C-RUI Collaborative Project: Intracellular and Structural Analysis of Roles of a Cytokine Precursor in Aminoacyl-tRNA Synthetase Complexes

氨酰-tRNA合成酶复合物在蛋白质生物合成中起关键作用。了解这些复合物及其变体的结构和分布对于理解这一基本生物过程的机制和调控至关重要，这对细胞生长，维持和死亡产生了重要影响。核心多合成酶复合物含有9种氨酰-tRNA合成酶活性和3种辅助蛋白，p43，p38和p18。一种tRNA结合蛋白，p43增强复合物内特定合成酶的氨酰化活性。代谢应激诱导p43在半胱天冬酶样位点的裂解以及蛋白质的C-末端一半（EMAPII）的释放和分泌，其作为炎性细胞因子发挥作用。由于其在核心多酶氨酰-tRNA合成酶复合物中的中心位置，p43的可能功能是维持该颗粒的结构完整性。该项目将确定代谢应激对p43裂解和复合物稳定性的影响。具体地，哺乳动物和非哺乳动物细胞将在各种不利条件下生长。将从这些培养物中纯化多合成酶复合物，并表征其组成和结构。复合物、标记的p43及其加工组分的细胞定位和分布将通过亚细胞级分的免疫印迹分析来定量。这个合作研究项目将主要由本科生进行，他们将接受分子和结构生物学的培训。

Aminoacyl-tRNA synthetase complexes play a key role in protein biosynthesis. An understanding of the structure and distribution of these complexes and their variants is fundamental to understanding the mechanisms and regulation of this basic biological process, which critically impacts cell growth, maintenance and death. The core multisynthetase complex contains nine aminoacyl-tRNA synthetase activities and three auxiliary proteins, p43, p38, and p18. A tRNA binding protein, p43 enhances aminoacylation activity of specific synthetases within the complex. Metabolic stress induces cleavage of p43 at a caspase-like site as well as release and secretion of the C-terminal half of the protein (EMAPII), which functions as an inflammatory cytokine. Due to its central location in the core multienzyme aminoacyl-tRNA synthetase complex, a likely function of p43 is to maintain structural integrity of this particle. This project will determine the effect of metabolic stress on p43 cleavage and stability of the complex. Specifically, mammalian and non-mammalian cells will be grown under various adverse conditions. Multisynthetase complexes will be purified from these cultures and their compositions and structures characterized. The cellular localization and distribution of complexes, of tagged p43 and of its processed components will be quantitated by immunoblot analysis of subcellular fractions. This collaborative research project will be performed primarily by undergraduate students who will receive training in molecular and structural biology.