Structural and Functional Studies of LysRS in Mast Cell Activation

LysRS 在肥大细胞激活中的结构和功能研究

• 批准号: 8371444
• 负责人: Min Guo
• 金额: $ 39.6万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-20 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8371444
• 关键词: Active Sites; Affinity; Allergic; Amino Acyl-tRNA Synthetases; Binding; Binding Sites; Biochemical; Biological Assay; Cell Nucleus; Cells; Complex; Cytoplasmic Protein; Data; Deuterium; Disease; Dissociation; Enzymes; Gene Targeting; Genetic Transcription; Goals; Human; Hydrogen; In Vitro; Inflammation Mediators; Investigation; Ions; Link; Lysine-tRNA Ligase; MAPK14 gene; Mediator of activation protein; Medical; Monitor; Mutagenesis; Mutation; P(1),P(5)-di(adenosine-5' -)pentaphosphate; Pathway interactions; Phosphorylation; Production; Protein Biosynthesis; Proteins; Reporter; Research; Role; Scaffolding Protein; Signaling Molecule; Solutions; Structure; System; Testing; Translations; United States; Work; X-Ray Crystallography; allergic response; base; diadenosine tetraphosphate; diadenosine triphosphate; in vivo; insight; mast cell; mimetics; mutant; novel strategies; novel therapeutic intervention; reconstitution; transcription factor

DESCRIPTION (provided by applicant): The goals of the proposed research are to determine the structural basis for a phosphorylation-directed crosstalk between the eukaryotic translation and transcription machineries. By releasing potent pro-inflammatory mediators, mast cells are essential effectors in the elicitation of allergic responses. Activation of a specific transcriptio factor - MITF - is a critical step for the production of these mediators. Transcription of MITF target genes depends on a co-activator, lysyl-tRNA synthetase (LysRS), an essential component of the cytoplasmic protein synthesis machinery. Phosphorylation of Ser207 of LysRS (to give LysRSp207) directs release of this enzyme from the cytoplasmic multi-tRNA synthetase complex (MSC). LysRSp207 then translocates to the nucleus where it catalyzes the synthesis of a critical signaling molecule, diadenosine tetraphosphate (Ap4A). In the nucleus, nascent Ap4A disrupts the interaction of MITF with the HINT1 suppressor of MITF's function. With the disruption of the MITF-HINT1 complex, LysRSp207 binds to and activates MITF to promote transcription of target genes. In mast cells, a S207D phosphor-mimetic LysRS mutant (LysRSS207D) induces the production of Ap4A and can recapitulate the activity of LysRSp207, which promotes transcription of MITF target genes. This pathway is the first example of phosphorylation-directed crosstalk between the translation and transcription machineries. Our central goal is to understand the structural basis for the axis of the MSC-LysRS-MITF/HINT1 pathway. Our preliminary work showed that the S207D phosphor-mimetic mutation triggers a dynamic structural opening that completely switches off the translational function of LysRS. Thus, phosphorylation acts as a functional switch. Our aim is to determine the mechanism by which phosphorylation of Ser207 leads to dissociation of LysRS from the MSC scaffold protein p38 and therefore from the MSC. We will also attempt to understand how LysRS generates Ap4A and dissociates the suppressor HINT1 from MITF. Lastly, we will investigate the structural basis for formation of the LysRS-MITF complex. These investigations can result in the first structural understanding of the crosstalk between eukaryotic translation and transcription machineries. In addition, the insights gained by these studies could open up new therapeutic approaches to disrupting MITF function for treating allergic disorders, a common medical condition that afflicts more than one in five people in the United States. PUBLIC HEALTH RELEVANCE: The goals of the proposed research are to determine the structural basis for the axis of the MSC-LysRS-MITF/HINT1 pathway in controlling mast cell activation. Combining X-ray crystallography, HDX-MS and various functional analyses, the proposed studies will result in the first structural understanding of the phosphorylation-directed crosstalk between eukaryotic translation and transcription machineries. In addition, the insights gained by these studies could open up new therapeutic approaches to disrupting MITF function for treating allergic disorders, a common medical condition that afflicts more than one in five people in the United States.

描述（由申请人提供）：拟议研究的目标是确定真核翻译和转录机器之间磷酸化定向串扰的结构基础。通过释放有效的促炎介质，肥大细胞是引发过敏反应的重要效应器。特定转录因子 MITF 的激活是产生这些介质的关键步骤。 MITF 靶基因的转录依赖于共激活剂赖氨酰-tRNA 合成酶 (LysRS)，它是细胞质蛋白质合成机制的重要组成部分。 LysRS Ser207 的磷酸化（生成 LysRSp207）引导该酶从细胞质多 tRNA 合成酶复合物 (MSC) 中释放。然后 LysRSp207 转移到细胞核，在那里催化关键信号分子四磷酸二腺苷 (Ap4A) 的合成。在细胞核中，新生的 Ap4A 会破坏 MITF 与 MITF 功能的 HINT1 抑制因子的相互作用。随着 MITF-HINT1 复合物的破坏，LysRSp207 结合并激活 MITF 以促进靶基因的转录。在肥大细胞中，S207D 磷模拟 LysRS 突变体 (LysRSS207D) 诱导 Ap4A 的产生，并可以重现 LysRSp207 的活性，从而促进 MITF 靶基因的转录。该途径是翻译和转录机器之间磷酸化定向串扰的第一个例子。我们的中心目标是了解 MSC-LysRS-MITF/HINT1 通路轴的结构基础。我们的初步工作表明，S207D 磷模拟突变会触发动态结构开放，从而完全关闭 LysRS 的翻译功能。因此，磷酸化充当功能开关。我们的目的是确定 Ser207 磷酸化导致 LysRS 从 MSC 支架蛋白 p38 解离，从而从 MSC 解离的机制。我们还将尝试了解 LysRS 如何生成 Ap4A 并将抑制器 HINT1 从 MITF 解离。最后，我们将研究 LysRS-MITF 复合物形成的结构基础。这些研究可以导致对真核翻译和转录机器之间串扰的首次结构理解。此外，这些研究获得的见解可能会开辟新的治疗方法，以破坏 MITF 功能来治疗过敏性疾病，这是一种常见的疾病，困扰着美国五分之一以上的人。 公共健康相关性：拟议研究的目标是确定 MSC-LysRS-MITF/HINT1 通路轴控制肥大细胞激活的结构基础。结合 X 射线晶体学、HDX-MS 和各种功能分析，拟议的研究将首次对真核翻译和转录机器之间的磷酸化定向串扰进行结构理解。此外，这些研究获得的见解可能会开辟新的治疗方法，以破坏 MITF 功能来治疗过敏性疾病，这是一种常见的疾病，困扰着美国五分之一以上的人。