François Traincard

Stress-activated protein kinases are negatively regulated by cell density.

Stimulation by UV irradiation, TNFα, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts. This results in the phosphorylation of the N‐terminal domain of c‐Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c‐Jun phosphorylated at Ser63, we show that culture confluency drastically inhibited c‐Jun N‐terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G‐proteins cdc42 and Rac1. In contrast, the classical MAPK pathway was insensitive to confluency. The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c‐Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress‐dependent signal cascade by confluency will restrict c‐Jun N‐terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area.

NEMO oligomerization in the dynamic assembly of the IκB kinase core complex

NF‐κB essential modulator (NEMO) plays an essential role in the nuclear factor κB (NF‐κB) pathway as a modulator of the two other subunits of the IκB kinase (IKK) complex, i.e. the protein kinases, IKKα and IKKβ. Previous reports all envision the IKK complex to be a static entity. Using glycerol‐gradient ultracentrifugation, we observed stimulus‐dependent dynamic IKK complex assembly. In wild‐type fibroblasts, the kinases and a portion of cellular NEMO associate in a 350‐kDa high‐molecular‐mass complex. In response to constitutive NF‐κB stimulation by Tax, we observed NEMO recruitment and oligomerization to a shifted high‐molecular‐mass complex of 440 kDa which displayed increased IKK activity. This stimulus‐dependent oligomerization of NEMO was also observed using fluorescence resonance energy transfer after a transient pulse with interleukin‐1β. In addition, fully activated, dimeric kinases not bound to NEMO were detected in these Tax‐activated fibroblasts. By glycerol gradient ultracentrifugation, we also showed that: (a) in fibroblasts deficient in IKKα and IKKβ, NEMO predominantly exists as a monomer; (b) in NEMO‐deficient fibroblasts, IKKβ dimers are present that are less stable than IKKα dimers. Intriguingly, in resting Rat‐1 fibroblasts, 160‐kDa IKKα–NEMO and IKKβ–NEMO heterocomplexes were observed as well as a significant proportion of NEMO monomer. These results suggest that most NEMO molecules do not form a tripartite IKK complex with an IKKα–IKKβ heterodimer as previously reported in the literature but, instead, NEMO is able to form a complex with the monomeric forms of IKKα and IKKβ.

The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein

Summary Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa—animals, plants, and protists (including important human pathogens like Plasmodium)—suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.

A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes

T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI5–24 and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

Une technique immunoenzymatique pour la mise en évidence de l'hybridation moléculaire entre acides nucléiques

Summary An immunoenzymatic procedure has been developed based on the use of monoclonal antibodies specific for 5-bromodeoxyuridine (BdUr). It allows the detection of BdUr-labelled DNA immobilized on a nitrocellulose filter. Using this procedure, it was possible to detect up to 0.5 pg of mammalian DNA labelled in vivo with BdUr, 5 pg of nick-translated BdUr-labelled PBR-322 and, using this latter probe and dot-blot hybridization, 50 pg of native unlabelled PBR-322.

5-Bromodeoxyuridine in vivo labelling of M13 DNA, and its use as a non-radioactive probe for hybridization experiments.

We describe the in vivo production of 5-bromodeoxyuridine- (5-BUdR) labelled M13 DNA by a thymine-requiring Escherichia coli strain. We show that the 5-BUdR-labelled M13 single-stranded DNA is not extruded into the culture medium, but accumulates inside the bacterial cells. On the basis of this observation, a procedure involving FPLC gel filtration already reported and used for the isolation of plasmid DNA has been adapted for the isolation of at least 90% pure 5-BUdR-labelled single-stranded DNA. An M13 probe, containing part of the Hepatitis B Virus (HBV) genome was constructed, and the corresponding 5-BUdR-labelled single-stranded DNA was used in hybridization experiments to detect homologous HBV target DNA. Picogram amounts (10(-19) moles) of the probe itself or the target DNA could be detected, by monoclonal anti-5-BUdR antibodies in an immunoenzymatic assay.

