Bacterial origins of thymidylate metabolism in Asgard archaea and Eukarya.

Asgard archaea include the closest known archaeal relatives of eukaryotes. Here, we investigate the evolution and function of Asgard thymidylate synthases and other folate-dependent enzymes required for the biosynthesis of DNA, RNA, amino acids and vitamins, as well as syntrophic amino acid utilization. Phylogenies of Asgard folate-dependent enzymes are consistent with their horizontal transmission from various bacterial groups. We experimentally validate the functionality of thymidylate synthase ThyX of the cultured 'Candidatus Prometheoarchaeum syntrophicum'. The enzyme efficiently uses bacterial-like folates and is inhibited by mycobacterial ThyX inhibitors, even though the majority of experimentally tested archaea are known to use carbon carriers distinct from bacterial folates. Our phylogenetic analyses suggest that the eukaryotic thymidylate synthase, required for de novo DNA synthesis, is not closely related to archaeal enzymes and might have been transferred from bacteria to protoeukaryotes during eukaryogenesis. Altogether, our study suggests that the capacity of eukaryotic cells to duplicate their genetic material is a sum of archaeal (replisome) and bacterial (thymidylate synthase) characteristics. We also propose that recent prevalent lateral gene transfer from bacteria has markedly shaped the metabolism of Asgard archaea.

Synthesis and Structure-Activity Relationship Studies of Pyrido [1,2-e]Purine-2,4(1H,3H)-Dione Derivatives Targeting Flavin-Dependent Thymidylate Synthase in Mycobacterium tuberculosis.

In 2002, a new class of thymidylate synthase (TS) involved in the de novo synthesis of dTMP named Flavin-Dependent Thymidylate Synthase (FDTS) encoded by the thyX gene was discovered; FDTS is present only in 30% of prokaryote pathogens and not in human pathogens, which makes it an attractive target for the development of new antibacterial agents, especially against multi-resistant pathogens. We report herein the synthesis and structure-activity relationship of a novel series of hitherto unknown pyrido[1,2-e]purine-2,4(1H,3H)-dione analogues. Several synthetics efforts were done to optimize regioselective N1-alkylation through organopalladium cross-coupling. Modelling of potential hits were performed to generate a model of interaction into the active pocket of FDTS to understand and guide further synthetic modification. All those compounds were evaluated on an in-house in vitro NADPH oxidase assays screening as well as against Mycobacterium tuberculosis ThyX. The highest inhibition was obtained for compound 23a with 84.3% at 200 µM without significant cytotoxicity (CC50 > 100 µM) on PBM cells.

tRNA-dependent addition of amino acids to cell wall and membrane components.

The objective of the present review is to provide an insight into modifications of microbial cell walls and membrane constituents by using the aminoacyl-tRNA as amino acid donor. In bacteria, phospholipids are modified by Multiple peptide resistance Factor enzymes and peptidoglycan precursors by so called fem ligases. Although these modifications were thought to be restricted to procaryotes, we discovered enzymes that modify ergosterol (the main component of fungal membrane) with glycine and aspartate. The focus of this review is to present the molecular mechanisms underlying all these processes together with the structure of the enzymes and their substrates. This article also reviews how substrates are recognized and modified and how the products are subsequently exported in various organisms. Finally, the physiological outcome and the discoveries of each family of enzymes is also discussed.

Loss of seryl-tRNA synthetase (SARS1) causes complex spastic paraplegia and cellular senescence.

