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：
10531540
负责人：
Ananya Mukundan
金额：
$ 5.18万
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/10531540
关键词：
Adaptive Immune System Address Affinity Amino Acid Motifs Animals Antiparasitic Agents Binding Biochemical Biological Assay Biological Products 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 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.

项目摘要 Helminth寄生虫对全球健康构成威胁，目前有几乎三分之一的人口被感染，构成人类和动物之间的高疾病燃烧。高流行蠕虫可以归因于他们使用分泌的能力操纵和避免宿主的免疫系统免疫调节分子。这是理解免疫进化背后的机制是关键的关键治疗干预。小鼠胃肠道寄生虫heligmosomoides polygyrus是一个模型寄生免疫调节；感染导致幼稚的CD4+ T细胞转化为Foxp3+调节性T- 细胞（Tregs）及其膨胀，这些细胞可广泛抑制宿主免疫系统。仅在减少时 Treg人群中的小鼠能够驱逐寄生虫，表明寄生虫需要此 FOXP3+ Treg的人口增加了其宿主的持久性。通过与 Maizels实验室，我们的实验室确定寄生虫正在使用一组分泌的完成控制蛋白（CCP）通过模仿转化生长因子β同工型来促进宿主免疫低估性（TGF-）。 TGF-β同工型在维持自适应免疫系统中具有重要作用，TGF-β 已知是负责将幼稚的CD4+ T细胞转化为Treg和膨胀的主要细胞因子 Treg人口。这种免疫抑制对于维持免疫耐受性很重要，而且也很重要通过实现免疫系统的致癌演化来促进软组织癌的进展。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TGM1已显示与哺乳动物TGF-β受体直接结合，并与 TGM2和TGM4的两个紧密的同源物上调Treg人群。识别残留物和蛋白质负责结合TGF-受体的主题可用于设计TGM的形式：1）抗寄生虫这会阻止TGM家族成员与TGF-β家族受体之间的相互作用，以及2）TGF- 受体激酶抑制剂用于癌症免疫疗法。在此提案中，我们将确定如何 TGM家族TGM1和TGM4结合并组装TGF-β受体以激活TGF-途径，提供有关寄生分子模仿的洞察力。 TGM1域（AIM 1）和TGM4（AIM 2）的结构单独的结构域与其同源TGF-β家族受体将通过NMR确定射线晶体学和ITC/SPR结合研究。对受体结合最大的残留物将是通过特定于居住的替代和ITC/SPR结合研究确定（AIM 1，2）。此外，我们将利用此结构信息来设计具有选择性结合的TGM的FC融合结构 TGF-βI型受体，并测试通过TGF-βSMAD信号传导和Foxp3+ Treg诱导的抑制在培养的TGF-报告基细胞系和鼠脾脏衍生的Treg中的功能研究（AIM 3）作为用作癌症免疫疗法的辅助。