title: Camptothecin (CPT) directly binds to human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and inhibits the hnRNP A1/topoisomerase I interaction.
abstract: Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a K(D) value of 82.7 nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (K(D): 260 nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5-50 μM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster.
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Manita
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Toba
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takakusagi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Tomoe
authors.initials: T
authors.lastname: Kusayanagi
authors.forename: Kaori
authors.initials: K
authors.lastname: Takakusagi
authors.forename: Senko
authors.initials: S
authors.lastname: Tsukuda
authors.forename: Kazunori
authors.initials: K
authors.lastname: Takada
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kanai
authors.forename: Shinji
authors.initials: S
authors.lastname: Kamisuki
authors.forename: Kengo
authors.initials: K
authors.lastname: Sakaguchi
authors.forename: Fumio
authors.initials: F
authors.lastname: Sugawara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004331:Drosophila melanogaster; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding / Q000187:drug effects; D058525:Quartz Crystal Microbalance Techniques; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology*
chemical_list: D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D019151:Peptide Library; D059004:Topoisomerase I Inhibitors; C000619656:hnRNPA1 protein, human; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin