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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]

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20-/- MEFs exhibited reduced responsiveness to an extrinsic pro-apoptotic stimulus, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway appears to impact apoptosis regulation, providing new perspectives in the field including in therapeutic interventions. [ABSTRACT FROM AUTHOR]

N-myristoylation (MYR) is a crucial fatty acylation catalyzed by N-myristoyltransferases (NMTs) that is likely to have appeared over 2 billion years ago. Proteome-wide approaches have now delivered an exhaustive list of substrates undergoing MYR across approximately 2% of any proteome, with constituents, several unexpected, associated with different membrane compartments. A set of <10 proteins conserved in eukaryotes probably represents the original set of N-myristoylated targets, marking major changes occurring throughout eukaryogenesis. Recent findings have revealed unexpected mechanisms and reactivity, suggesting competition with other acylations that are likely to influence cellular homeostasis and the steady state of the modification landscape. Here, we review recent advances in NMT catalysis, substrate specificity, and MYR proteomics, and discuss concepts regarding MYR during evolution. MYR is an essential protein modification found in all eukaryotes, with the catalysts involved validated as drug targets in a number of human diseases. New studies on MYR at the proteome scale have revealed novel MYR features, compartments, and targets, as well as roles in quality control, the immune system, cell motility, and mitochondrial components. Detailed target analysis reveals that myristoylated substrates mark all eukaryotic compartments, including those only arising in specific organisms or resulting from cellular differentiation. Less than ten N-myristoylated targets are conserved throughout evolution and are associated with the earliest events recapitulating eukaryogenesis, suggesting MYR accompanied eukaryotic evolution. New structural and biochemical data on N-myristoyltransferases illuminate novel catalytic mechanism and substrate specificity. [ABSTRACT FROM AUTHOR]

N-myristoyltransferases (NMTs) catalyze essential acylations of N-terminal alpha or epsilon amino groups of glycines or lysines. Here, we reveal that peptides tightly fitting the optimal glycine recognition pattern of human NMTs are potent prodrugs relying on a single-turnover mechanism. Sequence scanning of the inhibitory potency of the series closely reflects NMT glycine substrate specificity rules, with the lead inhibitor blocking myristoylation by NMTs of various species. We further redesigned the series based on the recently recognized lysine-myristoylation mechanism by taking advantage of (1) the optimal peptide chassis and (2) lysine side chain mimicry with unnatural enantiomers. Unlike the lead series, the inhibitory properties of the new compounds rely on the protonated state of the side chain amine, which stabilizes a salt bridge with the catalytic base at the active site. Our study provides the basis for designing first-in-class NMT inhibitors tailored for infectious diseases and alternative active site targeting. [Display omitted] • Optimal octapeptide substrates of Gly-myristoylation are potent prodrug NMT inhibitors • Potent Lys-myristoylation-based inhibitors were derived from the substrate series • Inhibition relies on lysine protonation and a salt bridge with the catalytic base • Potent inhibition requires a secondary salt bridge stabilizing a disordered loop of NMT Peptides fitting the optimal human NMT Gly-myristoylation recognition pattern act as potent prodrugs via a single-turnover mechanism. Rivière et al. derived Lys-myristoylation-based inhibitors from these peptides. Despite tight structural similarities, each series' inhibitory properties are unique, relying on distinct interactions with the catalytic base at the active site. [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.

N-terminal myristoylation, a major eukaryotic protein lipid modification, is difficult to detect in vivo and challenging to predict in silico. We developed a proteomics strategy involving subfractionation of cellular membranes, combined with separation of hydrophobic peptides by mass spectrometry-coupled liquid chromatography to identify the Arabidopsis thaliana myristoylated proteome. This approach identified a starting pool of 8837 proteins in all analyzed cellular fractions, comprising 32% of the Arabidopsis proteome. Of these, 906 proteins contain an N-terminal Gly at position 2, a prerequisite for myristoylation, and 214 belong to the predicted myristoylome (comprising 51% of the predicted myristoylome of 421 proteins). We further show direct evidence of myristoylation in 72 proteins; 18 of these myristoylated proteins were not previously predicted. We found one myristoylation site downstream of a predicted initiation codon, indicating that posttranslational myristoylation occurs in plants. Over half of the identified proteins could be quantified and assigned to a subcellular compartment. Hierarchical clustering of protein accumulation combined with myristoylation and S -acylation data revealed that N-terminal double acylation influences redirection to the plasma membrane. In a few cases, MYR function extended beyond simple membrane association. This study identified hundreds of N -acylated proteins for which lipid modifications could control protein localization and expand protein function. [ABSTRACT FROM AUTHOR]

Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp 16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp 16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp 16 PDF specific activity to values similar to those of bacterial PDFs. We next show that Vp 16 PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp 16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp 16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness. [ABSTRACT FROM AUTHOR]

[Display omitted] • Elements guiding NMT to G- or K-myristoylation were searched and identified.. • NMT–dependent K-myristoylation occurs only on lysines when at positions 1, 2, or 3. • K-myristoylation is two orders of magnitude slower than G-myristoylation. • Protease IpaJ may unmask novel K-myristoylation sites in a dozen human proteins. • NMT-dependent K-myristoylation motifs are depleted in the human proteome. N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for cell survival and a range of pathophysiological processes. Originally thought to modify only N-terminal glycine α-amino groups (G-myristoylation), NMTs were recently shown to also modify lysine ε-amino groups (K-myristoylation). However, the clues ruling NMT-dependent K-myristoylation and the full range of targets are currently unknown. Here we combine mass spectrometry, kinetic studies, in silico analysis, and crystallography to identify the specific features driving each modification. We show that direct interactions between the substrate's reactive amino group and the NMT catalytic base promote K-myristoylation but with poor efficiency compared to G-myristoylation, which instead uses a water-mediated interaction. We provide evidence of depletion of proteins with NMT-dependent K-myristoylation motifs in humans, suggesting evolutionary pressure to prevent this modification in favor of G-myristoylation. In turn, we reveal that K-myristoylation may only result from post-translational events. Our studies finally unravel the respective paths towards K-myristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group. [ABSTRACT FROM AUTHOR]

The proteostasis network and associated protein quality control (PQC) mechanisms ensure proteome functionality and are essential for cell survival. A distinctive feature of eukaryotic cells is their high degree of compartmentalization, requiring specific and adapted proteostasis networks for each compartment. The nucleus, essential for maintaining the integrity of genetic information and gene transcription, is one such compartment. While PQC mechanisms have been investigated for decades in the cytoplasm and the endoplasmic reticulum, our knowledge of nuclear PQC pathways is only emerging. Recent developments in the field have underscored the importance of spatially managing aberrant proteins within the nucleus. Upon proteotoxic stress, misfolded proteins and PQC effectors accumulate in various nuclear membrane-less organelles. Beyond bringing together effectors and substrates, the biophysical properties of these organelles allow novel PQC functions. In this review, we explore the specificity of the nuclear compartment, the effectors of the nuclear proteostasis network, and the PQC roles of nuclear membrane-less organelles in metazoans., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Protein acetylation is a key co- and post-translational modification. However, how different types of acetylation respond to environmental stress is still unknown. To address this, we investigated the role of a member of the newly discovered family of plastid acetyltransferases (GNAT2), which features both lysine- and N-terminal acetyltransferase activities. Our study aimed to provide a holistic multi-omics acetylation-dependent view of plant acclimation to short-term light changes. We found that both the yield and coverage of the N-terminal acetylome remained unchanged in WT and gnat2-KO backgrounds after 2 h of exposure to high light or darkness. Similarly, no differences in transcriptome or adenylate energy charge were observed between the genotypes under the tested light conditions. In contrast, the lysine acetylome proved to be sensitive to the changes in light conditions, especially in the gnat2 background. This suggests unique strategies of plant acclimation for quick responses to environmental changes involving lysine, but not N-terminal, GNAT2-mediated acetylation activity., Competing Interests: Conflicts of interests The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein N-acetylation is one of the most abundant co- and post-translational modifications in eukaryotes, extending its occurrence to chloroplasts within vascular plants. Recently, a novel plastidial enzyme family comprising eight acetyltransferases that exhibit dual lysine and N-terminus acetylation activities was unveiled in Arabidopsis. Among these, GNAT1, GNAT2, and GNAT3 reveal notable phylogenetic proximity, forming a subgroup termed NAA90. Our study focused on characterizing GNAT1, closely related to the state transition acetyltransferase GNAT2. In contrast to GNAT2, GNAT1 did not prove essential for state transitions and displayed no discernible phenotypic difference compared to the wild type under high light conditions, while gnat2 mutants were severely affected. However, gnat1 mutants exhibited a tighter packing of the thylakoid membranes akin to gnat2 mutants. In vitro studies with recombinant GNAT1 demonstrated robust N-terminus acetylation activity on synthetic substrate peptides. This activity was confirmed in vivo through N-terminal acetylome profiling in two independent gnat1 knockout lines. This attributed several acetylation sites on plastidial proteins to GNAT1, reflecting a subset of GNAT2's substrate spectrum. Moreover, co-immunoprecipitation coupled with mass spectrometry revealed a robust interaction between GNAT1 and GNAT2, as well as a significant association of GNAT2 with GNAT3 - the third acetyltransferase within the NAA90 subfamily. This study unveils the existence of at least two acetyltransferase complexes within chloroplasts, whereby complex formation might have a critical effect on the fine-tuning of the overall acetyltransferase activities. These findings introduce a novel layer of regulation in acetylation-dependent adjustments in plastidial metabolism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The entire 4.6-Mb genome of Vibrio sp. 16, encoding 4,270 genes, best matches with Vibrio rotiferianus . A 46-kb plasmid (pVDT1), alongside two circular chromosomes, showcases parAB / repB partition genes and three toxin/antitoxin systems potentially linked to phage infection., Competing Interests: The authors declare no conflict of interest.

