Accepted Manuscript Effect of each guanidinium group on the RNA recognition and cellular uptake of Tat-derived peptides Cheng-Hsun Wu, Ming-Huei Weng, Hsien-Chen Chang, Jhe-Hao Li, Richard P. Cheng PII: DOI: Reference:
S0968-0896(14)00222-3 http://dx.doi.org/10.1016/j.bmc.2014.03.037 BMC 11484
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
10 February 2014 19 March 2014 21 March 2014
Please cite this article as: Wu, C-H., Weng, M-H., Chang, H-C., Li, J-H., Cheng, R.P., Effect of each guanidinium group on the RNA recognition and cellular uptake of Tat-derived peptides, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.03.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical Abstract
Effect of each guanidinium group on the RNA recognition and cellular uptake of Tatderived peptides
Leave this area blank for abstract info.
Cheng-Hsun Wu, Ming-Huei Weng, Hsien-Chen Chang, Jhe-Hao Li, and Richard P. Cheng* Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan
Bioorganic & Medicinal Chemistry jo u r n a l h o m e p a g e : w w w .e ls e v ie r .c o m
Effect of each guanidinium group on the RNA recognition and cellular uptake of Tatderived peptides Cheng-Hsun Wu, Ming-Huei Weng, Hsien-Chen Chang, Jhe-Hao Li and Richard P. Cheng∗ d
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan
A R T IC LE IN F O
A B S TR A C T
Article history: Received Received in revised form Accepted Available online
The six arginine (Arg) residues in the human immunodeficiency virus transactivator of transcription protein (HIV Tat protein) basic region (residues 47-57) are crucial for two bioactivities: RNA recognition and cellular uptake. Herein, we report a systematic study to investigate the role of the guanidinium group on Arg at each position in Tat-derived peptides for the two bioactivities. Tat-derived peptides, in which each guanidinium-bearing arginine was replaced with a urea-bearing citrulline (Cit) or an ammonium-bearing Lys, were synthesized by solid phase peptide synthesis. RNA recognition of the peptides was studied by electrophoretic mobility shift assays, and cellular uptake into Jurkat cells was determined by flow cytometry. Our results showed that removing the positive charge and altering the hydrogen bonding capacity of Arg affect the two biological functions differently. Furthermore, the effects are position dependent. These findings should be useful for the development of functional molecules containing guanidinium, urea, and ammonium groups for RNA recognition to affect biological processes and for cellular uptake for drug delivery.
Keywords: Tat-derived peptide Arginine Guanidinium group RNA recognition Cellular uptake
1. Introduction The human immunodeficiency virus transactivator of transcription protein (HIV Tat protein) is essential for HIV proliferation.1, 2 The Tat protein consists 101 amino acids with a basic region containing six arginines, which are highly conserved for RNA recognition3-5 and cell penetration.6-8 This 11-amino acid basic region of HIV Tat protein, Tat(47-57), specifically interacts with the bulge region of the transactivator response element (TAR) RNA.3-5 During HIV transcription, Tat protein binds TAR RNA to enable the mRNA transcription elongation9 and production of full-length RNA transcripts. As such, the TatTAR interaction is crucial for HIV proliferation,1, 2 serving as a target for potential development of anti-HIV therapeutics.10-20 The cell penetration capability of Tat enables the initiation of viral proliferation in infected neighboring cells21 and apoptosis of nearby healthy immune cells.22, 23 Exploiting this cell penetration characteristic, Tat-derived peptides have been used as molecular transporters to access intracellular targets for potential drug delivery.24-29 The six Arg residues in Tat(47-57) are critical for both TAR RNA recognition30 and cell penetration.31 The atomic resolution structure of the HIV Tat-TAR complex remains elusive, even though the structures of the bovine immunodeficiency virus and the equine infectious anemia virus Tat-TAR complexes showed a ———
2009 Elsevier Ltd. All rights reserved.
β-hairpin and an α-helical structure for the Tat-derived peptide, respectively.32, 33 Nonetheless, replacing the Arg residues at positions 52, 53, 55, 56 in HIV Tat-derived peptides with an uncharged Ala individually reduced the TAR RNA binding affinity by two fold.30 However, the positive charges on the Tatderived peptide should facilitate the binding to the poly-anionic RNA through electrostatic interactions. Accordingly, attenuation of the RNA binding affinity in the alanine scanning study30 could be attributed to just the removal of the positive charge and thus the corresponding electrostatic interaction. Also, replacing the Lys residues in Lys9 with Arg suggested that the Arg residues at positions 52 and 53 may be important for TAR RNA recognition.34 The importance of the guanidinium side chain of Arg for cellular uptake has also been explored in homopeptides.35 The homopeptides Lys9 , Cit9, and His9 all exhibited significantly lower cellular uptake compared to Arg9.35 Furthermore, simultaneously mono- or di-methylating all the guanidinium groups in Arg8 resulted in significantly reduced cellular uptake into Jurkat cells,36 suggesting the importance of the hydrogen bond donating capacity of Arg for cellular uptake. However, Tat(47-57) is distinctly different from homopeptides such as Arg8, Arg9, or Lys9.37 The aforementioned pioneering studies were important for identifying the crucial Arg positions for RNA binding30, 34 and for demonstrating the importance of the Arg guanidinium group for
∗ Corresponding author. Tel.: +886-2-3366-9789; fax: +886-2-3366-8671; e-mail: [email protected]
1
the cellular uptake. To provide further insight into the role of the Arg guanidinium groups in Tat(47-57), herein we report the TAR RNA binding and cellular uptake of Tat-derived peptides in which each guanidinium-bearing arginine in Tat(47-57) was replaced with a urea-bearing citrulline (Cit)38 or an ammonium bearing Lys individually. 2. Results 2.1. Peptide design and synthesis The Tat-derived peptides were designed based on the sequence of wild-type Tat protein residues 47-57 (Fig. 1). The native Tat(47-57) sequence was capped at both termini to give peptide ArgTat. To provide further insight into the role of the Arg guanidinium group in the TAR RNA binding of Tat(47-57), each Arg residue in ArgTat was replaced with citrulline (Cit) and Lys individually to give the TatCitN and TatLysN peptides, respectively (Fig. 1). Although both Arg and Lys are positively charged, the charge on the guanidinium group of Arg is more diffuse compared to the charge on the ammonium group of Lys. Furthermore, the guanidinium group is capable of forming multiple hydrogen bonds in a multidentate fashion, which is critical for various bioactivities.30, 35 Replacing one of the terminal nitrogens of Arg with oxygen gives the neutral ureabearing citrulline (Fig. 1). The overall shape of the Cit side chain is similar to the Arg side chain, however the net charge and hydrogen bonding profiles are different. To enable the detection of the peptides in cellular uptake experiments, the peptides were capped with 6-carboxyfluorescein at the N-terminus with an intervening βAla39 to give Flu-ArgTat, and the Flu-TatCitN and Flu-TatLysN peptides (Fig. 1). The peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry.40, 41 Upon cleavage with concomitant side chain deprotection, the peptides were confirmed by MALDI-TOF mass spectroscopy. All peptides were purified by reverse-phase high performance liquid chromatography to greater than 95% purity. The
concentration of the peptides was determined by UV-vis 42-44 spectroscopy. 2.2. Investigating RNA binding by electrophoretic mobility shift assays Electrophoretic mobility shift assays (EMSA) were performed to determine the binding of the Tat-derived peptide to TAR RNA. 45 The HIV TAR RNA was labeled with fluorescein at the 3’-terminus to enable these experiments. The bands corresponding to the presence of fluorescein-label RNA were monitored upon adding varying amounts of Tat-derived peptide (Figs. S1 and S7). The fraction of RNA bound to peptide was derived from the ratio of the peptide-RNA complex to the total RNA. The binding isotherm was fit globally assuming a 1:1 binding stoichiometry using the full quadratic equation39, 46 to obtain the apparent dissociation constant (KD) (Figs. S2 and S8). All the peptides bound TAR RNA with sub-micromolar affinity (Fig. 2 and Table S1). All the apparent KD values for the peptideTAR RNA complexes increased upon replacing Arg with Cit (Fig. 2A). For the RNA binding of the TatLysN peptides, the apparent KD also increased upon replacing Arg with Lys except for TatLys57, which exhibited an apparent KD similar to that for ArgTat (Fig. 2B).
