Daniele Derossi

The 60-amino acid long homeodomain of Antennapedia crosses biological membranes by an energy-independent mechanism, a phenomenon abolished by directed mutagenesis within the polypeptide C-terminal region. This finding led us to study the internalization of several chemically synthesized peptides derived from the third helix of the homeodomain. We report here that a polypeptide of 16 amino acids in length corresponding to the third helix of the homeodomain deleted of its N-terminal glutamate is still capable of translocating through the membrane. A longer peptide of 20 amino acids also translocates, whereas shorter peptides (15 amino acids) are not internalized by the cells. As is also the case for the entire homeodomain, the 20- and 16-amino acid long peptides are internalized at 4 degrees C, suggesting an energy-independent mechanism of translocation not involving classical endocytosis. The two translocated peptides can be recovered, intact, within the cells, strongly suggesting that they are not targeted to the lysosomal compartment. Finally, substitution of two tryptophans by two phenylalanines strongly diminishes translocation, raising the possibility that the internalization of the third helix is not solely based on its general hydrophobicity.

We have recently reported that a 16-amino acid long polypeptide corresponding to the third helix of the DNA binding domain (homeodomain) of Antennapedia, a Drosophila transcription factor, is internalized by cells in culture (Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) J. Biol. Chem. 269, 10444-10450). The capture of the homeodomain and of its third helix at temperatures below 10°C raised the problem of the mechanism of internalization. The present demonstration, that a reverse helix and a helix composed of D-enantiomers still translocate across biological membranes at 4 and 37°C strongly suggests that the third helix of the homeodomain is internalized by a receptor-independent mechanism. The finding that introducing 1 or 3 prolines in the structure does not hamper internalization also demonstrates that the α-helical structure is not necessary. The data presented are compatible with a translocation process based on the establishment of direct interactions with the membrane phospholipids. The third helix of the homeodomain has been used successfully to address biologically active substances to the cytoplasm and nucleus of cells in culture (Théodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P., and Prochiantz, A. (14Le Roux I. Duharcourt S. Volovitch M. Prochiantz A. Ronchi E. FEBS Lett. 1995; 368: 311-314Google Scholar) J. Neurosci. 15, 7158-7167). Therefore, in addition to their physiological implications (Prochiantz, A., and Théodore, L. (1995) BioEssays 17, 39-45), the present results open the way to the molecular design of cellular vectors. We have recently reported that a 16-amino acid long polypeptide corresponding to the third helix of the DNA binding domain (homeodomain) of Antennapedia, a Drosophila transcription factor, is internalized by cells in culture (Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) J. Biol. Chem. 269, 10444-10450). The capture of the homeodomain and of its third helix at temperatures below 10°C raised the problem of the mechanism of internalization. The present demonstration, that a reverse helix and a helix composed of D-enantiomers still translocate across biological membranes at 4 and 37°C strongly suggests that the third helix of the homeodomain is internalized by a receptor-independent mechanism. The finding that introducing 1 or 3 prolines in the structure does not hamper internalization also demonstrates that the α-helical structure is not necessary. The data presented are compatible with a translocation process based on the establishment of direct interactions with the membrane phospholipids. The third helix of the homeodomain has been used successfully to address biologically active substances to the cytoplasm and nucleus of cells in culture (Théodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P., and Prochiantz, A. (14Le Roux I. Duharcourt S. Volovitch M. Prochiantz A. Ronchi E. FEBS Lett. 1995; 368: 311-314Google Scholar) J. Neurosci. 15, 7158-7167). Therefore, in addition to their physiological implications (Prochiantz, A., and Théodore, L. (1995) BioEssays 17, 39-45), the present results open the way to the molecular design of cellular vectors.

We previously showed that the downregulation of Cu/Zn superoxide dismutase (SOD1) activity in PC12 cells by exposure to an appropriate antisense oligonucleotide causes their apoptotic death. In this report, we used this model to examine the pathways by which SOD1 downregulation leads to death and to compare these pathways with those responsible for death caused by withdrawal of trophic support. To improve delivery of the SOD1 antisense oligonucleotide, we coupled it to a carrier "vector" peptide homologous to the third helix of the Drosophila Antennapedia homeodomain. This caused not only efficient cellular uptake even in the presence of serum, but also inhibition of SOD1 activity and promotion of apoptosis at 100-fold lower concentrations of oligonucleotide. Death induced by SOD1 downregulation appeared to require the reaction of superoxide with nitric oxide (NO) to form peroxynitrite. In support of this, inhibitors of NO synthase, the enzyme responsible for NO synthesis, blocked death in our experiments, whereas NO generators and donors accelerated cell death. N-Acetylcysteine and chlorophenylthiol cAMP, which rescue PC12 cells and neurons from the withdrawal of nerve growth factor and other forms of trophic support, did not protect PC12 cells from SOD1 downregulation. In contrast, overexpression of bcl-2, which also rescues these cells form loss of trophic support, was equally effective in saving the cells in the SOD1 downregulation paradigm. Taken together with past findings, such observations suggest that SOD1 downregulation and withdrawal of trophic support trigger apoptosis via distinct initial mechanisms but may utilize a common final pathway to bring about death. Our findings may be relevant to the causes and potential amelioration of neuronal degenerative disorders caused by impaired regulation of cellular levels of NO and superoxide.

The third helix of antennapedia homeodomain pAntp-(43-58) can translocate through cell membrane and has been used as an intracellular vehicle for delivering peptides and oligonucleotides. The conformational and associative behaviour of two peptidic vectors pAntp-(43-58) and [Pro50] pAntp-(43-58) has been analyzed by different biophysical methods. pAntp-(43-58) adopts an amphipathic helical structure in 30% (by vol.) hexafluoroisopropanol, in perfluoro-tert-butanol and in the presence of SDS micelles. CD spectra indicate that the conformation of [Pro50]pAntp-(43-58) in contrast to pAntp-(43-58) is independent of the media used. 1H-NMR spectroscopy in SDS micelles or in perfluoro-tert-butanol allows detection of aggregated peptides probably in a ribbon 2(7) type conformation. These conformations became the predominant structure when Gln50 was replaced by Pro50. Interproton-distance restraints derived from NOE measurements have been classified in two groups corresponding to two types of structures: alpha-helix and essentially extended structures. Consecutive CH alpha (i)/ CH alpha (i + 1) NOEs are only compatible with aggregates. Simulated annealing calculation of dimeric structure agrees with phi and psi angles in the beta-sheet and gamma-turn regions. Fluorescence spectroscopy analysis has shown that the indole groups of both peptides penetrate into SDS micelles; both peptides also induce the formation of micelles at very low concentration of SDS (20 microM). Similar interaction was observed with reverse-phase micelles made of bis(2-ethyhexyl) sodium sulfosuccinate and small unilamellar vesicles (SUV) made of a mixture of phosphatidylcholine/phosphatidylserine. 31P-NMR of vesicles (SUV and large unilamellar vesicles) indicated that the addition of pAntp analogues did not affect the size of phosphatidylcholine/phosphatidylserine vesicles. The addition of pAntp analogues to lipidic dispersions modulates lipid polymorphism in different ways depending on the mixtures of acidic lipids.