Prion infection-impaired functional blocks identified by proteomics enlighten the targets and the curing pathways of an anti-prion drug.

Prion-induced neurodegeneration results from multiple cellular alterations among which the accumulation of a modified form of the host protein PrP is but a hallmark. Drug treatments need understanding of underlying mechanisms. Proteomics allows getting a comprehensive view of perturbations leading to neuronal death. Heparan sulfate mimetics has proved to be efficient to clear scrapie protein in cultured cells and in animals. To investigate the mechanisms of drug attack, protein profiles of the neuronal cell line GT1 and its chronically Chandler strain infected counterpart were compared, either in steady state cultures or after a 4-day drug treatment. Differentially expressed proteins were associated into functional blocks relevant to neurodegenerative diseases. Protein structure repair and modification, proteolysis, cell shape and energy/oxidation players were affected by infection, in agreement with prion biology. Unexpectedly, novel affected blocks related to translation, nucleus structure and DNA replication were unravelled displaying commonalities with proliferative processes. The drug had a double action in infected cells by reversing protein levels back to normal in some blocks and by heightening survival functions in others. This study emphasizes the interest of a proteomic approach to unravel novel networks involved in prion infection and curing.

Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns.

One of the unsolved problems in prion diseases relates to the physiological function of cellular prion protein (PrP), of which a misfolded isoform is the major component of the transmissible spongiform encephalopathies agent. Knowledge of the PrP-binding molecules may help in elucidating its role and understanding the pathological events underlying prion diseases. Because nucleic acids are known to bind PrP, we attempted to identify the preferred RNA sequences that bind to the ovine recombinant PrP. An in vitro selection approach (SELEX) was applied to a pool of 80-nucleotide(nt)-long RNAs containing a randomised 40-nt central region. The most frequently isolated aptamer, RM312, was also the best ligand (20 nM KD value), according to both surface plasmon resonance and filter binding assays. The fast rates of association and dissociation of RM312 with immobilized PrP, which are reminiscent of biologically relevant interactions, could point to a physiological function of PrP towards cellular nucleic acids. The minimal sequence that we found necessary for binding of RM312 to PrP presents a striking similarity with one previously described PrP aptamer of comparable affinity. In addition, we here identify the two lysine clusters contained in the N-terminal part of PrP as its main nucleic-acid binding sites.

Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor.

The binding domain of the chicken leptin receptor [chLBD (chicken leptin-binding domain)], subcloned from the full-size chicken leptin receptor and prepared in an Escherichia coli system, was subjected to site-directed mutagenesis to identify the amino acids involved in leptin binding. A total of 22 electrophoretically pure, >90% monomer-containing mutants were expressed, refolded and purified. The effects of the mutations were tested by the ability to form complexes with ovine leptin, and the kinetic parameters of interaction were determined by surface plasmon resonance. Six mutants were used to determine whether mutations of several amino acids that differ between chLBD and mammalian LBDs will affect affinity: none showed any such effect, except the mutant A105D (Ala(105)-->Asp), which exhibited some decrease in affinity. Surface plasmon resonance analysis identified six mutants in which binding activity was totally abolished (F73A, Y14A/F73A, V76A/F77A, L78A/L79A, V76A/F77A/L78A/L79A and A105D/D106V) and six mutants (Y14A, R41A, R41A/S42A/K43A, V103A, V135A/F136A and F136A) in which affinity for the hormone was reduced, mainly by increased dissociation rates. Gel-filtration experiments indicated the formation of a 1:1 ovine or human leptin-chLBD complex with a molecular mass of approx. 41 kDa. Gel-filtration experiments yielded 1:1 complexes with those mutants in which affinity had decreased, but not with the six mutants, which had totally lost their binding capacity. Modelling the leptin-chLBD complex indicated that the binding domain of the latter is located mainly in the L3 loop, which contributes nine amino acid residues interacting with leptin. Contact-surface analysis identified the residues having the highest contribution to the recognition site to be Phe73, Phe77 and Leu79.

CD4+ T cell-mediated immunity against prion proteins.

