Cell Biology of Homeoproteins

Alain JOLIOT

Homeoproteins are a class of transcription factors that share the unusual property of intercellular transfer. Homeoprotein internalization and secretion rely on unconventional mechanisms. Our research is centred on the analysis of the cellular mechanisms responsible for this secretion and internalisation, focusing on both physiological and biotechnological aspects.

Sequence requirement for homeoprotein intercellular transfer
The homeodomain DNA-binding motif is necessary and sufficient for homeoprotein transfer. We have characterized minimal internalization and secretion sequences within the homeodomain of the Engrailed homeoprotein, based on the ability of synthetic peptides to cross a tight junction epithelium. This process is highly specific for homeoproteins. Although intercellular transfer is a shared feature of most homeodomains, we have identified several members that have lost this property.

Mechanism of internalisation
Homeoprotein interaction with the external face of the plasma membrane is the initial step of the internalisation. In collaboration with F. Pincet (LPS, ENS), we have adapted a new technique (Biomembrane Force Probe) for analysis of the interaction of Penetratin (patented cell-penetrating peptide) or homeodomains with the cell surface in a live cell context, contrasting with the most common models based on artificial liposomes. The presence of negatively charged carbohydrates is critical for cell interaction with basic peptides such as Penetratin or Tat (derived from the HIV Tat protein), and with homeodomains. Only in the latter case, the interaction strongly depends on the nature of the carbohydrate. Cholesterol depletion dramatically impairs cell surface/Penetratin interaction with minimal effects on other basic peptides, suggesting a specific involvement of hydrophobic interactions in Penetratin internalisation.

Mechanism of secretion
There exists a correlation between homeodomain nuclear accumulation and secretion. Indeed, mutation of a nuclear localization signal (nls) within the homeodomain impairs its nuclear accumulation and secretion ability, both of which are restored upon addition of an ectopic canonical nls. The identification of three putative nuclear transporters (one importin and two exportins) for Engrailed has provided new tools for analyzing the role of nucleo/cytoplasmic exchanges in the secretion process. We have recently designed cellular models allowing for quantification of homeoprotein secretion in distinct experimental conditions.

Biotechnological applications
The crossing of biological membranes is a limiting for the development of therapeutic molecules. A non-viral strategy allowing the cellular delivery and intracellular biological action of hydrophilic cargoes has been successfully developed based on the fusion to small peptide sequences, known as Cell-Penetrating peptides (CPP). We have performed a comparative analysis of different CPPs for the intracellular delivery of an entire protein (CRE recombinase), and we are currently involved in the improvement of transfection strategies based on a combination of new cationic polymers and CPP.

Selected Publications 2004-2009

- A. Joliot and A. Prochiantz, Transduction peptides: from technology to physiology. Nat Cell Biol 6, 189-196 (2004).
- B. Christiaens, J. Grooten, M. Reusens, A. Joliot, M. Goethals, J. Vandekerckhove, A. Prochiantz, M. Rosseneu, Membrane interaction and cellular internalization of penetratin peptides. Eur J Biochem 271, 1187-1197 (2004).
- Kim SW, Moon JY, Jung JH, Chen X, Shi C, Rim YG, Kwon H, Jackson D, Datla R, Joliot A, Kim JY. Intercellular Trafficking of Homeodomain Proteins. The Plant Pathology Journal vol. 21 (1) pp. 21-26 (2005).
- M. Tassetto, A. Maizel, J. Osorio and A. Joliot, Plant and animal homeodomains use convergent mechanisms for intercellular transfer. EMBO Rep 6, 885-890 (2005).
- A. Joliot, Transduction peptides within naturally occurring proteins. Sci STKE 2005, pe54 (2005).
- E. Dupont, A. Prochiantz and A. Joliot. Identification of a Signal Peptide for Unconventional Secretion J Biol Chem, 282, 8994-9000 (2007)
- B. Lesaffre, A. Joliot, A. Prochiantz and M. Volovitch Direct non-cell autonomous Pax6 activity regulates eye development in the zebrafish. Neural Develop 2: 1-12 (2007)
- Aussedat B, Dupont E, Sagan S, Joliot A, Lavielle S, Chassaing G, Burlina F. Modifications in the chemical structure of Trojan carriers: impact on cargo delivery. Chem Commun (Camb) 12 pp. 1398-400 (2008)

People

Director:
Alain JOLIOT, DR2 CNRS

Technical staff:
Edmond DUPONT, IR CNRS
Val LEBLED, AJT CNRS

Postdocs and students:
Julie MIRALVES, Postdoc ANR-blanche
Christelle MOGES, Student
Lorenzo TIBALDI, Student