RÉSUMÉ
Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.
Sujet(s)
Protéines de transport/métabolisme , Hepacivirus/métabolisme , Hépatite C chronique/virologie , Protéines virales non structurales/métabolisme , Protéines adaptatrices de la transduction du signal/métabolisme , Protéines adaptatrices du transport vésiculaire/métabolisme , Antiviraux/pharmacologie , Antiviraux/usage thérapeutique , Protéines de transport/antagonistes et inhibiteurs , Protéines de transport/composition chimique , Protéines de transport/génétique , Résistance virale aux médicaments/génétique , Hepacivirus/enzymologie , Hepacivirus/génétique , Hépatite C chronique/traitement médicamenteux , Humains , Protéines et peptides de signalisation intracellulaire , Protéines mitochondriales/antagonistes et inhibiteurs , Protéines mitochondriales/composition chimique , Protéines mitochondriales/génétique , Protéines mitochondriales/métabolisme , Mutation , Nucleoside-triphosphatase/antagonistes et inhibiteurs , Nucleoside-triphosphatase/composition chimique , Nucleoside-triphosphatase/génétique , Nucleoside-triphosphatase/métabolisme , Protein Tyrosine Phosphatase, Non-Receptor Type 2/métabolisme , RNA helicases/antagonistes et inhibiteurs , RNA helicases/composition chimique , RNA helicases/génétique , RNA helicases/métabolisme , Protéases à sérine/composition chimique , Protéases à sérine/génétique , Protéases à sérine/métabolisme , Inhibiteurs de la sérine protéinase/pharmacologie , Inhibiteurs de la sérine protéinase/usage thérapeutique , Transduction du signal , Protéines virales non structurales/antagonistes et inhibiteurs , Protéines virales non structurales/composition chimique , Protéines virales non structurales/génétique , Réplication viraleRÉSUMÉ
Chronic infection with hepatitis C virus (HCV) affects 130 million people worldwide and is a major cause of liver cirrhosis and liver cancer. After translation of the HCV RNA genome into a polyprotein, 2 viral proteases process its non-structural protein (NS) region. While the essential chymotrypsin-like serine protease NS3-4A mediates all cleavages downstream of NS3, the NS2-3 cysteine protease catalyzes a vital cleavage at the NS2/3 site. Protease activity of NS2-3 has been described to require, besides NS2, the N-terminal 181 aa of NS3. The latter domain corresponds to the NS3 serine protease domain and contains a structural Zn(2+)-binding site with functional importance for both viral proteases. The catalytic triad of the NS2-3 protease resides in NS2; the role of the NS3 part in proteolysis remained largely undefined. Here we report a basal proteolytic activity for NS2 followed by only 2 amino acids of NS3. Basal activity could be dramatically enhanced by the NS3 Zn(2+)-binding domain (NS3 amino acids 81-213) not only in cis but also in trans which, however, required a more extended N-terminal part of NS3 downstream of NS2 in cis. Thus, this study defines for the first time (i) NS2 as a bona fide protease, (ii) NS3 as its regulatory cofactor, and (iii) functional subdomains in NS3 that cooperate in NS2 protease activation. These findings give new mechanistic insights into function and regulation of the NS2 protease and have important implications for the development of anti-HCV therapeutics.
Sujet(s)
Cysteine endopeptidases/métabolisme , Hepacivirus/composition chimique , Protéines virales non structurales/métabolisme , Sites de fixation , Structure tertiaire des protéines , ZincRÉSUMÉ
A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein-protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFbeta pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.
