Exposure to the Methylselenol Precursor Dimethyldiselenide Induces a Reductive Endoplasmic Reticulum Stress in .

Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in , MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH.

Methylselenol Produced In Vivo from Methylseleninic Acid or Dimethyl Diselenide Induces Toxic Protein Aggregation in .

Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide.

Spermidine strongly increases the fidelity of CRISPR Cas1-Cas2 integrase.

Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described However, this nonspecific integration activity is rare Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by Cas1-Cas2 integrase.

On the Relation between Chemical Oscillations and Self-Replication.

One proposed scenario for the emergence of biochemical oscillations is that they may have provided the basic mechanism behind cellular self-replication by growth and division. However, alternative scenarios not requiring any chemical oscillation have also been proposed. Each of the various protocell models proposed to support one or another scenario comes with its own set of specific assumptions, which makes it difficult to ascertain whether chemical oscillations are required or not for cellular self-replication.

Recent advances in the mechanism of selenoamino acids toxicity in eukaryotic cells.

Selenium is an essential trace element due to its incorporation into selenoproteins with important biological functions. However, at high doses it is toxic. Selenium toxicity is generally attributed to the induction of oxidative stress.

Exposure to selenomethionine causes selenocysteine misincorporation and protein aggregation in Saccharomyces cerevisiae.

Selenomethionine, a dietary supplement with beneficial health effects, becomes toxic if taken in excess. To gain insight into the mechanisms of action of selenomethionine, we screened a collection of ≈5900 Saccharomyces cerevisiae mutants for sensitivity or resistance to growth-limiting amounts of the compound. Genes involved in protein degradation and synthesis were enriched in the obtained datasets, suggesting that selenomethionine causes a proteotoxic stress.

DNA binding specificities of Escherichia coli Cas1-Cas2 integrase drive its recruitment at the CRISPR locus.

Prokaryotic adaptive immunity relies on the capture of fragments of invader DNA (protospacers) followed by their recombination at a dedicated acceptor DNA locus. This integrative mechanism, called adaptation, needs both Cas1 and Cas2 proteins. Here, we studied in vitro the binding of an Escherichia coli Cas1-Cas2 complex to various protospacer and acceptor DNA molecules.

Studies of the B-Z transition of DNA: The temperature dependence of the free-energy difference, the composition of the counterion sheath in mixed salt, and the preparation of a sample of the 5'-d[T-(m(5) C-G)12 -T] duplex in pure B-DNA or Z-DNA form.

It is often envisioned that cations might coordinate at specific sites of nucleic acids and play an important structural role, for instance in the transition between B-DNA and Z-DNA. However, nucleic acid models explicitly devoid of specific sites may also exhibit features previously considered as evidence for specific binding. Such is the case of the "composite cylinder" (or CC) model which spreads out localized features of DNA structure and charge by cylindrical averaging, while sustaining the main difference between the B and Z structures, namely the better immersion of the B-DNA phosphodiester charges in the solution.

Redesigning the stereospecificity of tyrosyl-tRNA synthetase.

D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity.

Trans-sulfuration Pathway Seleno-amino Acids Are Mediators of Selenomethionine Toxicity in Saccharomyces cerevisiae.

Toxicity of selenomethionine, an organic derivative of selenium widely used as supplement in human diets, was studied in the model organism Saccharomyces cerevisiae. Several DNA repair-deficient strains hypersensitive to selenide displayed wild-type growth rate properties in the presence of selenomethionine indicating that selenide and selenomethionine exert their toxicity via distinct mechanisms. Cytotoxicity of selenomethionine decreased when the extracellular concentration of methionine or S-adenosylmethionine was increased.

Neutralization by metal ions of the toxicity of sodium selenide.

Inert metal-selenide colloids are found in animals. They are believed to afford cross-protection against the toxicities of both metals and selenocompounds. Here, the toxicities of metal salt and sodium selenide mixtures were systematically studied using the death rate of Saccharomyces cerevisiae cells as an indicator.

Sodium selenide toxicity is mediated by O2-dependent DNA breaks.

Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)).

RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase.

