Department of Bioorganic Chemistry, Faculty of Chemistry , Wroclaw University of Science and Technology , Wyb. Wyspianskiego 27 , 50-370 Wroclaw , Poland.
Program in Cell Death and Survival Networks , Sanford Burnham Prebys Medical Discovery Institute , La Jolla , California 92037 , United States.
J Med Chem. 2018 Jun 28;61(12):5222-5234. doi: 10.1021/acs.jmedchem.8b00026. Epub 2018 Jun 9.
The proteasome is an enzyme complex critical for maintaining protein homeostasis. Perturbed proteasome function leads to pathologies including cancer and autoimmune and neurodegenerative disease. Therefore, the proteasome constitutes an excellent molecular target for pharmaceutical development. Here, using the HyCoSuL approach, we designed and synthesized novel and selective fluorogenic substrates for each of these three constitutive 20S proteasome activities and applied them to assess inhibition of proteasome subunits by MG-132 and a clinically used inhibitor bortezomib. Our results confirm the utility of designed substrates in biochemical assays. Furthermore, selective peptide sequences obtained in this manner were used to construct fluorophore-labeled activity-based probes and then utilized to detect each constitutive 20S proteasome subunit simultaneously in lysates of HEK-293F cells and red blood cells. Overall, we describe a simple and rapid method useful to measure constitutive 20S proteasome activity in whole human blood samples that could enable early diagnosis of pathological states associated with aberrantly upregulated proteasome activity.
蛋白酶体是一种对于维持蛋白质内稳态至关重要的酶复合物。蛋白酶体功能紊乱会导致多种疾病，包括癌症、自身免疫性疾病和神经退行性疾病。因此，蛋白酶体是药物开发的一个极好的分子靶点。在这里，我们使用 HyCoSuL 方法，为这三种组成型 20S 蛋白酶体活性中的每一种设计和合成了新型的、选择性的荧光底物，并将其用于评估 MG-132 和一种临床使用的抑制剂硼替佐米对蛋白酶体亚基的抑制作用。我们的结果证实了设计的底物在生化测定中的实用性。此外，通过这种方式获得的选择性肽序列被用于构建荧光标记的活性基探针，然后用于同时检测 HEK-293F 细胞和红细胞裂解物中的每种组成型 20S 蛋白酶体亚基。总的来说，我们描述了一种简单快速的方法，可用于测量全血样本中的组成型 20S 蛋白酶体活性，这可能有助于早期诊断与蛋白酶体活性异常上调相关的病理状态。
