Transcript-selective translational control of Th17 cell development and function

项目摘要 将幼稚的CD4+T辅助细胞(TH)分化为效应或调节亚群决定了 免疫反应是保护性的、无效的或致病性的。通过与马尔科姆博士长期合作 惠特曼，我们已经证明了发烧的植物天然产物家族-包括合成的 衍生物常青藤酮(HF)-通过以下方式有效和选择性地阻断人和小鼠Th17细胞的分化 激活一条保守的应激反应途径，称为氨基酸反应(AAR)。我们进一步展示了 该HF通过与哺乳动物Prolyl-tRNA合成酶的催化域Eprs结合来激活AAR， 抑制Prolyl-tRNA氨酰化(即荷电)。未充电的tRNA在细胞周期中积累 氨基酸限制，并通过核糖体信号顺序的翻译和转录反应- 相关蛋白激酶，GCN2，减少氨基酸需求，增加氨基酸供应，促进 回归动态平衡。尽管HF介导的Eprs抑制诱导未带电的Pro-1迅速积累- TRNAs和GCN2激活，我们已经证明了HF介导的EPRS抑制作用不依赖于已知的 氨基酸敏感通路，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蛋白的合成(即翻译)，诱导Th17细胞的“致病”功能。这 提案询问了我们假设的两条途径，这两条途径是HF/EPRS介导的STAT3翻译的基础 沉默。首先，Eprs(HF的细胞受体)有一种辅助功能，即“兼职”，作用是RNA-- 转录选择性翻译调节复合体的结合亚单位，称为步态(伽马干扰素- 激活的翻译抑制物)。组装的步态复合体结合并抑制选定的几组步态的平移 炎症相关转录本含有结构保守的3‘端未翻译的茎环基序 区域(UTRs)，但尚未在TH单元中进行正式评估。第二，我们已经证明了两种HF- 介导的AAR激活(GCN2依赖)和HF依赖的抗炎重编程(GCN2- 在基质细胞中)需要另一种核糖体相关蛋白Gcn1。尽管已知Gcn1 仅为了增强GCN2与未充电的tRNAs的结合，我们的数据表明Gcn1可以在功能上 从GCN2解偶联，并可能作为反应细胞中典型的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，并着眼于设计新的类似药物的小分子，保留STAT3的翻译 为了在不抑制必需酶(Eprs)的情况下抑制沉默活性，我们将：(I)使用Eprs免疫沉淀 在发育中的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； 和(Ii)使用可诱导的RNAi与Ribo-Tag小鼠耦合来确定HF/EPRS介导的STAT3是否翻译 抑制需要Gcn1。同时，这些研究将揭示对翻译调控的新见解 TH细胞分化，并提供新的、更具选择性的方法来治疗Th17驱动的免疫病理。

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.