Nuclear Translocation Peptides as Antibiotics
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《抗菌试剂及化学方法》
LETTER
Human immunodeficiency virus (HIV) Tat and antimicrobial peptides, such as LL-37, which transfer extraneous DNA to nucleus of host cells are known (5, 6). We performed experiments to find an antimicrobial function for nuclear localization signals (NLS) and nuclear export signals (NES), which are nuclear translocation signals required for shuttling cellular proteins between the nucleus and the cytoplasm (3, 4, 7). Consequently, we obtained evidence that the peptides, which consist of NLS tandems or combinations of NLS and NES, are effective antimicrobial peptides. The antimicrobial activity of these combination peptides for both gram-negative and gram-positive bacteria is very high, and antimicrobial peptides comprised of NLS and NES have the potential to become a new base for peptide antibiotics.
The different combinations of NLS and NES within the peptidic design principle comprise eight types of results: the NLS simple sequence, the NLS tandem sequences (NLS+NLS and NLS+NLS+NLS), the NES simple sequence, the NES tandem sequence (NES+NES), the sequence composed of NES linked to an NLS C terminus (NLS+NES), the sequence composed of NLS linked to an NES C terminus (NES+NLS), and the sequence composed of NLS linked to an NLS+NES C terminus (NLS+NES+NLS). The eight types of peptides were synthesized by following the 9-fluorenylmethoxycarbonyl solid-phase synthesis method and purified by reverse-phase high-performance liquid chromatography on a C18 column (purity of 95%), and their antimicrobial function against the gram-positive bacterium Staphylococcus aureus IFO12732 and the gram-negative bacterium Escherichia coli IFO12713 was tested using the microbroth dilution method to evaluate their MICs. The results are shown in Table 1. The antimicrobial activity for four types of NLS simple sequence (simian virus 40 [SV40] large T antigen, p54, SOX9, and NS5A) was present but weak. However, it was found that antimicrobial function increased remarkably when NLS was made into a tandem sequence (NLS+NLS). More specifically, the antimicrobial performance improved from 30 to 100 times that of NLS on its own. Moreover, the antimicrobial performance results against both gram-positive and gram-negative bacteria further improved several times by making NLS into the three NLS tandem sequence (NLS+NLS+NLS) and the NLS+NES+NLS tandem sequence. Also, NLS-linked NES (NLS+NES, NES+NLS) demonstrated a particularly strong antimicrobial effect against S. aureus. Compared to these results, the NES simple sequence and the NES tandem sequence (NES+NES) did not demonstrate a significant antimicrobial effect.
To evaluate the peptidic antimicrobial spectrum, MICs for five gram-positive bacterial strains and nine gram-negative bacterial strains were measured for the two types of NLS and NES combination peptides, and the results are shown in Table 2. The highly efficient antimicrobial function of the peptide RKKKRKV RKKKRKV, the tandem sequence composed of the modified peptide from SV40 (Table 2) NLS (PKKKRKV), was also confirmed. It is a sequence pair where the Pro in the N terminus was replaced with Arg, showing that some rearrangement could produce better antimicrobial function in NLS/NES combination peptides.
In this study, it was discovered that the antimicrobial function is inherent in the nuclear translocation signal peptide although this peptide does not originally exist for the purpose of self-defense. This is a significant difference from conventional antimicrobial peptides based on biomaterials that exist solely for self-defense (1, 2).
ACKNOWLEDGMENTS
We thank the Kourai Laboratory (group A3) at the University of Tokushima for providing bacterial strains.
EFERENCES
Albrethsen, B., et al. 2005. Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumors: a biomarker study. BMC Cancer 5:8.
Dutton, C. J., M. A. Haxell, H. A. I. McArthur, and R. G. Wax. 2002. Peptide antibiotics. Marcel Dekker, New York, N.Y.
Gorlich, D., and I. W. Mattaj. 1996. Nucleocytoplasmic transport. Science 271:1513-1516.
ost, B., R. Nair, and M. Cokol. 2000. Predict NLS is an automated tool for the analysis and determination of nuclear localization signals (NLS). Department of Biochemistry and Molecular Biophysics, Columbia University, New York, N.Y. http://cubic.bioc.columbia.edu/predictNLS/.
Sandgren, S., F. Cheng, and M. Belting. 2002. Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. J. Biol. Chem. 277:38877-38883.
Sandgren, S., A. Wittrup, F. Cheng, M. Jonsson, E. Eklund, S. Busch, and M. Belting. 2004. The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis. J. Biol. Chem. 279:17951-17956.
Truant, R., and B. R. Cullen. 1999. The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin -dependent nuclear localization signals. Mol. Cell. Biol. 19:1210-1217.
