[Prions and their biology]. / Los priones y su biología.

Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative disorders featured by an aberrant metabolism of a cellular membrane glycoprotein, the prion protein (PrP-C). PrP-C is being related to Cu (II) homeostasis and postulated as candidate for cell signaling and cell adhesion functions. Under pathological conditions PrP-C converts into a conformational isomer (PrP*, PrP-res, PrP-Sc, PrP-Creutzfeldt-Jakob disease, etc.). In addition to divergent biochemical characteristics, the latter form displays the property of recognizing the normal protein and transforming it into its homologue. Conversion process is ill tangled and participation of yet unidentified partners has been postulated.

Los priones y su biología / Prions and their biology

Las prionopatías o encefalopatías espongiformes transmisibles son un conjunto de neurodegeneraciones letales de mamíferos cuyo denominador común es el metabolismo aberrante de una glicoproteína celular de membrana, denominada proteína del prion (PrP-C). Esta proteína, implicada hipotéticamente en la homeostasis del Cu (II) y candidata a desempeñar un papel activo en procesos de señalización y de adhesión celular, en condiciones patológicas se transforma en un isómero conformacional (PrP*, PrP-res, PrP-Sc, PrP-enfermedad de Creutzfeldt-Jakob, etc.). Este último, además de presentar propiedades bioquímicas divergentes, exhibe la propiedad novel de reconocer a la forma normal y transformarla en su homólogo en un proceso en el cual se postula la participación de factores aún no identificados (AU)

Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major methyl donor for transmethylation reactions. Attempts to perform structural studies using rat liver MAT have met with problems because the protein purified from cellular extracts is heterogeneous. Overexpression of the enzyme in Escherichia coli rendered most of the protein as inclusion bodies. These aggregates were purified by specific washes using urea and Triton X-100 and used for refolding. Maximal activity was obtained when chaotropic solubilization included the structural cation Mg(2+), the protein concentration was kept below 0.1 mg/ml, and denaturant removal was carried out in a two-step process, namely, a fast dilution followed by dialysis in the presence of 10 mM DTT or GSH/GSSG redox buffers. Refolding by this procedure generated the oligomeric forms, MAT I and III, which were basically indistinguishable from the purified rat liver forms in secondary structure and catalytic properties.

Biophysical study of the perturbation of model membrane structure caused by seminal plasma protein PDC-109.

PDC-109, the major heparin-binding protein of bull seminal plasma, binds specifically to sperm choline lipids at ejaculation and mediates capacitation by stimulating cholesterol and phospholipid efflux. We carried out a biophysical study to investigate the membrane perturbation effect caused by PDC-109. Binding of PDC-109 to phosphatidylcholine model membranes was maximal at a 12:1 phosphatidylcholine to protein molar ratio. The process was independent of the membrane structure and involved a slight conformational change of the protein, compatible with an increased exposure to the solvent. PDC-109 binding to dimyristoylphosphatidylcholine prevented lipid molecules from participating in the gel-to-liquid phase transition, due to enhancement of both acyl chain disorder and interfacial hydration. Visualization of the lipid-protein complexes by electron microscopy showed surface irregularities and the presence of 10-nm particles. Permeability assays confirmed the PDC-109-induced disruption of the vesicles. This effect was not modified by heparin. However, presence of cholesterol inhibited the process in a concentration-dependent manner.

An optimized amphiphilic cationic peptide as an efficient non-viral gene delivery vector.

BACKGROUND: Due to their chemical definition and reduced size, the use of peptides as gene delivery systems is gaining interest as compared to the more common polymeric non-viral vectors. To achieve gene transfer efficiencies that would make peptides a realistic alternative to existing methods, we have evaluated and attempted to concert those properties with a direct impact on the activity of the system. These considerations have led to the design, synthesis and characterization of a 23-residue cationic peptide which we term RAWA. METHODS: We have characterized RAWA biophysically and functionally. Biophysical studies include evaluation of DNA condensation and membrane perturbing activities. DNA transfer activity has been evaluated in cell culture at controlled DNA-to-peptide stoichiometries, using a luciferase gene as reporter. Requirements for additional effectors such as chloroquine and peptide cofactors have also been considered. RESULTS: RAWA displays in vitro DNA condensing activity similar to that of protamines, reaching maximum effect at a peptide-to-DNA molar charge ratio (CR) of 4 (+/-). The reduced membrane perturbing activity diminishes its cytotoxic potential. In COS-7 cells, transfection efficiency with RAWA peptiplexes, compares favorably with well-recognized systems, including Lipofectamine Plus, Superfect, GenePorter and FuGene. The peptide-associated activity between free and DNA-bound species has been mapped by analyzing dependency on chloroquine treatment. The lack of significant serum inhibition and low toxicity make this system advantageous for potential in vivo application. A ternary complex including the acid-triggered fusogenic JTS-1 peptide is presented as a potential strategy for further in vivo studies. CONCLUSIONS: We have developed a gene delivery system based on an amphipathic cationic peptide with improved DNA condensation ability and reduced cytotoxicity, which maintains membrane binding and perturbing activities. Observed efficiency with this molecule is very high and compares favorably with currently available transfection systems.

