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首页> 外文学位 >Cloning and characterization of citrin: The aspartate/glutamate carrier mutated in adult-onset type II citrullinemia.
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Cloning and characterization of citrin: The aspartate/glutamate carrier mutated in adult-onset type II citrullinemia.

机译：柠檬酸的克隆和特征：天冬氨酸/谷氨酸携带者在成年发作的II型瓜氨酸血症中突变。

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摘要

Citrullinemia is a human metabolic deficiency of argininosuccinate synthetase (ASS); the enzyme that catalyzes the formation of argininosuccinate from citrulline and aspartate during the operation of the urea cycle. Two forms of citrullinemia have been described previously: CTLN1 and CTLN2. Unlike CTLN1 that is caused by mutations in the ASS gene, linkage analysis has excluded the ASS locus as the primary defect in CTLN2. Subsequently, homozygosity mapping has localized the CTLN2 locus to human chromosome 7q21.3.; This thesis describes the identification of the gene responsible for CTLN2, SLC25A13, and characterization of its protein product citrin in mice. Following its cloning, citrin was shown by others (Palmieri et al. [2001] EMBO J. 20, 5060-9) to be the electrogenic aspartate/glutamate (Asp/Glu) carrier studied decades previously. The function of citrin as an Asp/Glu carrier implicate it in not just urea-cycle function and maintenance, but also in pathways of the malate-aspartate (Mal-Asp) NADH shuttle, gluconeogenesis and cellular redox state.; Towards understanding the role of citrin in both metabolism and disease, the orthologue of SLC25A13 was studied in mouse. Following cloning of Slc25a13 and investigation of its mRNA expression pattern pre- and postnatally, a targeted gene disruption of Slc25a13 exon 10 was undertaken. The resultant Slc25a13-‘knock-out’ (Ctrn-deficient) mice were analyzed for a variety of molecular, biochemical and physiological parameters. Assays using isolated liver mitochondria revealed decreased activities in both aspartate transport and in Mal-Asp shuttle activity. Liver perfusion experiments showed quantitative deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate to pyruvate ratio in hepatocytes. However, the Ctrn −/− mice, up to one year of age, failed to show symptoms of CTLN2 due to normal hepatic ASS activity. Additionally, no evidence of physiological deficiencies in glucose homeostasis or amino-acid metabolism were observed. By using various nitrogen-loading treatments, the Ctrn−/− mice displayed only small changes in liver ammonium metabolism and hepatic amino-acid levels. Overall, the results indicate that citrin deficiency in mice alone may not be sufficient for the onset of a CTLN2-like phenotype, suggesting differences in citrin-dependent biochemical pathways between mice and humans.
机译：瓜氨酸血症是人的精氨酸琥珀酸合成酶（ASS）的代谢缺陷。在尿素循环操作过程中催化由瓜氨酸和天冬氨酸形成精氨酸琥珀酸酯的酶。先前已描述了两种形式的瓜氨酸血症：CTLN1和CTLN2。与由ASS基因突变引起的CTLN1不同，连锁分析排除了ASS基因座作为CTLN2的主要缺陷。随后，纯合性作图已将CTLN2基因座定位于人类染色体7q21.3。本文描述了小鼠CTLN2基因 SLC25A13 的鉴定及其蛋白产物柠檬酸的特性。柠檬酸克隆后，其他人（Palmieri et al 。[2001] EMBO J 。 20 ，5060-9）显示为柠檬酸。数十年来研究过的天冬氨酸/谷氨酸（Asp / Glu）载体。柠檬酸作为Asp / Glu载体的功能不仅使其具有尿素循环功能和维持功能，而且还涉及苹果酸-天冬氨酸（Mal-Asp）NADH穿梭，糖异生和细胞氧化还原状态的途径。为了了解柠檬酸在代谢和疾病中的作用，在小鼠中研究了 SLC25A13 的直向同源物。克隆 Slc25a13 并对其出生前和产后的mRNA表达模式进行研究后，进行了 Slc25a13 外显子10的靶向基因破坏。分析了所得的 Slc25a13 -“敲除”（ Ctrn 缺陷）小鼠的各种分子，生化和生理参数。使用分离的肝线粒体的分析显示，天冬氨酸转运和Mal-Asp穿梭活性均降低。肝灌注实验显示，氨尿素生成，乳酸盐糖异生和肝细胞中乳酸与丙酮酸之比的增加存在定量缺陷。然而，由于正常的肝脏ASS活性， Ctrn -/-小鼠直到1岁时仍未显示CTLN2症状。另外，未观察到葡萄糖稳态或氨基酸代谢中生理缺陷的证据。通过使用各种氮负荷处理， Ctrn -/-小鼠在肝铵代谢和肝氨基酸水平上仅表现出很小的变化。总体而言，结果表明，仅小鼠体内的柠檬酸不足可能不足以引发类似CTLN2的表型，这表明小鼠与人之间依赖于柠檬酸的生化途径存在差异。

著录项

  • 作者

    Sinasac, David Steven.;

  • 作者单位

    University of Toronto (Canada).;

  • 授予单位 University of Toronto (Canada).;
  • 学科 Biology Genetics.; Biology Molecular.; Health Sciences Pathology.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 202 p.
  • 总页数 202
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 遗传学;分子遗传学;病理学;
  • 关键词

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