Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin-26 S proteasome system in the living rat

Abstract

Mammalian metabolism of ingested cysteine is conducted principally within the liver. The liver tightly regulates its intracellular cysteine pool to keep levels high enough to meet the many catabolic and anabolic pathways for which cysteine is needed, but low enough to prevent toxicity. One of the enzymes the liver uses to regulate cysteine levels is CDO (cysteine dioxygenase). Catalysing the irreversible oxidation of cysteine, CDO protein is up-regulated in the liver in response to the dietary intake of cysteine. In the present study, we have evaluated the contribution of the ubiquitin-26 S proteasome pathway to the diet-induced changes in CDO half-life. In the living rat, inhibition of the proteasome with PS1 (proteasome inhibitor 1) dramatically stabilized CDO in the liver under dietary conditions that normally favour its degradation. Ubiquitinated CDO intermediates were also seen to accumulate in the liver. Metabolic analyses showed that PS1 had a significant effect on sulphoxidation flux secondary to the stabilization of CDO but no significant effect on the intracellular cysteine pool. Finally, by a combination of in vitro hepatocyte culture and in vivo whole animal studies, we were able to attribute the changes in CDO stability specifically to cysteine rather than the metabolite 2-mercaptoethylamine (cysteamine). The present study represents the first demonstration of regulated ubiquitination and degradation of a protein in a living mammal, inhibition of which had dramatic effects on cysteine catabolism.

Figure 1. A metabolic flowchart illustrating the position of CDO within the many pathways of cysteine catabolism

CDO catalyses the first step in the CSAD pathway, cysteine→CSA→hypotaurine→taurine (highlighted in boldface), and shunts cysteine towards the production of CSA, pyruvate, sulphate, hypotaurine and taurine. For the purpose of clarity, multistep pathways apart from the CSAD route have been condensed to a single arrow. ‘?’, unidentified enzyme.

Figure 2. Effects of dietary manipulation and proteasome inhibition on levels of liver CDO protein in rats

For SDS/PAGE analysis, 60 μg of soluble liver protein was loaded per lane on to a 12% (w/v) polyacrylamide gel. (A) A representative Western blot, probed first for CDO and then for actin as a loading control, is shown for one such analysis. (B) Average relative hepatic CDO protein levels. To determine the relative changes in protein levels for all samples, the absorbance (A) of each CDO band was measured by densitometry. Absorbances were then normalized by HP 0 h control CDO values (run on each gel) and expressed as a percentage of these controls in the bar graph (B). LP+CYS, LP diet +8.12 g of cysteine/kg of diet; LP+MEA, LP diet +7.2 g of MEA/kg of diet; LP+PS1, LP diet+intraperitoneal injection of PS1 (17 mg/kg). *P<0.01 versus initial (0 h HP) value. N=three rats per group.

Figure 3. Effects of dietary manipulation and proteasome inhibition on levels of ubiquitinated CDO

A Western blot showing the effects of dietary manipulation and proteasome inhibition on steady-state levels of ubiquitinated liver CDO. Liver homogenate (125 μg) was loaded into each lane. A molecular mass ladder (MW) is shown. Ubiquitinated species are designated CDO-Ub, with the total number of attached ubiquitin moieties indicated by a subscript numeral and their corresponding molecular masses.

Figure 4. A comparative evaluation of cysteine-like compounds to prevent CDO degradation in primary hepatocyte cultures

(A) Structures of the cysteine analogues tested. (B) The effect of each of these compounds on CDO protein, determined by SDS/PAGE and Western blotting. Lactacystin, a specific proteasome inhibitor, was used at a final concentration of 5 μM in the medium for comparison purposes. All other compounds were tested at a concentration of 1 mM. Results shown in (B) are presented as means±S.E.M.