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. We also performed circular dichroism study and propose a hypothetical structural model explaining the most part of observed activities of GLP-1 analogs on RIN T3 cells.

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. Conversely, replacement of any residue of the cyclic region significantly reduced the contractile activity of the molecule. The octapeptide UII(4-11) was 4 times more potent than UII, indicating that the C-terminal region of the molecule possesses full biological activity. Alanine or D-isomer substitutions in UII(4-11) or in UII(4-11)-NH2, respectively, showed a good correlation with the results obtained for UII-NH2. Disulfide bridge disruption or replacement of the cysteine residues by their D-enantiomers markedly reduced the vasoconstrictor effect of UII and its analogues. In contrast, acetylation of the N-terminal residue of UII and UII-NH2 enhanced the potency of the peptide. Finally, monoiodination of the Tyr6 residue in UII(4-11) increased by 5 fold the potency of the peptide in the aortic ring bioassay. This structure-activity relationship study should provide useful information for the rational design of selective and potent UII receptor agonists and antagonists.

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 1.74 A data set has been collected on a single orthorhombic crystal with unit cell parameters a = 25.25, b = 44.35, c = 68.58 A. Successful molecular replacement shows that GemH-37 is a dimeric parallel coiled-coil. Structural analysis is now in progress.

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. Cys124 and His124 mutated peptide preparations contained significantly higher amounts of amyloid than the native peptide. Blocking the SH group of Cys124 and deleting the first four NH2-terminal amino acids including Val112-Val113 resulted in a decrease in amyloid fibril formation while deletion of the nine CONH2-terminal residues increased amyloid fibril concentration. Fourrier transformed-infrared spectroscopy analysis of the different peptide solutions showed an increase in beta-pleated sheet structures in those with enhanced amyloid yielding. We designed a peptide (BB1) likely to counteract the role of Val112-Val113 in amyloid fibril formation. Incubation of Cys124 peptide with BB1 indeed resulted in a 35% inhibition of amyloid fibril formation. Our results are in keeping with the clinical observations of Arg124 mutation-linked amyloidosis and show the importance of Val112-Val113, disulfide and hydrogen bonding in increasing the beta-pleated conformation and amyloid formation. These findings shed new light on the molecular mechanisms of TGFBI protein amyloidogenesis and encourage further research on the use of specifically designed peptides as putative therapeutic agents for these disabling diseases.

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. The His tag was then removed by thrombin cleavage. In addition, the complete proprotegrin-3 (ProS-PG-3) (120 residues) was overexpressed in baculovirus-infected insect cells. As it contained the antibacterial peptide protegrin-3 in its C-terminal part, ProS-PG-3 contained four disulfide bonds. At neutral pH, ProS and ProS-PG-3 adopted two slowly exchanging conformations that existed in a ratio of 55/45. This ratio was progressively modified at acidic pH to reach a 90/10 value at pH 3.0, suggesting that electrostatic interactions are involved in such a conformational change. Therefore, the structural study of the main conformer was undertaken at pH 3.0 by circular dichroism, mass spectrometry, and homo- and heteronuclear NMR. In parallel, a model for the ProS structure was built from the X-ray structure of the chicken cystatin. ProS and the chicken cystatin share two conserved disulfide bonds as well as a high conservation of hydrophobic residues. The ProS model features the conservation of a hydrophobic core made of the interface between the N-terminal helix and the wrapping beta-sheet. Although the full assignment of the main conformer of ProS could not be obtained, available NMR data validated the presence of the N-terminal helix and of a four-stranded beta-sheet, in agreement with the cystatin fold. Moreover, we clearly demonstrated that ProS and ProS-PG-3 share the same global structure, suggesting that the presence of the highly constrained beta-hairpin of protegrin does not significantly modify the structure of the cathelicidin motif of the protegrin precursor.