Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type.

With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the "mycoides cluster'.

TGF-βRI kinase activity mediates Emdogain-stimulated in vitro osteoclastogenesis.

OBJECTIVES Emdogain, containing an extract of fetal porcine enamel matrix proteins, is a potent stimulator of in vitro osteoclastogenesis. The underlying molecular mechanisms are, however, unclear. MATERIAL AND METHODS Here, we have addressed the role of transforming growth factor-beta receptor type 1 (TGF-βRI) kinase activity on osteoclastogenesis in murine bone marrow cultures. RESULTS Inhibition of TGF-βRI kinase activity with SB431542 abolished the effect of Emdogain on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand or tumor necrosis factor-alpha. SB431542 also suppressed the Emdogain-mediated increase of OSCAR, a co-stimulatory protein, and dendritic cell-specific transmembrane protein and Atp6v0d2, the latter two being involved in cell fusion. Similar to transforming growth factor-beta1 (TGF-β), Emdogain could not compensate for the inhibition of IL-4 and IFNγ on osteoclast formation. When using the murine macrophage cell line RAW246.7, SB431542 and the smad-3 inhibitor SIS3 blocked Emdogain-stimulated expression of the transcription factor NFATc1. CONCLUSIONS Taken together, the data suggest that TGF-βRI kinase activity is necessary to mediate in vitro effects of Emdogain on osteoclastogenesis. CLINICAL RELEVANCE Based on these in vitro data, we can speculate that at least part of the clinical effects of Emdogain on osteoclastogenesis is mediated via TGF-β signaling.

Irisin – a myth rather than an exercise-inducible myokine

The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes

Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.

SNARE-fusion mediated inserton of membrane proteins into native and artificial membranes

Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE-protein based method for incorporation of multiple membrane proteins into membranes, and for delivery of large water-soluble substrates into closed membrane vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0-ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.

Current concepts: host-pathogen interactions in cystic fibrosis airways disease.

Chronic infection and inflammation are defining characteristics of cystic fibrosis (CF) airway disease. Conditions within the airways of patients living with CF are conducive to colonisation by a variety of opportunistic bacterial, viral and fungal pathogens. Improved molecular identification of microorganisms has begun to emphasise the polymicrobial nature of infections in the CF airway microenvironment. Changes to CF airway physiology through loss of cystic fibrosis transmembrane conductance regulator functionality result in a wide range of immune dysfunctions, which permit pathogen colonisation and persistence. This review will summarise the current understanding of how CF pathogens infect, interact with and evade the CF host.

A component of the mitochondrial outer membrane proteome of T. brucei probably contains covalent bound fatty acids

A subclass of eukaryotic proteins is subject to modification with fatty acids, the most common of which are palmitic and myristic acid. Protein acylation allows association with cellular membranes in the absence of transmembrane domains. Here we examine POMP39, a protein previously described to be present in the outer mitochondrial membrane proteome (POMP) of the protozoan parasite Trypanosoma brucei. POMP39 lacks canonical transmembrane domains, but is likely both myristoylated and palmitoylated on its N-terminus. Interestingly, the protein is also dually localized on the surface of the mitochondrion as well as in the flagellum of both insect-stage and the bloodstream form of the parasites. Upon abolishing of global protein acylation or mutation of the myristoylation site, POMP39 relocates to the cytosol. RNAi-mediated ablation of the protein neither causes a growth phenotype in insect-stage nor bloodstream form trypanosomes.

Ephrin-B1 transduces signals to activate integrin-mediated migration, attachment and angiogenesis.

Ephrin-B/EphB family proteins are implicated in bidirectional signaling and were initially defined through the function of their ectodomain sequences in activating EphB receptor tyrosine kinases. Ephrin-B1-3 are transmembrane proteins sharing highly conserved C-terminal cytoplasmic sequences. Here we use a soluble EphB1 ectodomain fusion protein (EphB1/Fc) to demonstrate that ephrin-B1 transduces signals that regulate cell attachment and migration. EphB1/Fc induced endothelial ephrin-B1 tyrosine phosphorylation, migration and integrin-mediated (alpha(v)beta(3) and alpha(5)beta(1)) attachment and promoted neovascularization, in vivo, in a mouse corneal micropocket assay. Activation of ephrin-B1 by EphB1/Fc induced phosphorylation of p46 JNK but not ERK-1/2 or p38 MAPkinases. By contrast, mutant ephrin-B1s bearing either a cytoplasmic deletion (ephrin-B1DeltaCy) or a deletion of four C-terminal amino acids (ephrin-B1DeltaPDZbd) fail to activate p46 JNK. Transient expression of intact ephin-B1 conferred EphB1/Fc migration responses on CHO cells, whereas the ephrin-B1DeltaCy and ephrin-B1DeltaPDZbd mutants were inactive. Thus ephrin-B1 transduces 'outside-in' signals through C-terminal protein interactions that affect integrin-mediated attachment and migration.

