Allele-Specific Chemical Rescue of Histone Demethylases Using Abiotic Cofactors.
Allele-Specific Chemical Rescue of Histone Demethylases Using Abiotic Cofactors.
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DOI：
10.1021/acschembio.1c00335
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发表时间：
2022-12-16
影响因子：
4
通讯作者：
Islam, Kabirul
中科院分区：
文献类型：
--
作者：
Scott, Valerie;Dey, Debasis;Kuwik, Jordan;Hinkelman, Kathryn;Waldman, Megan;Islam, Kabirul
从共同祖先基因进化而来的密切相关的蛋白质家族由于其折叠和功能的相似性而在开发成员和亚型特异性化学探针方面存在重大障碍。在这项工作中，我们探索了一种等位基因特异性化学拯救策略，使用合成辅因子激活野生型蛋白质的“死亡”变体，并证明其成功应用于 2-酮戊二酸 (2KG) 依赖性组蛋白去甲基酶 4 (KDM4) 家族的成员。我们表明，催化位点中特定残基的突变使变体对天然共底物失去活性。相比之下，具有适当立体电子特征的2KG衍生物赋予突变体类似天然的脱甲基酶活性，同时对一组野生型双加氧酶保持耐药性。正交酶-辅因子对证明了细胞环境中全长染色体组蛋白的位点和程度特异性赖氨酸去甲基化。我们的工作提供了一种通过识别和改造保守的苯丙氨酸残基来调节特定组蛋白去甲基化酶的策略，该残基充当 KDM4 亚家族中的看门人，以使该酶对一组新型 2KG 衍生物敏感。本文开发的正交对将作为探针来研究特定程度的赖氨酸去甲基化在哺乳动物基因表达中的作用。此外，这种克服活性位点简并性的方法预计将在所有人类 2KG 依赖性双加氧酶中得到普遍应用。
Closely related protein families evolved from common ancestral genes present a significant hurdle in developing member- and isoform-specific chemical probes, owing to their similarity in fold and function. In this piece of work, we explore an allele-specific chemical rescue strategy to activate a ‘dead’ variant of a wild type protein using synthetic cofactors and demonstrate its successful application to the members of the 2-ketoglutarate (2KG)-dependent histone demethylase 4 (KDM4) family. We show that a mutation at a specific residue in the catalytic site renders the variant inactive towards the natural co-substrate. In contrast, 2KG derivatives bearing appropriate stereoelectronic features endowed the mutant with native-like demethylase activity while remaining refractory to a set of wild type dioxygenases. The orthogonal enzyme-cofactor pairs demonstrated site- and degree-specific lysine demethylation on full-length chromosomal histone in cellular milieu. Our work offers a strategy to modulate a specific histone demethylase by identifying and engineering a conserved phenylalanine residue, which acts as a gatekeeper in the KDM4 sub-family, to sensitize the enzyme towards a novel set of 2KG derivatives. The orthogonal pairs developed herein will serve as probes to study the role of degree-specific lysine demethylation in mammalian gene expression. Furthermore, this approach to overcome active site degeneracy is expected to have general application among all human 2KG-dependent dioxygenases.
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影响因子：
3.7
作者：
Lohse B;Helgstrand C;Kristensen JB;Leurs U;Cloos PA;Kristensen JL;Clausen RP
通讯作者：
Clausen RP
影响因子：
15
作者：
Deckert, Katelyn;Budiardjo, S. Jimmy;Karanicolas, John
通讯作者：
Karanicolas, John
影响因子：
2.9
作者：
Krishnan, Swathi;Collazo, Evys;Trievel, Raymond C.
通讯作者：
Trievel, Raymond C.
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.