Structure-function relationship of the nuclear localization signal sequence of parathyroid hormone-related protein.
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.
Medical Genetics Division, Shizuoka Cancer Center Research Institute, Shizuoka 411-8777, Japan. k.ohshima@scchr.jp
Keiichic
K
Ohshima
Sachiyo
S
Takeda
Mariko
M
Hirose
Yasuto
Y
Akiyama
Kazuaki
K
Iguchi
Minoru
M
Hoshino
Ken
K
Yamaguchi
Tohru
T
Mochizuki
D000595:Amino Acid Sequence; D002272:Carcinoembryonic Antigen / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000378:metabolism; D044162:Parathyroid Hormone-Related Protein / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D002272:Carcinoembryonic Antigen; D057846:Cell-Penetrating Peptides; D000939:Epitopes; D019913:Nuclear Localization Signals; D044162:Parathyroid Hormone-Related Protein; D010455:Peptides