Fission yeast-based high-throughput screens for PKA pathway inhibitors and activators.
Fission yeast-based high-throughput screens for PKA pathway inhibitors and activators.
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基于裂变酵母的 PKA 途径抑制剂和激活剂高通量筛选。
DOI：
10.1007/978-1-4939-2269-7_6
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Hoffman,CharlesS
中科院分区：
文献类型：
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作者：
deMedeiros,AnaSantos;Kwak,Grace;Vanderhooft,Jordan;Rivera,Sam;Gottlieb,Rachel;Hoffman,CharlesS
裂殖酵母裂殖酵母AMP/PKA途径的特性对于cAMP途径蛋白的异源表达，如GαS亚基及其同源的腺苷环化酶、PKA催化和调节亚基以及环核苷酸磷酸二酯酶的异源表达尤为敏感。我们构建了两个PKA抑制的报告，用于高通量筛选，以检测提高或降低PKA活性的化合物，从而促进发现这些靶蛋白的抑制剂和激活剂。在这里，我们描述了构建筛选菌株的步骤以及优化和进行这些筛选的步骤。
Features of the fission yeastSchizosaccharomyces pombecAMP/PKA pathway makeS. pombeparticularly amenable for heterologous expression of cAMP pathway proteins such as GαSsubunits and their cognate adenylyl cyclases, PKA catalytic and regulatory subunits, and cyclic nucleotide phosphodiesterases. We have constructed two PKA-repressed reporters for use in high-throughput screens to detect compounds that elevate or reduce PKA activity, thus facilitating the discovery of both inhibitors and activators of these target proteins. Here, we describe steps to construct screening strains and to optimize and conduct these screens.