Preparation of Neocarzinostatin Apoprotein Mutants and the Randomized Library on the Chromophore-Binding Cavity

Tomioka, Y.; Kisara, Shigeki; Yoshizawa, Saki; Ozawa, Mika; Suzuki, Naoto; Yamaguchi, Hiroaki; Hishinuma, Takanori; Mizugaki, Michinao; Goto, Junichi

doi:10.1248/bpb.29.1010

Cited by 4 publications

(3 citation statements)

References 26 publications

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“…Among the synthetic ligands, EtBr exhibits a relatively high binding constant (8.57 × 10 −7 M) [32] and has been frequently used to examine the binding properties of NCS mutants [37,38]. NMR study also suggests that the EtBr binds to the NCS protein in the same cleft where the natural enediyne chromophore binds [32].…”

Section: Thermal Stability Of Ncs Protein With and Without Etbrmentioning

confidence: 99%

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Jayachithra¹,

Hariharan²,

Kumar³

et al. 2008

Analytical Biochemistry

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Most conjugate proteins undergo both conformational and stability changes on ligand removal. When architecture remains unchanged in the protein holo and apo forms, it is uncertain whether the protein stability also remains unaltered in both of the forms. Neocarzinostatin (NCS), a chromoprotein possessing a potent enediyne chromophore stands for such an instance. Protein-chromophore interaction has not been thoroughly explored previously due to a lack of strategies to independently and simultaneously monitor changes in the NCS conjugates. Here we report a method by which one can detect the signal exclusively from only one of the NCS conjugates without the spectral interference from the other. Stability of the NCS protein is significantly correlated to the proteinbound chromophore, irrespective of denaturation by heat, pH, urea, or ethanol. Despite the similarity in protein backbone conformation, protein stability of the NCS holo form diminishes and equalizes to that of the apo form when the chromophore is released and degraded. Although the enediyne chromophore is highly unstable, it intriguingly protects the protein by which it is protected. Significant mutual reliance between the carrier protein and its naturally associated ligand unveils important information on the NCS drug stability. KeywordsEnediyne; Neocarzinostatin; Chromophore; Ligand binding; Holoprotein; Protein stability Recently, much attention has been focused on the protein-ligand conjugation. Most conjugate proteins undergo changes in their conformation and subsequently, in stability on ligand removal [1][2][3]. For example, binding of heme to cytochrome changes the protein conformation and consequently, increases the protein stability [4]. It is rather uncommon for conjugate proteins to adopt a like conformation with or without the ligand. This includes azurin (β protein with a copper ion), flavodoxin (α/β protein with a flavin mononucleotide) [5], neocarzinostatin (NCS, 1 β protein with an enediyne chromophore) [6,7] and so on. In spite of structural similarity, the holo form of azurin is more stable than its apo form [8]. The flavodoxin holo form, in contrast, has little changes in either conformation or stability on its cofactor removal *Corresponding author. Fax: +886 4 22862547. E-mail address: chdhchin@dragon.nchu.edu.tw (D.-H. Chin). 1 Abbreviations used: NCS, neocarzinostatin; holoNCS, neocarzinostatin chromoprotein conjugate complex; apoNCS, apoprotein of neocarzinostatin; NCS-C, neocarzinostatin chromophore; NCS-C(b), protein-bound neocarzinostatin chromophore; CD, circular dichroism; HPLC, high-performance liquid chromatography; EtBr, ethidium bromide; NOESY, nuclear Overhauser effect spectroscopy; NMR, nuclear magnetic resonance; T m , temperature at which half of the protein is unfolded; T r , temperature at which half of the NCS-C(b) is released from NCS complex; C m , concentration of denaturant at which half of the protein is unfolded; C r , concentration of denaturant at which half of the NCS-C(b) is released from NCS com...

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Section: Thermal Stability Of Ncs Protein With and Without Etbrmentioning

confidence: 99%

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Jayachithra¹,

Hariharan²,

Kumar³

et al. 2008

Analytical Biochemistry

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“…F-41, which has ever been performed preliminary observation as antitumor candidate. 18,19) The NCS is a 1 : 1 noncovalent complex of an apoprotein and a chromophore, in which the chromophore plays the antitumor activity by DNA damaging activity. 18,20) The higher efficacy of enediyne antibiotics has intrigued intensive researches on cancer inhibition.…”

mentioning

confidence: 99%

“…18,19) The NCS is a 1 : 1 noncovalent complex of an apoprotein and a chromophore, in which the chromophore plays the antitumor activity by DNA damaging activity. 18,20) The higher efficacy of enediyne antibiotics has intrigued intensive researches on cancer inhibition. However, for most enediyne antibiotics, although the strong biological activities, especially anticancer capacity can be observed, their application are often limited by high toxicity and also low efficacy in vivo.…”

mentioning

confidence: 99%

Synergistic Anti-glioma Effects in Vitro and in Vivo of Enediyne Antibiotic Neocarzinostatin and Paclitaxel via Enhanced Growth Delay and Apoptosis-Induction

Tianqin

Chen²,

Wang

2016

Biological & Pharmaceutical Bulletin

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Neocarzinostatin (NCS) is a member of enediyne antibiotics with high anticancer potential. Our study was performed to explore the synergistic anti-glioma effects of NCS and paclitaxel (PTX) in vitro and in vivo. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicities of the drugs to human glioma cells U87MG and rat glioma cells C6 were evaluated. The results showed that the combinations of NCS and PTX can synergistically inhibit glioma cells survival. Cell apoptosis was detected by flow cytometry, and the results showed that the combinations of NCS and PTX synergistically enhanced apoptosis ratio of glioma cells. Western blot revealed that the cell signaling pathways of proliferation and apoptosis were synergistically regulated, in which Akt was synergistically inactivated, p53 was up-regulated with down-regulation of bcl-2. Meanwhile, with the subcutaneous model of U87MG cells and intracerebral implantation model of C6 cells, the combination strategy could synergistically delay the glioma growth and significantly prolong the survival of rats bearing orthotopic glioma. This study demonstrates that the combination of NCS and PTX can potentiate the effect on survival and apoptosis of glioma cells via suppression of Akt, bcl-2, and activations of p53; Meanwhile, the in vivo studies also confirmed that the combination of NCS and PTX synergistically inhibit the gliom growth. Our data about the combinational effects of NCS with PTX may provide an alternative strategy for glioma therapy.

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Probing the biophysical interaction between Neocarzinostatin toxin and EpCAM RNA aptamer

Athyala

Kanwar

Alameen

et al. 2016

Biochemical and Biophysical Research Communications

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Preparation of Neocarzinostatin Apoprotein Mutants and the Randomized Library on the Chromophore-Binding Cavity

Cited by 4 publications

References 26 publications

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Synergistic Anti-glioma Effects <i>in Vitro</i> and <i>in Vivo</i> of Enediyne Antibiotic Neocarzinostatin and Paclitaxel <i>via</i> Enhanced Growth Delay and Apoptosis-Induction

Probing the biophysical interaction between Neocarzinostatin toxin and EpCAM RNA aptamer

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