Communication Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Targeted Subcellular Protein Delivery Using Cleavable Cyclic CellPenetrating Peptides Anselm F. L. Schneider,†,# Antoine L. D. Wallabregue,† Luise Franz,†,# and Christian P. R. Hackenberger*,†,‡ †
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany # Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustrasse 3, 14189 Berlin, Germany Downloaded via IOWA STATE UNIV on January 15, 2019 at 07:32:06 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: The delivery of entire functional proteins into living cells is a long-sought goal in science. Cyclic cell-penetrating peptides (cCPPs) have proven themselves to be potent delivery vehicles to carry proteins upon conjugation into the cytosol of living cells with immediate bioavailability via a non-endosomal uptake pathway. With this strategy, we pursue the cytosolic delivery of mCherry, a medium-sized fluorescent protein. Afterward, we achieve subcellular delivery of mCherry to different intracellular loci by genetic fusion of targeting peptides to the protein sequence. We show efficient transport into a membranebound compartment, the nucleus, as well as targeting of the actin cytoskeleton, marking one of the first ways to label actin fluorescently in genetically unmodified living cells. Furthermore, we demonstrate that only by conjugation of cCPPs via a disulfide bond, is flawless localization to the target area achieved. This finding underlines the importance of using a cCPP-based delivery vehicle that is cleaved inside cells, for the precise intracellular localization of a protein of interest.
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INTRODUCTION
While some of these methods rely on delivery into endosomes, others achieve direct transport into the cytosol of cells. In order to achieve delivery and immediate bioavailability of functional proteins, we believe that the transport into the cells should ideally occur through a nonendocytic pathway, as proteins can be degraded in endosomes due to their low pH and the presence of proteases. While the specifics of cellular uptake of cell-penetrating peptides are still not fully understood, it has been shown that in addition to an endosomal uptake they can cross membranes in an energy- and temperature-independent manner,20−22 commonly referred to as transduction. It has been previously demonstrated that the cyclization of cell-penetrating peptides can have significant effects on their potential to mediate uptake of small cargoes.23−25 Most importantly, however, cyclization strongly increases the kinetics of this transduction through the
Delivering large biomolecules into cells has been a challenging but rewarding field of research in the biomedical sciences.1 The intracellular delivery of functional proteins, that can exert their function in targeted cells, is especially promising, enabling powerful functional studies alongside potential therapeutic applications.2 Many different approaches have been developed for the cellular delivery of biomolecules, including the use of supercharged proteins,3,4 and lipid-,5−8 polymer-9−11 and nanoparticle12-mediated delivery systems as well as the use of the anthrax toxin.13 Recently, a peptide was shown to lyse endosomal membranes while keeping the cell membrane intact, providing a powerful method for proteins to be delivered after macropinocytosis.14 A recent and intriguing concept is exploiting thiols on the surface of cells using reactive disulfides to achieve highly efficient uptake of probes into cells.15−18 There are also approaches to develop delivery vehicles for biomolecules that are activated under certain circumstances,19 an important feature for future therapeutic applications. © XXXX American Chemical Society
Special Issue: Delivery of Proteins and Nucleic Acids: Achievements and Challenges Received: November 27, 2018 Revised: December 22, 2018 Published: January 8, 2019 A
DOI: 10.1021/acs.bioconjchem.8b00855 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry membrane.25 We, and others, have previously demonstrated that by using cyclic cell-penetrating peptides (cCPPs) entire functional proteins can be delivered directly into the cytosol of cells, also in a temperature-independent fashion.26−28 We could also establish that a relatively simple cyclic R10 peptide, containing ten arginine residues, improved uptake efficiency in comparison to both a linear and cyclic derivative of the commonly used transactivator of transcription (TAT) cellpenetrating peptide.28 One particular goal in designing cellular delivery approaches is the ability to direct cargoes to a specific localization within the cell. For example, the delivery of the therapeutically relevant Indium-111 radionuclide to the nucleus of cells using a nuclear localization signal increased its antiproliferative effect, when compared to delivery into the cytosol.29 In another study, delivering the drug paclitaxel to mitochondria improved its potency as an anticancer agent.30 With this aspect in mind, we attempt to expand our cCPP-mediated delivery approach in this direction by directing a functional protein into specific loci within the cell, which we monitor using confocal microscopy. As a model protein, we chose mCherry, a 27 kDa fluorescent protein that is commonly used as a fluorescence marker in cell microscopy. Witihin our study, we first generate conjugates of mCherry with different cCPPs. Afterward, we compare the use of a cleavable and noncleavable linker between the protein of interest and the cell-penetrating peptide, which became evident for the successful delivery of nanobodies to an antigen in a previous publication.21,28,31 Finally, we attempt targeting mCherry to different cellular loci by genetic fusion to targeting sequences.
