Structural requirements of the N-terminal region of GLP-1-[7-37]-NH2 for receptor interaction and cAMP production.

A series of GLP-1-[7-36]-NH(2) (tGLP-1) and GLP-1-[7-37] analogs modified in position 7, 8, 9 and 36, have been designed and evaluated on murine GLP-1 receptors expressed in RIN T3 cells for both their affinity and activity. Ten of the synthesized peptides were found full agonists with activities superior or at least equal to that of the native hormone. Five of them were investigated for their plasmatic stability and the most stable, [a(8)-desR(36)]GLP-1-[7-37]- NH(2) (Compound 8), evaluated in vivo in a glucose tolerance test which confirmed a clearly longer activity than that of the native hormone.

Structure-activity relationships of human urotensin II and related analogues on rat aortic ring contraction.

The sequence of human urotensin II (UII) has been recently established as H-Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH, and it has been reported that UII is the most potent mammalian vasoconstrictor peptide identified so far. A series of UII analogues was synthesized, and the contractile activity of each compound was studied in vitro using de-endothelialised rat aortic rings. Replacement of each amino acid by an L-alanine or by a D-isomer showed that the N- and C-terminal residues flanking the cyclic region of the amidated peptide were relatively tolerant to substitution.

Crystallization and preliminary X-ray crystallographic analysis of human Geminin coiled-coil domain.

Initially discovered in Xenopus laevis, Geminin is a DNA replication initiation inhibitor found in higher eukaryotes. The coiled-coil domain of Human Geminin (termed GemH-37) has been crystallized by the vapor-diffusion sitting-drop method. A complete 1.

BIGH3 (TGFBI) Arg124 mutations influence the amyloid conversion of related peptides in vitro.

Amyloid deposits with Arg124 mutated TGFBI protein have been identified in autosomal dominant blinding corneal dystrophies. We assessed in vitro the mechanisms determining TGFBI protein amyloid transformation involving mutations of Arg124. Eight peptides synthesized following the TGFBI protein sequence, centered on codon Arg124 holding the previously reported amyloidogenic mutations and the respective controls were studied.

Overexpression and structural study of the cathelicidin motif of the protegrin-3 precursor.

Numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence of 96-101 residues, referred to as the cathelicidin motif. The structure of this widespread motif has not yet been reported. The cathelicidin motif of protegrin-3 (ProS) was overexpressed in Escherichia coli as a His-tagged protein to facilitate its purification.

Expression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the protegrin-3 precursor.

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.

Enhanced delivery of doxorubicin into the brain via a peptide-vector-mediated strategy: saturation kinetics and specificity.

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.

Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1).

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.

In vitro creation of amyloid fibrils from native and Arg124Cys mutated betaIGH3((110-131)) peptides, and its relevance for lattice corneal amyloid dystrophy type I.

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.

Peptides quantitative structure-function relationships: an automated mutation strategy to design peptides and pseudopeptides from substitution matrices.

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.

Evidence that luteinizing hormone-releasing hormone statin from ovine rete testis fluid is immunologically related to alphaC inhibin.

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.

The interface between caldesmon domain 4b and subdomain 1 of actin studied by nuclear magnetic resonance spectroscopy.

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).

Computer-assisted rational design of immunosuppressive compounds.

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.

Solution structure of the antimicrobial peptide ranalexin and a study of its interaction with perdeuterated dodecylphosphocholine micelles.

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.

Oligomerization of protegrin-1 in the presence of DPC micelles. A proton high-resolution NMR study.

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.

Synthetic rat V1a vasopressin receptor fragments interfere with vasopressin binding via specific interaction with the receptor.

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.

Synthesis and solution structure of the antimicrobial peptide protegrin-1.

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.

Change in membrane permeability induced by protegrin 1: implication of disulphide bridges for pore formation.

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.

Endothelin receptor synthetic N-terminal fragment interacts with the receptor itself.

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.

Variable mapping of structure-activity relationships: application to 17-spirolactone derivatives with mineralocorticoid activity.

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.

Analysis of the ion binding sites of calmodulin by electrospray ionization mass spectrometry.

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.

Antigenic analysis of HIV type 1 external envelope (Env) glycoprotein C2 region: implication for the structure of Env.

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.

Characterization of the regulatory domain of gizzard calponin. Interactions of the 145-163 region with F-actin, calcium-binding proteins, and tropomyosin.

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.