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Record Count: 7
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DESCRIPTION (provided by applicant): Carboxylesterases represent a large class of hydrolytic enzymes that play important roles in the metabolism of endogenous lipids, pharmacological determination of therapeutic agents and detoxication of organophosphorus insecticides. The focus of the current grant period has been on molecular cloning, enzyme kinetics and xenobiotic regulation. Enzymatic characterization and molecular modeling have revealed that carboxylesterases contain several functional subsites (structures) that likely play determinant roles in substrate recognition and inhibitor reactivity. Carboxylesterases, with the same functional subsites exhibit the same substrate preference and a similar sensitivity to serine enzyme inhibitors. Studies on xenobiotic regulation have demonstrated that dexamethasone suppresses the expression of rat hydrolase A, B/C and S, whereas the expression of human HCE-1 and HCE-2 is induced by this drug. Suppression requires only nanomolar whereas induction requires micromolar levels. The proposed studies are designed to test the hypotheses that non-conserved residues assumed to form functional subsites among carboxylesterases contribute significantly to the observed differences on the substrate preference and that the dexamethasone-directed suppression of rat carboxylesterases is mediated by the glucocorticoid receptor whereas the induction of human carboxylesterases is mediated by the pregnane X receptor. The specific aims of the proposed studies are: (1) to characterize functional subsites determining substrate and inhibitor selectivity; and (2) to elucidate molecular basis for species-dependent regulation by dexamethasone. Site-direct mutagenesis will be performed to replace a single residue or an entire putative subsite, and the mutants will be tested for the altered hydrolytic activity toward structurally dissimilar substrates. Studies on cell proliferation and toxicity will be performed to determine whether changes in catalytic properties translate into actual perturbations in biological functions. In order to specify the receptor involved in the species-dependent regulation by dexamethasone, antiglucocorticoids and dominant regulators will be used to selectively block or alter either pathway and the role of each receptor will be established in regulating carboxylesterase expression. Establishment of the importance of the subsites for substrate recognition will provide information to elucidate the catalytic action of carboxylesterases and to ascertain isoform-based pharmacological and toxicological relevance. Specification of a receptor involved in dexamethasone-mediated regulation will provide molecular mechanisms to predict potential drug-drug interactions as a result of altered expression of carboxylesterases.
Crisp Terms/Key Words: dexamethasone, laboratory rat, carboxylic ester hydrolase, catalyst, polymerase chain reaction, enzyme inhibitor, enzyme mechanism, site directed mutagenesis, reporter gene, liver cell, chemical model, high performance liquid chromatography, corticosteroid receptor, toxicology, cytotoxicity, toxin metabolism, SDS polyacrylamide gel electrophoresis, gel mobility shift assay, cell proliferation
DESCRIPTION (provided by applicant): The proposed work focuses on three molybdenum-containing enzymes of environmental relevance: DMSO reductase, arsenite oxidase and sulfite oxidase. The first of these catalyzes the reduction of DMSO to the anti-greenhouse gas DIMS, and as such plays an important role not simply in the global sulfur cycle but in modulating climate as well. The second enzyme catalyzes the oxidation of arsenite to arsenate, an important step in the biotransformation of arsenic in the environment that represents a detoxification mechanism for those microorganisms in which it is found. It is a member of the same family of molybdenum-containing enzymes as DMSO reductase, but has an active site structure that represents a variation on that seen in DMSO reductase. Sulfite oxidase from higher eukaryotes (both vertebrates and plants) catalyzes the final step in sulfur catabolism, the oxidation of sulfite to sulfate, and prevents the deleterioius accumulation of the highly reactive sulfite in vivo. The overall goal of the proposed work is to gain a more complete understanding of the mechanism of action of these enzymes in the context of their structures, comparing and contrasting their behavior. The guiding hypothesis behind the approach is that enzyme function and catalytic power are dictated by the physical and electronic structure of the active site. The Specific Aims include rapid kinetic studies as well as spectroscopic and computational work aimed at determining the electronic structures of the enzyme active sites. In the cases of DMSO reductase and sulfite oxidase, site-directed mutants targeting specific active site amino acid residues will also be examined to evaluate their roles in catalysis.
Crisp Terms/Key Words: protein protein interaction, enzyme activity, Arabidopsis, iron sulfur protein, active site, plant protein, oxidoreductase, circular magnetic dichroism, molybdenum, site directed mutagenesis, enzyme structure, enzyme mechanism, Raman spectrometry, electron spin resonance spectroscopy, chemical kinetics
DESCRIPTION (provided by applicant): Organophosphorus (OP) pesticide poisoning is a leading cause of premature death in many developing countries, killing an estimated 200,000 people every year in the Asia-Pacific region alone. In North America and Europe, the situation is quite different. While pesticide poisoning does occur, the main risk of OP poisoning is from terrorist attacks on civilian populations - through the release of OP nerve gases in crowded spaces or perhaps introduction of highly toxic pesticides into water supplies. The acute toxicity of OPs is primarily due to inhibition of acetylcholinesterase (AChE). Current therapy for OP poisoning requires resuscitation and use of atropine, followed by administration of oximes to reactivate AChE. However, these antidotes have limited effectiveness and between 10 and 40% of patients, depending on the responsible OP, still die even with intensive care support. Although OP pesticides have been a clinical problem for 50 years, no new therapies have been introduced since the 1960s. Because early therapeutic interventions lead to improved outcomes after OP poisoning, a treatment that is safe and highly effective, and that can be given by first responders at the site of poisoning, should markedly improve outcome. Both bacteria and humans make enzymes that hydrolyze OP compounds. Recombinant bacterial OP hydrolases have the potential to provide an affordable, widely available, and safe treatment that is rapidly effective against a wide variety of OPs. CSIRO, Entomology, in Australia has developed a bacterial enzyme, called OpdA, with excellent in vitro catalytic activity against many currently used OPs. In proof-of-concept studies, we have shown that OpdA has excellent efficacy when used alone or with 2- PAM in rat models of parathion and dichlorvos poisoning. However, a number of further steps, including the proposed non-human primate studies, are required before clinical trials in humans with OP poisoning. The purposes of this grant are to develop a new non-human primate (NHP) model of parathion poisoning and to test the safety and efficacy of OpdA in this NHP model. Proof that the enzyme is safe and effective against parathion should provide the necessary impetus for further development for human use. Our central hypothesis is that OpdA is safe and improves survival after poisoning with parathion. If successful, the proposed research promises to improve public health by mitigating the acute toxic effects of pesticides after accidental, intentional, or suicidal poisoning. The research also has implications for the treatment of military personnel and civilians after nerve gas poisoning.
DESCRIPTION (provided by applicant): The vicinal haloalkenes are toxicants commonly found at many Superfund sites. Of the 30 most common toxicants detected at Superfund sites, five are nephrotoxic vicinal haloalkenes. Unlike other halogenated hydrocarbons, vicinal haloalkenes uniquely damage the kidney by destroying proximal tubule cells and induce renal carcinomas. It is believed that the nephrotoxic and nephrocarcinogenic effects of vicinal haloalkenes stems from their conversion in hepatic microsomes by the enzyme microsomal glutathione transferase-1 (MGST1) to GSH S-conjugates, which are transported to intestine and then converted to the corresponding cysteine S-conjugates. These cysteine S-conjugates are then transported to the kidney and cleaved by renal cysteine beta-lyases to form toxic haloalkylthiols that damage mitochondria in renal proximal tubular cells. This hypothesis is controversial as there are competing theories that do not include a role for either the liver or for MGST1. Confirmation (or disproof) of this hypothesis has been difficult due to the complex interaction between multiple organ systems and a lack of in vitro models. Complicating the issue is the recent finding that there are multiple human microsomal glutathione transferases capable of conjugating halogenated hydrocarbons. Our objectives are [1] To definitively determine the role of MGST1 in modulating the toxicity of these Superfund vicinal haloalkene contaminates. This will be accomplished by producing both MGST1 overexpressing animals and two types of MGST1 -deficient animals (complete MGST1 nulls versus liver-deficient only) and determining their sensitivity/resistance to the prototype vicinal haloalkenes trichloroethylene (TCE) and hexachlorobutadiene (HCBD). Our studies will confirm the tissue and subcellular distribution of MGST1, which is also controversial, and also determine if deletion of MGST1 results in compensatory changes in other cytosolic and microsomal GST isoforms and in select antioxidant systems. We will also investigate the stress-induced regulation of MGST1 by examining variation in mRNA transcripts that are produced by alternative start sites, and monitoring changes in MGST1 protein content in various organs. [2] To determine if other members of the MGST family are capable of conjugating vicinal haloalkenes and thereby have a potential role in bioactivation of these toxins. [3] To determine whether recombinant MGST proteins could assist in bioremediation of HCBD. [4] To determine the relative contribution of MGST1 and MGST2 to cellular antioxidant capacity through studies utilizing human MGST1 and MGST2 null cells.
