美国科罗拉多州立大学Robert E. Cohen研究小组取得一项新突破。他们的最新研究提出了高亲和力游离泛素传感器，能够准确定量泛素稳态和去泛素化。 相关论文于2019年8月2日发表在《自然—方法学》上。

该研究组描述了一种基于亲和力的荧光传感器，该传感器能够特异性结合游离泛素分子，解离常数Kd值小于60pM，加上新开发的工作流，使得研究者能够在细胞裂解物中区分和量化游离的、连有蛋白质的和巯基酯形式的泛素。另外，通过传感器染色，研究者通过显微镜在固定细胞内观测到了游离泛素。通过传感器实时分析，能够十分灵活且精密地测定任何（多）泛素连接物的去泛素化。

据介绍，泛素(Ub)连接是一种十分重要的翻译后修饰，几乎影响真核生物内的所有蛋白质。泛素化的功能和机制得到了广泛地研究，然而其动力学和游离（未连接的）泛素分子调节机制还知之甚少，其中一个主要障碍是缺乏简单可靠的技术来量化细胞内泛素化水平，及监控结合物中泛素分子的释放。

附：英文原文

Title: High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination

Author: Yun-Seok Choi, Sarah A. Bollinger, Luisa F. Prada, Francesco Scavone, Tingting Yao, Robert E. Cohen

Issue&Volume:  Volume 16 Issue 8

Abstract: 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 60pM 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.

DOI: 10.1038/s41592-019-0469-9

Source:https://www.nature.com/articles/s41592-019-0469-9