The Role of Zinc Fingers in H2S Signaling

锌指在 H2S 信号传导中的作用

• 批准号: 10621493
• 负责人: SARAH L MICHEL
• 金额: $ 5.79万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10621493
• 关键词: Affect; Amino Acids; Anisotropy; Antibodies; Attention; Binding; Biochemical; Biological; Biological Assay; Biological Process; Biology; Biomimetics; Biophysics; Cardiovascular system; Cell Culture Techniques; Cells; Chemistry; Coupled; Cysteine; Data; Disease; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Electrons; Fluorescence; Fluorescence Anisotropy; Fractionation; Genetic Transcription; Geometry; Health; Histidine; Human; Hydrogen Peroxide; Hydrogen Sulfide; In Vitro; Inductively Coupled Plasma Mass Spectrometry; Inflammation; Inflammatory; Ions; Knowledge; Label; Life; Ligands; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Metabolic Diseases; Metabolism; Methods; Micronutrients; Modeling; Modification; NMR Spectroscopy; Neurologic; Oxidation-Reduction; Pathway interactions; Peptides; Physiological; Planet Earth; Play; Post-Translational Protein Processing; Process; Proteins; Proteome; Proteomics; RNA Binding; Reporting; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Spectrometry, Mass, Electrospray Ionization; Stable Isotope Labeling; Structure; TIS11 protein; Transcriptional Regulation; Translational Regulation; Work; Zinc; Zinc Fingers; analog; base; biophysical techniques; cellular targeting; cofactor; dimedone; insight; interest; mutant; neuroregulation; novel; persulfides; protein function

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.

该提案的重点是了解锌指 (ZF) 蛋白在 H2S 信号传导中的作用。 ZF 高度 丰富的蛋白质在转录和翻译调节中发挥关键作用。 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 因为一种新的范例正在出现，其中锌离子扮演着更具反应性的角色。该证据包括 发现硫化氢 (H2S) 以 ZF 蛋白为目标。 H2S 是一种内源性气体递质，已获得 近年来作为半胱氨酸过硫化的生物信号分子受到关注。这个过程现在是 被认为是一种蛋白质翻译后修饰，在细胞信号传导中发挥着关键作用。它一直 研究表明，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 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 有限的。 H2S的大分子靶点尚未明确，H2S的作用机制尚未明确。 大多数情况下已经确定。我们获得了令人兴奋的蛋白质组学数据，将许多 ZF 识别为 H2S 的细胞目标。我们还报道了一种特定的 ZF，三四脯氨酸 (TTP)，与锌中的 H2S 发生反应。 和O2依赖性方式产生过硫化半胱氨酸中间体并调节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”。在这个提案中 我们的目标是 (i) 使用过硫化物选择性蛋白质组学策略识别细胞中 H2S 靶向的 ZF (ii) 通过应用一系列生化和生物物理方法确定 H2S 与 ZF 的反应性和机制 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）到 ZF 蛋白、ZF 肽和合成模型复合物以及 (iii) 通过使用组合的 LC-ICP-MS、蛋白质组学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了解很大程度上 ZF 蛋白在 H2S 信号传导中的作用尚未被探索，并且有可能改变我们对 H2S 的理解 发信号。