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Abstract
Homocysteine is a thiol-containing amino acid that is generated by the hydrolysis of S-adenosylhomocysteine in methylation metabolism. Elevated levels of homocysteine (10--100 μM) are associated with cardiovascular diseases, neural tube defects, Alzheimer's disease. The transsulfuration pathway represents an important route for homocysteine detoxification and provides a limiting reagent for glutathione synthesis, cysteine, thus connecting transmethylation and redox metabolism. The first step in the transsulfuration pathway is catalyzed by cystathionine β-synthase (CBS). Despite its significance, the in vivo regulation at the homocysteine junction is not well understood.
In this study, we have characterized the impact of regulating CBS activity on homocysteine and redox homeostasis. First, we investigated a potential mechanistic link between the transsulfuration pathway and the methionine dependence of cancer cells, which prevents growth when methionine is replaced by homocysteine. We found that methionine restriction in vitro resulted in a reversible >10-fold downregulation of CBS levels, which was caused by destabilization of CBS in the absence of a methionine metabolite, AdoMet. Lower CBS levels were accompanied by reduced cell viability under oxidative stress conditions. The AdoMet-related CBS downregulation was also observed in vivo, in a murine model for steatohepatitis and in human hepatocarcinoma. These findings provide a mechanistic link between the coordinate changes in methylation (low AdoMet) and redox (low GSH) metabolism seen in liver disease.
Further, we employed a mathematical model to assess the effects of allosteric regulation of CBS by AdoMet. The model predicts an increase in homocysteine levels upon the loss of allosteric activation, thus explaining hyperhomocysteinemia observed in CBS D444N patients.
Finally, we studied the basis of a sexual dimorphism in CBS regulation in mouse kidneys. We found that in mice, testosterone upregulates CBS mRNA, protein and activity levels resulting in ∼2-fold higher CBS in males than females. Lower CBS in females is correlated with an elevated plasma homocysteine, which is further amplified by a high methionine/low folate diet. These data suggest that the kidney transsulfuration pathway plays a major role in plasma homocysteine clearance.