In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced.

Selenodiglutathione uptake by the Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p.

The Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p is involved in heavy metal detoxification by mediating the ATP-dependent transport of glutathione-metal conjugates to the vacuole. In the case of selenite toxicity, deletion of YCF1 was shown to confer increased resistance, rather than sensitivity, to selenite exposure [Pinson B, Sagot I & Daignan-Fornier B (2000) Mol Microbiol36, 679-687]. Here, we show that when Ycf1p is expressed from a multicopy plasmid, the toxicity of selenite is exacerbated.

NMR-based substrate analog docking to Escherichia coli peptidyl-tRNA hydrolase.

Escherichia coli peptidyl-tRNA hydrolase activity is inhibited by 3'-(L-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine, a stable mimic of the minimalist substrate 2'(3')-O-(L-[N,N-diacetyl-lysinyl)adenosine. The complex of this mimic with the enzyme has been analyzed by NMR spectroscopy, enabling experimental mapping of the catalytic center for the first time. Chemical shift variations point out the sensitivity of residues Asn10, Met67, Asn68, Gly111, Asn114, Leu116, Lys117, Gly147, Phe148, and Val149 to complex formation.

Computational protein design with a generalized Born solvent model: application to Asparaginyl-tRNA synthetase.

Computational Protein Design (CPD) is a promising method for high throughput protein and ligand mutagenesis. Recently, we developed a CPD method that used a polar-hydrogen energy function for protein interactions and a Coulomb/Accessible Surface Area (CASA) model for solvent effects. We applied this method to engineer aspartyl-adenylate (AspAMP) specificity into Asparaginyl-tRNA synthetase (AsnRS), whose substrate is asparaginyl-adenylate (AsnAMP).

Uptake of selenite by Saccharomyces cerevisiae involves the high and low affinity orthophosphate transporters.

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake.

2D label-free imaging of resonant grating biochips in ultraviolet.

2D images of label-free biochips exploiting resonant waveguide grating (RWG) are presented. They indicate sensitivities on the order of 1 pg/mm2 for proteins in air, and hence 10 pg/mm2 in water can be safely expected. A 320x256 pixels Aluminum-Gallium-Nitride-based sensor array is used, with an intrinsic narrow spectral window centered at 280 nm.

Widespread distribution of cell defense against D-aminoacyl-tRNAs.

Several l-aminoacyl-tRNA synthetases can transfer a d-amino acid onto their cognate tRNA(s). This harmful reaction is counteracted by the enzyme d-aminoacyl-tRNA deacylase. Two distinct deacylases were already identified in bacteria (DTD1) and in archaea (DTD2), respectively.

Detection of biological macromolecules on a biochip dedicated to UV specific absorption.

This work describes an ultraviolet biosensing technique based on specific molecular absorption detected with a previously developed spectrally selective aluminum gallium nitride (AlGaN) based detector. Light absorption signal of DNA and proteins, respectively at 260 nm and 280 nm, is used to image biochips. To allow detection of protein or DNA monolayers at the surface of a biochip, we develop contrast-enhancing multilayer substrates.

Probing electrostatic interactions and ligand binding in aspartyl-tRNA synthetase through site-directed mutagenesis and computer simulations.

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further.

Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes.

Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.

Ammonium scanning in an enzyme active site. The chiral specificity of aspartyl-tRNA synthetase.

D-amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Aspartyl-tRNA synthetase (AspRS) can misacylate tRNA(Asp) with D-aspartate instead of its usual substrate, L-Asp. We investigate how the preference for L-Asp arises, using molecular dynamics simulations.

GEK1, a gene product of Arabidopsis thaliana involved in ethanol tolerance, is a D-aminoacyl-tRNA deacylase.

GEK1, an Arabidopsis thaliana gene product, was recently identified through its involvement in ethanol tolerance. Later, this protein was shown to display 26% strict identity with archaeal d-Tyr-tRNA(Tyr) deacylases. To determine whether it actually possessed deacylase activity, the product of the GEK1 open reading frame was expressed in Escherichia coli from a multi-copy plasmid.