conjugated to the protein via Michael-addition in slightly basic HEPES buffer at pH 8.5 overnight at room temperature (Scheme 1b). The conjugation efficiency for this reaction was very efficient as seen by the high conversion of mCherry 3 to the protein conjugate 4 by SDS-PAGE (Figure 1b). Mass
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RESULTS AND DISCUSSION Our first aim was to achieve the cytosolic delivery of mCherry into cells. We planned to synthesize mCherry-cCPP conjugates containing either a cleavable (disulfide) or noncleavable (maleimide) linker. For this, we synthesized a maleimidefunctionalized cCPP with an R10 sequence (1, Scheme 1a, SI Figure 1; for synthesis details see SI) along with our previously published cysteine-functionalized R10 cCPP (2, Scheme 1a, SI Figure 2). mCherry 3 was expressed in E. coli with a cleavable Histidine-tag for purification and an additional cysteine near the C-terminus for conjugation of the cCPP. After protein purification, the maleimide-functionalized cCPP 1 was
Figure 1. Uptake experiments for cell-permeable cleavable and noncleavable mCherry-conjugates. (a) Concept of intracellular cleavage for conjugates 4 and 5. (b) SDS-PAGE gel showing the conversion for the conjugation reactions according to Scheme 1b and 1c (for synthetic details, see SI). (c,d) Deconvoluted ESI-MS spectra of mCherry-conjugates 4 and 5. (e,f) Confocal microscopy images of HeLa cells incubated with 50 μM 4 or 5, respectively, for 1 h at 37 °C, subsequent washing and counterstaining with Hoechst 33342. Nucleoli of one cell are highlighted with yellow arrowheads.
Scheme 1. (a) Structure of the Cell-Penetrating Peptides 1 and 2 Used in This Study. (b,c) Conjugation of 1 and 2 to mCherry 3 to Yield Noncleavable and Cleavable mCherrycCPP Conjugates 4 and 5
spectrometry analysis further revealed that the imide bond of the maleimide was mostly hydrolyzed to yield a stable conjugate even in the presence of thiols (Figure 1b,c).32,33 Excess cCPP was removed by desalting the protein mixture using a spin desalting column and rebuffering into HEPES buffer at pH 7.5. The disulfide-linked mCherry-cCPP conjugate was obtained by reaction of the cysteine-containing cyclic R10 peptide 2 with the mCherry protein 1, carrying an electrophilic disulfide, activated beforehand by Ellman’s reagent following a previously published protocol (Scheme 1c). We envisioned that the disulfide-conjugate 5 produced this way should be reduced by glutathione or other thiols present in the cytosol of living cells, yielding the unmodified mCherry protein (Figure 1a) after transduction.28,34 Analogously, the conversion of the disulfide conjugation to 5 was also high as elucidated by SDS-PAGE and mass spectrometry (Figure 1b,d). It should be noted that full conversion is B
DOI: 10.1021/acs.bioconjchem.8b00855 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry difficult to achieve because of a competing formation of the mCherry disulfide homodimer as a side product. With mCherry-cCPP conjugates 4 and 5 in hand, we then probed the cellular uptake of both conjugates into live HeLa CCL-2 cells with counterstaining of the nuclei. For this, we incubated the cells with the protein-conjugates in HEPES buffer at pH 7.5 for 1 h at 37 °C, as published previously.26,28 We found very efficient cellular uptake of the protein into cells at a 50 μM concentration. At a lower concentration of 30 μM we also observed successful cellular internalization of the disulfide-conjugate 5 into the cell; however, the