The prion protein (PrP(C)) is essential for susceptibility to transmissible spongiform encephalopathies. A specific conformer of this protein (PrP(Sc)) is, according to the 'protein only' hypothesis, the principal or only component of the infectious agent, designated prion. Transmission of prions between species is often inefficient, resulting in low attack rates and/or prolonged incubation times and is ascribed to a 'species barrier' caused by differences in the amino acid sequence of PrP between recipient and donor. In this report, we demonstrate that these differences in amino acid sequence result in presentation of distinct peptides on major histocompatibility complex class II molecules. These peptides result in activation of specific CD4+ T cells which leads to the induction of an effective immune response against foreign PrP as demonstrated by antibody production. Therefore, CD4+ T cells represent a crucial component of the immune system to distinguish between foreign and self PrP.

Selective prion protein binding to synaptic components is modulated by oxidative and nitrosative changes induced by copper(II) and peroxynitrite in cholinergic synaptosomes, unveiling a role for calcineurin B and thioredoxin.

Choline acetyltransferase (ChAT) and choline transport are decreased after nitrosative stress. ChAT activity is altered in scrapie-infected neurons, where oxidative stress develops. Cellular prion protein (PrPc) may play a neuroprotective function in participating in the redox control of neuronal environment and regulation of copper metabolism, a role impaired when PrPc is transformed into PrPSc in prion pathologies. The complex cross-talk between PrPc and cholinergic neurons was analyzed in vitro using peroxynitrite and Cu2+ treatments on nerve endings isolated from Torpedo marmorata, a model of the motoneuron pre-synaptic element. Specific interactions between solubilized synaptic components and recombinant ovine prion protein (PrPrec) could be demonstrated by Biacore technology. Peroxynitrite abolished this interaction in a concentration-dependent way and induced significant alterations of neuronal targets. Interaction was restored by prior addition of peroxynitrite trapping agents. Cu2+ (in the form of CuSO4) treatment of synaptosomes triggered a milder oxidative effect leading to a bell-shaped increase of PrPrec binding to synaptosomal components, counteracted by the natural thiol agents, glutathione and thioredoxin. Copper(II)-induced modifications of thiols in several neuronal proteins. A positive correlation was observed between PrPrec binding and immunoreactive changes for calcineurin B and its partners, suggesting a synergy between calcineurin complex and PrP for copper regulation.

Conformational dynamics underlie the activity of the auxin-binding protein, Nt-abp1.

The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.

Evidence of rainbow trout prolactin interaction with its receptor through unstable homodimerisation.

This study aims to characterise Prolactin receptor (PRLR) in rainbow trout for which no information is available despite the availability of Salmonid PRL preparations. By screening a freshwater rainbow trout intestine cDNA library with a probe corresponding to the extracellular domain (ECD) of tilapia PRLR, we have cloned a 2.5 kb insert coding for the PRLR. The mature protein of 614 amino acid residues is similar to PRLR isolated in tilapia and also the long form of mammalian PRLR. Analysis of PRLR gene expression in osmoregulatory organs revealed the presence of a unique transcript, thus confirming the involvement of this hormone in the control of osmoregulation in this fish species. By using surface plasmon resonance (SPR) technology, kinetic measurement of interaction between trout PRL and its receptor ECD was studied. This approach allowed us to demonstrate the formation of a transient, unstable homodimeric complex. This unstability could explain the inability to perform binding experiments using homologous PRL. In contrast, heterologous lactogenic ligands were able to interact through a more stable complex. Whether these characteristics of PRL-receptor interaction in rainbow trout are different to what occurs in tilapia where a homologous radioreceptor assay was developed would require further studies.

Purification and structural analysis of a soluble human chorionogonadotropin hormone-receptor complex.

Receptors for the luteotropin/human chorionogonadotropin hormone belong to the G-protein-coupled receptor family by their membrane-anchoring domains. They also possess a large extracellular domain (ECD) responsible for most of the hormone-receptor interactions. Structure-function studies identified several contacts between hormone and receptor ECD, but the precise topology of the complex is still unknown because of the lack of suitable heterologous expression means. Receptor ECDs exhibit leucine repeats and have been modelized on the basis of the three-dimensional structure of the porcine ribonuclease inhibitor, the first structurally known leucine-rich repeats protein. Here we report overexpression (up to 20 mg per liter) and purification to homogeneity of a soluble human chorionogonadotropin-ECD receptor complex secreted by stably cotransfected Chinese hamster ovary cells. Biochemical analysis and surface plasmon resonance data were in favor of a unique dimer with a 1:1 ligand-receptor stoichiometry. Immunopurified complex was submitted to circular dichroism characterization; CD spectra deconvolution indicated more than 25% alpha helices contributed by the receptor, in agreement with the porcine ribonuclease inhibitor-based modelization.

