A novel cell-penetrating peptide sequence derived by structural minimization of a snake toxin exhibits preferential nucleolar localization

Article abstract of DOI:10.1021/jm8009475

Structural simplification of a 42-residue venom peptide by N-to-C-terminal splicing led to two sequences [YKQCHKKG-GXKKGSG, where X = nil (1) or 6-aminohexanoyl (2)], both efficiently uptaken by HeLa cells and, most interestingly, specifically localized at the nucleolus. Retro-2 was uptaken less efficiently, but a single (His → Ile) replacement recovered the translocation ability. None of the peptides were cytotoxic up to 100 μM. Enantio-1 did not translocate, suggesting that peptide uptake was receptor-mediated.

Full text of DOI:10.1021/jm8009475

J. Med. Chem. 2008, 51, 7041–7044
7041
Letters
A Novel Cell-Penetrating Peptide Sequence
Derived by Structural Minimization of a
Snake Toxin Exhibits Preferential Nucleolar
Localization
Gandhi Ra´dis-Baptista,†,‡,# Beatriz G. de la Torre,† and
David Andreu*,†
Department of Experimental and Health Sciences, Pompeu Fabra
UniVersity, Dr Aiguader, 80, E-08003 Barcelona, Spain, and
Department of Biochemistry, Center for Biological Sciences,
Federal UniVersity of Pernambuco, Cidade UniVersita´ria,
50732-970 Recife-PE, Brazil
ReceiVed July 28, 2008
Figure 1. 3D structure of crotamine (PDB entry no. 1h5o)29,30 showing
proximity between the N- (R-helix, residues 1-9, blue) and C-terminal
(random, residues 28-32) regions (both highlighted in blue), which is
the basis for the design of peptides 1 and 2 and retro versions 3 and 4.
Abstract: Structural simplification of a 42-residue venom peptide
by N-to-C-terminal splicing led to two sequences [YKQCHKKG-
GXKKGSG, where X ) nil (1) or 6-aminohexanoyl (2)], both
efficiently uptaken by HeLa cells and, most interestingly, specifically
localized at the nucleolus. Retro-2 was uptaken less efficiently, but a
single (His f Ile) replacement recovered the translocation ability. None
of the peptides were cytotoxic up to 100 µM. Enantio-1 did not
translocate, suggesting that peptide uptake was receptor-mediated.
from the structural similarities (cationic character, amphipathic
nature) that underlie their ability to interact with lipid bilayer
components, and eventually penetrate cells and target specific
organelles.17 Examples along these lines include the amphibian,
histone 2A-derived peptide buforin II18 or salivary histatin 5,
which translocates into the cytoplasm of Leishmania and
accumulates into the mitochondrion.19 Hybrid buforin 2-ma-
gainin 2 sequences have been shown to translocate efficiently
across the bacterial membrane and deliver into the cytoplasm
the antimicrobial R-helical portion of magainin 2.20
Animal venoms are a very rich source of bioactive polypep-
tides that in several cases have been turned into actual or
candidate drugs. Snakes,21 scorpions,22 spiders,23 or sea snails,24
to name just a few, produce and secrete a valuable diversity of
toxins capable of interacting and interfering with distinct
molecular targets in the cell. Hitherto, a very limited number
of animal toxins able to translocate the cytoplasmic membrane
and localize into distinct cell compartments are known. Two
examples are maurocalcine,25 a scorpion toxin of 33 amino acids
and three disulfide bonds that localizes in the cytoplasm and
release calcium ions, and crotamine,26 a rattlesnake toxin of 42
amino acids and also three disulfide bonds that selectively
translocates into actively proliferating cells, both in vivo and
in vitro, during the G1/S phase of the cell cycle and localizes
to the nucleus. For both peptides, the uptake mechanism appears
to involve initial binding to heparan sulfate proteoglycans,
followed by endocytosis for crotamine27 and macropinocytosis
for maurocalcine.28 In a solution NMR study, crotamine was
shown to adopt an Rꢀꢀꢀ fold29,30 (Figure 1), similar to that of
the human antimicrobial peptide ꢀ-defensin 2 or to scorpion
toxins targeting sodium channels.
Cell penetrating peptides (CPPsa) are short cationic amphi-
pathic sequences with high affinity for lipid membranes and
cell-surface proteoglycans.1,2 CPPs can translocate membrane
systems, localize in different cell compartments, and often
mediate the intracellular delivery of assorted cargos, including
drugs, imaging agents, biopolymers (nucleic acids, polypep-
tides), liposomes, and nanoparticles3-5 and have for this reason
raised considerable interest as therapeutic tools. The first
recognized CPP sequences were the transactivator of transcrip-
tion from HIV-1, named Tat protein,6 and the antennapedia
(Antp) homeodomain from Drosophila.7 Later, short fragments
of Tat [e.g., Tat(47-57) or Tat(48-60)]8 and a 16-amino acid
segment of Antp [Antp(43-58), named “penetratin”]9 were
demonstrated to be sufficient to translocate through the plasma
membranes. Since then, an increasing number of natural and
synthetic CPPs have been identified and utilized to translocate
and deliver into the cell cytoplasm and nucleus distinct types
of cargos both in vitro and in vivo.10-13 Transduction peptides,
containing or not containing homing nuclear domains, have also
been identified in other proteins like VP22 from Herpes simplex
virus type I,14 fibroblast growth factors 1 and 2,15 or the opioid
neuropeptide dynorphin.16 Some eukaryotic antimicrobial pep-
tides also display cell-penetrating behavior, somehow expected
* To whom correspondence should be addressed. Phone: +34-933160868.
Fax: +34-933160901. E-mail: david.andreu@upf.edu.
†
Pompeu Fabra University.
Federal University of Pernambuco.
Structure-guided deconstruction of complex, highly folded
bioactive peptides has been advantageously used to identify the
structural traits essential for their biological activity,31,32 i.e.,
their pharmacophore, and in turn to design minimalist, thera-
peutically useful versions of such peptides.33 Although identi-
fication of which particular section of a protein will translocate
still relies more on experimental than on predictive approaches,34
it seemed worthwhile to perform a structural dissection of
‡
#
Present address: Institute for Marine SciencessLabomar, Federal
University of Ceará, Avenida Abolição, 3207, Fortaleza-CE, 60165-081,
Brazil.
a Abbreviations: Ahx, 6-aminohexanoic acid; CPP, cell-penetrating
peptide; DIC, differential interference contrast; FITC, fluorescein isothio-
cyanate; Fmoc, 9-fluorenylmethyloxycarbonyl; HPLC, high performance
liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption
ionization time-of-flight mass spectrometry.
10.1021/jm8009475 CCC: $40.75
2008 American Chemical Society
Published on Web 11/05/2008

