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Fig. 2. Effect of L-MCa andD-MCa on Ca 2+ release throughRyR.Fig. 3. Cell penetration of D-MCa-FAM in CHO cells.enantiomeric form of the ligand. D-MCa should nolonger bind onto RyR and thus ineffectively triggerCa 2+ release from the endoplasmic reticulum(Figure 2). This was indeed observed as publishedearlier [7]. As expected from a peptide synthesizedwith D amino acids, D-MCa is fully protease resistant(to trypsin and chymotrypsin), even after 24 hrs ofincubation, at times and concentrations where L-MCais fully degraded. The objective of the study was todevelop an analogue that preserved cell penetrationproperties. Earlier studies on CPPs had demonstratedthat D-Tat or D-penetratin kept normal cellpenetration properties. A similar behavior wasexpected for D-MCa. This conserved property can beexplained by intact interaction withglycoaminoglycans (GAGs) and negatively chargedlipids [8]. D-MCa was labeled on its N-terminus with5,6-carboxyfluorescein first. Dose-response curves forcell penetration of FAM-D-MCa, as assessed by flowcytometry, demonstrate that FAM-D-MCa penetrateswith a similar dose-dependent relationship than FAM-L-MCa [7]. Also, cell penetration was evidenced byconfocal microscopy and images reflected eitherdiffuse staining (likely by a process of membranetranslocation), indicating a predominant cytoplasmiclocalization, or more punctuate staining (indicating aform of endocytosis), that is reminiscent of lateendosomes (Figure 3). An earlier study had pointedout that depending on the nature of the cargo, L-MCacould preferentially choose between membranetranslocation [9] and macropinocytosis [8] for cellentry. This question was sorted out by applying inhibitors of macropinocytosis andquantifying by FACS the amount of cell entry of FAM-D-MCa. We found out thatendocytosis represents only a minor fraction of D-MCa entry, suggesting that D-MCawould be an excellent vector for the delivery of compounds within the cytoplasm.Concluding remarksWe were able to produce a D-analogue of L-MCa that folded remarkably well andpreserved the original disulfide pattern of L-MCa. The resulting peptide shows a total lackof pharmacological activity since it no longer recognizes RyR or produces Ca 2+ releasefrom the endoplasmic reticulum. The data demonstrate however that D-MCa is as efficientas L-MCa for cell penetration and cargo delivery. It has the added benefit to be proteaseresistant.For all these reasons, we predict that it has a bright future for the cell delivery ofcompounds in vivo for applications where the half-life of the vector / cargo couple matters.AcknowledgmentsThis work was supported by grants from technology pour la santé (Program TIMOMA2 of theCommissariat à l’Energie Atomique) and from ANR PNANO (SYNERGIE and NanoFret programs).References1. Mosbah, A., et al. Proteins 40 (3), 436-442 (2000).2. Fajloun, Z., et al. FEBS Lett. 469(2-3), 179-185 (2000).3. Merrifield, R.B. Mol. Biol. 32, 221-296 (1969).4. Altafaj, X., et al. J. Biol. Chem. 280(6), 4013-4016 (2005).5. Mabrouk, K., et al. Biochim Biophys Acta 1768(10), 2528-2540 (2007).6. Ram, N., et al. J. Biol. Chem. 283(40), 27048-270056 (2008).7. Poillot, C., et al. J. Biol. Chem. in press (2010).8. Ram, N., et al. J. Biol. Chem. 283(35), 24274-24284(2008).9. Aroui, S., et al. Pharm. Res. 26(4), 836-845(2009).403