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S. Biswas, F. Harris, S. Dennison, J. Singh, D. Phoenix; Biophysical Studies Into the Role of Lipid/ Membranes in the Activation of an Eye Lens Calpain . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1871.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Calpain 2 is a heterodimeric calcium–dependent protease and both animal and human models suggest a role for the enzyme in cataract formation and the induction of age–induced changes. In vitro studies had shown that levels of calcium required for enzyme activation far exceed normal intracellular levels. More recent studies have shown that the binding of lipid to calpain 2 can lower the calcium requirements and have suggested that the activity of the enzyme may be modulated by membrane interaction involving a segment, GTAMRILGGVI, located in domain V of the protein's small unit. Here, we have used a range of biophysical techniques to investigate the conformational behaviour of this segment in the presence of lipid vesicles and its ability to penetrate lipid monolayer/ bilayer assemblies. Methods: A peptide homologue of the calpain 2 segment, GTAMRILGGVI, was synthesised. FTIR spectroscopy was used to analyse the levels of secondary structure whilst Langmuir–Blogett were used to study the interaction of the peptide with lipid monolayers. Additionally, neutron diffraction was used to monitor the ability of the peptide to penetrate the core of bilayer structures. Results: FTIR conformational analysis showed the peptide homologue of the GTAMRILGGVI segment to adopt high levels of alpha–helical structure in the presence of lipid vesicles. The homologue also strongly interacted with anionic and zwitterionic monolayers. Neutron lamellar diffraction studies showed the peptide to interact with the acyl chain region of bilayers of similar lipid composition. Conclusions: The results of the present study clearly suggests that the GTAMRILGGVI segment of calpain 2 interacts with membranes via the formation of a lipid–interactive, amphiphilic alpha helix, which penetrates deeply into the bilayer hydrophobic core. These results strongly support a role for lipid/ membrane interactions in the modulation of eye lens calpain 2 activity, and thereby, a role for the enzyme in cataractogenesis.
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