Structural basis of TLR and NLR recognition and signaling

描述(由申请人提供)：先天免疫提供了抵御病原体或危险相关分子模式的第一道防线。在已知的先天受体中，Toll样受体(TLRs)和Nod样受体(NLRs)具有共同的结构基序，由多个富含亮氨酸的重复序列(LRR)组成，每个重复序列的氨基酸残基和长度存在一定的变异性。无颌鱼可变淋巴细胞受体(VLRs)的LRR结构研究也表明，LRR结构域在配体识别中起着重要作用。因此，TLR和NLR的LRR在与激动剂配体的相互作用中是必不可少的，以启动先天免疫中的细胞信号转导。此外，当LRR结构域从信号域中分离出来时，它可以通过竞争性地与配体结合来负向调节天然免疫。因此，本研究的最终目的是进一步了解LRRs在TLRs中的结构和功能，以及NLRs在配体诱导的激活和信号转导中的结构和功能。包括我们自己在内的几个研究小组已经完成了TLR1/2/3/4/5/6中LRR结构域的结构和生物物理表征，但关于TLR7/8/9如何与核酸和其他配体相互作用并受其调节以响应微生物病原体的主要问题仍未解决。与TLRs相比，NLRs的结构研究非常有限。到目前为止，只有一种NLR LRR结构被解决，即NLRX1，我们还证明了它直接与RNA配体结合。凭借我们在表达、纯化、结晶和鉴定各种LRR蛋白方面的丰富专业知识，我们将研究TLR7/8/9和NLRC3/4/5中LRR结构域的结构、生物物理和功能特性，并推进我们对TLR5和NLRX1的已有成果的研究。这些发现将解决TLR7/8/9和NLRC3/4/5与其同源配体的特定相互作用所产生的结构和功能机制，并有助于理解天然免疫中受体的激活和调节。我们将利用我们之前对TLR5和NLRX1的研究来回答关于它们的信号机制以及外部因素如何调控它们的关键问题。与Andrei Gudkov博士、Cynthia Leifer博士和Jenny Ting博士在这些受体的细胞方面的合作，以及与Andrew Ward博士和David Baker博士在专门生物物理方法上的合作，将补充我们的生物物理和功能研究，并刺激激动剂和拮抗剂的开发，将其作为免疫功能受损或高炎症性疾病的治疗药物。

DESCRIPTION (provided by applicant): Innate immunity provides a first line of defense against pathogen- or danger-associated molecular patterns. Among known innate receptors, Toll-like receptors (TLRs) and NOD-like receptors (NLRs) share a common structural motif that consists of multiple leucine-rich repeats (LRRs) with some variability in amino- acid residues and length of each repeat. Structural studies of LRRs of variable lymphocyte receptors (VLRs) in jawless fish have also shown that LRR domains are important in ligand recognition. Thus, the LRRs of TLRs and NLRs are essential for interaction with agonist ligands in order to initiate cellular signaling in innate immunity. Furthermore, when the LRR domain is disengaged from the signaling domains, it can negatively regulate innate immunity, by competitively binding to ligands. Thus, the ultimate goal of this study is to advance understanding of the structure and function of LRRs in TLRs and NLRs in ligand-induced activation and signaling. Structural and biophysical characterization of LRR domains in TLR1/2/3/4/5/6 have been accomplished by several groups, including our own, but major unresolved questions remain concerning how TLR7/8/9 interact with and are regulated by nucleic acid and other ligands in response to microbial pathogens. Compared to TLRs, structural investigation of NLRs has been extremely limited. To date, only one NLR LRR structure, that of NLRX1, has been solved and we have also shown that it directly binds RNA ligands. With our extensive expertise in expression, purification, crystallization and characterization of diverse LRR proteins, we will investigate structural, biophysical and functional properties of LRR domains in TLR7/8/9 and NLRC3/4/5, as well as advance our already productive studies of TLR5 and NLRX1. Such findings will address the structural and functional mechanisms that arise from the specific interactions of TLR7/8/9 and NLRC3/4/5 with their cognate ligands and lead to understanding of receptor activation and regulation in innate immunity. We will capitalize on our previous studies of TLR5 and NLRX1 to answer key questions about their signaling mechanisms and how they might be regulated by outside agents. Collaborations on cellular aspects on these receptors with Drs. Andrei Gudkov, Cynthia Leifer and Jenny Ting and on specialized biophysical approaches with Drs. Andrew Ward and David Baker will complement our biophysical and functional studies as well as stimulate development of agonists and antagonists as therapeutics in compromised immunity or in hyper- inflammatory diseases.