Figure 2. Apparent dissociation constant (KD) for the TAR RNA complexes with ArgTat, TatCitN and TatLysN peptides determined by EMSA using 100 nM fluorescein-labeled HIV TAR RNA.
Figure 1. Sequences of Tat(47-57) and Tat-derived peptides.
Many negatively charged entities exist in the cell, which may bind non-specifically to positively charged peptides such as the Tat-derived peptides. To investigate the specific binding of the peptides to TAR RNA, EMSA was performed in the presence of the competing negatively charged poly(dI-dC)47 to obtain the apparent dissociation constant (KD) with attenuated nonspecific binding, i. e., specificity (Fig. 3 and Table S1). The KD for the binding between the peptides and poly(dI-dC) was not determined because poly(dI-dC) is an alternating copolymer with a range of molecular weights, multiple non-specific peptide binding sites, and unknown binding stoichiometry to the peptides. The binding of ArgTat to TAR RNA was not significantly affected by the presence of poly(dI-dC) (Figs. 2 and 3; Table S1). However, the binding affinity of the TatCitN peptides to TAR RNA was significantly reduced upon adding poly(dI-dC) (Figs. 2A and 3A; Table S1). The apparent KD of the TatCitN-TAR complexes in the presence of poly(dI-dC) followed the trend: ArgTat < TatCit57 ~ TatCit56 ~ TatCit52 ~ TatCit49 ≤ TatCit55 ~ TatCit53 (Fig 3A), which was the same as the apparent KD trend in the absence of the non-specific competitor poly(dI-dC) (Figs. 2A and 3A). However, the binding affinity of TatLysN peptides in the presence of poly(dI-dC) followed the trend: ArgTat ~ TatLys57 ~ TatLys52 ~ TatLys49 > TatLys56 ~ TatLys55 > TatLys53 (Fig. 3B) which was different from the trend in the absence of the non-specific competitor poly(dI-dC) (Figs. 2B and 3B). Furthermore, the affinity of TatLys55 and TatLys53 for TAR RNA was significantly decreased compared to
2
ArgTat (Fig. 3B). Interestingly, the affinity of TatLys57, TatLys56, TatLys52, and TatLys49 for TAR RNA was not affected by the presence of poly(dI-dC) (p value > 0.05).
should be particularly important for TAR RNA binding. 2.3. Cellular uptake determined by flow cytometry
Figure 3. Apparent dissociation constant (KD) for the TAR RNA complexes with ArgTat, TatCitN and TatLysN peptides using 100 nM fluorescein-labeled HIV TAR RNA in the presence of 10 µg/mL poly(dI-dC).
Figure 5. Flow cytometry results for cellular uptake of Flu-ArgTat and Flu-TatCitN Tat peptides into Jurkat cells in the presence of fetal bovine serum at 37 °C. Mean cellular fluorescence upon incubation with 30 µM peptide (A) and various peptide concentrations (B) for 15 minutes.
A more stringent non-specific competitor (than poly(dI-dC)) would be the bulk transfer ribonucleic acids (tRNA) from Escherichia coli.48 This mixture of various tRNAs would contain various types of RNA secondary structures with some sequence variation, making the direct determination of the KD for the complexes between the peptides and such tRNAs impractical. Nonetheless, the binding between the peptide and TAR RNA in the presence of bulk tRNA would be more representative of intracellular conditions and should screen out non-specific binding more effectively compared to poly(dI-dC). Indeed, the binding affinity for TAR RNA was attenuated more in the presence of bulk E.coli tRNA compared to poly(dI-dC) (Figs. 3 and 4; Table S1). Upon replacing Arg with Cit, all the apparent KD values for the peptide-TAR RNA complexes in the presence of E.coli tRNA increased significantly (Fig. 4A). Similarly, the apparent KD for TatLys53 binding to TAR RNA increased dramatically compared to ArgTat (Fig 4B). However, the apparent KD of TatLys55 and TatLys52 for binding TAR RNA was similar to ArgTat (Fig 4B). Surprisingly, the apparent KD for binding peptides TatLys57, TatLys56, and TatLys49 to TAR RNA in the presence of tRNA was lower than ArgTat (Fig 4B). These results showed that replacing Arg with Cit at any position decreased both the binding affinity and specificity of Tat for TAR RNA. However, the effect of replacing Arg with Lys in Tat-derived peptides on Tat-TAR RNA binding affinity and specificity was position dependent. Since the apparent KD increased dramatically upon replacing the Arg at position 53 with either Cit or Lys, the guanidinium group of Arg at this position
Figure 6. Flow cytometry results for cellular uptake into Jurkat cells in the presence of fetal bovine serum upon incubation with 120 µM FluArgTat, Flu-TatCitN (A) and Flu-TatLysN (B) peptides for 15 minutes at 37 °C.
Figure 4. Dissociation constant (KD) for the TAR RNA complexes with ArgTat, TatCitN and TatLysN peptides using 100 nM fluoresceinlabeled HIV TAR RNA in the presence of 10 µg/mL bulk E.coli tRNA.
Cellular uptake experiments were performed on Jurkat cells, because these cells are a CD4+ helper T cell cancer cell line, and CD4+ helper T cells are the target of HIV-1. Jurkat cells were incubated separately with various concentrations (30, 60, 90, and 120 µM) of Flu-TatCitN for 15 minutes at 37°C in the presence of fetal bovine serum, and then treated with trypsin to remove cell-surface bound peptide.7 The amount of peptide uptake into the cells was quantified by flow cytometry (Figs. 5 and S13-S16). Only live cells were included in these studies. The fluorescence intensity increased significantly upon increasing the peptide concentration. Incubating with 30 µM peptide, all the FluTatCitN peptides exhibited lower uptake compared to Flu-ArgTat (Fig. 5A). At concentrations higher than 60 µM, peptides FluTatCit52 and Flu-TatCit49 clearly exhibited higher cellular uptake compared to Flu-ArgTat (p value < 0.05) (Fig. 5B). The cellular uptake of peptides Flu-TatCit56, Flu-TatCit55, and FluTatCit53 were similar to Flu-ArgTat at concentrations higher than 60 µM (Fig. 5B). Interestingly, Flu-TatCit57 had the lowest cellular uptake efficiency regardless of peptide concentration. In contrast to the range of uptake efficiencies upon replacing Arg with Cit, the cellular uptake efficiency was not affected upon replacing Arg with Lys at any of the Arg positions in the FluArgTat peptide, even up to 120 µM peptide (Fig. 6B). Based on these results, replacing the Arg at positions 52 and 49 with Cit increased the cellular uptake of the peptides at higher peptide concentrations (> 60 µM) and the positive charge on Arg at position 57 was more important compared to the other positions for cellular uptake.
