Understanding of a Family of Bacterial Transcription Factors

描述(申请人提供)：大肠杆菌cAMP受体蛋白(CRP)一直是我们理解配体感应、DNA识别和转录调控的模型转录因子。在序列和结构上与CRP相似的蛋白质广泛存在于真细菌的整个结构域中，形成了CRP-FNR超家族(这里称为CRP同源物)。CRP同源物使每个细菌能够通过感知环境线索和调节必要基因的表达来快速适应环境变化，通常是在全球范围内。在细菌病原体中发现的同系物作为毒力因子，因此对家族蛋白的研究也具有生物医学意义。近年来，细菌基因组不断被测序，已经产生了超过7000个蛋白质家族成员。这项研究的长期目标是在全基因组水平上系统地了解这些未知的CRP同源物。这将揭示超家族更广泛的功能范围，其中包括一些成员在人类疾病中的关键毒力作用。不幸的是，传统的实验方法缺乏在实际时间框架内表征压倒性数量的同源物的手段。虽然生物信息学可以为我们提供更快的同源同源性估计，但它不能提供通过实验数据获得的准确信息集。研究任何未知的CRP同源物的内在障碍来自于家族成员共享的双管齐下的功能(配体结合和DNA结合)。如果想要识别配基，就必须事先知道目标DNA，以便进行功能分析。相反，如果不知道激活蛋白质的配体，就不能自信地激活蛋白质，这是识别或评估DNA靶标的先决条件。这项建议的具体目标是开发实验工具，绕过固有的问题，系统地表征未研究的CRP同源物。因此，这一建议的方法学核心在于使用各种类型的嵌合蛋白，这些嵌合蛋白将大肠杆菌CRP的一部分与各种未知的CRP同源物的另一部分融合在一起。由于大肠杆菌CRP在两个结构域(一个用于配体结合，另一个用于DNA结合)中都有很好的特性，该方法使我们能够激活嵌合体(通过CRP的配体cAMP)或功能分析蛋白质(通过已知的CRP的靶DNA)。这一想法是基于我们成功的嵌合蛋白先例，虽然该方法尚未建立，但可能为未知CRP同源物的功能阐明提供无与伦比的优势。然后，嵌合式方法将与其他新概念和一般方法相结合。这些方法将被系统地应用于几个CRP同源物，以测试它们对未知的CRP同源物的可行性。具体目标是(I)开发新的策略来识别未知的CRP同源物的配体，(Ii)阐明CRP同源物的DNA-蛋白质相互作用规则，以及(Iii)将功能增益策略应用于CRP同源物的全基因组功能。此外，拟议的实验将为少数族裔服务机构的研究生和本科生提供一个极好的研究机会。 公共卫生相关性：在铜绿假单胞菌和单核细胞增多性李斯特菌等细菌病原体中发现的CRP同系物是毒力因子。这项建议将系统地表征已知或潜在毒力因子的三个铜绿假单胞菌CRP同源物。这种病原体是医院感染的常见原因，因为它对许多抗生素具有天然和获得性耐药性。这些蛋白质可以成为很好的治疗靶点；因此，从这项研究中获得的知识可能会导致开发一种替代的治疗策略来控制病原体。

DESCRIPTION (provided by applicant): The Escherichia coli cAMP receptor protein (CRP) has been the model transcription factor for our understanding of ligand sensing, DNA recognition and transcriptional regulation. Proteins sequentially and structurally similar to CRP are widespread throughout the domain of the eubacteria and form the CRP-FNR superfamily (termed CRP homologs here). CRP homologs allow each bacterium to quickly adapt to environmental changes by sensing environmental cues and modulating the expression of the necessary genes, often on a global scale. The homologs found in bacterial pathogens serve as virulence factors, so the study of the family proteins is also of biomedical importance. In recent years, bacterial genomes have continuously been sequenced, which has generated more than 7,000 protein family members. The long-term goal of this study is to understand these unknown CRP homologs systematically and at the genome-wide level. This will reveal a wider functional scope of the superfamily, which includes pivotal virulence roles of some members in human disease. Unfortunately, conventional experimental approaches lack the means to characterize the overwhelming number of homologs within a practical time frame. While bioinformatics may provide us a more rapid estimate of homolog identity, it cannot provide the accurate set of information made available through experimental data. Intrinsic hurdles to studying any unknown CRP homologs originate from the two-pronged function (ligand binding and DNA binding) shared by the family members. If one wants to identify the ligand, one has to know the target DNA in advance for functional assay. Conversely, without knowing the ligand that activates the protein, one cannot confidently activate the protein, which is a prerequisite for identifying or evaluating the DNA targets. The specific goal of this proposal is to develop experimental tools bypassing the intrinsic problem to systematically characterize unstudied CRP homologs. Thus, the methodological core of this proposal lies in the use of various types of chimeric proteins that fuse a portion of the E. coli CRP to another portion of various unknown CRP homologs. Since the E. coli CRP is well characterized in each of the two domains (one for ligand binding and the other for DNA binding) is well characterized, the approach enables us to either activate the chimera (via cAMP, the ligand of CRP) or to functionally assay the protein (via the known target DNA of CRP). The idea is based on our successful precedents of chimeric proteins, and the approach, while not established, potentially provides an unmatched advantage for the functional elucidation of unknown CRP homologs. Then, the chimeric approach will be combined with other novel concepts and also with general methodologies. These methods will be systematically applied to several CRP homologs to test their feasibility to unknown CRP homologs. Specific aims are (i) developing new strategies for identifying the ligands of unknown CRP homologs, (ii) elucidating DNA-protein interaction rules for CRP homologs and (iii) applying gain-of-function strategies to CRP homologs for their genome-wide functions. In addition, the proposed experiments will provide an excellent research opportunity for graduate and undergraduate students in a minority-serving institution. PUBLIC HEALTH RELEVANCE: The CRP homologs found in bacterial pathogens such as Pseudomonas aeruginosa and Listeria monocytogenes act as virulence factors. This proposal will systematically characterize three P. aeruginosa CRP homologs of known or potential virulence factors. The pathogen is a frequent cause of nosocomial infections due to its natural and acquired resistance to many antibiotics. The proteins can be good therapeutic targets; therefore, the knowledge gained from this study might lead to the development of an alternative therapeutic strategy to control the pathogen.