Activity Guided Azide-methyllysine Photo-trapping for Substrate Profiling of Lysine Demethylases.
Activity Guided Azide-methyllysine Photo-trapping for Substrate Profiling of Lysine Demethylases.
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DOI：
10.1021/jacs.3c07299
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发表时间：
2023-09-27
影响因子：
15
通讯作者：
Islam, Kabirul
中科院分区：
文献类型：
--
作者：
Kuwik, Jordan;Hinkelman, Kathryn;Waldman, Megan;Stepler, Kaitlyn E.;Wagner, Shana;Arora, Simran;Chernenkoff, Sasha;Cabalteja, Chino;Sidoli, Simone;Robinson, Rena A. S.;Islam, Kabirul
可逆的翻译后修饰（PTM）是建立蛋白质-蛋白质和蛋白质-核酸相互作用的关键，这些相互作用控制着细胞中的大多数信号传导途径。序列特异性PTM由转移酶催化，并且它们的去除由一类称为“去转移酶”的反向作用酶进行。目前可用的化学蛋白质组学方法在表征转移酶的底物方面是有价值的。然而，蛋白质组范围的编目的底物的脱转移酶是具有挑战性的，主要是由于表位的损失，使免疫沉淀和活性为基础的方法无效。在此，我们开发了一种称为交联辅助底物识别（CASI）的一般化学蛋白质组学策略，用于系统表征脱转移酶的细胞靶点，并成功地将其应用于赖氨酸脱甲基酶（KDM），该酶催化去除组蛋白中赖氨酸侧链的甲基以调节基因转录。通过在KDM 4的活性位点深处建立一个有针对性的叠氮基-甲氨基光反应，我们揭示了一种新的“去甲基化”，这种去甲基化已经逃脱了传统的方法。蛋白质组学研究发现了KDM 4的一系列非组蛋白底物，将KDM 4的生物足迹扩展到其在基因转录中的典型功能之外。KDM 4A介导的真核生物翻译起始因子中进化上保守的赖氨酸残基的去甲基化的一个值得注意的发现证明了KDM 4A在核糖体过程中具有更广泛的作用。CASI通过将焦点从简单的基于肽的探针转移到采用全长光活化脱甲基酶，代表了与早期方法的实质性偏离，准备应用于>400种人类脱转移酶，其中许多由于缺乏对其细胞靶标的知识而仍然知之甚少。
Reversible post-translational modifications (PTMs) are key to establishing protein–protein and protein–nucleic acid interactions that govern a majority of the signaling pathways in cells. Sequence-specific PTMs are catalyzed by transferases, and their removal is carried out by a class of reverse-acting enzymes termed “detransferases”. Currently available chemoproteomic approaches have been valuable in characterizing substrates of transferases. However, proteome-wide cataloging of the substrates of detransferases is challenging, mostly due to the loss of the epitope, rendering immunoprecipitation and activity-based methods ineffective. Herein, we develop a general chemoproteomic strategy called crosslinking-assisted substrate identification (CASI) for systematic characterization of cellular targets of detransferases and successfully apply it to lysine demethylases (KDMs) which catalyze the removal of methyl groups from lysine sidechain in histones to modulate gene transcription. By setting up a targeted azido-methylamino photo-reaction deep inside the active site of KDM4, engineered to carry p-azido phenylalanine, we reveal a novel “demethylome” that has escaped the traditional methods. The proteomic survey led to the identification of a battery of nonhistone substrates of KDM4, extending the biological footprint of KDM4 beyond its canonical functions in gene transcription. A notable finding of KDM4A-mediated demethylation of an evolutionarily conserved lysine residue in eukaryotic translational initiation factor argues for a much broader role of KDM4A in ribosomal processes. CASI, representing a substantive departure from earlier approaches by shifting focus from simple peptide-based probes to employing full-length photo-activatable demethylases, is poised to be applied to >400 human detransferases, many of which have remained poorly understood due to the lack of knowledge about their cellular targets.
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影响因子：
11.2
作者：
Cho, Hyun-Soo;Suzuki, Takehiro;Hamamoto, Ryuji
通讯作者：
Hamamoto, Ryuji
影响因子：
2.9
作者：
Grunbeck, Amy;Huber, Thomas;Sakmar, Thomas P.
通讯作者：
Sakmar, Thomas P.
影响因子：
5.7
作者：
Krishnan, Swathi;Trievel, Raymond C.
通讯作者：
Trievel, Raymond C.
影响因子：
16.8
作者：
Couture, Jean-Francois;Collazo, Evys;Trievel, Raymond C.
通讯作者：
Trievel, Raymond C.
DOI：
10.1016/j.bbagrm.2015.07.001
发表时间：
2015-08-01
影响因子：
4.7
作者：
Jung, Eun-Shil;Sim, Ye Ji;Kim, Kye-Seong
通讯作者：
Kim, Kye-Seong