Rotavirus assembly: an alternative model that utilizes an atypical trafficking pathway.

We review here recent advances in our knowledge on trafficking and assembly of rotavirus and rotaviral proteins in intestinal cells. Assembly of rotavirus has been extensively studied in nonpolarized kidney epithelial MA104 cells, where several data indicate that most if not all the steps of rotavirus assembly take place within the endoplasmic reticulum (ER) and that rotavirus is release upon cell lysis. We focus here on data obtained in intestinal cells that argue for another scheme of rotavirus assembly, where the final steps seem to take place outside the ER with an apically polarized release of rotavirus without significant cell lysis. One of the key observations made by different groups is that VP4 and other structural proteins interact substantially with specialized membrane microdomains enriched in cholesterol and sphingolipids termed rafts. In addition, recent data point to the fact that VP4 does not localize within the ER or the Golgi apparatus in infected intestinal cells. The mechanisms by which VP4, a cytosolic protein, may be targeted to the apical membrane in these cells and assembles with the other structural proteins are discussed. The identification of cellular proteins such as Hsp70, flotillin, rab5, PRA1 and cytoskeletal components that interact with VP4 may help to define an atypical polarized trafficking pathway to the apical membrane of intestinal cells that will be raft-dependent and by-pass the classical exocytic route.

[Present possibilities and future development of clinical proteomics]. / Présent et futur de la Protéomique clinique.

This review focuses on "clinical proteomics" which represents an emerging discipline in biomedical research. "Clinical proteomics" relies on the analysis of the proteome, i.e. the entire set of peptides and proteins present in a biological sample, to provide relevant data for diagnosis, prognosis or therapeutic strategies of human pathologies. This new type of approach has tremendous potential for the diagnosis of complex pathologies or for the early detection of cancers. This article reports the conclusions of a workgroup of the French Society for Clinical Biology (SFBC) 2004-2006 which evaluated the status, the impact and the future development of proteomics in the clinical field. It provides therefore a broad view going from the methods already present in the clinical laboratories (multiplex technologies...), to the tools for clinical and basis research including bioinformatics.

On the mode of action of basic phospholipase A2 from Naja nigricollis venom.

A study has been made of the cytotoxic activity of basic phospholipase A2 of venom from Naja nigricollis on different types of cells and of the participation of esterase activity in this cytotoxic activity. It was previously shown that the cytotoxicity observed is not due to a contaminant, since the cytotoxic action vanished after immunoprecipitation of basic phospholipase A2 by specific monoclonal antibodies. All eukaryotic cells tested were sensitive to the cytotoxic action of basic phospholipase A2. In contrast, Escherichia coli K-12 wild strain was resistant. Participation of cell membranes in the sensitive or resistant character of cells to the phospholipase A2 attack was investigated using E. coli K-12 membrane mutants. Some membrane mutants were sensitive and the sensitivity or resistance to basic phospholipase A2 was found to be related to a single mutation in the locus envA. The requirement for esterase activity of phospholipase A2 in cytotoxic attack has been shown to be dependent on the cell line tested. Indeed, when the esterase activity of basic phospholipase A2 was eliminated by treatment with p-bromophenacyl bromide, the enzyme retained a cytotoxic potency inducing necrosis of certain tumoral cells grown in vitro, but not other cells such as erythrocytes, for which concomitant esterase activity was also necessary. In vivo studies of toxicity showed that the loss of either cytotoxic potency or esterase activity eliminates the lethal character of basic phospholipase A2. This leads us to propose that in vivo toxicity of phospholipase A2 depends on simultaneous expression of esterase activity and a non-enzymatic property, manifested by in vitro cytotoxic potency.

A monoclonal antibody recognizing a conserved epitope in a group of phospholipases A2.

