Blanc SVSE 4 - Sciences de la vie, de la santé et des écosystèmes : Neurosciences

Homeoprotein Paracrine Activity and Translocation – ParaHP

Proteins «Passe-Murailles«:A journey through cell membranes

Understanding the language used by cells to communicate with each other is a major challenge for both physiology and pathology. Our goal is to dissect the mechanisms and functions of a new mode of communication involving the direct transfer of proteins between two cells.

Mechanism and function of homeoprotein intercellular transfer

Homeoproteins are a family of proteins of a similar nature originally identified for their role in organizing embryo development, through the regulation of gene expression. These proteins have additional functions in adults and their mutation is often associated with pathologies. Recently, a new mode of action has been identified for these proteins, involving their unexpected ability to transfer between two cells. Through this way, a cell expressing a homeoprotein can instruct neighbouring cells lacking the protein and therefore influence their fate. Our goal is to dissect this mode of communication that raises several questions and disrupts certain dogmas of biology. Indeed, the transfer of a protein between two cells involves its passage through cell membranes whose primary function is to provide a rampart towards the external environment. A second aspect of our study focuses on the functional consequences of this transfer. Although it is now proven that the transfer property is a key component to homeoprotein function, the extent of this mode of communication and its effects in the recipient cell remains to be determined.

The study of homeoprotein tranfer has several levels of analysis related to the physics, chemistry and biology, which can not be addressed by a multidisciplinary approach. With physico-chemical approaches, we analyse how homeoproteins interact with each component of the cell membrane to understand how these hydrophilic molecules (soluble in water), are able to cross the hydrophobic barrier (water insoluble) of the cell membrane, containing lipids. In addition, the role of these interactions in the process of homeoprotein transfer is analysed in a simplified biological context of cell culture. The physiological effects resulting from the transfer of homeoprotein requires the use of animal models in which the protein is put in its natural context.

We have shown in previous works that the domain that defines the homeoprotein family (the homeodomain) has an essential role for crossing cell membranes. We now show that the structure of this domain is strongly modified upon contact with a hydrophobic environment that mimics cell membranes. We also identified preferential interactions of the homeodomain or its derivatives with specific components of the membrane such as certain types of lipids or such as long sugar chains called glycosaminoglycans. The latter are abundant on the cell surface but their precise composition greatly varies. During the study of the physiological function of homeoproteins, we demonstrated that a given homeoprotein interacts only with few types of glycosaminoglycan chains, allowing restricting its action to cells that exhibit this particular sugar pattern.

Beyond our understanding of the mechanism homeoprotein transfer and its associated functions, this project opens the way to wider prospects, especially for therapeutic purposes. Indeed, the intracellular environment is an infinite source of therapeutic targets, but access to these targets, so the crossing of cell membranes, is a major obstacle to the development of new drugs. Our work could lead to new strategies to cross these barriers. Note that due their ability to enter cells and their multiple functions, homeoproteins themselves could be promising therapeutic agents by direct addition of the protein in the extracellular medium.

1. Tryptophan within basic peptide sequences triggers glycosaminoglycan-dependent endocytosis. Bechara C, Pallerla M, Zalstman Y, Burlina F, Alves ID, Lequin O, Sagan S, FASEBJ (sous presse). This study highlights the role of interactions between fragments homeoproteins responsible for membrane crossing s and sugar motifs present at the cell surface
2. Dupras G., Bernard C., Perrot A., Cattiaux L., Prochiantz A., Lortat-Jacob H., Mallet J.-M. Toward libraries of biotinylated chondroitin sulfate analogs: from synthesis to in vivo studies. Chemitry a European Journal., in press. This study describes the characterization of sugar motifs interacting specifically with a given homeoprotein

Homeoproteins (HPs) have important cell autonomous developmental functions. Many HPs are also expressed throughout adulthood suggesting a role in animal physiology. In addition to being transcription factors, several HPs regulate translation and have recently been shown to transfer between cells in vitro and in vivo. HP transfer involves secretion and internalization through not fully understood mechanisms. To limit the discussion to internalization, the driving force for HP internalization is the third helix (16 amino acids in length) of the DNA-binding domain or Homeodomain (HD). The 60 amino acid-long HD and its third helix (Penetratin) have been widely used as vectors for the intracellular addressing of hydrophilic reagents. Several HP non-cell autonomous functions have recently been described, including neuroepithelium compartmentalization, axon guidance and regulation of the critical period during visual cortex post-natal maturation. The present project will explore some aspects of HP activity as signalling entities. It gathers chemists, cell biologists, developmental biologists and physiologists that have accumulated unequalled experience in the field. Most studies will be done on Engrailed1 (En1), a HP primarily expressed in the developing mesencephalon and also showing a strong expression in adult mesencephalic dopaminergic (mDA) neurons. Due to the high conservation of the sequences necessary for secretion and internalization, and the large number of HPs that transfer between cells, it is anticipated that several conclusions derived from this study on En1 will also apply to other HPs.
A first task will consist in using the HD to verify if internalization modifies HD structure through its association with molecular entities, in priority lipids or complex sugars that will be identified. This work will involve NMR studies in vitro and in live cells. Such modifications would allow the cell to distinguish between cell autonomous and non-cell autonomous (internalized) HP functions. Once the residues involved in such interactions will have been identified, they will be mutated and the resulting En1 variants will be tested in two physiological read-out.
A second task consists in using biochemical strategies to identify En1 “interactors”. This will be done thanks to pull-down experiments followed by mass spectrometry an biochemical identification of lipids and sugars. The sites of interaction of En1 with “interactors” will be identified by classical molecular genetic approaches and mutated to produce En1 variants. The tools will be constructed to produce gain and loss of functions of selected “interactors” and to test their physiological importance as well as that of a selected number of En1 variants. Internalization and translation deficient En1 variant are already available that will be immediately tested. The role of complex sugars will also be tested without delay thanks to the existence of specific sugar-degrading enzymes.
The third task describes the physiological read outs. A first read out is the collapse of growth cones from temporal retinal axons in the presence of En1. This collapse is regulated by En1 internalization and dependent on local mRNA translation. Our preliminary results demonstrate that En1 application is immediately followed by an enhanced ATP extracellular concentration necessary for En1 collapsing activity and acting trough AR1 purinergic receptors following ATP degradation into Adenosine.
A second read out will be based on single or multi patch-clamp recordings associated to fast-cyclic voltametry on mice brain slices. We shall primarily focus on midbrain neurons from the substancia nigra but other experiments involving Otx2 and the cortex will also be pursued (se main text for details). In preliminary experiments we have observed a significant increase of T-type calcium current following an Engrailed1 acute application on identified dopaminergic neurons.

Project coordinator

Monsieur Alain Joliot (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS A) – alain.joliot@curie.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CDF - CNRS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS A
LBM-UPMC UNIVERSITE PARIS VI [PIERRE ET MARIE CURIE]
LBM-UPMC UNIVERSITE PARIS VI [PIERRE ET MARIE CURIE]
inserm U667 INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION PARIS XII
BCHP - CNRS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS A

Help of the ANR 834,199 euros
Beginning and duration of the scientific project: - 36 Months

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