Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 48
Filter
Add more filters










Publication year range
1.
Acta Crystallogr D Biol Crystallogr ; 57(Pt 11): 1677-9, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11679742

ABSTRACT

Numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence which belongs to the cathelicidin family of proteins. The three-dimensional structure of this cathelicidin motif, which contains two disulfide bonds, has not yet been reported. The cathelicidin motif (ProS) of the protegrin-3 precursor was overexpressed in Escherichia coli as a His-tagged protein. The His(6) tag was removed by thrombin cleavage. ProS was purified to homogeneity and single crystals were obtained by the hanging-drop vapour-diffusion method at pH 3-4. Preliminary X-ray diffraction analysis indicated that these crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 51.42, c = 134.25 A. These crystals diffracted beyond 2.75 A (1.9 A at ESRF) and contain one molecule per asymmetric unit.


Subject(s)
Proteins/chemistry , Amino Acid Motifs , Antimicrobial Cationic Peptides , Blood Proteins/chemistry , Crystallization , Crystallography, X-Ray , Protein Conformation , Protein Precursors/chemistry , Recombinant Proteins/chemistry
2.
J Pharmacol Exp Ther ; 296(1): 124-31, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11123372

ABSTRACT

Doxorubicin delivery to the brain is often restricted because of the poor transport of this therapeutic molecule through the blood-brain barrier (BBB). To overcome this problem, we have recently developed a technology, Pep:trans, based on short natural-derived peptides that are able to cross efficiently the BBB without compromising its integrity. In this study, we have used the in situ mouse brain perfusion method to evaluate the brain uptake of free and vectorized doxorubicin. Doxorubicin was coupled covalently to small peptide vectors: L-SynB1 (18 amino acids), L-SynB3 (10 amino acids), and its enantio form D-SynB3. We first confirmed the very low brain uptake of free radiolabeled doxorubicin, which is most likely due to the efflux activity of the P-glycoprotein at the level of the BBB. Vectorization with either L-SynB1, L-SynB3, or D-SynB3 significantly increased the brain uptake of doxorubicin (about 30-fold). We also investigated the mechanism of transport of vectorized doxorubicin. We show that vectorized doxorubicin uses a saturable transport mechanism to cross the BBB. The effect of poly(L-lysine) and protamine, endocytosis inhibitors, on the transport across the brain was also investigated. Both inhibitors reduced the brain uptake of vectorized doxorubicin in a dose-dependent manner. These studies indicate that the transport of vectorized doxorubicin appears to occur via an adsorptive-mediated endocytosis.


Subject(s)
Brain/metabolism , Doxorubicin/analogs & derivatives , Peptides/pharmacokinetics , Algorithms , Amino Acid Sequence , Animals , Blood-Brain Barrier , Brain/blood supply , Doxorubicin/administration & dosage , Doxorubicin/metabolism , Doxorubicin/pharmacokinetics , Endocytosis/drug effects , Functional Laterality , In Vitro Techniques , Kinetics , Male , Mice , Microcirculation , Molecular Sequence Data , Peptides/administration & dosage , Peptides/metabolism , Perfusion , Polylysine/pharmacology , Stereoisomerism
3.
Biochemistry ; 39(47): 14436-47, 2000 Nov 28.
Article in English | MEDLINE | ID: mdl-11087396

ABSTRACT

MGD-1 is a 39-residue defensin-like peptide isolated from the edible Mediterranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synthesis and an easy way to improve the yield of the four native disulfide bonds. Synthetic and native MGD-1 display similar antibacterial activity, suggesting that the hydroxylation of Trp28 observed in native MGD-1 is not involved in the antimicrobial effect. The three-dimensional solution structure of MGD-1 has been established using (1)H NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta-strands (Arg20-Cys25 and Cys33-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabilized alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 disulfide bond which is solvent-exposed, the three others belong to the particularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positive activity, suggesting that the fourth disulfide bond of MGD-1 is not essential for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typical distribution of positively charged and hydrophobic side chains. The positively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that the rather well conserved location of certain positively charged residues and of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.


Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Bivalvia/chemistry , Defensins , Disulfides/chemistry , Proteins/chemistry , Proteins/physiology , Amino Acid Motifs , Animals , Anti-Infective Agents/chemical synthesis , Arginine/chemistry , Circular Dichroism , Computer Simulation , Cysteine/chemistry , Cystine/chemistry , Disulfides/chemical synthesis , Glycine/chemistry , Microbial Sensitivity Tests , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Oxidation-Reduction , Proline/chemistry , Protein Conformation , Protein Structure, Secondary , Proteins/chemical synthesis , Sequence Alignment , Solutions
4.
Biochem Biophys Res Commun ; 273(2): 649-53, 2000 Jul 05.
Article in English | MEDLINE | ID: mdl-10873659

ABSTRACT

BetaIGH3 protein has been recently involved in the pathogenesis of blinding corneal diseases, some of which have characteristic amyloid corneal deposits. The 124 codon of the betaig-h3 gene seems to be crucial for the amyloidogenicity of the protein product. We presently report an in vitro system that reproducibly forms amyloid fibrils from betaIGH3((110-131)) derived peptides. We also assessed the differences in fibril formation of two 22-amino acid peptides centered on the 124 residue: the native form and the Arg124Cys peptide (mutation linked to lattice corneal amyloid dystrophy type 1). After dialysis of Arg124Cys peptide against PBS 1/15 M pH 7.4 for 72 hours, Congo red staining and electron microscopy demonstrated the presence of abundant material fulfilling the criteria of amyloid. Quantitative analysis with thioflavine T fluorescence studies confirmed the high capacity of Arg124Cys peptide to form amyloid fibrils when compared to the native form.


Subject(s)
Amyloid/biosynthesis , Corneal Dystrophies, Hereditary/genetics , Extracellular Matrix Proteins , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Transforming Growth Factor beta , Amino Acid Sequence , Chemical Precipitation , Corneal Dystrophies, Hereditary/etiology , Corneal Dystrophies, Hereditary/metabolism , Humans , In Vitro Techniques , Microscopy, Electron , Molecular Sequence Data , Mutagenesis, Site-Directed , Neoplasm Proteins/chemistry , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism
5.
Biol Reprod ; 62(6): 1551-63, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10819755

ABSTRACT

LHRH Statin is a putative gonadal protein that increases the interval between two consecutive LHRH pulses. The present work was aimed at analyzing the immunological homology between LHRH Statin and the N-terminal region of the alphaC subunit of inhibin. Thus, rete testis fluid (RTF) proteins were purified by immunoaffinity chromatography using antibodies against residues 1-7 plus 7-30 (experiment 1, A-fractions) and 14-28 of the alphaC inhibin subunit (experiment 2, B-fractions), and the LHRH Statin activity of the fractions was examined by intracerebroventricular administration in castrated rams followed by RIA of plasma LH levels in 15-min blood samples. Fractions that bound to the immunoaffinity column with low affinity were eluted with 0.5 M NaCl, pH 7.4 (-F2); then highly bound fractions were eluted sequentially in acidic (pH 2.5, -F3) followed by basic conditions (pH 11.5, -F4). In experiment 1, RTF (40 microg, n = 4) and highly bound fractions (A-F3, 30 ng, n = 8, 150 ng, n = 3; A-F4, 120 ng, n = 5) decreased LH mean plasma levels between 4 and 6 h after injection by 39%, 29%, 43%, and 37%, respectively (P<0.001 to 0.01), while the weakly bound fractions (A-F2, 180 ng, n = 4) and albumin control (40 microg, n = 4) had no activity. In experiment 2, RTF (100 microg, n = 4) and B-F3 (100 ng, n = 3) decreased plasma LH levels by 48% and 38%, respectively (P<0.001 to 0.05), whereas B-F4 (100 ng, n = 4) and albumin control (100 microg, n = 4) had no effect. A fraction obtained from B-F3 by gel filtration had significant LHRH Statin activity (63%, n = 6, P<0.001). PAGE with colloidal gold staining revealed 3 high molecular weight bands and 5 low molecular weight bands in B-F3. The 3 high molecular weight bands were shown to belong to the clusterin family and did not appear to have LHRH Statin activity. The 5 low molecular weight bands were all labeled by anti-alphaC inhibin antibodies. Collectively, these results strongly suggest that LHRH Statin has some homology with the 14-28 alphaC inhibin sequence.


