Search

Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents [NAD(P)H] and they provide the cell with intermediates for a range of biosynthetic pathways. In addition to this, mitochondria contribute to a number of specialized functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of mitochondria and general biochemical properties such as oxidative phosphorylation. We will then mention a few adaptive properties in response to water stress, seed maturation and germination, and the ability to function under hypoxic conditions. The discussion will mainly focus on Arabidopsis cell cultures, etiolated germinating rice seedlings and potato tubers as model plants. It will cover the general proteome as well as the posttranslational modification protein phosphorylation. To date 64 phosphorylated mitochondrial proteins with a total of 103 phosphorylation sites have been identified. [ABSTRACT FROM AUTHOR]

RATIONALE Some large-scale proteomics studies in which strong cation exchange chromatography has been applied are used to determine proteomes and post-translational modification dynamics. Although such datasets favour the characterisation of thousands of modified peptides, e.g., phosphorylated and N-α-acetylated, a large fraction of the acquired spectra remain unexplained by standard proteomics approaches. Thus, advanced data processing allows characterisation of a significant part of these unassigned spectra. METHODS Our recent investigation of the N-α-acetylation status of plant proteins gave a dataset of choice to investigate further the in-depth characterisation of peptide modifications using Mascot tools associated with relevant validation processes. Such an approach allows to target frequently occurring modifications such as methionine oxidation, phosphorylation or N-α-acetylation, but also the less usual peptide cationisation. Finally, this dataset offers the unique opportunity to determine the overall influence of some of these modifications on the identification score. RESULTS Although methionine oxidation has no influence and tends to favour the characterisation of protein N-terminal peptides, peptide alkalinisation shows an adverse effect on peptide average score. Nevertheless, peptide cationisation appears to favour the characterisation of protein C-terminal peptides with a limited to no direct influence on the identification score. Unexpectedly, our investigation reveals the unfortunate combination of the molecular weight of N-α-acetylation and potassium cation that mimics the mass increment of a phosphorylation group. CONCLUSIONS Since these characterisations rely upon computational treatment associated with statistical validation approaches such as 'False discovery rates' calculation or post-translational modification position validation, our investigation highlights the limitation of such treatment which is biased by the initial searched hypotheses. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The article presents a study which explores how HYPK promotes the activity of the N-alpha-acetyltransferase A complex to determine proteostasis of nonAc-X2/N-degron–containing proteins. It demonstrate that HYPK is a critical regulator of global proteostasis by facilitating masking of the recently identified nonAc-X2/N-degron.

Abstract: N-terminal-ubiquitinylation (NTU) is a newly discovered protein degradation pathway initiated by ubiquitin-tagging of the N-terminal α-amino group. We have used data from recent genomic studies, especially those on humans, to up-date and re-interpret biochemical data to identify the sequence features associated with NTU. We compared a mini-proteome for which experimental protein sequence is available with large-scale genomic data. We conclude that N-α-acetylation involves less than 30%, and not the widely assumed 90%, of the proteins encoded by any higher eukaryote genome, greatly increasing thereby the number of possible targets for NTU-mediated degradation. Next, straightforward rules linking the first N-terminal residues of any nascent polypeptides to the nature of their processed N-termini are established and dedicated prediction tool is made available at http://www.isv.cnrs-gif.fr/Terminator/. We provide strong arguments indicating that the nature of the processed N-terminus is a major determinant factor of the half-life of the protein. We finally reveal that one third of the nuclear-encoded proteins starting with an unprocessed and unblocked methionine are at least one order of magnitude less stable than is average in higher eukaryotes. This appears to be the first common feature of proteins undergoing N-terminal ubiquitinylation. Hence, a pool of about 3000 proteins in each proteome could be unstable per se and tagged for rapid degradation via NTU. [Copyright &y& Elsevier]