Sujet(s)
Hépatite C/métabolisme , Protéines virales/métabolisme , Hepacivirus/métabolisme , Hepacivirus/physiologie , Humains , Liaison aux protéines , Techniques de double hybrideRÉSUMÉ
A clinical study was carried out to compare the response rate of two groups of non-responder (NR) hepatitis C virus (HCV) genotype 1 chronically infected patients treated with interferon and ribavirin, with or without amantadine. The viral load decreased more markedly in the group treated by tritherapy including amantadine, but the response rate at the end of treatment was not significantly different between bitherapy and tritherapy. As amantadine could have an antiviral effect on the ion channel activity of the p7 HCV protein, the p7 quasispecies was characterized by cloning and sequencing. Sequence data were analyzed to determine the pattern and significance of p7 genetic heterogeneity and a possible relationship with therapy. Subtype differences were confirmed between p7 HCV genotypes 1a and 1b, and quasispecies analysis showed a reduction of genetic diversity in subtype 1a, but not 1b, during tritherapy. However, the absence of changes at numerous positions, as well as the conservative changes at other positions, indicated the high conservation of the p7 structure. Residue His-17, proposed to interact with amantadine, was fully conserved in both subtypes 1a and 1b, independently of amantadine administration. In conclusion, although the analysis of the p7 sequences revealed a selective pressure during therapy, no specific residues appeared to be linked to the effect of amantadine on viral decline. These results suggest that the potential antiviral effect of amantadine might be non-specific and related to a reduction in endosomal acidification and therefore reduced viral entry of HCV via its pH-dependent pathway.
Sujet(s)
Amantadine , Antiviraux , Variation génétique , Hepacivirus/effets des médicaments et des substances chimiques , Hépatite C chronique/traitement médicamenteux , Interféron alpha , Ribavirine , Protéines virales/effets des médicaments et des substances chimiques , Adulte , Sujet âgé , Amantadine/administration et posologie , Amantadine/pharmacologie , Amantadine/usage thérapeutique , Séquence d'acides aminés , Antiviraux/administration et posologie , Antiviraux/pharmacologie , Antiviraux/usage thérapeutique , Association de médicaments , Femelle , Hepacivirus/classification , Hepacivirus/génétique , Hépatite C chronique/virologie , Humains , Interféron alpha-2 , Interféron alpha/administration et posologie , Interféron alpha/pharmacologie , Interféron alpha/usage thérapeutique , Mâle , Adulte d'âge moyen , Données de séquences moléculaires , Protéines recombinantes , Ribavirine/administration et posologie , Ribavirine/pharmacologie , Ribavirine/usage thérapeutique , Analyse de séquence d'ADN , Résultat thérapeutique , Protéines virales/composition chimique , Protéines virales/génétiqueRÉSUMÉ
48 year old man with chronic obstructive pulmonary disease (COPD) secondary to pulmonary hypertension with domiciliary non-invasive ventilation was seen. He came to the emergency department with acute exacerbation of COPD. The patient was admitted to the Cardiology Service with the diagnosis of congestive heart failure. Diagnostic imaging (chest X-ray, transthoracic Doppler-echocardiography, multidetector row spiral CT and myocardial perfusion imaging) revealed an enlarged right ventricle. ECG was consistent with right ventricular failure. The heart perfusion imaging (pharmacologic stress testing with dobutamine) showed cor pulmonale and right ventricle ischemia induced by drug stress with dobutamine. Although right ventricle myocardial chronic dysfunction rarely causes right ventricular failure, it can occur when cor pulmonale and ischemia heart disease are present.
