Houston Kaiulani M, Melvin Adam T, Woss Gregery S, Fayer Effrat L, Waters Marcey L, Allbritton Nancy L
Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599, United States.
Cain Department of Chemical Engineering, Louisiana State University , Baton Rouge, Louisiana 70803, United States.
ACS Omega. 2017 Mar 31;2(3):1198-1206. doi: 10.1021/acsomega.7b00109. Epub 2017 Mar 29.
Regulation of the ubiquitin-proteasome system (UPS) to treat select types of cancer has become a popular area of drug discovery research. The FDA approval of proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma has led to an increased need for chemical reporters capable of detecting and quantifying protein ubiquitination and the activity of members of the UPS including E3 ubiquitin ligases and the proteasome in the tumor cells of the patients. One limitation of peptide-based reporters is their rapid degradation in the cellular environment by cytosolic peptidases. Conversely, β-hairpin "protectides" exhibit a pronounced secondary structure that significantly increases their lifetime under cellular conditions. The goal of this work was to develop a family of novel, ornithine-rich protectides that could act as primary degrons serving as substrates for in vitro ubiquitination. The fluorescent peptide-based reporters were demonstrated to be highly resistant to degradation in multiple myeloma cell lysates. The most stable β-hairpin primary degron, containing a single ornithine residue at the N-terminus, OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH], demonstrated rapid ubiquitination kinetics and a 20-fold increase in stability when compared with an unstructured primary degron. A screen of E1 and E3 enzyme inhibitors in cell lysates showed that ubiquitination of OWRWR was significantly impaired by inhibitors of the SCF family of E3 ligases. Furthermore, this is the first report demonstrating the use of an ornithine residue on a primary degron as a ubiquitination site. This study serves as a strong foundation for the development of stable, fluorescent, peptide-based reporters capable of quantifying protein ubiquitination and the enzymatic activity of members of the UPS.
调节泛素-蛋白酶体系统（UPS）以治疗特定类型的癌症已成为药物发现研究中一个热门的领域。FDA批准蛋白酶体抑制剂硼替佐米和卡非佐米用于治疗多发性骨髓瘤，这使得对能够检测和定量蛋白质泛素化以及UPS成员（包括E3泛素连接酶和蛋白酶体）在患者肿瘤细胞中的活性的化学报告分子的需求增加。基于肽的报告分子的一个局限性是它们在细胞环境中会被胞质肽酶快速降解。相反，β-发夹“保护肽”具有明显的二级结构，可显著延长其在细胞条件下的寿命。这项工作的目标是开发一系列新型的、富含鸟氨酸的保护肽，它们可以作为主要降解子，作为体外泛素化的底物。基于荧光肽的报告分子在多发性骨髓瘤细胞裂解物中被证明对降解具有高度抗性。最稳定的β-发夹主要降解子，在N端含有一个鸟氨酸残基，即OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH]，与无结构的主要降解子相比，显示出快速的泛素化动力学，稳定性提高了20倍。在细胞裂解物中对E1和E3酶抑制剂的筛选表明，E3连接酶SCF家族的抑制剂显著损害了OWRWR的泛素化。此外，这是第一份证明在主要降解子上使用鸟氨酸残基作为泛素化位点的报告。这项研究为开发能够定量蛋白质泛素化和UPS成员酶活性的稳定、荧光、基于肽的报告分子奠定了坚实的基础。
