Solution structure of humanin, a peptide against Alzheimer’s disease-related neurotoxicity

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Abstract

Humanin is a newly identified 24-residue peptide that suppresses neuronal cell death caused by a wide spectrum of familial Alzheimer’s disease genes and the β-amyloid peptide. In this study, NMR and circular dichroism studies of synthetic humanin in aqueous and 30% 2,2,2-trifluoroethanol (TFE) solutions are reported. In aqueous solution, humanin exists predominantly in an unstructured conformation in equilibrium with turn-like structures involving residues Gly5 to Leu10 and Glu15 to Leu18, providing indication of nascent helix. In the less polar environment of 30% TFE, humanin readily adopts helical structure with long-range order spanning residues Gly5 to Leu18. Comparative 3D modeling studies and topology predictions are in qualitative agreement with the experimental findings in both environments. Our studies reveal a flexible peptide in aqueous environment, which is free to interact with possible receptors that mediate its action, but may also acquire a helical conformation necessary for specific interactions and/or passage through membranes.

Section snippets

Materials and methods

Preparation of humanin. Humanin was prepared by Fmoc-solid phase synthesis [16] on an in-house prepared o-Cl-trityl-amidomethyl polystyrene resin. The peptide was purified to 95% with semi-preparative RP-HPLC and suitably characterized.

CD spectropolarimetry. CD spectra of humanin in the range 250–180 nm were recorded on a Jasco J715 spectropolarimeter using 1 mm quartz cells, 1 nm bandwidth, 0.2 nm resolution, 1 s response, and an average of 8 scans for each spectrum. The temperature of the samples

CD study

The CD spectra of humanin (0.19 mg/mL, 0.07 mM) in aqueous solutions in the presence of various concentrations of TFE at 25 °C are shown in Fig. 1A. In the absence of TFE, the CD spectrum shows strong negative dichroism below 200 nm, indicative of a small degree of ordering in the peptide [26]. On addition of TFE, changes diagnostic of conformational equilibrium between unordered structures and helical conformations of humanin in solution take place in the CD spectra [27]. The mean residue

Discussion

The potent in vitro neuroprotective activity of humanin against the insults of the AD-related genes and the neurotoxic β-amyloid peptide has spurred new hopes for the development of a pharmaceutical therapy against AD. To establish therapeutic strategies, as well as to clarify the mechanism of AD pathogenesis, the mode of humanin action must be understood. Our CD, NMR, and 3D comparative modeling data demonstrate that in aqueous solution humanin possesses essentially no stable secondary

Acknowledgments

M. Pelecanou wishes to acknowledge financial support by the National Bank of Greece and C. Zikos wishes to acknowledge a financial grant offered by Biomedica Life Sciences S.A.

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