Peptide deformylase (PDF) inhibitors have a strong potential to be used as a new class of antibiotics. However, recent studies have shown that the mitochondria of most eukaryotes, including humans, contain an essential PDF, PDF1A. The crystal structure of the Arabidopsis thaliana PDF1A (AtPDF1A), considered representative of PDF1As in general, has been determined. This structure displays several similarities to that of known bacterial PDFs. AtPDF1A behaves as a dimer, with the C-terminal residues responsible for linking the two subunits. This arrangement is similar to that of Leptospira interrogans PDF, the only other dimeric PDF identified to date. AtPDF1A is the first PDF for which zinc has been identified as the catalytic ion. However, the zinc binding pocket does not differ from the binding pockets of PDFs with iron rather than zinc. The crystal structure of AtPDF1A in complex with a substrate analog revealed that the substrate binding pocket of PDF1A displays strong modifications. The S1′ binding pocket is significantly narrower, due to the creation of a floor from residues present in all PDF1As but not in bacterial PDFs. A true S3′ pocket is created by the residues of a helical CD-loop, which is very long in PDF1As. Finally, these modified substrate binding pockets modify the position of the substrate in the active site. These differences provide guidelines for the design of bacterial PDF inhibitors that will not target mitochondrial PDFs. [ABSTRACT FROM AUTHOR]

N-terminal myristoylation is an essential eukaryotic modification crucial for cellular homeostasis in the context of many physiological processes. Myristoylation is a lipid modification resulting in a C14 saturated fatty acid addition. This modification is challenging to capture due to its hydrophobicity, low abundance of target substrates, and the recent discovery of unexpected NMT reactivity including myristoylation of lysine side chains and N-acetylation in addition to classical N-terminal Gly-myristoylation. This chapter details the high-end approaches developed to characterize the different features of N-myristoylation and its targets through in vitro and in vivo labeling., (Copyright © 2023 Elsevier Inc. All rights reserved.)

N-myristoyltransferases (NMTs) are members of the large family of GCN5-related N-acetyltransferases (GNATs). NMTs mainly catalyze eukaryotic protein myristoylation, an essential modification tagging protein N-termini and allowing successive subcellular membrane targeting. NMTs use myristoyl-CoA (C14:0) as major acyl donor. NMTs were recently found to react with unexpected substrates including lysine side-chains and acetyl-CoA. This chapter details the kinetic approaches that have allowed the characterization of the unique catalytic features of NMTs in vitro., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Global acetylation profiling (GAP) consists of heterologous expression of a given N-acetyltransferase (NAT) in Escherichia coli to assess its specificity. The remarkable sensitivity and robustness of the GAP pipeline relies on the very low frequency of known N-terminal acetylated proteins in E. coli, including their degree of N-terminal acetylation. Using the SILProNAQ mass spectrometry strategy on bacterial protein extracts, GAP permits easy acquisition of both qualitative and quantitative data to decipher the impact of any putative NAT of interest on the N-termini of newly acetylated proteins. This strategy allows rapid determination of the substrate specificity of any NAT., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

N α -terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. In plants, the biological function of NTA remains enigmatic. The dominant N -acetyltransferase (Nat) in Arabidopsis ( Arabidopsis thaliana ) is NatA, which cotranslationally catalyzes acetylation of ∼40% of the proteome. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In human ( Homo sapiens ), fruit fly ( Drosophila melanogaster ), and yeast ( Saccharomyces cerevisiae ), this core NatA complex interacts with NAA50 to form the NatE complex. While in metazoa, NAA50 has N -acetyltransferase activity, yeast NAA50 is catalytically inactive and positions NatA at the ribosome tunnel exit. Here, we report the identification and characterization of Arabidopsis NAA50 (AT5G11340). Consistent with its putative function as a cotranslationally acting Nat, AtNAA50-EYFP localized to the cytosol and the endoplasmic reticulum but also to the nuclei. We demonstrate that purified AtNAA50 displays N α -terminal acetyltransferase and lysine-ε-autoacetyltransferase activity in vitro. Global N -acetylome profiling of Escherichia coli cells expressing AtNAA50 revealed conservation of NatE substrate specificity between plants and humans. Unlike the embryo-lethal phenotype caused by the absence of AtNAA10 and AtNAA15, loss of NAA50 expression resulted in severe growth retardation and infertility in two Arabidopsis transfer DNA insertion lines ( naa50-1 and naa50-2 ). The phenotype of naa50-2 was rescued by the expression of HsNAA50 or AtNAA50. In contrast, the inactive ScNAA50 failed to complement naa50-2 Remarkably, loss of NAA50 expression did not affect NTA of known NatA substrates and caused the accumulation of proteins involved in stress responses. Overall, our results emphasize a relevant role of AtNAA50 in plant defense and development, which is independent of the essential NatA activity., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences.

