Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.
Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.
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DOI：
10.1242/jcs.241406
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发表时间：
2020-01-30
影响因子：
4
通讯作者：
Balasubramanian MK
中科院分区：
文献类型：
--
作者：
Hatano T;Sivashanmugam L;Suchenko A;Hussain H;Balasubramanian MK
肌动蛋白是最丰富的真核细胞骨架聚合物形成蛋白之一，其丝状形式调节许多生理过程，从细胞分裂和迁移到发育和组织功能。肌动蛋白在不同的生物体中进行了不同的翻译后修饰 (PTM)，其中包括 Met、Ala、Asp 和 Glu N-乙酰化、N-精氨酸化和第 73 个 His 残基 (His-73) 甲基化，不同的生物体表现出不同的 PTM 特征。目前还没有方法可以生产具有生物体特异性 PTM 谱的肌动蛋白同工型。在这里，我们报告了 Pick-ya 肌动蛋白，这是一种表达来自任何真核生物的肌动蛋白亚型的方法，具有其自己的关键特征 PTM 模式。我们在酵母菌株中使用合成生物学策略来实现这一目标，该酵母菌株表达 1. 通过泛素融合具有所需 N 端的肌动蛋白同工型和 2. 促进乙酰化和甲基化的哺乳动物酶。 Pick-ya 肌动蛋白应该极大地促进肌动蛋白细胞骨架及其 PTM 的生化、结构和生理学研究。
Actin is one of the most abundant eukaryotic cytoskeletal polymer-forming proteins, which in the filamentous form regulates a number of physiological processes, ranging from cell division and migration to development and tissue function. Actins are differentially post-translationally modified (PTMs) in different organisms, which include Met, Ala, Asp, and Glu N-acetylation, N-arginylation, and the 73th His residue (His-73) methylation, with different organisms displaying a distinct signature of PTMs. Currently methods are not available to produce actin isoforms with organism specific PTM profile. Here we report Pick-ya actin, a method to express actin isoforms from any eukaryote with its own key characteristic PTM pattern. We achieve this using a synthetic biology strategy in a yeast strain that expresses 1. actin isoforms with the desired N-end via ubiquitin fusion and 2. mammalian enzymes that promote acetylation and methylation. Pick-ya actin should greatly facilitate biochemical, structural, and physiological studies of the actin cytoskeleton and its PTMs.