The granulocyte orphan receptor CEACAM4 is able to trigger phagocytosis of bacteria.

Human granulocytes express several glycoproteins of the CEACAM family. One family member, CEACAM3, operates as a single-chain phagocytic receptor, initiating the detection, internalization, and destruction of a limited set of gram-negative bacteria. In contrast, the function of CEACAM4, a closely related protein, is completely unknown. This is mainly a result of a lack of a specific ligand for CEACAM4. By generating chimeric proteins containing the extracellular bacteria-binding domain of CEACAM3 and the transmembrane and cytoplasmic part of CEACAM4 (CEACAM3/4) we demonstrate that this chimeric receptor can trigger efficient phagocytosis of attached particles. Uptake of CEACAM3/4-bound bacteria requires the intact ITAM of CEACAM4, and this motif is phosphorylated by Src family PTKs upon receptor clustering. Furthermore, SH2 domains derived from Src PTKs, PI3K, and the adapter molecule Nck are recruited and associate directly with the phosphorylated CEACAM4 ITAM. Deletion of this sequence motif or inhibition of Src PTKs blocks CEACAM4-mediated uptake. Together, our results suggest that this orphan receptor of the CEACAM family has phagocytic function and prompt efforts to identify CEACAM4 ligands.

Size does matter: 18 amino acids at the N-terminal tip of an amino acid transporter in Leishmania determine substrate specificity

Long N-terminal tails of amino acid transporters are known to act as sensors of the internal pool of amino acids and as positive regulators of substrate flux rate. In this study we establish that N-termini of amino acid transporters can also determine substrate specificity. We show that due to alternative trans splicing, the human pathogen Leishmania naturally expresses two variants of the proline/alanine transporter, one 18 amino acid shorter than the other. We demonstrate that the longer variant (LdAAP24) translocates both proline and alanine, whereas the shorter variant (∆18LdAAP24) translocates just proline. Remarkably, co-expressing the hydrophilic N-terminal peptide of the long variant with ∆18LdAAP24 was found to recover alanine transport. This restoration of alanine transport could be mediated by a truncated N-terminal tail, though truncations exceeding half of the tail length were no longer functional. Taken together, the data indicate that the first 18 amino acids of the negatively charged N-terminal LdAAP24 tail are required for alanine transport and may facilitate the electrostatic interactions of the entire negatively charged N-terminal tail with the positively charged internal loops in the transmembrane domain, as this mechanism has been shown to underlie regulation of substrate flux rate for other transporters.

Peptide-based interactions with calnexin target misassembled membrane proteins into endoplasmic reticulum-derived multilamellar bodies.

Oligomeric assembly of neurotransmitter transporters is a prerequisite for their export from the endoplasmic reticulum (ER) and their subsequent delivery to the neuronal synapse. We previously identified mutations, e.g., in the gamma-aminobutyric acid (GABA) transporter-1 (GAT1), which disrupted assembly and caused retention of the transporter in the ER. Using one representative mutant, GAT1-E101D, we showed here that ER retention was due to association of the transporter with the ER chaperone calnexin: interaction with calnexin led to accumulation of GAT1 in concentric bodies corresponding to previously described multilamellar ER-derived structures. The transmembrane domain of calnexin was necessary and sufficient to direct the protein into these concentric bodies. Both yellow fluorescent protein-tagged versions of wild-type GAT1 and of the GAT1-E101D mutant remained in disperse (i.e., non-aggregated) form in these concentric bodies, because fluorescence recovered rapidly (t(1/2) approximately 500 ms) upon photobleaching. Fluorescence energy resonance transfer microscopy was employed to visualize a tight interaction of GAT1-E101D with calnexin. Recognition by calnexin occurred largely in a glycan-independent manner and, at least in part, at the level of the transmembrane domain. Our findings are consistent with a model in which the transmembrane segment of calnexin participates in chaperoning the inter- and intramolecular arrangement of hydrophobic segment in oligomeric proteins.

Mimicking respiratory phosphorylation using purified enzymes

The enzymes of oxidative phosphorylation are a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.