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.
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