Production and analysis of titin kinase: Exploiting active/inactive kinase homologs in pseudokinase validation
Production and analysis of titin kinase: Exploiting active/inactive kinase homologs in pseudokinase validation
复制标题
DOI：
10.1016/bs.mie.2022.03.028
复制
发表时间：
2022-01-01
期刊：
影响因子：
--
通讯作者：
Mayans, Olga
中科院分区：
文献类型：
--
作者：
Bogomolovas, Julius;Gravenhorst, Peter;Mayans, Olga
蛋白假激酶是真核细胞的关键调控因子。理解它们的非常规分子机制依赖于破译它们进行磷转移的假定潜力，它们的脚手架性质和它们的调节性质。肌氨酸假激酶（TK）是一个不太明确的肌肉特异性激酶家族的定义成员，被认为是肌节机械信号的传感器和传感器。由于传统知识的产生和分析所面临的挑战，传统知识的作用机制尚不清楚。在这里，我们为TK的研究提供了指导和量身定制的研究方法，包括利用其与秀丽隐杆线虫的催化活性同源抽动激酶（TwcK）密切的结构-功能关系。我们描述了一种生产重组TK和TwcK样品的方法管道；设计，优先考虑和验证突变和截断的变体；评估样品稳定性并进行活性分析。该策略可导出到tk样激酶家族的其他假激酶成员。
Protein pseudokinases are key regulators of the eukaryotic cell. Understanding their unconventional molecular mechanisms relies on deciphering their putative potential to perform phosphotransfer, their scaffolding properties and the nature of their regulation. Titin pseudokinase (TK) is the defining member of a family of poorly characterized muscle-specific kinases thought to act as sensors and transducers of mechanical signals in the sarcomere. The functional mechanisms of TK remain obscure due to the challenges posed by its production and analysis. Here, we provide guidelines and tailored research approaches for the study of TK, including profiting from its close structure-function relationship to the catalytically active homolog twitchin kinase (TwcK) from C. elegans. We describe a methodological pipeline to produce recombinant TK and TwcK samples; design, prioritize and validate mutated and truncated variants; assess sample stability and perform activity assays. The strategy is exportable to other pseudokinase members of the TK-like kinase family.