Sujet(s)
Ischémie myocardique/complications , Broncho-pneumopathie chronique obstructive/complications , Coeur pulmonaire/étiologie , Diabète de type 2/complications , Dobutamine , Échocardiographie-doppler , Échocardiographie de stress , Coeur/imagerie diagnostique , Défaillance cardiaque/diagnostic , Défaillance cardiaque/étiologie , Ventricules cardiaques , Humains , Hypercholestérolémie/complications , Hypertrophie ventriculaire droite/complications , Hypertrophie ventriculaire droite/diagnostic , Mâle , Adulte d'âge moyen , Ischémie myocardique/diagnostic , Coeur pulmonaire/diagnostic , Scintigraphie , Insuffisance respiratoire/étiologie , Fumer/effets indésirables , Débit systolique , Tomodensitométrie hélicoïdaleRÉSUMÉ
UNLABELLED: The purpose of this study was to compare helical CT with lung perfusion scintigraphy (LPS) as the initial investigations of patients with suspected pulmonary embolism (PE). PATIENTS AND METHOD: A total of 54 patients with clinically suspected acute PE were studied retrospectively. Each patient was assigned a pretest clinical probability of acute PE (very likely, 90%; possible, 50%; unlikely, 10%). Within 72 hours of presentation, patients underwent LPS and contrast material-enhanced helical CT. Perfusion LS was classified following the PISAPED criteria as normal or near normal; abnormal consistent with PE or abnormal not consistent with PE. Helical CT studies were categorized as positive for PE, negative for PE or non-diagnostic. The standard reference was a consensus based on LS, helical CT and clinical outcome. RESULTS: In 38 of the 54 patients, the results of LS a hCT were concordant, 13 with PE and 25 without. There were 4 indeterminate hCT. In 12 patients LS and hCT were discordant. There were 4 LS false negative; 2 with parenchyma damage and 2 chronic PE. There were 5 LS false negative; 3 extrinsic vascular compressions and one low clinical probability. There was 1 hCT false positive because of breathing artifact and 2 false negatives because of subsegmental emboli. CONCLUSION: Accurate diagnosis of acute PE is possible combining perfusion scanning and clinical probability. Helical CT has added information in patients with discordant clinical probability and perfusion lung scan results. Helical CT demonstrated lesions other than PE considered responsible for the patient's symptoms, but it was insensitive to embolism of subsegmental branches.
Sujet(s)
Embolie pulmonaire/imagerie diagnostique , Tomodensitométrie , Adulte , Sujet âgé , Sujet âgé de 80 ans ou plus , Femelle , Humains , Mâle , Adulte d'âge moyen , Scintigraphie , Études rétrospectivesRÉSUMÉ
The influence of viral factors on the severity of hepatitis C virus (HCV)-related liver disease is controversial. We studied 68 liver transplant patients with recurrent hepatitis C, of whom 53 were infected by genotype 1 strains. Relationships between core sequences, serum HCV RNA levels, and fibrosis scores for each patient were analyzed in pairwise fashion 5 years after transplantation. We used Mantel's test, a matrix correlation method, to evaluate the correspondence between measured genetic distances and observed phenotypic differences. No clear relationship was found when all 68 patients were analyzed. In contrast, when the 53 patients infected by genotype 1 strains were analyzed, a strong positive relationship was found between genetic distance and differences in 5-year fibrosis scores (P = 0.001) and differences in virus load (P = 0.009). In other words, the smaller the genetic distance between two patients' viral core sequences, the smaller the difference between the two patients' fibrosis scores and viral replication levels. No relationship was found between genetic distance and differences in age, sex, or immunosuppression. In multivariate analysis, the degree of fibrosis was negatively related to the virus load (r = -0.68; P = 0.003). In the particular setting of liver transplantation, and among strains with closely related phylogenetic backgrounds (genotype 1), this study points to a correlation between the HCV genetic sequence and the variability of disease expression.
Sujet(s)
Hepacivirus/génétique , Hépatite C chronique/physiopathologie , Hépatite C chronique/chirurgie , Transplantation hépatique , Famille multigénique , Génotype , Hepacivirus/isolement et purification , Hépatite C chronique/virologie , Humains , Analyse multifactorielle , Phénotype , Phylogenèse , Récidive , Charge viraleRÉSUMÉ
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.
Sujet(s)
Hepacivirus/enzymologie , RNA replicase/métabolisme , Séquence d'acides aminés , Séquence nucléotidique , Lignée cellulaire , Membrane cellulaire/métabolisme , Membrane cellulaire/virologie , Amorces ADN , Réticulum endoplasmique/métabolisme , Technique d'immunofluorescence , Humains , Données de séquences moléculaires , Biosynthèse des protéines , Similitude de séquences d'acides aminés , Tétracycline/pharmacologie , Transcription génétique , Protéines virales non structurales/composition chimique , Protéines virales non structurales/métabolismeRÉSUMÉ
Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.
