Structure-function relationship of the nuclear localization signal sequence of parathyroid hormone-related protein
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- Ohshima Keiichic
- Medical Genetics Division, Shizuoka Cancer Center Research Institute, Shizuoka 411-8777, Japan
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- Takeda Sachiyo
- Laboratory of Bioorganic Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan,
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- Hirose Mariko
- Laboratory of Bioorganic Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan,
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- Akiyama Yasuto
- Immunotherapy Division, Shizuoka Cancer Center Research Institute, Shizuoka 411-8777, Japan,
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- Iguchi Kazuaki
- Laboratory of Bioorganic Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan,
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- Hoshino Minoru
- Laboratory of Bioorganic Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan,
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- Yamaguchi Ken
- Shizuoka Cancer Center Hospital and Research Institute, Shizuoka 411-8777, Japan
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- Mochizuki Tohru
- Medical Genetics Division, Shizuoka Cancer Center Research Institute, Shizuoka 411-8777, Japan
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Abstract
Parathyroid hormone-related protein (PTHrP) contains a nuclear localization signal (NLS) sequence within 87-107. NLS sequences are generally capable of penetrating cellular membranes due to a richness of basic amino acid residues, and thus have been used as cell-penetrating peptides (CPPs) to translocate biologically active peptides/proteins into cells. The NLS sequence of PTHrP is not exception to this finding; however, PTHrP(87-107) contains 2 acidic glutamate residues at 99 and 101 within the basic amino acid stretch, which is not commonly observed in other CPPs such as HIV-1 Tat(48-60). In this study, we indicated structure-function relationship of the PTHrP NLS to understand the effect of acidic glutamate residues on cell permeability and intracellular localization. We chemically synthesized PTHrP(87-107) and its N-terminally truncated analogues. Their intracellular localization pattern was analyzed by microscopy, radioimmunoassay, and fluorescence-activated cell sorting. Although all analogues were translocated into cells, internalization by the cytoplasm and/or nucleus was length-dependent; specifically, PTHrP(97-107), PTHrP(95-107), and PTHrP(93-107) were more frequently localized in the cytoplasm. We assume that reduction in the net positive charge within PTHrP NLS analogues resulted in increased cytoplasm- translocation activity. We propose that PTHrP(97-107) is a useful carrier peptide for delivery and expression of cargo molecules in the cytoplasm.
Journal
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- Biomedical Research
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Biomedical Research 33 (3), 191-199, 2012
Biomedical Research Press