Biomedical subjects

Gilles Labesse

Publications and source records attributed to Gilles Labesse.

28 records · Page 2Linked to original sources

PubMed2003

Ssu72 is a phosphatase essential for transcription termination of snoRNAs and specific mRNAs in yeast.

Ssu72 is an essential yeast protein that is involved in transcription. It physically interacts with transcription initiation and termination complexes. In this report, we provide evidence that Ssu72 is a phosphatase that physically interacts with the CTD kinase Kin28 and functionally interacts with the CTD phosphatase Fcp1. A genome-wide expression analysis of mutant ssu72-ts69 during growth in complete medium revealed a number of defects, including the accumulation of a limited number of mRNAs and the read-through transcription of small nucleolar RNAs and of some mRNAs. We hypothesize that Ssu72 plays a key role in the transcription termination of certain transcripts, possibly by promoting RNA polymerase pausing and release. The possibility that the CTD of the largest subunit of RNA polymerase II is a substrate of Ssu72 is discussed.

Amino Acid Sequence↗

PubMed2003

Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain.

Focal adhesions (FAs) are large submembrane signaling complexes formed at sites of cellular attachment to the extracellular matrix. The interaction of LD motifs with their targets plays an important role in the assembly of FAs. We have determined the molecular basis for the recognition of two paxillin LD motifs by the FA targeting (FAT) domain of FA kinase using a combination of X-ray crystallography, solution NMR, and homology modeling. The four-helix FAT domain displays two LD binding sites on opposite sites of the molecule that bind LD peptides in a helical conformation. Threading studies suggest that the LD-interacting domain of p95PKL shares a common four-helical core with the FAT domain and the tail of vinculin, defining a structural family of LD motif binding modules.

Amino Acid Motifs↗

PubMed2003

Comparative sequence analysis and predictions for the envelope glycoproteins of insect endogenous retroviruses.

Insect endogenous retroviruses (IERVs) are present in the genome of several species. Previous studies have shown a relationship between the envelope glycoproteins (Envs) and fusion proteins (FPs) of several baculoviruses. We used this sequence similarity to predict fusion domains in the Envs of IERVs. We suggest that FPs and Envs share several specific sequence and structural motifs with other RNA viruses in the viral transmembrane protein superfamily.

Amino Acid Sequence↗

PubMed2003

UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity.

The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units.mg-1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate (Ant-dGTP) can increase catalysis significantly. Binding of Ant-dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.

Amino Acid Sequence↗

PubMed2002

No ligand binding in the GB2 subunit of the GABA(B) receptor is required for activation and allosteric interaction between the subunits.

The GABA(B) receptor plays important roles in the tuning of many synapses. Although pharmacological differences have been observed between various GABA(B)-mediated effects, a single GABA(B) receptor composed of two subunits (GB1 and GB2) has been identified. Although GB1 binds GABA, GB2 plays a critical role in G-protein activation. Moreover, GB2 is required for the high agonist affinity of GB1. Like any other family 3 G-protein-coupled receptors, GB1 and GB2 are composed of a Venus Flytrap module (VFTM) that usually contains the agonist-binding site and a heptahelical domain. So far, there has been no direct demonstration that GB2 binds GABA or another endogenous ligand. Here, we have further refined the GABA-binding site of GB1 and characterized the putative-binding site in the VFTM of GB2. None of the residues important for GABA binding in GB1 appeared to be conserved in GB2. Moreover, mutation of 10 different residues, alone or in combination, within the possible binding pocket of GB2 affects neither GABA activation of the receptor nor the ability of GB2 to increase agonist affinity on GB1. These data indicate that ligand binding in the GB2 VFTM is not required for activation. Finally, although in either GB1 or the related metabotropic glutamate receptors most residues of the binding pocket are conserved from Caenorhabditis elegans to human, no such conservation is observed in GB2. This suggests that the GB2 VFTM does not constitute a binding site for a natural ligand.

Allosteric Regulation↗

PubMed2002

Crystal structure of MabA from Mycobacterium tuberculosis, a reductase involved in long-chain fatty acid biosynthesis.

The fatty acid elongation system FAS-II is involved in the biosynthesis of mycolic acids, which are major and specific long-chain fatty acids of the cell envelope of Mycobacterium tuberculosis and other mycobacteria, including Mycobacterium smegmatis. The protein MabA, also named FabG1, has been shown recently to be part of FAS-II and to catalyse the NADPH-specific reduction of long chain beta-ketoacyl derivatives. This activity corresponds to the second step of an FAS-II elongation round. FAS-II is inhibited by the antituberculous drug isoniazid through the inhibition of the 2-trans-enoyl-acyl carrier protein reductase InhA. Thus, the other enzymes making up this enzymatic complex represent potential targets for designing new antituberculous drugs. The crystal structure of the apo-form MabA was solved to 2.03 A resolution by molecular replacement. MabA is tetrameric and shares the conserved fold of the short-chain dehydrogenases/reductases (SDRs). However, it exhibits some significant local rearrangements of the active-site loops in the absence of a cofactor, particularly the beta5-alpha5 region carrying the unique tryptophan residue, in agreement with previous fluorescence spectroscopy data. A similar conformation has been observed in the beta-ketoacyl reductase from Escherichia coli and the distantly related dehydratase. The distinctive enzymatic and structural properties of MabA are discussed in view of its crystal structure and that of related enzymes.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

PubMed2002

Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase.

