Miniglucagon (glucagon 19-29), a potent and efficient inhibitor of secretagogue-induced insulin release through a Ca2+ pathway

Using the MIN6 B-cell line, we investigated the hypothesis that miniglucagon, the C-terminal () fragment processed from glucagon and present in pancreatic A cells, modulates insulin release, and we analyzed its cellular mode of action. We show that, at concentrations ranging from 0.01 to 1000 pM, miniglucagon dose-dependently (ID50 = 1 pM) inhibited by 80-100% the insulin release triggered by glucose, glucagon, glucagon-like peptide-1-(7-36) amide (tGLP-1), or glibenclamide, but not that induced by carbachol. Miniglucagon had no significant effects on cellular cAMP levels. The increase in 45Ca2+ uptake induced by depolarizing agents (glucose or extracellular K+), by glucagon, or by the Ca2+channel agonist Bay K-8644 was blocked by miniglucagon at the doses active on insulin release. Electrophysiological experiments indicated that miniglucagon induces membrane hyperpolarization, probably by opening potassium channels, which terminated glucose-induced electrical activity. Pretreatment with pertussis toxin abolished the effects of miniglucagon on insulin release. It is concluded that miniglucagon is a highly potent and efficient inhibitor of insulin release by closing, via hyperpolarization, voltage-dependent Ca2+ channels linked to a pathway involving a pertussis toxin-sensitive G protein.     

Min-21 and min-23, the smallest peptides that fold like a cystine-stabilized beta-sheet motif: design, solution structure, and thermal stability.

Small disulfide-rich proteins provide examples of simple and stable scaffolds for design purposes. The cystine-stabilized beta-sheet (CSB) motif is one such elementary structural motif and is found in many protein families with no evolutionary relationships. In this paper, we present NMR structural studies and stability measurements of two short peptides of 21 and 23 residues that correspond to the isolated CSB motif taken from a 28-residue squash trypsin inhibitor. The two peptides contain two disulfide bridges instead of three for the parent protein, but were shown to fold in a native-like fashion, indicating that the CSB motif can be considered an autonomous folding unit. The 23-residue peptide was truncated at the N-terminus. It has a well-defined conformation close to that of the parent squash inhibitor, and although less stable than the native protein, it still exhibits a high T(m) of about 100 degrees C. We suggest that this peptide is a very good starting building block for engineering new bioactive molecules by grafting different active or recognition sites onto it. The 21-residue peptide was further shortened by removing two residues in the loop connecting the second and third cysteines. This peptide exhibited a less well-defined conformation and is less stable by about 1 kcal mol(-)(1), but it might be useful if a higher flexibility is desired. The lower stability of the 21-residue peptide is supposed to result from inadequate lengths of segments connecting the first three cysteines, thus providing new insights into the structural determinants of the CSB motif.     

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.     

Knottin cyclization: impact on structure and dynamics

Background  

Present in various species, the knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, knottins are considered as excellent leads or scaffolds in drug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack.     

Glucagon-(19-29) exerts a biphasic action on the liver plasma membrane Ca2+ pump which is mediated by G proteins

We have recently shown that nanomolar concentrations of glucagon-(19-29), which can derive from native glucagon by proteolytic cleavage of the dibasic doublet Arg17-Arg18, inhibit the Ca2+ pump in liver plasma membrane vesicles independently of adenylyl cyclase activation (Mallat, A., Pavoine, C., Dufour, M., Lotersztajn, S., Bataille, D., and Pecker, F. (1987) Nature 325, 620-622). We report here that the regulation of the Ca2+ pump by glucagon-(19-29) is dependent on guanine nucleotides. In the presence of 10 microM guanosine 5'-3-O-(thio) triphosphate (GTP gamma S) or 75 microM GTP, glucagon-(19-29) caused a biphasic regulation of the Ca2+ pump. ATP-dependent Ca2+ transport was inhibited in the presence of 10 pM to 1 nM glucagon-(19-29), while higher concentrations of the peptide (1-100 nM) reversed the inhibition caused by lower ones. GTP gamma S alone, at high concentrations (100 microM), reproduced the inhibitory effect of glucagon-(19-29) and induced a 40% inhibition of the basal activity of the Ca2+ pump which was reversed by low concentrations of glucagon-(19-29) (10 pM to 1 nM). Treatment of rats with cholera toxin resulted in a 70% increase in the basal activity of the Ca2+ pump, a loss of sensitivity to GTP gamma S and to the biphasic regulation by glucagon-(19-29). Treatment with pertussis toxin did not affect the response of the Ca2+ pump to GTP gamma S and glucagon-(19-29). We conclude that glucagon-(19-29) can exert a biphasic effect on the Ca2+ pump which is mediated by G protein(s) sensitive to cholera toxin.     

Conformational study of a native monodisulfide bridge analogue of EETI II

Trypsin inhibitor EETI II, possessing six cysteinesengaged in three disulfide bridges, shares a commonstructural motif with other proteins of differentorigins and functions. To understand the principlesthat govern folding of this largely distributed basicscaffold, mainly composed of a small triple-stranded-sheet, we have studied different stages in thefolding of EETI II. The conformational properties ofa synthetic analogue of EETI II possessing only onenative (15-27) disulfide bridge were investigated withthe combined use of ¹H NMR and molecularmodelling. Although two native-like reverse turns wereobserved, formation of -sheet could not beevidenced in the one disulfide analogue, while themotif has been shown to be present in a foldingintermediate with two native disulfide bridges (9-21and 15-27). These results suggest that the structuralmotif requires stabilisation by two disulfide bridges     

Glucagon is processed to the (19-29) fragment at the level of the hepatocyte membrane

Upon incubation with hepatic plasma membranes, glucagon is processed into its (19-29) C-terminal fragment. This suggests that, in physiological conditions, glucagon is processed in a target tissue at the level of its Arg17-Arg18 basic doublet, leading to the production of a fragment which is known to display an original biological specificity, namely the modulation of the calcium pump present in hepatocyte plasma membrane.     