3
3. Discussion Our studies showed that replacing any of the Arg residues in the Tat-derived peptide with Cit greatly diminished the affinity and specificity for binding TAR RNA (Fig. 2A, 3A, and 4A). For positions 52 and 49, replacing Arg with Cit slightly increased the cell penetration at high peptide concentration (> 60 µM) (Fig. 5B). In contrast, replacing most of the Arg residues in the Tatderived peptide with Lys diminished the affinity for binding TAR RNA (Fig. 2B), however the binding specificity was position dependent (Fig. 3B and 4B). For positions 57, 56, and 49, replacing Arg with Lys increased the specific binding to TAR RNA (Fig. 4B). Nonetheless, there was minimal effect on cellular uptake upon replacing the Arg residues with Lys individually (Fig. 6B). The side chain functional group of citrulline is a neutral urea group, which is a chemical denaturant like guanidinium chloride.49 The urea group can form multiple hydrogen bonds with the base and backbone of nucleic acids.38, 50 In contrast, the side chain functional group of lysine is a positively charged ammonium group, which cannot form such a high number of hydrogen bonds compared to the guanidinium group. The binding affinity of the Tat-derived peptide for TAR RNA decreased upon introducing either Cit or Lys (to replace Arg) (Fig. 2). As such, the positive charge and the ability to form multiple hydrogen bonds in a multidentate fashion are both important for TAR RNA binding. Arg contains two primary amine groups (NH2 ) and one secondary amine group (NH), each of which can serve as a hydrogen bond donor. For Cit, oxygen is introduced at one of the terminal nitrogen positions (of Arg) and serves as a hydrogen bond acceptor. Accordingly, the difference in hydrogen bond donor/acceptor profile and charge between Arg and Cit may give rise to the significantly decreased specific binding of TAR RNA in the presence of negatively charged competitors (Fig. 3A and 4A). Introducing Lys at positions 57, 52, and 49 resulted in similar TAR RNA binding specificity compared to ArgTat in the presence of poly(dI-dC) (Fig. 3B), suggesting that charge is essential for these positions. However, introducing Lys at positions 56, 55, and 53 resulted in attenuated binding to TAR RNA (Fig. 3B). This suggests that the higher hydrogen bonding capacity of the guanidinium group on Arg at these positions is more important for interacting with TAR RNA compared to poly(dI-dC). Introducing Lys at positions 57, 56, and 49 resulted in higher binding specificity compared to ArgTat in the presence of bulk E.coli tRNA (Fig 4B). It is possible that ArgTat bound stronger to tRNA compared to TatLys57, TatLys56, and TatLys49, because the guanidinium group has more hydrogen bond donors than the ammonium group for binding. This would lower the specificity of ArgTat for binding TAR RNA, resulting in the relative higher binding specificity for Tat-derived peptide with Lys at positions 57, 56, and 49. Furthermore, the binding specificity of the Tat-derived peptide for TAR RNA diminished considerably upon introducing Cit or Lys individually at position 53, suggesting that the guanidinium group at position 53 is essential for the specific recognition of TAR RNA by Tat, consistent with the Ala scanning30 and Lys9 replacement studies.34 The first step for the cellular uptake of guanidinium rich transporters, including the Tat-derived peptide, is the association of the positively charged guanidinium groups with the negatively charged entities on the membrane.51 The positively charged guanidinium group features a rigidly planar hydrogen bond donors which can form a bidentate hydrogen bond with negatively charged functional groups on the membrane
constituents such as carboxylates, phosphates, and sulfates. 36 The urea group of Cit may also form such bidentate hydrogen bonds similar to the guanidinium group.38, 50 However, the cellular uptake decreased dramatically at 30 µM for the Flu-TatCitN peptides compared to ArgTat, demonstrating the importance of the positive charge on Arg for cellular uptake. Interestingly, the cellular uptake for two of the Flu-TatCitN peptides was slightly higher than Flu-ArgTat at 120 µM (Figs. 5B and 6A). Removing one positive charge would attenuate the electrostatic interaction between the peptide and the negatively charged moieties on the membrane during the association step, leading to diminished cellular uptake. However, the decreased association could be compensated by increasing the peptide concentration to increase the amount of cell surface binding, leading to similar overall cellular uptake efficiency compared to Flu-ArgTat. Furthermore, the positive charge at position 57 of the Tat-derived peptide was more sensitive to Cit incorporation compared to the other positions for cellular uptake. Significantly decreased uptake was previously shown upon simultaneously replacing all the Arg residues with Lys in Arg9.35 In contrast, our results on FluTatLysN peptides showed that replacing Arg with Lys individually has minimal effect for cellular uptake (Fig. 6B). This result suggests that the effect of the number and form of hydrogen bond donors on cellular uptake was less significant compared to removing the positive charge at any of the six positions of Tat-derived peptide except for position 57. The results on TAR RNA binding and cellular uptake together suggest that Lys may be incorporated at positions 49, 56, or 57 (individually) in the Tat basic region without attenuating either specific binding of TAR RNA or cell penetration. As such, these three substitutions may be natural mutations in viable HIV Tat proteins. 4. Conclusions The guanidinium group on the six Arg residues is important for TAR RNA recognition and cellular uptake of Tat-derived peptides. The effect of replacing the guanidinium-bearing Arg with either the urea-bearing Cit or ammonium-bearing Lys on these two biological functions were investigated. Our results showed that removing the positive charge and altering the hydrogen bonding capacity of Arg affected the two biological functions differently. Furthermore, the effects were position dependent. The positive charge of Arg was important for specific TAR RNA binding. However, for position 53, any change to the guanidinium group resulted in significant attenuation of the specific RNA binding. Cellular uptake was not affected significantly so long as the positive charge was retained. Interestingly, replacing the positively charged Arg with the neutral Cit at positions 49 or 52 enhanced the uptake at concentrations greater than 60 µM. The same replacement at position 57 resulted in reduced uptake. These findings should be useful for the development of functional molecules containing guanidinium, urea, and ammonium groups for RNA recognition to affect biological processes and for cell penetration for intracellular drug delivery. 5. Experimental section 5.1. Peptide synthesis The peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry. 40, 41 After cleavage, the peptides were purified by reverse phase HPLC and confirmed by MALDI-TOF MS. 5.2. Electrophoretic mobility shift assay (EMSA)
4
The fluorescein-labeled RNA (100 nM) and peptide (at various concentrations) were incubated in pH 7.4 buffer (10 µL) containing Tris-HCl (50 mM), KCl (50 mM), 2% glycerol, and Triton X-100 (0.05%) in the presence of poly(dI-dC) (10 µg/mL) or bulk E.coli tRNA (10 µg/mL) at room temperature. The samples were analyzed by loading into 12% native polyacrylamide gels in 0.5X TB buffer and electrophoresis was performed with 140 V at room temperature. Bands corresponding to the free and bound RNA were used to determine to fraction bound RNA. The fraction bound RNA data was used to globally derive the apparent dissociation constants assuming a 1:1 binding stoichiometry39, 46 using the full quadratic equation (see SI). 5.3. Cellular uptake assay Jurkat cells (8 x 105) were incubated with the peptides at various concentrations (30, 60, 90, and 120 µM) at 37 °C with 5% CO2 for 15 minutes. The cells were then incubated with 0.05 % trypsin/EDTA in PBS for 5 minutes to remove the peptides which adhered to the cell surface rather than entry to the cell.7 Propidium iodide (PI) was added to all samples to stain the dead cells but should not stain the live cells. The cells were then transferred into the flow tube and analyzed by flow cytometry (FACScan, Becton Dickinson Bioscience). The minimum propidium iodide fluorescence intensity for the dead control cells (which were terminated by adding Triton-X 100) treated with propidium iodide was set as the threshold value. The mean 6carboxyfluorescein fluorescence intensity for 10,000 live cells (with appropriate forward scatter and side scatter values, and below the propidium iodide fluorescence threshold) was determined for each experiment. Each experiment was independently repeated at least three times.
9. 10.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
21.
22.