Differential localisation of an Eimeria tenella aspartyl proteinase during the infection process.

Aspartyl proteinases are essential for the survival of many pathogens. A single copy gene in species of Eimeria encodes an aspartyl proteinase, which we propose should be called eimepsin to conform to the commonly used names of this family of proteinases. An epitope map, constructed using BIAcore technology, confirmed the specificity of 14 mAbs for eimepsin and defined four antigenic domains, which were conserved between native and recombinant forms of eimepsin. In resting sporozoites, mAb defining antigenic domains I and II stained the refractile body organelles, whereas those defining antigenic domains III and IV stained cytoplasmic granules. During host cell invasion, the staining patterns of mAb defining antigenic domains I, III and IV changed dramatically with the apical tips of invading sporozoites becoming strongly stained. In contrast, mAb defining antigenic domain II continued to stain only the refractile bodies. During early schizogony, mAb to all four domains stained the single fused refractile body, but when schizonts matured, mAb to antigenic domains I, III and IV stained the apical tip of merozoites whereas those to antigenic domain II continued to follow the developmental redistribution of the refractile body. Irrespective of localisation, mAb to three antigenic domains recognised a polypeptide of 49 kDa, which from N-terminal sequencing corresponds to a mature form of eimepsin. Staining with fluorescent pepstatin localised a mature, active form of eimepsin to the refractile bodies of the sporozoite, schizont and first generation merozoite. It remains to be determined whether eimepsin has a catalytic function within the refractile body or whether the activated enzyme is stored in the refractile body so that it can be rapidly redistributed to the apical tip during parasite invasion.

High yield purification and physico-chemical properties of full-length recombinant allelic variants of sheep prion protein linked to scrapie susceptibility.

Sheep susceptibility to scrapie is governed by polymorphisms at two major sites, codons 136 and 171, of the prp gene. To get more insight into the prion protein (PrP) sequence-linked basis of differential scrapie susceptibility, a high yield one-step method for the purification (over 99% final purity) of the full-length recombinant sheep PrP was developed, based on the affinity of the conserved octapeptide repeats for transition-metal cations. Thermal and chemical denaturation experiments and limited proteolysis studies were performed on the natural variants (A136R171, V136Q171 and A136Q171) and a recombinant PrP mutated at position 136 (V136R171). Results revealed the influence of mutations in positions 136 and 171 on the folding thermodynamic parameters and on the conformation of the C-terminal domain. Together, our results show that the VQ cellular protein linked to higher scrapie susceptibility is intrinsically more compact and/or stable than the resistance-linked AR counterpart. This might lead to a lower in vivo clearance rate of VQ and a consequently higher probability of occurrence of pathological events.

Functional heterodimerization of prolactin and growth hormone receptors by ovine placental lactogen.

Although homo- or heterodimerization are common mechanisms for activation of cytokine receptors, cross-talk between two distinct receptors in this superfamily has been never shown. Here we show a physiologically relevant example indicating that such an interaction does occurs, thus raising the hypothesis that heterodimerization between distinct cytokine receptors may be a novel mechanism contributing to the diversity of cytokine signaling. These findings were documented using both surface plasmon resonance and gel filtration experiments and show that ovine placental lactogen (PL) heterodimerizes the extracellular domains (ECDs) of ruminant growth hormone receptor (GHR) and prolactin receptor (PRLR). We also show that PL or PL analogues that exhibit little or no activity in cells transfected with PRLRs and no activity in cells transfected with ovine GHRs exhibit largely enhanced activity in cells cotransfected with both PRLRs and GHRs. Furthermore, chimeric receptors consisting of cytosolic and transmembrane part of ovine GHR or ovine PRLR and ECDs of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha or beta were constructed. Upon transfection into Chinese hamster ovary cells along with reporter luciferase gene and stimulation by GM-CSF, a significant increase in luciferase activity occurred when GM-CSFR-alpha-PRLR and GM-CSFR-beta-GHR or GM-CSFR-alpha-GHR and GM-CSRR-beta-PRLR were cotransfected. In conclusion, we show that ovine PL is capable of functional heterodimerization of GHR and PRLR and that when their cytosolic parts, coupled to the ECD of GM-CSF receptors, are heterodimerized by GM-CSF, they are capable of transducing biological signal.

Monoclonal antibodies as probes for the changes in antigenicity of bovine and porcine aspartyl proteases with pH.

