This competitive renewal application requests 5 years of support to investigate the biochemistry and physiology of homocysteine metabolism and its control by cystathionine beta synthase (CBS). The applicant has previously developed a functional assay for human CBS by complementation of CBS-deficient yeast for the ability to grown on cysteine-deficient medium. In the previous funding cycle, experiments based on the yeast assay led to the unexpected finding that the carboxyl-terminal domain of CBS is a negative regulator of catalytic activity carried out by the amino-terminal domain. Previous work had implicated the carboxy-terminal domain as being required for allosteric control of CBS activity by the physiologic positive regulator S-adenosyl methionine (SAM). The current application proposes to build upon these observations.
In Aim 1, a structure-function analysis of the carboxy-terminal domain will be carried out to identify amino acid substitutions that will either enhance or suppress the ability of the carboxy-terminal domain to inhibit amino terminal enzymatic activity as inferred from the yeast complementation assay. The physiologic consequences of a mutation that suppresses inhibition (and therefore activates enzymatic activity) will be investigated in Aim 2 by constructing transgenic mice that express variant CBS proteins in a knockout background.
In Aim 3, the ability of small molecules to effect changes in regulatory activity of the carboxy-terminal domain will be investigated by screening peptide expression libraries in yeast. Finally, Aim 4 will examine the potential relevance of these findings to allosteric control by AdoMet by determining the AdoMet binding site using a derivatized form of AdoMet that can be covalently cross-linked after photo-activation.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Mammalian Genetics Study Section (MGN)
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Jaquish, Cashell E
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Fox Chase Cancer Center
United States
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Lee, Hyung-Ok; Wang, Liqun; Kuo, Yin-Ming et al. (2017) Lack of global epigenetic methylation defects in CBS deficient mice. J Inherit Metab Dis 40:113-120
Gupta, Sapna; Wang, Liqun; Anderl, Janet et al. (2013) Correction of cystathionine ?-synthase deficiency in mice by treatment with proteasome inhibitors. Hum Mutat 34:1085-93
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