Acknowledgments This work was supported by National Taiwan University (NTU-ERP-103R891302 and NTU-CESRP-103R7621) and the National Science Council in Taiwan (NSC-99-2113-M-002-002MY2, NSC-101-2113-M-002-006-MY2). Supplementary data
23. 24. 25. 26.
Supplementary data associated with this article can be found, in the online version, at
27.
References and notes
28.
1. 2. 3.
4. 5. 6. 7.
8.
Cullen, B. R. FASEB J. 1991, 5, 2361-2368. Stevens, M.; De Clercq, E.; Balzarini, J. Med. Res. Rev. 2006, 26, 595-625. Cordingley, M. G.; Lafemina, R. L.; Callahan, P. L.; Condra, J. H.; Sardana, V. V.; Graham, D. J.; Nguyen, T. M.; Legrow, K.; Gotlib, L.; Schlabach, A. J.; Colonno, R. J. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 8985-8989. Weeks, K. M.; Ampe, C.; Schultz, S. C.; Steitz, T. A.; Crothers, D. M. Science 1990, 249, 1281-1285. Rosen, C. A.; Sodroski, J. G.; Haseltine, W. A. Cell 1985, 41, 813-823. Rana, T. M.; Jeang, K. T. Arch. Biochem. Biophys. 1999, 365, 175-185. Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.; Verbeure, B.; Gait, M. J.; Chernomordik, L. V.; Lebleu, B. J. Biol. Chem. 2003, 278, 585-590. Vives, E.; Brodin, P.; Lebleu, B. J. Biol. Chem. 1997, 272, 16010-16017.
29.
30. 31.
32. 33.
34. 35.
Kao, S. Y.; Calman, A. F.; Luciw, P. A.; Peterlin, B. M. Nature 1987, 330, 489-493. Bryson, D. I.; Zhang, W. Y.; McLendon, P. M.; Reineke, T. M.; Santos, W. L. ACS Chem. Biol. 2012, 7, 210-217. Lee, S. J.; Hyun, S.; Kieft, J. S.; Yu, J. J. Am. Chem. Soc. 2009, 131, 2224-2230. Huq, I.; Ping, Y. H.; Tamilarasu, N.; Rana, T. M. Biochemistry 1999, 38, 5172-5177. Kumar, S.; Maiti, S. Biochimie 2013, 95, 1422-1431. Kumar, S.; Maiti, S. Plos One 2013, 8. Wang, X. L.; Huq, I.; Rana, T. M. J. Am. Chem. Soc. 1997, 119, 6444-6445. Tamilarasu, N.; Huq, I.; Rana, T. M. J. Am. Chem. Soc. 1999, 121, 1597-1598. Kesavan, V.; Tamilarasu, N.; Cao, H.; Rana, T. M. Bioconjugate Chem. 2002, 13, 1171-1175. Belousoff, M. J.; Gasser, G.; Graham, B.; Tor, Y.; Spiccia, L. J. Biol. Inorg. Chem. 2009, 14, 287-300. Belousoff, M. J.; Graham, B.; Spiccia, L.; Tor, Y. Org. Biomol. Chem. 2009, 7, 30-33. Staple, D. W.; Venditti, V.; Niccolai, N.; Elson-Schwab, L.; Tor, Y.; Butcher, S. E. ChemBioChem 2008, 9, 93102. Ensoli, B.; Buonaguro, L.; Barillari, G.; Fiorelli, V.; Gendelman, R.; Morgan, R. A.; Wingfield, P.; Gallo, R. C. J. Virol. 1993, 67, 277-287. Berman, J. W.; Eugenin, E. A.; King, J. E.; Nath, A.; Calderon, T. M.; Zukin, R. S.; Bennett, M. V. L. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 3438-3443. Misumi, S.; Takamune, N.; Ohtsubo, Y.; Waniguchi, K.; Shoji, S. AIDS Res. Hum. Retrov. 2004, 20, 297-304. Fischer, R.; Fotin-Mleczek, M.; Hufnagel, H.; Brock, R. ChemBioChem 2005, 6, 2126-2142. Wadia, J. S.; Stan, R. V.; Dowdy, S. F. Nat. Med. 2004, 10, 310-315. Torchilin, V. P. Adv. Drug Delivery Rev. 2008, 60, 548558. El-Sayed, A.; Futaki, S.; Harashima, H. AAPS J. 2009, 11, 13-22. Wu, C. H.; Chen, Y. P.; Wu, S. H.; Hung, Y.; Mou, C. Y.; Cheng, R. P. ACS Appl. Mater. Interfaces 2013, 5, 12244-12248. Erazo-Oliveras, A.; Muthukrishnan, N.; Baker, R.; Wang, T. Y.; Pellois, J. P. Pharmaceuticals 2012, 5, 1177-1209. Calnan, B. J.; Biancalana, S.; Hudson, D.; Frankel, A. D. Genes Dev. 1991, 5, 201-210. Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.; Rothbard, J. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003-13008. Puglisi, J. D.; Chen, L.; Blanchard, S.; Frankel, A. D. Science 1995, 270, 1200-1203. Anand, K.; Schulte, A.; Vogel-Bachmayr, K.; Scheffzek, K.; Geyer, M. Nat. Struct. Mol. Biol. 2008, 15, 12871292. Calnan, B. J.; Tidor, B.; Biancalana, S.; Hudson, D.; Frankel, A. D. Science 1991, 252, 1167-1171. Mitchell, D. J.; Kim, D. T.; Steinman, L.; Fathman, C. G.; Rothbard, J. B. J. Pept. Res. 2000, 56, 318-325.
5
36.
37. 38.
39.
40.
41. 42. 43. 44. 45. 46. 47. 48.
49. 50.
51.
Wender, P. A.; Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A. J. Am. Chem. Soc. 2004, 126, 95069507. Wu, C.-H.; Chen, Y.-P.; Mou, C.-Y.; Cheng, R. P. Amino Acids 2013, 44, 473-480. Priyakumar, U. D.; Hyeon, C.; Thirumalai, D.; MacKerell, A. D. J. Am. Chem. Soc. 2009, 131, 1775917761. Gelman, M. A.; Richter, S.; Cao, H.; Umezawa, N.; Gellman, S. H.; Rana, T. M. Org. Lett. 2003, 5, 35633565. Atherton, E.; Fox, H.; Harkiss, D.; Logan, C. J.; Sheppard, R. C.; Williams, B. J. J. Chem. Soc. Chem. Commun. 1978, 537-539. Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214. Edelhoch, H. Biochemistry 1967, 6, 1948-1954. Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. Protein Sci. 1995, 4, 2411-2423. Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals; Eugene, OR, USA, 1996. Wang, J. H.; Huang, S. Y.; Choudhury, I.; Leibowitz, M. J.; Stein, S. Anal. Biochem. 1995, 232, 238-242. Suryawanshi, H.; Sabharwal, H.; Maiti, S. J. Phys. Chem. B 2010, 114, 11155-11163. Zondlo, N. J.; Schepartz, A. J. Am. Chem. Soc. 1999, 121, 6938-6939. Athanassiou, Z.; Dias, R. L. A.; Moehle, K.; Dobson, N.; Varani, G.; Robinson, J. A. J. Am. Chem. Soc. 2004, 126, 6906-6913. Rashid, F.; Sharma, S.; Bano, B. Protein J. 2005, 24, 283-292. Guinn, E. J.; Schwinefus, J. J.; Cha, H. K.; McDevitt, J. L.; Merker, W. E.; Ritzer, R.; Muth, G. W.; Engelsgjerd, S. W.; Mangold, K. E.; Thompson, P. J.; Kerins, M. J.; Record, M. T. J. Am. Chem. Soc. 2013, 135, 5828-5838. Wender, P. A.; Galliher, W. C.; Goun, E. A.; Jones, L. R.; Pillow, T. H. Adv. Drug Delivery Rev. 2008, 60, 452-472.