Structural domain organization of gastric H+,K+-ATPase and its rearrangement during the catalytic cycle.

Differential scanning calorimetry has been used to characterize the thermal denaturation of gastric (H+,K+)-ATPase. The excess heat capacity function of (H+,K+)-ATPase in highly oriented gastric vesicles displays two peaks at 53.9 degrees C (Tm1) and 61.8 degrees C (Tm2). Its thermal denaturation is an irreversible process that does not exhibit kinetic control and can be resolved in two independent two-state processes. They can be assigned to two cooperative domains located in the cytoplasmic loops of the alpha-subunit, according to the disappearance of the endothermic signal upon removal of these regions by proteinase K digestion. Analysis of the thermal-induced unfolding of the enzyme trapped in different catalytic cycle intermediates has allowed us to get insight into the E1-E2 conformational change. In the E1 forms both transitions are always observed. As Tm1 is shifted to Tm2 by vanadate and ATP interaction, the unfolding mechanism changes from two independent to two sequential two-state transitions, revealing interdomain interactions. Stabilization of the E2 forms results in the disappearance of the second transition at saturation by K+, Mg2+-ATP, and Mg2+-vanadate as well as in significant changes in Tm2 and DeltaH1. The catalytic domain melts following a process in which intermolecular interactions either in the native or in the unfolded state might be involved. Interestingly, the E2-vanadate-K+ form displays intermediate properties between the E1 and E2 conformational families.

Conformational features and thermal stability of bovine seminal plasma protein PDC-109 oligomers and phosphorylcholine-bound complexes.

At ejaculation, PDC-109, the major heparin-binding protein of bull seminal plasma, binds to the phosphorylcholine group of sperm lipids and modulates capacitation promoted by glycosaminoglycans during sperm residence in the female genital tract. Combination of size-exclusion chromatography, analytical ultracentrifugation, circular dichroism, Fourier-transform infrared spectroscopy, and differential scanning calorimetry has allowed us to biophysically characterize PDC-109 and its interaction with phosphorylcholine. PDC-109 can be regarded as a polydisperse molecule whose aggregation state can be modulated by the solute composition of its solution environment. Dissociation of PDC-109 oligomers occurs upon increasing the concentration of either NaCl, EDTA, CaCl2, or phosphorylcholine, suggesting that both ionic and hydrophobic interactions are responsible for the aggregation tendency of PDC-109 monomers. Dissociation processes are accompanied by exposure of peptide bonds to the solvent, changes in the environment of tyrosine and tryptophan residues, and a slight increase in the turn content at the expense of non-regular structure. Analysis of the heat-induced denaturation of PDC-109 oligomers revealed two melting transitions at about 36 degrees C (irreversible) and 55 degrees C (partially reversible) characterized by calorimetric enthalpy changes of 42 kJ/mol and 217 kJ/mol, respectively. These transitions could be assigned to the dissociation of oligomers and to the cooperative unfolding of PDC-109 monomers, respectively. The modulation of the aggregation state of PDC-109 by its molecular environment and by phosphorylcholine binding suggests possible mechanisms for capacitation mediated by the seminal plasma protein.

Biochemical and conformational characterisation of HSP-3, a stallion seminal plasma protein of the cysteine-rich secretory protein (CRISP) family.

