Structure-function relationship of the nuclear localization signal sequence of parathyroid hormone-related protein

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.Peptide-mediated protein translocation is a popular method of delivering biologically active peptides/ proteins into cells. This transport system is conducted by short peptides (< 30 amino acids) that translocate large molecules into cells [reviewed in (14)]. These so-called cell-penetrating peptides (CPPs) are found in protein transduction domains or nuclear localization signals (NLSs) of viral proteins (5). CPPs have been applied to in vitro biological and clinical studies (6). For instance, short peptides from HIV-1 Tat protein including Tat(48-60) and Tat(47-57) have been widely used to deliver cargo molecules into cells (26,29). The amino acid sequences of CPPs are rich in basic amino acid residues. Interestingly, a short peptide containing only 8 arginine residues was translocatable (7). Parathyroid hormone-related protein (PTHrP) was identified as a tumor-derived secretory protein (17). The N-terminal 20 residues of the 141-amino acid peptide showed structural similarity to parathyroid

Functional Roles of the Nuclear Localization Signal of Parathyroid Hormone-Related Protein (PTHrP) in Osteoblastic Cells

“…This is the first amino acid of the osteostatin sequence (7,8), and it has also been included in the sequence constituting the NLS domain by most investigators (3,5,46,51) but not by others (19,20). We here show that deletion or mutation of this amino acid in the PTHrP molecule significantly hampers its stimulatory effects on MC3T3-E1 cell proliferation and several osteoblast differentiation markers, namely ALP activity and OC gene expression.…”

Types of nuclear localization signals and mechanisms of protein import into the nucleus

“…However, a large number of ncNLS do not have a regular characteristic structure, including those of PTHrP (parathyroid hormone-related protein) [ 29 ], Pho4 (phosphorylation regulates association of the transcription factor) [ 30 ], and rpL23a (ribosomal protein L23a) [ 31 ], among others (Table 1 ).…”

Cell membrane penetrating function of the nuclear localization sequence in human cytokine IL-1α

“…MPG is a fusion peptide of the HIV-1 gp41 derived sequence combined with the NLS of the SV40 large T antigen [20,21]. Also, NLS of PTHrP [37], NF-jB, TFIIE-b and OCT-6 showed cell membrane penetrating function [38]. In this study, the NLS of human cytokine IL-1a showed remarkable cell membrane penetrating ability in vitro and in vivo suggesting NLS could have cell membrane penetration ability.…”