2000 — 2003 |
Chen, Guangping |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Active Site Studies of Human Sulfotransferases @ University of Arkansas Med Scis Ltl Rock
Sulfotransferase (ST) catalyzed sulfation is an important pathway in the metabolism of hydroxyl (aryl or alkyl) containing drugs, carcinogens, xenobiotics and endogenous compounds. Sulfation of drugs and xenobiotics is mostly associated with detoxification by which a relatively hydrophobic compound is biotransformed into a more water-soluble sulfuric ester that is readily excreted. However, there are numerous important exceptions wherein the formation of chemically reactive sulfuric esters is an essential step in metabolic pathways leading to toxic or carcinogenic bioactivation. Detoxification or bioactivation is highly dependent upon the electrophilic reactivity of the individual sulfuric ester products formed. The long-term objective of the proposed research is to define the structural characteristics of the active sites of human STs and to investigate the catalytic mechanism of the sulfation reaction catalyzed by STs. An enzyme's catalytic mechanism and substrate specificity are determined by the micro-environment produced by specific amino acid residues localized within the active site and by the three dimensional structure of the protein molecule. According to our previous studies and reports in the literature, we hypothesize that the co-substrate PAPS binding site is composed of three highly conserved regions and other elements, and the substrate specificity of different STs is dictated by amino acid residues in several variable regions. Specific investigations to be carried out in this proposal are as follows: 1. Use photoaffinity probes to identify amino acid residues in both the substrate- binding site and PAPS binding site of human liver phenol- sulfating ST (P-PST-1), dehydroepiandrosterone ST (DHEA-ST), and estrogen ST (EST). 2. Use amino acid modification reagents to identify the essential amino acid residues in the catalytic active site of STs. 3. Mutagenesis of essential amino acids and/or functional domains of human STs. 4.Computational molecular structure studies of human STs. The computational molecular structure studies, combining the experimental results from specific aims 1-3, will help us to understand the catalytic mechanism and differing substrate specificity of human STs. The information on human ST active site structure, substrate specificity, and catalytic mechanism should have important implications for the prediction of biotransformation pathways and inter-individual differences in metabolism of endogenous compounds, xenobiotics, and carcinogens. The characterization of the active sites of STs will also help to find mechanism-based specific inhibitors/activators for use in studying the physiological functions of STs and designing new drugs.
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1 |
2006 — 2009 |
Chen, Guangping |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms and Functions of Human Sulfotransferases @ Oklahoma State University Stillwater
Phase II drug metabolizing enzymes sulfotransferases (SULTs) catalyzed sulfation is important in the regulation of different hormones and the detoxification of drugs and other xenobiotics. Sulfation also leads to bioactivation of procarcinogens leading to toxic effect. The long-term goal of this research project is to understand human SULT biological functions and to investigate their relevance to human health under physiological and pathological conditions. Specific aims in this proposal are as follows: 1. To Investigate Mechanisms of Catalysis, Substrate Inhibition, and Product Inhibition/Activation of Human SULTs. The proposed bypass ordered mechanism and related alternative mechanisms will be investigated using kinetic analysis, isotope exchange, sulfated active site amino acid residue identification, and site-directed mutagenesis. The information will have important implications for the prediction of biotransformation pathways. 2. To Investigate the Effect of Sulfated Drugs on Human SULT Catalytic Activities. The inhibition and activation effect of clinically important drug sulfates on catalytic activities of human SULTs will be investigated. E. coli expressed and purified human SULTs;human intestinal cytosols;and human Hep G2 and Caco-2 cells will be used for these investigations. The effect of clinical drugs on human SULT activities may interfere SULT normal biological functions in hormone regulation and xenobiotic detoxification. 3. To Define Oxidative Regulation Mechanisms of Human SULT1E1. Oxidative regulation of human SULT1E1 in Hep G2 and Caco-2 cells and redox thiol regulation mechanisms of purified human SULT1E1 will be investigated using enzyme assay, Western blot, RT-PCR, amino acid modification, site-directed mutagenesis, kinetic analysis, crystal structure analysis, and computer modeling methods. Knowledge on oxidative regulation of certain human SULT is important in understanding the ability of SULT functioning under physiological and pathological conditions. This proposal studies human SULTs. These studies will be significant in understanding SULT biological functions including hormone regulation, drug metabolism, xenobiotic detoxification and procarcinogen bioactivation. The knowledge will be important in understanding drug side effect, drug-drug interaction, drug development, and the potential roles SULTs play in cancer prevention and causation.
