Structural and Functional Studies of LysRS in Mast Cell Activation

描述(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导向的串扰的结构基础。通过释放强有力的促炎介质，肥大细胞是诱发过敏反应的重要效应者。激活一种特定的转录因子--MITF--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)，LysRS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(得到LysRSp207)直接从细胞质的多tRNA合成酶复合体(MSC)中释放该酶。然后LysRSp207转位到细胞核，在那里它催化合成关键的信号分子二腺苷四磷酸(Ap4A)。在细胞核中，新生的Ap4A破坏了MITF与MITF功能的HINT1抑制子的相互作用。随着MITF-HINT1复合体的破坏，LysRSp207与MITF结合并激活MITF，促进靶基因的转录。在肥大细胞中，S207D类似磷的LysRS突变体(LysRSS207D)可以诱导Ap4A的产生，并可以概括LysRSp207的活性，促进MITF靶基因的转录。这一途径是第一个翻译和转录机制之间由磷酸化引导的串扰的例子。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模拟磷光突变触发了一个动态的结构开放，完全关闭了LysRS的翻译功能。因此，磷酸化起着功能开关的作用。我们的目的是确定Ser207的磷酸化导致LysRS从MSC支架蛋白p38解离从而从MSC解离的机制。我们还将试图了解LysRS是如何产生Ap4A并将抑制子HINT1从MITF中分离出来的。最后，我们将研究LysRS-MITF络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制之间的串扰的第一次结构性理解。此外，这些研究获得的见解可能会开辟新的治疗方法，扰乱MITF治疗过敏性疾病的功能。在美国，每五个人中就有一个以上患有过敏性疾病。 公共卫生相关性：拟议研究的目标是确定MSC-LysRS-MITF/HINT1途径轴在控制肥大细胞激活中的结构基础。结合X射线结晶学、HDX-MS和各种功能分析，所提出的研究将首次对真核细胞翻译和转录机制之间的磷酸化导向的串扰进行结构理解。此外，这些研究获得的见解可能会开辟新的治疗方法，扰乱MITF治疗过敏性疾病的功能。在美国，每五个人中就有一个以上患有过敏性疾病。

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.