Solution structure of the squash trypsin inhibitor MCoTI-II. A new family for cyclic knottins.

The "knottin" fold is a stable cysteine-rich scaffold, in which one disulfide crosses the macrocycle made by two other disulfides and the connecting backbone segments. This scaffold is found in several protein families with no evolutionary relationships. In the past few years, several homologous peptides from the Rubiaceae and Violaceae families were shown to define a new structural family based on macrocyclic knottin fold. We recently isolated from Momordica cochinchinensis seeds the first known macrocyclic squash trypsin inhibitors. These compounds are the first members of a new family of cyclic knottins. In this paper, we present NMR structural studies of one of them, MCoTI-II, and of a beta-Asp rearranged form, MCoTI-IIb. Both compounds display similar and well-defined conformations. These cyclic squash inhibitors share a similar conformation with noncyclic squash inhibitors such as CPTI-II, and it is postulated that the main effect of the cyclization is a reduced sensitivity to exo-proteases. On the contrary, clear differences were detected with the three-dimensional structures of other known cyclic knottins, i.e., kalata B1 or circulin A. The two-disulfide cystine-stabilized beta-sheet motif [Heitz et al. (1999) Biochemistry 38, 10615-10625] is conserved in the two families, whereas in the C-to-N linker, one disulfide bridge and one loop are differently located. The molecular surface of MCoTI-II is almost entirely charged in contrast to circulin A that displays a well-marked amphiphilic character. These differences might explain why the isolated macrocyclic squash inhibitors from M. cochinchinensis display no significant antibacterial activity, whereas circulins and kalata B1 do.     

Glucagon-(19-29), a Ca2+ pump inhibitory peptide, is processed from glucagon in the rat liver plasma membrane by a thiol endopeptidase

Glucagon-(19-29) is 1000-fold more potent that glucagon as an inhibitor of the liver plasma membrane calcium pump, which suggests that this peptide fragment is naturally occurring. Since glucagon-(19-29) is undetectable in plasma, the processing of glucagon into its (19-29) fragment may occur upon interaction of glucagon with its target tissues. The use of a specific radioimmunoassay for glucagon-(19-29) in association with the separation and identification of peptides by high performance liquid chromatography revealed that, upon incubation at 37 degrees C with hepatic plasma membranes, glucagon is processed into its (19-29) C-terminal fragment. The identity of the fragment was confirmed by amino acid sequencing. The processing activity was inhibited by reagents of the thiol group and by 1,10-phenanthroline, suggesting that a thiol endopeptidase containing a catalytically active metal is involved in this processing. Following its production, glucagon-(19-29) was degraded with a half-life of less than 10 s. This degradation was inhibited by bacitracin and by the aminopeptidase inhibitors bestatin and amastatin. When glucagon was incubated with liver plasma membranes in the absence of inhibitors, the accumulation of glucagon-(19-29) reached a maximum at 2 min (1% of initial glucagon), followed by a slow decline. In the presence of bacitracin and bestatin, the amounts of glucagon-(19-29) obtained from glucagon increased continuously, 1 and 2% of glucagon being transformed after 10 and 30 min, respectively. The production of glucagon-(19-29) did not appear to be associated with the binding of glucagon to its receptors, since (i) guanosine 5'-(3-O-thio)triphosphate, a compound which decreases the glucagon-receptor interaction, could not decrease the conversion of glucagon into glucagon-(19-29); (ii) a glucagon analogue which displays a strongly decreased affinity for the hepatic glucagon receptors was processed similarly to glucagon. The conversion also occurs upon incubation with intact hepatoma cells in monolayer culture. These observations suggest that, under physiological conditions, glucagon is processed in liver by cleavage of the Arg17-Arg18 basic doublet, leading to the production of a fragment which is known to display an original biological specificity, namely the modulation of the hepatocyte plasma membrane calcium pump.     

Computational reverse-engineering of a spider-venom derived peptide active against Plasmodium falciparum SUB1

Background

Psalmopeotoxin I (PcFK1), a protein of 33 aminoacids derived from the venom of the spider Psalmopoeus Cambridgei, is able to inhibit the growth of Plasmodium falciparum malaria parasites with an IC in the low micromolar range. PcFK1 was proposed to act as an ion channel inhibitor, although experimental validation of this mechanism is lacking. The surface loops of PcFK1 have some sequence similarity with the parasite protein sequences cleaved by PfSUB1, a subtilisin-like protease essential for egress of Plasmodium falciparum merozoites and invasion into erythrocytes. As PfSUB1 has emerged as an interesting drug target, we explored the hypothesis that PcFK1 targeted PfSUB1 enzymatic activity.

Findings

Molecular modeling and docking calculations showed that one loop could interact with the binding site of PfSUB1. The calculated free energy of binding averaged −5.01 kcal/mol, corresponding to a predicted low-medium micromolar constant of inhibition. PcFK1 inhibited the enzymatic activity of the recombinant PfSUB1 enzyme and the in vitro P.falciparum culture in a range compatible with our bioinformatics analysis. Using contact analysis and free energy decomposition we propose that residues A14 and Q15 are important in the interaction with PfSUB1.

Conclusions

Our computational reverse engineering supported the hypothesis that PcFK1 targeted PfSUB1, and this was confirmed by experimental evidence showing that PcFK1 inhibits PfSUB1 enzymatic activity. This outlines the usefulness of advanced bioinformatics tools to predict the function of a protein structure. The structural features of PcFK1 represent an interesting protein scaffold for future protein engineering.