The Role of Zinc Fingers in H2S Signaling

该提案的重点是了解锌指（ZF）蛋白在H2S信号转导中的作用。ZF高度 在转录和翻译调控中发挥关键作用的丰富蛋白质。ZF富含半胱氨酸， 使用锌作为辅助因子。长期以来，锌在ZF中的作用一直被认为仅仅是结构性的;然而，证据表明， 因为一种新的范例正在出现，其中锌离子起着更反应性的作用。这些证据包括 发现硫化氢（H2S）靶向ZF蛋白。H2S是一种内源性气体递质， 近年来作为通过半胱氨酸过硫化的生物信号分子受到关注。这一过程现在 被认为是一种在细胞信号传导中起关键作用的蛋白质翻译后修饰。已经 显示H2S信号传导与炎症、神经调节、转录和代谢相关， 异常的H2S活性与心血管、神经、炎症和代谢疾病有关。尽管 随着人们越来越认识到H2S在生物学中的重要性，我们对H2S生物学作用的理解是 有限公司H2S的大分子靶点尚未明确，H2S的作用机制也不清楚 在大多数情况下都是确定的。我们已经获得了令人兴奋的蛋白质组学数据，确定了许多ZFs， H2S的细胞目标。我们还报道了一种特殊的ZF，tristetraprolin（TTP），与锌中的H2S反应， 和O2依赖的方式产生过硫化半胱氨酸中间体和调节TTP结构。这些 结构变化通过消除TTP的RNA结合能力来传递来自H2S的“信号”。本提案中 我们的目标是（i）使用过硫化物选择性蛋白质组学策略鉴定细胞中H2S靶向的ZF（ii） 通过应用一套生物化学和生物物理方法， 方法（包括冷冻-ESI-质谱、荧光猝灭、EPR、XAS和NMR光谱， 和荧光各向异性结合测定）与ZF蛋白、ZF肽和合成的模型复合物的结合， (iii)通过使用LC-ICP-MS和蛋白质组学相结合，确定锌形态如何影响ZF的H2S靶向 和抗体/微阵列策略。这种“从分子到蛋白质组学”的方法将使我们能够在很大程度上了解 ZF蛋白在H2S信号传导中的作用尚未探索，并有可能改变我们对H2S的理解 发信号。

This proposal focuses on understanding the role of zinc finger (ZF) proteins in H2S signaling. ZFs are highly abundant proteins that play critical roles in transcriptional and translational regulation. ZFs are cysteine rich and use zinc as a cofactor. The role of zinc in ZFs has long been considered to be solely structural; however, evidence for a new paradigm is emerging in which the zinc ion plays a more reactive role. This evidence includes the finding that hydrogen sulfide (H2S) targets ZF proteins. H2S is an endogenous gasotransmitter that has gained attention in recent years as a biological signaling molecule via cysteine persulfidation. This process is now proposed as a type of protein post-translational modification that plays a critical role in cell signaling. It has been shown that H2S signaling is associated with inflammation, neuromodulation, transcription and metabolism, with aberrant H2S activity linked to cardiovascular, neurological, inflammatory and metabolic diseases. Despite the growing appreciation for the importance of H2S in biology, our understanding of the biological roles of H2S is limited. The macromolecular targets of H2S are not clearly defined, and the mechanism of H2S action has not been determined in most cases. We have obtained exciting proteomics data that identifies numerous ZFs as cellular targets for H2S. We have also reported that a specific ZF, tristetraprolin (TTP), reacts with H2S in a zinc and O2 dependent manner to give persulfidated cysteine intermediates and modulate TTP structure. These structural changes transmit the `signal' from H2S by abrogating the RNA binding ability of TTP. In this proposal we aim to (i) identify the ZFs that are targeted by H2S in cells using a persulfide selective proteomics strategy (ii) determine the reactivity and mechanism of H2S with ZFs by applying a suite of biochemical and biophysical methods (including cryo-ESI-mass spectrometry, fluorescence quenching, EPR, XAS and NMR spectroscopies, and fluorescence anisotropy binding assays) to ZF proteins, ZF peptides and synthetic model complexes and (iii) determine how zinc speciation impacts H2S targeting of ZFs by using a combined LC-ICP-MS, proteomics and antibody/microarray strategy. This `molecules to proteomics' approach will allow us to understand the largely unexplored role of ZF proteins in H2S signaling, and has the potential to transform our understanding of H2S signaling.