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Protease Activity Profiling via Programmable Phage Display of Comprehensive Proteome-Scale Peptide Libraries
Cell Systems ( IF 9.0 ) Pub Date : 2020-09-23 , DOI: 10.1016/j.cels.2020.08.013
Gabriel D Román-Meléndez 1 , Thiagarajan Venkataraman 1 , Daniel R Monaco 1 , H Benjamin Larman 1
Affiliation  

Endopeptidases catalyze the internal cleavage of proteins, playing pivotal roles in protein turnover, substrate maturation, and the activation of signaling cascades. A broad range of biological functions in health and disease are controlled by proteases, yet assays to characterize their activities at a proteomic scale do not exist. To address this unmet need, we developed Sensing EndoPeptidase Activity via Release and recapture using flAnking Tag Epitopes (SEPARATE), which uses a monovalent phage display of the human proteome at a 90-aa peptide resolution. We demonstrate that SEPARATE is compatible with several human proteases from distinct catalytic classes, including caspase-1, ADAM17, and thrombin. Both well-characterized and newly identified substrates of these enzymes were detected in the assay. SEPARATE was used to discover a non-canonical caspase-1 substrate, the E3 ubiquitin ligase HUWE1, a key mediator of apoptotic cell death. SEPARATE enables efficient, unbiased assessment of endopeptidase activity by using a phage-displayed proteome. A record of this paper’s Transparent Peer Review process is included in the Supplemental Information.



中文翻译:

通过综合蛋白质组规模肽库的可编程噬菌体展示进行蛋白酶活性分析

内肽酶催化蛋白质的内部裂解,在蛋白质周转、底物成熟和信号级联激活中起关键作用。蛋白酶在健康和疾病中的广泛生物学功能受蛋白酶控制,但尚不存在在蛋白质组学尺度上表征其活性的分析方法。为了解决这一未满足的需求,我们开发了通过释放和重新捕获使用侧翼标签表位 (SEPARATE) 来感知内肽酶活性,该表位使用人类蛋白质组的单价噬菌体展示,分辨率为 90-aa。我们证明 SEPARATE 与来自不同催化类别的几种人类蛋白酶兼容,包括 caspase-1、ADAM17 和凝血酶。在测定中检测到这些酶的充分表征和新鉴定的底物。SEPARATE 用于发现非经典 caspase-1 底物,即 E3 泛素连接酶 HUWE1,它是凋亡细胞死亡的关键介质。SEPARATE 通过使用噬菌体展示的蛋白质组,能够对内肽酶活性进行有效、无偏见的评估。本文的透明同行评审过程的记录包含在补充信息中。

更新日期:2020-10-30
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