Transcript-selective translational control of Th17 cell development and function

Th17 细胞发育和功能的转录选择性翻译控制

基本信息

批准号：
10753258
负责人：
Mark Scott Sundrud
金额：
$ 19.13万
依托单位：
DARTMOUTH-HITCHCOCK CLINIC
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：
2021-09-27 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10753258
关键词：
3&apos Untranslated Regions Acute Adaptor Signaling Protein Amino Acids Amino Acyl-tRNA Synthetases Anti-Inflammatory Agents Antiinflammatory Effect Autoimmune Binding Biology CD4 Positive T Lymphocytes Catalytic Domain Cell Differentiation process Cell Lineage Cell physiology Cells Charge Chemicals Collaborations Complex Coupled Data Development Elements Engineering Enzymes Epigenetic Process FRAP1 gene Family GCN2 protein kinase Genetic Genetic Transcription Halofuginone Helper-Inducer T-Lymphocyte Homeostasis Human IL17 gene Immune Immune response Immunoprecipitation Inflammation Inflammatory Interferon Type II Interleukin-6 Link Macrophage Maps Mediating Messenger RNA Mus Natural Products Pathogenicity Pathology Pathway interactions Plants Property Protein Biosynthesis Protein Family Protein Kinase Proteins RNA Binding RNA Interference Regulatory T-Lymphocyte Reporter Reporting Repression Research RiboTag Ribosomal Proteins Ribosomes Role Signal Transduction Starvation Stat3 protein Stromal Cells System Therapeutic Transcript Transfer RNA Transfer RNA Aminoacylation Translational Regulation Translational Repression Translations Yeasts acid stress biological adaptation to stress chronic inflammatory disease cytokine deprivation experimental study febrifugine immunoregulation in vivo inhibitor insight interleukin-23 medical schools natural product derivative novel novel drug class novel strategies novel therapeutics pharmacologic proline-tRNA receptor response scaffold sensor small molecule stem transcriptome sequencing translatome treatment strategy

项目摘要

Project Summary Differentiation of naïve CD4+ T helper (TH) cells into effector or regulatory subsets determines whether an immune response is protective, ineffective or pathogenic. Through a standing collaboration with Dr. Malcolm Whitman, we have shown that the febrifugine family of plant natural products—including the synthetic derivative halofuginone (HF)—potently and selectively blocks human and mouse Th17 cell differentiation by activating a conserved stress response pathway, called the amino acid response (AAR). We further showed that HF activates the AAR by binding to the catalytic domain of the mammalian prolyl-tRNA synthetase, EPRS, and inhibiting prolyl-tRNA aminoacylation (i.e., charging). Uncharged tRNAs accumulate in cells during periods of amino acid restriction, and signal sequential translational and transcriptional responses via the ribosome- associated protein kinase, Gcn2, to reduce amino acid demand, increase amino acid supply and facilitate a return to homeostasis. Although HF-mediated EPRS inhibition induces rapid accumulation of uncharged prolyl- tRNAs and Gcn2 activation, we have shown that HF-mediated EPRS inhibition acts independent of known amino acid sensing pathways, including Gcn2, to repress both IL-6-driven Th17 cell differentiation and IL-23- induced ‘pathogenic’ Th17 cell function by selectively repressing Stat3 protein synthesis (i.e., translation). This proposal interrogates two pathways that we hypothesize underlie HF/EPRS-mediated Stat3 translational silencing. First, EPRS (the cellular receptor for HF) has an auxiliary, or ‘moonlighting’, function as the RNA- binding sub-unit of a transcript-selective translational regulatory complex, called GAIT (gamma interferon- activated inhibitor of translation). Assembled GAIT complexes bind and repress translation of select sets of inflammation-associated transcripts containing structurally conserved stem-loop motifs in their 3’ untranslated regions (UTRs), but has yet to be formally evaluated in TH cells. Second, we have shown that both HF- mediated AAR activation (Gcn2-dependent) and HF-dependent anti-inflammatory reprogramming (Gcn2- independent) in stromal cells requires another ribosome-associated protein, Gcn1. Although Gcn1 is known only for enhancing Gcn2-binding to uncharged tRNAs, our data suggest that Gcn1 can be functionally uncoupled from Gcn2, and may serve as a branch point from the canonical AAR pathway in cells responding to amino acid stress. To elucidate the contribution of these pathways to cellular responses to amino acid deprivation, and with an eye towards engineering new drug-like small molecules that retain Stat3 translational silencing activity without inhibiting an essential enzyme (EPRS), we will: (i) employ EPRS immunoprecipitation experiments to assess HF-driven GAIT complex assembly and Stat3 mRNA-binding in developing Th17 cells; and (ii) use inducible RNAi coupled with Ribo-tag mice to determine if HF/EPRS-mediated Stat3 translational repression requires Gcn1.Together, these studies will reveal novel insights into the translational regulation of TH cell differentiation, and inform new, more selective approaches to treat Th17-driven immune pathologies.

