Department of Chemistry , Wake Forest University , Winston-Salem , North Carolina 27101 , United States.
Department of Internal Medicine , Wake Forest School of Medicine , Winston-Salem , North Carolina 27157 , United States.
Chem Res Toxicol. 2019 Mar 18;32(3):526-534. doi: 10.1021/acs.chemrestox.8b00385. Epub 2019 Mar 4.
Redox-mediated protein modifications control numerous processes in both normal and disease metabolism. Protein sulfenic acids, formed from the oxidation of protein cysteine residues, play a critical role in thiol-based redox signaling. The reactivity of protein sulfenic acids requires their identification through chemical trapping, and this paper describes the use of the triphenylphosphonium (TPP) ion to direct known sulfenic acid traps to the mitochondria, a verified source of cellular reactive oxygen species. Coupling of the TPP group with the 2,4-(dioxocyclohexyl)propoxy (DCP) unit and the bicyclo[6.1.0]nonyne (BCN) group produces two new probes, DCP-TPP and BCN-TPP. DCP-TPP and BCN-TPP react with C165A AhpC-SOH, a model protein sulfenic acid, to form the expected adducts with second-order rate constants of k = 1.1 M s and k = 5.99 M s, respectively, as determined by electrospray ionization time-of-flight mass spectrometry. The TPP group does not alter the rate of DCP-TPP reaction with protein sulfenic acid compared to dimedone but slows the rate of BCN-TPP reaction compared to a non-TPP-containing BCN-OH control by 4.6-fold. The hydrophobic TPP group may interact with the protein, preventing an optimal reaction orientation for BCN-TPP. Unlike BCN-OH, BCN-TPP does not react with the protein persulfide, C165A AhpC-SSH. Extracellular flux measurements using A549 cells show that DCP-TPP and BCN-TPP influence mitochondrial energetics, with BCN-TPP producing a drastic decrease in basal respiration, perhaps due to its faster reaction kinetics with sulfenylated proteins. Further control experiments with BCN-OH, TPP-COOH, and dimedone provide strong evidence for mitochondrial localization and accumulation of DCP-TPP and BCN-TPP. These results reveal the compatibility of the TPP group with reactive sulfenic acid probes as a mitochondrial director and support the use of the TPP group in the design of sulfenic acid traps.
氧化还原介导的蛋白质修饰控制着正常和疾病代谢中的许多过程。蛋白质亚磺酸，由蛋白质半胱氨酸残基的氧化形成，在基于硫醇的氧化还原信号中起着关键作用。蛋白质亚磺酸的反应性需要通过化学捕获来鉴定，本文描述了使用三苯基膦（TPP）离子将已知的亚磺酸捕获剂引导到线粒体，线粒体是细胞活性氧的已知来源。TPP 基团与 2,4-（二氧环己基）氧基（DCP）单元和双环[6.1.0]壬炔（BCN）基团的偶联产生了两种新的探针，DCP-TPP 和 BCN-TPP。DCP-TPP 和 BCN-TPP 与 C165A AhpC-SOH（一种模型蛋白质亚磺酸）反应，形成预期的加合物，其二级速率常数 k 分别为 1.1 M s 和 5.99 M s，这是通过电喷雾电离飞行时间质谱法确定的。与二酮相比，TPP 基团不会改变 DCP-TPP 与蛋白质亚磺酸的反应速率，但与不含 TPP 的 BCN-OH 对照相比，TPP 基团会使 BCN-TPP 的反应速率减慢 4.6 倍。疏水性 TPP 基团可能与蛋白质相互作用，阻止 BCN-TPP 进行最佳的反应取向。与 BCN-OH 不同，BCN-TPP 不与蛋白质过硫化物 C165A AhpC-SSH 反应。使用 A549 细胞进行的细胞外通量测量表明，DCP-TPP 和 BCN-TPP 影响线粒体能量学，BCN-TPP 使基础呼吸急剧下降，这可能是由于其与亚磺酰化蛋白质的更快反应动力学。与 BCN-OH、TPP-COOH 和二酮的进一步对照实验提供了 TPP 基团作为线粒体导向剂与 DCP-TPP 和 BCN-TPP 线粒体定位和积累的强证据。这些结果表明，TPP 基团与反应性亚磺酸探针的兼容性作为线粒体导向剂，并支持在亚磺酸捕获剂的设计中使用 TPP 基团。
