Edward Morgan - US grants

The goals of this project are evaluate the roles of the sexually differentiated pituitary growth hormone secretory pattern (GHSP), and of other hormones, in the regulation of multiple hepatic genes in the rat, and to clone cDNAs and genomic genes for hepatic mRNAs regulated by the GHSP. This will constitute a major step towards the elucidation of the molecular mechanism(s) of growth hormone action in the liver. The GHSP is distinguished by a highly regular pulsatile rhythm in males, and a more continuous, irregular pattern in females. Several sex-dependent hepatic gene products are regulated by the sexually differentiated GHSP, including two isozymes of cytochrome P-450 (h and i) for which I have cloned cDNAs. Various lines of evidence indicate that although the GHSP has a major role in regulation of these function, other hormones also control the expression of at least some of them. One specific aim of this project is to evaluate the relative roles of the GHSP and other hormones in regulation of sexually differentiated hepatic gene expression, using P-450h and i as a model. The roles of glucocorticoids and thyroid hormones will be investigated in vivo by administering them to hypophysectomized rats, alone or in combination with growth hormone. The molecular level at which these hormones act will be investigated by measuring hormone effects on the hepatic proteins, their mRNAs, and the rates of nuclear transcription of their genes. Similarly, the role of insulin will be evaluated using a streptozotocin diabetes model. The modulation of P-450h and i during acute inflammatory stress, and the humoral factors involved, will also be examined. Another aim is to clone cDNAs and genomic genes for multiple hepatic GHSP-regulated mRNAs using a hybridization-subtraction method to obtain cDNA libraries and probes enriched for such sequences. The possibility that P- 450 isozyme DEa is GHSP-regulated will be studied by purifying it by a published method, preparing specific antibodies to it, and by cloning its cDNA. It is anticipated that the cloned GHSP- responsive genes and their 5'-flanking regions will be valuable and essential tools in the gaining a better understanding of the mechanism of action of this important hormone, and of peptide hormones in general.

DESCRIPTION (provided by applicant): The primary purpose of this broadly based multidisciplinary Training Program is to prepare students for biomedical research careers in schools of medicine, dentistry and pharmacy, in research institutes, and in government or industrial laboratories. A major goal is to provide trainees with a program in which they will be represented by the 35 training faculty include transmembrane signaling, cell growth control and molecular recognition, substance abuse, and molecular toxicology. Training faculty represent 11 departments and three Centers at Emory. The most important component of training is laboratory research, first as a series of three research rotations, then in the dissertation laboratory. This training is complemented by core courses in pharmacology that emphasize quantitative analysis of drug action, including receptor structure and function, courses in biochemistry/molecular biology and biostatistics, and advanced courses in specialty areas; by seminar courses and by journal clubs. Emphasis throughout is placed on oral presentation skills; students make formal oral presentations of their own work or that in the literature on numerous occasions before their dissertation defense. The Program is designed to support six students each year from a total population of about 25 eligible students. The student who completes the predoctoral training program will have acquired broad familiarity with pharmacology, knowledge in depth in the area of dissertation research, the ability to search, read critically and report on the literature of the biomedical sciences, mastery of a variety of laboratory techniques useful in modern biomedical research, skill in planning and executing a research project, ability to write clear, accurate scientific reports for publication, and ability to present effectively the results of research. A large percentage of the previous trainees of the core faculty have obtained postdoctoral training and then secured desirable positions in academic or industrial institutions; they continue to be productive in biomedical sciences.

DESCRIPTION (provided by applicant): Nitric oxide (NO) acts as an intra- and inter-cellular signaling molecule in many tissues, where it has important physiological and pathophysiological functions. NO regulates the activities and functions of cellular proteins by a diversity of mechanisms. Modification of critical amino acids on proteins, such as S-nitrosylation and oxidation of cysteines, or nitration of tyrosines, by NO or reactive nitrogen species derived from NO, is emerging as an important mechanism by which NO regulates cellular function. We have recently obtained compelling evidence that NO generated during an inflammatory response causes a rapid, NO-dependent down-regulation of rat liver CYP2B proteins (members of the cytochrome P450 superfamily). Here, we propose to investigate the hypothesis that modification of CYP2B proteins by reactive nitrogen species results in their targeted proteolytic degradation, and to identify the specific amino acids targeted on human CYP2B6. This will be accomplished by pulse-chase studies on CYP2B enzyme turnover in rat hepatocytes, and on degradation of wild-type and mutant CYP2B6 enzymes expressed in human hepatoma cells via retroviral transduction. Amino acid adducts will be identified by a variety of methods including immunoptecipitation with adduct-specific antibodies, chemical modification, protein chip adsorption and mas spectrometry. Since little is known about the regulation of other human CYP enzymes by NO, we will also investigate the regulation of human CYP3A4, 2D6, 2E1 and 2C enzymes by NO in cultured human hepatocytes. These studies investigate a highly novel mechanism of regulation of CYP proteins. As such, they have implications not only for the function of CYP2B enzymes in metabolism of diverse therapeutic agents, detoxification and bioactivation of environmental chemicals, and in gene therapy, but also for the toxicological, pharmacological, and physiological functions of other CYPs that are regulated in the same way. They also have important implications for the broader field of NO biology, since knowledge in the area of NO-stimulated protein degradation is limited.