Sujet(s)
Hepacivirus/physiologie , Hepacivirus/pathogénicité , Protéines de l'enveloppe virale/composition chimique , Protéines virales/composition chimique , Protéines virales/génétique , Séquence d'acides aminés , Séquence conservée , Hepacivirus/génétique , Hépatite C/traitement médicamenteux , Hépatite C/virologie , Humains , Interféron gamma/usage thérapeutique , Données de séquences moléculaires , Conformation des protéines , Analyse de séquence d'ADN , Relation structure-activitéRÉSUMÉ
The Bacillus subtilis catabolite repression HPr (Crh) exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system. We report here that Crh preparations contain a mixture of monomers and homodimers, whereas HPr is known to be monomeric in solution. The dissociation rate of dimers is very slow (t1/2 of about 10 hours), and the percentage of dimers in Crh preparations increases with rising temperature or protein concentration. However, at temperatures above 25 degrees C and a protein concentration of 10 mg/ml, Crh dimers slowly aggregate. Typically, NMR spectra recorded at 25 degrees C showed the coexistence of both forms of Crh, while in Crh solutions kept at 35 degrees C, almost exclusively Crh monomers could be detected. Circular dichroism analysis revealed that the monomeric and dimeric forms of Crh are well folded and exhibit the same overall structure. The physiological significance of the slow Crh monomer/dimer equilibrium remains enigmatic.
Sujet(s)
Bacillus subtilis/métabolisme , Phosphoenolpyruvate-fructose phosphotransferase/composition chimique , Phosphoprotéines/composition chimique , Phosphoprotéines/métabolisme , Protéines bactériennes/composition chimique , Dichroïsme circulaire , Dimérisation , Résonance magnétique nucléaire biomoléculaire , Phosphoenolpyruvate-fructose phosphotransferase/métabolisme , Conformation des protéines , Protéines recombinantes/composition chimiqueRÉSUMÉ
Sodium dodecyl sulfate (SDS) has consistently been shown to induce secondary structure, particularly alpha-helices, in polypeptides, and is commonly used to model membrane and other hydrophobic environments. However, the precise mechanism by which SDS induces these conformational changes remains unclear. To examine the role of electrostatic interactions in this mechanism, we have designed two hydrophilic, charged amphipathic alpha-helical peptides, one basic (QAPAYKKAAKKLAES) and the other acidic (QAPAYEEAAEELAKS), and their structures were studied by CD and NMR. The design of the peptides is based on the sequence of the segment of residues 56-70 of human platelet factor 4 [PF4(56-70), QAPLYKKIIKKLLES]. Both peptides were unstructured in water, and in the presence of neutral, zwitterionic, or cationic detergents. However, in SDS at neutral pH, the basic peptide folded into an alpha-helix. By contrast, the pH needed to be lowered to 1.8 before alpha-helix formation was observed for the acidic peptide. Strong, attractive electrostatic interactions, between the anionic groups of SDS and the cationic groups of the lysines, appeared to be necessary to initiate the folding of the basic peptide. NMR analysis showed that the basic peptide was fully embedded in SDS-peptide micelles, and that its three-dimensional alpha-helical structure could be superimposed on that of the native structure of PF4(56-70). These results enabled us to propose a working model of the basic peptide-SDS complex, and a mechanism for SDS-induced alpha-helical folding. This study demonstrates that, while the folding of peptides is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of SDS-induced structure.
