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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., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The emergence of Neisseria meningitidis serogroup X (NmX) in the African meningitis belt has urged the development of diagnostic tools and vaccines for this serogroup, especially following the introduction of a conjugate vaccine against N. meningitidis serogroup A (NmA). We have developed and evaluated a new rapid diagnostic test (RDT) for detecting the capsular polysaccharide (cps) antigen of this emerging serogroup. Whole inactivated NmX bacteria were used to immunize rabbits. Following purification by affinity chromatography, the cpsX-specific IgG antibodies were utilized to develop an NmX-specific immunochromatography dipstick RDT. The test was validated against purified cpsX and meningococcal strains of different serogroups. Its performance was evaluated against that of PCR on a collection of 369 cerebrospinal fluid (CSF) samples obtained from patients living in countries within the meningitis belt (Cameroon, Côte d'Ivoire, and Niger) or in France. The RDT was highly specific for NmX strains. Cutoffs of 10(5) CFU/ml and 1 ng/ml were observed for the reference NmX strain and purified cpsX, respectively. Sensitivity and specificity were 100% and 94%, respectively. A high agreement between PCR and RDT (Kappa coefficient, 0.98) was observed. The RDT gave a high positive likelihood ratio and a low negative likelihood (0.07), indicating almost 100% probability of declaring disease or not when the test is positive or negative, respectively. This unique NmX-specific test could be added to the available set of RDT for the detection of meningococcal meningitis in Africa as a major tool to reinforce epidemiological surveillance after the introduction of the NmA conjugate vaccine., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Hypomorphic mutations in the X-linked human NEMO gene result in various forms of anhidrotic ectodermal dysplasia with immunodeficiency. NEMO function is mediated by two distal ubiquitin binding domains located in the regulatory C-terminal domain of the protein: the coiled-coil 2-leucine zipper (CC2-LZ) domain and the zinc finger (ZF) domain. Here, we investigated the effect of the D406V mutation found in the NEMO ZF of an ectodermal dysplasia with immunodeficiency patients. This point mutation does not impair the folding of NEMO ZF or mono-ubiquitin binding but is sufficient to alter NEMO function, as NEMO-deficient fibroblasts and Jurkat T lymphocytes reconstituted with full-length D406V NEMO lead to partial and strong defects in NF-κB activation, respectively. To further characterize the ubiquitin binding properties of NEMO ZF, we employed di-ubiquitin (di-Ub) chains composed of several different linkages (Lys-48, Lys-63, and linear (Met-1-linked)). We showed that the pathogenic mutation preferentially impairs the interaction with Lys-63 and Met-1-linked di-Ub, which correlates with its ubiquitin binding defect in vivo. Furthermore, sedimentation velocity and gel filtration showed that NEMO ZF, like other NEMO related-ZFs, binds mono-Ub and di-Ub with distinct stoichiometries, indicating the presence of a new Ub site within the NEMO ZF. Extensive mutagenesis was then performed on NEMO ZF and characterization of mutants allowed the proposal of a structural model of NEMO ZF in interaction with a Lys-63 di-Ub chain.

NEMO (NF-kappaB essential modulator) is a regulatory protein essential to the canonical NF-kappaB signaling pathway, notably involved in immune and inflammatory responses, apoptosis, and oncogenesis. Here, we report that the zinc finger (ZF) motif, located in the regulatory C-terminal half of NEMO, forms a specific complex with ubiquitin. We have investigated the NEMO ZF-ubiquitin interaction and proposed a structural model of the complex based on NMR, fluorescence, and mutagenesis data and on the sequence homology with the polymerase eta ubiquitin-binding zinc finger involved in DNA repair. Functional complementation assays and in vivo pull-down experiments further show that ZF residues involved in ubiquitin binding are functionally important and required for NF-kappaB signaling in response to tumor necrosis factor-alpha. Thus, our findings indicate that NEMOZFisa bona fide ubiquitin-binding domain of the ubiquitin-binding zinc finger type.

NF-kappaB essential modulator (NEMO) plays an essential role in the nuclear factor kappaB (NF-kappaB) pathway as a modulator of the two other subunits of the IkappaB kinase (IKK) complex, i.e. the protein kinases, IKKalpha and IKKbeta. 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-kappaB 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-1beta. 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 IKKalpha and IKKbeta, NEMO predominantly exists as a monomer; (b) in NEMO-deficient fibroblasts, IKKbeta dimers are present that are less stable than IKKalpha dimers. Intriguingly, in resting Rat-1 fibroblasts, 160-kDa IKKalpha-NEMO and IKKbeta-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 IKKalpha-IKKbeta heterodimer as previously reported in the literature but, instead, NEMO is able to form a complex with the monomeric forms of IKKalpha and IKKbeta.