BACKGROUND: Aminoacyl-tRNA synthetases (ARS) are key enzymes catalysing the first reactions in protein synthesis, with increasingly recognised pleiotropic roles in tumourgenesis, angiogenesis, immune response and lifespan. Germline mutations in several ARS genes have been associated with both recessive and dominant neurological diseases. Recently, patients affected with microcephaly, intellectual disability and ataxia harbouring biallelic variants in the seryl-tRNA synthetase encoded by seryl-tRNA synthetase 1 (SARS1) were reported. METHODS: We used exome sequencing to identify the causal variant in a patient affected by complex spastic paraplegia with ataxia, intellectual disability, developmental delay and seizures, but without microcephaly. Complementation and serylation assays using patient's fibroblasts and an Saccharomyces cerevisiae model were performed to examine this variant's pathogenicity. RESULTS: A de novo splice site deletion in SARS1 was identified in our patient, resulting in a 5-amino acid in-frame insertion near its active site. Complementation assays in S. cerevisiae and serylation assays in both yeast strains and patient fibroblasts proved a loss-of-function, dominant negative effect. Fibroblasts showed an abnormal cell shape, arrested division and increased beta-galactosidase staining along with a senescence-associated secretory phenotype (raised interleukin-6, p21, p16 and p53 levels). CONCLUSION: We refine the phenotypic spectrum and modes of inheritance of a newly described, ultrarare neurodevelopmental disorder, while unveiling the role of SARS1 as a regulator of cell growth, division and senescence.

Visualizing Mitochondrial Importability of a Protein Using the Yeast Bi-Genomic Mitochondrial-Split-GFP Strain and an Ordinary Fluorescence Microscope.

Proving with certainty that a GFP-tagged protein is imported inside mitochondria by visualizing its fluorescence emission with an epifluorescence microscope is currently impossible using regular GFP-tagging. This is particularly true for proteins dual localized in the cytosol and mitochondria, which have been estimated to represent up to one third of the established mitoproteomes. These proteins are usually composed of a surpassingly abundant pool of the cytosolic isoform compared to the mitochondrial isoform. As a consequence, when tagged with a regular GFP, the fluorescence emission of the cytosolic isoform will inevitably eclipse that of the mitochondrial one and prevent the detection of the mitochondrial echoform. To overcome this technical limit, we engineered a yeast strain expressing a new type of GFP called Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). In this strain, one moiety of the GFP is encoded by the mitochondrial DNA while the second moiety of the GFP can be tagged to any nuclear-encoded protein (suspected to be dual localized or bona fide mitochondrial). By doing so, only mitochondrial proteins or echoforms of dual localized proteins, regardless of their organismal origin, trigger GFP reconstitution that can be visualized by regular fluorescence microscopy. The strength of the BiG Mito-Split-GFP system is that proof of the mitochondrial localization of a given protein rests on a simple and effortless microscopy observation.

RNA-dependent synthesis of ergosteryl-3ß-O-glycine in Ascomycota expands the diversity of steryl-amino acids.

A wide range of bacteria possess virulence factors such as aminoacyl-tRNA transferases (ATTs) that are capable of rerouting aminoacyl-transfer RNAs away from protein synthesis to conjugate amino acids onto glycerolipids. We recently showed that, although these pathways were thought to be restricted to bacteria, higher fungi also possess ergosteryl-3ß-O-L-aspartate synthases (ErdSs), which transfer the L-Asp moiety of aspartyl-tRNAAsp onto the 3ß-OH group of ergosterol (Erg), yielding ergosteryl-3ß-O-L-aspartate (Erg-Asp). Here, we report the discovery, in fungi, of a second type of fungal sterol-specific ATTs, namely, ergosteryl-3ß-O-glycine (Erg-Gly) synthase (ErgS). ErgS consists of a freestanding DUF2156 domain encoded by a gene distinct from and paralogous to that of ErdS. We show that the enzyme only uses Gly-tRNAGly produced by an independent glycyl-tRNA synthetase (GlyRS) to transfer glycine onto the 3ß-OH of Erg, producing Erg-Gly. Phylogenomics analysis also show that the Erg-Gly synthesis pathway exists only in Ascomycota, including species of biotechnological interest, and more importantly, in human pathogens, such as Aspergillus fumigatus. The discovery of a second type of Erg-aa not only expands the repertoire of this particular class of fungal lipids but suggests that Erg-aa synthases might constitute a genuine subfamily of lipid-modifying ATTs.

Assembly-dependent translation of subunits 6 (Atp6) and 9 (Atp9) of ATP synthase in yeast mitochondria.

