetylation studied by N-terminomics

Cotranslational protein N-terminal modifications, including proteolytic maturation such as initiator methionine excision by methionine aminopeptidases and N-terminal blocking, occur universally. Protein alpha-N-acetylation, or the transfer of the acetyl moiety of acetyl-coenzyme A to nascent protein N-termini, catalysed by multisubunit N-terminal acetyltransferase complexes, generally takes place during protein translation. Nearly all protein modifications are known to influence different protein aspects such as folding, stability, activity and localization, and several studies have indicated similar functions for protein alpha-N-acetylation. However, until recently, protein alpha-N-acetylation remained poorly explored, mainly due to the absence of targeted proteomics technologies. The recent emergence of N-terminomics technologies that allow isolation of protein N-terminal peptides, together with proteogenomics efforts combining experimental and informational content have greatly boosted the field of alpha-N-acetylation. In this review, we report on such emerging technologies as well as on breakthroughs in our understanding of protein N-terminal biology.     

Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguis ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.     

Golgi targeting of ARF-like GTPase Arl3p requires its Nalpha-acetylation and the integral membrane protein Sys1p

Myristoylation of ARF family GTPases is required for their association with Golgi and endosomal membranes, where they regulate protein sorting and the lipid composition of these organelles. The Golgi-localized ARF-like GTPase Arl3p/ARP lacks a myristoylation signal, indicating that its targeting mechanism is distinct from myristoylated ARFs. We demonstrate that acetylation of the N-terminal methionine of Arl3p requires the NatC N(alpha)-acetyltransferase and that this modification is required for its Golgi localization. Chemical crosslinking and fluorescence microscopy experiments demonstrate that localization of Arl3p also requires Sys1p, a Golgi-localized integral membrane protein, which may serve as a receptor for acetylated Arl3p.     

Arabidopsis calcium-binding mitochondrial carrier proteins as potential facilitators of mitochondrial ATP-import and plastid SAM-import

Chloroplasts and mitochondria are central to crucial cellular processes in plants and contribute to a whole range of metabolic pathways. The use of calcium ions as a secondary messenger in and around organelles is increasingly appreciated as an important mediator of plant cell signaling, enabling plants to develop or to acclimatize to changing environmental conditions. Here, we have studied the four calcium-dependent mitochondrial carriers that are encoded in the Arabidopsis genome. An unknown substrate carrier, which was previously found to localize to chloroplasts, is proposed to present a calcium-dependent S-adenosyl methionine carrier. For three predicted ATP/phosphate carriers, we present experimental evidence that they can function as mitochondrial ATP-importers.     

A direct spectrophotometric assay for peptide deformylase.

A direct UV-VIS spectrophotometric assay has been developed for peptide deformylase. This assay employs a novel class of peptide mimetics as deformylase substrates which, upon enzymatic removal of the N-terminal formyl group, rapidly release free thiols. The released thiols are quantitated using Ellman's reagent. A variety of peptide analogues that contain beta-thiaphenylalanine or beta-thiamethionine as the N-terminal residue were synthesized and found to be excellent substrates of the peptide deformylase from Escherichia coli (k(cat)/K(M) = 6.9 x 10(5) M(-1) s(-1) for the most reactive substrate). The deformylase reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion. The versatility of the assay has been demonstrated by its application to kinetic characterization of the deformylase, pH profile studies, and enzyme inhibition assays. The assay can also be performed in an end-point fashion. The results demonstrate that this assay is a simple, highly sensitive, and rapid method to study kinetic properties of deformylases without the use of any coupling enzymes.     

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.     

Alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe operates both in nucleus and in mitochondria

The fission yeast, Schizosaccharomyces pombe, possesses a UV-damaged DNA endonuclease-dependent excision repair (UVER) pathway in addition to nucleotide excision repair pathway for UV-induced DNA damage. We examined cyclobutane pyrimidine dimer removal from the myo2 locus on the nuclear genome and the coI locus on the mitochondrial genome by the two repair pathways. While nucleotide excision repair repairs damage only on the nuclear genome, UVER efficiently removes cyclobutane pyrimidine dimers on both nuclear and mitochondrial genomes. The ectopically expressed wild type UV-damaged DNA endonuclease was localized to both nucleus and mitochondria, while modifications of N-terminal methionine codons restricted its localization to either of two organelles, suggesting an alternative usage of multiple translation initiation sites for targeting the protein to different organelles. By introducing the same mutations into the chromosomal copy of the uvde(+) gene, we selectively inactivated UVER in either the nucleus or the mitochondria. The results of UV survival experiments indicate that although UVER efficiently removes damage on the mitochondrial genome, UVER in the mitochondria hardly contributes to UV resistance of S. pombe cells. We suggest a possible UVER function in mitochondria as a backup system for other UV damage tolerance mechanisms.