HSP-3 is a member of the cysteine-rich secretory protein (CRISP) family from stallion seminal plasma. We report a large-scale purification protocol for native HSP-3. This protein is a non-glycosylated polypeptide chain with a pI of 8-9 and an isotope-averaged molecular mass of 24987 +/- 3 Da. The molecular mass of HSP-3, determined by equilibrium sedimentation, is 26 kDa, showing that the protein exists in solution as a monomer. The concentration of HSP-3 in the seminal plasma of different stallions ranged from 0.3 to 1.3 mg/ml. On average, 0.9-9 million HSP-3 molecules/cell coat the postacrosomal and mid-piece regions of an ejaculated, washed stallion spermatozoon, suggesting a role in sperm physiology. Conformational characterisation of purified HSP-3 was assessed by combination of circular dichroism and Fourier-transform infrared spectroscopies and differential scanning microcalorimetry. Based on secondary structure assignment, HSP-3 may belong to the alpha+beta class of proteins. Thermal denaturation of HSP-3 is irreversible and follows a non-two state transition characterised by a Tm of 64 degrees C, an enthalpy change of 75 kcal/mol, and a van 't Hoff enthalpy of 184 kcal/mol. Analysis of the spectroscopic and calorimetric data indicates the occurrence of aggregation of denatured HSP-3 molecules and suggests the monomer as the cooperative unfolding unit.

Structural characterization of the unligated and choline-bound forms of the major pneumococcal autolysin LytA amidase. Conformational transitions induced by temperature.

The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I' band, results in 19% of alpha-helix and tight loops, 47% of beta-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module.

Structural organization of the major autolysin from Streptococcus pneumoniae.

LytA amidase is the best known bacterial autolysin. It breaks down the N-acetylmuramoyl-L-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae and requires the presence of choline residues in the cell-wall teichoic acids for activity. Genetic experiments have supported the hypothesis that its 36-kDa chain has evolved by the fusion of two independent modules: the NH2-terminal module, responsible for the catalytic activity, and the COOH-terminal module, involved in the attachment to the cell wall. The structural organization of LytA amidase and of its isolated COOH-terminal module (C-LytA) and the variations induced by choline binding have been examined by differential scanning calorimetry and analytical ultracentrifugation. Deconvolution of calorimetric curves have revealed a folding of the polypeptide chain in several independent or quasi-independent cooperative domains. Elementary transitions in C-LytA are close but not identical to those assigned to the COOH-terminal module in the complete amidase, particularly in the absence of choline. These results indicate that the NH2-terminal region of the protein is important for attaining the native tertiary fold of the COOH terminus. Analytical ultracentrifugation studies have shown that LytA exhibits a monomer <--> dimer association equilibrium, through the COOH-terminal part of the molecule. Dimerization is regulated by choline interaction and involves the preferential binding of two molecules of choline per dimer. Sedimentation velocity experiments give frictional ratios of 1.1 for C-LytA monomer and 1.4 for C-LytA and LytA dimers; values that deviated from that of globular rigid particles. When considered together, present results give evidence that LytA amidase might be described as an elongated molecule consisting of at least four domains per subunit (two per module) designated here in as N1, N2, C1, and C2. Intersubunit cooperative interactions through the C2 domain in LytA dimer occur under all experimental conditions, while C-LytA requires the saturation of low affinity choline binding sites. The relevance of the structural features deduced here for LytA amidase is examined in connection with its biological function.

Characterization of the antifungal protein secreted by the mould Aspergillus giganteus.

An antifungal polypeptide (AFP) of 51 amino acid residues, secreted by the mould Aspergillus giganteus, has been purified to homogeneity and characterized. The inhibitory effect of this protein on the growth of different microorganisms has been studied. Whereas the growth of many of the filamentous fungi assayed is inhibited, no effect has been observed against yeasts or bacteria. The minimal concentration for total inhibition of the growth is in the range 6 to 25 microM. The antifungal polypeptide does not produce any effect on the growth of the producing mould. The polypeptide promotes aggregation of acidic phospholipid vesicles. A remarkable resistance to proteolysis and a low hydrogen x deuterium exchange have been observed for this protein. The protein does not show any thermal transition up to 80 degrees C when studied by differential scanning calorimetry and infrared spectroscopy. The uv absorbance, fluorescence emission, and circular dichroism (CD) characteristics of this protein have been studied. The protein exhibits a strong positive band at 230 nm as a prominent feature of the CD spectrum in the far uv region. All the spectroscopical properties of the antifungal protein are highly influenced by the abundance of tyrosine residues. These can be grouped in two different populations, buried and exposed, based on the results of pH-titration experiments. Fourier-transform infrared spectroscopy reveals a high content of beta-structure in AFP. Reduction and carboxy-amidomethylation produces a rather unstructured polypeptide as deduced from its spectroscopical properties.

