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Molecular dynamic of the methionyl-tRNA synthetase and its novel non-canonical functions in the yeast S. cerevisiae
Abstract
Aminoacyl-tRNA synthetases (aaRSs) are essential and ubiquitous enzymes catalyzing formation of aminoacyl- tRNAs (aa-tRNAs) during protein synthesis. However, aaRSs are not limited to aa-tRNAs formation. Indeed, they evolved to form multl-protein complexes that acquired additional functions. S. cerevisiae contains the simplest eukaryotic multi-synthetase complex which is formed by the association of methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRS) to the cytosolic anchoring protein Arc1.This complex (named AME) is highly dynamic depending on the nutritional conditions that the cells are facing, and the two associated aaRSs harbor additional functions that are essential for cell survival. In this PhD thesis, I have studied three different aspect of theyeast MetRS: (i) I created a new bifluorescent reporter to quantify endogenous mismethionylation mediated by the yeast MetRS. I also (ii) characterized a new truncated yeast MetRS isoform produced in vivo, and (iii) I analysed the relative importance of Arc1for cell surviving during the diauxic shift from fermentation to respiration.
... cerevisiae AspRS (pdb: 1ASY), is characterized by 7 -strands and contains characteristic motifs 1, 2 and 3. Class II aaRSs bind the extended 3'CCA extremity binding domain but their structural organization differs between aaRS subclasses. Inspired from Debard, 2019. Catalytic domain anticodon binding domain 23 code) which are essential motifs of the catalytic site (Eriani et al., 1990). ...
... Alternatively, the ND-aaRSs, the AdT and the tRNA can also form a complex called transamidosome ( Figure I-5C) in which the non-cognate aa-tRNA is directly channeled from one enzyme to the other (Bailly et al., 2007;Rathnayake et al., 2017) preventing Asp-tRNA Asn and Glu-tRNA Gln of reaching the ribosome. (Debard 2019) ...
... In addition to the active site, the catalytic domain of some aaRSs, contains an editing site that proofreads the edited within the catalytic domain of the aaRS. After the second step of aminoacylation (transfer), the (B) The editing aaRSs with their corresponding edited aa are listed in the lower panel, as well as trans-editing factors with α-aminobuty-Ling, Reynolds and Rubio andIbba, 2020 andDebard, 2019 order to discriminate it from other RNAs and macromolecules. The "initial" interactions also participate to the discrimination of class II tRNA due to their large V-(stem-)loop for example (Asahara et al., 1993;Park and Schimmel 1988;Dock-Bregeon et al., 1990). ...
Aminoacyl-tRNAs (aa-tRNA) play a central role in protein synthesis but can also be rerouted to other biological pathways. In bacteria, aa-tRNA transferases (AAT) having a DUF2156 domain catalyze the aminoacid (aa) transfer onto glycerolipids to improve their drug resistance or virulence. Such a mechanism was never described in eukaryotes. My thesis work revealed that numerous fungi species have DUF2156/AAT enzymes that aminoacylate sterols. Thus, ergosteryl-aspartate and ergosteryl-glycine are the founding members of a new class of lipids, namely aminoacylated sterols (AS). Moreover, we also identified a specific hydrolase that removes the aa from several AS. Considering the central role of sterols, we propose that AS might participate to cell surface remodeling, trafficking, resistance to stresses and/or pathogenicity.
Recent studies suggested an essential role for seryl-tRNA synthetase (SerRS) in vascular development. This role is specific to SerRS among all tRNA synthetases and is independent of its well-known aminoacylation function in protein synthesis. A unique nucleus-directing domain, added at the invertebrate-to-vertebrate transition, confers this novel non-translational activity of SerRS. Previous studies showed that SerRS, in some unknown way, controls VEGFA expression to prevent vascular over-expansion. Using in vitro, cell and animal experiments, we show here that SerRS intervenes by antagonizing c-Myc, the major transcription factor promoting VEGFA expression, through a tandem mechanism. First, by direct head-to-head competition, nuclear-localized SerRS blocks c-Myc from binding to the VEGFA promoter. Second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to erase prior c-Myc-promoted histone acetylation. Thus, vertebrate SerRS and c-Myc is a pair of 'Yin-Yang' transcriptional regulator for proper development of a functional vasculature. Our results also discover an anti-angiogenic activity for SIRT2.
As the central dogma of molecular biology, genetic information flows from DNA through transcription into RNA followed by translation of the message into protein by transfer RNAs (tRNAs). However, mRNA translation is not always perfect, and errors in the amino acid composition may occur. Mistranslation is generally well tolerated, but once it reaches super-physiological levels, it can give rise to a plethora of diseases. The key causes of mistranslation are errors in translational decoding of the codons in mRNA. Such errors mainly derive from tRNA misdecoding and misacylation, especially when certain codon-paired tRNA species are missing. Substantial progress has recently been made with respect to the mecha-nistic basis of erroneous mRNA decoding as well as the resulting consequences for physiology and pathology. Here, we aim to review this progress with emphasis on viral evolution and cancer development.
Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research.