项目概要幼稚 CD4+ T 辅助细胞 (TH) 分化为效应子或调节子集决定了是否通过与马尔科姆博士的长期合作，免疫反应是保护性的、无效的或致病性的。惠特曼，我们已经证明，常古碱家族的植物天然产物——包括合成的衍生物卤常酮 (HF)——有效且选择性地阻断人类和小鼠 Th17 细胞分化我们进一步表明，激活一种保守的应激反应途径，称为氨基酸反应（AAR）。 HF通过与哺乳动物脯氨酰-tRNA合成酶EPRS的催化结构域结合来激活AAR，并抑制脯氨酰-tRNA 氨酰化（即充电）。氨基酸限制，并通过核糖体发出顺序翻译和转录反应的信号相关蛋白激酶 Gcn2，可减少氨基酸需求、增加氨基酸供应并促进尽管 HF 介导的 EPRS 抑制会诱导不带电的脯氨酰的快速积累。 tRNA 和 Gcn2 激活，我们已经证明 HF 介导的 EPRS 抑制作用独立于已知的氨基酸传感通路，包括 Gcn2，抑制 IL-6 驱动的 Th17 细胞分化和 IL-23- 通过选择性抑制 Stat3 蛋白合成（即翻译）诱导“致病性”Th17 细胞功能。该提案询问了我们在 HF/EPRS 介导的 Stat3 翻译基础上所追求的两条途径首先，EPRS（HF 的细胞受体）具有辅助或“兼职”功能，就像 RNA 一样。转录选择性翻译调节复合体的结合亚单位，称为 GAIT（γ 干扰素- 激活的翻译抑制剂）。炎症相关转录本的 3' 非翻译区含有结构保守的茎环基序区域（UTR），但尚未在 TH 细胞中进行正式评估其次，我们已经证明 HF- 介导的 AAR 激活（Gcn2 依赖性）和 HF 依赖性抗炎重编程（Gcn2- 基质细胞中的独立）需要另一种核糖体相关蛋白 Gcn1，尽管 Gcn1 是已知的。仅用于增强 Gcn2 与不带电荷的 tRNA 的结合，我们的数据表明 Gcn1 可以在功能上与 Gcn2 解偶联，并且可能作为响应细胞中典型 AAR 途径的分支点阐明这些途径对细胞对氨基酸反应的贡献。剥夺，并着眼于设计新的药物样小分子，保留 Stat3 的翻译能力为了在不抑制必需酶 (EPRS) 的情况下沉默活性，我们将： (i) 采用 EPRS 免疫沉淀评估 Th17 细胞发育过程中 HF 驱动的 GAIT 复合体组装和 Stat3 mRNA 结合的实验； (ii) 使用诱导型 RNAi 与 Ribo-tag 小鼠结合来确定 HF/EPRS 介导的 Stat3 翻译是否有效抑制需要 Gcn1。这些研究将共同揭示对 Gcn1 翻译调控的新见解 TH 细胞分化，并为治疗 Th17 驱动的免疫病理学提供新的、更具选择性的方法。