Two monoclonal antibodies, 918(4) and 139(7), directed against either bovine or porcine pepsin, respectively, were retained among 365 positive hybridoma clones. These monoclonal antibodies were characterized by using both indirect and sandwich ELISA. Characterization of these monoclonal antibodies was further performed by the biospecific interaction analysis (BIA-core analysis). Then, they were used as antigenic probes to study the changes in antigenicity of both bovine and porcine pepsins induced by pH. The results demonstrated the importance of the conformational change in both catalytic activities and antigenic determinant accessibility of bovine and porcine pepsins. Furthermore, our results suggest that changes in the conformation due to pH can be detected by specific monoclonal antibodies.

A novel immunological approach establishes that the auxin-binding protein, Nt-abp1, is an element involved in auxin signaling at the plasma membrane.

Interactions of a collection of monoclonal antibodies (mAbs) to the recombinant Nicotiana tabacum auxin-binding protein 1 (Nt-abp1) were extensively characterized using surface plasmon resonance. Dynamic interaction studies using combinations of Nt-abp1, synthetic peptides corresponding to conserved sequences within auxin-binding proteins, and the mAbs have shown that a number of the mAbs recognized discontinuous epitopes revealing the junction of distinct domains in the folded protein. In particular, the two putative auxin binding domains and the C terminus of the protein were shown to interact with each other in the folded protein. Using the auxin-induced electrical response of tobacco protoplasts as a functional assay, all the mAbs exhibited either auxin antagonist or hormonomimetic properties. These effects, measured for the first time in homologous conditions, confirm that Nt-abp1 is present at the plasma membrane and is involved in the activation of the auxin-dependent electrical response of tobacco protoplasts. Based on our surface plasmon resonance data, we propose that the key event leading to the activation of this auxin electrical response consists of a conformational change in Nt-abp1.

Identification of the RNA-binding, dimerization, and eIF4GI-binding domains of rotavirus nonstructural protein NSP3.

The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3' end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.

Partial antigenic characterization of different potato virus Y NTN strain isolates.

Eight isolates of potato virus Y NTN strain (PVY-NTN) of different origin were studied by means of monoclonal antibodies (MAbs) in non-competitive and competitive enzyme-linked immunosorbent assay (ELISA), and by immunoblot analysis of the viral coat protein (CP). As the MAbs reacted with the denatured viral CP, their epitopes must be continuous. The ELISA data demonstrate that the epitopes are topologically different. The epitopes may be located on the N-terminal part of CP as showed its partial amino acid sequencing and the immunoblot analysis.

Novel recombinant analogues of bovine placental lactogen. G133K and G133R provide a tool to understand the difference between the action of prolactin and growth hormone receptors.

Two new analogues of bovine placental lactogen (bPL), bPL(G133K) and bPL(G133R), were expressed in Escherichia coli, refolded, and purified to a native form. Binding experiments, which are likely to represent the binding to site 1 only, to intact FDC-P1 cells transfected with rabbit (rb) growth hormone receptor (GHR) or with human (h) GHR, to Nb2 rat lymphoma cells, or to rabbit mammary gland membranes prolactin receptor (PRLR), revealed only small or no reduction in binding capacity. The complex formation between these analogues and receptor extracellular domains (R-ECD) of various hormones was determined by gel filtration. Wild type bPL yielded 1:2 complex with hGHR-ECD, rat PRLR-ECD, and rbPRLR-ECD, whereas both analogues formed only 1:1 complexes with all R-ECDs tested. Real time kinetics experiments demonstrated that the ability of the analogues to form homodimeric complexes was compromised in both PRLR- and GHR-ECDs. The biological activity transduced through lactogenic receptors in in vitro bioassays in rabbit mammary gland acini culture and in Nb2 cells was almost fully retained, whereas the activity transduced through somatogenic receptors in FDC-P1 cells transfected with rbGHRs or with hGHRs was abolished. Both analogues exhibited antagonistic activity in the latter cells. To explain the discrepancy between the effect of the mutation on the signal transduced by PLR versus GHRs we suggest that: 1) the mutation impairs the ability of site 2 of bPL to form a stable homodimeric complex with both lactogenic and somatogenic receptors by a drastic shortening of the half-life of 2:1 complex; 2) the transient existence of the homodimeric complex is still sufficient to initiate the signal transduced through lactogenic receptors but not through somatogenic receptors; and 3) one possible reason for this difference is that JAK2, which serves as a mediator of both receptors, is already associated with lactogenic receptors prior to hormone binding-induced receptor dimerization, whereas in somatogenic receptors the JAK2 receptor association occurs subsequently to receptor dimerization.

