Hetfeld Bettina K J, Helfrich Annett, Kapelari Barbara, Scheel Hartmut, Hofmann Kay, Guterman Adi, Glickman Michael, Schade Rüdiger, Kloetzel Peter-Michael, Dubiel Wolfgang
Division of Molecular Biology, Department of Surgery, Charité, Universitätsmedizin Berlin, Monbijoustrasse 2, 10117 Berlin, Germany.
Curr Biol. 2005 Jul 12;15(13):1217-21. doi: 10.1016/j.cub.2005.05.059.
The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.
COP9信号小体（CSN）是一种在所有真核细胞中都存在的保守蛋白复合物，参与泛素（Ub）/26S蛋白酶体系统的调节。它能结合众多蛋白质，包括Ub E3连接酶和去泛素化酶Ubp12p，后者是人类USP15在粟酒裂殖酵母中的同源物。我们发现USP15与人类CSN复合物共纯化。分离出的CSN复合物表现出对多聚Ub底物进行去泛素化的蛋白酶活性，并且被金属螯合剂邻菲罗啉（OPT）完全抑制。令人惊讶的是，在OPT存在的情况下，重组USP15也无法切割多聚Ub链的异肽键。对USP序列的详细分析导致在USP15和相关USP中发现了一种新型锌（Zn）指结构。预测的Zn结合基序中单个保守半胱氨酸残基的突变导致USP15降解多聚Ub底物的能力丧失，表明Zn指结构对于切割多聚Ub链至关重要。此外，下拉实验表明四聚Ub与突变型USP15的结合减少。USP15和Ub连接酶Rbx1的共转染显示，野生型去泛素化酶而非具有缺陷Zn指结构的USP15突变体使Rbx1对Ub系统稳定，最有可能是通过逆转多聚/自泛素化实现的。总之，需要USP15的功能性Zn指结构来维持拆解多聚Ub链所必需的构象，这是拯救E3连接酶Rbx1的先决条件。