Notexin and nigexine are monomeric phospholipases A2(PLA2s) from the venoms of Notechis scutatus scutatus and Naja nigricollis, respectively. Polyclonal antibodies raised in mice against these antigenic proteins displayed non-reciprocal cross-reactivity; anti-notexin antibodies recognized notexin but not nigexine, whereas anti-nigexine antibodies recognized both antigens. Polyclonal antibodies raised by successive immunization with nigexine and notexin contained cross-reacting antibodies with affinities for both antigens that differed from those of antibodies present in anti-nigexine antiserum. A monoclonal antibody has been obtained from a mouse immunized with both PLA2s. This monoclonal antibody, called MN1, recognized notexin and nigexine with comparable high affinity (Kd = 10(-9) M). It also recognized most purified PLA2s from elapid snake venoms and all PLA2-containing venoms from cobras and sea-snakes. This offers the first demonstration that most PLA2s from cobras and sea-snakes share a fine structure which is not restricted to the common catalytic site.

Unsaturated acyl chains dramatically enhanced cellular uptake by direct translocation of a minimalist oligo-arginine lipopeptide.

The recurring issue with cell penetrating peptides is how to increase direct translocation vs. endocytosis, to avoid premature degradation. Acylation by a cis unsaturated chain (C22:6) of a short cationic peptide provides a new rational design to favour diffuse cytosolic and dense Golgi localisations.

Ubiquitin is physiologically induced by interferons in luminal epithelium of porcine uterine endometrium in early pregnancy: global RT-PCR cDNA in place of RNA for differential display screening.

Early in the course of pregnancy, at the preimplantation stage, the pig embryo is likely to exert a paracrine effect on the tissue intended to receive it, via the secretion of interferons. Our observations show that trophoblastic interferons induce an increase of some mRNAs in the epithelial cells of the gilt endometrium, which would illustrate this phenomenon. The increase of four mRNAs, whose corresponding cDNAs are dD1, dD2, dD3 and dD4, has been examined in this study. The method used is similar to Northern blot analysis except that mRNAs in the blot are replaced by cDNAs produced from total cellular poly(A)+ mRNAs by global reverse-transcription polymerase chain reaction (RT-PCR). Northern blot hybridization requires a considerable quantity of starting material--which we estimate in this study to be several million porcine endometrium cells--whereas the RT-PCR-based method gives comparable results starting with only a few cells--about 200. Using this method, the differential nature of dD1, dD2, dD3 and dD4 was shown. dD2 and dD3 correspond to genes already identified as interferon-induced: the beta2-microglobulin and Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously secreted protein (FAU). dD1 corresponds to a still unidentified gene. dD4 encodes for the porcine UbA52 ubiquitin. Up to now, the increase in ubiquitin mRNA as a result of interferon effect has not been reported and is discussed in view of recent publications.

Evidence that the anti-coagulant and lethal properties of a basic phospholipase A2 from snake venom are unrelated.

The basic phospholipase A2 (PLA2) from venom of the African elapid Naja nigricollis was previously shown to have anti-coagulant and lethal properties, both of which were abolished by treatment with p-bromophenacyl bromide (pBP). In the present paper we first report that pBP-treated PLA2 is capable of inhibiting the anti-coagulant activity but not the lethal activity of native PLA2, thus suggesting that both properties might be independent. We then confirm this evidence using PLA2-specific monoclonal immunoglobulins. One of these, called HSF, neutralized the lethal activity but not the anti-coagulant activity, whereas another antibody, called HSP2, inhibited the anti-coagulant activity but not the lethal activity of the PLA2. The data presented in this paper are taken as evidence that the anti-coagulant activity is not implicated in the lethal effects of basic PLA2 from Naja nigricollis.

Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus.

Neutralizing antibodies were raised in mice against notexin, the most toxic phospholipase A2 (PLA2) from Notechis scutatus scutatus venom, without the necessity of detoxifying the toxin prior to immunization. Using a sensitive radioimmunoassay we demonstrated that anti-notexin antibodies recognized (i) the parent antigen, (ii) closely related isoforms of notexin and (iii) venoms from Notechis genus snakes. In contrast, they failed to recognize other purified PLA2 or PLA2-containing venoms from other origins. Substitutions or chemical modifications occurring in the C-terminal part of the polypeptide chain of notexin altered the binding affinity for antibodies, implying that this region constitutes an antigenic domain of notexin.

