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.

Identification of the active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified. The enzyme was labeled by [3H]DFP, treated by CNBr and the resulting peptides were separated by reverse-phase-HPLC. The main radiolabeled peptide was sequenced. Ser-348, in the following sequence, Gly-Lys-Ser-Tyr-Leu-Gly, was identified as the active site serine. A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area. The consensus sequence surrounding the active site serine in the three known X-prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G-X-S-Y-X-G, where X is a non-conserved amino acid.

Comparison of mitochondrial cationic channels in wild-type and porin-deficient mutant yeast.

Bilayers were formed at the tip of microelectrodes from a suspension of proteoliposomes derived from wild-type and porin-deficient mutant yeast mitochondria. In both preparations, identical cationic channels of large conductance were recorded. This result rules out any relationship between this channel and the outer membrane voltage-dependent anion channel, the activity of which is carried by porin. The ionic selectivity and the voltage-dependence of the yeast cationic channel suggest that it is related to that recently described in mammalian mitochondria. This hypothesis is further supported by the fact that both channels are blocked by a mitochondrial addressing peptide.

A 28 kDa mitochondrial protein is radiolabelled by crosslinking with a 125I-labelled presequence.

A 13-residue peptide containing the first 12 amino acids of the N-terminal part of the signal sequence of yeast cytochrome c oxidase subunit IV is shown by chemical crosslinking to interact with a mitochondrial protein. This result is obtained with mitochondria from four different origins. Submitochondrial localization experiments suggest that the 28 kDa labelled component is present on the outer face of the inner membrane. Since such addressing peptides are imported into mitochondria through the same machinery as protein precursors, the 28 kDa protein might be a component of the translocation apparatus.

Ionic mitochondrial channels: characteristics and possible role in protein translocation.

Most of the mitochondrial proteins are synthesized in the cytoplasm as precursors which are then translocated into the organelle. These precursors have a NH2-terminal extension which functions as a mitochondrial targeting signal. The import process through mitochondrial membranes is voltage-dependent; its mechanism is still unknown. Translocation has been proposed to occur through specific channels, thus, indicating the interest of the study of mitochondrial ionic channels. Two anion channels with different electrical characteristics have been described in the outer and the inner membranes. Using the technique of "Tip-Dip", we have shown the existence of a cation channel of large conductance in mitochondria. The characteristics of this channel differ from that of the other mitochondrial anion channels. A positively charged 13-residue synthetic peptide, with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of yeast cytochrome C oxidase, induces a blockade of the cationic channel. From the characteristics of the blockade, it is likely that the channel could be permeable to the peptide. The specificity of this effect suggests that this channel might be involved in protein translocation.

Vesicle permeabilization by purified soluble oligomers of prion protein: a comparative study of the interaction of oligomers and monomers with lipid membranes.

The conversion of normal cellular prion protein (PrP) into its pathological isoform, scrapie PrP, may occur at the cell surface or, more probably, in late endosomes. The early events leading to the structural conversion of PrP appear to be related to the presence of more or less stable soluble oligomers, which might mediate neurotoxicity. In the current study, we investigate the interaction of alpha-rich PrP monomers and beta-rich size-exclusion-chromatography-purified PrP oligomers with lipid membranes. We compare their structural properties when associated with lipid bilayers and study their propensities to permeabilize the membrane at physiological pH. We also study the influence of the N-terminal flexible region (residues 24-103) by comparing full-length PrP(24-234) and N-terminally truncated PrP(104-234) oligomers. We showed that both 12-subunit oligomers cause an immediate and large increase in the permeability of the membrane, whereas equivalent amounts of monomeric forms cause no detectable leakage. Although the two monomeric PrP constructs undergo an alpha-to-beta conformational change when bound to the negatively charged membrane, only the full-length form of monomeric PrP has a weak fusogenic effect. Finally, the oligomers affect the integrity of the membrane differently from the monomers, independently of the presence of the N-terminal flexible domain. As for other forms of amyloidogenesis, a reasonable mechanism for the toxicity arising from PrP fibrillization must be associated with low-molecular-weight oligomeric intermediates, rather than with mature fibrils. Knowledge of the mechanism of action of these soluble oligomers would have a high impact on the development of novel therapeutic targets.

Preparation of bacterial X-prolyl dipeptidyl aminopeptidase and its stabilization by organic cosolvents.

To obtain large amounts of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp lactis (PepX, E.C. 3.4.14.5), PepX was purified from a commercial L. lactis cell extract. The enzyme was purified in only three steps and the last one was performed by HPLC on a C4 reverse-phase column using acetonitrile as an eluent. Despite its high molecular mass (175 kDa), the enzyme was recovered with a good activity yield (75%). Advantages and drawbacks of this technique compared to the classical ones are discussed. The stability of the enzyme in aqueous solutions and in the presence of 10 water-miscible solvents was also investigated. PepX was found to be stabilized by dimethyl sulfoxide, triglyme, and glycerol.

A peptide-sensitive channel of large conductance is localized on mitochondrial outer membrane.