Human immunodeficiency virus (HIV) Tat and antimicrobial peptides, such as LL-37, which transfer extraneous DNA to nucleus of host cells are known (5, 6). We performed experiments to find an antimicrobial function for nuclear localization signals (NLS) and nuclear export signals (NES), which are nuclear translocation signals required for shuttling cellular proteins between the nucleus and the cytoplasm (3, 4, 7). Consequently, we obtained evidence that the peptides, which consist of NLS tandems or combinations of NLS and NES, are effective antimicrobial peptides. The antimicrobial activity of these combination peptides for both gram-negative and gram-positive bacteria is very high, and antimicrobial peptides comprised of NLS and NES have the potential to become a new base for peptide antibiotics.
The different combinations of NLS and NES within the peptidic design principle comprise eight types of results: the NLS simple sequence, the NLS tandem sequences (NLS+NLS and NLS+NLS+NLS), the NES simple sequence, the NES tandem sequence (NES+NES), the sequence composed of NES linked to an NLS C terminus (NLS+NES), the sequence composed of NLS linked to an NES C terminus (NES+NLS), and the sequence composed of NLS linked to an NLS+NES C terminus (NLS+NES+NLS). The eight types of peptides were synthesized by following the 9-fluorenylmethoxycarbonyl solid-phase synthesis method and purified by reverse-phase high-performance liquid chromatography on a C18 column (purity of 95%), and their antimicrobial function against the gram-positive bacterium Staphylococcus aureus IFO12732 and the gram-negative bacterium Escherichia coli IFO12713 was tested using the microbroth dilution method to evaluate their MICs. The results are shown in Table 1. The antimicrobial activity for four types of NLS simple sequence (simian virus 40 [SV40] large T antigen, p54, SOX9, and NS5A) was present but weak. However, it was found that antimicrobial function increased remarkably when NLS was made into a tandem sequence (NLS+NLS). More specifically, the antimicrobial performance improved from 30 to 100 times that of NLS on its own. Moreover, the antimicrobial performance results against both gram-positive and gram-negative bacteria further improved several times by making NLS into the three NLS tandem sequence (NLS+NLS+NLS) and the NLS+NES+NLS tandem sequence. Also, NLS-linked NES (NLS+NES, NES+NLS) demonstrated a particularly strong antimicrobial effect against S. aureus. Compared to these results, the NES simple sequence and the NES tandem sequence (NES+NES) did not demonstrate a significant antimicrobial effect.
To evaluate the peptidic antimicrobial spectrum, MICs for five gram-positive bacterial strains and nine gram-negative bacterial strains were measured for the two types of NLS and NES combination peptides, and the results are shown in Table 2. The highly efficient antimicrobial function of the peptide RKKKRKV RKKKRKV, the tandem sequence composed of the modified peptide from SV40 (Table 2) NLS (PKKKRKV), was also confirmed. It is a sequence pair where the Pro in the N terminus was replaced with Arg, showing that some rearrangement could produce better antimicrobial function in NLS/NES combination peptides.
In this study, it was discovered that the antimicrobial function is inherent in the nuclear translocation signal peptide although this peptide does not originally exist for the purpose of self-defense. This is a significant difference from conventional antimicrobial peptides based on biomaterials that exist solely for self-defense (1, 2).
ACKNOWLEDGMENTS
We thank the Kourai Laboratory (group A3) at the University of Tokushima for providing bacterial strains.
EFERENCES
Albrethsen, B., et al. 2005. Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumors: a biomarker study. BMC Cancer 5:8.
Dutton, C. J., M. A. Haxell, H. A. I. McArthur, and R. G. Wax. 2002. Peptide antibiotics. Marcel Dekker, New York, N.Y.
Gorlich, D., and I. W. Mattaj. 1996. Nucleocytoplasmic transport. Science 271:1513-1516.
ost, B., R. Nair, and M. Cokol. 2000. Predict NLS is an automated tool for the analysis and determination of nuclear localization signals (NLS). Department of Biochemistry and Molecular Biophysics, Columbia University, New York, N.Y. http://cubic.bioc.columbia.edu/predictNLS/.
Sandgren, S., F. Cheng, and M. Belting. 2002. Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. J. Biol. Chem. 277:38877-38883.
Sandgren, S., A. Wittrup, F. Cheng, M. Jonsson, E. Eklund, S. Busch, and M. Belting. 2004. The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis. J. Biol. Chem. 279:17951-17956.
Truant, R., and B. R. Cullen. 1999. The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin -dependent nuclear localization signals. Mol. Cell. Biol. 19:1210-1217.