Du Jiale, Strieter Eric R
Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA.
Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, 01003, USA.
Anal Biochem. 2018 Jun 1;550:84-89. doi: 10.1016/j.ab.2018.04.018. Epub 2018 Apr 23.
Ubiquitin chains regulate distinct signaling events through cooperative interactions with effector proteins and deubiquitinases. Measuring the strength of these interactions is often challenging; either large amounts of material are required or one of the binding partners must be labeled for detection. We sought to develop a label-free method for measuring binding of ubiquitin chains to the proteasome-associated deubiquitinase UCH37 and its binding partner RPN13. The method we describe here is based on a fluorescence polarization competition (FP) assay in which fluorescent monoubiquitin is competed off the UCH37•RPN13 complex by the addition of unlabeled ubiquitin chains. We show that the UCH37•RPN13 complex displays higher affinity toward chains with more than two ubiquitin subunits. Removing the ubiquitin-binding PRU domain of RPN13 does not change affinities. These results suggest UCH37•RPN13 acts to selectively recruit proteins modified with long chains (>2 subunits) to the proteasome for degradation. We also demonstrate that the FP assay is suitable for high-throughput screening, which is important considering both UCH37 and RPN13 are potential targets for cancer therapy.
泛素链通过与效应蛋白和去泛素化酶的协同相互作用来调节不同的信号事件。测量这些相互作用的强度通常具有挑战性；要么需要大量材料，要么必须对其中一个结合伴侣进行标记以进行检测。我们试图开发一种无标记方法来测量泛素链与蛋白酶体相关去泛素化酶UCH37及其结合伴侣RPN13的结合。我们在此描述的方法基于荧光偏振竞争（FP）测定法，其中通过添加未标记的泛素链，荧光单泛素从UCH37•RPN13复合物上被竞争掉。我们表明UCH37•RPN13复合物对具有两个以上泛素亚基的链表现出更高的亲和力。去除RPN13的泛素结合PRU结构域不会改变亲和力。这些结果表明UCH37•RPN13的作用是将用长链（>2个亚基）修饰的蛋白质选择性地募集到蛋白酶体进行降解。我们还证明FP测定法适用于高通量筛选，考虑到UCH37和RPN13都是癌症治疗的潜在靶点，这一点很重要。