Subject(s)
Body Fluids/chemistry , Epitopes/immunology , Gonadotropin-Releasing Hormone/immunology , Inhibins , Peptides/immunology , Rete Testis/metabolism , Sheep , Animals , Antibody Specificity , Chromatography, Affinity , Epitopes/analysis , Epitopes/chemistry , Gonadotropin-Releasing Hormone/isolation & purification , Gonadotropin-Releasing Hormone/pharmacology , Immunologic Techniques , Luteinizing Hormone/blood , Male , Peptides/analysis , Peptides/isolation & purification , Sequence Homology
6.
Biochemistry ; 38(47): 15459-69, 1999 Nov 23.
Article in English | MEDLINE | ID: mdl-10569928

ABSTRACT

The ability of caldesmon to inhibit actomyosin ATPase activity involves the interaction of three nonsequential segments of caldesmon domain 4 (amino acids 600-756) with actin. Two of these contacts are located in the C-terminal half of this region of caldesmon which has been designated domain 4b (658-756). To investigate the spatial relationship between the two sites and to determine whether their corresponding contacts on actin are sequentially distinct, we have used NMR spectroscopy to compare the actin binding properties of the minimal inhibitory peptide LW30 comprising residues 693-722 with those of the recombinant domain 4b constructs 658C (658-756) and Cg1 (a mutant of 658C in which the sequence (691)Glu-Trp-Leu-Thr-Lys-Thr(696) is changed to Pro-Gly-His-Tyr-Asn-Asn). Cg1 retains dual-sited actin attachment but displays lowered actin affinity. In the presence of tropomyosin, domain 4b-actin contacts were stronger but not qualitatively different, indicating that tropomyosin affected the conformational equilibrium of caldesmon binding. Simultaneous dual-sited attachment of domain 4b to actin is enabled by the conformational properties of the site-spanning sequence common to 658C, Cg1, and LW30 as reflected in the corresponding NOE and other NMR spectral parameters. A backbone turn region ((713)Gly-Asp-Val-Ser(716)) preceded by an extended segment (Ser(702)-Pro-Ala-Pro-Lys-Pro) acts to constrain the relative disposition of the flanking actin contact sites of domain 4b. In tests with a library of actin peptides, only the C-terminus, 350-375, bound to 658C and LW30. The use of Cu(2+) as a paramagnetic spectral probe bound to the unique His-371 provided evidence of a well-defined geometry for the complex between LW30 and actin residues 350-375 with the N-terminal, site B of domain 4b close to the C-terminal residues of actin. The data are discussed in the context of the potentiation of inhibitory activity by tropomyosin.


Subject(s)
Actins/chemistry , Calmodulin-Binding Proteins/chemistry , Peptide Fragments/chemistry , Actins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Chickens , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular/methods , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation , Protein Structure, Tertiary/genetics , Rabbits , Tropomyosin/chemistry , Tropomyosin/metabolism
7.
J Mol Graph Model ; 17(5-6): 292-309, 1999.
Article in English | MEDLINE | ID: mdl-10840689

ABSTRACT

The process by which analogs in peptide chemistry are currently designed does not include any quantitative basis for amino acid substitutions from pharmacological leads. Here, we show that substitution matrices such as PAM 250 can provide quantitative constraints compatible with biological activity. This article describes its use in a strategy of rational amino acid substitution in peptides and proteins: we have computed a chemically derived matrix equivalent to the well-known PAM 250 matrix, reflecting the natural mutability rates of amino acids in protein evolutions but that can be extended to all the noncoded amino acids. Some of these noncoded amino acids are widely used to mimic secondary structure, to constrain backbone conformation, or to evade protease degradation. An automated sequence mutation (ASM) strategy has been defined to generate mutations within constraints. Application of such a substitution matrix to quantitative structure-function relationship studies will be of use in the design of proteins and peptides destined to become pharmaceutical drugs. In particular, issues such as which functionally conserved substitutions are able to satisfy conformational restrictions, oral bioavailability, or formulation demands can be quantitatively addressed.