DESCRIPTION (provided by applicant): Inflammation and infection are known to down-regulate the hepatic expression of various cytochrome P450 (CYP) enzymes, and this has important consequences for clinical drug therapy. Our laboratory and others have extensively characterized the acute effects of bacterial lipopolysaccharide (LPS) on basal and drug-induced hepatic CYP expression as a model of bacterial sepsis. In preliminary studies, we found that infection of mice with Citrobacter rodentium, a rodent model of enteropathogenic E.coli (EPEC) infection and of inflammatory bowel disease (IBD) in humans caused relatively selective effects on CYP expression in mouse liver. These effects were found to occur in the absence of a functional toll-like receptor-4 (TLR4), suggesting that they are independent of bacterial LPS. We hypothesize that differential regulation of CYP expression is regulated by multiple host and pathogen factors during C. rodentium infection. Understanding these factors is crucial to predicting clinical drug responses in disease states. We further hypothesize that modulation of CYPs play specific roles in the host response to C. rodentium infection. To address these hypotheses, we will characterize the time course of regulation of hepatic CYP expression in mice infected with C. rodentium and compare it to the progression and resolution of infection and pathology. We will also compare it to a model of chemically-induced IBD. Then, we will define bacterial and host factors involved in CYP regulation during C. rodentium infection, using bacterial and mouse genetics as well as pharmacological approaches. Finally, we will determine whether global or liver-specific modulation of CYP activity can regulate the hepatic, gastrointestinal or systemic responses to C. rodentium infection, using the nonspecific CYP inhibitor 1-aminobenzotriazole, and mice in which the hepatic NADPH-cytochrome P450 reductase (CPR) gene has been selectively deleted.

DESCRIPTION (provided by applicant): Modern drug discovery and development require the training of scientists who understand the molecular, physiological and quantitative basis of drug action and specificity, and who can apply modern technologies and concepts to the development of novel therapeutic strategies. This multidisciplinary doctoral training program in the Pharmacological Sciences is designed to help meet that demand by preparing students for biomedical research careers in schools of medicine, dentistry and pharmacy, in research institutes, and in governmental or industrial laboratories. The most important component of training is laboratory research, first as a series of research rotations, then in the dissertation laboratory. This training is complemented by a core course that integrates the theoretical and experimental foundations of modern biological sciences;core courses in pharmacology that emphasize quantitative analysis of drug action, pharmacokinetics, drug disposition, biostatistics and experimental design;advanced courses in specialty areas;seminar courses and journal clubs. Emphasis throughout is placed on development and refinement of communication and analytical skills. The 43 training faculty represent 8 basic science and 6 clinical departments or Centers at Emory, and two Departments at Georgia Tech., providing a wealth of diverse research training opportunities. Their research foci can be loosely grouped in four broad areas: transmembrane signaling, cell growth control and molecular recognition, substance abuse and behavioral pharmacology, and molecular toxicology. This Program currently supports six students each year, who are selected mainly from a pool of approximately 35 eligible students in the Molecular and Systems Pharmacology (MSP) Program. Six slots are requested in this renewal. Graduates will have acquired broad familiarity with pharmacology, knowledge in depth in the area of dissertation research, and the technical, communicative and analytical skills necessary to pursue an independent research career. Students graduate an average of 5.2 years after matriculation. The research conducted by the trainees in this program will advance our knowledge of disease processes and contribute to development of novel and improved therapeutic strategies that will benefit the health of our citizens. By preparing young scientists to contribute to and lead the nation's efforts in these areas, this training program will help to ensure that our ability to improve the nation's health remains strong in the future.