6
S0968-0896(14)00222-3 http://dx.doi.org/10.1016/j.bmc.2014.03.037 BMC 11484
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
10 February 2014 19 March 2014 21 March 2014
Please cite this article as: Wu, C-H., Weng, M-H., Chang, H-C., Li, J-H., Cheng, R.P., Effect of each guanidinium group on the RNA recognition and cellular uptake of Tat-derived peptides, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.03.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical Abstract
Effect of each guanidinium group on the RNA recognition and cellular uptake of Tatderived peptides
Leave this area blank for abstract info.
Cheng-Hsun Wu, Ming-Huei Weng, Hsien-Chen Chang, Jhe-Hao Li, and Richard P. Cheng* Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan
Bioorganic & Medicinal Chemistry jo u r n a l h o m e p a g e : w w w .e ls e v ie r .c o m
Effect of each guanidinium group on the RNA recognition and cellular uptake of Tatderived peptides Cheng-Hsun Wu, Ming-Huei Weng, Hsien-Chen Chang, Jhe-Hao Li and Richard P. Cheng∗ d
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan
A R T IC LE IN F O
A B S TR A C T
Article history: Received Received in revised form Accepted Available online
The six arginine (Arg) residues in the human immunodeficiency virus transactivator of transcription protein (HIV Tat protein) basic region (residues 47-57) are crucial for two bioactivities: RNA recognition and cellular uptake. Herein, we report a systematic study to investigate the role of the guanidinium group on Arg at each position in Tat-derived peptides for the two bioactivities. Tat-derived peptides, in which each guanidinium-bearing arginine was replaced with a urea-bearing citrulline (Cit) or an ammonium-bearing Lys, were synthesized by solid phase peptide synthesis. RNA recognition of the peptides was studied by electrophoretic mobility shift assays, and cellular uptake into Jurkat cells was determined by flow cytometry. Our results showed that removing the positive charge and altering the hydrogen bonding capacity of Arg affect the two biological functions differently. Furthermore, the effects are position dependent. These findings should be useful for the development of functional molecules containing guanidinium, urea, and ammonium groups for RNA recognition to affect biological processes and for cellular uptake for drug delivery.
Keywords: Tat-derived peptide Arginine Guanidinium group RNA recognition Cellular uptake
1. Introduction The human immunodeficiency virus transactivator of transcription protein (HIV Tat protein) is essential for HIV proliferation.1, 2 The Tat protein consists 101 amino acids with a basic region containing six arginines, which are highly conserved for RNA recognition3-5 and cell penetration.6-8 This 11-amino acid basic region of HIV Tat protein, Tat(47-57), specifically interacts with the bulge region of the transactivator response element (TAR) RNA.3-5 During HIV transcription, Tat protein binds TAR RNA to enable the mRNA transcription elongation9 and production of full-length RNA transcripts. As such, the TatTAR interaction is crucial for HIV proliferation,1, 2 serving as a target for potential development of anti-HIV therapeutics.10-20 The cell penetration capability of Tat enables the initiation of viral proliferation in infected neighboring cells21 and apoptosis of nearby healthy immune cells.22, 23 Exploiting this cell penetration characteristic, Tat-derived peptides have been used as molecular transporters to access intracellular targets for potential drug delivery.24-29 The six Arg residues in Tat(47-57) are critical for both TAR RNA recognition30 and cell penetration.31 The atomic resolution structure of the HIV Tat-TAR complex remains elusive, even though the structures of the bovine immunodeficiency virus and the equine infectious anemia virus Tat-TAR complexes showed a ———
2009 Elsevier Ltd. All rights reserved.
β-hairpin and an α-helical structure for the Tat-derived peptide, respectively.32, 33 Nonetheless, replacing the Arg residues at positions 52, 53, 55, 56 in HIV Tat-derived peptides with an uncharged Ala individually reduced the TAR RNA binding affinity by two fold.30 However, the positive charges on the Tatderived peptide should facilitate the binding to the poly-anionic RNA through electrostatic interactions. Accordingly, attenuation of the RNA binding affinity in the alanine scanning study30 could be attributed to just the removal of the positive charge and thus the corresponding electrostatic interaction. Also, replacing the Lys residues in Lys9 with Arg suggested that the Arg residues at positions 52 and 53 may be important for TAR RNA recognition.34 The importance of the guanidinium side chain of Arg for cellular uptake has also been explored in homopeptides.35 The homopeptides Lys9 , Cit9, and His9 all exhibited significantly lower cellular uptake compared to Arg9.35 Furthermore, simultaneously mono- or di-methylating all the guanidinium groups in Arg8 resulted in significantly reduced cellular uptake into Jurkat cells,36 suggesting the importance of the hydrogen bond donating capacity of Arg for cellular uptake. However, Tat(47-57) is distinctly different from homopeptides such as Arg8, Arg9, or Lys9.37 The aforementioned pioneering studies were important for identifying the crucial Arg positions for RNA binding30, 34 and for demonstrating the importance of the Arg guanidinium group for
∗ Corresponding author. Tel.: +886-2-3366-9789; fax: +886-2-3366-8671; e-mail: [email protected]
1
the cellular uptake. To provide further insight into the role of the Arg guanidinium groups in Tat(47-57), herein we report the TAR RNA binding and cellular uptake of Tat-derived peptides in which each guanidinium-bearing arginine in Tat(47-57) was replaced with a urea-bearing citrulline (Cit)38 or an ammonium bearing Lys individually. 2. Results 2.1. Peptide design and synthesis The Tat-derived peptides were designed based on the sequence of wild-type Tat protein residues 47-57 (Fig. 1). The native Tat(47-57) sequence was capped at both termini to give peptide ArgTat. To provide further insight into the role of the Arg guanidinium group in the TAR RNA binding of Tat(47-57), each Arg residue in ArgTat was replaced with citrulline (Cit) and Lys individually to give the TatCitN and TatLysN peptides, respectively (Fig. 1). Although both Arg and Lys are positively charged, the charge on the guanidinium group of Arg is more diffuse compared to the charge on the ammonium group of Lys. Furthermore, the guanidinium group is capable of forming multiple hydrogen bonds in a multidentate fashion, which is critical for various bioactivities.30, 35 Replacing one of the terminal nitrogens of Arg with oxygen gives the neutral ureabearing citrulline (Fig. 1). The overall shape of the Cit side chain is similar to the Arg side chain, however the net charge and hydrogen bonding profiles are different. To enable the detection of the peptides in cellular uptake experiments, the peptides were capped with 6-carboxyfluorescein at the N-terminus with an intervening βAla39 to give Flu-ArgTat, and the Flu-TatCitN and Flu-TatLysN peptides (Fig. 1). The peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry.40, 41 Upon cleavage with concomitant side chain deprotection, the peptides were confirmed by MALDI-TOF mass spectroscopy. All peptides were purified by reverse-phase high performance liquid chromatography to greater than 95% purity. The
concentration of the peptides was determined by UV-vis 42-44 spectroscopy. 2.2. Investigating RNA binding by electrophoretic mobility shift assays Electrophoretic mobility shift assays (EMSA) were performed to determine the binding of the Tat-derived peptide to TAR RNA. 45 The HIV TAR RNA was labeled with fluorescein at the 3’-terminus to enable these experiments. The bands corresponding to the presence of fluorescein-label RNA were monitored upon adding varying amounts of Tat-derived peptide (Figs. S1 and S7). The fraction of RNA bound to peptide was derived from the ratio of the peptide-RNA complex to the total RNA. The binding isotherm was fit globally assuming a 1:1 binding stoichiometry using the full quadratic equation39, 46 to obtain the apparent dissociation constant (KD) (Figs. S2 and S8). All the peptides bound TAR RNA with sub-micromolar affinity (Fig. 2 and Table S1). All the apparent KD values for the peptideTAR RNA complexes increased upon replacing Arg with Cit (Fig. 2A). For the RNA binding of the TatLysN peptides, the apparent KD also increased upon replacing Arg with Lys except for TatLys57, which exhibited an apparent KD similar to that for ArgTat (Fig. 2B).
