Structure and Mechanism of Class II Aminoacyl-tRNA Synthetase

描述(由申请人提供)： 项目摘要/II类氨基酰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，并允许有序的细胞分裂和调节的分化。翻译装置的酶的突变和表达变化可能在人类疾病中呈现复杂的表型，这代表着知识的重大差距。这些疾病有限的组织和器官特异性(例如大脑)也是一个挑战。这项工作的长期目标是发现翻译机构保守成分的功能在遗传和表达水平上的特定变化如何导致人类疾病。更好地理解这种与翻译因素相关的基因表型关系将对人类健康产生重大影响。这项应用的直接目标是了解两种氨基酰基tRNA合成酶-组氨酰-tRNA合成酶(HARS)和苏氨酰-tRNA合成酶(TARS)-如何在蛋白质合成中的典型作用和在其他生理过程中的辅助作用之间进行功能转换。HARS和TARS中这种转换的变化可能分别与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，我们假设与疾病相关的突变替换降低了Histidyl-tRNAHis水平，导致近密码子抑制或营养缺陷依赖的应激反应。或者，与以前未发现的次级功能相关的蛋白质-蛋白质相互作用或信号通路可能会受到干扰。对于TARS，我们假设它与癌症的关系需要切换到非规范的血管生成相关功能，并随后通过外切体依赖的途径从细胞中输出。本申请包含两个多部分目标：(1)通过(I)测量HisRS酰化不足/编码错误对Usher IIIB表型的贡献，确定Y454S人组氨酸-tRNA合成酶与III型Usher综合征之间联系的分子基础；(Ii)确定IIIb型Usher综合征在多大程度上是HARS-蛋白质相互作用丢失的结果；以及(Iii)通过测定HARS的磷酸化状态，确定Y454S HisRS与信号改变的关联。(2)确定人类苏氨酰-tRNA合成酶在其典型的氨酰化功能和其新的血管生成功能之间切换的机制，方法是：(I)确定TARS的细胞内位置如何随着药物、缺氧、营养缺陷和诱导分泌的细胞因子(TNF-α)的变化而改变；(Ii)确定TARS的分泌和促血管生成功能与外体相关的程度；以及(Iii)确定TARS的隐蔽GTP酶功能在促进血管生成功能中的作用。这项研究对于获得关于蛋白质功能多样性和灵活性的潜在基础的知识，以及获得对感觉神经性疾病和癌症机制的进一步见解的可能性很高，具有重要意义。

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.