[Fluctuations of serotonin and its metabolites in the lung and intestinal mucosa in the rat infected with Nippostrongylus brasiliensis]. / Fluctuations de la sérotonine et de ses métabolites au niveau du poumon et du mucus intestinal chez le rat infesté par Nippostrongylus brasiliensis.

The influence of parasitism on host biogenic amine levels was investigated in Nippostrongylus brasiliensis infected rats. Amine levels were estimated in tissues surrounding Nematods in their biological environment: the lung and intestinal mucus. D0 being the day of infestation, tissues were obtained at 24, 30 and 45 hrs, and every day between D4 and D14 (when the rat was completely deparasited by the self-cure phenomenon). Biogenic amines belonging to the serotoninergic pathway were quantified by HPLC with electrochemical detection. In the lungs and mucus, parasitism resulted in an important decrease in serotonine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels, as opposed to the immediate 5-HT precursor, the 5-hydroxy-tryptophane (5-HTP). Host response to parasitism is translated by serotoninergic pathway levels. This leads to two hypotheses: 5-HT turn-over may be accelerated, but the inhibition of 5-HT synthetic enzyme, 5-hydroxytryptophane hydroxylase, by the parasite present in the host seems more probable.

Levels of biogenic amines in larvae and adults of the rat hookworm, Nippostrongylus brasiliensis (Nematoda).

Norepinephrine, 5-hydroxytryptophan, octopamine, dihydroxyphenylacetic acid, N-acetyldopamine, dopamine, 5-hydroxyindoleacetic acid, N-acetylserotonin, tyramine, tryptophan and serotonin in larvae (third free stage and parasitic stages) and adult males and females (at defined ages during the intestinal phase) of the parasitic nematode Nippostrongylus brasiliensis were quantified simultaneously by high-performance liquid chromatography with electrochemical detection. Biogenic amine levels depended on the stage, the age and the sex of parasites and on environmental conditions. Their physiological roles in reproductively competent adults of this nematode are discussed in relation to exuviation and egg laying. Parallel fluctuations in free ecdysteroids and norepinephrine were observed in females from the same worm populations.

Site-directed mutagenesis of recombinant bovine placental lactogen at lysine-73 leads to selective attenuation of its somatogenic activity.

Bovine placental lactogen (bPL) is capable of binding and transducing biological activity via somatogenic and lactogenic receptors. To modify this capability, three analogs, bPL(K73D), bPL(K73F) and bPL(K73A), mutated at position 73, and corresponding to R64 in human GH (hGH), were produced in Escherichia coli. Circular dichroic spectrum analyses indicated proper refolding in all cases. Biological activity of these analogs was tested in vitro. In a lactogenic-receptor-mediated Nb2 rat lymphoma cell bioassay, bPL and its analogs acted similarly. In another lactogenic bioassay that measures beta-casein synthesis by HC-11 mouse mammary-gland cells, the analogs were 30-40% as potent as bPL. In contrast, somatogenic receptor-mediated bioactivity in FDC-P1 cells transfected with either rabbit (rb) or hGH receptor (R) was almost completely abolished in these analogs. In receptor binding assays, the effect was more conspicuous and the mutations affected not only somatogenic but also lactogenic binding. Binding to rat (r) and rabbit PRL receptor extracellular domains (ECDs) or membrane-embedded receptors was only slightly changed, except for bPL (K73D), which displayed very low affinity. In somatogenic binding assays to intact IM-9 human lymphocytes, hGHR-ECD or bovine liver membranes, bPL (K73D) did not bind at all, and bPL(K73F) or bPL(K73A) binding was drastically reduced. Binding experiments performed in real time using a BIAcore apparatus revealed that the decreased binding could be mainly attributed to increased k(off) rather than decreased k(on) values. The complex with hGHR-ECD revealed a 2:1 stoichiometry with bPL, bPL(K73F) and bPL(K73A), although the complex with these analogs was less stable than with bPL, whereas bPL(K73D) scarcely assembled a 1:1 complex. In contrast, bPL and the three analogs formed stable 1:2 complexes with rPRL-ECD. These results suggest that position 73 in bPL is more important for somatogenic than lactogenic properties and concurs with results from other groups, which have shown that R64, the analogous amino acid in hGH holds the same differential importance with respect to somatogenic binding.