Analysis of the structural organization and thermal stability of two spermadhesins. Calorimetric, circular dichroic and Fourier-transform infrared spectroscopic studies.

The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel beta-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5 degrees C and 78.6 degrees C, respectively. The calorimetric enthalpy changes (delta Hcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that beta-structure and turns are the major structural element of both PSP-I/PSP-II (53% of beta-sheet, 21% of turns) and aSFP (44% of beta-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, alpha-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-beta-structure proteins, and would support a beta-barrel topology like that predicted for the CUB domain. Other beta-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.

Thermal unfolding of the cytotoxin alpha-sarcin: phospholipid binding induces destabilization of the protein structure.

The effect of membrane binding on the structure and stability of the cytotoxin alpha-sarcin has been studied by differential scanning calorimetry, Fourier-transform infrared and fluorescence spectroscopic techniques. The thermal unfolding of alpha-sarcin in aqueous solution fits into a two-state transition characterized by a transition temperature (Tm) of 52.6 degrees C and a calorimetric enthalpy (delta Hcal) of 136 kcal/mol. Upon interaction with phosphatidylglycerol vesicles, alpha-sarcin undergoes conformational changes, as deduced from the FTIR and fluorescence emission spectra. These changes result in a decreased Tm and delta Hcal values for the thermal unfolding of phospholipid-bound alpha-sarcin. The lower Tm value for lipid-bound alpha-sarcin is also observed at the level of secondary and tertiary structures, based on analyses of both the amide I' infrared spectrum and the tryptophan emission of the protein as a function of temperature, respectively. The results obtained indicate a protein destabilization promoted by the phospholipid interaction.

Spectroscopic characterization of the alkylated alpha-sarcin cytotoxin: analysis of the structural requirements for the protein-lipid bilayer hydrophobic interaction.

alpha-Sarcin is a ribosome-inactivating protein that translocates across lipid bilayers, these two abilities explaining its cytotoxic character. This protein is composed of a single polypeptide chain with two disulfide bridges. Reduction and carboxyamidomethylation of alpha-sarcin results in protein unfolding, based on the results of the spectroscopic characterization of the chemically modified protein. The absorption and fluorescence emission bands of the tryptophan residues of the modified protein appear blue- and red-shifted, respectively. Far-UV circular dichroism analysis reveals the presence of residual secondary structure (beta-strands and turns) in the alkylated protein. This retains its ability to interact with lipid bilayers. It promotes vesicle aggregation, lipid-mixing between bilayers and leakage of the intravesicular aqueous contents. The modified protein tends to abolish the phase transition of acid phospholipids as detected by differential scanning calorimetry and depolarization measurements of fluorescence-labelled vesicles. The protein gain access to vesicle-entrapped trypsin. The fluorescence emission of the tryptophan residues is blue-shifted upon interaction of the protein with the bilayers, and anthracene incorporated into the hydrophobic core of the membranes quenches the tryptophan fluorescence emission of the protein. The secondary structure of the alkylated protein interacting with lipid vesicles has been studied by infrared spectroscopy. An increase in the alpha-helix and turn contents and a concomitant decrease in the beta-structure content are observed upon interaction with the bilayers. The results obtained are discussed in terms of the structural requirements for the interaction of alpha-sarcin with lipid membranes.

Escherichia coli JA221 can suppress the UAG stop signal.

The Escherichia coli strain JA221 can suppress the UAG stop codon, although the existence of an amber suppressor tRNA has not previously been described for this strain. When using a plasmid to express alpha-sarcin, which has TAG as its stop signal, two proteins were obtained: a smaller protein corresponding in size to that of the expected protein, and a larger protein, which could be accounted for by the presence of a second stop codon (TGA) 18 base pairs downstream of the original. This feature of strain JA221 must therefore be considered when using this strain as a host for the production of recombinant proteins.

Substitution of histidine-137 by glutamine abolishes the catalytic activity of the ribosome-inactivating protein alpha-sarcin.

