Use of the Noncanonical Amino Acid Mutagenesis Technique in Combination with Other Approaches to Study Functions of Posttranslational Lysine Modifications in Proteins
使用非常规氨基酸诱变技术与其他方法相结合来研究蛋白质翻译后赖氨酸修饰的功能
基本信息
- 批准号:10406602
- 负责人:
- 金额:$ 31.04万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2022
- 资助国家:美国
- 起止时间:2022-04-01 至 2027-03-31
- 项目状态:未结题
- 来源:
- 关键词:AcademiaAddressAmberAmino AcidsAmino Acyl-tRNA SynthetasesBindingBiologicalBiologyCellsChargeChemicalsChromatinCodon NucleotidesComplexCryoelectron MicroscopyCyclic GMPDiseaseEngineeringEnzymesEpigenetic ProcessEukaryotic CellFruitGeneticGoalsHDAC1 geneHalf-LifeHealth PromotionHistonesHumanIndustryInnate Immune ResponseKDM1A geneLysineMass Spectrum AnalysisMethodsMissionModificationMolecularMutagenesisNucleosomesPolyubiquitinationPost-Translational RegulationProkaryotic CellsProtein BiosynthesisProtein IsoformsProteinsProteomicsPublic HealthRegulationResearchRoleSiteSystemTechniquesUbiquitinUbiquitin Like ProteinsUnited States National Institutes of Healthbasebiological researchds-DNAmutantnovelpathogenpyrrolysinereconstitutionsensortherapeutic targettool
项目摘要
PROJECT SUMMARY/ABSTRACT
As a naturally existing amber suppression system, the pyrrolysine (Pyl) incorporation machinery has turned
into an enormous tool for undergoing amber suppression-based noncanonical amino acid (ncAA) mutagenesis
in both prokaryotic and eukaryotic cells. By ectopically expressing tRNAPyl and pyrrolysyl-tRNA synthetase
(PylRS) or a PylRS mutant that charges tRNAPyl with an ncAA, about 200 ncAAs have been genetically encoded
by the amber codon in various cells. As one of the original pioneers in the research field of engineering the Pyl
system for the genetic incorporation of ncAAs, the PI’s group has contributed more than one third of the total
encoded ncAAs. These ncAAs contain a large variety of functionalities, allowing a myriad of applications in both
academia and industry possible. After more than a decade of engineering the Pyl system for the ncAA
incorporation, the field is now able to use the Pyl system-based ncAA mutagenesis technique to conduct grand
explorations to address fundamentally important biological questions. In the past, the PI’s lab has devised a
variety of ncAA mutagenesis-based approaches for the synthesis of proteins with posttranslational lysine
modifications (lysine PTMs). A novel method that allows direct functionalization of ubiquitin and ubiquitin like
proteins for their conjugation with other proteins has also been developed in the PI’s lab. With all these methods
coming to fruition, the PI’s lab is shifting its research focus to use their developed techniques to study basic and
fundamentally important biological questions. Five specific directions will be pursued. The first direction is to use
the ncAA mutagenesis technique to produce designer nucleosomes (nucleosomes with defined lysine PTMs) for
probing histone lysine sites and PTM types targeted by epigenetic erasers including SIRT6, SIRT7, HDAC1, and
LSD1 (HDAC1 and LSD1 in their native complexes). The second direction is to use reconstituted designer
nucleosomes as probes to enrich their binding partners from cells whose identities can be determined using
mass spectrometry-based proteomic analysis. The third direction is to conduct cryo-EM analysis of designer
nucleosomes bound with SIRT6, SIRT7, HDAC1, and LSD1 (HDAC1 and LSD1 in their native complexes) to
elucidate the structural basis of the four enzymes in their recognition of targeted lysine sites and PTMs in
chromatin. The fourth direction is to synthesize different triubiquitin isoforms and use them as probes to enrich
binding partners from cells whose identities will be confirmed with mass spectrometry-based proteomic analysis.
The last but not least direction is to synthesize cyclic GMP-AMP synthase (cGAS), which is a frontline sensor in
human cells that detect double-stranded DNA from pathogens and triggers innate immune responses, with lysine
PTMs and to study the functional roles of lysine PTMs in regulating activity, cellular localization and cellular half-
life of cGAS. A long-term goal of the PI’s research is to push applications of the Pyl system-based ncAA
mutagenesis technique in combination with other chemical biology techniques to enhance basic biological
research.
