Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling
Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling
复制标题
通过生物正交标记鉴定和分析组蛋白乙酰转移酶底物
DOI：
10.1002/cpz1.497
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Zheng, Y. George
中科院分区：
文献类型：
--
作者：
Song, Jiabao;Han, Zhen;Zheng, Y. George
组蛋白乙酰转移酶（HAT，也称为赖氨酸乙酰转移酶，KAT）使用乙酰辅酶A（Ac-CoA）作为辅因子催化其同源蛋白质底物的乙酰化，并参与各种生理和病理过程。基于质谱的蛋白质组学的进展已经允许发现数千种乙酰化蛋白质和特定的乙酰化赖氨酸位点。然而，由于HAT活性的快速动力学和功能冗余，以及使用抗体捕获乙酰化赖氨酸的限制，系统地和精确地定义给定HAT直接乙酰化的底物和位点是具有挑战性的。在这里，我们描述了一种化学蛋白质组学方法来识别和配置文件的蛋白质底物的个人HAT酶的蛋白质组规模。该方法涉及蛋白质工程以扩大目标HAT的Ac-CoA结合口袋，从而产生突变形式，其可以使用官能化酰基-CoA作为辅因子替代物来生物正交标记其蛋白质底物。酰化的蛋白质底物可以与荧光探针（用于成像检测）或生物素手柄（用于链霉亲和素下拉和化学蛋白质组学鉴定）化学选择性缀合。这种模块化化学生物学方法已成功用于鉴定p300、GCN 5和HAT 1的蛋白质底物，预计这种方法可用于分析和鉴定许多其他HAT酶的亚乙酰基组。© 2022 Wiley Periodicals LLC.基本方案1：用叠氮化物/炔-生物素标记HAT蛋白质底物替代方案：用叠氮化物/炔-塔姆拉标记HAT的蛋白质底物用于凝胶内可视化支持方案1：HAT突变体的表达和纯化支持方案2：Ac-CoA替代物的合成基本方案2：生物素化HAT底物的链霉亲和素富集基本方案3：HAT底物的化学蛋白质组学鉴定基本方案4：用蛋白质印迹法验证特异性HAT底物
Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl‐CoA (Ac‐CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry‐based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac‐CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl‐CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub‐acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC.Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne‐biotinAlternate Protocol: Labeling protein substrates of HATs with azide/alkyne‐TAMRA for in‐gel visualizationSupport Protocol 1: Expression and purification of HAT mutantsSupport Protocol 2: Synthesis of Ac‐CoA surrogatesBasic Protocol 2: Streptavidin enrichment of biotinylated HAT substratesBasic Protocol 3: Chemoproteomic identification of HAT substratesBasic Protocol 4: Validation of specific HAT substrates with western blotting
登录
查看更多内容
影响因子：
29
作者：
Wagner GR;Bhatt DP;O'Connell TM;Thompson JW;Dubois LG;Backos DS;Yang H;Mitchell GA;Ilkayeva OR;Stevens RD;Grimsrud PA;Hirschey MD
通讯作者：
Hirschey MD
用于分析短链脂肪酰化的生物正交前代谢物† †提供电子补充信息 (ESI)，请参阅 DOI：10.1039/c7sc00247e。
影响因子：
8.4
作者：
Wilson R. Sinclair;Jonathan H. Shrimp;Thomas T. Zengeya;Rhushikesh A. Kulkarni;Julie M. Garlick;Hans F. Luecke;A. Worth;I. Blair;N. Snyder;J. Meier
通讯作者：
J. Meier
影响因子：
15
作者：
Carter-O'Connell I;Jin H;Morgan RK;David LL;Cohen MS
通讯作者：
Cohen MS
影响因子：
62.1
作者：
Huang H;Lin S;Garcia BA;Zhao Y
通讯作者：
Zhao Y
影响因子：
2.7
作者：
Yu-Ying, Yang;Markus, Grammel;Howard, Hang C.
通讯作者：
Howard, Hang C.