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：
10373280
负责人：
Mark Scott Sundrud
金额：
$ 9.14万
依托单位：
SCRIPPS FLORIDA
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 2022-04-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10373280
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 Eye FRAP1 gene Family GCN2 protein kinase Gait Genetic Genetic Transcription Halofuginone Helper-Inducer T-Lymphocyte Homeostasis Human Immune Immune response Immunoprecipitation Inflammation Inflammatory Interferon Type II Interleukin-17 Interleukin-6 Link Maps Mediating Messenger RNA Mus Natural Products Pathogenicity Pathology Pathway interactions Pharmacology Plants Property Protein Biosynthesis Protein Family Protein Kinase Proteins RNA Binding RNA Interference Regulatory T-Lymphocyte Reporter Reporting Research RiboTag Ribosomal Proteins Ribosomes Role Signal Transduction Starvation Stat3 protein Stromal Cells Structure System Therapeutic Transcript Transfer RNA Transfer RNA Aminoacylation Translational Regulation Translational Repression Translations Yeasts acid stress base biological adaptation to stress chronic inflammatory disease cytokine deprivation experimental study febrifugine immunoregulation in vivo inhibitor/antagonist insight interleukin-23 macrophage medical schools novel novel drug class novel strategies novel therapeutics 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.

项目摘要将幼稚的CD4+ T辅助（Th）细胞分化为效应子或调节子集，确定是否确定免疫反应受到保护，无效或致病性。通过与Malcolm博士的长期合作惠特曼（Whitman），我们已经证明了植物天然产品的Febrifugine家族 - 包括合成衍生物卤素（HF） - 通过，有选择地阻止人和小鼠Th17细胞分化激活一个称为氨基酸反应（AAR）的保守应力反应途径。我们进一步显示 HF通过与哺乳动物脯氨酰-TRNA合成酶EPRS的催化结构域结合而激活AAR 并抑制丙基-TRNA氨基酰化（即充电）。未充电的TRNA在周期内积聚在细胞中氨基酸限制，以及通过核糖体的信号顺序翻译和转录响应相关蛋白激酶GCN2，以减少氨基酸的需求，增加氨基酸供应并促进尽管HF介导的EPRS抑制作用诱导未加成的prlelyl-迅速积累 TRNA和GCN2激活，我们已经表明HF介导的EPR抑制作用与已知的无关包括GCN2在内的氨基酸传感途径，以反映IL-6驱动的Th17细胞分化和IL-23- 通过选择性反映STAT3蛋白合成（即翻译），诱导的“致病” Th17细胞功能。这提案询问我们假设HF/EPRS介导的STAT3转化基础的两种途径沉默。首先，EPRS（HF的细胞受体）具有辅助或“月光”功能作为RNA- 转录选择性翻译调节复合物的结合子单位称为步态（伽马干扰素 - 激活的翻译抑制剂）。组装的步态配合物结合并反映了精选集的翻译与炎症相关的转录本，其中包含结构构成的茎环图案的3'未翻译中区域（UTRS），但尚未在TH细胞中进行正式评估。其次，我们已经证明了两个HF- 介导的AAR激活（依赖GCN2依赖性）和HF依赖性抗炎重编程（GCN2-- 独立）在基质细胞中需要另一种与核糖体相关的蛋白GCN1。虽然GCN1是已知的我们的数据仅用于增强gcn2结合到未充电的trNA，我们的数据表明GCN1可以在功能上与GCN2取消耦合，可以用作响应的细胞中规范AAR途径的分支点氨基酸应激。为了阐明这些途径对细胞对氨基酸的反应的贡献剥夺，并着眼于工程新药样的小分子，以保留STAT3翻译沉默活动而不抑制必需酶（EPRS），我们将：（i）员工EPRS免疫沉淀在开发Th17细胞中评估HF驱动的步态复合体组装和STAT3 mRNA结合的实验；（ii）使用可诱导的RNAi与Ribo-Tag小鼠相结合来确定HF/EPRS介导的STAT3转换是否抑制需要gcn1。这些研究将揭示对翻译调节的新见解细胞分化，并为治疗TH17驱动的免疫病理学的新的，更有选择性的方法提供信息。