The ribosome-tethered N-terminal acetyltransferase A (NatA) acetylates 52% of soluble proteins in Arabidopsis thaliana. This co-translational modification of the N terminus stabilizes diverse cytosolic plant proteins. The evolutionary conserved Huntingtin yeast partner K (HYPK) facilitates NatA activity in planta, but in vitro, its N-terminal helix α1 inhibits human NatA activity. To dissect the regulatory function of HYPK protein domains in vivo, we genetically engineer CRISPR-Cas9 mutants expressing a HYPK fragment lacking all functional domains (hypk-cr1) or an internally deleted HYPK variant truncating helix α1 but retaining the C-terminal ubiquitin-associated (UBA) domain (hypk-cr2). We find that the UBA domain of HYPK is vital for stabilizing the NatA complex in an organ-specific manner. The N terminus of HYPK, including helix α1, is critical for promoting NatA activity on substrates starting with various amino acids. Consequently, deleting only 42 amino acids inside the HYPK N terminus causes substantial destabilization of the plant proteome and higher tolerance toward drought stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by 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.)

Protein myristoylation is a C14 fatty acid modification found in all living organisms. Myristoylation tags either the N-terminal alpha groups of cysteine or glycine residues through amide bonds or lysine and cysteine side chains directly or indirectly via glycerol thioester and ester linkages. Before transfer to proteins, myristate must be activated into myristoyl coenzyme A in eukaryotes or, in bacteria, to derivatives like phosphatidylethanolamine. Myristate originates through de novo biosynthesis (e.g., plants), from external uptake (e.g., human tissues), or from mixed origins (e.g., unicellular organisms). Myristate usually serves as a molecular anchor, allowing tagged proteins to be targeted to membranes and travel across endomembrane networks in eukaryotes. In this review, we describe and discuss the metabolic origins of protein-bound myristate. We review strategies for in vivo protein labeling that take advantage of click-chemistry with reactive analogs, and we discuss new approaches to the proteome-wide discovery of myristate-containing proteins. The machineries of myristoylation are described, along with how protein targets can be generated directly from translating precursors or from processed proteins. Few myristoylation catalysts are currently described, with only N-myristoyltransferase described to date in eukaryotes. Finally, we describe how viruses and bacteria hijack and exploit myristoylation for their pathogenicity., (Copyright © 2021. Published by Elsevier Ltd.)

N terminal protein α-acetylation (NTA) is a pervasive protein modification that has recently attracted renewed interest. Early studies on NTA were mostly conducted in yeast and metazoans, providing a detailed portrait of the modification, which was indirectly applied to all eukaryotes. However, new findings originating from photosynthetic organisms have expanded our knowledge of this modification, revealing strong similarities as well as idiosyncratic features. Here, we review the most recent advances on NTA and its dedicated machinery in photosynthetic organisms. We discuss the cytosolic and unique plastid NTA machineries and their critical biological roles in development, stress responses, protein translocation, and stability. These new findings suggest that the multitasking plastid and cytosolic machineries evolved to support the specific needs of photoautotrophs., (Copyright © 2020 Elsevier Ltd. 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.