Hameed Dharjath S, Ovaa Huib, van der Heden van Noort Gerbrand J, Sapmaz Aysegul
Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, Netherlands.
Front Mol Biosci. 2022 May 26;9:866467. doi: 10.3389/fmolb.2022.866467. eCollection 2022.
The ubiquitin-proteasome system is an essential regulator of many cellular processes including controlling protein homeostasis. The degradation of proteins by the multi-subunit proteasome complex is tightly regulated through a series of checkpoints, amongst which are a set of deubiquitinating proteases (DUBs). The proteasome-associated DUBs, UCH-L5 (Ubiquitin carboxyl-terminal hydrolase isozyme L5) and USP14 (Ubiquitin-specific protease 14), and the integral-DUB in the proteasome, Rpn11, is known to regulate proteasomal degradation by deubiquitination of distinct substrates. Although selective inhibitors for USP14 and Rpn11 have been recently developed, there are no known inhibitors that selectively bind to UCH-L5. The X-ray structure of the Ubiquitin (Ub) bound to UCH-L5 shows a β-sheet hairpin in Ub that contains a crucial hydrophobic patch involved in the interaction with UCH-L5. Herein, we designed and developed both a Ub sequence-based linear- and cyclic- β-sheet hairpin peptide that was found to preferably inhibit UCH-L5. We show that these peptides have low micromolar IC values and the cyclic peptide competes with the activity-based UbVME (Ubiquitin-Vinyl-Methyl-Ester) probe for UCH-L5, binding in a concentration-dependent manner. We further establish the selectivity profile of the cyclic peptide for UCH-L5 compared to other members of the UCH-DUB family and other cysteine DUBs in cell lysate. Furthermore, the cyclic peptide infiltrated cells resulting in the accumulation of polyUb chains, and was found to be non-toxic at the concentrations used here. Taken together, our data suggest that the cyclic peptide permeates the cell membrane, inhibits UCH-L5 by possibly blocking its deubiquitinating function, and contributes to the accumulation of polyubiquitinated substrates. The implications of inhibiting UCH-L5 in the context of the 26S proteasome render it an attractive candidate for further development as a potential selective inhibitor for therapeutic purposes.
泛素-蛋白酶体系统是包括控制蛋白质稳态在内的许多细胞过程的重要调节因子。多亚基蛋白酶体复合物对蛋白质的降解通过一系列检查点进行严格调控，其中包括一组去泛素化蛋白酶（DUBs）。已知蛋白酶体相关的DUBs，UCH-L5（泛素羧基末端水解酶同工酶L5）和USP14（泛素特异性蛋白酶14），以及蛋白酶体中的整合DUB，Rpn11，通过对不同底物的去泛素化来调节蛋白酶体降解。尽管最近已经开发出针对USP14和Rpn11的选择性抑制剂，但尚无已知的选择性结合UCH-L5的抑制剂。与UCH-L5结合的泛素（Ub）的X射线结构显示Ub中有一个β-折叠发夹结构，其中包含一个与UCH-L5相互作用的关键疏水区域。在此，我们设计并开发了一种基于Ub序列的线性和环状β-折叠发夹肽，发现其可优先抑制UCH-L5。我们表明，这些肽具有低微摩尔IC值，并且环状肽与基于活性的UbVME（泛素-乙烯基-甲基-酯）探针竞争UCH-L5的活性，以浓度依赖性方式结合。我们进一步确定了环状肽与细胞裂解物中UCH-DUB家族的其他成员和其他半胱氨酸DUB相比对UCH-L5的选择性特征。此外，环状肽渗透到细胞中导致多聚泛素链的积累，并且发现在此处使用的浓度下无毒。综上所述，我们的数据表明环状肽可穿透细胞膜，可能通过阻断其去泛素化功能来抑制UCH-L5，并导致多聚泛素化底物的积累。在26S蛋白酶体的背景下抑制UCH-L5的意义使其成为作为潜在治疗目的选择性抑制剂进一步开发的有吸引力的候选物。
