Develop General Methods for the Synthesis of Proteins with Posttranslational Lysine Modifications

开发具有翻译后赖氨酸修饰的蛋白质合成的通用方法

• 批准号: 10307614
• 负责人: Wenshe Ray Liu
• 金额: $ 33.4万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-15 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10307614
• 关键词: Acylation; Affect; Alkylation; Amber; Amines; Amino Acids; Amino Acyl-tRNA Synthetases; Bacteriophages; Biochemical; Catalysis; Cells; Chemicals; Complex; Cysteine; DNA biosynthesis; Development; Disease; Engineering; Escherichia coli; Eukaryota; Evolution; Gene Expression; Genetic; Genus Mycobacterium; Health; Health Promotion; Human; Investigation; Life; Ligation; Lysine; Methods; Mission; Modification; Mutagenesis; Organism; Post-Translational Protein Processing; Protein Biosynthesis; Proteins; Public Health; Reaction; Recombinants; Regulation; Research; Side; Signal Transduction; Site; Technology; Testing; Transfer RNA; Ubiquitin; Ubiquitin Like Proteins; Ubiquitination; United States National Institutes of Health; amidation; base; biomaterial compatibility; cellular development; deep sequencing; enzyme activity; improved; protein metabolism; proteostasis; small molecule

PROJECT SUMMARY/ABSTRACT Among all 20 native amino acids in proteins, lysine (Lys) undergoes the most diverse forms of posttranslational modifications (PTMs). The unique nucleophilicity of its side chain amine allows Lys to be selectively modified with several types of alkylation and a number of small-molecule and protein acylations. These PTMs, especially in eukaryotes, regulate enzyme activities, interactions of proteins with their partners, cellular localization of proteins, and protein metabolism. Abnormality of these PTMs correlates with the development of many diseases. Although important, biochemical studies of Lys PTMs are cumbersome due to the difficulty to synthesize proteins with them. Several methods have been developed for the synthesis of proteins with Lys PTMs. However, they cannot be generally applied. With an overall objective to formulate straightforward methods that can be generally applied for the synthesis of proteins with Lys PTMs, the current application will focus on the development of amber suppression-based noncanonical amino acid (ncAA) mutagenesis methods in combination with chemical transformation for the installation of Lys PTMs into proteins. Three specific aims will be pursued: 1) Develop enhanced amber suppression-based ncAA mutagenesis methods for the recombinant synthesis of proteins with Lys alkylations and small-molecule acylations; 2) Develop amber suppression-based ncAA mutagenesis methods for the synthesis of proteins with ubiquitin and ubiquitin-like protein modifications; and 3) Formulate recombinant strategies in conjunction with biocompatible reactions to synthesize proteins installed site-specifically with two different Lys PTMs. The successful completion of the proposed study will make available straightforward methods for the synthesis of proteins with most Lys PTMs for their functional investigation.

项目概要/摘要 在蛋白质的所有 20 种天然氨基酸中，赖氨酸 (Lys) 经历的形式最为多样 翻译后修饰 (PTM)。其侧链胺独特的亲核性使得Lys可以 通过几种类型的烷基化和一些小分子和蛋白质酰化进行选择性修饰。 这些 PTM，特别是在真核生物中，调节酶活性、蛋白质与其伴侣的相互作用、 蛋白质的细胞定位和蛋白质代谢。这些 PTM 的异常与 许多疾病的发展。尽管重要，但 Lys PTM 的生化研究却很麻烦，因为 用它们合成蛋白质的困难。已开发出多种合成方法 带有 Lys PTM 的蛋白质。然而，它们不能被普遍应用。制定总体目标 简单的方法通常可用于合成具有 Lys PTM 的蛋白质，目前 申请将重点开发基于琥珀抑制的非规范氨基酸（ncAA） 诱变方法与化学转化相结合，将 Lys PTM 安装到 蛋白质。将追求三个具体目标：1）开发基于增强琥珀抑制的 ncAA 用于重组合成赖氨酸烷基化和小分子蛋白质的诱变方法 酰化； 2) 开发基于琥珀抑制的 ncAA 诱变方法，用于合成蛋白质 泛素和泛素样蛋白修饰； 3) 联合制定重组策略 合成具有两种不同 Lys PTM 的位点特异性安装的蛋白质的生物相容性反应。这 拟议研究的成功完成将为合成提供直接的方法 具有大多数 Lys PTM 的蛋白质用于其功能研究。

项目成果

Taf2 mediates DNA binding of Taf14.

Taf2 介导 Taf14 的 DNA 结合。

• 发表时间: 2022-06-08
• 影响因子: 16.6
• 作者: Klein, Brianna J;Feigerle, Jordan T;Zhang, Jibo;Ebmeier, Christopher C;Fan, Lixin;Singh, Rohit K;Wang, Wesley W;Schmitt, Lauren R;Lee, Thomas;Hansen, Kirk C;Liu, Wenshe R;Wang, Yun;Strahl, Brian D;Anthony Weil, P;Kutateladze, Tatiana G
• 通讯作者: Kutateladze, Tatiana G

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease.

SARS-CoV-2 类木瓜蛋白酶的药物再利用。

• 发表时间: 2022
• 影响因子: 3.4
• 作者: Cho, Chia;Li, Shuhua G;Lalonde, Tyler J;Yang, Kai S;Yu, Ge;Qiao, Yuchen;Xu, Shiqing;Ray Liu, Wenshe
• 通讯作者: Ray Liu, Wenshe

The Pyrrolysyl-tRNA Synthetase Activity can be Improved by a P188 Mutation that Stabilizes the Full-Length Enzyme.

吡咯赖氨酰-tRNA 合成酶活性可以通过稳定全长酶的 P188 突变来提高。

• DOI: 10.1016/j.jmb.2022.167453
• 发表时间: 2022-04-30
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Cho, Chia-Chuan;Blankenship, Lauren R.;Ma, Xinyu;Xu, Shiqing;Liu, Wenshe
• 通讯作者: Liu, Wenshe

Covalent Inhibition in Drug Discovery.

药物发现中的共价抑制。

• 发表时间: 2019
• 影响因子: 3.4
• 作者: Ghosh, Avick Kumar;Samanta, Indranil;Mondal, Anushree;Liu, Wenshe Ray
• 通讯作者: Liu, Wenshe Ray

A Designed, Highly Efficient Pyrrolysyl-tRNA Synthetase Mutant Binds o-Chlorophenylalanine Using Two Halogen Bonds.

一种设计的高效吡咯赖氨酰-tRNA 合成酶突变体使用两个卤素键结合邻氯苯丙氨酸。

• DOI: 10.1016/j.jmb.2022.167534
• 发表时间: 2022-04-30
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Vatansever, Erol C.;Yang, Kai S.;Geng, Zhi Zachary;Qiao, Yuchen;Li, Pingwei;Xu, Shiqing;Liu, Wenshe Ray
• 通讯作者: Liu, Wenshe Ray