Structure and Mechanism of Class II Aminoacyl-tRNA Synthetase

描述（由申请人提供）： II类氨酰-tRNA合成酶的结构和机制所有细胞都需要蛋白质合成来维持其核心生理功能，以及允许有序的细胞分裂和调节分化。翻译装置中的酶的突变和表达变化可以在人类疾病中呈现复杂的表型，这代表了知识上的主要差距。这些疾病的有限的组织和器官特异性性质（例如，脑）也是一个挑战。这项工作的长期目标是发现特定的变化，无论是在遗传和表达水平，在翻译装置的保守组件的功能带来人类疾病。更好地理解这种基因表型关系，因为它涉及到翻译因子将对人类健康产生重大影响。本申请的直接目标是了解两种氨酰-tRNA合成酶-组氨酰- tRNA合成酶（HARS）和苏氨酰-tRNA合成酶（TARS）-如何在它们在蛋白质合成中的典型作用和在其他生理过程中的次要作用之间进行功能转换。HARS和TARS中这种转换的改变可能分别与IIIb型Usher综合征和癌症有关。在这里，我们将测试特定的假设来解释这些次要功能。对于HARS和IIIb型Usher综合征，我们假设与该疾病相关的突变取代降低了组氨酰-tRNACis水平，导致接近密码子抑制或营养缺陷依赖性应激反应。或者，蛋白质-蛋白质相互作用或与先前未发现的次要功能相关的信号传导途径可能受到干扰。对于TARS，我们假设其与癌症的关联需要转换为非经典的血管生成相关功能，并随后通过外泌体依赖性途径从细胞中输出。该申请包含两个多部分目的：（1）通过（i）测量HisRS酰化不足/错误编码对Usher IIIB表型的贡献;（ii）确定IIIb型Usher综合征是HARS-蛋白质相互作用丧失的结果的程度，确定Y 454 S人组氨酰-tRNA合成酶和III型Usher综合征之间联系的分子基础;和（iii）通过确定HARS的磷酸化状态来确定Y 454 S HisRS与改变的信号传导的关联。(2)确定人苏氨酰-tRNA合成酶在其经典氨酰化功能和其新的血管生成功能之间转换的机制，通过（i）确定TARS的细胞内位置如何响应于药物、缺氧、营养缺乏和引起分泌的细胞因子（TNF-α）而变化;（ii）确定TARS分泌和促血管生成功能与外来体相关的extnt;和（iii）确定TARS的隐蔽GT3功能在促血管生成功能中的作用。这项研究对于获得有关蛋白质功能多样性和灵活性的基础知识，以及进一步深入了解感觉神经疾病和癌症机制的可能性很大。

DESCRIPTION (provided by applicant): Project Summary/Abstract Structure and Mechanism of Class II Aminoacyl-tRNA Synthetases All cells require protein synthesis to maintain their core physiology, as well as permit orderly cel division and regulated differentiation. Mutations and expression changes in enzymes of the translation apparatus can present with complex phenotypes in human diseases, representing a major gap in knowledge. The limited tissue and organ specific nature (e,g, the brain) of these diseases is also a challenge. The long term objective of this work is to discover how specific changes, both genetic and at the level of expression, in the function of conserved components of the translational apparatus bring about human disease. A better understanding of this gene phenotype relationship as it relates to translation factors will have major implications for human health. The immediate goal of this application is to understand how two aminoacyl-tRNA synthetases- histidyl- tRNA synthetase (HARS) and threonyl-tRNA synthetase (TARS)- undergo functional switching between their canonical role in protein synthesis and secondary roles in other physiological processes. Alterations in this switching in HARS and TARS may be linked to Type IIIb Usher syndrome and cancer, respectively. Here, we will test specific hypotheses to account for these secondary functions. For HARS and the Type IIIb Usher syndrome, we hypothesize that the mutant substitution linked to the disease decreases histidyl-tRNAHis levels, leading to near codon suppression or nutritional deficit dependent stress responses. Alternatively, protein- protein interactions or signaling pathways associated with a previously undiscovered secondary function may be perturbed. For TARS, we hypothesize that its association with cancer requires a switch to a non-canonical angiogenesis related function and its subsequent export from cells via an exosome dependent pathway. The application contains two multi-part Aims: (1) Determine the molecular basis of the link between Y454S human histidyl-tRNA synthetase and type III Usher syndrome by (i) measuring the contribution of HisRS under-acylation/miscoding to the Usher IIIB phenotype; (ii) determining the extent to which Type IIIb Usher Syndrome is the result of a loss a HARS-protein protein interaction; and (iii) determining the association of Y454S HisRS with altered signaling by determining the phosphorylation state of HARS. (2) Determine the mechanism by which human threonyl-tRNA synthetase switches between its canonical aminoacylation function and its novel angiogenic function by (i) Determining how the intracellular location of TARS changes in response to drug, hypoxia, nutritional deficit, and cytokines that elicit secretion (TNF-a); (ii) determining the extnt to which TARS secretion and pro-angiogenic functios are associated with exosomes; and (iii), determining the role of TARS's cryptic GTPase function in pro-angiogenic functions. The research is significant for the knowledge gained regarding the underlying basis of protein functional diversity and flexibility, and for the high likelihood that further insights into the mechanisms of sensorineural disease and cancer will be gained.