Coiled-coil mediated activation of oligo-arginine
cell-penetrating peptides
Citation for published version (APA):
Bode, S. A., Kruis, I. C., Adams, P. J. H. M., Boelens, W. C., Pruijn, G., van Hest, J. C. M., & Löwik, D. W. P. M.
(2017). Coiled-coil mediated activation of oligo-arginine cell-penetrating peptides. ChemBioChem, 18(2),
185–188 . https://doi.org/10.1002/cbic.201600614
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Coiled-Coil-Mediated Activation of Oligoarginine
Cell-Penetrating Peptides
Saskia A. Bode,
[a]Ilmar C. Kruis,
[a, b]Hans P. J. H. M. Adams,
[a]Wilbert C. Boelens,
[b]Ger J. M. Pruijn,
[b]Jan C. M. van Hest,
[a]and Dennis W. P. M. Lçwik*
[a]A supramolecular approach was undertaken to create function-ally activatable cell-penetrating peptides. Two tetra-arginines were assembled into an active cell-penetrating peptide by het-erodimerizing leucine zippers. Three different leucine-zipper pairs were evaluated: activation was found to depend on the association constant of the coiled-coil peptides. The weaker-binding peptides required an additional disulfide linkage to induce cell-penetrating capability, whereas for the most-stable coiled-coil no additional stabilization was needed. The latter zipper pair was used to show that the induced formation of the coiled coils allows control over the uptake of an oligoargi-nine CPP-conjugated cargo protein.
The efficacy of drugs with intracellular targets is dependent on their ability to cross the cellular membrane, which represents an impermeable barrier to many pharmaceutically relevant compounds.[1]Cell-penetrating peptides (CPPs) have the ability
to quickly enter living cells, and are therefore regarded as promising candidates for the delivery of bioactive cargoes into cells.[2]Extensive research has been carried out to identify
effi-cient, non-toxic CPPs that can facilitate the transport of almost any type of cargo through the cellular membrane.[3–7] One of
the main limitations of CPPs, however, is their lack of specifici-ty: regular CPPs enter almost any cell type at any given moment, and will thus deliver drugs to both healthy and dis-eased cells.[8–10]To overcome this lack of control, several
strat-egies have been developed to regulate the activity of CPPs. In general, inert peptides are converted into CPPs upon a specific environmental trigger, such as a pH change, exposure to UV light, or by enzymatic reaction.[11–16] By applying such
strat-egies, the peptide and its cargo will only be taken up at target locations where the conversion into active CPPs takes place. Previously, we reported an activation strategy for oligoarginine CPPs; this is independent of the microenvironment and is based on the reconstitution of two short, inert oligoarginines, both functionalized with a cysteine residue.[17] Upon disulfide
formation, an active CPP of eight to nine arginine residues was obtained. Nonetheless, this approach is not optimal, as non-specific reactions with thiol groups in biological systems are prone to affect the efficiency of this approach.
Here, we demonstrate how the specific interaction between heterodimerizing leucine zippers can be employed to reconsti-tute selectively two inert tetra-arginines into a functional octa-arginine CPP (Figure 1). This process of coiled-coil formation is highly selective and bio-orthogonal, as there are no competing binding partners in vivo.[18–21]As the strength of the interaction
can be modulated (even up to the association constant of biotin–streptavidin binding), this method is also to be expect-ed to work under dilute (i.e., physiologically relevant) condi-tions.
For such a strategy, monomeric leucine zippers N-terminally
elongated with tetra-arginine must not induce cellular uptake, whereas upon coiled-coil formation with the complementary leucine zipper (also containing a tetra-arginine motif), cellular uptake is induced. In order to investigate the efficiency of coiled-coil-induced uptake, three leucine-zipper pairs were considered; all form heterodimeric coiled coils with parallel di-rectionality, but differ in coiled-coil stability and amino acid composition. For further stabilization of the coiled-coil struc-ture, an optional disulfide bond between cysteine residues in-corporated near both tetra-arginine extensions can be formed (Figure 1).
