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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, 2010Targeting the Nuclear Pore Complex with Proteomimetic CellPenetrating PeptidesSarah Jones and John HowlResearch Institute in Healthcare Science, University of Wolverhampton,Wolverhampton, WV1 1LY, UKIntroductionThe past decade has witnessed a resurgent interest in the therapeutic utilizations of cellpenetrating peptides (CPPs). Accordingly, CPP technologies have successfully beenemployed for the modulation of intracellular signal transduction. Moreover, CPPs withintrinsic signal transduction modulatory properties offer a novel strategy for the modulationof intracellular biological events [1-3]. QSAR analysis was employed to identify putativeCPP sequences within human Cytochrome c [4]. Two such sequences located within the C-terminal helix, Cyt c 77-101 and Cyt c 86-101 induced apoptosis of malignant astrocytoma whenexogenously applied, thus mimicking the well-documented role(s) of Cyt c as a keyregulator of programmed cell death [1]. Whilst Cyt c 86-101 preferentially demonstrated anuclear localization [1], Cyt c 77-101 proved to be an extremely efficient CPP and was thusselected for modification to incorporate additional bioactive peptide sequences.Results and DiscussionN-terminal extension of Cyt c 77-101 , via a flexible aminohexanoic acid linker, with a targetmimetic of FxFG nucleoporins (Nup153 980-987 , CH 3 CO-NFKFGLSS) yielded a chimericpeptide (Ac-Nup153-Cyt c) that displayed a significantly enhanced apoptogenic potency(LD 50 = 0.73 μM) compared to Cyt c 77-101 alone and a scrambled chimeric analogue,(Ac-ScrNup153-Cyt c) (Table 1). Significantly, the apoptogenic potency of Ac-Nup153-Cytc (0.73 μM) would also indicate in vivo applicability. The detection of intra-nucleosomalDNA fragmentation by in situ TUNEL analysis and a specific activation of caspase-3confirmed that apoptosis was indeed the mechanism of cell death employed by Ac-Nup153-Cyt c in the U373MG astrocytoma model cell line.To gain insight into the possible mechanisms employed by this novel chimericconstruct, Nup153 980-987 was translocated into U373MG cells as a fluorescein-labelleddisulphide-linked cargo, using the inert CPP M918. Following a subsequent intracellularreduction of cysteine, liberated Fluo-Nup153 980-987 assumed a preferential co-localizationwith the nuclear pore complex (NPC). To refine these results, U373MG cells were treatedwith the rhodamine-labelled chimera Rho-Nup153-Cyt c (3 M) and scrambled analoguefor 1 hour. Whilst the scrambled chimera demonstrated a diffuse and generalisedcytoplasmic distribution, Rho-Nup153-Cyt c strongly co-localised with the nuclear poreprotein nucleoporin 153 (Table 1). Moreover, after 4 hours of treatment, exogenousapplication of Ac-Nup153-Cyt c (3 M) further facilitated the dramatic redistribution ofimmuno-labelled NPC proteins into the nucleoplasm and cytoplasm, a phenomenon thatwas not a downstream consequence of caspase activation since this redistribution event alsooccurred in the presence of the pan-caspase inhibitor Q-VD-Oph (20 M).Studies with scrambled peptides confirmed that the observed apotogenic action wasboth translocation- and sequence-dependent. Notably, translocation efficacies of bothchimeras, Rho-Nup153-Cyt c and Rho-ScrNup153-Cyt c, were significantly enhanced(408.5 + 15.8 fold and 412.5 + 5.20 fold, respectively) compared to that of Cyt c 77-101 alone(105.9 + 0.42 fold) and thus indicates a sequence-dependent specificity of action for theAc-Nup153-Cyt c chimera. We conclude that Ac-Nup153-Cyt c demonstrates therapeuticpotential as a potent inducer of apoptosis, whilst propounding the nuclear pore complex asan accessible, and therefore druggable, intracellular target.416