Thierry Meinnel - Academia.edu (original) (raw)

Papers by Thierry Meinnel

Research paper thumbnail of Kinetic and catalytic features of N-myristoyltransferases

Methods in Enzymology, 2023

Research paper thumbnail of Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases

Plant Physiology, Jul 10, 2023

The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their a... more The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3-ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multi-omics approaches to characterise potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3-ligases in the Nt-acetylation-(NTA-) dependent turnover of proteins at global and protein-specific scales. Arabidopsis has two ER-localised DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wildtype, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localised SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdomspecific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.

Research paper thumbnail of eNergiome and N-TerPred: novel tools to improve the prediction of plastidic and mitochondrial mature N-termini

HAL (Le Centre pour la Communication Scientifique Directe), Jul 7, 2018

Research paper thumbnail of Crystal structure of PDF from the Vibrio parahaemolyticus bacteriophage VP16T in complex with actinonin - crystal form II

Research paper thumbnail of Peptide Deformylase Catalytic Core (Residues 1-147), NMR, 20 Structures

Research paper thumbnail of HsNMT1 in complex with CoA and acetylated-NCFSKPK peptide

Research paper thumbnail of Crystal structure of type 2 PDF from Streptococcus agalactiae, crystallized in cacodylate buffer

Research paper thumbnail of Biochemical and structural analysis of N-myristoyltransferase mediated protein tagging

Methods in Enzymology, 2023

Research paper thumbnail of Structural and Large-scale Analysis Unveil the Intertwined Paths Promoting NMT-catalyzed Lysine and Glycine Myristoylation

Journal of Molecular Biology, Nov 1, 2022

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for ... more 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 NMTdependent 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 Kmyristoylation 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 Kmyristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.

Research paper thumbnail of HYPK promotes the activity of the <i>N</i> <sup>α</sup> -acetyltransferase A complex to determine proteostasis of nonAc-X <sup>2</sup> /N-degron–containing proteins

Science Advances, Jun 17, 2022

HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Distributed under a Creative Commons Attribution-NonCommercial 4.0 International License HYPK promotes the activity of the N-alpha acetyltransferase A complex to determine proteostasis of nonAc-X 2 /N-degron-containing proteins

Research paper thumbnail of Kinetic and catalytic features of N-myristoyltransferases

Research paper thumbnail of Structural and Large-scale Analysis Unveil the Intertwined Paths Promoting NMT-catalyzed Lysine and Glycine Myristoylation

Journal of Molecular Biology

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for ... more 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 NMTdependent 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 Kmyristoylation 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 Kmyristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.

Research paper thumbnail of N-acetylation of secreted proteins in Apicomplexa is widespread and is independent of the ER acetyl-CoA transporter AT1

Journal of Cell Science

Acetyl-CoA participates in post-translational modification of proteins and in central carbon and ... more Acetyl-CoA participates in post-translational modification of proteins and in central carbon and lipid metabolism in several cell compartments. In mammals, acetyl-CoA transporter 1 (AT1, also known as SLC33A1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with the activity of dedicated acetyltransferases such as NAT8. However, the involvement of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. Here, we identified homologs of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei parasites. Proteome-wide analyses revealed widespread N-terminal acetylation of secreted proteins in both species. Such extensive acetylation of N-terminally processed proteins has not been observed previously in any other organism. Deletion of AT1 homologs in both T. gondii and P. berghei resulted in considerable reductions in parasite fitness. In P. bergh...

Research paper thumbnail of Synthèse du livre blanc des sciences de la vie du département des sciences de la vie de l'université Paris Saclay

HAL (Le Centre pour la Communication Scientifique Directe), 2015

Research paper thumbnail of Additional file 3: of EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples

Mascot Distiller processing method for protein N-terminal acetylation quantitation. (PDF 24Â kb)

Research paper thumbnail of Additional file 2: Table S2. of EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples

Results obtained after EnCOUNTer processing applied to a large A. thaliana LC-MS/MS dataset (XLSX... more Results obtained after EnCOUNTer processing applied to a large A. thaliana LC-MS/MS dataset (XLSX 381Â kb)

