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

• 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
• 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' 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

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 幼稚的CD 4 + T辅助细胞（TH）分化为效应细胞或调节细胞亚群决定了免疫应答是否是免疫应答。 免疫应答是保护性的、无效的或致病的。通过与马尔科姆博士的长期合作 惠特曼，我们已经表明，febrifugine家族的植物天然产品-包括合成的 卤夫酮衍生物（HF）-通过以下途径有效并选择性地阻断人和小鼠Th 17细胞分化： 激活保守的应激反应途径，称为氨基酸反应（AAR）。我们进一步研究发现 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）。不带电荷的tRNA在细胞内积累， 的氨基酸限制，并通过核糖体的信号顺序翻译和转录反应- 相关蛋白激酶Gcn 2，以减少氨基酸需求，增加氨基酸供应，并促进 恢复体内平衡虽然HF介导的EPRS抑制诱导不带电荷的脯氨酰的快速积累， tRNAs和Gcn 2激活，我们已经表明HF介导的EPRS抑制作用不依赖于已知的 氨基酸传感途径，包括Gcn 2，以抑制IL-6驱动的Th 17细胞分化和IL-23驱动的Th 17细胞分化。 通过选择性抑制Stat 3蛋白合成诱导“致病性”Th 17细胞功能（即，翻译）。这 我们假设HF/EPRS介导的Stat 3翻译的两条途径 沉默首先，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物的结合亚单位，称为GAIT（γ干扰素- 翻译抑制剂（inhibitor of translation）。组装的GAIT复合物结合并抑制选择的GAIT基因组的翻译。 在其3'非翻译区含有结构保守的茎环基序的炎症相关转录物 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（Gcn 2依赖性）和HF依赖性抗炎重编程（Gcn 2- 独立）在基质细胞中需要另一种核糖体相关蛋白Gcn 1。虽然GCN 1已知 我们的数据表明，Gcn 1可以在功能上增强Gcn 2与不带电荷的tRNA的结合， 与Gcn 2解偶联，并且可以作为响应于Gcn 2的细胞中经典AAR途径的分支点。 氨基酸胁迫为了阐明这些途径对细胞对氨基酸反应的贡献， 剥夺，并着眼于工程新的药物样小分子，保留Stat 3翻译 沉默活性而不抑制必需酶（EPRS），我们将：（i）采用EPRS免疫沉淀 评估HF驱动的GAIT复合物组装和发育中的Th 17细胞中的Stat 3 mRNA结合的实验; 和（ii）使用与Ribo-标签小鼠偶联的诱导型RNAi来确定HF/EPR介导的Stat 3翻译 总之，这些研究将揭示新的见解的翻译调控， TH细胞分化，并告知新的，更具选择性的方法来治疗Th 17驱动的免疫病理学。