Figure 2. Apparent dissociation constant (KD) for the TAR RNA complexes with ArgTat, TatCitN and TatLysN peptides determined by EMSA using 100 nM fluorescein-labeled HIV TAR RNA.
Figure 1. Sequences of Tat(47-57) and Tat-derived peptides.
Many negatively charged entities exist in the cell, which may bind non-specifically to positively charged peptides such as the Tat-derived peptides. To investigate the specific binding of the peptides to TAR RNA, EMSA was performed in the presence of the competing negatively charged poly(dI-dC)47 to obtain the apparent dissociation constant (KD) with attenuated nonspecific binding, i. e., specificity (Fig. 3 and Table S1). The KD for the binding between the peptides and poly(dI-dC) was not determined because poly(dI-dC) is an alternating copolymer with a range of molecular weights, multiple non-specific peptide binding sites, and unknown binding stoichiometry to the peptides. The binding of ArgTat to TAR RNA was not significantly affected by the presence of poly(dI-dC) (Figs. 2 and 3; Table S1). However, the binding affinity of the TatCitN peptides to TAR RNA was significantly reduced upon adding poly(dI-dC) (Figs. 2A and 3A; Table S1). The apparent KD of the TatCitN-TAR complexes in the presence of poly(dI-dC) followed the trend: ArgTat < TatCit57 ~ TatCit56 ~ TatCit52 ~ TatCit49 ≤ TatCit55 ~ TatCit53 (Fig 3A), which was the same as the apparent KD trend in the absence of the non-specific competitor poly(dI-dC) (Figs. 2A and 3A). However, the binding affinity of TatLysN peptides in the presence of poly(dI-dC) followed the trend: ArgTat ~ TatLys57 ~ TatLys52 ~ TatLys49 > TatLys56 ~ TatLys55 > TatLys53 (Fig. 3B) which was different from the trend in the absence of the non-specific competitor poly(dI-dC) (Figs. 2B and 3B). Furthermore, the affinity of TatLys55 and TatLys53 for TAR RNA was significantly decreased compared to
2
ArgTat (Fig. 3B). Interestingly, the affinity of TatLys57, TatLys56, TatLys52, and TatLys49 for TAR RNA was not affected by the presence of poly(dI-dC) (p value > 0.05).
should be particularly important for TAR RNA binding. 2.3. Cellular uptake determined by flow cytometry
Figure 3. Apparent dissociation constant (KD) for the TAR RNA complexes with ArgTat, TatCitN and TatLysN peptides using 100 nM fluorescein-labeled HIV TAR RNA in the presence of 10 µg/mL poly(dI-dC).
Figure 5. Flow cytometry results for cellular uptake of Flu-ArgTat and Flu-TatCitN Tat peptides into Jurkat cells in the presence of fetal bovine serum at 37 °C. Mean cellular fluorescence upon incubation with 30 µM peptide (A) and various peptide concentrations (B) for 15 minutes.
A more stringent non-specific competitor (than poly(dI-dC)) would be the bulk transfer ribonucleic acids (tRNA) from Escherichia coli.48 This mixture of various tRNAs would contain various types of RNA secondary structures with some sequence variation, making the direct determination of the KD for the complexes between the peptides and such tRNAs impractical. Nonetheless, the binding between the peptide and TAR RNA in the presence of bulk tRNA would be more representative of intracellular conditions and should screen out non-specific binding more effectively compared to poly(dI-dC). Indeed, the binding affinity for TAR RNA was attenuated more in the presence of bulk E.coli tRNA compared to poly(dI-dC) (Figs. 3 and 4; Table S1). Upon replacing Arg with Cit, all the apparent KD values for the peptide-TAR RNA complexes in the presence of E.coli tRNA increased significantly (Fig. 4A). Similarly, the apparent KD for TatLys53 binding to TAR RNA increased dramatically compared to ArgTat (Fig 4B). However, the apparent KD of TatLys55 and TatLys52 for binding TAR RNA was similar to ArgTat (Fig 4B). Surprisingly, the apparent KD for binding peptides TatLys57, TatLys56, and TatLys49 to TAR RNA in the presence of tRNA was lower than ArgTat (Fig 4B). These results showed that replacing Arg with Cit at any position decreased both the binding affinity and specificity of Tat for TAR RNA. However, the effect of replacing Arg with Lys in Tat-derived peptides on Tat-TAR RNA binding affinity and specificity was position dependent. Since the apparent KD increased dramatically upon replacing the Arg at position 53 with either Cit or Lys, the guanidinium group of Arg at this position
Figure 6. Flow cytometry results for cellular uptake into Jurkat cells in the presence of fetal bovine serum upon incubation with 120 µM FluArgTat, Flu-TatCitN (A) and Flu-TatLysN (B) peptides for 15 minutes at 37 °C.
Figure 4. Dissociation constant (KD) for the TAR RNA complexes with ArgTat, TatCitN and TatLysN peptides using 100 nM fluoresceinlabeled HIV TAR RNA in the presence of 10 µg/mL bulk E.coli tRNA.
Cellular uptake experiments were performed on Jurkat cells, because these cells are a CD4+ helper T cell cancer cell line, and CD4+ helper T cells are the target of HIV-1. Jurkat cells were incubated separately with various concentrations (30, 60, 90, and 120 µM) of Flu-TatCitN for 15 minutes at 37°C in the presence of fetal bovine serum, and then treated with trypsin to remove cell-surface bound peptide.7 The amount of peptide uptake into the cells was quantified by flow cytometry (Figs. 5 and S13-S16). Only live cells were included in these studies. The fluorescence intensity increased significantly upon increasing the peptide concentration. Incubating with 30 µM peptide, all the FluTatCitN peptides exhibited lower uptake compared to Flu-ArgTat (Fig. 5A). At concentrations higher than 60 µM, peptides FluTatCit52 and Flu-TatCit49 clearly exhibited higher cellular uptake compared to Flu-ArgTat (p value < 0.05) (Fig. 5B). The cellular uptake of peptides Flu-TatCit56, Flu-TatCit55, and FluTatCit53 were similar to Flu-ArgTat at concentrations higher than 60 µM (Fig. 5B). Interestingly, Flu-TatCit57 had the lowest cellular uptake efficiency regardless of peptide concentration. In contrast to the range of uptake efficiencies upon replacing Arg with Cit, the cellular uptake efficiency was not affected upon replacing Arg with Lys at any of the Arg positions in the FluArgTat peptide, even up to 120 µM peptide (Fig. 6B). Based on these results, replacing the Arg at positions 52 and 49 with Cit increased the cellular uptake of the peptides at higher peptide concentrations (> 60 µM) and the positive charge on Arg at position 57 was more important compared to the other positions for cellular uptake.