On the purification of notexin. Isolation of a single amino acid variant from the venom of Notechis scutatus scutatus.

Venom of the Australian tiger snake, Notechis scutatus scutatus was fractionated by conventional ion-exchange chromatography. The fraction containing notexin, a well-known single-chain toxic phospholipase A2, was further purified by reverse-phase high-performance liquid chromatography. Two main components were isolated and the major one corresponded to notexin. The other component, designated as notechis Ns, was an isoform of notexin. Notechis Ns and notexin possessed similar in vitro esterase activity, in vitro neuromuscular activity and in vivo lethality. Amino acid composition and sequence of the Staphylococcus aureus V8-protease peptides demonstrated that primary structures of notechis Ns and notexin differed from each other by a single substitution amongst 119 amino acids: Lys----Arg at position 16.

[Lethal in vivo and cytotoxic in vitro properties of a basic phospholipase A2 modified with para-bromophenacyl bromide]. / Propriétés létale in vivo et cytotoxique in vitro d'une phospholipase A2 basique modifiée par le bromure de para-bromophénacyle.

Nigexine is a basic phospholipase A2 isolated from the venom of Naja nigricollis (Institut Pasteur). Previous works have shown that nigexine is (i) cytotoxic in vitro toward epithelial cells of FL strain and, (ii) toxic to mice (LD50 = 29 nmoles/kg). We have investigated the properties of nigexine treated with p-bromophenacyl bromide, a reagent which specifically destroys the enzymatic activity of phospholipases A2. We report that pure derivatized nigexine has no lethal detectable activity to mice whereas it still possesses a residual 20% cytotoxic activity, as compared with the parental compound.

[Basic phospholipase from the venom of Naja nigricollis is cytotoxic]. / La phospholipase basique du venin de Naja nigricollis est cytotoxique.

The basic phospholipase A2 purified from the venom of Naja nigricollis (Institut Pasteur), possesses an intense cytotoxic activity toward fetal cells from FL strain. At a concentration equal to 1.6 x 10(-6) M, the PLA2 lyses 50% of the cells present in a suspension containing 3.5 x 10(6) cells per millilitre. Other PLA2 from various origins do not exhibit such cytotoxic activity.

Dimeric character of a basic phospholipase A2 from cobra venom: experimental and modelling study.

When it is gel filtered on Sephadex in the absence of calcium ions, basic phospholipase A2 from Naja nigricollis venom elutes as a dimer. In order to study the possibility of this dimerization from a structural point of view, three-dimensional models of both monomeric and dimeric N. nigricollis phospholipases A2 have been graphically built on the basis of homologies with the phospholipases A2 from pancreatic bovine and Crotalus atrox venom. The building of a dimeric model is made possible by the deletion of a particular loop of the bovine structure. The predicted models of N. nigricollis phospholipase A2 have been checked using molecular mechanics and molecular dynamics techniques according to a suitable protocol which has been developed starting from refined X-ray structures of phospholipases A2 as the test case. The observed stability of the dimeric model, in the absence of calcium, agrees with the hypothesis of the dimerization of the basic phospholipase A2. Particularly, Arg31, which replaces the hydrophobic residue present in pancreatic bovine and C.atrox venom phospholipases A2, contributes to this stability.

Nigexine, a phospholipase A2 from cobra venom with cytotoxic properties not related to esterase activity. Purification, amino acid sequence, and biological properties.

