Structure-function studies of the H. polygyrus TGF-beta, TGM

总结 蠕虫寄生虫仍然是世界上人类和动物健康面临的最大挑战之一， 全世界有20亿人被感染。寄生虫控制仅限于低成本的短期抑制 由于缺乏有效的疫苗，蠕虫在人类和动物体内的持续存在证明了 它们高度进化的逃避免疫系统的能力，因此了解逃避机制是关键。 开发新的干预措施。在这项提案中，重点是小鼠寄生虫Heligmosomoides 多脑回病毒，以L3幼虫形式感染动物，并在肠道中定植和成熟。感染小鼠 H.在多脑回中，Foxp3+调节性T细胞（Tcells）的数量显著上调， 并有效地介导免疫抑制。感染小鼠中Treg功能的干扰导致 H. polygyrus，这表明Foxp3 + T细胞的扩增库对于持久性是必不可少的。合作 与Maizels小组，我们已经表明，分泌的五域补体控制蛋白（CCP）蛋白， 它模拟哺乳动物TGF-β，一种通过刺激免疫系统而有效抑制免疫系统的细胞因子。 T细胞的增殖和分化是H.多脑回感染 动物尽管与哺乳动物TGF-β缺乏任何同源性，但这种称为TGF-β模拟物或TGM的蛋白质结合 直接作用于哺乳动物TGF-β受体T β RI和T β RII，以诱导信号传导和下游效应， 你好在这个提议中，我们将确定TGM如何结合T β RI和T β RII并将其组装成信号复合物， 目的是利用这些信息更好地了解TGM如何发出信号并识别同源蛋白质 在利用TGF-β途径逃避宿主免疫反应的其他寄生虫中。此外，这些信息 可用于工程化TGM形式，用于：1）治疗自身免疫性疾病，如类风湿性关节炎， 多发性硬化症或哮喘，2）抑制器官移植中的免疫系统，以及3）发展 通过阻断TGM和TGF-β受体之间的相互作用发挥作用的抗寄生虫药。为支持 提出的研究，我们表明，使用NMR和ITC/SPR结合研究，TGM结构域3（TGM-D3）是 负责结合TbRII的主要结构域，而TGM结构域D2（TGM-D2），连同更次要的 来自TGM结构域1（TGM-D1）的贡献负责结合TbRI。为了实现目标， 在所提出的研究中，我们将单独确定TGM-D2和TGM-D3的结构，以及TGM-D2：TbRI的结构。 TGM-D123：TbRII：TbRI复合物结构的结构， 在Aim 2中使用X射线晶体学合成复合物。为了鉴定对受体功能贡献最大的残基， 结合，定点诱变和ITC-和SPR为基础的结合研究将被使用，连同功能 在培养的TGF-b报告细胞系和TGF-b中的研究。

Summary Helminth parasites remain one of the world’s greatest challenges to human and animal health, with more than two billion infected humans world-wide. Parasite control is restricted to short-term suppression with low cost drugs due the unavailability of effective vaccines. Persistence of helminths in humans and animals is testament to their highly evolved ability to evade the immune system, thus understanding the evasion mechanisms is key to developing new interventions. In this proposal, the focus is on the mouse parasite Heligmosomoides polygyrus, which infects animals as L3 larvae and colonizes and matures in the intestine. In mice infected with H. polygyrus, there is a dramatic upregulation in the number of Foxp3+ regulatory T-cells (Tregs), which broadly and potently mediate immune suppression. Interference of Treg function in infected mice results in expulsion of H. polygyrus, demonstrating that the expanded pool of Foxp3+ Tregs is essential for persistence. In collaboration with the Maizels group, we have shown that a secreted five-domain complement control protein (CCP) protein that mimics mammalian TGF-b, a cytokine that potently suppresses the immune system by stimulating the proliferation and differentiation of Tregs, is critical for immune hyporesponsiveness in H. polygyrus infected animals. In spite of lacking any homology to mammalian TGF-β, this protein termed TGF-b mimic or TGM, binds directly to the mammalian TGF-β receptors, TβRI and TβRII, to induce signaling and downstream effects on Tregs. In this proposal, we will determine how TGM binds and assembles TβRI and TβRII into a signaling complex, with the goal of using this information to better understand how TGM signals and identify homologous proteins in other parasites that exploit the TGF-b pathway to evade host immune responses. In addition, this information can be leveraged to engineer forms of TGM for: 1) treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, or asthma, 2) suppressing the immune system in organ transplantation, and 3) developing anti-parasitics that function by blocking the interaction between TGM and the TGF-β receptors. In support of the proposed studies, we show using NMR and ITC/SPR binding studies that TGM domain 3 (TGM-D3) is the primary domain responsible for binding TbRII, while TGM domain D2 (TGM-D2), together with a more minor contribution from TGM domain 1 (TGM-D1), is responsible for binding TbRI. In order to accomplish the objectives of the proposed research, we will determine the structures of TGM-D2 and TGM-D3 alone, and the TGM-D2:TbRI and TGM-D3:TbRII complex structures, using NMR in Aim 1, and the structure of the TGM-D123:TbRII:TbRI complex using X-ray crystallography in Aim 2. In order to identify the residues that contribute greatest to receptor binding, site-directed mutagenesis and ITC- and SPR-based binding studies will be used, together with functional studies in cultured TGF-b reporter cell lines and Tregs.