The Role of Zinc Fingers in H2S Signaling

本研究旨在了解锌指(ZF)蛋白在硫化氢信号转导中的作用。ZF是高度 丰富的蛋白质，在转录和翻译调控中发挥关键作用。ZF富含半胱氨酸， 使用锌作为辅因子。长期以来，锌在锌铁中的作用一直被认为是纯结构性的；然而，有证据表明 因为一种新的范式正在出现，在这种范式中，锌离子扮演着更具反应性的角色。这一证据包括 发现硫化氢(H2S)以ZF蛋白为目标。硫化氢是一种内源性气体递质，它已经获得了 近年来作为一种生物信号分子半胱氨酸过硫化而备受关注。这一过程现在正在进行 被认为是一种蛋白质翻译后修饰，在细胞信号转导中发挥关键作用。一直以来 研究表明，硫化氢信号与炎症、神经调节、转录和代谢有关，与 硫化氢活性异常与心血管、神经、炎症和代谢性疾病有关。尽管 随着人们对硫化氢在生物学中的重要性的认识日益加深，我们对硫化氢的生物学作用的理解是 有限的。硫化氢的大分子靶标还没有明确定义，硫化氢的作用机理也还没有明确。 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，这些数据确定了许多ZF是 硫化氢的细胞靶标。我们还报道了一种特定的ZF，Tristetraprolin(TTP)，与锌中的硫化氢反应 以氧依赖的方式产生过硫化半胱氨酸中间体，调节TTP结构。这些 结构变化通过取消TTP的RNA结合能力来传递来自硫化氢的“信号”。在本建议书中 我们的目标是(I)使用过硫化物选择性蛋白质组学策略(Ii)确定细胞中被硫化氢靶向的ZF。 应用一套生化和生物物理方法确定硫化氢与氟化锌的反应活性和反应机理 方法(包括冷冻-电喷雾质谱、荧光猝灭、EPR、XAS和核磁共振光谱， 和荧光各向异性结合分析)与ZF蛋白、ZF多肽和合成的模型络合物以及 (Iii)利用LC-ICP-MS、蛋白质组学相结合的方法，确定锌形态如何影响硫化氢对零氟化锌的靶向作用 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理解 ZF蛋白在硫化氢信号转导中的作用未知，有可能改变我们对硫化氢的理解 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.