br Conclusion In conclusion we discovered a pair

Conclusion In conclusion, we discovered a pair of novel epimers CBC and CBD from plant C. bungei. These two natural compounds inhibit Hh pathway by blocking signaling at the level of Gli. They are effective in suppressing Hh pathway-dependent medulloblastoma growth in vitro and in vivo. Furthermore, they exhibited potential for overcoming primary and acquired resistance to current Smo inhibitors. These results highlight the potential of CBC and CBD as effective lead compounds in the treatment of medulloblastoma and other Hh-dependent malignancy.
Authors' contributions
Conflicts of interest
Acknowledgments We thank Prof. Zhen Yang and Shengchang Xin from Peking University (China) for kind provision of Shh-Light 2 cell and Shh-N cell. We are grateful to Dr. Jian Yang from Wellcome Trust Sanger Institute (UK) for professional English editing service. We also appreciated helpful discussions with Dr. Tian-Hui Chen from Zhejiang Academy of Medical Sciences and Prof. Hong-Xiang Sun from Zhejiang University (China). The work was supported by Grant-in-Aid from the National Natural Science Foundation of China (No. 21472175), Zhejiang Provincial Natural Science Foundation (No. LY15H280001 and LQ16H300002), Zhejiang Provincial Science and Technology Council (2017F10005 and LGF18H310003), Zhejiang Provincial Medicinal Health Program (201472465 and 2014KYA037) and Zhejiang Provincial Education Council (Y201738521).
Introduction Drosophila cubitus interruptus (Ci) and its vertebrate homologues, the glioblastoma (Gli) protein family (Gli1, Gli2, and Gli3), are critical regulators in animal development (Huangfu and Anderson, 2006; ; Jiang and Hui, 2008; Lum and Beachy, 2004; Stecca and Ruiz i Altaba, 2010). They are key transcription factors (TFs) in the hedgehog (Hh) signal transduction pathway. Structurally, Gli1–3 and Ci are 75–150kDa zinc finger (ZF) Firefly Luciferase mRNA with a single ZF domain composed of five tandemly repeated C2H2 ZFs. The ZF domain of Gli1 is known to bind DNA (consensus sequence 5′-TGGGTGGTC-3′) in a sequence-specific manner (Kinzler and Vogelstein, 1990) and is similar to the ZF domains of the Glis and Zic families, which are also animal development-controlling TFs. Together, these proteins comprise the Gli/Glis/Zic ZF protein superfamily.
Nuclear Localization Signal and Nuclear Export Signal In eukaryotes, trafficking between the cytoplasm and the nucleus is restricted by the nuclear membrane. Macromolecules are transported into and out of the nucleus through large proteinaceous structures called nuclear pore complexes (NPCs) (; ). Although small macromolecules (<40kDa) can passively diffuse through NPCs, larger molecules must be imported via active carrier-mediated transport. The Importin α-mediated pathway is one of the most well-characterized pathways for nuclear import (). Importin α binds to the nuclear localization signal (NLS) of target cargo proteins (; ). The Importin α–NLS cargo protein complex then binds an additional component, Importin β, and the resultant heterotrimeric protein is transported into the nucleus with the support of the RanGDP–RanGTP gradient from the cytoplasm to the nucleus (). After import to the nucleus, the complex is disassembled and the cargo protein is released (Fig. 4.1) (; Gorlich and Mattaj, 1996; ). Protein localization is also affected by nuclear export signals (NESs), which mediate the binding of adaptor proteins (e.g., CRM1) involved in the export of proteins from the nucleus through NPCs. Thus, NLSs and NESs are major intrinsic determinants of nucleus–cytoplasm protein distribution. Previous studies have addressed the conserved features of NLS-constituting amino acid sequences. Some NLSs contain one or two clusters of basic amino acid residues (). These NLSs were initially identified in SV40 large T-antigen (PKKKRKV) and Xenopus nucleoplasmin (KRPAATKKAGQAKKKK), and they are collectively referred to as classical NLSs (). Single cluster (monopartite) classical NLSs contain a three to four basic amino acid stretch as is found in SV40 large T, whereas bipartite classical NLSs contain two clusters of basic amino acids that are separated by 10–12 intervening amino acids as is found in Xenopus nucleoplasmin (Dingwall and Laskey, 1991; ). The binding of classical NLSs to Importin α has been well studied (Conti and Kuriyan, 2000; ; ); however, recent studies indicate the presence of another pathway that employs proline–tyrosine (PY)-NLSs and the receptor transportin-1/karyopherin-β2 besides the Importin α/karyopherin-α and Importin β/karyopherin-β1 that may target classical NLSs (). Thus, the study of the mechanisms and molecular determinants of nuclear import is still a developing research field.