Thorne Curtis A, Lafleur Bonnie, Lewis Michelle, Hanson Alison J, Jernigan Kristin K, Weaver David C, Huppert Kari A, Chen Tony W, Wichaidit Chonlarat, Cselenyi Christopher S, Tahinci Emilios, Meyers Kelly C, Waskow Emily, Orton Darren, Salic Adrian, Lee Laura A, Robbins David J, Huppert Stacey S, Lee Ethan
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8240, USA.
J Biomol Screen. 2011 Oct;16(9):995-1006. doi: 10.1177/1087057111416657. Epub 2011 Aug 21.
Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.
Wnt信号通路的失调已被证明与多种人类疾病有关，最显著的是癌症。针对该信号通路抑制剂的筛选几乎完全是在培养的哺乳动物细胞中或使用纯化蛋白进行的。我们之前开发了一种利用非洲爪蟾卵提取物的生化检测方法，以重现Wnt信号通路中的关键细胞质事件。利用这个生化系统，我们发现Wnt共受体LRP6的重组形式以相反的方式调节Wnt信号通路的两个关键成分（β-连环蛋白和轴蛋白）的稳定性。我们现在分别将β-连环蛋白和轴蛋白与萤火虫荧光素酶和海肾荧光素酶融合，并证明融合蛋白的行为与其野生型对应物相似。利用这种双荧光素酶读数，我们将非洲爪蟾提取物系统应用于高通量筛选。这些筛选结果显示的信号分布曲线反映了所筛选文库的复杂性。在几种被鉴定为Wnt信号通路细胞质调节剂的化合物中，有一种在培养的哺乳动物细胞和非洲爪蟾胚胎中被进一步确认为Wnt信号通路的真正抑制剂。我们表明，其他胚胎信号通路也可能适用于在非洲爪蟾卵提取物中筛选抑制剂/调节剂。
