基于细胞的 Smoothened 泛素化和 sumoylation 检测分析。
Cell-Based Assays for Smoothened Ubiquitination and Sumoylation.
机构信息
Department of Molecular Biology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA.
出版信息
Methods Mol Biol. 2022;2374:139-147. doi: 10.1007/978-1-0716-1701-4_12.
The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis by regulating the abundance, localization, and activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from Drosophila to mammals. Smo ubiquitination is dynamically regulated by E3 ubiquitin ligases and deubiquitinases (dubs) and is opposed by Hh signaling. By contrast, Smo sumoylation is stimulated by Hh, which counteracts Smo ubiquitination by recruiting the dub USP8. We describe cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligases in Drosophila. We also describe assays for Smo sumoylation in both Drosophila and mammalian cultured cells.
刺猬（Hh）家族的分泌蛋白通过调节 G 蛋白偶联受体（GPCR）家族蛋白 Smoothened（Smo）的丰度、定位和活性来控制胚胎发育和成年组织的内稳态。Smo 的运输和亚细胞积累受到多种翻译后修饰（PTM）的控制，包括磷酸化、泛素化和 sumoylation，这似乎在从果蝇到哺乳动物的过程中是保守的。Smo 的泛素化受到 E3 泛素连接酶和去泛素酶（dub）的动态调节，并且受到 Hh 信号的拮抗。相比之下，Smo 的 sumoylation 受到 Hh 的刺激，它通过招募 dub USP8 来抵消 Smo 的泛素化。我们描述了用于 Smo 泛素化及其在果蝇中受 Hh 和 E3 连接酶调节的细胞基础测定法。我们还描述了在果蝇和哺乳动物培养细胞中 Smo sumoylation 的测定法。
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