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

基本信息

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
项目类别：
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

项目摘要

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）：我们将建立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。 DSBA将蛋白质的半胱氨酸直接加入二硫键，而DSBB则重新氧化，从而再生活跃的DSBA。许多其他细菌，包括结核分枝杆菌（MTB），似乎使用了人类维生素K环氧化物还原酶VKOR的同源物进行二硫键键形成而不是DSBB。 MTB VKOR可以在大肠杆菌中代替DSBB。我们将通过定义哪种基因产物包含该氧化途径，表达在大肠杆菌和分枝杆菌Smegmatis中表达候选基因（比MTB更具拖动）来表征分生不清的二硫键键形成系统。在某些古细菌中，我们获得了细胞质中二硫键形成的另一种异常途径的证据。这些古细菌包含两个VKOR，一个胞质面向且显然参与细胞质二硫键形成。我们将验证该途径并识别细胞质底物（细胞质“ DSBA”？），即该VKOR氧化。我们将通过表达和操纵大肠杆菌和solfataricus古老的候选基因来测试该VKOR的拟议作用。在我们的实验室中开发的一系列测定和遗传技术，用于分析细胞质和周质中的二硫键形成，这将极大地促进这些研究以及在其他实验室中进行的研究。在我们的实验室中，我们发展了大肠杆菌的菌株，这些大肠杆菌使用新型途径进行二硫键形成或还原。我们假设还有其他原核生物自然使用这些相同的电子转移途径。我们将测试该假设如下：我们将使用已开发的生物信息学分析来评估其他生物体建立二硫键键的能力。然后，我们将使用已经测序的细菌基因组生物信息学的生物素来鉴定可能缺乏大肠杆菌用途的途径基因的原核生物，但包含其产物可能构成新途径之一的基因。然后，当这些候选基因在大肠杆菌中和本地生物本身中表达时，我们将研究这些候选基因的性质。我们将使用化学生物学方法研究DSBB和VKOR功能，以获得DSBB和VKOR抑制剂的小分子。使用高度敏感的测定法进行二硫键形成，我们将筛选一个大型化学物质库，以抑制大肠杆菌DSBB和MTB VKOR。这些化学物质将用于剖析这两种蛋白质作用的步骤，以通过抗性突破来定义作用部位，并对VKOR和DSBB进行比较研究。这些抑制剂还将被筛选为他们作为候选候选抗生素的活性和抗凝剂的潜在抗生素。（人VKOR是血液凝结途径的组成部分。）公共卫生相关性：在高吞吐量筛查中获得的抑制剂也可能是发展为医学上有用的化合物，例如针对结核病的抗生素和新型血液稀释剂的抗生素。我们的研究可能会产生细菌菌株，可用于产生大量具有二硫键且在医学上很重要的蛋白质，例如肽激素和免疫球蛋白。同样，由于在许多最突出的细菌毒力因子中发现了二硫键蛋白质，因此这些研究可能有助于抗生素发育。