Research paper thumbnail of RESEARCH ARTICLE Molecular

identification and functional characterization of the first N-acetyltransferase in plastids by gl... more identification and functional characterization of the first N-acetyltransferase in plastids by global acetylome profiling

Research paper thumbnail of eNergiome and N-TerPred: novel tools to improve the prediction of plastidic and mitochondrial mature N-termini

Proteins N-terminus and related modifications influence protein fate and half-life. The most freq... more Proteins N-terminus and related modifications influence protein fate and half-life. The most frequent co-translational N-terminal protein modifications are linked to initial methionine excision and protein N-alpha-acetylation. Another modification occurring next to the protein N-term corresponds to the excision of the transit peptide of nuclear-encoded proteins targeted to the mitochondria and/or the plastids. Only few bioinformatics tools are currently available to predict mature proteins N-term such as TermiNator3 (processing and modifications at position 1-2) and TargetP/ChloroP/SignalP tools that provide transit-peptide length predictions and protein subcellular localization. Unfortunately, the reliability of these tools suffer minor inconsistencies to major defects especially for the transit-peptide length prediction with the TargetP suite. Here, we present a dedicated database (eNergiomeDB) which data were collected from large scale N-terminomics studies. Experimental data rel...

Research paper thumbnail of Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2018

Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genom... more 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 Vp16 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 Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 specific activity to values similar to those of bacterial PDFs. We next show that Vp16PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.

Research paper thumbnail of Myristoylation, an Ancient Protein Modification Mirroring Eukaryogenesis and Evolution

Trends in Biochemical Sciences, 2020

N-myristoylation (MYR) is a crucial fatty acylation catalyzed by N-myristoyltransferases (NMTs) t... more N-myristoylation (MYR) is a crucial fatty acylation catalyzed by N-myristoyltransferases (NMTs) that is likely to have appeared over two 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. Lipidated proteins Plasma membranes (PMs) are composed of extrinsic and intrinsic proteins (52%) and lipids (40%), the latter sustaining the overall cellular architecture. Membrane-penetrating extrinsic proteins often possess covalently linked lipids, usually fatty acids, which allow the protein to contact other intra-and extracellular proteins [1]. Protein lipidation involves amides (i.e. N-αmyristoylation, MYR, see Glossary and glycosylphosphatidylinositol (GPI) anchors), thioesters (i.e. S-palmitoylation, PAL), and thioethers (i.e. isoprenylation and farnesylation) [2]. Of these, MYR is a frequent and conserved modification specific to eukaryotes that targets major cellular components. Mapping the proteins undergoing MYR has proven challenging due to their difficult handling characteristics and amphiphilic, chimeric nature. Recently, high-end technologies have allowed the first lipidated proteome, the myristoylome, to be described in detail in various organisms recapitulating the tree of life [3]. These and other studies on myristoylome composition and genesis have also revealed (i) an unexpected novel mechanism of action of Nmyristoyltransferase (NMT), (ii) NMT substrate selectivity, and (iii) the capacity of NMT to act on N-terminal lysines (Lys) as well as the usual glycines (Gly). This review highlights the series of groundbreaking discoveries that have recently significantly advanced our knowledge about this long-studied enzyme. This includes an overview of a new NMT catalytic mechanism, substrate specificity, and proteomics, and a discussion of how a reduced set of targets is closely related to eukaryogenesis and eukaryote evolution. How NMTs catalyze MYR with high selectivity NMTs are GNAT members closely related to Nα-acetyltransferases Seventy-four crystal structures have now revealed that the C-terminal 400 amino acid-long NMT catalytic core displays a conserved 3D GCN5-related N-acetyltransferase (GNAT) core. GNATs also include the catalytic subunits of Nα-acetyltransferases (NAAs) [4], with Naa10 being the closest to NMT as it modifies N-terminal Gly [5-7]. NMTs have two adjacent GNAT domains, most likely to have arisen through duplication of a Naa10-like GNAT domain [8]. Prokaryotes do not possess NMTs, which seem to have arisen as eukaryotes evolved from the last archaeal

Research paper thumbnail of Kinetic and catalytic features of N-myristoyltransferases

Methods in Enzymology, 2023

Research paper thumbnail of Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases

Plant Physiology, Jul 10, 2023

The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their a... more The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3-ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multi-omics approaches to characterise potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3-ligases in the Nt-acetylation-(NTA-) dependent turnover of proteins at global and protein-specific scales. Arabidopsis has two ER-localised DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wildtype, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localised SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdomspecific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.

