Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis.

Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.

Sex differences in nuclear receptor-regulated liver metabolic pathways.

Liver metabolism is markedly sex-dimorphic; accordingly, the prevalence of liver diseases is different between sexes. The superfamily of nuclear receptors (NRs) governs the proper expression of key liver metabolism genes by sensing lipid-soluble hormones and dietary lipids. When the expression of those genes is deregulated, disease development is favored. However, we lack a comprehensive picture of the differences between NR actions in males and females. Here, we reviewed explorative studies that assessed NR functions in both sexes, and we propose a first map of sex-dimorphic NR expression in the liver. Our analysis suggested that NRs in the female liver exhibited cross-talk with more liver-protective potential than NRs in male liver. This study provides empirical support to the hypothesis that women are more resilient to some liver diseases than men, based on a more compensative NR network. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Quantifying degradation rates of transmembrane receptor kinases.

Transmembrane receptor-kinases are widespread throughout eukaryotes and their activities are known to regulate all kinds of cellular responses in diverse organs and cell types. In order to guarantee the correct amplitude and duration of signals, receptor levels at the cellular surface need to be tightly controlled. The regulation of receptor degradation is the most direct way to achieve this and elaborate mechanisms are in place to control this process. Therefore, the rate of receptor degradation is a parameter of central importance for understanding the dynamics of a signal transduction cascade. Unfortunately, degradation of transmembrane receptors is a complicated multistep process that involves internalization from the plasma membrane, invagination into the lumen of endosomal compartments, and finally fusion with the vacuole for degradation by vacuolar proteases. Therefore, degradation should be measured in an as noninvasive way as possible, such as not to interfere with the complicated transport processes. Here, a method for minimally invasive, in vivo turn-over measurements in intact organs is provided. This technique was used for quantifying the turn-over rates of the Brassinosteroid receptor kinase BRI1 (BRASSINOSTEROID INSENSITIVE 1) in Arabidopsis thaliana root meristems. Pulse-chase expression of a fluorescently labeled BRI1 variant was used and its turn-over rate was determined by quantitative confocal microscopy. This method is well suited to measure turn-over of transmembrane kinases, but can evidently be extended to measure turn-over of any types of transmembrane proteins.

Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the alpha 1B-adrenergic receptor.

The alpha 1B-adrenergic receptor (alpha 1BAR) and its truncated mutant T368 lacking the last 147 amino acids were stably expressed in Rat1 fibroblasts. The wild type alpha 1BAR was rapidly phosphorylated upon exposure to the agonist epinephrine as well as to phorbol ester as assessed by immunoprecipitation of the receptor with antiserum raised against its amino-terminal portion. Exposure of cells expressing the wild type alpha 1BAR to epinephrine resulted also in rapid homologous desensitization of receptor-mediated response on polyphosphoinositide hydrolysis. On the other hand, truncation of the serine- and threonine-rich carboxyl portion of the alpha 1BAR abolished agonist-induced phosphorylation and greatly impaired homologous desensitization of the receptor. The truncated receptor T368 could undergo agonist-induced decrease of cell surface receptors but to a lesser extent, as compared with the wild type alpha 1BAR. These results demonstrate that the carboxyl portion of the alpha 1BAR plays a crucial role in the regulation of receptor function. They also suggest a strong relationship between agonist-induced phosphorylation and desensitization of the alpha 1BAR, which were both insensitive to the inhibitor of protein kinase C RO-318220. Our findings support the emerging hypothesis that the biochemical mechanisms involved in rapid agonist-dependent regulation of G protein-coupled receptors, which activate polyphosphoinositide hydrolysis, do not primarily involve protein kinase C.

Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase.

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.

Oncogenic Ras inhibits Fas ligand-mediated apoptosis by downregulating the expression of Fas.

Tumor growth is the result of deregulated tissue homeostasis which is maintained through the delicate balance of cell growth and apoptosis. One of the most efficient inducers of apoptosis is the death receptor Fas. We report here that oncogenic Ras (H-Ras) downregulates Fas expression and renders cells of fibroblastic and epitheloid origin resistant to Fas ligand-induced apoptosis. In Ras-transformed cells, Fas mRNA is absent. Inhibition of DNA methylation restores Fas expression. H-Ras signals via the PI 3-kinase pathway to downregulate Fas, suggesting that the known anti-apoptotic effect of the downstream PKB/Akt kinase may be mediated, at least in part, by the repression of Fas expression. Thus, the oncogenic potential of H-ras may reside on its capacity not only to promote cellular proliferation, but also to simultaneously inhibit Fas-triggered apoptosis.

The beta-catenin--TCF-1 pathway ensures CD4(+)CD8(+) thymocyte survival.