Subject(s)
Endothelin-1/chemistry , Oxytocin/chemistry , Amino Acid Substitution , Animals , Automation , Endothelin-1/analogs & derivatives , Endothelin-1/genetics , Mutagenesis , Oligopeptides/chemistry , Oxytocin/genetics , Peptides/chemistry , Rats , Structure-Activity Relationship , Swine
8.
Nat Biotechnol ; 16(8): 748-52, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9702773

ABSTRACT

We describe the rational design of immunosuppressive peptides without relying on information regarding their receptors or mechanisms of action. The design strategy uses a variety of topological and shape descriptors in combination with an analysis of molecular dynamics trajectories for the identification of potential drug candidates. This strategy was applied to the development of immunosuppressive peptides with enhanced potency. The lead compounds were peptides, derived from the heavy chain of HLA class I, that modulate immune responses in vitro and in vivo. In particular, a peptide derived from HLA-B2702, amino acids 75-84 (2702.75-84) prolonged skin and heart allograft survival in mice. The biological activity of the rationally designed peptides was tested in a heterotopic mouse heart allograft model. The molecule predicted to be most potent displayed an immunosuppressive activity approximately 100 times higher than the lead compound.


Subject(s)
Computer-Aided Design , Drug Design , Graft Survival/drug effects , Immunosuppressive Agents , Peptides , Animals , Computer Simulation , Consensus Sequence , Drug Evaluation, Preclinical , Heart Transplantation , Histocompatibility Antigens Class I , Humans , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/therapeutic use , Ligands , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Peptide Library , Peptides/chemistry , Peptides/pharmacology , Peptides/therapeutic use , Protein Conformation , T-Lymphocytes, Cytotoxic/immunology , Transplantation, Homologous/immunology
9.
Eur J Biochem ; 253(1): 221-8, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9578480

ABSTRACT

Ranalexin, a 20-residue peptide isolated from the skin of the bullfrog Rana catesbeiana displays antimicrobial activity. This peptide contains two cysteine residues in positions 14 and 20 linked by a disulphide bridge. Ranalexin was chemically synthesised and close antimicrobial activities were measured for the reduced and oxidised forms. The solution structure of ranalexin was determined by using circular dichroism, proton NMR spectroscopy and molecular modelling techniques. The reduced and oxidised forms of ranalexin are mainly unstructured in water but display an alpha-helical structure spanning residues 8-15 and 8-17, respectively, in a trifluoroethanol/water mixture (3:7, by vol.). Ranalexin was found to interact with micelles of dodecylphosphocholine and to adopt a similar helical structure. Moreover, slow-exchanging amide protons located on the same side of the helix suggest that the hydrophobic face of the helix lies on the micelle surface. Hydrophobic residues of the poorly structured N-terminal part which are important for the biological activity are also involved in the interaction with micelles. Taken together, the results suggest that the disulphide bond does not strongly affect either the conformation or the antimicrobial activity of ranalexin.


Subject(s)
Anti-Bacterial Agents/chemistry , Peptides, Cyclic/chemistry , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Circular Dichroism , Cysteine/chemistry , Deuterium , Escherichia coli/drug effects , Magnetic Resonance Spectroscopy , Micelles , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Phosphorylcholine/analogs & derivatives , Protein Conformation , Protein Structure, Secondary , Rana catesbeiana , Skin/chemistry , Solutions , Staphylococcus epidermidis/drug effects , Trifluoroethanol , Water
10.
FEBS Lett ; 421(3): 263-7, 1998 Jan 16.
Article in English | MEDLINE | ID: mdl-9468319

ABSTRACT

Protegrins are members of a family of five Cys-rich naturally occurring cationic antimicrobial peptides. The NMR solution structure of protegrin-1 (PG-1) has been previously determined as a monomeric beta-hairpin both in water and in dimethylsulfoxide solution. Protegrins are bactericidal peptides but their mechanism of action is still unknown. In order to investigate the structural basis of their cytotoxicity, we studied the effect of lipid micelles on the structure of PG-1. The NMR study reported in the present work indicates that PG-1 adopts a dimeric structure when it binds to dodecylphosphocholine micelles. Moreover, the amide proton exchange study suggests the possibility of an association between several dimers.


