Nutritional Regulation of y-Glutamylcysteine Synthetase

γ-谷氨酰半胱氨酸合成酶的营养调节

• 批准号: 7082073
• 负责人: MARTHA H STIPANUK
• 金额: $ 21.23万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7082073
• 关键词: cystathionine beta synthase; dietary aminoacid; dietary proteins; dietary supplements; enzyme activity; gene expression; genetic transcription; high performance liquid chromatography; laboratory rat; northern blottings; nuclear runoff assay; nutrient interaction; nutrition related tag; oxidative stress; polymerase chain reaction; posttranslational modifications; protein glutamine gamma glutamyltransferase; protein quantitation /detection; tissue /cell culture; transcription factor; western blottings

DESCRIPTION (provided by applicant): The rate-limiting enzyme for glutathione (GSH) synthesis is gamma-glutamylcysteine synthetase (GCS). Despite much study over the past two decades, the molecular and signaling mechanisms involved in regulation of GCS activity in cells are still poorly understood. Nutrient-induced regulation of GCS gene expression has not been reported except for preliminary work done in our laboratory over the past few years. Our long-term goal is to elucidate the mechanisms by which dietary sulfur amino acid or protein level induces changes in levels of the two subunits of GCS and changes in GCS activity state as well as the role of cellular cysteine status in this process. Specific objectives of this project are: (1) to determine the effect of cysteine supplementation on the expression of the modifier subunit of GCS and to determine the effect of cysteine supplementation on the ratio of the catalytic and modifier subunits in liver cells; (2) to determine the effect of dietary protein and sulfur amino acid supplementation on the expression of the modifier subunit of GCS and the ratio of the catalytic and modifier subunits of GCS in liver of intact rats; (3) to determine if the level of the GCS modifier subunit is limiting for GCS holoenzyme formation in HepG2 cells; (4) to determine the roles of GCS gene transcription and message stability in regulation of GCS subunit mRNA levels in response to cysteine concentration; (5) to determine if GCS subunit degradation rates are regulated; (6) to assess the ability of cysteine analogs (esp. cysteamine) to downregulate GCS subunit expression and/or GCS activity; (7) to determine if expression of GCS subunit genes and GCS activity are regulated in response to cysteine concentration vs. GSH concentration or thiol/disulfide redox state; and (8) to determine if transcription factor Nrf2 is necessary for activation of GCS subunit gene transcription. Capacity for GSH synthesis, availability of the precursor amino acid cysteine, and steady-state GSH levels all contribute to the body's ability to defend itself against oxidative or chemical stressors.

描述（由申请人提供）：谷胱甘肽（GSH）合成的限速酶是γ -谷氨酰半胱氨酸合成酶（GCS）。尽管在过去的二十年中进行了大量的研究，但对细胞中GCS活性调节的分子和信号机制仍然知之甚少。除了我们实验室在过去几年做的初步工作外，还没有关于营养诱导的GCS基因表达调控的报道。我们的长期目标是阐明饲粮含硫氨基酸或蛋白质水平诱导GCS两个亚基水平变化和GCS活性状态变化的机制，以及细胞半胱氨酸状态在这一过程中的作用。该项目的具体目标是：(1)确定补充半胱氨酸对GCS修饰亚基表达的影响，并确定补充半胱氨酸对肝细胞中催化亚基和修饰亚基比例的影响；(2)测定饲粮中添加蛋白质和含硫氨基酸对正常大鼠肝脏中GCS修饰亚基表达及GCS催化亚基与修饰亚基比值的影响；(3)确定GCS修饰亚基的水平是否限制HepG2细胞中GCS全酶的形成；(4)确定GCS基因转录和信息稳定性在半胱氨酸浓度对GCS亚基mRNA水平的调控中的作用；(5)确定GCS亚基降解速率是否受到调控；(6)评估半胱氨酸类似物（尤其是半胱胺）下调GCS亚基表达和/或GCS活性的能力；(7)确定GCS亚基基因的表达和GCS活性是否受到半胱氨酸浓度与GSH浓度或硫醇/二硫氧化还原状态的调节；(8)确定转录因子Nrf2是否为激活GCS亚基基因转录所必需。谷胱甘肽的合成能力、前体氨基酸半胱氨酸的可用性和稳定的谷胱甘肽水平都有助于身体抵御氧化或化学应激源的能力。