Structural and Functional Studies of LysRS in Mast Cell Activation

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制之间磷酸化指导的串扰的结构基础。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因子赖氨酰-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Ser 207的磷酸化（以产生LysRSp 207）指导该酶从细胞质多tRNA合成酶复合物（MSC）释放。然后LysRSp 207易位到细胞核，在那里它催化关键信号分子二腺苷四磷酸（Ap 4A）的合成。在细胞核中，新生Ap 4A破坏MITF与MITF功能的HINT 1抑制因子的相互作用。随着MITF-HINT 1复合物的破坏，LysRSp 207结合并激活MITF以促进靶基因的转录。在肥大细胞中，S207 D拟磷LysRS突变体（LysRSS 207 D）诱导Ap 4A的产生，并且可以重现LysRSp 207的活性，其促进MITF靶基因的转录。该途径是翻译和转录机制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/HINT 1通路轴的结构基础。我们的初步工作表明，S207 D磷酸模拟突变触发了一个动态的结构开放，完全关闭LysRS的翻译功能。因此，磷酸化作为一个功能开关。我们的目的是确定Ser 207磷酸化导致LysRS从MSC支架蛋白p38上解离并因此从MSC上解离的机制。我们还将试图了解LysRS如何产生Ap 4A并将抑制因子HINT 1从MITF中分离出来。最后，我们将研究LysRS-MITF复合物形成的结构基础。这些调查可以导致在第一个结构的理解真核生物的翻译和转录机器之间的串扰。此外，这些研究所获得的见解可以开辟新的治疗方法来破坏MITF功能，以治疗过敏性疾病，这是一种常见的医学疾病，在美国有超过五分之一的人患有这种疾病。 公共卫生相关性：本研究的目的是确定MSC-LysRS-MITF/HINT 1通路控制肥大细胞活化的轴的结构基础。结合X射线晶体学，HDX-MS和各种功能分析，拟议的研究将导致第一次结构理解的磷酸化指导的串扰之间的真核翻译和转录机器。此外，这些研究所获得的见解可以开辟新的治疗方法来破坏MITF功能，以治疗过敏性疾病，这是一种常见的医学疾病，在美国有超过五分之一的人患有这种疾病。

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.