Bioinorganic Explorations of Host-defense Proteins

项目摘要 这项更新申请的首要目标是阐明人钙卫蛋白如何在细胞内表达的分子基础。 (CP，S100 A8/S100 A9寡聚体）在金属抑制先天性免疫应答中起作用，并评估其 对尿路致病性大肠杆菌（UPEC）的生理影响，该大肠杆菌引起大多数尿路感染， 人类感染。过渡金属是所有生物体的必需营养素， 营养素在微生物感染期间起着关键作用。因此，人类先天免疫系统 启动金属抑制反应，并将金属螯合宿主防御蛋白部署到 细胞外空间限制金属的可用性并阻碍病原体生长。CP是一种丰富的功能性 多功能金属抑制蛋白质;它螯合多种金属营养素，包括Mn（II），Fe（II），Ni（II）和 Zn（II）。我们实验室和其他人最近的研究提供了令人信服的证据，表明分子物种形成 细胞外CP是不同物种的异质系综，来自不同的金属结合 形式以及氧化翻译后修饰。我们假设这种复杂的分子 CP的形态，包括蛋氨酸氧化和二硫键的发生，具有深刻的影响。 影响其细胞外功能和寿命。我们实验室和其他机构最近的研究也表明， 证明CP是一种铜抑制蛋白。我们假设CP螯合Cu（II）和Cu（I）， 这种功能影响了各种细菌病原体的生理学和金属稳态， UPEC。在目标1中，我们将研究CP异二聚体内部和之间的二硫键形成， 这些二硫键连接的物种的生物物理性质，以及它们从细菌中螯合金属的能力， 病原体在目标2中，我们将评估CP的Cu（II/I）结合特性以及多重结合的后果。 作为案例研究，通过CP对UPEC的金属螯合。我们希望这些调查将推动 CP如何有助于金属螯合先天免疫反应的分子模型，强调了 考虑氧化翻译后修饰产生的CP物质的重要性，并阐明 CP抑制铜的分子基础。此外，我们希望我们对合作原则和 UPEC将为宿主和病原体如何竞争Cu和其他营养金属提供新的见解。我们 进一步期望该倡议的结果可以指导新诊断的设计和开发， 用于微生物感染和其它病理学如炎症的预防和治疗方法 涉及CP的疾病。

PROJECT SUMMARY The overarching goal of this renewal application is to elucidate the molecular basis for how human calprotectin (CP, S100A8/S100A9 oligomer) functions in the metal-withholding innate immune response, and to evaluate its impact on the physiology of uropathogenic Escherichia coli (UPEC), which cause the majority of urinary tract infections in humans. Transition metals are essential nutrients for all organisms, and the availability of these nutrients plays a critical role during microbial infection. Consequently, the human innate immune system launches a metal-withholding response and deploys metal-sequestering host-defense proteins into the extracellular space to limit metal availability and hinder pathogen growth. CP is an abundant and functionally versatile metal-withholding protein; it sequesters multiple metal nutrients including Mn(II), Fe(II), Ni(II) and Zn(II). Recent studies by our laboratory and others provide compelling evidence that the molecular speciation of extracellular CP is a heterogenous ensemble of different species that arises from different metal-bound forms as well as oxidative post-translational modifications. We hypothesize that this complex molecular speciation of CP, including the occurrence of methionine oxidation and disulfide bonding, has profound consequences for its extracellular function and lifetime. Recent studies by our laboratory and others also demonstrate that CP is a Cu-withholding protein. We hypothesize that CP sequesters both Cu(II) and Cu(I) and that this function impacts the physiology and metal homeostasis in diverse bacterial pathogens including UPEC. In Aim 1, we will examine disulfide bond formation within and between CP heterodimers, the biophysical properties of these disulfide-linked species, and their ability to sequester metals from bacterial pathogens. In Aim 2, we will evaluate the Cu(II/I)-binding properties of CP and the consequences of multi- metal sequestration by CP on UPEC as a case study. We expect that these investigations will advance the molecular model for how CP contributes to the metal-sequestering innate immune response, underscore the importance of considering CP species that result from oxidative posttranslational modification, and elucidate the molecular basis for Cu withholding by CP. Moreover, we expect that our studies of the interplay of CP and UPEC will provide new insight into how the host and pathogen compete for Cu and other nutrient metals. We further expect that the outcomes of this initiative may guide the design and development of novel diagnostic, preventative and therapeutic approaches for microbial infections and other pathologies such as inflammatory diseases that involve CP.