PMC0
10.1093%2Fprotein%2Fgzs104
introduction
The extensively studied neocarzinostatin (NCS) complex produced by Streptomyces carzinostaticus is a 12-kDa protein (Kuromizu et al., >>1986<<) bound to an enediyne chromophore ligand (Napier et al., 1979).
The extensively studied neocarzinostatin (NCS) complex produced by Streptomyces carzinostaticus is a 12-kDa protein (Kuromizu et al., 1986) bound to an enediyne chromophore ligand (Napier et al., >>1979<<). The enediyne chromophore is toxic (Kappen et al., 1980), which has been attributed to binding of the chromophore to DNA, upon which it induces double-stranded DNA cleavage (D'Andrea and Haseltine, 1978; Hensens et al., 1994).
The enediyne chromophore is toxic (Kappen et al., >>1980<<), which has been attributed to binding of the chromophore to DNA, upon which it induces double-stranded DNA cleavage (D'Andrea and Haseltine, 1978; Hensens et al., 1994).
The enediyne chromophore is toxic (Kappen et al., 1980), which has been attributed to binding of the chromophore to DNA, upon which it induces double-stranded DNA cleavage (D'Andrea and Haseltine, >>1978<<; Hensens et al., 1994).
The enediyne chromophore is toxic (Kappen et al., 1980), which has been attributed to binding of the chromophore to DNA, upon which it induces double-stranded DNA cleavage (D'Andrea and Haseltine, 1978; Hensens et al., >>1994<<). The SMANCS construct, in which NCS is conjugated to poly(styrene comaleic acid) (Maeda et al., 1979; Oda and Maeda, 1987) has been used clinically to treat hepatomas in humans (Konno, 1992; Konno et al., 1994).
The SMANCS construct, in which NCS is conjugated to poly(styrene comaleic acid) (Maeda et al., >>1979<<; Oda and Maeda, 1987) has been used clinically to treat hepatomas in humans (Konno, 1992; Konno et al., 1994).
The SMANCS construct, in which NCS is conjugated to poly(styrene comaleic acid) (Maeda et al., 1979; Oda and Maeda, >>1987<<) has been used clinically to treat hepatomas in humans (Konno, 1992; Konno et al., 1994).
The SMANCS construct, in which NCS is conjugated to poly(styrene comaleic acid) (Maeda et al., 1979; Oda and Maeda, 1987) has been used clinically to treat hepatomas in humans (Konno, >>1992<<; Konno et al., 1994).
The SMANCS construct, in which NCS is conjugated to poly(styrene comaleic acid) (Maeda et al., 1979; Oda and Maeda, 1987) has been used clinically to treat hepatomas in humans (Konno, 1992; Konno et al., >>1994<<).
The NMR (Takashima et al., >>2005<<; Caddick et al., 2006) and crystal (Kim et al., 1993; Teplyakov et al., 1993) structures of NCS have been elucidated, which reveal a hydrophobic groove that binds the chromophore with nanomolar affinity (Povirk and Goldberg, 1980).
The NMR (Takashima et al., 2005; Caddick et al., >>2006<<) and crystal (Kim et al., 1993; Teplyakov et al., 1993) structures of NCS have been elucidated, which reveal a hydrophobic groove that binds the chromophore with nanomolar affinity (Povirk and Goldberg, 1980).
The NMR (Takashima et al., 2005; Caddick et al., 2006) and crystal (Kim et al., >>1993<<; Teplyakov et al., 1993) structures of NCS have been elucidated, which reveal a hydrophobic groove that binds the chromophore with nanomolar affinity (Povirk and Goldberg, 1980).
The NMR (Takashima et al., 2005; Caddick et al., 2006) and crystal (Kim et al., 1993; Teplyakov et al., >>1993<<) structures of NCS have been elucidated, which reveal a hydrophobic groove that binds the chromophore with nanomolar affinity (Povirk and Goldberg, 1980).
al., 2005; Caddick et al., 2006) and crystal (Kim et al., 1993; Teplyakov et al., 1993) structures of NCS have been elucidated, which reveal a hydrophobic groove that binds the chromophore with nanomolar affinity (Povirk and Goldberg, >>1980<<). The considerable stability of apo-NCS to reduction and denaturation (Meienhofer et al., 1972; Sudhahar and Chin, 2006), and its ability to bind a wide range of synthetic ligands have stimulated efforts to develop ligands for NCS for use
The considerable stability of apo-NCS to reduction and denaturation (Meienhofer et al., >>1972<<; Sudhahar and Chin, 2006), and its ability to bind a wide range of synthetic ligands have stimulated efforts to develop ligands for NCS for use as chemotherapeutics (Caddick et al., 2006).