DESCRIPTION (provided by applicant): Cytochrome P450 (P450) enzymes play crucial roles in the clearance of drugs from the circulation, and therefore changes in their activities can significantly influence the therapeutic and adverse effects of a drug on an individual. In addition some P450 enzymes have crucial roles in metabolism of physiological substrates e.g. CYP2C enzymes in the metabolism of arachidonic acid to bioactive epoxyeicosatrienoic acids. Infectious and inflammatory diseases cause the down-regulation of many P450s and other drug metabolizing enzymes in the liver, causing impairment of drug clearances. Our laboratory has shown that nitric oxide (NO) formed in hepatocyte during an inflammatory response selectively directs the ubiquitin-dependent proteasomal degradation of rat CYP2B1 and CYP3A2, but not CYP2C11. Our preliminary data demonstrate that not only are human CYP2B6 and CYP2C enzymes down-regulated by NO, but they are more sensitive than the rat enzymes to degradation caused by NO-releasing chemicals. Here, we propose to use proteomic methods to define the scope of NO-mediated degradation in human hepatocytes. We will characterize the proteolytic enzymes involved in the NO-mediated degradation of CYP2B6 and 2C9, and elucidate the mechanism whereby NO regulates these processes in an enzyme-specific manner

ABSTRACT: Pilot Project Program The HERCULES Center at Emory is based upon the emerging scientific interest in exposome- related approaches and the substantial research base in the Environmental Health Sciences (EHS) on campus. The other components of HERCULES address critical infrastructure needs that are essential for growth of our program. The Pilot Project Program perfectly complements the overall theme of HERCULES and provides an opportunity to broadly promote Environmental Health Science research at Emory University and Georgia Tech, while specifically advancing the concept of the exposome within the field.

Project Summary Human evolution has created complex metabolism systems to transform and eliminate potentially harmful chemicals to which we are exposed. Available evidence indicates that these systems generate a million or more different chemical metabolites, most of which are completely uncharacterized. Widespread use of mass spectrometry-based metabolomics methods shows that many unidentified mass spectral features are significantly associated with human diseases. Substantial epidemiological research implicates environmental contributions to many disease processes, and we believe that many of the unidentified mass spectral features are metabolites of environmental chemicals. We have an established and successful human exposome research center focused on improving the understanding of environmental contributions to disease. The present proposal is to build upon this foundation to develop powerful new chemical identification tools that can be scaled to identify hundreds of thousands of foreign chemical metabolites in the human body. We have assembled an exposome research team of analytical scientists with expertise in mass spectrometry, xenobiotic metabolism, computational chemistry and robotic methods, to develop and test new chemical identification tools to identify hundreds of thousands of foreign chemical metabolites. Our approach relies upon expertise in 1) computational chemistry to predict possible xenobiotic metabolites, respective adduct forms and ion dissociation patterns in mass spectrometry, 2) use of enzymatic and cellular xenobiotic biotransformation systems, which allows creation of multi-well panels containing specific biotransformation systems to generate xenobiotic metabolites, 3) ion fragmentation mass spectrometry and NMR spectroscopy methods to confirm chemical identities and 4) expertise with robotic systems which can be used to scale the approach to identify hundreds of thousands of metabolites of environmental chemicals. An Administrative Core will maintain an organizational structure and coordinate activities between the Experimental Core and the Computational Core, NIH and the Stakeholder Engagement and Program Coordination Center (SEPCC). The Experimental Core will develop and provide compound identification capability with ultra-high-resolution mass spectrometry support. The Computational Core will develop a predicted xenobiotic metabolite database to support metabolite identification. The Administrative Core will maintain interactions with HERCULES Exposome Research Center and support interactions with prospective Core users. Milestones are established to monitor progress toward goals to establish tools for compound identification that can be scaled to identify hundreds of thousands of foreign chemical metabolites. The results will catalyze metabolomics research by providing new ways to identify unknown metabolites of environmental chemicals, and also support identification of a broader range of metabolites of drugs, food, microbiome, dietary supplements and commercial products.

