Kathryn S. Prickett

Efforts toward deriving the CD spectrum of a 310 helix in aqueous medium

There have been two recent reports suggesting that 310 helices can be distinguished from α helices by circular dichroism. The differentiating feature is stated to be a [θ]222:[θ]208 ratio (R2) distinctly smaller than unity. This has been reported for a Cα,α′‐disubstituted homooctamer [Toniolo et al. (1996), J. Am. Chem. Soc. 118, 2744–27451 and for alanine‐rich systems of 16–21 residue length with modest fractional helicity [Millhauser (1995) Biochemistry 34, 3873–38771. We report here the changes in the CD spectrum produced by inserting aminoisobutyric acid (Aib) residues into the helical domain of human pancreatic amylin. In order to examine this effect at comparable net fractional helicities, CD spectra were measured for each species during the course of a helicity titration by trifluoroethanol addition. The addition of five Aib residues gave results of particular interest. At low net fractional helicity, this Aib‐rich system displays a diminished (circa 208 nm) rotational strength versus the less Aib‐rich species. However, NMR data and comparisons of CD difference spectra suggest that fluoroalcohol‐induced extension of the short Aib‐rich helix is in the form of an α helix. Given the diminished intensity of the minimum at 208 nm at low net helicity when 310 conformations should contribute, we urge extreme caution in using a [θ]222:[θ]208 ratio smaller than unity as a diagnostic for 310 helices.

Medium-dependence of the secondary structure of exendin-4 and glucagon-like-peptide-1.

Exendin-4 is a natural, 39-residue peptide first isolated from the salivary secretions of a Gila Monster (Heloderma suspectum) that has some pharmacological properties similar to glucagon-like-peptide-1 (GLP-1). This paper reports differences in the structural preferences of these two peptides. For GLP-1 in aqueous buffer (pH 3.5 or 5.9), the concentration dependence of circular dichroism spectra suggests that substantial helicity results only as a consequence of helix bundle formation. In contrast, exendin-4 is significantly helical in aqueous buffer even at the lowest concentration examined (2.3 microM). The pH dependence of the helical signal for exendin-4 indicates that helicity is enhanced by a more favorable sequence alignment of oppositely charged sidechains. Both peptides become more helical upon addition of either lipid micelles or fluoroalcohols. The stabilities of the helices were assessed from the thermal gradient of ellipticity (partial differential theta(221)/partial differential T values); on this basis, the exendin helix does not melt appreciably until temperatures significantly above ambient. The extent of helix formation for exendin-4 in aqueous buffer (and the thermal stability of the resulting helix) suggests the presence of a stable helix-capping interaction which was localized to the C-terminal segment by NMR studies of NH exchange protection. Solvent effects on the thermal stability of the helix indicate that the C-terminal capping interaction is hydrophobic in nature. The absence of this C-capping interaction and the presence of a flexible, helix-destabilizing glycine at residue 16 in GLP-1 are the likely causes of the greater fragility of the monomeric helical state of GLP-1. The intramolecular hydrophobic clustering in exendin-4 also appears to decrease the extent of helical aggregate formation.

