Control of Renal Glutaminase Induction During Acidosis

酸中毒期间肾谷氨酰胺酶诱导的控制

• 批准号: 6473265
• 负责人: NORMAN P. CURTHOYS
• 金额: $ 30.05万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-10-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6473265
• 关键词: DNA footprinting; NAD(P)H dehydrogenase; RNA biosynthesis; acidity /alkalinity; acidosis; binding proteins; biological signal transduction; cell line; enzyme activity; enzyme induction /repression; gel mobility shift assay; genetic promoter element; genetic regulation; glutaminase; isozymes; kidney metabolism; laboratory rat; messenger RNA; tissue /cell culture; transcription factor

During chronic metabolic acidosis, increased renal ammoniagenesis and gluconeogenesis from glutamine are sustained, in part,, by a cell specific increase in expression of the mitochondrial glutaminase (GA) that results from the selective stabilization of the GA mRNA. Previous experiments identified an 8-base AU- sequence within the 3'- non-translated region of the GA mRNA that functions as a pH-response element (pHRE) when introduced into a chimeric reporter mRNA. This sequence was used as an affinity ligand to purify and identify zeta-crystallin/NADPH; quinone reductase as the pHRE-binding protein. The functional characterization of the pHRE was carried out in LLC-PK1-FBPase+ cells, a porcine proximal tubule-like cell line that expresses multiple forms of GA mRNAs. The corresponding porcine GA cDNAs were cloned and sequenced. Only the 4.5-kb GA mRNA contains pHREs that are identical to the sequence identified in the rat GA mRNA and only this form of GA mRNA is stabilized and increased by incubating the cells in acidic medium (Ph=6.9, 10 mM HCO3). Thus this cell lines provides a system to further characterized the molecular mechanism of GA mRNA stabilization and the associated signal transduction pathway. Finally, the 4.50kb GA mRNA is the ortholog of a newly identified isoform of the human kidney-type GA that is generated by alternative splicing of exons within the GA gene. This isoform contains a unique C-terminal domain of unknown function. The specific aims of the proposed research are: to express and characterize the isoforms of the kidney-type GA; to characterize the mechanism of GA mRNA turnover; to characterize the role of zeta-crystallin/NADPH; quinone reductase in the stabilization of the GA mRNA; and to identify the signal transduction pathway that leads to enhanced binding of zeta-crystallin/NADPH; quinone reductase to the pHRE. The results of the proposed experiments should significantly increase the understanding of the molecular mechanism that regulates this essential adaptive response and may provide insight to improve the clinical treatment of chronic acidosis.

在慢性代谢性酸中毒期间，肾氨生成和谷氨酰胺糖异生的增加在一定程度上是通过线粒体谷氨酰胺酶 (GA) mRNA 选择性稳定所导致的细胞特异性表达增加来维持的。先前的实验鉴定了 GA mRNA 3'-非翻译区域内的 8 个碱基 AU 序列，当引入嵌合报告基因 mRNA 时，该序列充当 pH 响应元件 (pHRE)。该序列用作亲和配体来纯化和鉴定zeta-晶状体蛋白/NADPH；醌还原酶作为 pHRE 结合蛋白。 pHRE 的功能表征是在 LLC-PK1-FBPase+ 细胞中进行的，LLC-PK1-FBPase+ 细胞是一种表达多种形式 GA mRNA 的猪近端小管样细胞系。克隆并测序了相应的猪 GA cDNA。只有 4.5-kb GA mRNA 含有与大鼠 GA mRNA 中鉴定的序列相同的 pHRE，并且只有这种形式的 GA mRNA 通过在酸性介质（Ph=6.9，10 mM HCO3）中孵育细胞来稳定和增加。因此，该细胞系提供了一个系统来进一步表征 GA mRNA 稳定的分子机制和相关的信号转导途径。最后，4.50kb GA mRNA 是新鉴定的人肾型 GA 亚型的直系同源物，该亚型是通过 GA 基因内的外显子选择性剪接产生的。该亚型包含一个功能未知的独特 C 端结构域。本研究的具体目标是：表达和表征肾型 GA 的亚型；表征 GA mRNA 周转的机制；表征 zeta-晶状体蛋白/NADPH 的作用；醌还原酶稳定 GA mRNA；并确定导致 zeta-晶状体蛋白/NADPH 结合增强的信号转导途径；醌还原酶至 pHRE。所提出的实验结果应显着增加对调节这种基本适应性反应的分子机制的理解，并可能为改善慢性酸中毒的临床治疗提供见解。