The alpha-sarcin cytotoxin is an extracellular fungal protein that inhibits protein biosynthesis by specifically cleaving one phosphodiester bond of the 28 S rRNA. The His137 residue of alpha-sarcin is suggested to be involved in the catalytic activity of this protein, based on the observed sequence similarity with some fungal ribonucleases. Replacement of this residue by Gln (H137Q mutant variant of alpha-sarcin) abolishes the ribonuclease activity of the protein. This has been demonstrated for an homogeneous preparation of the H137Q alpha-sarcin by measuring its effect against both intact rabbit ribosomes and the homopolymer poly(A). The conformation of H137Q alpha-sarcin is highly similar to that of the wild-type protein, which has been analysed by CD and fluorescence spectroscopy. Both H137Q and wild-type alpha-sarcin exhibit identical CD spectra in the peptide-bond region, indicating that no changes at the level of the secondary structure are produced upon mutation. Only minor differences are observed in both near-UV CD and fluorescence emission spectra in comparison to those of the wild-type protein. Moreover, H137Q alpha-sarcin interacts with phospholipid vesicles, promoting the same effects as the native cytotoxin. Therefore, we propose that His137 is part of the ribonucleolytic active site of the cytotoxin alpha-sarcin.

Membrane interaction of a beta-structure-forming synthetic peptide comprising the 116-139th sequence region of the cytotoxic protein alpha-sarcin.

alpha-Sarcin is a cytotoxic protein that strongly interacts with acid phospholipid vesicles. This interaction exhibits a hydrophobic component although alpha-sarcin is a highly polar protein. A peptide comprising the amino acid sequence corresponding to the 116-139th segment of the alpha-sarcin cytotoxin has been synthesized by a standard fluoren-9-yl-methoxycarbonyl-based solid phase method. Its primary structure is: (116)-NPGPARVIYTYPNKVFCGIIAHTK-(139). Two beta-strands have been predicted in this region of alpha-sarcin, where the less polar stretches of the protein are found. The synthetic peptide interacts with negatively charged large unilamellar vesicles of either natural or synthetic phospholipids. An apparent fragmentation of the vesicles is produced by the peptide based on electron microscopy studies. The peptide promotes leakage of the intravesicular aqueous contents and lipid mixing of bilayers. The packing of the phospholipid molecules is greatly perturbed by the peptide, as deduced from the drastic changes induced by the peptide in cooperative properties associated with the phase transition of the bilayers. At saturating peptide/phospholipid ratios, the phase transition of dimyristoylphosphatidylglycerol vesicles is abolished. All of these effects are saturated at about 0.3 peptide/lipid molar ratio. The peptide adopts a mostly random structure in aqueous solution. A conformation composed of a high proportion of antiparallel beta-sheet is induced as a consequence of the interaction with the phospholipid vesicles in opposition to trifluoroethanol that promotes alpha-helical peptide structures, as deduced from circular dichroism measurements. The obtained results are discussed in terms of the potential involvement of the region comprising residues 116-139 of alpha-sarcin in the hydrophobic interactions of this cytotoxic protein with membranes.

Predictive study of the conformation of the cytotoxic protein alpha-sarcin: a structural model to explain alpha-sarcin-membrane interaction.

Alpha-sarcin is a cytotoxic protein composed of a single polypeptide chain. This protein shows a significant degree of amino acid sequence similarity with a group of several phylogenetically related fungal ribonucleases. The leading member of such a group is ribonuclease T1. Three proteins of this group, ribonucleases T1, Ms and F1, are well known in terms of their crystal structures. These data have been used to propose a conformation for alpha-sarcin. The secondary structure of the cytotoxin would contain one alpha-helix segment as well as around six beta-strands and 14 beta-turns. The folding of these structural motifs is proposed by comparison with the three-dimensional structure of the three proteins from the ribonuclease T1 subfamily. The four longest beta-strands of alpha-sarcin would define an antiparallel beta-sheet structure resulting in a highly hydrophobic domain. The predicted folding for alpha-sarcin is discussed in terms of the ability of this protein to electrostatically and hydrophobically interact with phospholipid vesicles. The proposed conformation would explain how a highly polar protein, such as alpha-sarcin, can produce membrane destabilization resulting in protein translocation across lipid bilayers.