Development and Application of a Sensitive Peptide Reporter to Discover 20S Proteasome Stimulators,ACS Combinatorial Science

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Development and Application of a Sensitive Peptide Reporter to Discover 20S Proteasome Stimulators
ACS Combinatorial Science Pub Date : 2018-03-19 00:00:00 , DOI: 10.1021/acscombsci.7b00193
Rachel A. Coleman ₁ , Darci J. Trader ₁

Affiliation

To attenuate an overabundance of cellular protein, it has been hypothesized that the 20S core particle (20S CP) of the proteasome can be chemically stimulated to degrade proteins into nontoxic peptides more quickly. Screening for small molecule 20S CP stimulators is typically performed with a reporter peptide composed of four amino acids and a coumarin group that is released upon proteasome-mediated hydrolysis to generate a fluorescent signal. Screening with this small reporter can lead to false negatives because the reporter peptide is rapidly turned-over without stimulation. To improve the screening for 20S CP stimulators, we have developed a peptide FRET reporter nearly four times more sensitive to stimulation but still amenable for high throughput screening. Through application of our FRET reporter, we have discovered two 20S CP gate-opening stimulators and also a molecule that elicits its mechanism of action through an interaction with a 20S CP active site.

中文翻译：

发现20S蛋白酶体刺激物的灵敏肽报道分子的开发与应用

为了减轻细胞蛋白质的过量，已经假设可以化学刺激蛋白酶体的20S核心颗粒（20S CP）以更快地将蛋白质降解为无毒肽。小分子20S CP刺激物的筛选通常使用由四个氨基酸和香豆素基团组成的报告肽进行，该肽在蛋白酶体介导的水解过程中释放以产生荧光信号。用这种小的报道分子进行筛选可能会导致假阴性，因为报道分子肽无需刺激即可快速翻转。为了改善20S CP刺激物的筛选，我们开发了一种肽FRET报告基因，其对刺激的敏感性提高了近四倍，但仍然适合高通量筛选。通过我们的FRET记者的应用，

更新日期：2018-03-19

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