Structure and Functionof a Parasitic TGF-beta Mimic, TGM

寄生 TGF-β 模拟物 (TGM) 的结构和功能

• 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
• 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

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.

项目摘要 寄生虫是对全球健康的威胁，目前世界上近三分之一的人口 感染，在人和动物中都构成了很高的疾病负担。艾滋病的高流行率 蠕虫可以归因于它们利用分泌物操纵和逃避宿主免疫系统的能力 免疫调节分子。因此，了解免疫逃避背后的机制是 治疗性干预。小鼠胃肠道寄生虫多回螺旋体是一种 寄生免疫调节；感染导致幼稚的CD4+T细胞转化为Foxp3+调节性T细胞- 细胞(树突状细胞)及其扩张，其作用是广泛抑制宿主免疫系统。仅限在减少时 其中的Treg种群是能够驱逐寄生虫的老鼠，这表明寄生虫需要这一点 增加了Foxp3+Tregs的种群，以保持其在宿主中的持久性。通过与 Maizels实验室，我们的实验室已经确定这种寄生虫正在使用一套分泌的补体控制蛋白 (Ccp)通过模仿转化生长因子β亚型激发宿主免疫低反应 (转化生长因子)。转化生长因子-β亚型与转化生长因子-β在维持适应性免疫系统中具有重要作用。 已知是导致幼稚的CD4+T细胞转化为树突状细胞和扩增的主要细胞因子 特雷格人的数量。这种免疫抑制在维持免疫耐受方面很重要，但也 通过促进免疫系统的致癌逃逸来促进软组织癌症的进展。虽然缺乏，但 与寄生蛋白家族创始成员转化生长因子-β的序列和结构相似性 Maizels组，TGM1，已被证明直接与哺乳动物的转化生长因子-β受体结合，并与 两个接近的同源基因TGM2和TGM4上调Treg群体。残基和蛋白质的鉴定 负责结合转化生长因子-受体的基序可用于设计TGM的形式，用于：1)抗寄生虫 阻断TGM家族成员与转化生长因子-β家族受体的相互作用；2)转化生长因子- 用于癌症免疫治疗的受体激酶抑制剂。在这项提案中，我们将确定两名成员如何 TGM家族，TGM1和TGM4，结合和组装转化生长因子-β受体激活转化生长因子-途径， 提供了对寄生分子模仿的洞察力。TGM1结构域(Aim 1)和TGM4结构(Aim 2) 单独的结构域和与其同源的转化生长因子-β家族受体的复合体将通过核磁共振，X-射线光电子能谱确定。 射线结晶学和ITC/SPR结合研究。对受体结合贡献最大的残基将是 通过残基特定替代和ITC/SPR结合研究确定(目标1、2)。此外，我们还将 利用这些结构信息来设计TGM的Fc融合结构，并选择性地与 转化生长因子-βI型受体及其对转化生长因子-βSmad信号和Foxp3+Treg诱导的抑制作用 转化生长因子-报告细胞系和小鼠脾来源Tregs(Aim 3)的功能研究 癌症免疫治疗中的辅助物。