Lin Hong-Yu, Haegele Joseph A, Disare Michael T, Lin Qishan, Aye Yimon
†Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
§Proteomics/Mass Spectrometry Facility, Center for Functional Genomics, University of Albany, Rensselaer, New York 12144, United States.
J Am Chem Soc. 2015 May 20;137(19):6232-44. doi: 10.1021/ja5132648. Epub 2015 May 5.
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 and 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.
尽管已知小分子亲电试剂易于与众多半胱氨酸活性蛋白发生反应，但单个信号诱导剂的生物学作用已显示出具有化学型特异性。为了精确确定和量化在整个蛋白质组中修饰一个靶点的影响，我们开发了一种针对目标蛋白的个性化“亲电试剂工具箱”，利用它可以在特定时间通过特定的反应性信号选择性地修饰特定的细胞内靶点。这种通用方法，即T-REX（可靶向的反应性亲电试剂和氧化剂），是通过以下方式建立的：（1）构建一个能够将一系列电子和空间结构不同的生物活性脂质衍生的信号亲电试剂递送至细胞内特定蛋白质的平台；（2）探究靶向递送概念的动力学，结果表明细胞中的靶向效率在很大程度上由烷基化的初始结合速率驱动；（3）评估蛋白质靶点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能够对Nrf2转录因子依赖性抗氧化反应元件（ARE）信号传导在何种程度上被Keap1蛋白上的选择性亲电修饰激活进行定量研究，Keap1蛋白是Nrf2-ARE轴的几种氧化还原敏感调节因子之一。结果证明Keap1是一种混杂的亲电试剂响应传感器，能够以相似的效率对离散的亲电信号作出反应，并促进Nrf2-ARE诱导的可比强度。在全细胞亲电试剂泛滥在导致细胞毒性之前未能刺激ARE诱导的情况下，T-REX也能够引发细胞激活。该平台提供了一个以前无法获得的机会，以阐明在其他未受影响的细胞背景下小分子信号和蛋白质靶点特异性亲电修饰的功能后果。
