Exploring the role of the CXXC motif in the regulation of some enzymes involved in redox signaling.

探索 CXXC 基序在调节参与氧化还原信号传导的一些酶中的作用。

• 批准号: RGPIN-2017-04925
• 负责人: Mutus, Bulent
• 金额: $ 2.91万
• 依托单位: University of Windsor
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748474
• 关键词: Exploring; role; CXXC; motif; regulation

The CXXC-motif refers to a protein sequence where two cysteine residues are separated by intervening residues, denoted by 'X' (X=any amino acid). This motif is found in many enzyme active-sites i.e. thioredoxin and protein disulfide isomease (PDI). In addition, the CXXC-motif is found peripheral to the active sites, yet performing crucial functional and regulatory roles. Studies have also shown that variation in the XX residues or those N- or C- terminal to the CXXC-motif can modulate enzyme activity. In the short term, we will determine the role of the CXXC-motif and flanking residues in the regulation of PDI, S-nitrosoglutathione reductase (GSNOR) and cystathionine-gamma-lyase (CSE), some key enzymes involved in thiol redox, nitric oxide (NO) and hydrogen sulfide (H2S) signaling. The work on PDI and GSNOR will indicate whether a lysine (K) residue at the C-terminal end of the CXXC-motif or other lysine residues within PDI and GSNOR are the sites for post translational modification by acetylation. We will assess the consequences of lysine-acetylation on the regulation, secretion and extra-cellular and intra-cellular localization of PDI and GSNOR. CSE contains 2 CXXC-motifs. We will test CSE's CXXC-motifs for: a) a previously undiscovered PDI-like thiol reductase activity role; and b) thiol-disulfide mediated regulatory role of its activity. In addition, we will use CSE-generated (homo)cysteine persulfides to determine whether H2S directly participates in sulfide signaling. Our long term goals are to investigate the cross-talk between enzymes that participate in thiol-mediated signaling.In terms of impact to the field, the research program will introduce acetylation as a newly discovered mode of regulation for PDI and GSNOR, key enzymes involved in thiol-mediated signaling and implicated in several pathologies. PDI-work will yield information on the regulation of its secretion, cell surface attachment and thiol reductase and chaperone activities. GSNOR will be characterized with respect to: a new allosteric site; the role of acetylation on its catalytic/allosteric behaviour; interaction with eNOS; and intracellular localization. The work on CSE will yield information on its redox regulation and a new PDI-like thiol reductase activity. The CSE-generated homocysteine persulfides will be used to answer the fundamental question of whether sulfide participates in protein signaling or is a bystander molecule. Furthermore, information gained with respect to the control of the activities of these enzymes will be invaluable for the development of pharmaceutical agents to control these enzymes and the signaling pathways they modulate.

CXXC基序是指其中两个半胱氨酸残基被插入残基分开的蛋白质序列，插入残基由“X”表示（X=任何氨基酸）。 该基序存在于许多酶活性位点，即硫氧还蛋白和蛋白质二硫键异构酶（PDI）中。此外，CXXC基序被发现外围的活性位点，但执行关键的功能和调节作用。研究还表明，XX残基或CXXC基序的N-或C-末端的那些残基的变化可以调节酶活性。 在短期内，我们将确定CXXC-基序和侧翼残基在PDI，S-亚硝基谷胱甘肽还原酶（GSNOR）和胱硫醚-γ-裂解酶（CSE）的调节中的作用，这些酶参与硫醇氧化还原，一氧化氮（NO）和硫化氢（H2S）信号传导。对PDI和GSNOR的研究将表明CXXC基序C末端的赖氨酸（K）残基或PDI和GSNOR内的其他赖氨酸残基是否是乙酰化翻译后修饰的位点。 我们将评估赖氨酸乙酰化对PDI和GSNOR的调节、分泌以及细胞外和细胞内定位的后果。 CSE含有2个CXXC基序。 我们将测试CSE的CXXC基序：a）以前未发现的PDI样巯基还原酶活性作用;和B）巯基-二硫化物介导的其活性调节作用。 此外，我们将使用CSE产生的（同型）半胱氨酸过硫化物来确定H2S是否直接参与硫化物信号传导。我们的长期目标是研究参与硫醇介导的信号传导的酶之间的串扰。就对该领域的影响而言，该研究计划将引入乙酰化作为一种新发现的PDI和GSNOR的调节模式，PDI和GSNOR是参与硫醇介导的信号传导的关键酶，并与多种病理学有关。 PDI工作将产生关于其分泌、细胞表面附着和巯基还原酶和伴侣活性的调节的信息。 GSNOR的特征在于：一个新的变构位点;乙酰化对其催化/变构行为的作用;与eNOS的相互作用;以及细胞内定位。 CSE的工作将产生关于其氧化还原调节和新的PDI样巯基还原酶活性的信息。CSE产生的同型半胱氨酸过硫化物将用于回答硫化物是否参与蛋白质信号传导或作为旁观者分子的基本问题。 此外，关于控制这些酶的活性所获得的信息对于开发控制这些酶及其调节的信号传导途径的药剂将是非常宝贵的。