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1 |
2008 — 2010 |
Chen, Guangping |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Isolation and Characterization of Rat Kidney Active Urea Transporter
DESCRIPTION (provided by applicant): Urea plays an important role in the urinary concentrating mechanisms. In the past decade, significant progress has been made in understanding urea reabsorption and recycling in kidney including the cloning of the two facilitated urea transporter genes, UT-A and UT-B. However, strong evidence from physiological studies demonstrated that the existence of a sodium- dependent active transporter in mammalian kidney, which can uphill move urea in some nephron segments. Though the active urea transporter was proposed over 40 years ago, none has yet been characterized. The overall objective of this project is to clone this novel active urea transporter from kidney inner medulla (IM) by using two independent approaches: 1) modified two-tester suppression subtraction hybridization (ttSSH) and 2) expression screening using a urea uptake defective yeast strain. After having successfully identified the novel urea transporter, we will investigate the nature of the active urea transporter. We hope the two approaches we apply here will lead us to discover the new genes. We believe the success of this project will fill in the gap with the molecular evidence how urea is actively transported in kidney and will extend our understanding on the urinary concentrating mechanism and water homeostasis. PUBLIC HEALTH RELEVANCE Strong physiological evidence demonstrated the existence of active urea transport activity in mammalian kidney over 40 years. The overall objective of this project is to clone the novel active urea transporter from kidney IM by using two independent approaches. The success of this project will extend our understanding on the urinary concentrating mechanism and water homeostasis.
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0.966 |
2011 — 2015 |
Chen, Guangping |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Small Proteins and Renal Urea Transport Regulation
DESCRIPTION (provided by applicant): Urea plays a critical role in the urinary concentrating mechanism and therefore in the regulation of water balance. Although the first UT was cloned in 1993 and significant progress was achieved, the molecular mechanisms for UT-A1 regulation in cells are still unclear. We recently identified a number of accessory proteins which might play critical regulatory roles in UT-A1 maturation, trafficking, recycling and degradation. The overall goal of the current application is to investigate how the protein- protein interaction regulates UT-A1 activity in cells. We are particularly interested in a group of small molecular weight proteins, such as caveolin-1 and 14-3-3 proteins. Our preliminary data show that these proteins can physically interact with UT-A1. We choose these three candidates since they represent three kinds of proteins that regulate UT-A1 at different steps with different mechanisms. Understanding these interactions is a critical step in dissecting the processes of UT-A1 intracellular translocation, membrane trafficking, as well as retrieval/degradation that all contribute to the urea transport in kidney under physiological and pathological conditions. Our study will not only provide us new insights to understand UT-A1 cellular regulation (trafficking, recycling and degradation) but also provide new insights in the entire transporter research field. In collaboration with Dr. Haian Fu and the Emory Chemical Biology Drug Discovery Center, our long-term project aim is to screen and develop small-molecule modulators of these proteins (first 14- 3-3) that could affect UT-A1 function in vivo for potential therapeutic interventions for the diseases with total body fluid overload such as hypertension, congestive heart failure, cirrhosis, and nephrotic syndrome. PUBLIC HEALTH RELEVANCE: Sodium and urea are the two major solutes that contribute to the medullary osmolarity gradient in kidney. Urea reabsorption in kidneys is mainly mediated by urea transporter UT-A1 in the inner medullary collecting duct (IMCD) epithelial cells. Although the first urea transporter was cloned in 1993 and significant progress has been achieved, the mechanisms for its regulation are still unclear. The aim of the current application is to explore the molecular mechanisms of how UT-A1 is regulated by accessory proteins. We are particularly interested in a group of small molecular weight proteins (20~30kDa) such as caveolin and 14-3-3. We have preliminary data suggesting that these proteins may play important roles in the UT-A1 urea transporter trafficking, recycling, activation, and degradation. Our long-term goal is to screen and develop small-molecule modulators of these proteins that could affect UT-A1 function in vivo for potential therapeutic interventions for total body fluid overloaded diseases such as hypertension, congestive heart failure, cirrhosis, and nephrotic syndrome.
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0.966 |