Applying the technique of 'tip-dip' to mitochondria, we have shown the existence in this organelle of a cationic channel of large conductance, which is blocked by a 13-residue peptide possessing the sequence of the N-terminal extremity of the cytochrome c oxidase subunit IV precursor. To study the submitochondrial localization of the channel, the effect of trypsin on isolated channels and on entire mitochondria were compared. One side of isolated channels is sensitive to trypsin, which eliminates the voltage dependence. Channels isolated from trypsinized mitochondria were devoid of voltage dependence and were blocked by the peptide. This suggests a localization of the channel on the outer membrane. Consistent with this hypothesis, the channel was observed with the highest frequency in outer membrane fractions purified by different procedures, either from bovine adrenal cortex or from rat liver mitochondria. Such a localization is also consistent with digitonin solubilization experiments. The channel was solubilized before the inner membrane marker, cytochrome c oxidase. The orientation of the channel was inferred from its trypsin sensitivity and its potential dependence: a transmembrane potential (inside negative) will close the channel.

Incorporation in lipid bilayers of a large conductance cationic channel from mitochondrial membranes.

Membranes from subcellular fractions of adrenal medulla were incorporated in phospholipid bilayers formed at the tip of microelectrodes. Current fluctuations recorded in the presence of a transmembrane potential revealed the existence of a voltage-dependent channel of large conductance. This channel is characterized by fast kinetics and four conductance levels separated by jumps of 100, 220 and 220 pS in 150 mM NaCl. It is permeant to Na+,K+, tetraethylammonium, Cl- and acetate and has some cation selectivity. Exposure to trypsin or pronase abolished the voltage-dependence. Upon subcellular fractionation, the activity was found to be associated with mitochondria. A similar activity was observed in mitochondrial fractions from other organs. By its kinetics, its selectivity and its potential-dependence, this channel differs from the voltage-dependent anion channel of outer mitochondrial membranes.

[Demonstration of a large-conduction ion channel in subcellular fractions of the adrenal medulla]. / Mise en évidence d'un canal ionique de grande conductance dans des fractions sub-cellulaires de la médullo-surrénale.

The "tip-dip" technique (formation of a lipid bilayer at the tip of a microelectrode) allows the electrophysiological study of organelle membranes. An ionic channel of large conductance has been found by this technique in membrane preparations from adrenal medulla. This channel is voltage-sensitive and it has 4 levels of conductance. Its subcellular origin is to be determined; it is borne by a structure which sediments between 1,500 and 25,000 X g.

Blockade of a mitochondrial cationic channel by an addressing peptide: an electrophysiological study.

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed at the tip of microelectrodes from proteoliposomes derived from mitochondrial membranes. This channel was blocked by a 13-residue peptide with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of cytochrome c oxidase. The blockade was reversible, voltage- and dose-dependent. The peptide did not affect the activity of a Torpedo chloride channel observed under the same conditions. From experiments with phospholipid monolayers, it is unlikely that the peptide inserts into bilayers under the experimental conditions used. The blockade was observed from both sides of the membrane, being characterized by more frequent transitions to the lower conductance states, and a maximum effect was observed around 0 mV. Channels, the gating mechanism of which had been eliminated by exposure to trypsin, were also blocked by the peptide. For trypsinized channels, the duration of the closure decreased and the blockade saturated at potentials below -30 mV. These observations are consistent with a translocation of the peptide through the channel. Dynorphin B, which has the same length and charge as the peptide, had some blocking activity. Introduction of negative charges in the peptide by succinylation suppressed the activity.

[Blockage of a mitochondrial cationic channel by a mitochondrial addressing peptide]. / Blocage d'un canal cationique de mitochondrie par un peptide d'adressage mitochondrial.

A 13-residue peptide, including the sequence of the amino terminal end of cytochrome c oxidase subunit IV precursor, blocks a cationic channel from mitochondrial membranes. The effect is reversible and voltage-dependent. The blocking properties suggest that the peptide plugs the pore. Furthermore, when the transmembrane potential favours transfer, the peptide appears to be able to cross the channel.

Towards a proteomic map of Lactococcus lactis NCDO 763.

Lactococcus lactis is a widely used bacteria in dairy industry, specially in cheese ripening. Numerous lactococcal enzymes and proteins are involved in this process. Proteomics makes it possible to deal with a high number of proteins and identify modification of their patterns in two-dimensional (2-D) gels. However, an annotated reference map is necessary prior to analyzing protein variations. We have begun to construct such a map in easily reproducible conditions and identify proteins.

Purification, crystallization, and preliminary X-ray analysis of PepX, an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 A, b = 102.6 A, and c = 101.6 A, space group P2(1)2(1)2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 A and are suitable for high-resolution structural analysis.

Vesicular stomatitis virus produced from infected LSTRA lymphoma cells bear tyrosine protein kinase activity (p56).

Murine LSTRA lymphoma cells contain a very active tyrosine protein kinase of 56 kDa (p56) which is not related to any of the other known tyrosine kinases. In the past the purification and characterization of the p56 have been hampered because of the low amount of this protein in LSTRA membranes. In this study, we have utilized a different approach for purification which consisted of trapping the protein in the membrane of vesicular stomatitis virus. Incubation of the virions with [gamma-32P]ATP resulted in the phosphorylation of p56 on tyrosine residues. Moreover, the phosphopeptide digest profile of vesicular stomatitis virus-p56 was identical to that observed with authentic LSTRA-p56. The p56 from such virions could be resolved from other proteins by two-dimensional gels, and furthermore, such virions have been used to prepare several antisera directed against the p56.