Dung Le-Nguyen

Active site chemical mutagenesis of ecballium elaterium trypsin inhibitor ii new microproteins inhibiting elastase and chymotrypsin

Seven microproteins analogous to Ecballium elaterium Trypsin Inhibitor II. were synthesized. The study of their inhibiting power showed a change in selectivity from trypsin to elastase for 5 of the compounds and to alpha-chymotrypsin for another one. A striking characteristic that appeared during this synthetic approach was the ability of the 28 amino acid peptides to refold and close correctly the 3 disulfide bridges, giving in each case an active compound.

Renin substrates. Part 2. Rapid solid phase synthesis of the ratine sequence tetradecapeptide using BOP reagent

A successful synthesis of Asp1-Arg2-Val3-Tyr4-IIe5-His6-Pro7-Phe6-His9-Leu10-Leu11-Tyr12-Tyr13-Ser14 has been achieved by the classical stepwise solid-phase method using 1% crosslinked Merrifield resin and benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) respectively as solid support and coupling reagent. The coupling protocol included in situ neutralization of the amino partners after Boc-deprotection and the coupling times seldom exceeded 30 min. For His6 and His9, Boc-His(Boc). DCHA was neutralized in situ and allowed to couple. The peptide, purified by reparative h.p.l.c., was obtained in 29% overall yield based on the first residue attached to the solid support.

Immunological detection of prohormone convertases in two different proglucagon processing cell lines

The distribution of the prohormone convertases, PCI/3, PC2 and PC5/6, was determined by immunoblotting in two cell lines. In αTC1–6 cells, the proglucagon processing occured according to the pancreatic A‐cell type. In STC‐1 cells, proglucagon was processed in a manner reminiscent of the intestinal L‐cell type. PC1/3 was undetectable in both proglucagon processing cell lines whereas PC2 displayed a strong immunostaining in the αTC1–6 cells and was barely detectable in the STC‐1 cells. PC5/6 was detected as a 70 kDa protein in both cell lines. These results suggest a possible role of PC2 in the processing of proglucagon into glucagon in the A‐cells, whereas in L‐cells it would require still undetermined endoproteases.

1H NMR study of the solution structure of sarafotoxin-S6b

Sarafotoxin-S6b has been synthesized and studied by (1)H NMR in 50 50 acetonitrile/water mixture. All spin systems were identified and assigned with the aid of 2D experiments. On the basis of these data, a 3D structure of sarafotoxin is proposed and compared to that of [Nle(7)]endothelin obtained in the same conditions. From this study, it appeared that sarafotoxin-S6b and [Nle(7)]endothelin roughly share the same 3D structure, the main differences being located in the 4-7 loop bearing the sequence variation.

Conformational study of a native monodisulfide bridge analogue of EETI II

Trypsin inhibitor EETI II, possessing six cysteines engaged in three disulfide bridges, shares a common structural motif with other proteins of different origins and functions. To understand the principles that govern folding of this largely distributed basic scaffold, mainly composed of a small triple-stranded β-sheet, we have studied different stages in the folding of EETI II. The conformational properties of a synthetic analogue of EETI II possessing only one native (15–27) disulfide bridge were investigated with the combined use of1H NMR and molecular modelling. Although two native-like reverse turns were observed, formation of β-sheet could not be evidenced in the one disulfide analogue, while the motif has been shown to be present in a folding intermediate with two native disulfide bridges (9–21 and 15–27). These results suggest that the structural motif requires stabilisation by two disulfide bridges.

Miniglucagon: A Local Regulator of Islet Physiology

Abstract: Miniglucagon, or glucagon‐[19–29], is partially processed from glucagon in its target tissues where it modulates the glucagon action. In the islets of Langerhans, the glucagon‐producing A cells contain miniglucagon at a significant level (2–5% of the glucagon content). We studied a possible control of insulin release by miniglucagon using as a model the MIN6 cell line. Miniglucagon, in the 10−14 to 10−9 M range, inhibited insulin release induced by glucose, glucagon, tGLP‐1, or glibenclamide by 85–100% with an IC50 close to 1 pM. While no change in the cyclic AMP content was noted, Ca2+ influx was reduced in parallel with the inhibition of insulin release. Use of pharmacological modulators of L‐type voltage‐sensitive Ca2+ channels and bacterial toxins indicates that miniglucagon blocks insulin release by closing this type of channel via a pertussis toxin‐sensitive G protein. Miniglucagon is a novel, possibly physiologically relevant, local regulator of islet function.

Solution Structure of the Macrocyclic Squash Trypsin Inhibitor MCoTI-II, the First Member of a New Family of Cyclic Knottins

Squash trypsin inhibitors are small disulfide-rich proteins (28–30 residues, 6 cysteines). Their 3D structures revealed that one disulfide crosses the macrocycle formed by the other two disulfide bridges and the interconnecting backbone, hence the term “knottins”. This particular topology has been found in a number of cystine-rich peptides with diverse biological activities isolated from plants or animals. Several of these peptides, such as kalata Bl, circulin A and B and Cycloviolacin 01, display macrocyclic structures. These cyclic knottins were grouped in two subfamilies following sequence comparison [1]. To date, all the reported squash trypsin inhibitors have a linear structure. We recently isolated several cyclic squash trypsin inhibitors from Momordica cochinchinensis seeds [2]. MCoTI-II is the most abundant and its primary structure has been identified as cyclo-(S1 G S D G G V C P K10 I L K K C R R D S D20 C P G A C I C R G N30 G Y C G). Here we report the NMR and molecular modeling studies of this peptide.