An oligonucleotide microarray for the monitoring of repair enzyme activity toward different DNA base damage

Characterization of DNA-N-glycosylase activities in cell extract is a challenging problem and could represent a major concern for medical applications. Synthetic oligonucleotides which contain base lesions located on specific sites constitute suitable substrates for their study. An in vitro miniaturized assay was developed that allows the measurement of cleavage activities of DNA repair enzymes on a set of oligonucleotides (ODNs) that contained different lesions. The modified ODNs were indirectly hybridized onto probes chemically fixed at defined sites on a circular format within each well of a 96-well microtiter plate (Oligo Sorbent Array, OLISA). The lesions were selected among oxidative damage (8-oxo-7,8-dihydroguanine, formylamine), deaminated bases (uracil, hypoxanthine) and alkylated base (N(6)-etheno-adenine). Cleavage specificity was checked using different enzymes: Fapy-DNA-N-glycosylase, 3-methyladenine DNA glycosylase II, uracil-N-glycosylase, endonuclease V and endonuclease VIII. The extent of excision could be monitored simultaneously for the selected base damage. For this purpose, we used automated fluorescence imaging analysis of the residual ODNs that contained lesions and remained on the support after release of the cleaved ODNs recognized by the repair enzymes. The results indicated that this assay could advantageously replace the analysis of glycosylase activities by PAGE techniques. Finally we show that this in vitro repair assay represents an interesting tool for the determination of cellular repair activities.     

IGF-II is up-regulated and myofibres are hypertrophied in regenerating soleus of mice lacking FGF6

Important functions in myogenesis have been proposed for FGF6, a member of the fibroblast growth factor family accumulating almost exclusively in the myogenic lineage. However, the use of FGF6(-/-) mutant mice gave contradictory results and the role of FGF6 during myogenesis remains largely unclear. Using FGF6(-/-) mice, we first analysed the morphology of the regenerated soleus following cardiotoxin injection and showed hypertrophied myofibres in soleus of the mutant mice as compared to wild-type mice. Secondly, to examine the function of the IGF family in the hypertrophy process, we used semiquantitative and real-time RT-PCR assays and Western blots to monitor the expression of the insulin-like growth factors (IGF-I and IGF-II), their receptors [type I IGF receptor (IGF1R) and IGF-II receptor (IGF2R)], and of a binding protein IGFBP-5 in regenerating soleus muscles of FGF6(-/-) knockout mice vs. wild-type mice. In the mutant, both IGF-II and IGF2R, but not IGF-I and IGF1R, were strongly up-regulated, whereas IGFBP5 was down-regulated, strongly suggesting that, in the absence of FGF6, the mechanisms leading to myofibre hypertrophy were mediated specifically by an IGF-II/IGF2R signalling pathway distinct from the classic mechanism involving IGF-I and IGF1R previously described for skeletal muscle hypertrophy. The potential regulating role of IGFBP5 on IGF-II expression is also discussed. This report shows for the first time a specific role for FGF6 in the regulation of myofibre size during a process of in vivo myogenesis.     

Cell-type specific modification of PII is involved in the regulation of nitrogen metabolism in the cyanobacterium Anabaena PCC 7120

In the heterocystous cyanobacterium Anabaena PCC 7120, the modification state of the signalling PII protein is regulated according to the nitrogen regime of the cells, as already observed in some unicellular cyanobacteria. However, during the adaptation to diazotrophic growth conditions, PII is phosphorylated in vegetative cells while unphosphorylated in heterocysts. Isolation of mutants affected on PII modification state and analysis of their phenotypes allow us to show the implication of PII in the regulation of molecular nitrogen assimilation and more specifically, the requirement of unmodified state of PII in the formation of polar nodules of cyanophycin in heterocysts.     

The PhoP-PhoQ two-component regulatory system of Photorhabdus luminescens is essential for virulence in insects

Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system (phoP) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection of a large number of the phoP bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the PhoPQ system mediated Mg(2+)-dependent modifications in lipopolysaccharides and controlled a locus (pbgPE) required for incorporation of 4-aminoarabinose into lipid A. Mg(2+)-regulated gene expression of pbgP1 was absent in the mutant and was restored when phoPQ was complemented in trans. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.     

