Bioinorganic Explorations of Host-defense Proteins

点击翻译按钮获取中文摘要

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.