Subject(s)
Anti-Infective Agents/chemistry , Oligopeptides/chemistry , Phosphorylcholine/analogs & derivatives , Proteins/chemistry , Anti-Infective Agents/metabolism , Antimicrobial Cationic Peptides , Micelles , Nuclear Magnetic Resonance, Biomolecular , Oligopeptides/metabolism , Phosphorylcholine/metabolism , Phosphorylcholine/pharmacology , Protein Conformation , Proteins/metabolism , Protons , Titrimetry
11.
J Biol Chem ; 272(34): 21027-36, 1997 Aug 22.
Article in English | MEDLINE | ID: mdl-9261104

ABSTRACT

To study the vasopressin receptor domains involved in the hormonal binding, we synthesized natural and modified fragments of V1a vasopressin receptor and tested their abilities to affect hormone-receptor interactions. Natural fragments mimicking the external loops one, two, and three were able to inhibit specific vasopressin binding to V1a receptor. In contrast, the natural N-terminal part of the V1a vasopressin receptor was found inactive. One fragment, derived from the external second loop and containing an additional C-terminal cysteine amide, was able to fully inhibit the specific binding of both labeled vasopressin agonist and antagonist to rat liver V1a vasopressin receptor and the vasopressin-sensitive phospholipase C of WRK1 cells. The peptide-mediated inhibition involved specific interactions between the V1a receptor and synthetic V1a vasopressin receptor fragment since 1) it was dependent upon the vasopressin receptor subtype tested (Ki(app) for the peptide: 3.7, 14.6, and 64.5 microM for displacing [3H]vasopressin from rat V1a, V1b, and V2 receptors, respectively; 2) it was specific and did not affect sarcosin 1-angiotensin II binding to rat liver membranes; 3) it was not mimicked by vasopressin receptor unrelated peptides exhibiting putative detergent properties; and 4) no direct interaction between [3H]vasopressin and synthetic peptide linked to an affinity chromatography column could be observed. Such an inhibition affected both the maximal binding capacity of the V1a vasopressin receptor and its affinity for the labeled hormone, depending upon the dose of synthetic peptide used and was partially irreversible. Structure-activity studies using a serie of synthetic fragments revealed the importance of their size and cysteinyl composition. These data indicate that some peptides mimicking extracellular loops of the V1a vasopressin receptor may interact with the vasopressin receptor itself and modify its coupling with phospholipase C.


Subject(s)
Arginine Vasopressin/metabolism , Receptors, Vasopressin/chemistry , 1-Sarcosine-8-Isoleucine Angiotensin II/metabolism , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , Cell Membrane/metabolism , Cysteine/chemistry , Female , Inositol Phosphates/metabolism , Kinetics , Liver/metabolism , Molecular Sequence Data , Peptide Fragments/metabolism , Rats , Rats, Wistar , Tumor Cells, Cultured , Type C Phospholipases/metabolism
13.
Eur J Biochem ; 237(3): 575-83, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8647100

ABSTRACT

Protegrins are members of a family of five Cys-rich, cationic antimicrobial peptides recently isolated from porcine cells. We have synthesised an 18-amino-acid peptide that corresponds to protegrin-1. After Cys oxidation, the peptide has bactericidal activity against gram-positive and gram-negative bacteria, similar to that described for the natural peptide. The solution structure of protegrin-1 was investigated by means of 1H-NMR spectroscopy in water and in (CD3)2SO, with distance-geometry and simulated-annealing calculations. The C6-C15 and C8-C13 disulfide pattern was determined on the basis of NMR-derived constraints. These two parallel disulfide bridges stabilised a beta-sheet structure which comprised two antiparallel strands (residues 5-9 and 12-16) linked by a distorted beta-turn (residues 9-12). The N-terminus and C-terminus were essentially disordered. The distribution of hydrophobic and hydrophilic residues at the peptide surface was found to be a structural feature shared with tachyplesin-1, a related peptide which displays cytolytic activity, and, to a lesser extent, with mammalian defensins. These findings led us to assume that the distribution pattern could be required for the cytolytic activity of these peptides.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/chemical synthesis , Proteins/chemistry , Proteins/chemical synthesis , Amino Acid Sequence , Animals , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Molecular Structure , Protein Conformation , Proteins/genetics , Rabbits , Sequence Homology, Amino Acid , Solutions , Swine , Thermodynamics
14.
FEBS Lett ; 383(1-2): 93-8, 1996 Mar 25.
Article in English | MEDLINE | ID: mdl-8612801

