? Elevations in plasma homocysteine levels are associated with an increased risk of a number of human diseases including vascular disease, neural tube defects, age-related dementia, and osteoporotic fractures. Homocysteine can be metabolized by the action of cystathionine (3-synthase (CBS) which condenses homocysteine with serine to form cystathionine. Individuals lacking functional CBS due to mutation have clinical CBS deficiency that is characterized by extremely elevated plasma homocysteine levels and phenotypes in the vascular, nervous, skeletal and visual systems. Some CBS-deficient patients respond clinically to pharmacologic doses of pyridoxine (vitamin B6). Most mutations in CBS are point mutations resulting in single amino acid substitutions in the CBS protein. We have previously shown that it is possible to restore function to a large percentage of mutant CBS proteins by truncating the C-terminal domain of CBS or by having specific missense mutations within this domain. More recently, we have found that chemical chaperones can also restore enzyme activity to mutant CBS proteins. The overall goal of this application is to modulate the activity of mutant CBS enzymes by chemical and genetic means. There are four specific aims. First, we will determine the mechanism by which chemical chaperones restore function to mutant CBS proteins. We will determine the number of different mutant CBS proteins that can be rescued by chemical chaperone treatment and we will determine how these treatments affect CBS function. We will also attempt to identify chaperone proteins whose overexpression can stabilize mutant CBS. In our second aim, we will identify and characterize new small molecules that can activate mutant CBS enzymes using a high- throughput screening strategy in S. cerevisiae. In our third aim, we will develop a mouse model to study pyridoxine-responsive homocystinuria. This will be done by making a transgenic mouse expressing the R266K mutant form of CBS and crossing this animal with a CBS knockout mouse. In the fourth aim, we will develop mouse models to study the effects of mutations in the regulatory domain of CBS. Two different mice will be characterized, one with a deletion in the regulatory domain of CBS and the other with a missense mutation in this domain. These studies may lead to new strategies to treat CBS-deficiency and other homocysteine related diseases. Lay abstract: Elevated plasma homocysteine is associated with a number of human diseases. Cystathionine beta-synthase (CBS) is a key enzyme in the metabolism of homocysteine. In this proposal we will develop new strategies and reagents to activate CBS thereby reducing plasma homocysteine levels. ? ? ? ? ? ?
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