3
3. Discussion Our studies showed that replacing any of the Arg residues in the Tat-derived peptide with Cit greatly diminished the affinity and specificity for binding TAR RNA (Fig. 2A, 3A, and 4A). For positions 52 and 49, replacing Arg with Cit slightly increased the cell penetration at high peptide concentration (> 60 µM) (Fig. 5B). In contrast, replacing most of the Arg residues in the Tatderived peptide with Lys diminished the affinity for binding TAR RNA (Fig. 2B), however the binding specificity was position dependent (Fig. 3B and 4B). For positions 57, 56, and 49, replacing Arg with Lys increased the specific binding to TAR RNA (Fig. 4B). Nonetheless, there was minimal effect on cellular uptake upon replacing the Arg residues with Lys individually (Fig. 6B). The side chain functional group of citrulline is a neutral urea group, which is a chemical denaturant like guanidinium chloride.49 The urea group can form multiple hydrogen bonds with the base and backbone of nucleic acids.38, 50 In contrast, the side chain functional group of lysine is a positively charged ammonium group, which cannot form such a high number of hydrogen bonds compared to the guanidinium group. The binding affinity of the Tat-derived peptide for TAR RNA decreased upon introducing either Cit or Lys (to replace Arg) (Fig. 2). As such, the positive charge and the ability to form multiple hydrogen bonds in a multidentate fashion are both important for TAR RNA binding. Arg contains two primary amine groups (NH2 ) and one secondary amine group (NH), each of which can serve as a hydrogen bond donor. For Cit, oxygen is introduced at one of the terminal nitrogen positions (of Arg) and serves as a hydrogen bond acceptor. Accordingly, the difference in hydrogen bond donor/acceptor profile and charge between Arg and Cit may give rise to the significantly decreased specific binding of TAR RNA in the presence of negatively charged competitors (Fig. 3A and 4A). Introducing Lys at positions 57, 52, and 49 resulted in similar TAR RNA binding specificity compared to ArgTat in the presence of poly(dI-dC) (Fig. 3B), suggesting that charge is essential for these positions. However, introducing Lys at positions 56, 55, and 53 resulted in attenuated binding to TAR RNA (Fig. 3B). This suggests that the higher hydrogen bonding capacity of the guanidinium group on Arg at these positions is more important for interacting with TAR RNA compared to poly(dI-dC). Introducing Lys at positions 57, 56, and 49 resulted in higher binding specificity compared to ArgTat in the presence of bulk E.coli tRNA (Fig 4B). It is possible that ArgTat bound stronger to tRNA compared to TatLys57, TatLys56, and TatLys49, because the guanidinium group has more hydrogen bond donors than the ammonium group for binding. This would lower the specificity of ArgTat for binding TAR RNA, resulting in the relative higher binding specificity for Tat-derived peptide with Lys at positions 57, 56, and 49. Furthermore, the binding specificity of the Tat-derived peptide for TAR RNA diminished considerably upon introducing Cit or Lys individually at position 53, suggesting that the guanidinium group at position 53 is essential for the specific recognition of TAR RNA by Tat, consistent with the Ala scanning30 and Lys9 replacement studies.34 The first step for the cellular uptake of guanidinium rich transporters, including the Tat-derived peptide, is the association of the positively charged guanidinium groups with the negatively charged entities on the membrane.51 The positively charged guanidinium group features a rigidly planar hydrogen bond donors which can form a bidentate hydrogen bond with negatively charged functional groups on the membrane
constituents such as carboxylates, phosphates, and sulfates. 36 The urea group of Cit may also form such bidentate hydrogen bonds similar to the guanidinium group.38, 50 However, the cellular uptake decreased dramatically at 30 µM for the Flu-TatCitN peptides compared to ArgTat, demonstrating the importance of the positive charge on Arg for cellular uptake. Interestingly, the cellular uptake for two of the Flu-TatCitN peptides was slightly higher than Flu-ArgTat at 120 µM (Figs. 5B and 6A). Removing one positive charge would attenuate the electrostatic interaction between the peptide and the negatively charged moieties on the membrane during the association step, leading to diminished cellular uptake. However, the decreased association could be compensated by increasing the peptide concentration to increase the amount of cell surface binding, leading to similar overall cellular uptake efficiency compared to Flu-ArgTat. Furthermore, the positive charge at position 57 of the Tat-derived peptide was more sensitive to Cit incorporation compared to the other positions for cellular uptake. Significantly decreased uptake was previously shown upon simultaneously replacing all the Arg residues with Lys in Arg9.35 In contrast, our results on FluTatLysN peptides showed that replacing Arg with Lys individually has minimal effect for cellular uptake (Fig. 6B). This result suggests that the effect of the number and form of hydrogen bond donors on cellular uptake was less significant compared to removing the positive charge at any of the six positions of Tat-derived peptide except for position 57. The results on TAR RNA binding and cellular uptake together suggest that Lys may be incorporated at positions 49, 56, or 57 (individually) in the Tat basic region without attenuating either specific binding of TAR RNA or cell penetration. As such, these three substitutions may be natural mutations in viable HIV Tat proteins. 4. Conclusions The guanidinium group on the six Arg residues is important for TAR RNA recognition and cellular uptake of Tat-derived peptides. The effect of replacing the guanidinium-bearing Arg with either the urea-bearing Cit or ammonium-bearing Lys on these two biological functions were investigated. Our results showed that removing the positive charge and altering the hydrogen bonding capacity of Arg affected the two biological functions differently. Furthermore, the effects were position dependent. The positive charge of Arg was important for specific TAR RNA binding. However, for position 53, any change to the guanidinium group resulted in significant attenuation of the specific RNA binding. Cellular uptake was not affected significantly so long as the positive charge was retained. Interestingly, replacing the positively charged Arg with the neutral Cit at positions 49 or 52 enhanced the uptake at concentrations greater than 60 µM. The same replacement at position 57 resulted in reduced uptake. These findings should be useful for the development of functional molecules containing guanidinium, urea, and ammonium groups for RNA recognition to affect biological processes and for cell penetration for intracellular drug delivery. 5. Experimental section 5.1. Peptide synthesis The peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry. 40, 41 After cleavage, the peptides were purified by reverse phase HPLC and confirmed by MALDI-TOF MS. 5.2. Electrophoretic mobility shift assay (EMSA)
4
The fluorescein-labeled RNA (100 nM) and peptide (at various concentrations) were incubated in pH 7.4 buffer (10 µL) containing Tris-HCl (50 mM), KCl (50 mM), 2% glycerol, and Triton X-100 (0.05%) in the presence of poly(dI-dC) (10 µg/mL) or bulk E.coli tRNA (10 µg/mL) at room temperature. The samples were analyzed by loading into 12% native polyacrylamide gels in 0.5X TB buffer and electrophoresis was performed with 140 V at room temperature. Bands corresponding to the free and bound RNA were used to determine to fraction bound RNA. The fraction bound RNA data was used to globally derive the apparent dissociation constants assuming a 1:1 binding stoichiometry39, 46 using the full quadratic equation (see SI). 5.3. Cellular uptake assay Jurkat cells (8 x 105) were incubated with the peptides at various concentrations (30, 60, 90, and 120 µM) at 37 °C with 5% CO2 for 15 minutes. The cells were then incubated with 0.05 % trypsin/EDTA in PBS for 5 minutes to remove the peptides which adhered to the cell surface rather than entry to the cell.7 Propidium iodide (PI) was added to all samples to stain the dead cells but should not stain the live cells. The cells were then transferred into the flow tube and analyzed by flow cytometry (FACScan, Becton Dickinson Bioscience). The minimum propidium iodide fluorescence intensity for the dead control cells (which were terminated by adding Triton-X 100) treated with propidium iodide was set as the threshold value. The mean 6carboxyfluorescein fluorescence intensity for 10,000 live cells (with appropriate forward scatter and side scatter values, and below the propidium iodide fluorescence threshold) was determined for each experiment. Each experiment was independently repeated at least three times.
9. 10.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
21.
22.
Acknowledgments This work was supported by National Taiwan University (NTU-ERP-103R891302 and NTU-CESRP-103R7621) and the National Science Council in Taiwan (NSC-99-2113-M-002-002MY2, NSC-101-2113-M-002-006-MY2). Supplementary data
23. 24. 25. 26.