ABSTRACT

Protegrin 1 (PG-1) is a naturally occurring cationic antimicrobial peptide that is 18 residues long, has an aminated carboxy terminus and contains two disulphide bridges. Here, we investigated the antimicrobial activity of PG-1 and three linear analogues. Then, the membrane permeabilisation induced by these peptides was studied upon Xenopus laevis oocytes by electrophysiological methods. From the results obtained, we concluded that protegrin is able to form anion channels. Moreover, it seems clear that the presence of disulphide bridges is a prerequisite for the pore formation at the membrane level and not for the antimicrobial activity.


Subject(s)
Cell Membrane Permeability/drug effects , Disulfides/pharmacology , Proteins/pharmacology , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides , Blood Proteins/pharmacology , Calcium/metabolism , Defensins , Disulfides/chemistry , Escherichia coli/drug effects , Ion Channels/drug effects , Ion Channels/metabolism , Microbial Sensitivity Tests , Molecular Sequence Data , Oocytes , Patch-Clamp Techniques , Peptides/chemistry , Peptides/pharmacology , Proteins/chemistry , Sequence Alignment , Staphylococcus/drug effects , Structure-Activity Relationship , Xenopus laevis
15.
Peptides ; 17(3): 521-6, 1996.
Article in English | MEDLINE | ID: mdl-8735982

ABSTRACT

Endothelin binds to receptors belonging to the family of G-protein-coupled receptors with an N-terminal extracellular domain that is suspected to be part of the binding site. We have synthesized different peptides of this N-terminal extracellular domain and analyzed the increase in calcium concentration ([Ca2+]i) induced by these peptides in the MEG-01 cell line and their influence on the ET-1 concentration-effect response. Nt (20-79) exhibited a partial agonistic effect on [Ca2+]i and blunted the functional response of ET-1 in MEG-01 cells, but was not able to compete with radiolabeled ET-1 binding. The agonist effect was inhibited by the ET receptor antagonists PD 142893 and BQ123, suggesting an interaction between Nt (20-79) and the ETA receptor at a site that could be different from the one of ET-1.


Subject(s)
Endothelin-1/antagonists & inhibitors , Megakaryocytes/metabolism , Peptide Fragments/metabolism , Receptors, Endothelin/metabolism , Amino Acid Sequence , Animals , Binding Sites , Calcium/metabolism , Cattle , Cell Line , Dose-Response Relationship, Drug , Humans , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Protein Binding , Protein Conformation
16.
J Mol Graph ; 13(6): 356-67, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8820304

ABSTRACT

Fifty-four steroid homologs, belonging to the series of 17-spirolactones, were modelled by molecular and quantum mechanics. We studied the affinity of these compounds for the cytosolic mineralocorticoid receptor by way of various parameters describing each structure and its molecular properties. After the failure of a classic preliminary QSAR study, demonstrating the nonlinear relationships between affinity and structural descriptors, we constructed a model allowing us to predict the affinity of new compounds. Our method is based on simple graphic tools coupled to a cluster significance analysis. A complementary study of the activity relating the prediction of the antagonist/agonist character of 37 high-affinity compounds was also carried out using the same methodology. The principal electronic and structural characteristics leading to a selective activity were revealed.


Subject(s)
Computer Simulation , Mineralocorticoids/pharmacology , Models, Molecular , Spironolactone/chemistry , Spironolactone/pharmacology , Animals , Cluster Analysis , Mineralocorticoid Receptor Antagonists , Mineralocorticoids/chemistry , Mineralocorticoids/metabolism , Rats , Receptors, Mineralocorticoid/agonists , Receptors, Mineralocorticoid/metabolism , Spironolactone/metabolism , Structure-Activity Relationship
17.
Biochemistry ; 34(42): 13825-32, 1995 Oct 24.
Article in English | MEDLINE | ID: mdl-7577976

