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 蠕虫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，构成人类和动物之间的高疾病负担。的高患病率 蠕虫可以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。 免疫调节分子。因此，了解免疫逃避背后的机制是关键， 治疗干预小鼠胃肠道寄生虫Heligmosomoides polygyrus是一种模型， 寄生虫免疫调节;感染导致幼稚CD 4 + T细胞转化为Foxp 3+调节性T细胞， 细胞（TcB）及其扩增，其作用是广泛抑制宿主免疫系统。只有在减少 这些Treg群体中的小鼠能够驱逐寄生虫，这表明寄生虫需要这种能力。 增加Foxp 3 + T细胞的数量以在其宿主中持久存在。通过不断与 梅泽尔实验室，我们的实验室已经确定，寄生虫是使用一套分泌补体控制蛋白 (CCP)通过模拟转化生长因子β亚型刺激宿主免疫低反应性 (TGF-）。TGF-β同种型在维持适应性免疫系统中具有重要作用，其中TGF-β 已知是负责幼稚CD 4 + T细胞转化为T细胞并扩增的主要细胞因子 Treg的人口。这种免疫抑制对于维持免疫耐受性很重要，但也 通过使免疫系统的致癌逃避促进软组织癌症进展。虽然缺乏 与TGF-β的序列和结构相似性，TGF-β是寄生蛋白家族的创始成员， Maizels组，TGM 1，已经显示直接结合哺乳动物TGF-β受体，并且沿着 两种接近的同源物，TGM 2和TGM 4，上调Treg群体。鉴定残留物和蛋白质 负责结合TGF-β受体的基序可用于设计TGM形式：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-β 用于癌症免疫治疗的受体激酶抑制剂。在这份提案中，我们将确定两名成员如何 TGM家族，TGM 1和TGM 4，结合并组装TGF-β受体以激活TGF-β途径， 提供了对寄生分子拟态的深入了解。TGM 1结构域（Aim 1）和TGM 4（Aim 2）的结构 结构域单独和与它们的同源TGF-β家族受体复合将通过NMR、X-射线衍射和透射电镜测定。 射线晶体学和ITC/SPR结合研究。对受体结合贡献最大的残基将是 通过残基特异性取代和ITC/SPR结合研究鉴定（目的1，2）。此外，我们会 利用该结构信息来工程化TGM的Fc融合构建体，其具有选择性结合TGM的Fc融合构建体。 TGF-β I型受体，并测试TGF-β Smad信号传导的抑制和Foxp 3 + Treg诱导， 在培养的TGF-β 1报告细胞系和鼠脾衍生的TGF-β 1（Aim 3）中的功能研究， 癌症免疫治疗的进展。