Establishment of a suite of assays that support the discovery of proteasome stimulators.
Establishment of a suite of assays that support the discovery of proteasome stimulators.
复制标题
DOI：
10.1016/j.bbagen.2017.01.003
复制
发表时间：
2017-04
期刊：
影响因子：
--
通讯作者：
Kodadek T
中科院分区：
文献类型：
--
作者：
Trader DJ;Simanski S;Dickson P;Kodadek T
蛋白酶体催化许多错误折叠蛋白质的降解，否则这些蛋白质是细胞毒性的。人们对发现蛋白酶体激动剂很感兴趣，但之前的努力都令人失望。小的荧光肽的裂解通常被用作筛选蛋白酶体调节剂的试验。我们已经开发了后续检测，使用更多的生理相关底物。为了证明该工作流程的有效性，对NIH临床收集（NCC）进行了筛选。虽然许多化合物刺激蛋白酶体介导的促荧光肽底物的蛋白水解，但大多数化合物在较大的肽或蛋白质底物的实验中没有表现出活性。我们还表明，先前声称是蛋白酶体激动剂的两种分子，橄榄苦苷和白桦酸，确实加速了荧光底物的水解，但对体外或纤维素中错误折叠蛋白质的周转没有影响。然而，来自NCC的两个小分子，MK-866和AM-404，刺激活细胞中蛋白酶体介导的错误折叠蛋白的3- 4倍的周转。监测蛋白酶体介导的大肽和蛋白质降解的试验可以区分真正的激动剂和只能刺激短的、非生理相关肽的切割的化合物。已经建立了一套检测方法，可以发现真正的蛋白酶体激动剂。AM-404和MK-866可以作为细胞培养实验的有用工具，并且可以作为支架来产生更有效的20S刺激剂。
The proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing. The cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators. We have developed follow-on assays that employ more physiologically relevant substrates. To demonstrate the efficacy of this workflow, the NIH Clinical Collection (NCC) was screened. While many compounds stimulated proteasome-mediated proteolysis of the pro-fluorogenic peptide substrates, most failed to evince activity in assays with larger peptide or protein substrates. We also show that two molecules claimed previously to be proteasome agonists, oleuropein and betulinic acid, indeed accelerate hydrolysis of the fluorogenic substrate, but have no effect on the turnover of a mis-folded protein in vitro or in cellulo. However, two small molecules from the NCC, MK-866 and AM-404, stimulate the proteasome-mediated turnover of a mis-folded protein in living cells by 3- to 4-fold. Assays that monitor the proteasome-mediated degradation of larger peptides and proteins can distinguish bona fide agonists from compounds only able to stimulate the cleavage of short, non-physiologically relevant peptides. A suite of assays has been established that allows the discovery of bona fide proteasome agonists. AM-404 and MK-866 can be useful tools for cell culture experiments, and can serve as scaffolds to generate more potent 20S stimulators.