ABSTRACT

The binding of Ca2+ and Mg2+ to four calmodulins (SynCaM 1, SynCaM 8, SynCaM 12A, and SynCaM 18A) has been studied by ESI-MS. The mass spectra were recorded by dissolving the apoproteins in methanol/water (20/80, v/v) containing 1 mM CaCl2 or 1 mM MgCl2 and the pH adjusted to 6.0 with ammonia. The carrier solvent was methanol/water (20/80, v/v). In the case of Ca2+ complexation, ESI-MS reveals the presence of three kinds of sites: the first of high affinity corresponding to those determined using flow and equilibrium dialysis techniques and two others with lower affinities. These results clearly confirm the conclusion of Milos et al. [Milos, M., Comte, M., Schaer, J. J., & Cox, J. A. (1989) J. Inorg. Biochem. 36, 11-25] that there should exist between four and six auxiliary sites for Ca2+. Concerning the complexation of magnesium, the four proteins are able to bind two Mg2+ almost certainly on auxiliary cationic sites.


Subject(s)
Calcium/metabolism , Calmodulin/metabolism , Magnesium/metabolism , Amino Acid Sequence , Binding Sites , Calmodulin/chemistry , Mass Spectrometry , Molecular Sequence Data , Mutation
18.
AIDS Res Hum Retroviruses ; 11(10): 1177-86, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8573373

ABSTRACT

To determine whether the amino acid sequence extending from residue 273 to residue 288 in the second conserved region C2 of the HIV-1 envelope glycoprotein represents a target for antibodies on monomeric and oligomerized HIV-1 gp120env, we characterized several antisera and monoclonal antibodies (MAb) raised against C2 synthetic peptides. A cross-reactive epitope was evidenced on HIV-1Lai and HIV-1Eli C2-derived peptides, but was not encountered on HIV-2 C2-derived peptide. This epitope was found to be expressed on the native monomeric gp120env but was not detected in the context of oligomeric Env, suggesting this region is sequestered in the oligomeric molecule. Preincubation of oligomeric Env with sCD4 apparently failed to expose this epitope. Our results suggest that the amino acid sequence extending from residue 273 to residue 288 in C2 of HIV-1 gp120env may be involved in intermolecular interaction within the oligomeric Env complex.


Subject(s)
HIV Antigens/chemistry , HIV Envelope Protein gp120/chemistry , HIV-1/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Cell Line , Female , HIV Antibodies/immunology , HIV Antigens/immunology , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Conformation
20.
J Biol Chem ; 270(15): 8867-76, 1995 Apr 14.
Article in English | MEDLINE | ID: mdl-7721794

ABSTRACT

Earlier, we proposed that the interaction of gizzard calponin with F-actin, promoting the inhibition of the actomyosin ATPase activity, involves the NH2-terminal portion of the calponin segment Ala145-Tyr182 (Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). In this work, we have directly probed this region for actin binding sites using five peptide analogs covering different stretches of the sequence Thr133-Ile163. Co-sedimentation with F-actin, actomyosin ATPase measurements, and zero-length cross-linking reactions demonstrated that the 19-residue sequence Ala145-Ile163 is essential for actin interaction and ATPase inhibition. Furthermore, each peptide was tested for binding to the Ca(2+)-dependent proteins, caltropin and calmodulin, in both an actomyosin ATPase assay and an affinity chromatographic assay. The results revealed the 11-residue segment Gln153-Ile163, representing the COOH-terminal moiety of the F-actin binding sequence, as a crucial region for the high affinity binding of these regulatory proteins with concomitant removal of the ATPase inhibition. The 153-163 stretch contained also interactive sites for tropomyosin as assessed by affinity chromatography and spectrofluorometry. Collectively, the data support our initial results and highlight the ability of the multifunctional 145-163 region to serve as a potent regulatory domain of the smooth muscle calponin.


Subject(s)
Actins/metabolism , Calcium-Binding Proteins/metabolism , Tropomyosin/metabolism , Amino Acid Sequence , Animals , Binding Sites , Calcium-Binding Proteins/chemistry , Cattle , Chickens , Chymotrypsin/metabolism , Hydrolysis , Microfilament Proteins , Molecular Sequence Data , Myosins/antagonists & inhibitors , Protein Binding , Rabbits , Turkeys , Calponins
SELECTION OF CITATIONS
SEARCH DETAIL
...