7042 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Letters
Figure 2. Nucleolar localization of peptide R1 with evidence of preserved nuclear envelope integrity. Slide-adhered live HeLa cells (see Supporting
Information for details) were incubated with 15 µM peptide, and cell uptake was monitored for 60 min. Then the vital dye DRAQ5 was added for
live nuclear visualization: (A) DIC image; (B) confocal microscopy image; (C) overlay of panel B image with DRAQ5 fluorescence. All panels are
at the same magnification, bar ) 50 µm. In panel B, symbols indicate cytokinesis ([), reassociating nucleoli (triangle), and interphasic nucleoli (/).
In panel C, the nucleus, the nuclear envelope and metaphasic chromosomes are clearly visible.
Table 1. Peptides Representing N-to-C-Terminal Splicing of Crotamine
the peptide remained essentially in monomeric (free thiol) form,
with e1% of dimer detectable by HPLC.
peptidea
amino acid sequenceb
residues spliced
(1-9)-(38-42)
In Figure 2 it can be seen that uptake of 1 and subsequent
localization at the nucleolus do not affect the integrity of the
nuclear envelope, as assessed by the DRAQ5 live nuclear dye.
For 2, a similar pattern of cell penetration and nucleolar
localization was observed (Supporting Information Figure SF2),
indicating that the additional flexibility provided by Ahx at the
splicing sites did not significantly alter the translocation efficacy.
By use of an FITC-labeled Tat(48-60) peptide as positive
control (i.e., 100% translocation), 1 and 2 were found to
penetrate about 50% of the live HeLa cells (compare panels A
and B in Supporting Information Figure SF2) in the 15-50 µM
interval. The peptides are seen (Figure 2 and Supporting
Information Figures SF1 and SF2) to bind various stages of
nucleolar organization, including premitotic and reassociating
nucleoli, self-organizing nucleoli, nucleoli of cytokinetic cells,
and also metaphasic chromosomes.
The partial chemical similarity of the two crotamine end
regions spliced into 1 and 2, namely, the presence of positively
charged residues at each moiety, made it worth exploring
whether sequence inversion would preserve the cell-penetrating
properties. Peptide 3, the retro version of 2 (Table 1), was thus
synthesized and shown to translocate to a much lower extent
than the previous two, though the nucleolar homing preference
remained (Supporting Information Figure SF3). A His f Ile
point mutation (analogue 4, Table 1) caused a relative increase
in uptake and nucleolar localization (Supporting Information
Figure SF4), albeit to a lesser degree than 1 or 2. Thus, for
both retro analogues a minimum 50 µM peptide concentration
was required for translocation to be observed, in contrast with
15 µM for 1 and 2.
1
2
3
4
5
YKQCHKKGGKKGSG
YKQCHKKGGXKKGSG (1-9)-Ahx-(38-42)
GSGKKGGKKHCQKY
GSGKKGGKKICQKY
ykqchkkGGkkGsG
(42-38)-(9-1)
as 3, with His f Ile replacement
D-enantiomer of 1
a Derivatives with an N-terminal rhodamine B fluorophore (labeled
R1-R5) were also prepared for each peptide. b Non-native residues are
underlined. X ) 6-aminohexanoic acid. Lower case describes D-amino acid
residues.
crotamine along the lines of the aforesaid strategies in an attempt
to define a minimal structural motif capable of membrane
translocation and internalization. Herein, we discuss some
relevant findings in this direction.
Our attention has focused particularly on the two ends of
crotamine, which are brought spatially rather close (5.09 Å
between the CR of Gly-9 and Lys-38, Figure 1) by the specific
Rꢀꢀꢀ folding of the peptide. As both these regions are quite
rich in the basic (His, Lys) residues commonly found in CPPs,
we hypothesized that their splicing into a linear sequence might
produce a peptide with translocating properties. Thus, peptide
1 (YKQCHKKGGKKGSG), spanning residues 1-9 and 38-42
of crotamine, and its analogue 2, where the N- and C-terminal
segments are spliced through a 6-aminohexanoic acid (Ahx)
flexible spacer residue (Table 1), were initially prepared.
Peptides 1 and 2 were readily synthesized by Fmoc solid
phase methods, purified by HPLC, and satisfactorily character-
ized by MALDI-TOF MS (see Supporting Information). To
examine their cell uptake, fluorescent derivatives with a
rhodamine B dye at the N-terminus were also prepared (R1 and
R2, respectively).
In all cases, the fluorescent signal of the internalized CPP
(R1-R4) vanished after 24 h of incubation of HeLa cells with
peptide-free culture medium, indicating relatively fast degrada-
tion of the internalized species. Last but not least, in an MTT
cytotoxicity assay, all four peptides were found to be harmless
to HeLa cells up to 100 µM.
As an initial exploration of the mechanism of translocation
of these CPPs, peptide 5, the all-D enantiomer of 1, was tested.
In contrast to the above results, on incubation of HeLa cells
with 5 no significant uptake was observed, the peptide localizing
exclusively in a diffuse fashion at the cytoplasmic membrane
(Supporting Information Figure SF5). While mechanisms used
The ability of R1 and R2 to translocate through eukaryotic
membranes was next investigated. HeLa cells were cultured to
60% confluence in standard conditions, then incubated with R1,
and the time course of peptide uptake was monitored by in vivo
time-lapsed imaging confocal microscopy. At 15 min incubation
time (data not shown), discrete fluorescent spots were visualized
on the membranes; then, between 45 and 60 min, the peptide
was seen to translocate through the membrane and to concentrate
preferentially in the nucleus and specifically in the nucleolus
(Supporting Information Figure SF1). The internalization process
was concentration-dependent and observable at peptide con-
centrations above 15 µM. We also ascertained that at pH 7.2