Bacterial UMP kinases do not exhibit any sequence homology with other nucleoside monophosphate kinases described so far, and appear under oligomeric forms, submitted to complex regulation by nucleotides. We propose here a structural model of UMP kinase from Escherichia coli based on the conservation of the fold of carbamate kinase whose crystal structure was recently solved. Despite sequence identity of only 18% over 203 amino acids, alignment of UMP kinase from E. coli with carbamate kinase from Enterococcus faecalis by hydrophobic cluster analysis and threading suggested the conservation of the overall structure, except for a small subdomain (absent in UMP kinase). The modelled dimer suggested conservation of the dimer interface observed in carbamate kinase while interaction of UMP kinase with a monoclonal antibody (Mab 44-2) suggests a three in-plane dimer subunit arrangement. The model was analyzed in light of various modified forms of UMP kinase obtained by site-directed mutagenesis.

Amino Acid Sequence↗

PubMed2002

Diacylglyceride kinases, sphingosine kinases and NAD kinases: distant relatives of 6-phosphofructokinases.

Diacylglyceride kinases, sphingosine kinases, NAD kinases and 6-phosphofructokinases are thought to be related despite large evolution of their sequences. Discovery of a common signature has led to the suggestion that they possess a similar phosphate-donor-binding site and a similar phosphorylation mechanism. The substrate- and allosteric-binding sites are much more divergent and their delineation remains to be determined experimentally.

Allosteric Site↗

PubMed2002

MabA (FabG1), a Mycobacterium tuberculosis protein involved in the long-chain fatty acid elongation system FAS-II.

The fatty acid elongation system FAS-II is involved in the biosynthesis of mycolic acids, which are very long-chain fatty acids of the cell envelope specific to Mycobacterium tuberculosis and other mycobacteria. A potential component of FAS-II, the protein MabA (FabG1), was overexpressed and purified. Sedimentation equilibrium analyses revealed that MabA undergoes a dimer to tetramer self-association with a dissociation constant of 22 microM. The protein was detected by Western blotting in a mycobacterial cell-wall extract that produces mycolic acids and in the FPLC FAS-II fraction. MabA was shown to catalyse the NADPH-specific reduction of beta-ketoacyl derivatives, equivalent to the second step of a FAS-II elongation round. Unlike the known homologous proteins, MabA preferentially metabolizes long-chain substrates (C(8)-C(20)) and has a poor affinity for the C(4) substrate, in agreement with FAS-II specificities. Molecular modelling of MabA structure suggested the presence of an unusually hydrophobic substrate-binding pocket holding a unique Trp residue, suitable for fluorescence spectroscopic analyses. In agreement with the enzyme kinetic data, the spectral properties of MabA were different in the presence of the C(8)-C(16) ligands as compared to the C(4) ligand. Altogether, these data bring out distinctive enzymic and structural properties of MabA, which correlate with its predilection for long-chain substrates, in contrast to most of the other known ketoacyl reductases.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

PubMed2002

Structural and nucleotide-binding properties of YajQ and YnaF, two Escherichia coli proteins of unknown function.

Structural genomics is a new approach in functional assignment of proteins identified via whole-genome sequencing programs. Its rationale is that nonhomologous proteins performing similar or related biological functions might have similar tertiary structure. We used dye pseudoaffinity chromatography, two-dimensional gel electrophoresis, and mass spectrometry to identify two novel Escherichia coli nucleotide-binding proteins, YnaF and YajQ. YnaF exhibited significant sequence identity with MJ0577, an ATP-binding protein from a hyperthermophile (Methanococcus jannaschii), and with UspA, a protein from Haemophilus influenzae that belongs to the Universal Stress Protein family. YnaF conserves the ATP-binding site and the dimeric structure observed in the crystal of MJ0577. The protein YajQ, present in many bacterial genomes, is missing in eukaryotes. In the absence of significant similarities of YajQ to any solved structure, we determined its structural and ligand-binding properties by NMR and isothermal titration calorimetry. We demonstrate that YajQ is composed of two domains, each centered on a beta-sheet, that are connected by two helical segments. NMR studies, corroborated with local sequence conservation among YajQ homologs in various bacteria, indicate that one of the beta-sheets is mostly involved in biological activity.

Adenosine Triphosphate↗