Chemistry, Structure and Biology of Thiol Transferases

硫醇转移酶的化学、结构和生物学

• 批准号: 8913981
• 负责人: CHARLES R SANDERS
• 金额: $ 33.76万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8913981
• 关键词: Address; Amides; Anabolism; Antibiotics; Arachidonic Acids; Bacillus anthracis; Bacillus cereus; Binding; Biodiversity; Biology; Catabolic Process; Cells; Chemicals; Chemistry; Dependence; Deuterium; Endoplasmic Reticulum; Environment; Enzymes; Fever; Fosfomycin; Genes; Glutathione; Glutathione Metabolism Pathway; Glutathione S-Transferase; Goals; Health; Human; Hydrogen; Inflammation; Institutes; Integral Membrane Protein; Ions; Leukotrienes; Lipoxygenase; Mammals; Mass Spectrum Analysis; Membrane; Membrane Proteins; Microbe; Oxidation-Reduction; Pain; Pain management; Pathway interactions; Positioning Attribute; Prostaglandins; Proteins; Regulation; Research; Resistance; Role; Source; Staphylococcus aureus; Structure; Sulfhydryl Compounds; Transferase; Vertebral column; allergic response; chemical kinetics; design; divalent metal; inhibitor/antagonist; insight; member; microbial; microorganism

DESCRIPTION (provided by applicant): Thiols, thiol-transferases and related proteins are essential to virtually all aspects of biology. In mammals and microbes thiols maintain redox status, protect cells against toxic electrophiles and participate in crucial biosynthetic, metaboli and catabolic processes. The general objectives of this renewal application address specific problems in the understanding of the involvement of thiol transferases and related proteins in mammalian and microbial biology. The specific aims are (i) to advance our understanding of the mechanisms and structures of three MAPEG proteins (Membrane Associated Proteins in Eicosinoid and Glutathione metabolism) and (ii) to provide previously inaccessible scientific insight into the chemistry and biology of bacillithiol (BSH); a newly discovered thiol found in Gram-positive microorganisms. Members of the MAPEG superfamily are involved in several aspects of the regulation of pain, fever, inflammation and allergic response in humans. The first specific aim focuses on crucial issues regarding three different MAPEG proteins, microsomal prostaglandin E synthase 1 (MPEGS1), 5-lipoxigenase activating protein (FLAP) and microsomal glutathione transferase 1 (MGST1). All of these proteins are integral membrane proteins. Specific Aim 1 relies heavily on hydrogen/deuterium exchange mass spectrometry (H/DEX MS) to probe the structural aspects of these proteins as they interact with substrates, inhibitors and protein partners. Specific Aim 1a will ascertain if it is possible to perform H/DEX MS on an integral membrane protein MGST1 in its native membrane environment, the endoplasmic reticulum. Specific Aim 1b will address the potential inhibition of MPGES1 by nitrosylation of C59 and the chemical mechanism of MPGES1, about which virtually nothing is known. Specific Aim 1c is will elucidate the structural details of the interaction of FLAP with arachidonic acid and its interaction with 5-lipoxigenase (5-LOX) in the initiation of the lipoxygenase pathway for the synthesis of leukotrienes. Specific aim 2 is directed at understanding the role of bacillithiol (BSH) in the resistance of Gram-positive pathogenic microorganisms to the antibiotic fosfomycin conferred by the enzyme FosB. This aim is leveraged by the fact that we have, in the last project period with help of the Vanderbilt Institut of Chemical Biology Synthesis Core, synthesized 1.3 grams bacillithiol and have obtained the genes, expressed and purified the FosB enzymes from several sources including Bacillus cereus, Bacillus anthracis and Staphylococcus aureus which puts us in an excellent position to accomplish this aim. Specific Aim 2a is to determine the crystal structure of one or more FosB proteins with fosfomycin, BSH or product bound. Specific Aim 2b will elucidate the chemistry, kinetics, and divalent metal ion-dependence of the FosB enzymes with both BSH and the alternative substrate L-Cys.

描述（由申请人提供）：硫醇、硫醇转移酶和相关蛋白质对于生物学的几乎所有方面都是必不可少的。在哺乳动物和微生物中，硫醇维持氧化还原状态，保护细胞免受有毒亲电子试剂的侵害，并参与重要的生物合成、代谢和分解代谢过程。该更新申请的总体目标是解决理解硫醇转移酶和相关蛋白质在哺乳动物和微生物生物学中的参与的具体问题。具体目标是 (i) 增进我们对三种 MAPEG 蛋白（类二十烷酸和谷胱甘肽代谢中的膜相关蛋白）的机制和结构的理解，以及 (ii) 为杆菌硫醇 (BSH) 的化学和生物学提供以前无法获得的科学见解;在革兰氏阳性微生物中发现的一种新发现的硫醇。 MAPEG 超家族的成员参与人类疼痛、发烧、炎症和过敏反应调节的多个方面。第一个具体目标侧重于有关三种不同 MAPEG 蛋白的关键问题：微粒体前列腺素 E 合酶 1 (MPEGS1)、5-脂氧合酶激活蛋白 (FLAP) 和微粒体谷胱甘肽转移酶 1 (MGST1)。所有这些蛋白质都是完整的膜蛋白。具体目标 1 在很大程度上依赖氢/氘交换质谱 (H/DEX MS) 来探测这些蛋白质与底物、抑制剂和蛋白质伴侣相互作用时的结构方面。具体目标 1a 将确定是否可以在其天然膜环境（内质网）中对整合膜蛋白 MGST1 进行 H/DEX MS。具体目标 1b 将解决 C59 亚硝基化对 MPGES1 的潜在抑制作用以及 MPGES1 的化学机制，而对此几乎一无所知。具体目标 1c 将阐明 FLAP 与花生四烯酸相互作用的结构细节，以及在白三烯合成的脂氧合酶途径启动过程中与 5-脂氧合酶 (5-LOX) 相互作用的结构细节。具体目标 2 旨在了解杆菌硫醇 (BSH) 在革兰氏阳性病原微生物对由 FosB 酶赋予的抗生素磷霉素产生的耐药性中的作用。这一目标是通过以下事实实现的：在上一个项目期间，我们在范德比尔特化学生物学合成核心研究所的帮助下，合成了 1.3 克杆菌硫醇，并从包括蜡状芽孢杆菌在内的多个来源获得了基因、表达和纯化了 FosB 酶、炭疽芽孢杆菌和金黄色葡萄球菌，这使我们处于实现这一目标的绝佳位置。具体目标 2a 是确定一种或多种与磷霉素、BSH 或产物结合的 FosB 蛋白的晶体结构。具体目标 2b 将阐明 FosB 酶与 BSH 和替代底物 L-Cys 的化学、动力学和二价金属离子依赖性。