For each heterodimerizing leucine-zipper pair, one peptide (A) was N-terminally elongated with the sequence Cys(Npys)-Arg4; Npys (3-nitro-2-pyridinesulfenyl) activates the thiol for
Figure 1. Coiled-coil-mediated activation strategy. X represents either an alanine or cysteine residue.
[a] Dr. S. A. Bode, I. C. Kruis, H. P. J. H. M. Adams, Prof. Dr. Ir. J. C. M. van Hest, Dr. D. W. P. M. Lçwik
Bio-Organic Chemistry, Radboud University Nijmegen Heyendaalseweg 135, 6525 AJ Nijmegen (Netherlands) E-mail: d.lowik@science.ru.nl
[b] I. C. Kruis, Dr. W. C. Boelens, Dr. G. J. M. Pruijn Biomolecular Chemistry, Radboud University Nijmegen Heyendaalseweg 135, 6525 AJ Nijmegen (Netherlands)
Supporting information and the ORCID identification numbers for the authors of this article can be found under http://dx.doi.org/10.1002/ cbic.201600614.
rapid disulfide bridge formation at neutral pH.[22] The
comple-mentary peptide (B) was extended with FITC-Ahx-Arg4
-Cys-Gly4; the tetraglycine spacer allows a semilinear presentation
of the arginine residues after complex formation between the two peptides, and fluorescein isothiocyanate (FITC) connected by an e-aminohexanoic acid (Ahx) spacer affords quantification of the cellular uptake (Figure 1). The three selected leucine-zipper pairs (termed Vin, Hod, and Jer) were reported by the groups of Vinson,[23] Hodges,[24] and Jerala,[25] respectively;
amino acid sequences in Table S1 in the Supporting Informa-tion). These leucine-zipper pairs have parallel directionality and differ in coiled-coil stability and affinity, with the Vin coiled coil having the highest binding affinity, and Jer having the lowest. The conformations of the monomeric leucine-zipper pep-tides and the heterodimerized complexes were evaluated by circular dichroism spectroscopy. The individual R4-JerA and R4
-JerB peptides exhibited no substantial helical content. How-ever, by mixing these peptides an a-helical structure was ob-tained, thus indicating coiled-coil formation (Figure S1). Both the R4-HodA and R4-HodB leucine zippers and the R4-VinA and
R4-VinB leucine zippers already showed a modest a-helical
con-tent in the monomeric form, and this strongly increased upon mixing of the corresponding pairs (Figure S1).
In order to investigate whether the tetra-arginine extension influenced the biophysical properties of the leucine-zipper in-teractions, the binding constants of the leucine zippers with and without the N-terminal extensions were measured by iso-thermal titration calorimetry (ITC). The affinity of each of the tetra-arginine leucine-zipper pairs was of the same order of magnitude as for the leucine-zipper peptides lacking the tetra-arginines, albeit slightly higher in all cases (Table 1).
Next, the cellular uptake of the FITC-labeled leucine zippers
into HeLa cells was quantified by flow cytometry and visual-ized by confocal laser scanning microscopy (CLSM). Cells were incubated with 10 mm monomeric FITC-labeled peptide for 30 min at 37 8C, briefly washed, and analyzed immediately. As expected, R4-JerB and R4-VinB did not enter the cells (Figure 2).
R4-HodB showed some membrane binding (Figure S2).
How-ever, this binding was provoked by the thiol group and not the zipper sequence, as blocking the thiol with iodoacetic acid prior to the cell experiment prevented membrane binding (*R4-HodB, Figure 2).
Next we studied whether coiled-coil formation with comple-mentary leucine zippers would induce cellular uptake. FITC-la-beled R4-functionalized peptides were mixed with the
comple-mentary zipper peptides (1:1, 10 mm) in serum-containing cell culture medium, and incubated for 30 min with HeLa cells at
378C. Zipper peptides lacking R4(e.g., VinA with R4-VinB) were
used as controls. No cellular uptake was observed for R4-JerA/
R4-JerB at 10 mm (data not shown), but at 20 mm modest
uptake by endocytosis was observed (Figure 2). At this concen-tration JerA/R4-JerB did not show cellular internalization, thus
indicating that both tetra-arginine stretches are required (Fig-ure S3). Coiled-coil R4-HodA/R4-HodB showed higher
cell-pene-trating activity than R4-JerA/R4-JerB, with the former taken up
by endocytosis at 10 mm (Figure 2). Again, the control (HodA/ R4-HodB) did not internalize (Figure S3), thus again indicating
that both tetra-arginine stretches are required. Importantly, Vin zippers showed a remarkable uptake pattern. R4-VinA/R4-VinB
was clearly observed in both the cytosol and the nucleus, and in a number of cells, enrichment in the nucleoli was found as bright spots inside the nuclei (Figure 2).