The considerable stability of apo-NCS to reduction and denaturation (Meienhofer et al., 1972; Sudhahar and Chin, >>2006<<), and its ability to bind a wide range of synthetic ligands have stimulated efforts to develop ligands for NCS for use as chemotherapeutics (Caddick et al., 2006).
reduction and denaturation (Meienhofer et al., 1972; Sudhahar and Chin, 2006), and its ability to bind a wide range of synthetic ligands have stimulated efforts to develop ligands for NCS for use as chemotherapeutics (Caddick et al., >>2006<<). Such an activity could be coupled with the ability of NCS to functionally display complementarity determining regions loops from antibodies (Nicaise et al., 2004).
Such an activity could be coupled with the ability of NCS to functionally display complementarity determining regions loops from antibodies (Nicaise et al., >>2004<<).
Analyses of DNA replication rates (Kappen et al., >>1980<<) and gene transcription (Schaus et al., 2001) following incubation of live cells with holo-NCS suggest that holo-NCS is able to induce chromosomal DNA damage, and hence that the chromophore is able to enter the nucleus.
Analyses of DNA replication rates (Kappen et al., 1980) and gene transcription (Schaus et al., >>2001<<) following incubation of live cells with holo-NCS suggest that holo-NCS is able to induce chromosomal DNA damage, and hence that the chromophore is able to enter the nucleus.
The chromophore is highly unstable in its ‘free’ form, and so it has been suggested that the chromophore is protected from degradation in the cytoplasm by the NCS protein (Kappen et al., >>1980<<). In addition, microscopy of live cells treated with fluorescently labelled holo-NCS suggests that holo-NCS is able to internalise, and accumulate in the nucleus (Takeshita et al., 1980; Oda and Maeda, 1987).
In addition, microscopy of live cells treated with fluorescently labelled holo-NCS suggests that holo-NCS is able to internalise, and accumulate in the nucleus (Takeshita et al., >>1980<<; Oda and Maeda, 1987).
In addition, microscopy of live cells treated with fluorescently labelled holo-NCS suggests that holo-NCS is able to internalise, and accumulate in the nucleus (Takeshita et al., 1980; Oda and Maeda, >>1987<<).
results and discussion
Apo-NCS contains four native cysteines that form two stable disulfide bonds, which are resistant to the commonly used thiol-reducing agents (Meienhofer et al., >>1972<<). We hence reasoned that by a using a point mutation strategy, we could introduce free cysteine residues into the NCS sequence that would allow facile and site-selective conjugation of either biotin or fluorophore labels.
These spectra are consistent with previously published CD spectra for apo-NCS (Jayachithra et al., >>2005<<; Sudhahar and Chin, 2006), Thermal denaturation (Fig.
These spectra are consistent with previously published CD spectra for apo-NCS (Jayachithra et al., 2005; Sudhahar and Chin, >>2006<<), Thermal denaturation (Fig.
In order to determine how efficiently apo-NCS is internalised into mammalian cells, we adapted a strategy (Burlina et al., >>2006<<) that has been used previously for quantifying the internalisation of small cell-penetrating peptides (CPPs).
Conditions were employed that have previously been shown to permit internalisation of holo-NCS (Oda and Maeda, >>1987<<). Cells incubated at 37°C are known to permit cell entry by endocytosis, pinocytosis, and direct translocation (Zorko and Langel, 2005). Mass spectra were obtained following the addition of internal standard (15N-NCS(+C)-biotin) and
Cells incubated at 37°C are known to permit cell entry by endocytosis, pinocytosis, and direct translocation (Zorko and Langel, >>2005<<). Mass spectra were obtained following the addition of internal standard (15N-NCS(+C)-biotin) and subsequent to cell lysis. The internal standard was detected clearly on the mass spectrum, but no signal corresponding to intact
The internalisation efficiency of NCS was compared with that of trans-activating transcriptional activator (Tat) from human immunodeficiency virus 1 (Vivès et al., >>1997<<), a peptide that is known to internalise relatively weakly compared with other CPPs, with an estimated intracellular concentration of 1 µM when incubated under similar conditions used in this experiment with CHO cells (Burlina et al.,
a peptide that is known to internalise relatively weakly compared with other CPPs, with an estimated intracellular concentration of 1 µM when incubated under similar conditions used in this experiment with CHO cells (Burlina et al., >>2005<<). As expected, Tat was internalised. In contrast, incubation with NCS(S14C)-TMR and NCS(+C)-TMR did not result in fluorescently labelled cells (Fig.
A conformational change between the previously reported NMR structures of holo-NCS (1O5P) (Takashima et al., >>2005<<) and apo-NCS (1j5H) (Urbaniak et al., 2002) is consistent with this hypothesis.
A conformational change between the previously reported NMR structures of holo-NCS (1O5P) (Takashima et al., 2005) and apo-NCS (1j5H) (Urbaniak et al., >>2002<<) is consistent with this hypothesis.