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Penetratins 25
TABLE 2.1
Behavior of AntpHD Mutants
Hoemodomain Sequence a Internalization
This was achieved through the mechanical internalization of FITC-labeled homeodomains
into live postmitotic neurons. 8-10 The addition of exogenous Drosophila
Antennapedia homeodomain induced strong neurite outgrowth that was attributed
to competition between the homeodomain and endogenous homeoproteins for their
binding sites. 10 During these experiments the translocation of the Antennapedia
homeodomain (AntpHD) across biological membranes, followed by its nuclear
addressing, was discovered. This observation was later extended to several homeodomains
and full-length homeoproteins, leading to the concept of messenger proteins. 11
In an attempt to analyze the neurite-promoting function of the homeodomain
and its mechanism of action, three different point mutations were introduced
(Table 2.1). 12-14 AntpHD 50A has a glutamine/alanine substitution in position 50 of
the homeodomain (position 9 in the third helix), a position important for the specificity
of protein and DNA interactions. In AntpHD 48S, a single serine residue
replaces three amino acids (tryptophan 48, phenylalanine 49, and glutamine 50). Trp
48 and Phe 49 are conserved in all homeodomains and important for the homeodomain
structure. AntpHD 40P2 has a substitution of two amino acids located in
the turn between helices 2 and 3 (leucine 40 and threonine 41) by two prolines. The
DNA-binding capacity of the three mutants is either decreased (AntpHD 50A) or
completely abolished (AntpHD 48S and AntpHD 40P2) and translocation into live
cells is lost only in the AntpHD 48S mutant. 13 Biological activity (neurite outgrowth
stimulation) is lost in all cases. 12-14
2.2.2 THE PENETRATIN-1 PEPTIDE
DNA
binding
Biological
effect
35 60
AntpHD -AHALCLTERQIKIWFQNRRMKWKKEN +++ +++ +++
AntpHD 50A -AYALCLTERQIKIWFANRRMKWKKEN +++ + –
AntpHD 40P2 -AHALCPPERQIKIWFQNRRMKWKKEN ++ – –
AntpHD 48S -AHALCLTERQIK------SNRRMKWKKEN – – –
a Only residues 35 to 60 are shown.
Note: The third helix is in italics; mutations and deletions are shown in bold.
The results with AntpHD 48S suggested the presence of a cell translocation sequence
in the third helix. The 16 amino acids of the helix (amino acids 43 to 58, Table 2.2)
were synthesized and internalization into live cells was followed and observed thanks
to a N-terminal biotin. 15 Shorter versions of the same peptide, with N-ter or C-ter
deletions, are not internalized, suggesting that this peptide, hereafter penetratin, is
necessary and sufficient for internalization. By deleting more amino acids starting from
the N terminus, Fischer et al. have more recently described a shorter (seven amino acids)
penetratin-derived peptide with internalization properties (see Section 2.3.1.4). 16