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

Even if a myriad of approaches has been developed to identify the subcellular localization of a protein, the easiest and fastest way remains to fuse the protein to Green Fluorescent Protein (GFP) and visualize its location using fluorescence microscopy. However, this strategy is not well suited to visualize the organellar pools of proteins that are simultaneously localized both in the cytosol and in organelles because the GFP signal of a cytosolic pool of the protein (cytosolic echoform) will inevitably mask or overlay the GFP signal of the organellar pool of the protein (organellar echoform). To solve this issue, we engineered a dedicated yeast strain expressing a Bi-Genomic Mitochondrial-Split-GFP.

AARS Online: A collaborative database on the structure, function, and evolution of the aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases (aaRS) are a large group of enzymes that implement the genetic code in all known biological systems. They attach amino acids to their cognate tRNAs, moonlight in various translational and non-translational activities beyond aminoacylation, and are linked to many genetic disorders. The aaRS have a subtle ontology characterized by structural and functional idiosyncrasies that vary from organism to organism, and protein to protein.

Synthesis and Structure-Activity Relationship Studies of Pyrido [1,2-]Purine-2,4()-Dione Derivatives Targeting in .

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.

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.

Loss of seryl-tRNA synthetase () 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 () were reported.

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.

RNA-dependent synthesis of ergosteryl-3β-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-3β-O-L-aspartate synthases (ErdSs), which transfer the L-Asp moiety of aspartyl-tRNA onto the 3β-OH group of ergosterol (Erg), yielding ergosteryl-3β-O-L-aspartate (Erg-Asp). Here, we report the discovery, in fungi, of a second type of fungal sterol-specific ATTs, namely, ergosteryl-3β-O-glycine (Erg-Gly) synthase (ErgS).

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.

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.

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.

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.

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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.

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).

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.

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.

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.

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.

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.

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.

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.

High-throughput sequencing reveals circular substrates for an archaeal RNA ligase.

It is only recently that the abundant presence of circular RNAs (circRNAs) in all kingdoms of Life, including the hyperthermophilic archaeon Pyrococcus abyssi, has emerged. This led us to investigate the physiologic significance of a previously observed weak intramolecular ligation activity of Pab1020 RNA ligase. Here we demonstrate that this enzyme, despite sharing significant sequence similarity with DNA ligases, is indeed an RNA-specific polynucleotide ligase efficiently acting on physiologically significant substrates.

The complex evolutionary history of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of >2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived.