Carmela Giglione

Paternal Control of Embryonic Patterning in Arabidopsis thaliana

The YODA (YDA) mitogen-activated protein kinase pathway promotes elongation of the Arabidopsis zygote and development of its basal daughter cell into the extra-embryonic suspensor. Here, we show that the interleukin-1 receptor–associated kinase (IRAK)/Pelle-like kinase gene SHORT SUSPENSOR (SSP) regulates this pathway through a previously unknown parent-of-origin effect. SSP transcripts are produced in mature pollen but do not appear to be translated. Instead, they are delivered via the sperm cells to the zygote and the endosperm, where SSP protein transiently accumulates. Ectopic expression of SSP protein in the leaf epidermis is sufficient to activate YDA-dependent signaling. We propose that SSP protein produced from paternal transcripts upon fertilization triggers zygotic YDA activity, providing an essential temporal cue for the regulation of the asymmetric first division.

Protein N-terminal methionine excision.

N-terminal methionine excision (NME) is the major proteolytic pathway responsible for the diversity of N-terminal amino acids in proteins. Dedicated NME components have been identified in all organisms, in all compartments in which protein synthesis occurs: cytoplasm, plastids and mitochondria. Recent studies have revealed that NME is regulated at various levels and plays an important role in controlling protein turnover. NME is essential in Eubacteria and lower eukaryotes and is the target of many natural and synthetic inhibitors. Such inhibitors have considerable potential for use in the treatment of various human diseases, from cancer to bacterial and parasitic infections.

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.

Peptide deformylase as a target for new generation, broad spectrum antimicrobial agents

Peptide deformylase was discovered 30u2003years ago, but as a result of its unusually unstable activity it was not fully characterized until very recently. The aim of this paper is to review the many recent data concerning this enzyme and to try to assess its potential as a target for future antimicrobial drugs.

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. Dis ruption 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 inhibi tion 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.

Unexpected protein families including cell defense components feature in the N-myristoylome of a higher eukaryote

N-Myristoylation is an irreversible modification that affects the membrane binding properties of crucial cytoplasmic proteins from signal transduction cascades. We characterized the two putative N-myristoyltransferases of Arabidopsis thaliana as a means of investigating the entire N-myristoylation proteome (N-myristoylome) in a higher eukaryote. AtNMT1 compensated for the nmt1 defect in yeast, whereas AtNMT2 and chimeras of the two genes did not. Only AtNMT1 modified known N-myristoylated proteins in vitro. AtNMT1 is therefore responsible for the A. thaliana N-myristoylome, whereas AtNMT2 does not seem to have usual myristoylation activity. We began with the whole set of N-myristoylated G proteins in the A. thaliana proteome. We then used a reiterative approach, based on the in vitro N-myristoylation of more than 60 different polypeptides, to determine the substrate specificity of AtNMT1. We found that the positive charge on residue 7 of the substrate was particularly important in substrate recognition. The A. thaliana N-myristoylome consists of 437 proteins, accounting for 1.7% of the complete proteome. We demonstrated the N-myristoylation of several unexpected protein families, including innate immunity proteins, thioredoxins, components of the protein degradation pathway, transcription factors, and a crucial regulatory enzyme of glycolysis. The role of N-myristoylation is discussed in each case; in particular, this process may underlie the “guard” hypothesis of innate immunity.

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.

Comparative metagenomics of microbial traits within oceanic viral communities

Viral genomes often contain genes recently acquired from microbes. In some cases (for example, psbA) the proteins encoded by these genes have been shown to be important for viral replication. In this study, using a unique search strategy on the Global Ocean Survey (GOS) metagenomes in combination with marine virome and microbiome pyrosequencing-based datasets, we characterize previously undetected microbial metabolic capabilities concealed within the genomes of uncultured marine viral communities. A total of 34 microbial gene families were detected on 452 viral GOS scaffolds. The majority of auxiliary metabolic genes found on these scaffolds have never been reported in phages. Host genes detected in viruses were mainly divided between genes encoding for different energy metabolism pathways, such as electron transport and newly identified photosystem genes, or translation and post-translation mechanism related. Our findings suggest previously undetected ways, in which marine phages adapt to their hosts and improve their fitness, including translation and post-translation level control over the host rather than the already known transcription level control.

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‐α‐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.

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.