The venoms of the Naja species are known to be cytotoxic. This toxicity has been attributed to the presence of small nonenzymatic polypeptides of 60 amino acid residues, designated as cardiotoxins or cytotoxins. We investigated the cytotoxic potency of Naja nigricollis venom fractions and isolated another type of cytotoxic component which is even more potent than cardiotoxins. This cytotoxic compound, which was designated as nigexine, was purified to homogeneity and its amino acid sequence was determined. Nigexine is a basic phospholipase A2 consisting of a single chain of 118 amino acids. A detailed investigation of the cytotoxic effects on epithelial FL cells, C-13T neuroblastoma cells, and promyelocytic leukemia HL 60 cells revealed that nigexine not only altered cell viability but also prevented cell proliferation. This is a property that was specific to nigexine since other phospholipases A2 from various sources had no detectable cytotoxic activity. The cytotoxic activity of nigexine was not dependent on the presence of divalent cations, unlike its enzymatic activity. In particular, the cytotoxic activity of nigexine was identical in the presence or absence of either 2 mM Ca2+ or Sr2+, or 6 mM EDTA. We also present evidence based on chemical modifications that cytotoxic activity was not correlated with enzymatic activity. Thus, modification with parabromophenacyl bromide totally abolished the enzymatic activity of nigexine, which nevertheless retained 6-20% of the cytotoxicity of native nigexine. Conversely, treatment with cyanogen bromide gave a compound that retained 7% of the enzymatic activity of the parent molecule but was devoid of detectable cytotoxicity.

Tryptophan 110, a residue involved in the toxic activity but not in the enzymatic activity of notexin.

We prepared two derivatives of notexin, a phospholipase A2 from Notechis scutatus scutatus venom, by modifying the protein with 2-nitrophenylsulfenylchloride, a tryptophan-specific reagent. One derivative was modified at both tryptophans 20 and 110 whereas the other was modified at tryptophan 20. Evidence based on circular dichroic analysis and antigenicity towards a notexin-specific monoclonal antibody indicated that derivatization at both tryptophans did not affect the tertiary structure of notexin. Concomitant modification of tryptophans 20 and 110 induced a marked decrease in the capacity of notexin to kill mice and to block neuromuscular transmission in the chick biventer cervicis preparation, whereas selective modification at tryptophan 20 had no effect on the lethal properties of notexin. This implies that the decrease in the lethal properties of notexin after derivatization was due to modification at tryptophan 110. However, the diderivatized notexin retained full enzymatic activity, implying that neither tryptophan 20 and tryptophan 110 are involved in the catalytic function of the molecule. We conclude that notexin harbours two functional sites. One of them corresponds to the enzymatic site, whereas the other, which includes tryptophan 110, provides specific toxic characteristics to notexin. By reference to previous crystallographic studies, the relative spatial positions of elements involved in toxicity and the catalytic site, we propose a possible orientation of notexin with respect to its putative membrane-bound target.

Electron microscopy of alpha 2-macroglobulin with a thiol ester bound ligand.

In order to covalently bind the hydrolyzed thiol ester groups of the human alpha 2-macroglobulin (alpha 2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M(r) = 13,000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed alpha 2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of alpha 2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in alpha 2M. They also suggest that the four thiol esters are located near the center of the alpha 2M molecule.

The cytoplasmic/transmembrane domain of dipeptidyl peptidase IV, a type II glycoprotein, contains an apical targeting signal that does not specifically interact with lipid rafts.

We investigated the signals involved in the apical targeting of dipeptidyl peptidase IV (DPP IV/CD26), an archetypal type II transmembrane glycoprotein. A secretory construct, corresponding to the DPP IV ectodomain, was first stably expressed in both the enterocytic-like cell line Caco-2 and the epithelial kidney MDCK cells. Most of the secretory form of the protein was delivered apically in MDCK cells, whereas secretion was 60% basolateral in Caco-2 cells, indicating that DPP IV ectodomain targeting is cell-type-dependent. A chimera (CTM-GFP) containing only the cytoplasmic and transmembrane domains of mouse DPP IV plus the green fluorescent protein was then studied. In both cell lines, this chimera was preferentially expressed at the apical membrane. By contrast, a secretory form of GFP was randomly secreted, indicating that GFP by itself does not contain cryptic targeting information. Comparison of the sequence of the transmembrane domain of DPP IV and several other apically targeted proteins does not show any consensus, suggesting that the apical targeting signal may be conformational. Neither the DPP IV nor the CTM-GFP chimera was enriched in lipid rafts. Together these results indicate that, besides the well-known raft-dependent apical targeting pathway, the fate of the CTM domain of DPP IV may reveal a new raft-independent apical pathway.