Bioinorganic Explorations of Host-defense Proteins

项目总结 这一更新应用的首要目标是阐明人类钙化保护蛋白的分子基础 (CP，S100A8/S100A9寡聚体)在金属抑制的先天免疫反应中的作用，并评价其 尿路致病性大肠埃希菌对生理学的影响 对人类的感染。过渡金属是所有生物体必不可少的营养物质，而这些营养物质的可用性 营养物质在微生物感染过程中起着至关重要的作用。因此，人类的先天免疫系统 启动抑制金属的反应并将隔离金属的宿主防御蛋白部署到 限制金属供应和阻碍病原体生长的细胞外空间。CP是一个内容丰富、功能齐全的 多功能金属抑制蛋白；它隔离多种金属营养物质，包括锰(II)、铁(II)、镍(II)和 锌(II)。我们实验室和其他实验室最近的研究提供了令人信服的证据，证明分子物种形成 胞外CP是不同物种的异质集合，来自不同的金属结合 形式以及氧化翻译后修饰。我们假设这个复杂的分子 CP的形态形成，包括蛋氨酸氧化和二硫键的发生，具有深远的意义 对其细胞外功能和寿命的影响。我们实验室和其他实验室最近的研究也 证明CP是一种含铜蛋白。我们假设CP对铜(II)和铜(I)都有隔离作用 这一功能影响不同细菌病原体的生理和金属动态平衡，包括 UPEC。在目标1中，我们将研究CP杂二聚体内部和之间的二硫键的形成， 这些二硫键连接物种的生物物理性质及其从细菌中隔离金属的能力 病原体。在目标2中，我们将评估CP与铜(II/I)结合的性质以及多重结合的后果。 以UPEC上CP对金属的封存为例。我们预计这些调查将推进 CP如何促进金属隔离的先天免疫反应的分子模型，强调 考虑氧化翻译后修饰产生的CP物种的重要性，并阐明 CP抑制铜的分子基础。此外，我们预计，我们对CP和Cp相互作用的研究 UPEC将为宿主和病原体如何竞争铜和其他营养金属提供新的见解。我们 进一步期望这一倡议的结果可以指导设计和开发新的诊断方法， 预防和治疗微生物感染和其他病理疾病的方法，如炎症 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.