项目总结/摘要
作为一种天然存在的琥珀抑制系统,吡咯赖氨酸(Pyl)掺入机制已转向
成为一个巨大的工具,进行琥珀抑制为基础的非规范氨基酸(ncAA)诱变
在原核和真核细胞中。通过异位表达tRNAPyl和吡咯赖氨酰-tRNA合成酶
例如,在使用pylRS(PylRS)或用ncAA装载tRNAPyl的PylRS突变体的情况下,已经遗传编码了约200个ncAA。
在不同的细胞中被琥珀密码子所控制作为工程学研究领域的先驱之一,
在ncAA基因整合系统中,PI小组贡献了总数的三分之一以上。
编码的ncAA。这些ncAA包含各种各样的功能,允许在两种环境中的无数应用程序
学术界和工业界都有可能。在为ncAA设计Pyl系统十多年后,
并入后,该领域现在能够使用基于Pyl系统的ncAA诱变技术进行大规模的
探索解决基本重要的生物学问题。在过去,PI的实验室已经设计了一个
用于合成具有翻译后赖氨酸的蛋白质的多种基于ncAA诱变的方法
修饰(赖氨酸PTM)。一种允许泛素和泛素样蛋白直接官能化的新方法,
PI的实验室还开发了用于与其他蛋白质结合的蛋白质。所有这些方法
即将实现,PI的实验室正在转移其研究重点,使用他们开发的技术来研究基础和
重要的生物学问题。将采取五个具体方向。第一个方向是利用
ncAA诱变技术产生设计的核小体(具有限定的赖氨酸PTM的核小体),
探测由表观遗传擦除器靶向的组蛋白赖氨酸位点和PTM类型,包括SIRT 6、SIRT 7、HDAC 1和
LSD 1(HDAC 1和LSD 1的天然复合物)。第二个方向是使用重构设计器
核小体作为探针从细胞中富集它们的结合配偶体,
基于质谱的蛋白质组学分析。第三个方向是对设计人员进行低温电磁分析
与SIRT 6、SIRT 7、HDAC 1和LSD 1结合的核小体(HDAC 1和LSD 1在其天然复合物中),
阐明了四种酶在识别靶向赖氨酸位点和PTM中的结构基础。
染色质第四个方向是合成不同的triubiquitin异构体,并将其作为探针富集
来自细胞的结合配偶体,其身份将通过基于质谱的蛋白质组学分析来确认。
最后但并非最不重要的方向是合成环GMP-AMP合酶(cGAS),其是细胞内的前线传感器。
检测来自病原体的双链DNA并触发先天免疫反应的人类细胞,含赖氨酸
目的:研究赖氨酸PTMs在调节细胞活性、细胞定位和细胞半定量中的功能作用。
CGAS的寿命PI研究的长期目标是推动基于Pyl系统的ncAA的应用
诱变技术与其他化学生物学技术相结合,
research.
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Wenshe Ray Liu其他文献
Wenshe Ray Liu的其他文献
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{{ truncateString('Wenshe Ray Liu', 18)}}的其他基金
Development of fluorogenic and chemiluminogenic main protease substrates for cellular and in vivo imaging of SARS-CoV-2
开发用于 SARS-CoV-2 细胞和体内成像的荧光和化学发光主要蛋白酶底物
- 批准号:
10432895 - 财政年份:2022
- 资助金额:
$ 31.04万 - 项目类别:
Use of the Noncanonical Amino Acid Mutagenesis Technique in Combination with Other Approaches to Study Functions of Posttranslational Lysine Modifications in Proteins
使用非常规氨基酸诱变技术与其他方法相结合来研究蛋白质翻译后赖氨酸修饰的功能
- 批准号:
10591531 - 财政年份:2022
- 资助金额:
$ 31.04万 - 项目类别:
Development of fluorogenic and chemiluminogenic main protease substrates for cellular and in vivo imaging of SARS-CoV-2
开发用于 SARS-CoV-2 细胞和体内成像的荧光和化学发光主要蛋白酶底物
- 批准号:
10611456 - 财政年份:2022
- 资助金额:
$ 31.04万 - 项目类别:
Targeting the ENL YEATS domain for the development of anti-leukemia agents
靶向 ENL YEATS 结构域用于开发抗白血病药物
- 批准号:
10357052 - 财政年份:2021
- 资助金额:
$ 31.04万 - 项目类别:
Targeting the ENL YEATS domain for the development of anti-leukemia agents
靶向 ENL YEATS 结构域用于开发抗白血病药物
- 批准号:
10534219 - 财政年份:2021
- 资助金额:
$ 31.04万 - 项目类别:
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 - 财政年份:2019
- 资助金额:
$ 31.04万 - 项目类别:
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:
10063529 - 财政年份:2019
- 资助金额:
$ 31.04万 - 项目类别:
Phage Display with Two Genetically Incorporated Noncanonical Amino Acids
具有两种基因掺入的非规范氨基酸的噬菌体展示
- 批准号:
8730263 - 财政年份:2011
- 资助金额:
$ 31.04万 - 项目类别:
Phage Display with Two Genetically Incorporated Noncanonical Amino Acids
具有两种基因掺入的非规范氨基酸的噬菌体展示
- 批准号:
8821041 - 财政年份:2011
- 资助金额:
$ 31.04万 - 项目类别:
Phage Display with Two Genetically Incorporated Noncanonical Amino Acids
具有两种基因掺入的非规范氨基酸的噬菌体展示
- 批准号:
8450284 - 财政年份:2011
- 资助金额:
$ 31.04万 - 项目类别:
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