The association of trans-acting T cell factors (TCFs) or lymphoid enhancer factor 1 (LEF-1) with their coactivator beta-catenin mediates transient transcriptional responses to extracellular Wnt signals. We show here that T cell maturation depends on the presence of the beta-catenin--binding domain in TCF-1. This domain is necessary to mediate the survival of immature CD4(+)CD8(+) double-positive (DP) thymocytes. Accelerated spontaneous thymocyte death in the absence of TCF-1 correlates with aberrantly low expression of the anti-apoptotic protein Bcl-x(L). Increasing anti-apoptotic effectors in thymocytes by the use of a Bcl-2 transgene rescued TCF-1-deficient DP thymocytes from apoptosis. Thus, TCF-1, upon association with beta-catenin, transiently ensures the survival of immature T cells, which enables them to generate and edit T cell receptor (TCR) alpha chains and attempt TCR-mediated positive selection.

Immune response to mouse mammary tumor virus in mice lacking the alpha/beta interferon or the gamma interferon receptor.

Mouse mammary tumor virus (MMTV) is a retrovirus which induces a strong immune response and a dramatic increase in the number of infected cells through the expression of a superantigen (SAg). Many cytokines are likely to be involved in the interaction between MMTV and the immune system. In particular, alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) exert many antiviral and immunomodulatory activities and play a critical role in other viral infections. In this study, we have investigated the importance of interferons during MMTV infection by using mice with a disrupted IFN-alpha/beta or IFN-gamma receptor gene. We found that the SAg response to MMTV was not modified in IFN-alpha/betaR(0/0) and IFN-gammaR(0/0) mice. This was true both for the early expansion of B and T cells induced by the SAg and for the deletion of SAg-reactive cells at later stages of the infection. In addition, no increase in the amount of proviral DNA was detected in tissues of IFN-alpha/betaR(0/0) and IFN-gammaR(0/0) mice, suggesting that interferons are not essential antiviral defense mechanisms during MMTV infection. In contrast, IFN-gammaR(0/0) mice had increased amounts of IL-4 mRNA and an altered usage of immunoglobulin isotypes with a reduced frequency of IgG2a- and IgG3-producing cells. This was associated with lower titers of virus-specific antibodies in serum early after infection, although efficient titers were reached later.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

The yellow fever 17D vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines

The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members being present in most continents. Among the most important are yellow fever (YF), dengue with its four serotypes and Japanese encephalitis virus. A live attenuated virus is used as a cost effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The rise of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. One new approach is the use of cDNAs encopassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated viruses. The use of infectious cDNA as a carrier for heterologous antigens is gaining importance as chimeric viruses are shown to be viable, immunogenic and less virulent as compared to the parental viruses. The use of DNA to overcome mutation rates intrinsic of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost for production of live attenuated vaccines. The YF virus despite a long period ignored by researchers probably due to the effectiveness of the vaccine has made a come back, both in nature as human populations grow and reach endemic areas as well as in the laboratory being a suitable model to understand the biology of flaviviruses in general and providing new alternatives for vaccine development through the use of the 17D vaccine strain.

D-JNKi, a peptide inhibitor of c-Jun N-terminal kinase, promotes functional recovery after transient focal cerebral ischemia in rats

The c-Jun-N-terminal kinase (JNK) pathway has been shown to play an important role in excitotoxic neuronal death and several studies have demonstrated a neuroprotective effect of D-JNKi, a peptide inhibitor of JNK, in various models of cerebral ischemia. We have now investigated the effect of D-JNKi in a model of transient focal cerebral ischemia (90 min) induced by middle cerebral artery occlusion (MCAo) in adult male rats. D-JNKi (0.1 mg/kg), significantly decreased the volume of infarct, 3 days after cerebral ischemia. Sensorimotor and cognitive deficits were then evaluated over a period of 6 or 10 days after ischemia and infarct volumes were measured after behavioral testing. In behavioral studies, D-JNKi improved the general state of the animals as demonstrated by the attenuation of body weight loss and improvement in neurological score, as compared with animals receiving the vehicle. Moreover, D-JNKi decreased sensorimotor deficits in the adhesive removal test and improved cognitive function in the object recognition test. In contrast, D-JNKi did not significantly affect the infarct volume at day 6 and at day 10. This study shows that D-JNKi can improve functional recovery after transient focal cerebral ischemia in the rat and therefore supports the use of this molecule as a potential therapy for stroke.

High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

A theoretical model of the tridimensional structure of Bacillus thuringiensis subsp. medellin Cry 11Bb toxin deduced by homology modelling

Cry11Bb is an insecticidal crystal protein produced by Bacillus thuringiensis subsp. medellin during its stationary phase; this ¶-endotoxin is active against dipteran insects and has great potential for mosquito borne disease control. Here, we report the first theoretical model of the tridimensional structure of a Cry11 toxin. The tridimensional structure of the Cry11Bb toxin was obtained by homology modelling on the structures of the Cry1Aa and Cry3Aa toxins. In this work we give a brief description of our model and hypothesize the residues of the Cry11Bb toxin that could be important in receptor recognition and pore formation. This model will serve as a starting point for the design of mutagenesis experiments aimed to the improvement of toxicity, and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

Promoter architecture of mouse olfactory receptor genes.

Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.