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188 Cell-Penetrating Peptides: Processes and Applications
9.1 INTRODUCTION
Homeoproteins define a huge group of transcription factors sharing the evolutionary
conserved homeodomain. 1 Representatives of this group of proteins are found in all
the eukaryotic phyla, including yeasts, where they fulfill a wide range of biological
functions. Among these functions, homeoproteins are involved in developmental
processes such as the mating type switch in yeast, the main body axis patterning of
animal embryos, limb development, regionalization of developing organs, and control
of cell proliferation and differentiation. 2,3
With a few exceptions, the homeodomain is 60 amino acids long; the structure
determination of several representatives has shown a globular fold made of two
antiparallel α-helices followed by a third, perpendicular one. 3-6 In some instances,
the third α-helix may be followed by a kink and a short, fourth α-helix. The wellrecognized
function of the homeodomain is to bind DNA in a sequence-specific
manner. Its third α-helix thus fits into the major groove of a cognate DNA site with
amino acids contacting the base pairs. Furthermore, the extended N-terminal extremity
of the homeodomain makes additional contacts within the DNA minor groove.
The binding of the homeodomain to DNA also involves charge interactions, and
basic residues in particular — namely, lysines and arginines — contribute by interacting
with the phosphate groups of the DNA backbone.
Recently, it appeared that the homeodomain contributes to various other functions.
It has been shown that, at least in some instances, homeodomains can act as
an RNA binding domain, 6 define a transcriptional activation or repression domain, 6-9
modulate the activity of the protein, 10,11 and take part in protein-to-protein interactions.
12-15 Among these new roles, the most unforeseen was an involvement in the
intracellular trafficking of homeoproteins as well as in cell-to-cell communication.
Prochiantz and collaborators reported that the Antennapedia homeoprotein can
indeed be taken up by live cells. 16 Internalization by cells was further shown to be
conferred by a 16 amino acid-long peptide corresponding to the third α-helix of the
homeodomain. 17 Furthermore, this α-helix also appeared to allow the protein to be
conveyed to the cell nucleus.
Later on, the group of Prochiantz reported that the Engrailed homeoprotein can
escape cells and that its commitment to the secretory pathway is provided by an
11-amino acid-long motif overlapping helices 2 and 3 of the homeodomain and
corresponding to a functional nuclear export signal. 18,19 It was therefore proposed
that the access of Engrailed to the secretory pathway may be conditioned by its
transit to the nucleus.
All these data strongly suggest that the homeodomain transcription factors may
act as signaling molecules and play a paracrine function. However, the biological
phenomena relying on this possible paracrine function remain to be identified. The
ability of Antennapedia to be taken up by cells has now been extended to other
homeoproteins 20,21 (Rezsohazy, unpublished data).
Biophysical and cytological studies provided evidence that the cellular entry of
homeoproteins, or of peptides corresponding to the third α-helix of their homeodomain
now called penetratins, relies on a receptor- and energy-independent mechanism.
22 Indeed, penetratin can reach the intracellular milieu at either 37 or 4°C.