A set of polyclonal and monoclonal antibodies reveals major differences in post-translational modification of the rat HNF1 and vHNF1 homeoproteins

The related homeodomain-containing transcription factors HNF1 (HNF1 alpha) and vHNF1 (HNF1 beta) recognise common target DNA sequences in the regulatory regions of many genes and are expressed in several parenchymal cell types, predominantly in liver, kidney, intestine and pancreas. HNF1-null mutant mice, with a wild-type vHNF1 gene, develop normally, but die within a few weeks of birth with severe liver and kidney failure. Humans with a mutation in the HNF1 alpha gene develop non-insulin dependent diabetes on maturity (MODY 3). To determine distinctive roles for each of these proteins we produced a set of polyclonal sera and monoclonal antibodies, directed against different parts of the rat HNF1 and vHNF1 proteins. These antibodies reveal that HNF1 is present in vivo as a heterogeneous mixture of 92-98 kDa molecular mass polypeptides, a mass higher than that expected from its amino acid sequence. vHNF1 is present in the form of two isoforms of roughly the expected molecular masses, 65 and 68 kDa. In addition, some antibodies prepared against bacterially-produced HNF1 recognise vHNF1 but not HNF1, in liver and kidney extracts. Hence, we present the first evidence for differential post-translational modification of HNF1 and vHNF1 proteins.

Inhibition of potato virus Y NIa activity: preparation of monoclonal antibody directed against PVY NI protein that inhibits cleavage of PVY polyprotein.

Summary. A partially purified nuclear inclusion (NI) fraction was obtained from tobacco plants infected by potato virus Y (PVY). Four monoclonal antibodies (MAbs) were produced and characterized using this semipurified fraction as antigen. Data showed that only one was directed against NIa whereas two were directed against cytoplasmic inclusion (CI) protein and the last one against coat protein (CP). These results were due to the fact that the semipurified NI fraction was usually contaminated with CI and CP proteins. When used on in situ immunofluorescence method the anti-NIa MAb showed accumulation of the NIa protein in both nucleus and cytoplasm. In vivo, this MAb was able to detect different forms of the NIa protein including precursors and cleavage products. It was also able to inhibit the cleavage of the polyprotein detected in the semipurified NI.

FUNCTIONAL ANALYSIS OF THE CATALYTIC SUBUNIT OF DICTYOSTELIUM PKA IN VIVO

The catalytic subunit of the cAMP-dependent protein kinase (PKA) from Dictyostelium discoideum contains several domains, including an unusually long N-terminal extension preceding a highly conserved catalytic core. We transformed the aggregationless PkaC-null strain with several deletion constructs of both domains. Strains transformed with genes expressing catalytically-inactive polypeptides could not rescue development. Cotransformation with constructs encoding the N-terminal extension and the catalytic core, both unable to rescue development by themselves, yielded transformants able to proceed to late development. A 27-amino acid long hydrophobic region, immediately upstream of the catalytic core, was found indispensable for PKA function. A putative role of this sequence in the acquisition of the active conformation of the protein is discussed.

Monoclonal anti-nucleoside antibodies: Characterization and application in an enzyme immunoassay of single-stranded DNA

Two mouse monoclonal antibodies were raised against adenosine and guanosine coupled to bovine serum albumin (BSA) by periodate oxidation. They were named A-16 and G-K21 respectively and selected for their ability to recognize single-stranded DNA. Their epitope specificities were assessed and their dissociation constants determined by an indirect ELISA method. The KD values for adenosine and guanosine coupled to BSA were 9.9 X 10(-7) M and 1.1 X 10(-10) M for G-K21 respectively, and 2.5 X 10(-8) M and 1.0 X 10(-6) M for A-16. These monoclonal anti-nucleoside antibodies were used to develop a sandwich enzyme immunoassay for single-stranded DNA. The purified IgG antibodies were coupled to beta-galactosidase and alkaline phosphatase by the one-step glutaraldehyde method and used in a test optimized for pH, saturating proteins, coating antibody, nature of the conjugate and protein concentrations. Less than 100 pg/well of single-stranded DNA could be detected, and the detection was linear over a DNA concentration ranging from 0.34 to 34 ng/ml. The assay could quantitate single-stranded DNAs of differing origin, but not RNAs. The test was compared to another titration method, and used to calibrate target DNA amounts in non-radioactive hybridization experiments.