Sujet(s)
Peptides/composition chimique , Séquence d'acides aminés , Dichroïsme circulaire , Humains , Techniques in vitro , Spectroscopie par résonance magnétique , Modèles moléculaires , Données de séquences moléculaires , Fragments peptidiques/composition chimique , Fragments peptidiques/effets des médicaments et des substances chimiques , Peptides/effets des médicaments et des substances chimiques , Facteur-4 plaquettaire/composition chimique , Facteur-4 plaquettaire/effets des médicaments et des substances chimiques , Pliage des protéines , Structure secondaire des protéines/effets des médicaments et des substances chimiques , Dodécyl-sulfate de sodium/pharmacologie , Électricité statique , Tensioactifs/pharmacologieRÉSUMÉ
Oligomerization of viral envelope proteins is essential to control virus assembly and fusion. The transmembrane domains (TMDs) of hepatitis C virus envelope glycoproteins E1 and E2 have been shown to play multiple functions during the biogenesis of E1E2 heterodimer. This makes them very unique among known transmembrane sequences. In this report, we used alanine scanning insertion mutagenesis in the TMDs of E1 and E2 to examine their role in the assembly of E1E2 heterodimer. Alanine insertion within the center of the TMDs of E1 or E2 or in the N-terminal part of the TMD of E1 dramatically reduced heterodimerization, demonstrating the essential role played by these domains in the assembly of hepatitis C virus envelope glycoproteins. To better understand the alanine scanning data obtained for the TMD of E1 which contains GXXXG motifs, we analyzed by circular dichroism and nuclear magnetic resonance the three-dimensional structure of the E1-(350-370) peptide encompassing the N-terminal sequence of the TMD of E1 involved in heterodimerization. Alanine scanning results and the three-dimensional molecular model we obtained provide the first framework for a molecular level understanding of the mechanism of hepatitis C virus envelope glycoprotein heterodimerization.
Sujet(s)
Membrane cellulaire/composition chimique , Membrane cellulaire/métabolisme , Alanine/composition chimique , Séquence d'acides aminés , Antigènes CD4/métabolisme , Lignée cellulaire , Chromatographie en phase liquide à haute performance , Dichroïsme circulaire , Dimérisation , Réticulum endoplasmique/métabolisme , Humains , Spectroscopie par résonance magnétique , Données de séquences moléculaires , Mutagenèse dirigée , Oligosaccharides/métabolisme , Peptides/synthèse chimique , Plasmides/métabolisme , Tests aux précipitines , Pliage des protéines , Structure tertiaire des protéines , Relation structure-activité , Facteurs temps , Cellules cancéreuses en culture , Rayons ultraviolets , Protéines de l'enveloppe virale/composition chimique , Protéines de l'enveloppe virale/physiologieRÉSUMÉ
For most membrane proteins, the transmembrane domain (TMD) is more than just an anchor to the membrane. The TMDs of hepatitis C virus (HCV) envelope proteins E1 and E2 are extreme examples of the multifunctionality of such membrane-spanning sequences. Indeed, they possess a signal sequence function in their C-terminal half, play a major role in endoplasmic reticulum localization of E1 and E2, and are potentially involved in the assembly of these envelope proteins. These multiple functions are supposed to be essential for the formation of the viral envelope. As for the other viruses of the family Flaviviridae, these anchor domains are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved charged residue. Replacement of these charged residues by an alanine in HCV envelope proteins led to an alteration of all of the functions performed by their TMDs, indicating that these functions are tightly linked together. These data suggest that the charged residues of the TMDs of HCV glycoproteins play a key role in the formation of the viral envelope.
Sujet(s)
Hepacivirus/métabolisme , Protéines de l'enveloppe virale/composition chimique , Protéines de l'enveloppe virale/métabolisme , Séquence d'acides aminés , Lignée cellulaire , Membrane cellulaire/composition chimique , Membrane cellulaire/métabolisme , Réticulum endoplasmique/métabolisme , Flaviviridae/composition chimique , Flaviviridae/génétique , Hepacivirus/génétique , Données de séquences moléculaires , Mutation , Structure tertiaire des protéines , Protéines de fusion recombinantes/composition chimique , Protéines de fusion recombinantes/métabolisme , Analyse de séquence d'ADN , Protéines de l'enveloppe virale/génétiqueRÉSUMÉ
The existence of an extrahepatic reservoir of hepatitis C virus (HCV) is suggested by differences in quasispecies composition between the liver, peripheral blood mononuclear cells, and serum. We studied HCV RNA compartmentalization in the plasma of nine patients, in CD19(+), CD8(+), and CD4(+) positively selected cells, and also in the negatively selected cell fraction (NF). HCV RNA was detected in all plasma samples, in seven of nine CD19(+), three of eight CD8(+), and one of nine CD4(+) cell samples, and in seven of eight NF cells. Cloning and sequencing of HVR1 in two patients showed a sequence grouping: quasispecies from a given compartment (all studied compartments for one patient and CD8(+) and NF for the other) were statistically more genetically like each other than like quasispecies from any other compartment. The characteristics of amino acid and nucleotide substitutions suggested the same structural constraints on HVR1, even in very divergent strains from the cellular compartments, and homogeneous selection pressure on the different compartments. These findings demonstrate the compartmental distribution of HCV quasispecies within peripheral blood cell subsets and have important implications for the study of extrahepatic HCV replication and interaction with the immune system.
