Study of heme uptake and transport proteins from P. aerugenosa and S. dysenteriae

描述(申请人提供)：最近对铜绿假单胞菌和志贺氏菌的细菌血红素吸收和运输系统的遗传特征提出了有趣的问题，涉及到在这些过程中血红素结合、转位和释放的蛋白质玩家、血红素结合蛋白(HBPs)和血红素运输受体(HTRs)的功能细节。许多革兰氏阴性细菌病原体使用其宿主中最丰富的可溶性铁来源--血红素，作为确定感染所必需的铁的来源。然而，血红素具有细胞毒性，在吸收和运输过程中必须高度隔离。这种针对血红素毒性的保护要求对参与这一过程的蛋白质施加了两个设计标准。首先，为了保护生物体免受游离亚铁血红素的毒性影响，所有亚铁血红素：HBP和亚铁血红素：HTR络合物必须表现出内在的高热力学稳定性。然而，在这些具有高血红素亲和力的蛋白质之间进行简单的血红素转移必须是可能的。因此，第二个设计标准是，必须能够通过与货物所传递给的伙伴的特定相互作用来调节血红素：高铁血红素和高铁血红素：羟色胺的高稳定性。血红素与靶蛋白相互作用的自由能必须被传递，以施加驱动力和动力学不稳定性来驱动血红素从供体到靶的转移，即血红素向HO释放铁的有效方向可以被视为是机械驱动的。关于细菌同化血红素中铁的工作以及我们最近对一些相关蛋白质的生物物理表征表明，HBPs可以分为三类：具有阴离子酪氨酸轴向配体的HBPs，具有轴向His配体的HBPs，以及具有轴向His和Met配体的HBPs。在这项研究中，将检测痢疾杆菌和铜绿假单胞菌的HBPs和HTRs。基于遗传学研究，已经确定了与血红素吸收和运输有关的蛋白质。这项拟议的研究要解决的一般假设是，上述轴向配体环境与上述生物体中血红素结合、运输和释放的不同机制相关。我们将确定(S)从血红素配位化学对血红素：HBP和血红素：HTR络合物的稳定性的贡献。我们将进一步研究这些贡献中的哪些可以通过血红素：HBP或血红素：HTR与其目标蛋白的络合来调节。我们研究血红素吸收和转运蛋白的长期目标是阐明控制血红素转移的特异性和有效性的机制参数。这项工作与人类健康的相关性在于，结果最终可能为细菌感染的治疗提供新的途径。在本项目中，将从分子机制水平上研究细菌病原体从宿主获得血红素并吸收建立感染所需的铁的方法。这项工作与人类健康的相关性在于，了解细菌病原体吸收血红素的可能性最终为治疗细菌感染提供新的途径。

DESCRIPTION (provided by applicant): Recent genetic characterization of bacterial heme uptake and transport systems in Pseudomonas aerugenosa and Shigella dysenteriae have raised interesting questions regarding the functional details of heme binding, translocation, and release by the protein players, the heme-binding proteins (HBPs) and the heme-transport receptors (HTRs), in these processes. Many gram negative bacterial pathogens use the most abundant source of soluble iron, heme from their host, as a source of the iron essential to establish infection. However, heme is cytotoxic and must be highly sequestered throughout the course of uptake and transport. This requirement for protection against the toxicity of heme imposes two design criteria on the proteins involved in the process. The first is that, in order to protect the organism from the toxic effects of free heme, all heme:HBP and heme:HTR complexes must exhibit intrinsically high thermodynamic stabilities. Yet, facile transfer of heme between these proteins having high heme affinities must be possible. Thus the second design criterion is that it must be possible to modulate the high stabilities of the heme:HBPs and heme:HTRs through specific interactions with the partners to which they pass their cargo. The free energy of interaction between heme-loaded proteins and their targets must be transduced to impose driving force and kinetic lability on the transfer of heme from the donor to its target, i.e. effective direction of heme to HO for liberation of its iron can be viewed as being mechanistically driven. Work on bacterial assimilation of the iron from heme along with our recent biophysical characterization of some of the proteins involved suggests that the HBPs can be classified in three groups; those having anionic tyrosinate axial ligands, those having axial His ligands, and those having axial His and Met ligands. In this study, the HBPs and HTRs from S. dysenteriae and P. aerugenosa will be examined. Based on genetic studies, the proteins involved in heme uptake and transport have been identified. The general hypothesis to be addressed by this proposed study is that the aforementioned axial ligand environments correlate with distinct mechanisms for heme binding, transport, and release in the aforementioned organisms. We will identify contribution(s) from heme coordination chemistry to the stabilities of the heme:HBP and heme:HTR complexes. We will further investigate which of those contributions could be modulated by complexation of heme:HBP or heme:HTR with its target protein for heme transfer. The long-term goal of our work with heme uptake and transport proteins is to elucidate the mechanistic parameters that govern the specificity and efficacy of heme transfer. The relevance of this work to human health lies in the potential of the results to ultimately provide new inroads into treatment of bacterial infections. In this project, the means by which bacterial pathogens acquire heme from their hosts and assimilate the iron necessary to establish infection will be investigated at the level of molecular mechanism. The relevance of this work to human health lies in the potential for knowledge of heme uptake by bacterial pathogens to ultimately provide new inroads into treatment of bacterial infections.