Nutritional Regulation of Cystein Dioxygenase

描述(由申请人提供)：维持较低的细胞半胱氨酸水平对于细胞完整性是必不可少的，但具有足够高的细胞半胱氨酸水平以确保足够的谷胱甘肽、辅酶A和蛋白质的合成速度也是至关重要的。肝脏半胱氨酸双加氧酶(CDO)的活性在调节半胱氨酸的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的低半胱氨酸水平。当大鼠从10%蛋白质饲料转换为40%蛋白质饲料后，肝脏CDO活性在几小时内增加了30多倍，而肝脏半胱氨酸水平保持在0.1mmoL/g以下。这在很大程度上是由于26S蛋白酶体泛素化和CDO降解减少所致。在培养的肝细胞系统中，半胱胺和半胱氨酸可以抑制CDO多泛素化，这表明半胱氨酸本身可能是调节分子。有证据表明CDO活性异常或缺乏，包括半胱氨酸升高和低硫酸盐浓度，已在各种非神经系统和神经系统疾病的个体中被报道，这表明CDO在人类群体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中所起的作用。这个项目的主要目标是进一步阐明CDO水平因饮食蛋白质或SaaS而发生显著变化的分子机制。拟议工作的具体目标是：(A)为了进一步研究CDO的两种异构体，它们的形成过程，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快速降解的催化机理和活性中心结构的细节以及所涉及的位置和构象。(c.)评价蛋白质降解，特别是泛素-蛋白酶体途径在CDO表达水平调节中的作用，并阐明半胱氨酸在CDO降解调节中的作用。(D)探讨CDO在调节细胞半胱氨酸(和谷胱甘肽)水平中的生理学意义。

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.