Microbial Diversity in Mechanisms of Disulfide Bond Formation and Reduction

二硫键形成和还原机制的微生物多样性

• 批准号: 8238337
• 负责人: JONATHAN BECKWITH
• 金额: $ 70.47万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-06-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8238337
• 关键词: Antibiotics; Anticoagulants; Archaea; Bacteria; Bacterial Genome; Bioinformatics; Biological Assay; Biology; Blood coagulation; Candidate Disease Gene; Chemicals; Coagulants; Comparative Study; Cysteine; Cytoplasm; Development; Drug Delivery Systems; Electron Transport; Electrons; Environment; Enzymes; Escherichia coli; Evolution; Genes; Genetic; Genetic Techniques; Genome; Glutathione; Homologous Gene; Human; Immunoglobulins; Laboratories; Membrane Proteins; Mutation; Mycobacterium smegmatis; Mycobacterium tuberculosis; NADH; NADP; Natural regeneration; Nature; Organism; Oxidation-Reduction; Pathway interactions; Process; Prokaryotic Cells; Property; Protein Disulfide Isomerase; Proteins; Research; Resistance; Ribonucleotide Reductase; Role; Site; Source; Sulfhydryl Compounds; Sulfolobus solfataricus; Sulfur; Surveys; System; Testing; Thioctic Acid; Thioredoxin; Tuberculosis; Virulence Factors; Warfarin; base; cell envelope; comparative; disulfide bond; experimental analysis; follow-up; genome sequencing; glutaredoxin; high throughput screening; inhibitor/antagonist; microbial; mutant; mycobacterial; novel; peptide hormone; periplasm; public health relevance; small molecule; small molecule libraries; vitamin K epoxide reductase

DESCRIPTION (provided by applicant): We will establish the nature of two novel protein disulfide bond-forming pathways found in certain bacteria and archaea. E. coli and many other bacteria use two enzymes to introduce disulfide bonds into proteins. DsbA directly joins the cysteines of proteins into disulfide bonds while DsbB reoxidizes and thus regenerates active DsbA. Many other bacteria, including Mycobacterium tuberculosis (Mtb), appear to use a homologue of human vitamin K epoxide reductase, VKOR, for disulfide bond formation instead of DsbB. Mtb VKOR can substitute for DsbB in E. coli. We will characterize the mycobacterial VKOR-based disulfide-bond forming system by defining which gene products comprise this oxidative pathway, expressing candidate genes in both E. coli and Mycobacterium smegmatis (which is more tractable than Mtb). In certain archaea, we have obtained evidence for another unusual pathway for disulfide bond formation in the cytoplasm. These archaea contain two VKORs, one cytoplasmically-oriented and apparently involved in cytoplasmic disulfide bond formation. We will verify this pathway and identify the cytoplasmic substrate (a cytoplasmic "DsbA"?) that this VKOR oxidizes. We will test the proposed role of this VKOR by expressing and manipulating candidate genes both in E. coli and the archaeon Sulfolobus solfataricus. The array of assays and genetic techniques developed in our lab for analyzing disulfide bond formation in the cytoplasm and periplasm will greatly facilitate these studies and those carried out in other laboratories. In our laboratory, we have evolved strains of E. coli that use novel pathways for disulfide bond formation or reduction. We hypothesize that there are other prokaryotes that use these same electron transfer pathways naturally. We will test this hypothesis as follows: we will use bioinformatic analyses that we have developed to assess the capacity of other organisms to make disulfide bonds. We will then use bioinformatic anlaysis of bacterial genomes that have been sequenced to identify prokaryotes that may lack the genes for the pathways E. coli uses for these purposes, but contain genes whose products might constitute one of the novel pathways. We will then study the properties of these candidate genes from other organisms when they are expressed in E. coli and within the native organism itself. We will study DsbB and VKOR function using a chemical biology approach to obtain small molecules that are inhibitors of DsbB and VKOR. Using a highly sensitive assay for disulfide bond formation, we will screen a large library of chemicals for inhibition of E. coli DsbB and Mtb VKOR. These chemicals will be used to dissect out the steps in the action of these two proteins, to define sites of action through resistant-mutations, and to do comparative studies of VKORs and DsbB. The inhibitors will also be screened for their activity as candidates for development as potential antibiotics against tuberculosis and as anti-coagulants. (Human VKOR is a component of the blood coagulation pathway.) PUBLIC HEALTH RELEVANCE: Inhibitors obtained in the high throughput screening may also be candidates for development into medically useful compounds such as antibiotics against tuberculosis and novel classes of blood thinners. Our studies may yield bacterial strains that could be used to produce large amounts of proteins that have disulfide bonds and are medically important such as peptide hormones and immunoglobulins. Also, since disulfide-bonded proteins are found in many of the most prominent bacterial virulence factors, these studies may contribute to antibiotic development.