Sujet(s)
Hepacivirus/classification , Hepacivirus/isolement et purification , Hépatite C/virologie , Agranulocytes/virologie , Séquence d'acides aminés , Acides aminés/analyse , Clonage moléculaire , Variation génétique , Hepacivirus/composition chimique , Hepacivirus/génétique , Hépatite C/sang , Humains , Foie/virologie , Sous-populations de lymphocytes/virologie , Données de séquences moléculaires , Phylogenèse , ARN viral/sang , RT-PCR , Analyse de séquence d'ADNRÉSUMÉ
Presentation of one case of ammonium-magnesium phosphate calculi breakdown using systemic medical treatment with oral Acetohydroxamine acid dosed at 125 mg/8 h and antibiotic therapy based on the antibiogram results. This type of treatment is generally used in an attempt to eradicate the infection, and as prophylaxis of relapse following surgical treatment or SWEL in these difficult to eradicate calculi due to their high tendency to recur.
Sujet(s)
Calculs rénaux/composition chimique , Calculs rénaux/traitement médicamenteux , Composés du magnésium/analyse , Phosphates/analyse , Sujet âgé , Humains , MâleRÉSUMÉ
Type XIV collagen, a fibril-associated collagen with interrupted triple helices (FACIT), interacts with the surrounding extracellular matrix and/or with cells via its binding to glycosaminoglycans (GAGs). To further characterize such interactions in the NC1 domain of chicken collagen XIV, we identified amino acids essential for heparin binding by affinity chromatography analysis after proteolytic digestion of the synthetic peptide NC1(84-116). The 3D structure of this peptide was then obtained using circular dichroism and NMR. The NC1(84-116) peptide appeared poorly structured in water, but the stabilization of its conformation by the interaction with hydrophobic surfaces or by using cosolvents (TFE, SDS) revealed a high propensity to adopt an alpha-helical folding. A 3D structure model of NC1(84-116), calculated from NMR data recorded in a TFE/water mixture, showed that the NC1-heparin binding site forms a amphipathic alpha-helix exhibiting a twisted basic groove. It is structurally similar to the consensus spatial alpha-helix model of heparin-binding [Margalit et al. (1993) J. Biol. Chem. 268, 19228-19231], except that the GAG binding domain of NC1 may be extended over 18 residues, that is, the NC1(94-111) segment. In addition, the formation of a hydrophobic groove upon helix formation suggests the contribution of additional sequences to ensure the stability of the GAG-binding domain. Overall the NC1(84-116) model exhibits a nativelike conformation which presents suitably oriented residues for the interaction with a specific GAG.
Sujet(s)
Collagène/composition chimique , Collagène/métabolisme , Glycoprotéines/composition chimique , Glycoprotéines/métabolisme , Héparine/composition chimique , Héparine/métabolisme , Fragments peptidiques/composition chimique , Fragments peptidiques/métabolisme , Séquence d'acides aminés , Animaux , Sites de fixation , Poulets , Dichroïsme circulaire , Simulation numérique , Cristallographie aux rayons X , Glycosaminoglycanes/composition chimique , Glycosaminoglycanes/métabolisme , Modèles moléculaires , Données de séquences moléculaires , Résonance magnétique nucléaire biomoléculaire/méthodes , Liaison aux protéines , Structure secondaire des protéines , Structure tertiaire des protéinesRÉSUMÉ
Collagen XIV is known to bind to the dermatan sulfate chain of decorin and to the heparan sulfate chain of perlecan. To study its possible interaction with glycosaminoglycans, the NC1 domain of chicken collagen XIV was overproduced in E. coli. Purified NC1*(6-119)* appears poorly organized (the asterisks indicate the presence of extension sequences), but V8-protease generated fragments containing the 84-108 basic sequence tend to fold into alpha-helix. These fragments interact specifically with heparin, which induces an alpha-helical fold with a maximum effect for equimolar heparin/peptide ratio. These data demonstrate the existence of a glycosaminoglycan binding site in NC1.