The yeast mitochondrial ATP synthase is an assembly of 28 subunits of 17 types of which 3 (subunits 6, 8, and 9) are encoded by mitochondrial genes, while the 14 others have a nuclear genetic origin. Within the membrane domain (FO) of this enzyme, the subunit 6 and a ring of 10 identical subunits 9 transport protons across the mitochondrial inner membrane coupled to ATP synthesis in the extra-membrane structure (F1) of ATP synthase. As a result of their dual genetic origin, the ATP synthase subunits are synthesized in the cytosol and inside the mitochondrion. How they are produced in the proper stoichiometry from two different cellular compartments is still poorly understood. The experiments herein reported show that the rate of translation of the subunits 9 and 6 is enhanced in strains with mutations leading to specific defects in the assembly of these proteins. These translation modifications involve assembly intermediates interacting with subunits 6 and 9 within the final enzyme and cis-regulatory sequences that control gene expression in the organelle. In addition to enabling a balanced output of the ATP synthase subunits, these assembly-dependent feedback loops are presumably important to limit the accumulation of harmful assembly intermediates that have the potential to dissipate the mitochondrial membrane electrical potential and the main source of chemical energy of the cell.

Synthesis of acyclic nucleoside phosphonates targeting flavin-dependent thymidylate synthase in Mycobacterium tuberculosis.

Flavin-Dependent Thymidylate Synthase (FDTS) encoded by ThyX gene was discovered as a new class of thymidylate synthase involved in the de novo synthesis of dTMP named only in 30 % of human pathogenic bacteria. This target was pursed for the development of new antibacterial agents against multiresistant pathogens. We have developed a new class of ANPs based on the mimic of two natural's cofactors (dUMP and FAD) as inhibitors against Mycobacterium tuberculosis ThyX. Several synthetic efforts were performed to optimize regioselective N1-alkylation, cross-coupling metathesis and Sonogashira cross-coupling. Compound 19c showed a poor 31.8% inhibitory effect on ThyX at 200 µM.

Synthesis of aminoacylated ergosterols: A new lipid component of fungi.

Aminoacylated ergosterol such as 1-ergosteryl aspartate (Erg-Asp) is a new lipid component recently discovered in fungi. In order to study physiological functions of this novel sterol derivative and to develop potential antifungal agents, we established the method to synthesize aminoacylated ergosterol derivatives. Herein, we report the synthesis of Erg-Asp as well as some other aminoacylated ergosterols (Erg-Gly, Erg-Ala, Erg-Leu, Erg-Ile, and Erg-Val) using Boc protected amino acids.

Cex1 is a component of the COPI intracellular trafficking machinery.

COPI (coatomer complex I) coated vesicles are involved in Golgi-to-ER and intra-Golgi trafficking pathways, and mediate retrieval of ER resident proteins. Functions and components of the COPI-mediated trafficking pathways, beyond the canonical set of Sec/Arf proteins, are constantly increasing in number and complexity. In mammalian cells, GORAB, SCYL1 and SCYL3 proteins regulate Golgi morphology and protein glycosylation in concert with the COPI machinery. Here, we show that Cex1, homologous to the mammalian SCYL proteins, is a component of the yeast COPI machinery, by interacting with Sec27, Sec28 and Sec33 (Ret1/Cop1) proteins of the COPI coat. Cex1 was initially reported to mediate channeling of aminoacylated tRNA outside of the nucleus. Our data show that Cex1 localizes at membrane compartments, on structures positive for the Sec33 α-COP subunit. Moreover, the Wbp1 protein required for N-glycosylation and interacting via its di-lysine motif with the Sec27 ß'-COP subunit is mis-targeted in cex1Δ deletion mutant cells. Our data point to the possibility of developing Cex1 yeast-based models to study neurodegenerative disorders linked to pathogenic mutations of its human homologue SCYL1.

La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société.

