Direct Proximity Tagging of Small Molecule Protein Targets Using an Engineered NEDD8 Ligase

doi:10.1021/jacs.6b06828

	Direct Proximity Tagging of Small Molecule Protein Targets Using an Engineered NEDD8 Ligase
	Hill, Zachary B.1; Pollock, Samuel B.1; Zhuang, Min1,3 ; Wells, James A.1,2
	2016-10-12
发表期刊	JOURNAL OF THE AMERICAN CHEMICAL SOCIETY (IF:14.4[JCR-2023],14.8[5-Year])
ISSN	0002-7863
卷号	138 期号:40 页码:13123-13126
发表状态	已发表
DOI	10.1021/jacs.6b06828
摘要	Identifying the protein targets of bioactive small molecules remains a major problem in the discovery of new chemical probes and therapeutics. While activity based probes and photo-cross-linkers have had success in identifying protein targets of small molecules, each technique has limitations. Here we describe a method for direct proximity tagging of proteins that bind small molecules. We engineered a promiscuous ligase based on the NEDD8 conjugating enzyme, Ubc12, which can be covalently linked to a small molecule of interest. When target proteins bind the small molecule, they are directly labeled on surface lysines with a biotinylated derivative of the small ubiquitin homologue, NEDD8. This unique covalent tag can then be used to identify the small molecule binding proteins. Utilizing the drug dasatinib, we have shown that dasatinib-directed NEDDylation occurs for known endogenous protein binders in complex cell lysates. In addition, we have been able to improve NEDDylation efficiency through rational mutagenesis. Finally, we have shown that affinity-directed NEDDylation can be applied to two other protein ligand interactions beyond kinases. Proximity tagging using this engineered ligase requires direct binding of the target and, thus, provides a useful and orthogonal approach to facilitate small molecule target identification.
收录类别	SCI ; EI
语种	英语
资助项目	NIH[R01 CA191018]
WOS研究方向	Chemistry
WOS类目	Chemistry, Multidisciplinary
WOS记录号	WOS:000385469600007
出版者	AMER CHEMICAL SOC
EI入藏号	20164302935931
EI主题词	Amino acids ; Bins ; Enzymes ; Molecules ; Probes
EI分类号	Storage:694.4 ; Organic Compounds:804.1 ; Atomic and Molecular Physics:931.3
WOS关键词	KINASE ; CELLS ; INHIBITORS ; DASATINIB ; CANCER
原始文献类型	Article
引用统计	正在获取...
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/1671
专题	生命科学与技术学院_PI研究组_庄敏组
通讯作者	Wells, James A.
作者单位	1.Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA 2.Univ Calif San Francisco, Dept Cellular & Mol Pharmacol, San Francisco, CA 94158 USA 3.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai, Peoples R China
推荐引用方式 GB/T 7714	Hill, Zachary B.,Pollock, Samuel B.,Zhuang, Min,et al. Direct Proximity Tagging of Small Molecule Protein Targets Using an Engineered NEDD8 Ligase[J]. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY,2016,138(40):13123-13126.
APA	Hill, Zachary B.,Pollock, Samuel B.,Zhuang, Min,&Wells, James A..(2016).Direct Proximity Tagging of Small Molecule Protein Targets Using an Engineered NEDD8 Ligase.JOURNAL OF THE AMERICAN CHEMICAL SOCIETY,138(40),13123-13126.
MLA	Hill, Zachary B.,et al."Direct Proximity Tagging of Small Molecule Protein Targets Using an Engineered NEDD8 Ligase".JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 138.40(2016):13123-13126.