A Yeast Bioassay System for the Identification of Compounds That Can Lower Plasma Homocysteine Levels

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Background:

Coronary artery disease (CAD) and stroke are the two leading causes of death world-wide, together accounting for over 10 million deaths annually. The human and economic costs of these diseases are staggering. To date, an enormous amount of clinical effort has gone toward targeting the commonly known risk factors for CAD and stroke (hypertension, hyperlipidemia, smoking, obesity, metabolic disease, gender, age). However, the necessity for clarification of the roles of other risk factors in the etiology of these complex conditions remains high. Further delineation of the mechanisms of action of all the risk factors will enable design of more effective interventions.

High plasma levels of homocysteine as a risk factor for CAD are engendering a great deal of interest in both the scientific and popular media. Through study of homocysteinuria, an autosomal recessive inborn error of metabolism, homocysteine's role in heart disease and stroke is becoming clearer. Individuals with this disorder exhibit a number of clinical phenotypes including ocular, skeletal, neurological, and cardiovascular defects that are believed to result primarily from elevated plasma homocysteine levels. A characteristic effect of this disorder is premature vascular disease, with nearly 50% of untreated patients experiencing thromboembolitic events. Mortality is about 20% before the age of thirty. Observations in these patients have led to the idea that elevated plasma homocysteine might be involved in the pathogenesis of atherosclerosis and vascular disease. More than 75 studies have shown high plasma homocysteine levels to be an independent risk factor for coronary artery disease, peripheral artery disease, stroke, and venous thrombosis.

Much attention has been given to dietary modification and administration of folic acid and other vitamins to reduce homocysteine levels. While simple and inexpensive, these methods are effective only to a point; a plateau is reached at which plasma levels remain unchanged. In addition, it is unknown whether there is a metabolic setpoint for homocysteine as that seen with cholesterol, where, if dietary levels drop below a certain point, the liver produces more cholesterol. Development of effective therapies would serve to ameliorate this risk factor to a great extent.

Description:

Cystathionine b-synthase (CBS), a key enzyme in cysteine metabolism is mutated and non-functional in the genetic form of homocysteinuria. It has been suggested that up-regulation of this enzyme will result in lowered plasma homocysteine levels, making this pathway an ideal target for therapeutic intervention.

Enzymatic studies show that deletion of the carboxy-terminus of the CBS gene results in increased CBS activity, suggesting that the carboxy-terminus of the protein acts as a negative regulator of enzymatic activity. Drugs designed to disrupt this interaction may offer a method for modulation of plasma homocysteine levels.

Researchers at the Fox Chase Cancer Center have developed a bioassay system for expression of human cystathionine b-synthase in the yeast Saccharomyces cerevisiae. In this system, expression of human CBS is able to functionally complement a yeast strain lacking endogenous CBS activity. This system can be utilized to assess the action of compounds designed to increase human CBS activity either through abrogation of the inhibitory activity of the C-terminus, or by other mechanisms. In addition, mutant forms of human CBS can be examined to determine mechanisms of action of the therapeutics and also to investigate regulation and function of the CBS gene. We envision this as being an ideal bioassay for high-throughput screening for drugs that will lower plasma homocysteine, and thus, reduce risk for coronary artery disease, heart attack, and stroke.