Dual Function of VPO1 in Pathogen Recognition and Killing

描述(由申请人提供)：先天免疫被广泛定义为抵御入侵病原体的第一道防线。天然免疫识别传统上是由模式识别受体(PRRs)和病原体相关分子模式(PAMP)相互作用介导的，触发一系列下游信号事件；或者，一些酶，如溶菌酶、消化酶和防御素，在酶遇到微生物时不分青红皂白地杀死细菌。在后一种情况下，这种酶不直接识别微生物，对入侵微生物的清除也是非特异性的。是否有PRR可以直接杀死入侵的微生物还有待阐明。血管过氧化物酶1(VPO1)是新发现的哺乳动物含血红素的过氧化物酶(HPX)。VPO1在HPX家族的成员中是独一无二的，因为它在C末端含有一个催化结构域，在N末端有一个大的N端区，包括5个亮氨酸富集区(LRR)和4个免疫球蛋白(Ig)C2型域。VPO1在心血管系统、肺、肝、胰腺和脾中高度表达，分泌到血液中的浓度是MPO的1000倍。然而，其生物学功能尚未确定。这项研究的中心假设是，VPO1可以识别并直接杀死入侵的微生物。我们的具体目标是(1)确定VPO1的LRR和Ig C2结构域是否与PAMPs结合；(2)确定VPO1是否通过LRR和Ig C2结合，通过产生低卤酸介导杀菌活性。解决这一问题的主要方法包括分子克隆与表达、蛋白质纯化、荧光偏振技术和表面等离子体共振技术。这一提议的成功完成将：(1)为天然免疫反应提供新的见解；(2)鉴定第一个具有病原体识别和杀伤双重功能的哺乳动物蛋白质；(3)通过新的双功能HPX的生理作用创建维持血流不育性的新范式；(4)阐明VPO1介导的病原体识别和杀灭的分子机制，为未来涉及这一新的固有免疫途径的诊断和/或治疗学的发展奠定基础。 与公共卫生相关：该项目寻求识别和表征一种新的天然免疫机制，即病原体识别和杀死都发生在含有血红素的过氧化物酶中，扩展了我们目前的天然免疫概念，填补了天然免疫系统的空白。对这一活动的表征和对先天免疫反应这一臂的阐明将为治疗传染病带来潜在的新靶点。

DESCRIPTION (provided by applicant): Innate immunity is broadly defined as the first line of defense against invading pathogens. Innate immune recognition is classically mediated by the interaction of pattern-recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs), triggering serial downstream signaling events; alternatively, a number of enzymes such as lysozyme, digestive enzymes and defensins indiscriminately kill bacteria when the enzyme encounters the microbes. In the latter case, the enzyme does not directly recognize microbes, and the elimination of invading microbes is non-specific. Whether there is a PRR to directly kill invading microorganisms remains to be elucidated. Vascular peroxidase 1 (VPO1) is a newly-discovered mammalian heme-containing peroxidase (hPx). VPO1 is unique among the members of hPx family in that it contains a catalytic domain at its C-terminus and a large N-terminal region including five leucine-rich regions (LRRs) and four immunoglobulin (Ig) C2 type domains. VPO1 is highly expressed in the cardiovascular system, lung, liver, pancreas and spleen, and is secreted into bloodstream at a 1000-fold higher concentration than is MPO. However, its biological function has not been established. The central hypothesis of this proposed research is that VPO1 can recognize and directly kill invading microbes. Our specific aims are to (1) determine whether the LRR and Ig C2 domains of VPO1 bind to PAMPs; (2) determine if the binding of VPO1, via LRR and Ig C2, mediates microbicidal activity via generation of hypohalous acids. The major methods for addressing the aims include molecular cloning and expressing, protein purification, fluorescence polarization technology and surface plasmon resonance technology. Successful completion of this proposal will: (1) provide novel insights into innate immune responses; (2) identify the first mammalian protein with dual functions of pathogen recognition and killing; (3) create a new paradigm in the maintenance of bloodstream sterility by the physiological action of a novel dual function hPx; (4) elucidate the molecular mechanisms of VPO1-mediated pathogen recognition and killing, setting the foundation for the future development of diagnostics and/or therapeutics involving this novel innate immune pathway. PUBLIC HEALTH RELEVANCE: This project seeks is to identify and characterize a new mechanism of innate immunity in that both pathogen-recognition and killing occur within a heme-containing peroxidase, expanding our current concepts of innate immunity and filling the gap in innate immune system. The characterization of this activity and elucidation of this arm of the innate immune response will lead to potential new targets for therapies against infectious diseases.