Cysteine and methionine residues are the amino acids most sensitive to oxidation by reactive oxygen species. However, in contrast to other amino acids, certain cysteine and methionine oxidation products can be reduced within proteins by dedicated enzymatic repair systems. Oxidation of cysteine first results in either the formation of a disulfide bridge or a sulfenic acid. Sulfenic acid can be converted to disulfide or sulfenamide or further oxidized to sulfinic acid. Disulfide can be easily reversed by different enzymatic systems such as the thioredoxin/thioredoxin reductase and the glutaredoxin/glutathione/glutathione reductase systems. Methionine side chains can also be oxidized by reactive oxygen species. Methionine oxidation, by the addition of an extra oxygen atom, leads to the generation of methionine sulfoxide. Enzymatically catalyzed reduction of methionine sulfoxide is achieved by either methionine sulfoxide reductase A or methionine sulfoxide reductase B, also referred as to the methionine sulfoxide reductases system. This oxidized protein repair system is further described in this review article in terms of its discovery and biologically relevant characteristics, and its important physiological roles in protecting against oxidative stress, in ageing and in regulating protein function.
Production of eukaryotic ribosomal subunits is a highly dynamic process; pre-ribosomes undergo numerous structural rearrangements that establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome assembly. Using in vivo crosslinking, we here identify the pre-ribosomal binding sites of three RNA helicases. Our data support roles for Has1 in triggering release of the U14 snoRNP, a critical event during early 40S maturation, and in driving assembly of domain I of pre-60S complexes. Binding of Mak5 to domain II of pre-60S complexes promotes recruitment of the ribosomal protein Rpl10, which is necessary for subunit joining and ribosome function. Spb4 binds to a molecular hinge at the base of ES27 facilitating binding of the export factor Arx1, thereby promoting pre-60S export competence. Our data provide important insights into the driving forces behind key structural remodelling events during ribosomal subunit assembly.
The folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, as non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by using molecular chaperones to safeguard folding cotranslationally2. However, although most of the cellular proteome forms oligomeric assemblies3, little is known about the final step of folding: the assembly of polypeptides into complexes. In prokaryotes, a proof-of-concept study showed that the assembly of heterodimeric luciferase is an organized cotranslational process that is facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences—such as the rarity of polycistronic mRNAs and different chaperone constellations—raise the question of whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of the assembly of protein complexes in eukaryotes using ribosome profiling. We determined the in vivo interactions of the nascent subunits from twelve hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find nine complexes assemble cotranslationally; the three complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5,6,7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit, thereby counteracting its propensity for aggregation. The onset of cotranslational subunit association coincides directly with the full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains and then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for the assembly of hetero-oligomers in yeast and indicates that translation, folding and the assembly of protein complexes are integrated processes in eukaryotes.
Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-aminoacyl-tRNA synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the aminoacyl-tRNA synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.
Amino acid biochemistry and nutrition spans a broad range of fields including biochemistry, metabolism, physiology, immunology, reproduction, pathology, and cell biology. In the last half-century, there have been many conceptual and technical advancements, from analysis of amino acids by high-performance liquid chromatography and mass spectrometry to molecular cloning of transporters for amino acids and small peptides. Amino Acids: Biochemistry and Nutrition presents comprehensive coverage of these scientific developments, providing a useful reference for students and researchers in both biomedicine and agriculture. The text begins with the discoveries and basic concepts of amino acids, peptides, and proteins, and then moves to protein digestion and absorption of peptides and amino acids. Additional chapters cover cell-, tissue-, and species-specific synthesis and catabolism of amino acids and related nitrogenous substances, as well as the use of isotopes to study amino acid metabolism in cells and the body. The book also details protein synthesis and degradation, regulation of amino acid metabolism, physiological functions of amino acids, and inborn errors of amino acid metabolism. The final chapter discusses dietary requirements of amino acids by humans and other animals. While emphasizing basic principles and classical concepts of amino acid biochemistry and nutrition, the author includes recent progress in the field. This book also provides concise coverage of major historical developments of the scientific discipline, so that readers will appreciate the past, understand the current state of the knowledge, and explore the future of the field. Each chapter contains select references to provide comprehensive reviews and original experimental data on the topics discussed.
Thylakoid membranes are far from being homogeneous in composition. On the contrary, compositional heterogeneity of lipid and protein content is well known to exist in these membranes. The mechanisms for the confinement of proteins at a particular membrane domain have started to be unveiled, but we are far from a thorough understanding and many issues remain to be elucidated. During the differentiation of heterocysts in filamentous cyanobacteria of the Anabaena and Nostoc genera, thylakoids undergo a thorough reorganization, separating in two membrane domains of different appearance and subcellular localization. Evidence also indicates different functionality and protein composition for these two membrane domains. In this work we have addressed the mechanisms that govern the specific localization of proteins at a particular membrane domain. Two classes of proteins were distinguished according to their distribution in the thylakoids. Our results indicate that the specific accumulation of proteins of the CURVATURE THYLAKOID 1 family (CURT1) and proteins containing the homologous CAAD domain at sub-polar honeycomb thylakoids is mediated by multiple mechanisms including a previously unnoticed phenomenon of thylakoid membrane migration.