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 Helminth寄生虫对全球健康有威胁，目前有将近三分之一的人口 被感染，构成人类和动物的高疾病负担。 蠕虫可以归因于他们使用分泌的能力操纵和逃避宿主的免疫系统 因此，免疫调节分子。 治疗性干预。 寄生的免疫调节； 细胞（Tregs）和扩展，仅在还原时才能广泛抑制宿主的免疫系统 这是寄生虫的Treg，表明寄生虫需要的就是这样。 通过与The的持续合作，FOXP3+ Treg的人口增加了。 Maizels实验室，我们的实验室确定了分泌补体控制蛋白的寄生虫液 （CCP）通过模仿转化生长因子β同工型来促进宿主免疫反应性。 （TGF-）。 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人群的免疫抑制作用对于维持免疫耐受性很重要。 通过缺乏免疫系统的肿瘤性e术来促进软组织癌的进展 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的两个近距离物理学上的近距离上调了Treg人群。 负责结合TGF-受体的主题可以是TGM的工程师形式：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- 受体激酶吸入了癌症免疫疗法。 TGM家族TGM1和TGM4结合并组装TGF-β受体以激活TGF-途径， 将洞察到寄生分子模仿中。 单独的结构域和与同源TGF-β家族受体的复杂性将通过NMR确定 射线晶体学和ITC/SPR结合研究。 通过残留的特异性变异和ITC/SPR结合研究（AM 1，2）。 利用此结构信息来设计TGM的FC融合结构，然后选择 TGF-β型受体，并测试抑制TGF-βSMAD信号传导和FOXP3+ Trug指示 培养的TGF-报告基细胞lin和鼠脾脏衍生的Treg（AIM 3）的功能研究（AIM 3） 癌症免疫疗法的辅助。