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: 8548364
• 负责人: Min Guo
• 金额: $ 35.83万
• 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/8548364
• 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.

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性介质，肥大细胞是引起过敏反应的重要效应子。特定转录因子的激活-MITF-是产生这些介体的关键步骤。 MITF靶基因的转录取决于共激活因子晶体基-TRNA合成酶（LYSRS），这是细胞质蛋白质合成机制的重要组成部分。 LYSRS的Ser207的磷酸化（给出LYSRSP207）指导该酶从细胞质多-TRNA合成酶配合（MSC）中释放。然后，LYSRSP207易位到核催化临界信号分子二磷酸二磷酸（AP4A）的合成。在细胞核中，新生的AP4A破坏了MITF与MITF功能的HINT1抑制剂的相互作用。随着MITF-HINT1复合物的破坏，LYSRSP207与MITF结合并激活MITF以促进靶基因的转录。在肥大细胞中，S207D磷光模拟LYSRS突变体（LYSRSS207D）诱导AP4A的产生，并可以概括LYSRSP207的活性，从而促进MITF靶基因的转录。该途径是翻译机器和转录机械之间磷酸化指导的串扰的第一个例子。我们的中心目标是了解MSC-Lysrs-Mitf/hint1途径轴的结构基础。我们的初步工作表明，S207D磷光模拟突变触发了动态结构开口，该开口完全关闭了LYSR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的磷酸化导致LYSR从MSC支架蛋白p38以及从MSC解离的机制。我们还将尝试了解LYSR如何生成AP4A并将抑制剂Hint1与MITF分离。最后，我们将研究LYSRS-MITF复合物形成的结构基础。这些研究可能会导致对真核翻译与转录机械之间的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功能治疗过敏性疾病，这是一种常见的医学疾病，遭受了美国五分之一的折磨。