The Role of Zinc Fingers in H2S Signaling

锌指在 H2S 信号传导中的作用

• 批准号: 10096833
• 负责人: SARAH L MICHEL
• 金额: $ 36.94万
• 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/10096833
• 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.

该建议的重点是了解锌指（ZF）蛋白在H2S信号传导中的作用。 ZF是高度的 在转录和翻译调节中起关键作用的丰富蛋白质。 ZFS富含半胱氨酸， 使用锌作为辅因子。长期以来，锌在ZFS中的作用被认为仅是结构性的。但是，证据 对于新的范式，锌离子起着更具反应性的作用。这些证据包括 发现硫化氢（H2S）靶向ZF蛋白。 H2S是一种已获得的内源性质递质 近年来，人们通过半胱氨酸过硫作为生物信号分子的注意。这个过程现在 提出是一种蛋白质后翻译后修饰，在细胞信号传导中起关键作用。它一直 表明H2S信号传导与炎症，神经调节，转录和代谢有关 与心血管，神经系统，炎症和代谢疾病有关的异常H2S活性。尽管有 人们对H2在生物学中的重要性的越来越多，我们对H2S的生物学作用的理解是 有限的。 H2S的大分子靶标未明确定义，H2S作用的机制尚未 在大多数情况下已确定。我们获得了令人兴奋的蛋白质组学数据，这些数据将许多ZF识别为 H2S的细胞靶标。我们还报道说，特定的ZF三翼烷（TTP）在锌中与H2S反应 和O2依赖性方式，以给出过硫化的半胱氨酸中间体并调节TTP结构。这些 结构变化通过废除TTP的RNA结合能力从H2S传递“信号”。在此提案中 我们的目的是（i）使用Persulfide选择性蛋白质组学策略（II）确定H2S在细胞中针对H2S的ZFS。 通过应用一套生化和生物物理学来确定H2s对ZF的反应性和机制 方法（包括冷冻 - 质量光谱法，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肽和合成模型复合物以及 （iii）通过使用组合的LC-ICP-MS，蛋白质组学来确定锌形成如何影响ZF的H2S靶向H2S 和抗体/微阵列策略。这种“蛋白质组学的分子”方法将使我们在很大程度上理解 ZF蛋白在H2S信号传导中未开发的作用，并且有可能改变我们对H2S的理解 信号。