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.
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|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|
|Zhang, Daqing; Fang, Pu; Jiang, Xiaohua et al. (2012) Severe hyperhomocysteinemia promotes bone marrow-derived and resident inflammatory monocyte differentiation and atherosclerosis in LDLr/CBS-deficient mice. Circ Res 111:37-49|
|Cheng, Zhongjian; Jiang, Xiaohua; Kruger, Warren D et al. (2011) Hyperhomocysteinemia impairs endothelium-derived hyperpolarizing factor-mediated vasorelaxation in transgenic cystathionine beta synthase-deficient mice. Blood 118:1998-2006|
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|Zhang, Daqing; Jiang, Xiaohua; Fang, Pu et al. (2009) Hyperhomocysteinemia promotes inflammatory monocyte generation and accelerates atherosclerosis in transgenic cystathionine beta-synthase-deficient mice. Circulation 120:1893-902|
|Kadariya, Yuwaraj; Yin, Bu; Tang, Baiqing et al. (2009) Mice heterozygous for germ-line mutations in methylthioadenosine phosphorylase (MTAP) die prematurely of T-cell lymphoma. Cancer Res 69:5961-9|
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