Nutritional Regulation of Cystein Dioxygenase

描述（由申请人提供）：维持低细胞半胱氨酸水平对细胞完整性至关重要，但具有足够高的细胞半胱氨酸水平以确保谷胱甘肽、辅酶A和蛋白质的合成速率也至关重要。肝脏半胱氨酸双加氧酶（CDO）活性在调节半胱氨酸的分配以满足各种代谢需求同时通过处置过量的半胱氨酸来维持体内低半胱氨酸水平中起核心作用。在大鼠从10%蛋白质饮食转换为40%蛋白质饮食后的数小时内，肝脏CDO活性增加超过30倍，而肝脏半胱氨酸水平保持低于0.1 mmol/g。CDO的这种上调主要是由于26 S蛋白酶体对CDO的泛素化和降解减少。在培养的肝细胞系统中，半胱胺以及半胱氨酸可以抑制CDO多聚泛素化，这表明半胱氨酸本身可能是调节分子。CDO活性异常或缺陷的证据，包括半胱氨酸升高和硫酸盐浓度低，已在患有各种疾病的个体中报道，包括非神经系统和神经系统疾病，表明人群中CDO表达的异质性以及CDO活性在与衰老相关的几种慢性疾病的病因学中的作用。本项目的主要目标是进一步阐明CDO水平发生显著变化的分子机制，这些变化是对膳食蛋白质或SAA的反应。拟议工作的具体目标是：（a）为了进一步表征CDO的两种异构体、它们的形成所涉及的过程以及它们的相对酶活性。（B.）确定CDO的物理结构，并阐明半胱氨酸保护CDO免于快速降解的催化机制和活性中心结构的细节以及参与作用的位点和构象。（c.）评价蛋白质降解，特别是泛素-蛋白酶体途径在调节表达的CDO水平中的作用，并阐明半胱氨酸在调节CDO降解中的作用。（d.）探讨CDO对细胞内半胱氨酸（和谷氨酰胺）水平的调节作用及其生理意义。

DESCRIPTION (provided by applicant): Maintenance of a low cellular cysteine level is essential for cellular integrity, but having a sufficiently high cellular cysteine level to ensure adequate rates of synthesis of glutathione, coenzyme A, and proteins is also critical. Hepatic cysteine dioxygenase (CDO) activity plays a central role in regulating the partitioning of cysteine to meet various metabolic demands while at the same time maintaining low cysteine levels in the body by disposing of excess cysteine. Hepatic CDO activity increases more than 30-fold within hours after rats are switched from a 10% protein diet to a 40% protein diet, while hepatic cysteine levels remain less than 0.1 mmol/g. This upregulation of CDO is largely due to decreased ubiquitination and degradation of CDO by the 26S proteasome. The inhibition of CDO polyubiquitination can be effected by cysteamine, as well as cysteine, in cultured hepatocyte systems, suggesting cysteine itself may be the regulatory molecule. Evidence of abnormal or deficient CDO activity, including elevated cysteine and low sulfate concentrations, has been reported in individuals with a variety of diseases, both non-neurological and neurological, suggesting heterogeneity in CDO expression in the human population and a role of CDO activity in the etiology of several chronic diseases associated with aging. The major goal of this project is to further elucidate the molecular mechanisms involved in the marked changes in CDO levels that occur in response to dietary protein or SAAs. The specific aims for the proposed work are: (a.) To further characterize the two isoforms of CDO, the processes involved in their formation, and their relative enzymatic activity. (b.) To determine the physical structure of CDO and to elucidate the catalytic mechanism and details of the active site structure as well as sites and conformations involved in the action of cysteine in protecting CDO from rapid degradation. (c.) To evaluate the role of protein degradation, in particular the ubiquitin-proteasome pathway, in the regulation of the level of expressed CDO and to elucidate the role of cysteine in the regulation of CDO degradation. (d.) To evaluate the physiological significance of CDO in the regulation of cellular cysteine (and gtutathione) level.