Research paper thumbnail of eNergiome and N-TerPred: novel tools to improve the prediction of plastidic and mitochondrial mature N-termini

HAL (Le Centre pour la Communication Scientifique Directe), Jul 7, 2018

Research paper thumbnail of Crystal structure of PDF from the Vibrio parahaemolyticus bacteriophage VP16T in complex with actinonin - crystal form II

Research paper thumbnail of Peptide Deformylase Catalytic Core (Residues 1-147), NMR, 20 Structures

Research paper thumbnail of HsNMT1 in complex with CoA and acetylated-NCFSKPK peptide

Research paper thumbnail of Crystal structure of type 2 PDF from Streptococcus agalactiae, crystallized in cacodylate buffer

Research paper thumbnail of Biochemical and structural analysis of N-myristoyltransferase mediated protein tagging

Methods in Enzymology, 2023

Research paper thumbnail of Structural and Large-scale Analysis Unveil the Intertwined Paths Promoting NMT-catalyzed Lysine and Glycine Myristoylation

Journal of Molecular Biology, Nov 1, 2022

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for ... more 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 NMTdependent 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 Kmyristoylation 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 Kmyristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.

Research paper thumbnail of HYPK promotes the activity of the <i>N</i> <sup>α</sup> -acetyltransferase A complex to determine proteostasis of nonAc-X <sup>2</sup> /N-degron–containing proteins

Science Advances, Jun 17, 2022

HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Distributed under a Creative Commons Attribution-NonCommercial 4.0 International License HYPK promotes the activity of the N-alpha acetyltransferase A complex to determine proteostasis of nonAc-X 2 /N-degron-containing proteins

Research paper thumbnail of Kinetic and catalytic features of N-myristoyltransferases

Research paper thumbnail of Structural and Large-scale Analysis Unveil the Intertwined Paths Promoting NMT-catalyzed Lysine and Glycine Myristoylation

Journal of Molecular Biology

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for ... more 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 NMTdependent 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 Kmyristoylation 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 Kmyristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.

Research paper thumbnail of N-acetylation of secreted proteins in Apicomplexa is widespread and is independent of the ER acetyl-CoA transporter AT1

Journal of Cell Science

Acetyl-CoA participates in post-translational modification of proteins and in central carbon and ... more Acetyl-CoA participates in post-translational modification of proteins and in central carbon and lipid metabolism in several cell compartments. In mammals, acetyl-CoA transporter 1 (AT1, also known as SLC33A1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with the activity of dedicated acetyltransferases such as NAT8. However, the involvement of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. Here, we identified homologs of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei parasites. Proteome-wide analyses revealed widespread N-terminal acetylation of secreted proteins in both species. Such extensive acetylation of N-terminally processed proteins has not been observed previously in any other organism. Deletion of AT1 homologs in both T. gondii and P. berghei resulted in considerable reductions in parasite fitness. In P. bergh...

Research paper thumbnail of Synthèse du livre blanc des sciences de la vie du département des sciences de la vie de l'université Paris Saclay

HAL (Le Centre pour la Communication Scientifique Directe), 2015

Research paper thumbnail of Additional file 3: of EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples

Mascot Distiller processing method for protein N-terminal acetylation quantitation. (PDF 24Â kb)

Research paper thumbnail of Additional file 2: Table S2. of EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples

Results obtained after EnCOUNTer processing applied to a large A. thaliana LC-MS/MS dataset (XLSX... more Results obtained after EnCOUNTer processing applied to a large A. thaliana LC-MS/MS dataset (XLSX 381Â kb)