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 PKI(5-24) 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.

NF-kappa B essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappa B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappa B activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappa B activation was quantified using an NF-kappa B-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappa B activation with an IC(50) in the mum range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappa B activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappa B pathway activation through targeting the oligomerization state of the NEMO protein.

The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.

NEMO (NF-kappaB essential modulator) plays a key role in the canonical NF-kappaB pathway as the scaffold/regulatory component of the IkappaB kinase (IKK) complex. The self-association of NEMO involves the C-terminal halves of the polypeptide chains containing two putative coiled-coil motifs (a CC2 and a LZ leucine zipper), a proline-rich region, and a ZF zinc finger motif. Using purified truncation mutants, we showed that the minimal oligomerization domain of NEMO is the CC2-LZ segment and that both CC2 and LZ subdomains are necessary to restore the LPS-dependent activation of the NF-kappaB pathway in a NEMO-deficient cell line. We confirmed the association of the oligomerization domain in a trimer and investigated the specific role of CC2 and LZ subdomains in the building of the oligomer. Whereas a recombinant CC2-LZ polypeptide self-associated into a trimer with an association constant close to that of the wild-type protein, the isolated CC2 and LZ peptides, respectively, formed trimers and dimers with weaker association constants. Upon mixing, isolated CC2 and LZ peptides associated to form a stable hetero-hexamer as shown by gel filtration and fluorescence anisotropy experiments. We propose a structural model for the organization of the oligomerization domain of activated NEMO in which three C-terminal domains associate into a pseudo-hexamer forming a six-helix bundle. This model is discussed in relation to the mechanism of activation of the IKK complex by upstream activators.

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.

Stimulation by UV irradiation, TNFalpha, 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.

The C subunit of Dictyostelium cAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI. Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence-comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.

Localization of virion-associated protein x (Vpx) of SIVmac 251 was studied in lymph nodes and liver of six SIVmac-infected monkeys. Vpx was found associated with the network of follicular dendritic cells and macrophages in lymph nodes and/or livers from five out of six animals by immunohistochemistry. Although the humoral response to Vpx occurs in only 50% of the animals, the presence of Vpx in target cell or antibodies to Vpx in all the monkeys studied, suggests that Vpx may be necessary for viral replication in vivo.

Twelve murine monoclonal antibodies (MAbs) to human immunodeficiency virus type 2 (isolate ROD) envelope glycoproteins have been generated and characterized. Nine MAbs were specific to the external gp125 and three reacted with the transmembrane gp36. A large majority of MAbs displayed a significant affinity for the native gp140 precursor and were shown to bind to viral antigens on the surface of fixed HIV-2-infected cells. In Western blot analysis, the 12 MAbs showed varying profiles of cross-reactivity, but none of the MAbs cross-reacted with the HIV-1LAI envelope. Six MAbs reacted exclusively with the homologous HIV-2ROD isolate whereas only two MAbs displayed cross-reactivity with HIV-2ROD, HIV-2EHO, and SIVmac251. The four other MAbs cross-reacted with either HIV-2EHO or SIVmac251. Results of competitive binding assays indicated that the three anti-gp36 MAbs shared the same competition group, whereas at least eight competition groups were defined with the nine anti-gp125 MAbs. The epitopes of the three anti-gp36 and four anti-gp125 MAbs have been delineated using synthetic peptides or by immunological screening of an SIVmac251 peptide library expressed in yeast. The anti-gp36 MAbs are directed against the same domain of the transmembrane gp36 corresponding to the major antigenic determinant of HIV-2 and HIV-1. The four anti-gp125 MAbs recognize four distinct epitopes localized in the V2, V3, and C1 domains. None of the 12 MAbs displayed neutralizing activity against HIV-2ROD, including the 2 MAbs directed against the V2 and V3 domains.(ABSTRACT TRUNCATED AT 250 WORDS)

The antigenicity of HIV-gag p17 and p25 proteins was analyzed using a panel of 52 monoclonal antibodies (mAb) derived from 17 independent fusion experiment protocols performed in 12 different laboratories. These mAb were tested for their capacity to bind peptides corresponding to sequences of HIV1-BRU-gag p17 and p25. Thirty-five overlapping peptides (P1 to P35) totally covering the p17 and p25 proteins were used. This study allowed us to identify four immunodominant regions inducing B cell response, two on p17 corresponding to P2 and P13 (amino acids 11-25 and 121-132, respectively) and two on p25 corresponding to P21 and P28-P29-P30 (a.a. 201-218 and 285-320 respectively). According to secondary structure predictions, peptides P2 and P21 contained hydrophilic alpha helix folded regions whereas P13 sequence presented a beta turn propensity. These regions and the P28-30 region were also predicted to be easily accessible to mAb. Several other p25-derived peptides: P15 (a.a. 142-156), P16 (a.a. 148-162), P19 (a.a. 176-192), P22 (a.a. 219-233) and P23 (a.a. 233-253) were recognized by mAb. No p17-derived peptide other than P2, P13 and P12 (a.a. 111-123) was found to react with mAb. Cross-blocking studies between mAb, suggested the existence of more than four distinct epitopic areas on p17 and eight on p25.