PROJECT SUMMARY The goal of this new Initiative to Maximize Student Diversity (IMSD) proposal is to build on an existing IMSD program at Emory University to increase the diversity of the scientific workforce. In spite of many years of increased focus on diversity, the rate of enrollment and graduation of under-represented (UR) students in undergraduate and graduate degree programs in the biological and behavioral sciences is markedly lower that of the general population. Emory University is a highly rated teaching and research institution with over 14,200 students that is located in Atlanta, Georgia. Emory?s record of recruiting, retaining and graduating diverse students is significant. Approximately 15% of each entering graduate class is underrepresented (UR); undergraduate classes are 20%, having doubled since the early 90?s. The biomedical and behavioral science departments at Emory awarded 1,667 BS, 805 MS and 149 PhD degrees to UR students in the most recent fifteen-year period. After receiving only three years of support in 2013, Emory?s Initiative for Maximizing Student Development (IMSD) program has made significant progress towards enhancing the mentoring environment and providing career development and support to undergraduate and graduate students. The long- term goal of the Emory IMSD is to increase the average number of UR students obtaining PhDs in the biomedical sciences by placing at least two-thirds of IMSD Undergraduate Scholars in graduate programs and graduating 90% of IMSD Graduate Fellows and placing them in strong postdoctoral positions. In this new IMSD proposal, we seek to continue our success and further enhance the impact of the Emory IMSD as we develop an inclusive community and a continuum of mentoring and career development programming via three specific aims and a structured plan to transition from the prior leadership to a new leadership team. Aim 1 will continue with efforts to build a learning community of IMSD Undergraduate Scholars. We will increase the number of Emory undergraduate UR students majoring in the biomedical/behavioral/quantitative sciences who receive a Ph.D. in a science-related field to 12 per year. This will more than double our ten-year average that existed when the Emory IMSD program was first proposed (4.8 per year from 2003-2012) and remove all demographic disparity. Aim 2 will enhance and build a learning community of IMSD Graduate Fellows. Through these efforts, we will recruit 40 additional UR PhD students over five years, with over 90% completing the PhD. Finally, Aim 3 will provide formal training in mentoring for both faculty and graduate students in the Emory community and the greater Atlanta area by continuing efforts to develop a community of mentors, the Atlanta Society of Mentors (ASOM), with transferable skills applicable at all levels and adept in issues of diversity. These aims will have significant impact by building on our prior success with creating an inclusive and integrated community of undergraduate scholars, graduate fellows and program faculty that enhances the culture of learning at Emory University and increases diversity within the scientific workforce.

Experimental Core ?Project Summary The goal of the Experimental Core is to be able to generate from thousands of chemicals a spectrum of their possible metabolites and simultaneously identify the metabolites and the enzyme(s) responsible. This will be achieved via development of 96-well, then 384-well and eventually 1536-well arrays of cells expressing individual xenobiotic metabolizing enzymes to generate the metabolites, which will be analyzed by high- resolution mass spectrometry. Enzymes will be expressed in human Huh7 hepatoma cells using lentiviral transduction, which we have used successfully to express several xenobiotic-metabolizing cytochrome P450 (P450) enzymes in catalytically active form. We will begin by expressing major enzymes in the P450, flavin monooxygenase, UDP-glucuronyltransferase, sulfotransferase and N-acetyltransferase families and continue to develop additional cell lines through the duration of the project. Arrays will be plated and screened using robotic facilities in the Miller laboratory and the Emory Chemical Biology Discovery Center. The Computational Core will generate a list of candidate xenobiotic metabolic products, allowing us to determine whether a product with accurate mass matching the predicted metabolite is formed. Using the predicted m/z of the predicted metabolite, we will be able to direct MS/MS acquisition for likely products and compare those to MS/MS predicted for the metabolite by the Computational Core, thereby providing two levels of chemical identification in an automated manner. Broad coverage of tissue metabolites will be obtained by hybrid arrays incorporating pooled S9 fractions from human tissues together with the cellular approach. Since we will have a cell line specifically expressing an enzyme that generates a predicted product, we will have a scalable system for biologically producing enough of the xenobiotic metabolite to perform more definitive chemical identification. The first-generation platform will utilize pooled human S9 tissue fractions in 96-well format to rapidly develop our capacity to generate metabolites of important environmental chemicals. The second-generation platform will be a 96-well array of cell lines developed in the first year. The third-generation platform will be in 384 well format with an expanded array of enzymes. All platforms will be tested against a panel of model environmental chemicals with known metabolites. In the fourth year, the 3rd generation platform will be used to catalogue the metabolites of >200 chemicals from the EPA's TOXCAST library, and work towards building a 1536-well platform. The approach is scalable to support analysis of dozens of metabolites of multiple chemicals per day, providing a prospect for mega-scale chemical identification.