Exocytotic insertion of TRPC6 channel into the plasma membrane upon Gq protein-coupled receptor activation

TRPC proteins are the mammalian homologues of the Drosophila transient receptor potential channel and are involved in calcium entry after agonist stimulation of non-excitable cells. Seven mammalian TRPCs have been cloned, and their mechanisms of activation and regulation are still the subject of intense research. TRPC proteins interact with the inositol 1,4,5-trisphosphate receptor, and the conformational coupling plays a critical role in the activation of calcium entry. Some evidence also supports an exocytotic mechanism as part of the activation of calcium entry. To investigate the possible involvement of exocytosis in TRPC6 activation, we evaluated the location of TRPC6 at the plasma membrane by biotinylation labeling of cell surface proteins and by indirect immunofluorescence marking of TRPC6 in stably transfected HEK 293 cells. We showed that when the muscarinic receptor was stimulated or the thapsigargin-induced intracellular calcium pool was depleted the level of TRPC6 at the plasma membrane increased. The carbachol concentration at which TRPC6 externalization occurred was lower than the concentration required to activate TRPC6. Externalization occurred within the first 30 s of stimulation, and TRPC6 remained at the plasma membrane as long as the stimulus was present. These results indicate that an exocytotic mechanism is involved in the activation of TRPC6.     

Directed mutagenesis in region 713-720 of human thyroperoxidase assigns 713KFPED717 residues as being involved in the B domain of the discontinuous immunodominant region recognized by human autoantibodies

Autoantibodies (aAbs) to thyroid peroxidase (TPO), the hallmark of autoimmune thyroid disease (AITD), recognize conformational epitopes restricted to an immunodominant region (IDR), divided into two overlapping domains A and B. Despite numerous efforts aimed at localizing the IDR and identifying aAb-interacting residues on TPO, only two critical amino acids, Lys(713) and Tyr(772), have been characterized. Precise and complete delineation of the other residues involved in the IDR remains to be defined. By using a recombinant anti-TPO aAb T13, we demonstrated that four regions on TPO are part of the IDR/B; one of them, located between amino acids 713 and 720, is particularly important for the binding of sera from patients suffering from AITD. To precisely define critical residues implicated in the binding of aAb to human TPO, we used directed mutagenesis and expressed the mutants in stably transfected CHO cells. Then we assessed the kinetic parameters involved in the interactions between anti-TPO aAbs and mutants by real-time analysis. We identified (i) the minimal epitope 713-717 recognized by mAb 47 (a reference antibody) and (ii) the amino acids used as contact points for two IDR-specific human monoclonal aAbs TR1.9 (Pro(715) and Asp(717)) and T13 (Lys(713), Phe(714), Pro(715), and Glu(716)). Using a rational strategy to identify complex epitopes on proteins showing a highly convoluted architecture, this study definitively identifies the amino acids Lys(713)-Asp(717) as being the key residues recognized by IDR/B-specific anti-TPO aAbs in AITD.     

The Vif protein of human immunodeficiency virus type 1 is posttranslationally modified by ubiquitin

The viral infectivity factor (Vif), one of the six HIV-1 auxiliary genes, is absolutely necessary for productive infection in primary CD4-positive T lymphocytes and macrophages. Vif overcomes the antiviral function of the host factor APOBEC3G. To better understand this mechanism, it is of interest to characterize cellular proteins that interact with Vif and may regulate its function. Here, we show that Vif binds to hNedd4 and AIP4, two HECT E3 ubiquitin ligases. WW domains present in those HECT enzymes contribute to the binding of Vif. Moreover, the region of Vif, which includes amino acids 20-128 and interacts with the hNedd4 WW domains, does not contain proline-rich stretches. Lastly, we show that Vif undergoes post-translational modifications by addition of ubiquitin both in cells overexpressing Vif and in cells expressing HIV-1 provirus. Vif is mainly mono-ubiquitinated, a modification known to address the Gag precursor to the virus budding site.