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和蛋白质的适当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分配以满足各种代谢需求的同时，通过处理多余的半胱氨酸来维持体内低半胱氨酸水平。在大鼠从10%蛋白质饮食切换到40%蛋白质饮食后，肝脏CDO活性在数小时内增加了30倍以上，而肝脏半胱氨酸水平仍低于0.1 mmol/g。这种CDO的上调主要是由于26S蛋白酶体降低了CDO的泛素化和降解。在培养的肝细胞系统中，半胱胺和半胱氨酸均可抑制CDO多泛素化，提示半胱氨酸本身可能是调控分子。在多种疾病（包括非神经系统疾病和神经系统疾病）患者中，已经报道了异常或缺乏CDO活性的证据，包括半胱氨酸升高和硫酸盐浓度低，这表明CDO在人群中表达的异质性以及CDO活性在几种与衰老相关的慢性疾病的病因学中所起的作用。该项目的主要目标是进一步阐明膳食蛋白质或SAAs引起的CDO水平显著变化的分子机制。建议工作的具体目标是：(a)进一步表征CDO的两种同工异构体，其形成的过程，以及它们的相对酶活性。(b)。确定CDO的物理结构，阐明半胱氨酸保护CDO快速降解所涉及的活性位点结构和构象的催化机理和细节。(c)。评估蛋白质降解，特别是泛素-蛋白酶体途径在调控CDO表达水平中的作用，并阐明半胱氨酸在调控CDO降解中的作用。(d)。评价CDO在调节细胞半胱氨酸（谷胱甘肽）水平中的生理意义。

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.