High-Throughput Discovery of Substrate Peptide Sequences for E3 Ubiquitin Ligases Using a cDNA Display Method.

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS
Kenwa Tamagawa, Robert E Campbell, Takuya Terai
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引用次数: 0

Abstract

Cells utilize ubiquitin as a posttranslational protein modifier to convey various signals such as proteasomal degradation. The disfunction of ubiquitylation or following proteasomal degradation can give rise to the accumulation and aggregation of improperly ubquitylated proteins, which is known to be a general causation of many neurodegenerative diseases. Thus, the characterization of substrate peptide sequences of E3 ligases is crucial in biological and pharmaceutical sciences. In this study, we developed a novel high-throughput screening system for substrate peptide sequences of E3 ligases using a cDNA display method, which enables covalent conjugation between peptide sequences and their corresponding cDNA sequences. First, we focused on the MDM2 E3 ligase and its known peptide substrate as a model to establish the screening method, and confirmed that cDNA display method was compatible with in vitro ubiquitylation. Then, we demonstrated identification of MDM2 substrate sequences from random libraries to identify a novel motif (VKFTGGQLA). Bioinformatics analysis of the hit sequences was performed to gain insight about endogenous substrate proteins.

利用 cDNA 显示法高通量发现 E3 泛素连接酶的底物多肽序列
细胞利用泛素作为蛋白质翻译后修饰因子来传递蛋白酶体降解等各种信号。泛素化功能失调或蛋白酶体降解后,会导致泛素化不当的蛋白质堆积和聚集,这是许多神经退行性疾病的常见病因。因此,E3 连接酶底物肽序列的表征在生物和制药科学中至关重要。在这项研究中,我们利用 cDNA 展示法开发了一种新型的高通量 E3 连接酶底物肽序列筛选系统,该方法可实现肽序列与其相应 cDNA 序列之间的共价连接。首先,我们以MDM2 E3连接酶及其已知多肽底物为模型建立了筛选方法,并证实了cDNA展示法与体外泛素化兼容。然后,我们展示了从随机文库中识别MDM2底物序列的方法,从而确定了一个新的基序(VKFTGGQLA)。我们对命中序列进行了生物信息学分析,以深入了解内源性底物蛋白。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
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