Founded in 1919, the Society of Biology of Strasbourg (SBS) is a learned society whose purpose is the dissemination and promotion of scientific knowledge in biology. Subsidiary of the Society of Biology, the SBS celebrated its Centenary on Wednesday, the 16th of October 2019 on the Strasbourg University campus and at the Strasbourg City Hall. This day allowed retracing the various milestones of the SBS, through its main strengths, its difficulties and its permanent goal to meet scientific and societal challenges. The common thread of this day was the transmission of knowledge related to the past, the present, but also the future. At the start of the 21st century, the SBS must continue to reinvent itself to pursue its objective of transmitting scientific knowledge in biology and beyond. Scientific talks performed by senior scientists and former SBS thesis prizes awardees, a round table, and informal discussions reflected the history and the dynamism of the SBS association. All SBS Centennial participants have set the first milestone for the SBS Bicentennial.

TITLE: La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société. ABSTRACT: Filiale de la Société de Biologie, la Société de Biologie de Strasbourg (SBS) est une société savante qui a pour objet la diffusion et la promotion du savoir scientifique en biologie et en médecine. Fondée en 1919, La SBS a célébré son Centenaire le mercredi 16 octobre 2019. Cette journée a permis de retracer les différents jalons de la SBS, à travers ses lignes de forces, ses difficultés et sa volonté permanente de mettre en exergue les défis scientifiques et sociétaux auxquels participent les recherches strasbourgeoises. Le fil rouge de cette journée a été la transmission d'un savoir en lien avec le passé, le présent, mais également le futur. En ce début du 21e siècle, la SBS se doit de continuer de se réinventer pour poursuivre son objectif de transmission des connaissances scientifiques en biologie et au-delà. L'ensemble des participants du Centenaire de la SBS a ainsi posé la première pierre du Bicentenaire de la SBS.

Mechanism of Naphthoquinone Selectivity of Thymidylate Synthase ThyX.

Naphthoquinones (NQs) are natural and synthetic compounds with a wide range of biological activities commonly attributed to their redox activity and/or chemical reactivity. However, genetic and biochemical experiments have recently demonstrated that 2-hydroxy-NQs (2-OH-NQs) act as highly specific noncovalent inhibitors of the essential bacterial thymidylate synthase ThyX in a cellular context. We used biochemical experiments and molecular dynamics simulations to elucidate the selective inhibition mechanism of NQ inhibitors of ThyX from Mycobacterium tuberculosis (Mtb). Free energy simulations rationalized how ThyX recognizes the natural substrate dUMP in the N3-ionized form using an arginine, Arg199, in Mtb. The results further demonstrated that 2-OH-NQ, similar to dUMP, binds to ThyX in the ionized form, and the strong and selective binding of 2-OH-NQ to ThyX is also explained by electrostatic interactions with Arg199. The stronger binding of the close analog 5F-dUMP to ThyX and its inhibitory properties compared with dUMP were explained by the stronger acidity of the uracil N3 atom. Our results, therefore, revealed that the ionization of 2-OH-NQs drives their biological activities by mimicking the interactions with the natural substrate. Our observations encourage the rational design of optimized ThyX inhibitors that ultimately may serve as antibiotics.

Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP.

A single nuclear gene can be translated into a dual localized protein that distributes between the cytosol and mitochondria. Accumulating evidences show that mitoproteomes contain lots of these dual localized proteins termed echoforms. Unraveling the existence of mitochondrial echoforms using current GFP (Green Fluorescent Protein) fusion microscopy approaches is extremely difficult because the GFP signal of the cytosolic echoform will almost inevitably mask that of the mitochondrial echoform. We therefore engineered a yeast strain expressing a new type of Split-GFP that we termed Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). Because one moiety of the GFP is translated from the mitochondrial machinery while the other is fused to the nuclear-encoded protein of interest translated in the cytosol, the self-reassembly of this Bi-Genomic-encoded Split-GFP is confined to mitochondria. We could authenticate the mitochondrial importability of any protein or echoform from yeast, but also from other organisms such as the human Argonaute 2 mitochondrial echoform.

RNA-dependent sterol aspartylation in fungi.

