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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL057299-05
Application #
6262684
Study Section
Mammalian Genetics Study Section (MGN)
Program Officer
Jaquish, Cashell E
Project Start
1996-12-15
Project End
2004-11-30
Budget Start
2001-01-16
Budget End
2001-11-30
Support Year
5
Fiscal Year
2001
Total Cost
$388,313
Indirect Cost
Name
Fox Chase Cancer Center
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
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
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
Wei, Qiong; Wang, Liqun; Wang, Qiang et al. (2010) Testing computational prediction of missense mutation phenotypes: functional characterization of 204 mutations of human cystathionine beta synthase. Proteins 78:2058-74
Singh, Laishram R; Gupta, Sapna; Honig, Nicholaas H et al. (2010) Activation of mutant enzyme function in vivo by proteasome inhibitors and treatments that induce Hsp70. PLoS Genet 6:e1000807
Tang, Baiqing; Mustafa, Aladdin; Gupta, Sapna et al. (2010) Methionine-deficient diet induces post-transcriptional downregulation of cystathionine ýý-synthase. Nutrition 26:1170-5
Singh, Laishram R; Kruger, Warren D (2009) Functional rescue of mutant human cystathionine beta-synthase by manipulation of Hsp26 and Hsp70 levels in Saccharomyces cerevisiae. J Biol Chem 284:4238-45
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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