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Solution state structures of human pancreatic amylin and pramlintide.

ABSTRACT: 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.

SUBMITTER: Cort JR

PROVIDER: S-EPMC2719500 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Solution state structures of human pancreatic amylin and pramlintide.

Cort John R JR Liu Zhihong Z Lee Gregory M GM Huggins K N L KN Janes Susan S Prickett Kathryn K Andersen Niels H NH

Protein engineering, design & selection : PEDS 20090712 8

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)); ...[more]

PMID: 19596697

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Project description:Human amylin-derived oligomers and aggregates are believed to play an important role in the pathogenesis of type II diabetes mellitus (T2DM). In addition to amylin-evoked cell attrition, T2DM is often accompanied by elevated serum copper levels. Although previous studies have shown that human amylin, in the course of its aggregation, produces hydrogen peroxide (H2O2) in solution, and that this process is exacerbated in the presence of copper(ii) ions (Cu(2+)), very little is known about the mechanism of interaction between Cu(2+) and amylin in pancreatic ?-cells, including its pathological significance. Hence, in this study we investigated the mechanism by which Cu(2+) and human amylin catalyze formation of reactive oxygen species (ROS) in cells and in vitro, and examined the modulatory effect of Cu(2+) on amylin aggregation and toxicity in pancreatic rat insulinoma (RIN-m5F) ?-cells. Our results indicate that Cu(2+) interacts with human and rat amylin to form metalo-peptide complexes with low aggregative and oxidative properties. Human and non-amyloidogenic rat amylin produced minute (nM) amounts of H2O2, the accumulation of which was slightly enhanced in the presence of Cu(2+). In a marked contrast to human and rat amylin, and in the presence of the reducing agents glutathione and ascorbate, Cu(2+) produced ?M concentrations of H2O2 surpassing the amylin effect by several fold. The current study shows that human and rat amylin not only produce but also quench H2O2, and that human but not rat amylin significantly decreases the amount of H2O2 in solution produced by Cu(2+) and glutathione. Similarly, human amylin was found to also decrease hydroxyl radical formation elicited by Cu(2+) and glutathione. Furthermore, Cu(2+) mitigated the toxic effect of human amylin by inhibiting activation of pro-apoptotic caspase-3 and stress-kinase signaling pathways in rat pancreatic insulinoma cells in part by stabilizing human amylin in its native conformational state. This sacrificial quenching of metal-catalyzed ROS by human amylin and copper's anti-aggregative and anti-apoptotic properties suggest a novel and protective role for the copper-amylin complex.

Dataset Information

Solution state structures of human pancreatic amylin and pramlintide.

Publications

Solution state structures of human pancreatic amylin and pramlintide.

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