Diverting aminoacyl-transfer RNAs (tRNAs) from protein synthesis is a well-known process used by a wide range of bacteria to aminoacylate membrane constituents. By tRNA-dependently adding amino acids to glycerolipids, bacteria change their cell surface properties, which intensifies antimicrobial drug resistance, pathogenicity, and virulence. No equivalent aminoacylated lipids have been uncovered in any eukaryotic species thus far, suggesting that tRNA-dependent lipid remodeling is a process restricted to prokaryotes. We report here the discovery of ergosteryl-3ß-O-l-aspartate (Erg-Asp), a conjugated sterol that is produced by the tRNA-dependent addition of aspartate to the 3ß-OH group of ergosterol, the major sterol found in fungal membranes. In fact, Erg-Asp exists in the majority of "higher" fungi, including species of biotechnological interest, and, more importantly, in human pathogens like Aspergillus fumigatus We show that a bifunctional enzyme, ergosteryl-3ß-O-l-aspartate synthase (ErdS), is responsible for Erg-Asp synthesis. ErdS corresponds to a unique fusion of an aspartyl-tRNA synthetase-that produces aspartyl-tRNAAsp (Asp-tRNAAsp)-and of a Domain of Unknown Function 2156, which actually transfers aspartate from Asp-tRNAAsp onto ergosterol. We also uncovered that removal of the Asp modifier from Erg-Asp is catalyzed by a second enzyme, ErdH, that is a genuine Erg-Asp hydrolase participating in the turnover of the conjugated sterol in vivo. Phylogenomics highlights that the entire Erg-Asp synthesis/degradation pathway is conserved across "higher" fungi. Given the central roles of sterols and conjugated sterols in fungi, we propose that this tRNA-dependent ergosterol modification and homeostasis system might have broader implications in membrane remodeling, trafficking, antimicrobial resistance, or pathogenicity.

Noncanonical inputs and outputs of tRNA aminoacylation.

The aminoacylation reaction is one of most extensively studied cellular processes. The so-called "canonical" reaction is carried out by direct charging of an amino acid (aa) onto its corresponding transfer RNA (tRNA) by the cognate aminoacyl-tRNA synthetase (aaRS), and the canonical usage of the aminoacylated tRNA (aa-tRNA) is to translate a messenger RNA codon in a translating ribosome. However, four out of the 22 genetically-encoded aa are made "noncanonically" through a two-step or indirect route that usually compensate for a missing aaRS. Additionally, from the 22 proteinogenic aa, 13 are noncanonically used, by serving as substrates for the tRNA- or aa-tRNA-dependent synthesis of other cellular components. These nontranslational processes range from lipid aminoacylation, and heme, aa, antibiotic and peptidoglycan synthesis to protein degradation. This chapter focuses on these noncanonical usages of aa-tRNAs and the ways of generating them, and also highlights the strategies that cells have evolved to balance the use of aa-tRNAs between protein synthesis and synthesis of other cellular components.

Diversity of circular RNAs and RNA ligases in archaeal cells.

Circular RNAs (circRNAs) differ structurally from other types of RNAs and are resistant against exoribonucleases. Although they have been detected in all domains of life, it remains unclear how circularization affects or changes functions of these ubiquitous nucleic acid circles. The biogenesis of circRNAs has been mostly described as a backsplicing event, but in archaea, where RNA splicing is a rare phenomenon, a second pathway for circRNA formation was described in the cases of rRNAs processing, tRNA intron excision, and Box C/D RNAs formation. At least in some archaeal species, circRNAs are formed by a ligation step catalyzed by an atypic homodimeric RNA ligase belonging to Rnl3 family. In this review, we describe archaeal circRNA transcriptomes obtained using high throughput sequencing technologies on Sulfolobus solfataricus, Pyrococcus abyssi and Nanoarchaeum equitans cells. We will discuss the distribution of circular RNAs among the different RNA categories and present the Rnl3 ligase family implicated in the circularization activity. Special focus is given for the description of phylogenetic distributions, protein structures, and substrate specificities of archaeal RNA ligases.

Mutations in KARS cause a severe neurological and neurosensory disease with optic neuropathy.

