Dynamics and regulation of cysteine S-sulfhydration in cellular functions

硫化氢（H2S）目前被认为是一种内源性气体递质，参与多种细胞功能和生理过程。半胱硫氨酸γ -裂解酶（CSE）是哺乳动物细胞中产生硫化氢的主要酶。有人提出H2S的信号机制之一是通过活性半胱氨酸残基在靶蛋白上的s -巯基化产生氢过硫化物片段（-SSH），具有赋予功能改变的潜力。s -巯基水化调节多种细胞途径，包括代谢途径、蛋白质降解过程、DNA损伤修复、蛋白质相互作用和定位以及应激反应途径。利用蛋白质组学技术，通过将特定富集策略（生物素转换试验和/或标签转换试验）与高通量质谱（MS）分析相结合，实现了s -巯基化对半胱氨酸的氧化还原修饰。我们目前对s -巯基水合作用作为一种蛋白质信号传导方式的理解仍处于起步阶段。人们对直接从半胱氨酸硫醇中添加或去除SH基团的酶促机制的性质甚至必要性知之甚少。我的长期目标是阐明H2S的生物学重要性，以提高我们对多种细胞功能调控的理解，并更好地培养该领域的下一代研究人员。通过这项研究计划，短期目标是确定半胱氨酸s -巯基化在h2s调节的肌动蛋白聚合和内皮细胞迁移/间隙连接方面的生物学和化学特性。缺乏CSE的小鼠出现内皮功能障碍，提示H2S在维持内皮完整性方面的重要性。我的初步数据显示，缺乏H2S会减弱肌动蛋白聚合和内皮细胞迁移。此外，硫氧还蛋白1 （Trx1）的抑制增强，而巯基氧化酶表达的下调抑制肌动蛋白s -巯基化。本项目的目的是：1)研究CSE/H2S系统在肌动蛋白聚合和内皮细胞迁移/间隙连接中的作用，探索H2S s -巯基化的肌动蛋白-cofilin-profilin复合物和潜在的半胱氨酸位点；2)研究Trx1和巯基氧化酶对肌动蛋白s -巯基化/脱硫的调控作用。*******大量蛋白质基本处于s -巯基化的事实表明s -巯基化是一个重要的生理和病理信号。该计划可能会导致对H2S信号在人类中的生物学相关性的理解取得突破，这种理解将有助于揭示蛋白质的一种新的生理翻译后修饰，这可能会影响多种生物途径。*********

Hydrogen sulfide (H2S) is now considered as an endogenous gasotransmitter in a variety of cellular functions and physiological processes. Cystathionine gamma-lyase (CSE) is a major H2S-producing enzyme in mammalian cells. It is proposed that one of the signaling mechanisms of H2S is through the S-sulfhydration of reactive cysteine residues on target proteins by yielding a hydropersulfide moiety (-SSH), with the potential to confer a functional change. S-sulfhydration modulates diverse cellular pathways, including metabolic pathways, protein degradative processes, DNA damage repair, protein interaction and localization, and stress response pathways. This particular redox modification of cysteine by S-sulfhydration has been achieved using proteomics techniques by coupling a specific enrichment strategy (biotin-switch assay and/or tag-switch assay) with high-throughput mass spectrometry (MS) analysis. Our current understanding of the role of S-sulfhydration as a protein signaling modality is still in its infancy. Little is known about the nature of or even necessity for enzymatic mechanisms that may directly add or remove SH groups from cysteine thiols. *** My long-term goal is to elucidate the biological importance of H2S in order to improve our understanding of the regulation of a multitude of cellular functions, and to better train the next generation of researchers in this field. The short-term goal, through this research program, is to determine the biology and chemistry characteristic of cysteine S-sulfhydration with respect to H2S-regulated actin polymerization and cell migration/gap junction in endothelial cells. Mice deficient for CSE showed endothelial dysfunction, suggesting the importance of H2S in maintenance of endothelial integrity. My preliminary data showed that lack of H2S attenuates actin polymerization and endothelial cell migration. In addition, inhibition of thioredoxin 1 (Trx1) strengthened but knockdown of sulfhydryl oxidase expression inhibited actin S-sulfhydration. The objectives of this program are to 1) investigate the effects of the CSE/H2S system in actin polymerization and endothelial cell migration/gap junction, and explore H2S S-sulfhydration of actin-cofilin-profilin complex and the potential cysteine resides; 2), examine the regulation of Trx1 and sulfhydryl oxidase on actin S-sulfhydration/desulfhydation.******* The fact that a very large number of proteins are basally S-sulfhydrated suggests that S-sulfhydration is an important physiologic and pathologic signal. This program may lead to a breakthrough in the understanding of the biological relevance of H2S signaling in humans, and such an understanding will help reveal a novel physiologic posttranslational modification for proteins, which potentially influences a multitude of biological pathways.*********