A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling.
A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling.
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DOI：
10.1021/ja5132648
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发表时间：
2015-05-20
影响因子：
15
通讯作者：
Aye Y
中科院分区：
文献类型：
--
作者：
Lin HY;Haegele JA;Disare MT;Lin Q;Aye Y
尽管已知小分子亲电试剂倾向于与许多半胱氨酸活性蛋白反应，但个体信号诱导剂的生物作用已出现化学型特异性。为了精确定位和量化修改整个蛋白质组中的一个靶标的影响，我们开发了一个靶标-蛋白质个性化的“亲电工具箱”，可以通过特定的反应信号在精确的时间选择性地修改特定的细胞内靶标。这种通用的方法- t - rex（靶向性反应性亲电试剂和氧化剂）-是通过以下方式建立的：(1)构建一个平台，可以将一系列电子和立体不同的生物活性脂质衍生的信号亲电试剂传递到细胞中的特定蛋白质；(2)探讨了靶向递送概念的动力学，揭示了细胞中的靶向效率在很大程度上取决于初始烷基化率；(3)评估蛋白靶标和小分子信号特异性修饰对下游信号强度的影响。这些数据表明，T-REX允许定量询问Nrf2转录因子依赖的抗氧化反应元件（ARE）信号通过Nrf2 - ARE轴的几个氧化还原敏感调节因子的Keap1蛋白上的选择性亲电修饰激活的程度。结果表明，Keap1是一种混杂的亲电响应传感器，能够以相似的效率响应离散的亲电信号，促进Nrf2-ARE诱导的强度相当。T-REX还能够在全细胞亲电剂泛洪在引起细胞毒性之前未能刺激ARE诱导的情况下引发细胞活化。该平台提供了一个以前无法获得的机会来阐明小分子信号和蛋白质靶标特异性亲电修饰在其他不受影响的细胞背景下的功能后果。
Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized “electrophile toolbox” with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology—T-REX (targetable reactive electrophiles & oxidants)—is established by: (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein—one of several redox-sensitive regulators of the Nrf2–ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2–ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.