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

描述（由申请人提供）：最近铜绿假单胞菌和痢疾志贺氏菌中细菌血红素摄取和运输系统的遗传特征提出了关于血红素结合、转运和释放的功能细节的有趣问题，这些过程中蛋白质参与者，血红素结合蛋白（HBPs）和血红素运输受体（HTRs）。许多革兰氏阴性细菌病原体利用宿主体内最丰富的可溶性铁——血红素，作为建立感染所必需的铁的来源。然而，血红素具有细胞毒性，在整个摄取和运输过程中必须高度隔离。这种对血红素毒性的保护要求对该过程中涉及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血红素的毒性作用，所有血红素：HBP和血红素：HTR复合物必须表现出内在的高热力学稳定性。然而，血红素在这些具有高血红素亲和力的蛋白质之间的快速转移必须是可能的。因此，第二个设计标准是，必须有可能通过与合作伙伴的特定相互作用来调节血红素：HBPs和血红素：htr的高稳定性。装载血红素的蛋白与其靶标之间的相互作用的自由能必须被转导，才能对血红素从供体到靶标的转移施加驱动力和动力学稳定性，即血红素到HO的有效方向以释放其铁可以被视为是机械驱动的。细菌从血红素中吸收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可以分为三组；具有阴离子酪氨酸盐轴向配体的，具有轴向His配体的，以及具有轴向His和Met配体的。本研究将对痢疾链球菌和铜绿假单胞菌的HBPs和HTRs进行检测。根据遗传学研究，已经确定了参与血红素摄取和运输的蛋白质。本研究提出的一般假设是，上述轴向配体环境与上述生物中血红素结合、运输和释放的不同机制相关。我们将确定血红素配位化学对血红素：HBP和血红素：HTR复合物稳定性的贡献。我们将进一步研究血红素：HBP或血红素：HTR与其靶蛋白的血红素转移络合可以调节哪些贡献。我们研究血红素摄取和转运蛋白的长期目标是阐明控制血红素转运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其结果有可能最终为细菌感染的治疗提供新的进展。在这个项目中，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途径将在分子机制水平上进行研究。这项工作与人类健康的相关性在于，有可能了解细菌病原体对血红素的摄取，最终为治疗细菌感染提供新的突破口。

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.