CIRB
CELL BIOLOGY OF HOMEOPROTEINS
Director: Alain JOLIOT
Homeoproteins have a unique cellular behaviour with strong functional consequences. In addition to their well-known nuclear functions through transcriptional regulation, these proteins also transfer between cells and thus act as intercellular messenger proteins. Both cell-autonomous (intracrine) and direct non-cell autonomous (paracrine) activities are indispensable to the physiological function of homeoproteins. Two distinct sequences required respectively for internalisation and secretion reside within the conserved DNA-binding domain of homeoproteins, the homeodomain, suggesting that this property is widely conserved among this protein family. Our main thematic is to unravel the cellular and molecular basis of the two steps of the intercellular transfer process, secretion and internalisation, both of which which rely on unconventional but ubiquitous pathways.
Intercellular transfer of homeoproteins: When ectopically expressed in COS-7 cells, the homeodomain of KN1 homeoclomain (green) is transferred to neuronal cells (red) in a co-culture model.
Homeoprotein secretion
Although homeoproteins are devoid of classical secretion signal sequence and mainly accumulate in the nucleus, an intracellular pool of homeoproteins is detected in vesicular compartments and released in the extracellular medium in a free and soluble form. We have demonstrated an unexpected correlation between secretion and nuclear addressing suggesting a new role of the nuclear compartment and nuclear export in a specific unconventional secretion pathway. Preliminary data suggests that the latter pathway might be shared with other proteins unrelated to homeoproteins such as the 18Kd isoform of FGF2, which is also predominantly nuclear in most cell lines but actively exported from the nucleus and secreted. In addition, both homeoproteins and FGF2 strongly interact with cell surface carbohydrates. We have produced dedicated cell line to dissect and compare FGF2 and homeoprotein secretion pathways, including the dynamic of their intracellular distribution and the analysis of their cellular partners.
Homeoprotein Internalisation
Once secreted In the extracellular space, homeoproteins strongly interact with cell surface carbohydrates. This event initiates homeoprotein internalization, which ultimately leads to their cytosolic and nuclear accumulation. The internalization pathway, still active at low temperature, does not rely on classical endocytosis but likely involves direct interaction of homeoproteins with the lipid component of the plasma membrane. This mechanism is currently under investigation at different molecular levels, from the minimal internalization motif derived from homeoprotein (Penetratin) to homeodomains and full length homeoproteins.
Secretion and cell-surface association of FGF2 : Following secretion, FGF2 (green) accumulates at the cell surface (red).
Protein and peptide transduction
The crossing of biological membranes is a main bottleneck for the development of therapeutic molecules. From our study on homeoproteins, we have pioneered a new non-viral strategy allowing the cellular delivery of biologically active hydrophilic molecules. The strategy is based on the ability of short peptide sequences to promote cellular internalisation, of attached cargoes. We are currently developing two lines of research focusing respectively on the design of new genration of peptide vectors allowing the crossing of cellular barriers and on the delivery of large cargo molecules, from proteins to plasmid DNA, in vitro and in vivo.
Homeoprotein Internalization: Extracellular FITC-labelled Engrailed homeoprotein (green) accumulates in cytosolic and nuclear compartments of live CHO cells.
In vivo transduction of CRE-Penetratin: In vivo delivery of a CRE-Penetratin recombinant protein through intra-veinous injection induces massive recombination events (green) in the choroid plexus.
SELECTED PUBLICATIONS 2004-2010
- Wizenmann A., Brunet I., Lam J.S.Y., Sonnier L., Beurdeley M., Zarbalis K., Weisenhorn-Vogt D., Weinl C., Dwivedy A., Joliot A., Wurst W., Holt C. & Prochiantz A. (2009), Extracellular engrailed participates in the topographic guidance of retinal axons in vivo. Neuron 64, 355-366.
- Gitton Y., Tibaldi L., Dupont E., Levi G. &Joliot A. (2009), Efficient CPP-mediated Cre protein delivery to developing and adult CNS tissues. BMC Biotechnol 9, 40.
- Dupont E., Prochiantz A. & Joliot A. (2007), Identification of a signal peptide for unconventional secretion. J Biol Chem 282, 8994-9000.
- Tassetto M., Maizel A., Osorio J. & Joliot A. (2005), Plant and animal homeodomains use convergent mechanisms for intercellular transfer. EMBO Rep 6, 885-890.
- Prochiantz A. & Joliot A. (2003), Can transcription factors function as cell-cell signalling molecules? Nat Rev Mol Cell Biol 4, 814-819.