maleimideconjugate 4 showed predominantly endosomal uptake (SI Figure 3). As such, we chose to keep the protein concentration fixed at 50 μM in subsequent experiments. The noncleavable conjugate 4 showed localization of the protein in the cytosol of the cell as expected, but also shows the distinct localization in the nucleoli (marked with yellow arrowheads) that we and others have previously observed (Figure 1 e, representative tile scan in SI Figure 4).28,35 In contrast, the disulfide-linked conjugate 5 revealed diffuse localization in the cytosol and nucleus of the cells (Figure 1 f, representative tile scan in SI Figure 5), indicating that reductive disulfide cleavage of the cCPP-conjugate occurred, so that the protein is not directed to the nucleoli any longer. Next, we envisioned delivering mCherry into a membranebound compartment, namely, the nucleus. The standard approach to obtaining a nuclear protein is expressing the protein in cells as a genetic fusion to a nuclear localization signal (NLS). We opted for a peptide-based localization signal as well and fused the NLS of the human c-myc protein (PAAKRVKLD) to the N-terminus of mCherry (Figure 2a), as this NLS has been shown to be particularly efficient in human cells.36 Analogously to our previous conjugation reactions, we were able to obtain conjugates of the pure protein NLSmCherry protein 6 to both the maleimide and the cysteinecontaining cCPP 1 and 2 with high conjugation efficiencies to yield conjugates 7 and 8 (Figure 2b,c,d). The delivery of the nuclear localization proteins into HeLa cells proved to be efficient with a large majority of cells showing internalization of the disulfide conjugate (8, Figure 2f, representative tile scan in SI Figure 7). Interestingly, the maleimide conjugate (7, Figure 2e, representative tile scan in SI Figure 6) once again performed less efficient in uptake, showing more endosomal uptake into the cells than the disulfide. Importantly, the maleimide conjugate 7 showed very strong enrichment in the nucleoli, with another fraction of the protein being enriched in the nucleus (Figure 2e). The disulfide conjugate 8 shows enrichment of the red fluorescent protein in the nucleus, with no nucleolar localization as can be seen by strong overlap of the nuclear Hoechst stain and the red fluorescence (Figure 2f). These results, taken together with our previous findings,28 demonstrate that while mCherry already shows some nuclear localization on its own (see Figure 1f), clear nuclear localization can be achieved with reductively cleavable cCPP-protein conjugates carrying an NLS sequences. In contrast, the corresponding noncleavable cCPP-conjugate shows nuclear and predominantly nucleolar presence. Encouraged by the desired transduction in a membranebound compartment, we next applied our subcellular localization strategy to an even more challenging target. Labeling the actin cytoskeleton in living, untransfected cells is no trivial task, as commonly used phalloidin−dye conjugates are toxic to
Figure 2. Uptake experiments for cell-permeable cleavable and noncleavable NLS-mCherry-conjugates. (a) Structure of NLSmCherry-cCPP conjugates 7 and 8. (b) SDS-PAGE gel showing the conversion of the cCPP-conjugation for the NLS-mCherry protein (for synthetic details, see SI). (c,d) Deconvoluted ESI-MS spectra of NLS-mCherry-cCPP conjugates 7 and 8. (e,f) Confocal microscopy images of HeLa cells incubated with 50 μM 7 or 8, respectively, for 1 h at 37 °C, subsequent washing and counterstaining with Hoechst 33342.