Supplementary data associated with this article can be found, in the online version, at
27.
References and notes
28.
1. 2. 3.
4. 5. 6. 7.
8.
Cullen, B. R. FASEB J. 1991, 5, 2361-2368. Stevens, M.; De Clercq, E.; Balzarini, J. Med. Res. Rev. 2006, 26, 595-625. Cordingley, M. G.; Lafemina, R. L.; Callahan, P. L.; Condra, J. H.; Sardana, V. V.; Graham, D. J.; Nguyen, T. M.; Legrow, K.; Gotlib, L.; Schlabach, A. J.; Colonno, R. J. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 8985-8989. Weeks, K. M.; Ampe, C.; Schultz, S. C.; Steitz, T. A.; Crothers, D. M. Science 1990, 249, 1281-1285. Rosen, C. A.; Sodroski, J. G.; Haseltine, W. A. Cell 1985, 41, 813-823. Rana, T. M.; Jeang, K. T. Arch. Biochem. Biophys. 1999, 365, 175-185. Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.; Verbeure, B.; Gait, M. J.; Chernomordik, L. V.; Lebleu, B. J. Biol. Chem. 2003, 278, 585-590. Vives, E.; Brodin, P.; Lebleu, B. J. Biol. Chem. 1997, 272, 16010-16017.
29.
30. 31.
32. 33.
34. 35.
Kao, S. Y.; Calman, A. F.; Luciw, P. A.; Peterlin, B. M. Nature 1987, 330, 489-493. Bryson, D. I.; Zhang, W. Y.; McLendon, P. M.; Reineke, T. M.; Santos, W. L. ACS Chem. Biol. 2012, 7, 210-217. Lee, S. J.; Hyun, S.; Kieft, J. S.; Yu, J. J. Am. Chem. Soc. 2009, 131, 2224-2230. Huq, I.; Ping, Y. H.; Tamilarasu, N.; Rana, T. M. Biochemistry 1999, 38, 5172-5177. Kumar, S.; Maiti, S. Biochimie 2013, 95, 1422-1431. Kumar, S.; Maiti, S. Plos One 2013, 8. Wang, X. L.; Huq, I.; Rana, T. M. J. Am. Chem. Soc. 1997, 119, 6444-6445. Tamilarasu, N.; Huq, I.; Rana, T. M. J. Am. Chem. Soc. 1999, 121, 1597-1598. Kesavan, V.; Tamilarasu, N.; Cao, H.; Rana, T. M. Bioconjugate Chem. 2002, 13, 1171-1175. Belousoff, M. J.; Gasser, G.; Graham, B.; Tor, Y.; Spiccia, L. J. Biol. Inorg. Chem. 2009, 14, 287-300. Belousoff, M. J.; Graham, B.; Spiccia, L.; Tor, Y. Org. Biomol. Chem. 2009, 7, 30-33. Staple, D. W.; Venditti, V.; Niccolai, N.; Elson-Schwab, L.; Tor, Y.; Butcher, S. E. ChemBioChem 2008, 9, 93102. Ensoli, B.; Buonaguro, L.; Barillari, G.; Fiorelli, V.; Gendelman, R.; Morgan, R. A.; Wingfield, P.; Gallo, R. C. J. Virol. 1993, 67, 277-287. Berman, J. W.; Eugenin, E. A.; King, J. E.; Nath, A.; Calderon, T. M.; Zukin, R. S.; Bennett, M. V. L. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 3438-3443. Misumi, S.; Takamune, N.; Ohtsubo, Y.; Waniguchi, K.; Shoji, S. AIDS Res. Hum. Retrov. 2004, 20, 297-304. Fischer, R.; Fotin-Mleczek, M.; Hufnagel, H.; Brock, R. ChemBioChem 2005, 6, 2126-2142. Wadia, J. S.; Stan, R. V.; Dowdy, S. F. Nat. Med. 2004, 10, 310-315. Torchilin, V. P. Adv. Drug Delivery Rev. 2008, 60, 548558. El-Sayed, A.; Futaki, S.; Harashima, H. AAPS J. 2009, 11, 13-22. Wu, C. H.; Chen, Y. P.; Wu, S. H.; Hung, Y.; Mou, C. Y.; Cheng, R. P. ACS Appl. Mater. Interfaces 2013, 5, 12244-12248. Erazo-Oliveras, A.; Muthukrishnan, N.; Baker, R.; Wang, T. Y.; Pellois, J. P. Pharmaceuticals 2012, 5, 1177-1209. Calnan, B. J.; Biancalana, S.; Hudson, D.; Frankel, A. D. Genes Dev. 1991, 5, 201-210. Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.; Rothbard, J. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003-13008. Puglisi, J. D.; Chen, L.; Blanchard, S.; Frankel, A. D. Science 1995, 270, 1200-1203. Anand, K.; Schulte, A.; Vogel-Bachmayr, K.; Scheffzek, K.; Geyer, M. Nat. Struct. Mol. Biol. 2008, 15, 12871292. Calnan, B. J.; Tidor, B.; Biancalana, S.; Hudson, D.; Frankel, A. D. Science 1991, 252, 1167-1171. Mitchell, D. J.; Kim, D. T.; Steinman, L.; Fathman, C. G.; Rothbard, J. B. J. Pept. Res. 2000, 56, 318-325.
5
36.
37. 38.
39.
40.
41. 42. 43. 44. 45. 46. 47. 48.
49. 50.
51.
Wender, P. A.; Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A. J. Am. Chem. Soc. 2004, 126, 95069507. Wu, C.-H.; Chen, Y.-P.; Mou, C.-Y.; Cheng, R. P. Amino Acids 2013, 44, 473-480. Priyakumar, U. D.; Hyeon, C.; Thirumalai, D.; MacKerell, A. D. J. Am. Chem. Soc. 2009, 131, 1775917761. Gelman, M. A.; Richter, S.; Cao, H.; Umezawa, N.; Gellman, S. H.; Rana, T. M. Org. Lett. 2003, 5, 35633565. Atherton, E.; Fox, H.; Harkiss, D.; Logan, C. J.; Sheppard, R. C.; Williams, B. J. J. Chem. Soc. Chem. Commun. 1978, 537-539. Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214. Edelhoch, H. Biochemistry 1967, 6, 1948-1954. Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. Protein Sci. 1995, 4, 2411-2423. Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals; Eugene, OR, USA, 1996. Wang, J. H.; Huang, S. Y.; Choudhury, I.; Leibowitz, M. J.; Stein, S. Anal. Biochem. 1995, 232, 238-242. Suryawanshi, H.; Sabharwal, H.; Maiti, S. J. Phys. Chem. B 2010, 114, 11155-11163. Zondlo, N. J.; Schepartz, A. J. Am. Chem. Soc. 1999, 121, 6938-6939. Athanassiou, Z.; Dias, R. L. A.; Moehle, K.; Dobson, N.; Varani, G.; Robinson, J. A. J. Am. Chem. Soc. 2004, 126, 6906-6913. Rashid, F.; Sharma, S.; Bano, B. Protein J. 2005, 24, 283-292. Guinn, E. J.; Schwinefus, J. J.; Cha, H. K.; McDevitt, J. L.; Merker, W. E.; Ritzer, R.; Muth, G. W.; Engelsgjerd, S. W.; Mangold, K. E.; Thompson, P. J.; Kerins, M. J.; Record, M. T. J. Am. Chem. Soc. 2013, 135, 5828-5838. Wender, P. A.; Galliher, W. C.; Goun, E. A.; Jones, L. R.; Pillow, T. H. Adv. Drug Delivery Rev. 2008, 60, 452-472.
6
