Laboratory of Cell Signalling, Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India.
Methods Mol Biol. 2020;2091:93-105. doi: 10.1007/978-1-0716-0167-9_8.
Protein pyrophosphorylation involves the transfer of a high-energy β-phosphate from inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP) to phosphorylated serine residues. Over a decade of research has established several proteins, involved in diverse physiological processes, as substrates of InsP-mediated pyrophosphorylation. However, the need for detection of this posttranslational modification on endogenous proteins is paramount. "Back-pyrophosphorylation" is a simple technique to test whether a native protein undergoes InsP-mediated pyrophosphorylation inside cells. The basis of this technique relies on the fact that a target protein isolated from cells with lower InsP levels exists in a hypo-pyrophosphorylated form as compared to the same protein isolated from cells with normal InsP levels. Hence, when radiolabeled InsP is added to a target protein immunoprecipitated from both these cell types, the hypopyrophosphorylated protein accepts a higher amount of radiolabeled phosphate when compared to the protein isolated from wild-type cells. This chapter provides detailed methods to identify an InsP target protein and conduct a back-pyrophosphorylation assay on a target protein immunoprecipitated from cells with normal versus reduced InsP levels, to confirm its endogenous pyrophosphorylation status.
蛋白焦磷酸化涉及高能β-磷酸从肌醇焦磷酸盐（如二磷酸肌醇五磷酸（InsP）转移到磷酸化丝氨酸残基。十多年的研究已经确定了几种参与多种生理过程的蛋白质是 InsP 介导的焦磷酸化的底物。然而，检测内源性蛋白质的这种翻译后修饰是至关重要的。“反向焦磷酸化”是一种简单的技术，可以测试内源性蛋白质在细胞内是否经历 InsP 介导的焦磷酸化。该技术的基础依赖于这样一个事实，即与从正常 InsP 水平的细胞中分离的相同蛋白质相比，从 InsP 水平较低的细胞中分离的靶蛋白处于低焦磷酸化形式。因此，当将放射性标记的 InsP 添加到从这两种细胞类型免疫沉淀的靶蛋白中时，与从野生型细胞中分离的蛋白质相比，低焦磷酸化的蛋白质接受更高量的放射性标记的磷酸。本章提供了详细的方法来鉴定 InsP 靶蛋白，并对从 InsP 水平正常和降低的细胞中免疫沉淀的靶蛋白进行反向焦磷酸化测定，以确认其内源焦磷酸化状态。