the cells and additionally suffer from poor cell permeability.37 One exception to this, albeit still requiring relatively long labeling times of up to 8 h, and exhibiting an effect on cell proliferation, is the recently published silicon rhodamine (SiR)-actin, an infrared actin stain for living cells.38 By utilizing a well-characterized F-actin binding peptide (Lifeact, GVADLIKKFESISKEE),39 we envisioned delivering a Lifeact-mCherry fusion protein to the actin cytoskeleton upon conjugation with a cCPP. For this, we expressed LifeactmCherry with a C-terminal cysteine fused to proteasecleavable maltose-binding-protein (MBP) as purification and solubility tag. After removal of MBP and purification of LifeactmCherry 9 we conjugated cell-penetrating peptides 1 and 2 as before (Figure 3a). Again, the conjugation efficiencies proved to be high (Figure 3b,c,d). Delivery of the noncleavable and cleavable mCherry-conjugates 10 and 11 into cells was again efficient, although for both constructs many cells also showed a punctate pattern, which could point to an increased amount of endocytosis. Nevertheless, a majority of cells showed internalization of the constructs. We observed that the noncleavable maleimide-conjugate 10 showed some localization at the actin cytoskeleton (Figure 3e, representative tile scan in SI Figure 8), with the red fluorescence from the mCherry once again C
DOI: 10.1021/acs.bioconjchem.8b00855 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
delivery of proteins via fusogenic liposomes8 or hypertonicity,40 and overcoming this limitation must be a future goal of work in the field of cell-penetrating peptides.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00855. UV-purity, Microscopy images; Tile scans; Supporting Methods (PDF) Colocalization analysis data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Christian P. R. Hackenberger: 0000-0001-7457-4742 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Martin Lehmann for his assistance with the microscopy. We thank Kristin Kemnitz-Hassanin and Ines Kretzschmar for technical support. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SPP1623) to C.P.R.H. (HA 4468/9-1), the Einstein Foundation Berlin (Leibniz-Humboldt Professorship) and the Boehringer-Ingelheim Foundation (Plus 3 award) to C.P.R.H., the Fonds der Chemischen Industrie (FCI) to C.P.R.H. and A.F.L.S. (Chemiefonds fellowship).
Figure 3. Uptake experiments for cell-permeable cleavable and noncleavable actin-targeted-mCherry-conjugates. (a) Structure of the Lifeact-mCherry-cCPP conjugates 10 and 11. (b) SDS-PAGE gel showing the conversion of the conjugation for the Lifeact-mCherry protein (for synthetic details see SI). (c,d) Deconvoluted ESI-MS spectra of Lifeact-mCherry-cCPP conjugates 10 and 11. (e,f) Confocal microscopy images of HeLa cells incubated with 50 μM 10 or 11, respectively, for 1 h at 37 °C, then incubated with 500 nM SiR-actin for 2 h (SiR-actin shown in green for easier visualization) and finally staining of the nuclei with Hoechst 33342.
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CONCLUSION In summary, we demonstrate the delivery of a fluorescent, moderately sized protein cargo into cells using cyclic cellpenetrating peptides. Using targeting peptides, we can direct this protein into a membrane-bound compartment and to the actin-cytoskeleton. By comparing a reductively cleavable disulfide linkage with a noncleavable maleimide, we can demonstrate the importance of a cleavable delivery vehicle to ensure correct localization of the cargo protein at the targeted area. Future steps include applying the targeted delivery approach shown here to proteins with therapeutic relevance, as well as using alternative targeting moieties other than peptides to reach other loci within the cell with high efficiency. Still, the concentrations required to achieve efficient cytosolic uptake are higher when compared to other methods such as the D
DOI: 10.1021/acs.bioconjchem.8b00855 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.bioconjchem.8b00855 Bioconjugate Chem. XXXX, XXX, XXX−XXX
