Functional Role of Zebrafish TLR Proteins

描述（由申请人提供）：我们假设斑马鱼的TLR信号在机制上与哺乳动物不同，并预测了解这些差异将导致在高等脊椎动物中发现新的TLR信号通路。这项研究的一个关键焦点涉及免疫系统本身，特别是其功能可以增强、扩展和延长的方式。这些研究将确定哺乳动物TLR通路的起源，并最终为开发新的研究靶点和人类健康的新疗法提供方向。在此提案中，我们打算实现以下具体目标：1。发现斑马鱼TLR信号通路的新成分a.鉴定识别病毒配体的斑马鱼TLR受体b.通过酵母双杂交筛选鉴定斑马鱼TLR通路的新相互作用c.确定在斑马鱼中发现的相互作用是否也发生在人类TLR通路中。这些实验将揭示斑马鱼TLR通路的新成分，并将为人类和斑马鱼的研究提供新的研究目标。此外，还将阐明斑马鱼TLR蛋白蛋白-蛋白相互作用的关键结构域。2. 确定斑马鱼抗病毒途径成分在体内保护宿主免受感染中的作用证明TLR途径与疾病抗性之间的联系至关重要。在体内方法中，斑马鱼插入突变和morpholino oligonucleotides （MO）将被用来敲除TLR通路蛋白的表达。然后将丧失功能的突变体或变形体（注射了morpholino的斑马鱼）暴露于病原体并检查以确定先天免疫功能的改变如何影响对病原体挑战的抵抗力。

DESCRIPTION (provided by applicant): We hypothesize that zebrafish TLR signaling is mechanistically divergent from mammals, and predict that understanding these differences will lead to the discovery of novel TLR pathways in higher vertebrates. A critical focus of this investigation involves the immune system itself, specifically ways in which its function can be augmented, extended, and prolonged. These studies will establish the origins of the mammalian TLR pathways and ultimately provide a direction for development of new targets of investigation and new therapies for human health. In this proposal, we intend to fulfill the following SPECIFIC AIMS: 1. Discover novel zebrafish TLR signaling pathway components a. Identify the zebrafish TLR receptors that recognize viral ligands b. Identify novel interactors of the zebrafish TLR pathways via a yeast two-hybrid screen c. Determine if the interactions identified in the zebrafish also occur in the human TLR pathways These experiments will reveal new components of the zebrafish TLR pathways and will provide new targets for investigation in both human and zebrafish studies. Furthermore, the critical domains for protein-protein interaction of zebrafish TLR proteins will be elucidated. 2. Determine the role of the zebrafish antiviral pathway components in protecting the host from infection in vivo It is essential to demonstrate the link between TLR pathways and resistance to disease. In an in vivo approach, zebrafish insertional mutations and morpholino oligonucleotides (MO) will be used to knockdown the expression of the TLR pathway proteins. The loss-of-function mutants or morphants (morpholino-injected zebrafish) will then be exposed to pathogens and examined to determine how alterations in innate immune function affect resistance to pathogen challenge. PUBLIC HEALTH RELEVANCE Infectious diseases continue to play a major role in the human condition worldwide and as a result vaccines, antibiotics, and intervention strategies are continually being developed. These methods have largely proven effective; however, due to microbial adaptation and environment- triggered emergence of new diseases, it is critical to investigate alternative methods for controlling infectious diseases. A critical focus of this investigation involves the immune system itself, specifically ways in which its function can be augmented, extended, and prolonged.