Escherichia coli leucyl-tRNA synthetase (LeuRS) is one of aminoacyl-tRNA synthetases (aaRSs) and belongs to class 1 aaRSs. The apparent steady-state kinetics of the aminoacylation reaction catalyzed by LeuRS in the pr...Escherichia coli leucyl-tRNA synthetase (LeuRS) is one of aminoacyl-tRNA synthetases (aaRSs) and belongs to class 1 aaRSs. The apparent steady-state kinetics of the aminoacylation reaction catalyzed by LeuRS in the presence of some RE3+ were studied. The results show that Mg2+ can be substituted by RE3+ for the aminoacylation reaction. The apparent K-m values for ATP and leucine are markedly different between native and Mg2+-free tRNA(1)(Leu). At high concentration of ATP there is inhibitory effect on Mg2+-free tRNA but not on native tRNA, which indicates that metal ions are a substrate of the aminoacylation reaction.展开更多
Dihydroxyacid dehydratase (DHAD), the rate-limiting enzyme in the synthesis of branched-chain (BCA) amino acids in bacteria and plants, is sensitive to oxyradical toxicity. Oxidant stress reversibly inactivates DHAD a...Dihydroxyacid dehydratase (DHAD), the rate-limiting enzyme in the synthesis of branched-chain (BCA) amino acids in bacteria and plants, is sensitive to oxyradical toxicity. Oxidant stress reversibly inactivates DHAD and causes starvation for BCA and reversible cessation of growth in Escherichia coli [1][2]. To better understand the underlying toxicity mechanisms, we have determined the cellular concentrations of charged-tRNAs for BCA, in E. coli treated with the redox-active chemical, paraquat. Contrary to expectation, in the paraquat-treated cells, the concentration of only charged leucyl-tRNA decreased dramatically;whereas, the concentrations of the other BCAs (valine and isoleucine) increased. This paradoxical result, the “paraquat effect” can be best explained if leucine is the most abundant amino acid in the E. coli proteins and therefore the rate-limiting building block in their synthesis. Based on this assumption, we investigated the concentration of free amino acids in E. coli and their relative abundances in E. coli proteins. Protein amino acid frequencies were determined by analyzing one-hundred gene bank protein sequences with software developed as described in Methods. Leucine is the most abundant amino acid in the E. coli proteins (10%) and consequently, the cellular free leucine concentration is smaller and the native charged-leucyl-tRNA levels are much higher than those of valine and isoleucine. This has relevance to humans because: leucine-deprivation was shown to be beneficial in tumor suppression [3], and leucine-supplementation was beneficial in the recovery from exercise-induced muscle loss [4][5], and leucine also occurs at a higher frequency in almost all human proteins. In three human protein categories, we examined it ranged from 9% to 17%. This predominance of leucine in proteins would make cells vulnerable to impairment of the leucine pools and could explain our results in E. coli and some of the biological effects of free leucine in humans.展开更多
TORC1(target of rapamycin complex 1)可整合营养、能量、生长因子及氨基酸等多种细胞外信号,调控基因转录、蛋白质翻译、核糖体合成等生物过程,在细胞生长和凋亡中发挥极为重要的作用。亮氨酰-tRNA合成酶(LeuRS)的经典功能是催化合成...TORC1(target of rapamycin complex 1)可整合营养、能量、生长因子及氨基酸等多种细胞外信号,调控基因转录、蛋白质翻译、核糖体合成等生物过程,在细胞生长和凋亡中发挥极为重要的作用。亮氨酰-tRNA合成酶(LeuRS)的经典功能是催化合成亮氨酰-tRNA直接参与遗传信息的解码合成蛋白质。最新研究结果表明人细胞质LeuRS除了经典功能外,还参与调控TORC1途径。综述了LeuRS的非经典功能,它是如何通过感受细胞内的亮氨酸浓度来调节TORC1活性的。这些结果表明了古老的氨基酰-tRNA合成酶家族在进化的过程中被赋予了新的功能。展开更多
文摘Escherichia coli leucyl-tRNA synthetase (LeuRS) is one of aminoacyl-tRNA synthetases (aaRSs) and belongs to class 1 aaRSs. The apparent steady-state kinetics of the aminoacylation reaction catalyzed by LeuRS in the presence of some RE3+ were studied. The results show that Mg2+ can be substituted by RE3+ for the aminoacylation reaction. The apparent K-m values for ATP and leucine are markedly different between native and Mg2+-free tRNA(1)(Leu). At high concentration of ATP there is inhibitory effect on Mg2+-free tRNA but not on native tRNA, which indicates that metal ions are a substrate of the aminoacylation reaction.
