Dissection of the Functions of N-terminal Arginylation

描述(由申请人提供)： 由泛素系统调节的蛋白质降解控制着许多调节蛋白质的浓度。作为Ub系统的一条途径，N-末端规则通路的靶标包括含有不稳定N-末端残基的蛋白质。N-端作用通路的功能包括染色体分离的保真度。因此，这一途径的干扰有望影响哺乳动物细胞谱系中恶性表型的出现频率。在N-末端作用的不稳定残基中，N-末端天冬氨酸、谷氨酸和半胱氨酸是独特的，因为它们在N-末端规则途径识别底物蛋白质之前与Arg结合。氨基末端天冬氨酸、谷氨酸和半胱氨酸的精氨酸化是由ATE1编码的Arg-tRNA蛋白转移酶介导的。瓦尔沙夫斯基实验室已经证明，由于心脏发育和血管新生重构方面的缺陷，缺乏N-末端精氨酸化的小鼠ATE1-/-品系会作为胚胎死亡。此外，发现N-末端半胱氨酸的精氨酸化需要预先氧化，这表明N-末端的精氨酸化分支可能作为氧感受器发挥作用。我将在小鼠身上进行分子遗传学研究，使用允许有条件地去除特定细胞系中N-末端精氨酸化的技术，详细剖析N-末端规则通路的这一主要分支的心血管和其他功能。我还将鉴定R-转移酶的生理底物，并分析ATE1-/-细胞的染色体稳定性。

DESCRIPTION (provided by applicant): Regulated protein degradation by the ubiquitin system controls the concentrations of many regulatory proteins. The targets of the N-end rule pathway, one pathway of the Ub system, include proteins bearing destabilizing N-terminal residues. The functions of the N-end role pathway include fidelity of chromosome segregation. Perturbations of this pathway are therefore expected to influence the frequency of emergence of malignant phenotypes in the mammalian cell lineages. Among destabilizing residues in the N-end role, N-terminal Asp, Glu, and Cys are unique in that they are conjugated to Arg before the recognition of a substrate protein by the N-end rule pathway. The arginylation of N-terminal Asp, Glu, and Cys is mediated by ATE1-encoded Arg-tRNA protein transferases. The Varshavsky lab has demonstrated that mouse ATE1 -/- strains, lacking N-terminal arginylation, die as embryos, owing to defects in heart development and angiogenic remodeling. In addition, arginylation of N-terminal Cys was discovered to require its prior oxidation, suggesting that the arginylation branch of the N-end role may function as an oxygen sensor. I will carry out molecular genetic studies in the mouse, using techniques that allow conditional ablation of N-terminal arginylation in specific cell lineages, to dissect, in detail, the cardiovascular and other functions of this major branch of the N-end rule pathway. I will also identify physiological substrates of R-transferases and analyze chromosome stability in ATE1 -/- ceils.