Sujet(s)
Collagène/composition chimique , Glycoprotéines/composition chimique , Héparine/métabolisme , Séquence d'acides aminés , Animaux , Sites de fixation , Poulets , Collagène/métabolisme , Glycoprotéines/métabolisme , Données de séquences moléculaires , Liaison aux protéinesRÉSUMÉ
P-glycoprotein (Pgp) is a plasma membrane protein known as an ATP-dependent drug-efflux pump that confers multidrug resistance to tumor cells. Structural analysis of Pgp was investigated by circular dichroism (CD) for the first time and in combination with amino acid sequence analysis. CD of highly purified Pgp from human, rat and murine Pgp-overexpressing drug resistant cells revealed slight variations in the spectral shape when recorded in the presence of dodecyl maltoside (DM). These species-dependent variations in CD shapes resulted from the interaction of the oligosaccharidic part with the protein core since they were abolished either in the presence of sodium dodecyl sulfate (SDS) or after deglycosylation, the latter not altering the Pgp ATP-dependent drug transport activity. Whatever the level of Pgp glycosylation and the detergent used (SDS or DM), the content in secondary structure deduced from deconvolution of CD spectra is almost the same for the three sources of Pgp and estimated to 43% alpha-helix, 16% beta-sheet, 15% beta-turn and 26% of other structures. These data, which constitute the first report of Pgp structure analysis by circular dichroism, are consistent with the 48% alpha-helix and 16% beta-sheets global contents predicted by using recently reported efficient secondary structure prediction methods. This consistency reinforces the reliability of the probable nature and localization of predicted Pgp secondary structure elements. This provides a good framework for precise 3D structure modeling of Pgp by homology with proteins of known 3D structure, as it is illustrated here for the A motifs of the ATP-binding domains of Pgp.
Sujet(s)
Glycoprotéine P/composition chimique , Structure secondaire des protéines , Analyse de séquence , Séquence d'acides aminés , Animaux , Carcinome hépatocellulaire , Dichroïsme circulaire , Glycosylation , Humains , Leucémie lymphoïde , Souris , Données de séquences moléculaires , Rats , Cellules cancéreuses en cultureRÉSUMÉ
Japanese studies have defined the discrete 2209-2248 amino acid region of the non-structural 5A protein (NS5A(2209-2248)) of hepatitis C virus genotype 1b (HCV 1b) isolates as the interferon sensitivity determining region (ISDR). European studies did not confirm these results since most of the ISDR sequences harboured an intermediate profile. Recently, a direct interaction between the NS5A protein, involving the ISDR, and the interferon-induced protein kinase (PKR) has been reported and presented as a possible explanation of HCV interferon resistance. In the present study, the entire NS5A amino acid sequence from 11 resistant and eight sensitive strains from European HCV 1b isolates was inferred from direct sequencing. The previously described important amino acid stretches and positions in NS5A were compared between the resistant and sensitive groups. Although some variations were observed, no clear differences could be directly correlated with the interferon sensitivity. However, sensitive strains were different, owing to more amino acid changes when compared to a consensus sequence from all strains. The carboxy-terminal region and especially the previously reported NS5A/V3 region showed most of the variations. Moreover, the conformational analysis of NS5A by secondary structure prediction allowed the differentiation of most sensitive strains from resistant ones. It was concluded that other regions different from ISDR were involved in resistance to interferon maybe via the interaction between NS5A and PKR.