The cells were incubated with ethidium homodimer-1 or propidium iodide to ensure that the cellular membrane was not disrupted by the internalization of R4-VinA/R4-VinB. As
these compounds cannot cross the plasma membrane, they are indicators of plasma membrane integrity. Reassuringly, no internalization was observed, thus suggesting the cellular membrane was not compromised (Figure S5). Moreover, the control coiled-coil VinA/R4-VinB did not show any uptake
(Fig-ure S3).
In order to investigate whether disulfide-bridge formation between the two cysteines on each leucine zipper is necessary to induce cellular uptake, zippers R4-JerA, R4-HodA, and R4
-Table 1. Binding constants for the leucine zipper interactions measured by ITC.
Leucine-zipper pair KA[m@1] Leucine-zipper pair KA[m@1]
JerA/JerB 1.8V105 R 4-JerA/R4-JerB 5.6V105 HodA/HodB 2.2V106 R 4-HodA/R4-HodB 3.2 V106 VinA/VinB 1.8V1011 R 4-VinA/R4-VinB 4.3V1011
Figure 2. CLSM micrographs of the FITC-channel for the indicated monomer-ic leucine zippers (upper row), complexes (middle row), and coiled coils for which disulfide-bridge formation was inhibited due to C!A mutation of one of the leucines in each pair (bottom row). Scale bar: 50 mm. *R4-HodB:
R4-HodB peptide with the thiol blocked by iodoacetic acid.
ChemBioChem 2017, 18, 185 – 188 www.chembiochem.org 186 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
VinA were synthesized with mutation Cys(Npys)!Ala (C!A) to preclude disulfide bridge formation. Interestingly, neither 20 mm C!A-R4-JerA/R4-JerB nor 10 mm C!A-R4-HodA/R4-HodB
were taken up (Figure 2), thus showing that for these two coiled coils the disulfide bridge is required to keep the two tetra-arginine parts close and induce cell-penetrating activity. Intriguingly, cellular internalization by C!A-R4-VinB/R4-VinB
was very similar to that for R4-VinB/R4-VinB (Figure 2), thus
implying that for this very high-affinity leucine-zipper pair, the disulfide bridge is not needed. In addition, in separate experi-ment the formation of a disulfide bridge was also blocked by reacting the thiols of R4-JerA, R4-HodA, and R4-VinA with
iodo-acetic acid. Analysis by flow cytometry (Figure S4) revealed similar cellular uptake as for the C!A mutation.
