High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination.,Nature Methods

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High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination.
Nature Methods ( IF 36.1 ) Pub Date : 2019-07-15 , DOI: 10.1038/s41592-019-0469-9
Yun-Seok Choi ₁ , Sarah A Bollinger ₁ , Luisa F Prada ₁ , Francesco Scavone ₁ , Tingting Yao ₁ , Robert E Cohen ₁

Affiliation

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive study, and yet the dynamics and regulation of even free (that is, unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here, we describe avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have dissociation constant Kd values down to 60 pM and, together with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.

中文翻译：

高亲和力的免费泛素传感器，用于定量泛素稳态和去泛素作用。

泛素（Ubquitin）（Ub）缀合是必不可少的翻译后修饰，可影响真核生物中的几乎所有蛋白质。泛素化的功能和机制是广泛研究的领域，但对游离的（即未结合的）Ub的动力学和调控知之甚少。一个主要的障碍是缺乏简单而健壮的技术来量化细胞中的Ub水平和监测结合物中Ub的释放。在这里，我们描述了解决这一需求的基于亲和力的荧光传感器。传感器与游离Ub特异性结合，具有低至60 pM的解离常数Kd值，并与新开发的工作流程一起，使我们能够区分和量化来自细胞裂解液的Ub的游离，蛋白质结合和硫酯化形式的库。或者，固定细胞中的游离Ub可通过传感器染色在显微镜下可视化。使用传感器进行的实时测定提供了前所未有的灵活性和精确度，可以测量几乎任何（poly）Ub共轭物的去泛素化。

更新日期：2019-07-15

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