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7043
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Taken together, the above results clearly demonstrate that
the spliced crotamine peptides 1 and 2, and to a lesser extent 4
and 3, not only comprise a new group of CPPs but, more
fittingly, outline a novel nucleolar localization signature with
potentially promising applications in therapeutics. Aside from
the Cajal bodies and the splicing speckles, the nucleolus is the
most prominent functional structure in the nucleus of eukaryotic
cells. In addition to its conventional role in transcription of
rRNAs and ribosome biogenesis,42 other nucleolar functions
have been unveiled in recent years that may be more attuned to
therapeutic developments. These include roles in viral infection,
regulation of tumor suppression and oncogenesis, stress sensing,
control of aging, and modulation of telomerase activity. These
functions are all related to the movement of nucleolar compo-
nents, sequestration of regulatory cell-cycle-dependent proteins,
and transit of proteins that leave the nucleolus after their
respective transactions.43 Given their ability to localize in
assembled and dissociated nucleoli, one might suggest a role
for peptides 1-4 in the investigation of biomolecular traffic in
subnuclear bodies.
Acknowledgment. This research was supported by Minis-
terio de Educacio´n y Ciencia (Grant BIO2005-07592-CO2-02
to D.A.) and by Generalitat de Catalunya (Grant SGR2005-
00494). We thank Xavier Sanjuan (Scientific-Technical Services,
Pompeu Fabra University) and Cristina Plata (Laboratory of
Molecular Physiology, Pompeu Fabra University) for expert
assistance in confocal microscopy and cell culture, respectively.
G.R.-B. was a Visiting Researcher at the Pompeu Fabra
University, sponsored by Fundacio´n Carolina, Spain.
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Supporting Information Available: Details of solid phase
synthesis, purification, and characterization of 1-5 and their
rhodamine B-labeled derivatives, including instrumentation, tabu-
lated data, and copies of HPLC chromatograms. This material is
available free of charge via the Internet at http://pubs.acs.org.
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JM8009475