Protein N-terminal modifications have recently been involved in overall proteostasis through their impact on cell fate and protein life time. This explains the development of new approaches to characterize more precisely the N-terminal end of mature proteins. Although few approaches are available to perform N-terminal enrichment based on positive or negative discriminations, these methods are usually restricted to the enrichment in N-terminal peptides and their characterization by mass spectrometry. Recent investigation highlights both (1) the knowledge of the N-terminal acetylation status of most cytosolic proteins and (2) post-translational addition of this modification on the N-terminus of nuclear coded chloroplast proteins imported in the plastid and after the cleavage of the transit peptide. The workflow involves stable isotope labeling to assess N-acetylation rates followed by Strong Cation eXchange (SCX ) fractionation of the samples to provide protein N-terminal enriched fractions. Combined with mass spectrometry analyses, the technology finally requires extensive data processing. This last step aims first at discriminating the most relevant mature N-termini from the characterized peptides, next at determining its experimental position and then at calculating the N-terminal acetylation yield. Stable-Isotope Protein N-terminal Acetylation Quantification (SILProNAQ) is a complete workflow combining wet-lab techniques together with dry-lab processing to determine the N-terminal acetylation yield of mature proteins for a clearly defined localization.

Protein lipidation is a crucial protein modification involving the attachment of hydrophobic carbon skeletons (C:14-C:60) of various lipid classes--fatty acids, sterols, glycero-, phospho- and glycolipids. The lipid-protein bond frequently (i) involves the N- or C-terminal ends of the target, (ii) requires amide, ether or ester bonds to a small aminoacid, usually Gly or Cys and (iii) depends on proteolytic events. Lipidation results in protein targeting to the membrane, with the protein behaving as a peripheral component and being oriented toward the inside or outside of the cell. The addition of a single lipid is not sufficient for membrane targeting and another signal, often involving additional lipidation, is required. The methods available for predicting lipid modifications to proteins highlight the importance of identifying short protein motifs in a field in which few data are currently available, due to the complex nature of the modification. Full proteome annotation is already feasible with these predictive tools. We show that lipidation may affect 2-4 percent of all proteins in a given proteome and that double-lipidation is widespread.

New classes of antibiotics are urgently needed to counter increasing levels of pathogen resistance. Peptide deformylase (PDF) was originally selected as a specific bacterial target, but a human homologue, the inhibition of which causes cell death, was recently discovered. We developed a dual-screening strategy for selecting highly effective compounds with low inhibition effect against human PDF. We selected a new scaffold in vitro that discriminated between human and bacterial PDFs. Analyses of structure-activity relationships identified potent antibiotics such as 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (6b) with the same mode of action in vivo as previously identified PDF inhibitors but without the apoptotic effects of these inhibitors in human cells.

Peptide deformylase inhibitors (PDFIs) appear to be one of the most exciting classes of antibacterial agents discovered to date. Rapid progress in the development of PDFIs has been possible because peptide deformylase is a metalloprotease, and this class of enzymes shows a high degree of structure-function conservation, and because the most potent PDFIs are hydroxamate derivatives, a well known category of pharmacophores. The current challenge in structure-activity relationship analysis is obtaining molecules with potent in vivo antibacterial activity against a range of drug-resistant pathogens. The PDFIs currently in clinical trials target community-based bacterial infections, with a potential major pharmaceutical market.