文摘Dihydroxyacid dehydratase (DHAD), the rate-limiting enzyme in the synthesis of branched-chain (BCA) amino acids in bacteria and plants, is sensitive to oxyradical toxicity. Oxidant stress reversibly inactivates DHAD and causes starvation for BCA and reversible cessation of growth in Escherichia coli [1][2]. To better understand the underlying toxicity mechanisms, we have determined the cellular concentrations of charged-tRNAs for BCA, in E. coli treated with the redox-active chemical, paraquat. Contrary to expectation, in the paraquat-treated cells, the concentration of only charged leucyl-tRNA decreased dramatically;whereas, the concentrations of the other BCAs (valine and isoleucine) increased. This paradoxical result, the “paraquat effect” can be best explained if leucine is the most abundant amino acid in the E. coli proteins and therefore the rate-limiting building block in their synthesis. Based on this assumption, we investigated the concentration of free amino acids in E. coli and their relative abundances in E. coli proteins. Protein amino acid frequencies were determined by analyzing one-hundred gene bank protein sequences with software developed as described in Methods. Leucine is the most abundant amino acid in the E. coli proteins (10%) and consequently, the cellular free leucine concentration is smaller and the native charged-leucyl-tRNA levels are much higher than those of valine and isoleucine. This has relevance to humans because: leucine-deprivation was shown to be beneficial in tumor suppression [3], and leucine-supplementation was beneficial in the recovery from exercise-induced muscle loss [4][5], and leucine also occurs at a higher frequency in almost all human proteins. In three human protein categories, we examined it ranged from 9% to 17%. This predominance of leucine in proteins would make cells vulnerable to impairment of the leucine pools and could explain our results in E. coli and some of the biological effects of free leucine in humans.
基金supported by the Natural Science Foundation of China (30170224 ,30270310 and 30330180) ,the Chinese Academy of Sciences (KSCX-2-2-04) ,Committee of Science and Technologyin Shanghai (02DJ140567)
基金supported by the National Natural Science Foundation of China (No. 31960192,31900842)the Natural Science Foundation of Jiangxi Province,China (No. 20192BAB205081,20202ACBL216004)+1 种基金the Science and Technology Foundation of Jiangxi Provincial Department of Education,China (No. GJJ180560)the Humanities and Social Sciences Foundation in Higher Institutions of Jiangxi Province,China (No. TY17210)。
文摘蛋白质代谢平衡紊乱是诱发骨骼肌萎缩的根本原因,蛋白质合成减少则直接导致衰老性骨骼肌萎缩的发生与发展。亮氨酰-tRNA合成酶(leucyl-tRNA synthetase, LeuRS)的经典功能是催化亮氨酸与其同工tRNA之间连接形成氨基酰,在生物体内遗传解码过程中具有重要作用。随着近年对LeuRS蛋白研究的深入,人们认为其可能通过行使非经典功能而在衰老骨骼肌蛋白质代谢稳态调节中发挥关键作用。本文综述了氨基酰-tRNA合成酶和LeuRS的结构与生物学特性,并重点对LeuRS作为细胞内亮氨酸传感器调控衰老骨骼肌细胞蛋白质合成的研究进展进行总结,分析了LeuRS响应运动与氨基酸摄入等合成代谢刺激,活化哺乳动物雷帕霉素靶蛋白复合物1 (mammalian target of rapamycin complex 1, mTORC1)信号转导通路的作用机制,以期为衰老性骨骼肌萎缩的预防和诊治提供新的思路。
文摘TORC1(target of rapamycin complex 1)可整合营养、能量、生长因子及氨基酸等多种细胞外信号,调控基因转录、蛋白质翻译、核糖体合成等生物过程,在细胞生长和凋亡中发挥极为重要的作用。亮氨酰-tRNA合成酶(LeuRS)的经典功能是催化合成亮氨酰-tRNA直接参与遗传信息的解码合成蛋白质。最新研究结果表明人细胞质LeuRS除了经典功能外,还参与调控TORC1途径。综述了LeuRS的非经典功能,它是如何通过感受细胞内的亮氨酸浓度来调节TORC1活性的。这些结果表明了古老的氨基酰-tRNA合成酶家族在进化的过程中被赋予了新的功能。