Flow cytometry was used to quantify and compare the cellu-lar uptake of the complexes incubated with cells (Figure 3). Two additional control peptides were used: R4and R4-R4. These
peptides and their cellular uptake behaviors were described previously.[17] R
4-R4 contains a disulfide bridge to link the two
tetra-arginine stretches, analogously to the possible link be-tween the zipper peptides, in order to investigate to what extent the coiled coils contribute to the uptake of R4-R4. The
results were similar to those from the CLSM experiments. None of the leucine zippers attached to a single R4 sequence was
taken up by cells (Figure 3). In contrast, zipper-mediated R4-R4
formation resulted in cellular uptake, and the level of uptake showed a correlation with the strength of the coiled-coil inter-action. The C!A substitutions prevented uptake of the Jer and Hod zippers, but not of the Vin zipper. R4-JerA/R4-JerB was
taken up slightly less efficiently than R4-R4, whereas 10 mm R4
-HodA/R4-HodB was taken up equally as well as R4-R4. Finally,
R4-VinA/R4-VinB showed strongly superior uptake efficacy
com-pared to R4-R4. This is consistent with the efficient cytosolic
uptake of this coiled-coil complex observed by CLSM (Figure 2), most likely mediated by an uptake mechanism
other than endocytosis (found for R4-R4, as reported
ear-lier).[17]
The efficient cellular uptake of R4-VinA/R4-VinB prompted us
to investigate zipper-mediated delivery of a large protein cargo into cells. Superfolder GFP (sfGFP),[26] linked to R
4-VinA
with either a cysteine or an alanine (R4-VinA-sfGFP and
C!A-R4-VinA-sfGFP), was expressed in bacteria and purified. The
complementary leucine zipper to form the Vin coiled-coil was the peptide Ac-RRRRCGGGG-VinB (Ac-R4-VinB; R4-VinB without
a fluorescent label; protein and peptide sequences in Table S2). It was expected that R4-VinA-sfGFP and C!A-R4
-VinA-sfGFP would only be taken up after coiled-coil formation with Ac-R4-VinB. HeLa cells were incubated for 90 min at 37 8C
with 5 mm protein/peptide. First, the cells were incubated with only R4-VinA-sfGFP or C!A-R4-VinA-sfGFP. As expected, we did
not observe any uptake. However, upon addition of Ac-R4
-VinB, both R4-VinA-sfGFP and C!A-R4-VinA-sfGFP were
inter-nalized (Figure 4). These results show that even with a large cargo attached, the coiled-coil-mediated strategy based on the Vin-zipper is efficient without the need for a disulfide bridge. The cellular-localized sfGFP showed a punctuate green fluores-cence pattern, thus suggesting that the protein complexes were taken up by endocytosis. This localization pattern is different from that for the zipper peptide alone (compare Fig-ures 2 and 4) and indicates that the uptake pathway of the R4-VinA/R4-VinB coiled-coil depends on the cargo, a common
observation for CPPs.[27–28]
In summary, we have shown that the activation of a cell-penetrating peptide can be established through coiled-coil for-mation of leucine-zipper peptides. Importantly, we were able to control the uptake of a protein by this activation strategy. By extending the N termini of heterodimerizing leucine zippers with tetra-arginine motifs, coiled-coil formation yielded a struc-ture similar to octa-arginine, thereby inducing cellular uptake. Three sets of coiled coils with varying binding affinities were tested, and each led to a different uptake behavior. We con-clude that the stability of the coiled-coil that is formed by these peptides has a considerable influence on the uptake mechanism. Importantly, the use of a highly stable coiled-coil (i.e., KA>1011) eliminates the need for a covalent linkage
be-tween the two tetra-arginine parts (to keep them in close
Figure 4. CLSM micrographs from uptake experiments into HeLa cells of Vin-zipper-conjugated sfGFP without (left) or with a complementary zipper pep-tide, either containing a cysteine (middle) or an alanine (right). Scale bar: 50 mm.
Figure 3. Flow cytometry analysis of cellular uptake of coiled-coil peptide pairs compared with reference peptides R4(10 mm) and R4-R4(10 mm). It was
previously shown that R4-R4is efficiently taken up by HeLa cells, while R4is
not.[17]The indicated peptides were incubated with 10000 cells; higher
proximity). We conclude that our strategy offers a useful tool for a noncovalent conversion of inert precursors of CPPs into active peptides. Importantly, this approach does not require a trigger induced by a specific environment, and therefore pro-vides a generic method to gain control over the uptake of oli-goarginine CPP-conjugated cargo proteins. This is especially true when this strategy is combined with a targeting moiety that can be connected to one of the zippers, thus conferring cell-specific activation. We are currently pursuing this strategy, and others have shown it to be feasible for coiled-coil pep-tides.[29]
Acknowledgements
Elisabeth Pierson is acknowledged for helpful discussions. The de-partment of General Instruments of the Radboud University Nij-megen is acknowledged for providing light microscopy services. Keywords: activatable cellular uptake · cell-penetrating peptides · drug delivery · fluorescent probes · leucine zippers · noncovalent conjugation
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Manuscript received: November 15, 2016 Accepted Article published: November 21, 2016 Final Article published: December 14, 2016
ChemBioChem 2017, 18, 185 – 188 www.chembiochem.org 188 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
