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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Cyclic PDZ-Binding Peptides as Neuroprotective Agents AgainstExcitotoxic Brain DamageBrian M Austen 1 , Kate Duberley 1 , Paul Turner 2 , and Ruth Empson 21 Dept of Biomedical Sciences, St George’s University of London, London, SW17 ORE, UK;2 Dept of Physiology, Otago University, Otago, New ZealandIntroductionPost-synaptic density protein 95, a scaffold protein, regulates signaling in glutaminergicneurons by providing attachment points for NMDA receptors, calcium channels andsignaling enzymes [1]. Interactions are made via SH3 and PDZ domains. PDZ domains,consisting of a 5-membered β-barrel and an α-helix, bind the C-termini of interactingproteins, in a grove between one of the β-strands and the α-helix (Figure 1). Excitotoxicneuronal death occurring by over-activation of NMDA receptors, and influx of calcium isinvolved in neurodegerative disease. We have synthesized stable cyclic peptide analoguesof the C-terminal sequences of PMCA2 and NR2B, containing additional cell penetrationsequences. Cell staining and immunoprecipitation show that the peptides are internalizedand bind PSD95. These peptides have potential as neuroprotective agents.Results and DiscussionA cyclised form of an analogue C-terminal sequence of PMCA2b Ser-Leu-Glu-Thr-Lys-Leu-COOH was synthesised, by incorporating a β-alanine bridge between Lys -2 and Glu -4in the extended sequence:, acetyl-DArg-DArg-DArg-DArg-DArg-DArg-Gly-Gly-Lys(Biotin)Ser-Leu-c[Glu(βAla)Thr-Lys]-Leu-COOH (R2). R2 was synthesized onFmocLeu Peg-PS resin (ABI) using temporary α-amino Fmoc protected residues. Thecyclised C-terminal sequence of PMCA2 was extended at the N-terminus by six D-Argresidues to act as a cell penetration sequence, and finally N-acetylated to block theN-terminus against protease degradation. Orthogonally protected residue FmocLys(ivDde)was coupled at step 2 and FmocGlu((2-PhiPr) at step 4. Pior to final deprotection, theivDde group was removed with 2% hydrazine in DMF for 3x3 min. Fmoc-β-Ala wascoupled with PyBOP. The 2-PhilPr group was then removed by treatment with 1% TFAand 0.5% ethanedithiol in dichloromethanefor 4x3 min, and the final Fmocgroup removed by 20% piperidine inDMF. Subsequent ring closure, asmonitored by ninhydrin reaction, wasperformed by treatment with 4 equivalentsof PyBOP in 0.9M diisopropylamine inDMF. The peptidyl-resin was deprotectedand cleaved in 90% TFA, 2.5% water,2.5% triisopropyl silane, and 0.5%ethanedithiol, for 2hrs. The peptide waspurified by HPLC and characterised byMALDI mass spectrometry (MH=2192.1;calc 2193). Affinity for PSD95 was shownby co-immunoprecipitation experimentsand co-localisation of the biotin tag on R2,with PSD-95 in human neuroblastomaSHSY-5Y cells using TRITC-avidin andFig. 1. R2 binding to PDZ domain of PSD95[2].FITC-anti-rabbit antibody with a Rbantibody to human PSD-95 (Abcam)(Figure 2).544