描述（由申请人提供）：我们将确定在某些细菌和古细菌中发现的两种新型蛋白质二硫键形成途径的性质。大肠杆菌和许多其他细菌使用两种酶将二硫键引入蛋白质中。 DsbA 直接将蛋白质的半胱氨酸连接成二硫键，而 DsbB 则重新氧化，从而重新生成活性 DsbA。许多其他细菌，包括结核分枝杆菌 (Mtb)，似乎使用人类维生素 K 环氧化物还原酶的同源物 VKOR 代替 DsbB 来形成二硫键。 Mtb VKOR 可以替代大肠杆菌中的 DsbB。我们将通过定义哪些基因产物构成该氧化途径，在大肠杆菌和耻垢分枝杆菌（比结核分枝杆菌更容易处理）中表达候选基因，来表征分枝杆菌基于 VKOR 的二硫键形成系统。在某些古细菌中，我们已经获得了细胞质中二硫键形成的另一种不寻常途径的证据。这些古细菌含有两种 VKOR，一种面向细胞质，并且显然参与细胞质二硫键的形成。我们将验证该途径并鉴定该 VKOR 氧化的细胞质底物（细胞质“DsbA”？）。我们将通过在大肠杆菌和古细菌硫磺矿硫化叶菌中表达和操作候选基因来测试该 VKOR 的拟议作用。我们实验室开发的一系列用于分析细胞质和周质中二硫键形成的测定和遗传技术将极大地促进这些研究以及其他实验室进行的研究。 在我们的实验室中，我们已经进化出使用新途径形成或还原二硫键的大肠杆菌菌株。我们假设还有其他原核生物自然地使用这些相同的电子传递途径。我们将按如下方式检验这一假设：我们将使用我们开发的生物信息学分析来评估其他生物体形成二硫键的能力。然后，我们将使用已测序的细菌基因组的生物信息学分析来识别原核生物，这些原核生物可能缺乏大肠杆菌用于这些目的的途径的基因，但含有其产物可能构成新途径之一的基因。然后，我们将研究来自其他生物体的这些候选基因在大肠杆菌和本地生物体本身中表达时的特性。 我们将使用化学生物学方法研究 DsbB 和 VKOR 功能，以获得作为 DsbB 和 VKOR 抑制剂的小分子。使用高度灵敏的二硫键形成测定法，我们将筛选用于抑制大肠杆菌 DsbB 和 Mtb VKOR 的大型化学物质库。这些化学物质将用于剖析这两种蛋白质的作用步骤，通过抗性突变确定作用位点，并对 VKOR 和 DsbB 进行比较研究。还将筛选这些抑制剂的活性，作为开发潜在抗结核抗生素和抗凝血剂的候选药物。 （人类 VKOR 是凝血途径的一个组成部分。） 公共健康相关性：在高通量筛选中获得的抑制剂也可能是开发成医学上有用的化合物的候选者，例如抗结核病抗生素和新型血液稀释剂。我们的研究可能会产生可用于生产大量具有二硫键且具有医学重要性的蛋白质的细菌菌株，例如肽激素和免疫球蛋白。此外，由于在许多最重要的细菌毒力因子中发现了二硫键蛋白，因此这些研究可能有助于抗生素的开发。