Research paper thumbnail of RESEARCH ARTICLE Molecular

identification and functional characterization of the first N-acetyltransferase in plastids by gl... more identification and functional characterization of the first N-acetyltransferase in plastids by global acetylome profiling

Research paper thumbnail of eNergiome and N-TerPred: novel tools to improve the prediction of plastidic and mitochondrial mature N-termini

Proteins N-terminus and related modifications influence protein fate and half-life. The most freq... more Proteins N-terminus and related modifications influence protein fate and half-life. The most frequent co-translational N-terminal protein modifications are linked to initial methionine excision and protein N-alpha-acetylation. Another modification occurring next to the protein N-term corresponds to the excision of the transit peptide of nuclear-encoded proteins targeted to the mitochondria and/or the plastids. Only few bioinformatics tools are currently available to predict mature proteins N-term such as TermiNator3 (processing and modifications at position 1-2) and TargetP/ChloroP/SignalP tools that provide transit-peptide length predictions and protein subcellular localization. Unfortunately, the reliability of these tools suffer minor inconsistencies to major defects especially for the transit-peptide length prediction with the TargetP suite. Here, we present a dedicated database (eNergiomeDB) which data were collected from large scale N-terminomics studies. Experimental data rel...

Research paper thumbnail of Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2018

Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genom... more 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 Vp16 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 Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 specific activity to values similar to those of bacterial PDFs. We next show that Vp16PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.

Research paper thumbnail of Myristoylation, an Ancient Protein Modification Mirroring Eukaryogenesis and Evolution

Trends in Biochemical Sciences, 2020

N-myristoylation (MYR) is a crucial fatty acylation catalyzed by N-myristoyltransferases (NMTs) t... more N-myristoylation (MYR) is a crucial fatty acylation catalyzed by N-myristoyltransferases (NMTs) that is likely to have appeared over two 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. Lipidated proteins Plasma membranes (PMs) are composed of extrinsic and intrinsic proteins (52%) and lipids (40%), the latter sustaining the overall cellular architecture. Membrane-penetrating extrinsic proteins often possess covalently linked lipids, usually fatty acids, which allow the protein to contact other intra-and extracellular proteins [1]. Protein lipidation involves amides (i.e. N-αmyristoylation, MYR, see Glossary and glycosylphosphatidylinositol (GPI) anchors), thioesters (i.e. S-palmitoylation, PAL), and thioethers (i.e. isoprenylation and farnesylation) [2]. Of these, MYR is a frequent and conserved modification specific to eukaryotes that targets major cellular components. Mapping the proteins undergoing MYR has proven challenging due to their difficult handling characteristics and amphiphilic, chimeric nature. Recently, high-end technologies have allowed the first lipidated proteome, the myristoylome, to be described in detail in various organisms recapitulating the tree of life [3]. These and other studies on myristoylome composition and genesis have also revealed (i) an unexpected novel mechanism of action of Nmyristoyltransferase (NMT), (ii) NMT substrate selectivity, and (iii) the capacity of NMT to act on N-terminal lysines (Lys) as well as the usual glycines (Gly). This review highlights the series of groundbreaking discoveries that have recently significantly advanced our knowledge about this long-studied enzyme. This includes an overview of a new NMT catalytic mechanism, substrate specificity, and proteomics, and a discussion of how a reduced set of targets is closely related to eukaryogenesis and eukaryote evolution. How NMTs catalyze MYR with high selectivity NMTs are GNAT members closely related to Nα-acetyltransferases Seventy-four crystal structures have now revealed that the C-terminal 400 amino acid-long NMT catalytic core displays a conserved 3D GCN5-related N-acetyltransferase (GNAT) core. GNATs also include the catalytic subunits of Nα-acetyltransferases (NAAs) [4], with Naa10 being the closest to NMT as it modifies N-terminal Gly [5-7]. NMTs have two adjacent GNAT domains, most likely to have arisen through duplication of a Naa10-like GNAT domain [8]. Prokaryotes do not possess NMTs, which seem to have arisen as eukaryotes evolved from the last archaeal