DESCRIPTION (provided by applicant): Recent studies demonstrate widespread organophosphate pesticide (OP) exposures to pregnant women and children. However, given the same exposure, some individuals may be more susceptible to the adverse effects of OPs depending on their genetic makeup and expression of genes encoding key metabolic enzymes. For example, the human enzyme paraoxonase (PON1) detoxifies various OPs with different efficiency depending on the main polymorphism at position 192 and others along promoter and coding regions. As part of the CHAMACOS longitudinal birth cohort study, we have investigated OP exposures and health effects in -500 pregnant Latina women and their children living in the agricultural community of the Salinas Valley, CA. Initial data suggest that OP exposure in this cohort exceed national reference levels, and that maternal OP urinary metabolite levels were associated with shortened gestation and abnormal reflexes in neonates. Preliminary data from 130 maternal and cord blood samples show that newborns had lower PON1 activity than their mothers, suggesting they may be more susceptible to the adverse effects of OPs. Thus, differences in genotype, enzyme activity, and age may contribute to differential sensitivity to OP exposures. In the proposed study, we will take advantage of an extensive biorepository and data on growth and neurodevelopment from the CHAMACOS cohort. Our objectives are: 1) to create a PON1 gene haplotype map for this Latino population; 2) to examine the ontogeny of PON1 enzyme activity in infants from birth through 24 months; 3) to establish whether PON1 genotype is associated with OP pesticides in maternal and cord blood; and 4) to determine whether PON1 modifies the relationship of OP exposure and fetal growth, length of gestation and neurodevelopment. To address these aims, we will genotype CHAMACOS mothers and children for five PON1 polymorphisms (192, 55, -108, -909, -162); measure four substrate-specific PON1 enzyme activities (arylesterase, paraoxonase, diazoxonase, chlorpyrifos oxonase) in maternal, cord and child blood at 12 and 24 months; and measure OPs in cord and maternal bloods. This study will help identify human subpopulations more susceptible to the health impact of OP exposure. These data will support planning for the National Children's Study, identify subpopulations susceptible to chemical warfare agents, and inform policy decisions for implementation of the Food Quality Protection Act.
DESCRIPTION (provided by applicant): Aromatase is the enzyme that converts androgen to estrogen. The applicant has hypothesized and demonstrated that aromatase is an important target of endocrine disrupting chemicals. These compounds have been found to inhibit aromatase activity, which results in both a decrease in the level of estrogen and an increase in the level of androgen in treated cells. Animal experiments have been performed that demonstrated the in vivo action of anti-aromatase chemicals. Additionally, environmental chemicals (both phytochemicals and xenochemicals) were found to modify the expression of aromatase in various tissues, which results in a change in the conversion ratio of androgen to estrogen. The compounds that inhibit aromatase or suppress aromatase expression behave as antiestrogens or androgen-like compounds in vivo. Conversely, compounds that either increase aromatase expression or enhance aromatase activity (or stability) may actually function as anti-androgens or estrogen-like compounds. Research conducted in the applicant's laboratory during the last four years demonstrated that estrogen-related receptor alpha (ERR-alpha) and estrogen receptor alpha (ER-alpha) can regulate the expression of aromatase. It has also been shown that endocrine disrupting chemicals, by acting as the ligands of these two receptors, can modify the expression of aromatase in treated cells.
