Microbial Diversity in Mechanisms of Disulfide Bond Formation and Reduction

描述(由申请人提供)：我们将确定在某些细菌和古菌中发现的两种新的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重新氧化，从而再生活性的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VKOR的同系物来形成二硫键，而不是DsbB。在大肠杆菌中，MTB VKOR可以替代DsbB。我们将通过定义哪些基因产物包括这一氧化途径，在大肠杆菌和污垢分枝杆菌(比Mtb更容易)中表达候选基因，来表征基于分枝杆菌VKOR的二硫键形成系统。在某些古生菌中，我们已经获得了细胞质中另一种不寻常的二硫键形成途径的证据。这些古菌含有两个VKOR，一个面向细胞质，显然参与了细胞质二硫键的形成。我们将验证这一途径并确定细胞质底物(细胞质“DsbA”？)这种VKOR被氧化了。我们将通过在大肠杆菌和古细菌Sulfolobus solfararicus中表达和操作候选基因来测试这种VKOR的拟议作用。我们实验室开发的用于分析细胞质和周质中二硫键形成的一系列分析和基因技术将极大地促进这些研究和在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的途径形成或还原二硫键的大肠杆菌菌株。我们假设有其他原核生物自然地使用这些相同的电子转移途径。我们将对这一假设进行如下检验：我们将使用我们开发的生物信息学分析来评估其他生物形成二硫键的能力。然后，我们将使用已经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，但包含其产物可能构成其中一种新途径的基因。然后，我们将研究这些来自其他生物体的候选基因在大肠杆菌和天然生物体中表达时的特性。我们将使用化学生物学方法研究DsbB和VKOR的功能，以获得DsbB和VKOR的抑制剂小分子。使用高灵敏的二硫键形成试验，我们将筛选大量抑制E.ColiDsbB和Mtb VKOR的化学物质。这些化学物质将被用来剖析这两种蛋白质的作用步骤，通过耐药突变确定作用部位，并对VKOR和DsbB进行比较研究。这些抑制剂还将被筛选出它们的活性，作为潜在的抗结核病抗生素和抗凝血剂开发的候选药物。(人类VKOR是凝血途径的一个组成部分。) 公共卫生相关性：在高通量筛查中获得的抑制剂也可能被开发成医学上有用的化合物，如抗结核病的抗生素和新型血液稀释剂。我们的研究可能会产生细菌菌株，这些菌株可以用来产生大量具有二硫键的、具有重要医学意义的蛋白质，如肽激素和免疫球蛋白。此外，由于在许多最突出的细菌毒力因子中发现了二硫键结合蛋白，这些研究可能有助于抗生素的开发。

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.