Temporally controlled targeting of 4-hydroxynonenal to specific proteins in living cells.
Temporally controlled targeting of 4-hydroxynonenal to specific proteins in living cells.
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DOI：
10.1021/ja405400k
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发表时间：
2013-10-02
影响因子：
15
通讯作者：
Aye Y
中科院分区：
文献类型：
--
作者：
Fang X;Fu Y;Long MJ;Haegele JA;Ge EJ;Parvez S;Aye Y
对复杂生物过程的深入化学理解取决于系统地干扰单个系统的能力。然而，目前研究生物相关活性小分子影响的方法涉及用过量的小分子浸泡整个细胞或分离的细胞器，从而导致脱靶效应。因此，缺乏生化特异性一直困扰着我们对生物亲电试剂如何介导信号转导或调节赋予细胞亲电应激防御机制的反应的理解。在这里，我们介绍了一种靶向性亲电试剂传递平台，该平台将最终为探究活性亲电试剂对细胞中特定靶蛋白的影响铺平道路。通过光诱导靶向递送4-羟基壬烯醛（HNE）到蛋白质Keap1和PTEN，证明了这种新方法。HaloTag®的HNE前体共价偶联融合到靶蛋白上，可以在光激活后直接递送HNE。该策略提供了在哺乳动物细胞中选择性递送反应性亲电试剂到个体亲电反应蛋白的概念证明。它开辟了一条新的途径，使更精确地确定hne诱导的化学修饰对细胞中特定靶蛋白的病理生理后果。
In-depth chemical understanding of complex biological processes hinges upon the ability to systematically perturb individual systems. However, current approaches to study impacts of biologically relevant reactive small molecules involve bathing of the entire cell or isolated organelle with excess amounts, leading to off-target effects. The resultant lack of biochemical specificity has plagued our understanding of how biological electrophiles mediate signal transduction or regulate responses that confer defense mechanisms to cellular electrophilic stress. Here we introduce a target-specific electrophile delivery platform that will ultimately pave the way to interrogate effects of reactive electrophiles on specific target proteins in cells. The new methodology is demonstrated by photoinducible targeted delivery of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent conjugation of the HNE-precursor to HaloTag® fused to the target proteins enables directed HNE delivery upon photoactivation. The strategy provides proof of concept of selective delivery of reactive electrophiles to individual electrophile-responsive proteins in mammalian cells. It opens a new avenue enabling more precise determination of the pathophysiological consequences of HNE-induced chemical modifications on specific target proteins in cells.