Aromatase, ER-alpha, and ERR-alpha are all important players in maintaining our endocrine function. To continue with and expand on the current efforts in the applicant's laboratory to identify endocrine disrupting chemicals, it is proposed in this competitive renewal application that they utilize their newly established high throughput computer-assisted virtual screening approach to search endocrine disrupting chemicals which serve as ligands/inhibitors of these proteins. It is also further proposed that they determine the structural characteristics of these chemicals by evaluating their structure activity relationship. Such structural information will be valuable for the identification of unanticipated endocrine disrupting chemicals. The proposed studies will focus on 1014 phytochemicals (including chalcones, chromones, coumarins, flavanones, flavones and isoflavones) that would be purchased from Indofine Co (Somerville, New Jersey). In addition, it is proposed that the endocrine disrupting actions of compounds (which have been identified by the high throughput screening method) be confirmed through both in vitro and in vivo experiments.
DESCRIPTION (provided by applicant): Protein malfolding plays an important role neurodegenerative conditions, such as Parkinson's Disease, Alzheimer's Disease and Motor Neuron Disease. Accumulating evidence suggests that environmental agents may contribute to the pathophysiology of these common disorders by perturbing protein folding, either directly or indirectly through their effects on cell metabolism. However, little is known about how cells adapt to the threat of environmentally-induced proteotoxicity. This study will exploit arsenic as a model for an environmental toxin that adversely affects protein folding and one that represents an important public health hazard affecting multiple organ systems. Two recently-identified adaptations to arsenic exposure will serve as this study's point of departure: (1) Regulated attenuation of new protein synthesis. (2) Modification of the cell's protein degradation apparatus to better accommodate it to arsenic-induced proteotoxicity. Stress-induced phosphorylation of translation initiation factor 2a (elF2a) attenuates protein synthesis and activates a salubrious gene expression program known as the Integrated Stress Response (ISR), which reduces the stress caused by arsenic-induced protein malfolding. Therefore, elF2a phosphorylation has emerged as an important component of cellular unfolded protein responses (UPR). Phosphatases that dephosphorylate elF2a will be characterized in an effort to identify specific biochemical steps whose inhibition activates the ISR. The physiological significance of inhibiting elF2a phosphatases will be tested in mouse models of neurodegenerative diseases. These studies will uncover the promise and potential limitations of therapeutic strategies to protect against proteotoxicity by inhibiting elF2a phosphatases. AIRAP, a novel arsenite induced protein, adapts the proteasome's regulatory cap to the conditions in cells experiencing arsenite-induced proteotoxicity and thereby promotes the cell's ability to deal with malfolded proteins. In an effort to understand how the intracellular protein degradation machinery adapts to proteotoxicity, arsenite-induced and AIRAP-dependent changes in the composition of the proteasome will be characterized by proteomic approaches. Gene knock out experiments in mouse and worms will be used to create experimental systems lacking AIRAP, and these will be applied as tools to identify arsenite-modified proteins whose degradation depends on AIRAP induction and AIRAP integration into the 19S proteasome regulatory particle. In vitro biochemical assays of purified proteasomes containing AIRAP will be used to characterize functionally proteasomal adaptation to environmentally-induced protein malfolding. The goal of this research program is to reduce the cellular adaptations to protein malfolding induced by environmental toxins to their molecular constituents. This will lay the groundwork for identifying relevant bio- markers of exposure and for future preventive and therapeutic interventions against neurodegeneration.
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