Two murine monoclonal antibodies (MAbs), designated MATG2014 and MATG2033, were generated. They are reactive with the external envelope glycoprotein gp130 of the simian immunodeficiency virus of macaque monkey (SIVmac251), and display a cell-free virus neutralizing activity in vitro. In addition, MATG2014 cross-reacts with HIV-2Rod gp140. Epitope mapping of these MAbs was performed by screening and SIVmac peptide library expressed in yeast and confirmed using synthetic peptides. MATG2014 and MATG2033 recognize two overlapping epitopes localized in an 18 residue domain between amino acid 171 and 188 of the SIVmac251 gp130. Sera from experimentally SIV-infected macaques are immunoreactive with this neutralizing domain. Sequence comparison with related SIV and HIV-2 viral strains indicates a low variability of this region, consistent with the cross-reactivity of MATG2014 with HIV-2Rod gp140. This domain should then be considered in designing experimental vaccines.

A cDNA clone corresponding to the gp41 gene fragment nucl. 7573-7730 of the human immunodeficiency virus type 1 (HIV-1) was selected from a random HIV-1 genomic library expressed in yeast. This clone encodes a 52-residue long peptide (amino acid (a.a.)) 591-642) bearing the major immunodominant domain (a.a. 598-609) of the HIV-1 transmembrane glycoprotein gp41. Expression of the recombinant peptide pSE-env591-642 was driven by the alpha-mating factor leader sequence contained in a plasmid pSE-x allowing the synthesis and secretion of foreign gene product in Saccharomyces cerevisiae. Time-course analysis of the secretion into culture medium revealed an optimal production of the glycoprotein fragment at 28-30 h with no observable cytotoxicity. The secreted peptide is highly glycosylated with NH2-terminal heterogeneity probably due to different post-translational modifications. The secreted peptide shows an extreme antigenicity since in ELISA assays, as few as 5 microliters/well of crude supernatant are sufficient to obtain a strong detection by monoclonal antibodies or by 100% of sera from HIV-infected individuals. The purified glycopeptide pSE-env591-642 binds to a monoclonal antibody directed against the immunodominant epitope (a.a. 603-609) with an affinity similar to that of the complete glycoprotein gp160 (Kd values within the 10(-10) M range) and with a 100-fold higher affinity than that of a linear peptide fragment SP-env584-609. These results indicate that overexpression in yeast can efficiently provide an abundant source of highly antigenic gp41 protein fragment pSE-env591-642 which retains the antigenic properties of the native gp160 protein. Such a recombinant peptide should therefore be considered as a good candidate for antigen in HIV detection tests.

Two anti-nucleoside monoclonal antibodies (A-16 and G-K21) were raised after immunizing mice with adenosine or guanosine coupled to bovine serum albumin by periodate oxidation. They were selected for their ability to detect these immunogens and single-stranded DNA in an enzyme-linked immunosorbent assay test. The antibodies were purified from ascitic fluids, their isotypes were determined and their ability to detect DNAs and RNAs on nitrocellulose membranes was tested. They belonged to the IgG1 subclass and were both able to recognize picogram amounts of single-stranded DNAs on nitrocellulose sheets, whatever the origin of the nucleic acid, but were unable to detect RNA efficiently. The same monoclonal antibodies were used to estimate minute amounts of target staphylococci DNAs to permit standardization of non-radioactive hybridization experiments for detection of antibiotic resistance genes.

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.

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.

The importance of cell culture contamination by mycoplasmas is well recognized, but the means used to detect such contamination still need improvement. Most mycoplasmas possess an enzyme, adenosine phosphorylase, which is not found in cell lines. We used the ultramicromethod of Uitendaal et al. to detect the presence of mycoplasmas in sera and in tissue culture medium. The absence of adenosine phosphorylase activity seems to be the best guarantee that a serum is not contaminated by mycoplasmas. This test is also most efficient for the detection of mycoplasmas in tissue or cell cultures in vitro.