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Penetratins 27
there a chiral recognition mechanism between penetratins and a membrane receptor?
Do helicity and amphiphilicity influence translocation? What is the minimal
sequence required for internalization? What is the relative importance of each residue
of penetratin-1 in the internalization process?
2.3.1.1 Chirality
Two peptides were synthesized, a 43–58 peptide composed of D-amino acids (Dpenetratin-1)
and an inverso form of the 43–58 peptide: the 58–43 peptide. These
peptides are internalized as efficiently as penetratin-1 at 4 and 37°C, demonstrating
that a chiral membrane receptor is not required for cellular translocation. 15,17 Additionally,
accumulation of D-penetratin-1 was increased because of the resistance of
D-peptides to proteolysis. Fluid phase endocytosis does not require a membrane
receptor, cannot be saturated, and is not inhibited at 4°C, three characteristics
resembling those of penetratin. However, peptide localization by electronic microscopy
showed no endocytotic figures and demonstrated an accumulation in the cytoplasm
and nucleus, thus precluding fluid phase endocytosis. 17
2.3.1.2 Helicity
Peptide helicity was broken by introducing one (Pro50) or three (3Pro) prolines
within the sequence (Table 2.2). 17 In Pro50, glutamine in position 50 is replaced by
a proline; in 3Pro, glutamine 50, isoleucine 45, and lysine 55 are replaced by prolines.
Neither modification hampered peptide internalization at 4 or 37°C, suggesting that
a helical structure is not required. However, the subcellular localization of 3Pro
differed from that of penetratin-1 as it was not conveyed to the cell nucleus. This
suggested that nuclear addressing and accumulation is sequence-dependent and does
not simply reflect the small size of the peptides.
Recently, Fischer et al. have analyzed the importance of the penetratin secondary
structure by constraining its conformation. 16 Cyclic peptides were obtained through
the addition of N- and C-terminal cysteines followed by air oxidation at an elevated
pH. The linear form of the mutant peptide is internalized, but the cyclic one is not,
suggesting that the secondary structure is important for translocation.
2.3.1.3 Amphiphilicity
To address the role of amphiphilicity a penetratin-1 mutant with two phenylalanines
in place of tryptophans 48 and 56 (Table 2.2) was tested. 15 This double mutant was
not internalized, demonstrating that amphiphilicity is not sufficient to mediate internalization.
This experiment also suggested that one or both tryptophans are crucial.
More recent data, 16,18 and the internalization of the homeodomain of Engrailed which
lacks tryptophan 56, 19 demonstrates that tryptophan 48 is a key residue. It is noteworthy
that this residue has been conserved in all homeodomains.
2.3.1.4 Sequence Length
Two shorter peptides encompassing amino acids 41 to 55 or 46 to 60 (Table 2.2) do
not translocate into live cells. 15,17 This suggested that the N- and C-terminal residues