The long-term objective of this proposal are to define how abnormal expression of the cystathionine beta-synthase (CBS) gene plays a role in human disease, in particular, homocystinuria and vascular occlusive disease due to hyperhomocysteinemia. CBS, a central enzyme at a branch pint in sulfur metabolism, is a tetramer of 63 kDa subunits; the enzyme binds two substrates, homocysteine and serine, and three ligands, PLP, AdoMet, and heme. It is encoded in humans on chromosome 21; it's inherited deficiency causes the most common form of homocystinuria. We have isolated cDNA and genomic clones for yeast, mouse, rat, and human CBS genes. We have recently obtained the full 28-kbp DNA sequence of the human gene including approximately 5kbp of sequence upstream of the promoter region. We cloned the human CBS cDNA into several fusion expression vectors and developed methods to purify large amounts of the full-length and truncated enzyme to homogeneity. We demonstrated that CBS is a heme is a heme-containing enzyme, and that the porphyrin is required for both PLP binding and activity. We have, together with our collaborators, identified 100 mutations in homocystinuria so far.
Our specific aims are 1) To study the commercially available CBS-knockout mouse model to identify the cause of early lethality in animals homozygous for the disrupted CBS gene and to improve their survival by treatment. If survival can be sufficiently improved we will investigate if the animals develop the clinical signals of homocystinuria; 2) to thoroughly characterize the transcriptional regulation of CBS; 3) to investigate the post-translational regulation of CBS; 4) to perform a detailed analysis of the tissue specific regulation of CBS in humans; 5) to determine if yeast is a suitable model system in which to investigate the role of heme in CBS; 6) to continue to optimize the expression, purification, and the crystallization conditions for solving the structure of CBS by X-ray diffraction. The role of this project is to extend our knowledge of the primary to the tertiary enzyme structure and to investigate the possibility that aberrant regulation of CBS is a factor in predisposition towards cardiovascular, cerebrovascular and neurodegenerative disease.
Showing the most recent 10 out of 30 publications