Solution state structures of human pancreatic amylin and pramlintide

We have employed pramlintide (prAM) as a surrogate for hAM in CD and NMR studies of the conformational preferences of the N-terminal portion of the structure in media which do not provide long-lived monomeric solutions of hAM due to its rapid conversion to preamyloid beta aggregate states. Direct comparison of hAM and prAM could be made under helix-formation-favoring conditions. On the basis of CD and NMR studies: (i) the Cys(2)-Cys(7) loop conformation has a short-span of helix (Ala(5)-Cys(7)); (ii) the extent to which this helix propagates further into the sequence is medium-dependent; a helix from Ala(5) through Ser(20) (with end fraying from His(18) onward) is observed in aqueous fluoroalcohol media; (iii) in 12+ vol.% HFIP, the amyloidogenic region of hAM forms a second helical domain (Phe(23)-Ser(29)); (iv) the two helical regions of hAM do not have any specific geometric relationship as they are connected by a flexible loop that takes different conformations and (v) although the extreme C-terminus is essential for bioactivity, it is found to be extensively randomized with conformer interconversions occurring at a much faster rate than that is observed in the remainder of the peptide sequence. Two NMR-derived structures of the 1-22 sequence fragment of hAM have been derived. The work also serves to illustrate improved methods for the NMR characterization of helices. A detailed quantitative analysis of the NOE intensities observed in aqueous HFIP revealed alternative conformations in the C-terminal portion of the common amylin helix, a region that is known to be involved in the biorecognition phenomena leading to amyloidogenesis. Even though the SNN sequence appears to be a flexible loop, the chemical shifts (and changes induced upon helix structuring) suggest some interactions between the loop and the amyloidogenic segment of hAM that occur on partial helix formation.

SPECIAL REPORT Regulation of muscle glycogen metabolism by CGRP and amylin: CGRP receptors not involved

The aim of the present study was to determine whether amylin and calcitonin gene‐related peptide (CGRP) act through shared or distinct receptors to inhibit insulin‐stimulated incorporation of [14C]‐glucose into glycogen. Rat amylin was 3 fold more potent than either rat αCGRP or rat βCGRP at reducing glycogen synthesis from [14C]‐glucose in insulin‐treated rat soleus muscle. This action was blocked by peptide antagonists, with the rank order of potency being AC187> salmon calcitonin 8–32 (sCT8.32) > h‐αCGRP8–37 for antagonism of either amylin or CGRP. The antagonist potency order correlated with affinity for amylin receptors measured in rat nucleus accumbens but not CGRP receptors measured in rat L6 muscle cells. Inhibition of glucose incorporation into glycogen by amylin and CGRP appears to be mediated by shared receptors that have the pharmacological characteristics of amylin receptors, and are distinct from previously described CGRP receptors.

Selective amylin antagonist suppresses rise in plasma lactate after intravenous glucose in the rat. Evidence for a metabolic role of endogenous amylin.

Data presented here provide the first demonstration that circulating amylin regulates metabolism in vivo, and support an endocrine hormonal role that is distinct from its autocrine action at pancreatic islets. When rats were pre‐treated with the potent amylin antagonist AC187 (n = 18), and then administered a 2 mmol glucose load, the rise in plasma lactate was less than in rats administered glucose only (n = 27; P < 0.02). When rats were treated so that plasma glucose and insulin profiles were similar (n = 8), the increase in plasma lactate in the presence of AC187 was only 50.3% as high as the increase when AC187 was absent (P < 0.001). These experimental results fit with the view that some of the lactate appearing in plasma after a glucose load comes from insulin‐sensitive tissues. The experiments also support the view that an important fraction of the increase in lactate depends on processes inhibited by a selective amylin antagonist, most likely amylin action in muscle.

Treatment of gestational diabetes

The question is not whether to treat, but how and who?

Section Review Oncologic, Endocrine & Metabolic: The amylin, CGRP and calcitonin family of peptides

The calcitonins are a group of 32 amino acid, disulphide bridged, C-terminally amidated peptides, first isolated from the thyroid and characterised as hormones which regulate blood calcium levels. Observations on alternative splicing of mRNA derived from the calcitonin gene resulted in the identification of the potent vasodilator: calcitonin gene-related peptide (CGRP), a 37 amino acid, disulphide bridged peptide, again with a C-terminal amide. More recently a third type of peptide, amylin, has been isolated from pancreases of Type 2 diabetics. This also is a 37 amino acid residue, disulphide bridged C-terminally amidated peptide, and acts as a regulator of blood glucose levels in conjunction with insulin. Two other moiecules may also be somewhat related to these: adrenomedullin, which has weak structural homology with CGRP and amylin and shows vasodilatory properties, and maxadilan, which is structurally unrelated but resembles CGRP biologically. This review summarises recent progress on the characterisa...