Mutations in genes encoding aminoacyl-tRNA synthetases have been reported in several neurological disorders. KARS is a dual localized lysyl-tRNA synthetase and its cytosolic isoform belongs to the multiple aminoacyl-tRNA synthetase complex (MSC). Biallelic mutations in the KARS gene were described in a wide phenotypic spectrum ranging from nonsyndromic deafness to complex impairments. Here, we report on a patient with severe neurological and neurosensory disease investigated by whole-exome sequencing and found to carry biallelic mutations c.683C>T (p.Pro228Leu) and c.871T>G (p.Phe291Val), the second one being novel, in the KARS gene. The patient presented with an atypical clinical presentation with an optic neuropathy not previously reported. At the cellular level, we show that cytoplasmic KARS was expressed at a lower level in patient cells and displayed decreased interaction with MSC. In vitro, these two KARS variants have a decreased aminoacylation activity compared with wild-type KARS, the p.Pro228Leu being the most affected. Our data suggest that dysfunction of cytoplasmic KARS resulted in a decreased level of translation of the nuclear-encoded lysine-rich proteins belonging to the respiratory chain complex, thus impairing mitochondria functions.

Synthesis and Structure-Activity Relationship Studies of Benzo[b][1,4]oxazin-3(4H)-one Analogues as Inhibitors of Mycobacterial Thymidylate Synthase†X.

Since the discovery of a flavin-dependent thymidylate synthase (ThyX or FDTS) that is absent in humans but crucial for DNA biosynthesis in a diverse group of pathogens, the enzyme has been pursued for the development of new antibacterial agents against Mycobacterium tuberculosis, the causative agent of the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous screening efforts and report herein an optimization campaign of a novel series of inhibitors with a unique inhibition profile. The inhibitors display competitive inhibition toward the methylene tetrahydrofolate cofactor of ThyX, enabling us to generate a model of the compounds bound to their target, thus offering insight into their structure-activity relationships.

Cytosolic aminoacyl-tRNA synthetases: Unanticipated relocations for unexpected functions.

Prokaryotic and eukaryotic cytosolic aminoacyl-tRNA synthetases (aaRSs) are essentially known for their conventional function of generating the full set of aminoacyl-tRNA species that are needed to incorporate each organism's repertoire of genetically-encoded amino acids during ribosomal translation of messenger RNAs. However, bacterial and eukaryotic cytosolic aaRSs have been shown to exhibit other essential nonconventional functions. Here we review all the subcellular compartments that prokaryotic and eukaryotic cytosolic aaRSs can reach to exert either a conventional or nontranslational role. We describe the physiological and stress conditions, the mechanisms and the signaling pathways that trigger their relocation and the new functions associated with these relocating cytosolic aaRS. Finally, given that these relocating pools of cytosolic aaRSs participate to a wide range of cellular pathways beyond translation, but equally important for cellular homeostasis, we mention some of the pathologies and diseases associated with the dis-regulation or malfunctioning of these nontranslational functions.

Discovery of a new Mycobacterium tuberculosis thymidylate synthase X inhibitor with a unique inhibition profile.

Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), is an infection that is responsible for roughly 1.5 million deaths per year. The situation is further complicated by the wide-spread resistance to the existing first- and second-line drugs. As a result of this, it is urgent to develop new drugs to combat the resistant bacteria as well as have lower side effects, which can promote adherence to the treatment regimens. Targeting the de novo synthesis of thymidylate (dTMP) is an important pathway to develop drugs for TB. Although Mtb carries genes for two families of thymidylate synthases (TS), ThyA and ThyX, only ThyX is essential for its normal growth. Both enzymes catalyze the conversion of uridylate (dUMP) to dTMP but employ a different catalytic approach and have different structures. Also, ThyA is the only TS found in humans. This is the rationale for identifying selective inhibitors against ThyX. We exploited the NADPH oxidation to NADP+ step, catalyzed by ThyX, to develop a spectrophotometric biochemical assay. Success of the assay was demonstrated by its effectiveness (average Z'=0.77) and identification of selective ThyX inhibitors. The most potent compound is a tight-binding inhibitor with an IC50 of 710nM. Its mechanism of inhibition is analyzed in relation to the latest findings of ThyX mechanism and substrate and cofactor binding order.