Structure and Functionof a Parasitic TGF-beta Mimic, TGM

寄生 TGF-β 模拟物 (TGM) 的结构和功能

基本信息

批准号：
10315068
负责人：
Ananya Mukundan
金额：
$ 5.1万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10315068
关键词：
Adaptive Immune System Address Affinity Amino Acid Motifs Animals Antiparasitic Agents Binding Biochemical Biological Biological Assay Biophysics CD4 Positive T Lymphocytes Calorimetry Cell Line Cellular Assay Chemicals Chimeric Proteins Clinical Collaborations Complement Complement Factor B Complex Data Development Disease Engineering Escherichia coli FOXP3 gene Family Family member Generations Goals Growth Factor Helminths High Prevalence Homologous Gene Human Immune Immune Evasion Immune Tolerance Immune system Immunosuppression In Vitro Individual Infection Intervention Isotope Labeling Knowledge Lead Learning Length Ligands Maintenance Malignant Neoplasms Mammalian Cell Matrix Metalloproteinases Mediating Methods Modeling Molecular Mimicry Mus Mutagenesis Nematospiroides dubius Oncogenic Parasites Pathway interactions Phosphotransferases Physicians Physiological Play Population Protein Engineering Protein Family Protein Isoforms Proteins Proteolysis Receptor Signaling Regulatory T-Lymphocyte Reporter Role Scientist Signal Pathway Signal Transduction Signaling Protein Site Specificity Spleen Structure Surface Plasmon Resonance T-Lymphocyte TGFBR2 gene Tertiary Protein Structure Testing Therapeutic Therapeutic Intervention Titrations Training Transforming Growth Factors X-Ray Crystallography burden of illness cancer immunotherapy career clinical practice cytokine design gastrointestinal global health immunoregulation improved in vivo inhibitor/antagonist insight interest kinase inhibitor member mimicry protein structure receptor receptor binding skills soft tissue structural biology targeted treatment tumor progression

项目摘要

Project Summary Helminth parasites are a threat to global health, with nearly one-third of the world’s population currently infected, constituting a high disease burden amongst both humans and animals alike. The high prevalence of helminths can be attributed to their ability to manipulate and evade their host’s immune system using secreted immunomodulatory molecules. Thus, understanding the mechanisms behind immune evasion is key to therapeutic intervention. The mouse gastrointestinal parasite Heligmosomoides polygyrus is a model for parasitic immunoregulation; infection results in the conversion of naïve CD4+ T-cells into Foxp3+ regulatory T- cells (Tregs) and their expansion, which act to broadly suppress the host immune system. Only upon reduction of this Treg population are mice able to expel the parasite, demonstrating that the parasite requires this increased population of Foxp3+ Tregs for persistence in its host. Through an ongoing collaboration with the Maizels lab, our labs have determined that the parasite is using a set of secreted complement control proteins (CCP) to incite host immune hyporesponsiveness through mimicry of the transforming growth factor β isoforms (TGF-). The TGF-β isoforms have essential roles in maintenance of the adaptive immune system, with TGF-β known to be the major cytokine responsible for the conversion of naïve CD4+ T-cells into Tregs and expansion of the Treg population. This immunosuppression is important in maintaining immune tolerance, but also promotes soft tissue cancer progression by enabling oncogenic evasion of the immune system. Though lacking sequence and structural similarity to TGF-β, the founding member of the parasitic protein family identified by the Maizels group, TGM1, has been shown to directly bind to the mammalian TGF-β receptors, and along with two close homologues, TGM2 and TGM4, upregulate the Treg population. Identifying the residues and protein motifs responsible for binding the TGF- receptors can be used to engineer forms of TGM for: 1) anti-parasitics that block the interaction between TGM family members and the TGF-β family receptors, and 2) TGF- receptor kinase inhibitors for cancer immunotherapy. In this proposal, we will determine how two members of the TGM family, TGM1 and TGM4, bind and assemble the TGF-β receptors to activate the TGF- pathway, providing insight into parasitic molecular mimicry. The structure of TGM1 domains (Aim 1) and TGM4 (Aim 2) domains alone and in complex with their cognate TGF-β family receptors will be determined through NMR, X- ray crystallography, and ITC/SPR binding studies. Residues that contribute greatest to receptor binding will be identified through residue-specific substitution and ITC/SPR binding studies (Aim 1, 2). In addition, we will leverage this structural information to engineer an Fc-fusion construct of TGM with selective binding to the TGF-β type I receptor, and test for inhibition of TGF-β Smad signaling and Foxp3+ Treg induction through functional studies in cultured TGF- reporter cell lines and murine spleen-derived Tregs (Aim 3) for use as adjuncts in cancer immunotherapy.

项目概要蠕虫寄生虫对全球健康构成威胁，目前世界上近三分之一的人口患有蠕虫寄生虫感染，对人类和动物造成很高的疾病负担。蠕虫可归因于它们利用分泌的免疫系统操纵和逃避宿主免疫系统的能力。因此，了解免疫逃避背后的机制是关键。小鼠胃肠道寄生虫 Heligmosomoides polygyrus 是一种模型。寄生免疫调节导致幼稚 CD4+ T 细胞转化为 Foxp3+ 调节性 T-细胞细胞（Treg）及其扩增，仅在减少时才能广泛抑制宿主免疫系统。该 Treg 群体中的小鼠能够排出寄生虫，这表明寄生虫需要这种能力通过与宿主的持续合作，增加了 Foxp3+ Tregs 的数量，以使其在宿主中持久存在。 Maizels 实验室，我们的实验室已确定寄生虫正在使用一组分泌的补体控制蛋白 (CCP) 通过模仿转化生长因子 β 亚型来引发宿主免疫反应低下 (TGF-β)。TGF-β 同种型在维持适应性免疫系统中具有重要作用。已知是负责将初始 CD4+ T 细胞转化为 Tregs 并扩增的主要细胞因子这种免疫抑制对于维持免疫耐受很重要，而且也很重要。尽管缺乏，但可以通过使致癌物质逃避免疫系统来促进软组织癌症的进展。与 TGF-β 的序列和结构相似，TGF-β 是通过鉴定的寄生蛋白家族的创始成员 Maizels 组 TGM1 已被证明可以直接与哺乳动物 TGF-β 受体结合，并且与两个密切的同源物 TGM2 和 TGM4 上调 Treg 群体的残基和蛋白质。负责结合 TGF-β 受体的基序可用于设计 TGM 的形式，用于：1) 抗寄生虫阻断 TGM 家族成员与 TGF-β 家族受体之间的相互作用，以及 2) TGF- 在这项提案中，我们将确定如何将受体激酶抑制剂用于癌症免疫治疗。 TGM 家族 TGM1 和 TGM4 结合并组装 TGF-β 受体以激活 TGF-β 通路，提供对寄生分子拟态的深入了解。TGM1 结构域（目标 1）和 TGM4（目标 2）的结构。单独的结构域和与其同源 TGF-β 家族受体复合的结构域将通过 NMR、X- 对受体结合贡献最大的残基将是射线晶体学和 ITC/SPR 结合研究。通过残基特异性取代和 ITC/SPR 结合研究（目标 1、2）确定。利用该结构信息来设计 TGM 的 Fc 融合构建体，并选择性结合 TGF-β I 型受体，并通过测试抑制 TGF-β Smad 信号传导和 Foxp3+ Treg 诱导在培养的 TGF-β 报告细胞系和小鼠脾源性 Tregs 中进行功能研究（目标 3），用作癌症免疫治疗的辅助剂。