Seiichi Matsuda - US grants

This research program concerns 15-oxygenated sterols wich have been found to be extraordinarily potent inhibitors of cholesterol synthesis in cultured mammalian cells. Several of these compounds have been shown to have significant hypocholesterolemic activity in rats. In the course of these studies we have obtained rather striking results in preliminary studies in two species of nonhuman primates. The potential importance of these findings, coupled with our continuing fundamental studies of the chemistry and mechanisms of action of these compounds, forms the basis and justification of the renewal request. Briefly, oral administration of 5Alpha-cholest-8(14)-en-3Beta-ol-15-one to baboons caused significant lowering of serum cholesterol levels and of the levels of cholesterol in lipoprotein fraction containing low density lipoproteins (LDL) and very low density lipoproteins (VLDL). Moreover, and elevation of the levels of high density lipoprotein (HDL) cholesterol was described in animals whose initial values of HDL cholesterol were low (or in which the percentage of total serum cholesterol in the HDL fraction was low). Even more striking findings have been observed upon administration of the compound to rhesus monkeys maintained on a diet of moderate cholesterol content. LDL cholesterol and protein levels reduced markedly while HDL cholesterol and protein levels were increased. The HDL profiles were shifted to one in which the HDL2 species dominated. We now seek funds required to extend and confirm these observations in baboons and monkeys and to undertake studies to explore the mechanisms involved in the actions of the Delta8(14)-15-ketosterol in lowering serum cholesterol levels and altering the distribution of cholesterol in lipoproteins in baboons. In addition, we propose to investigate the effects of the inhibitor on serum cholesterol and plasma lipoprotein in rhesus monkeys and most importantly, to study the effect of the compound on coronary artery atherosclerosis. We also propose to extend our studies on the effects, metabolism, and mechanisms of action of 15-oxygenated sterols in cultured mammalian cells and in intact small animals and to continue our studies on the chemistry of 15-oxygenated sterols and their derivatives. The proposal involves a collaborative effort between this laboratory and two NHLBI Primate Resource Centers with additional important collaborations with investigators at a number of other institutions.

The major emphasis of the total program is on the chemistry and metabolism of sterols and sphingolipids, two classes of compounds generally considered unique to the cell structure and function of eukaryotic organisms. The program includes 6 major categories. Project area I concerns sterol intermediates in the biosynthesis of cholesterol and includes detailed metabolic, enzymatic and mechanistic studies of the conversion of 14Alpha-methyl-, 14Alpha-hydroxymethyl-, and 14Alpha-formyl-substituted sterols to cholesterol and to intermediates in the biosynthesis of cholesterol; exploration of the possible role of 14Alpha-formoyloxy-substituted sterols as potential intermediates in the enzymatic removal of carbon atom C-32 of sterol precursors of cholesterol; and determination of the chemical nature of sterols of mouse LM fibroblasts; and studies of the mechanisms involved in the enzymatic conversion of Delta8(14)-sterols to cholesterol. Project area II includes: studies of the possible role of 14Alpha-hydroxymethyl- and 14Alpha-formyl-substituted sterols as regulators of sterol synthesis in cultured mammalian cells; studies of the role of oxygenated derivatives of cholesterol in the regulation of the biosynthesis of cholesterol including investigations of the occurrence and chemical nature of oxygenated sterols in: (a), low density lipoproteins of human plasma, and (b), commercial preparations of egg yolk and butter; and studies directed towards the purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from liver microsomes of normal rats. Project area III involves a number of studies of the effects of selected oxygenated sterols on developmental processes in insects. Project area IV involves studies of the chemistry and biosynthesis of phytosphingosine, and the possible coordination of the synthesis of sterols and of sphingolipids in cultured mammalian cells. Project area V involves stereochemical and mechanistic studies involved in the area of lipid metabolism. Project area VI involves general and specific applications of mass spectrometry and nuclear magnetic resonance spectroscopy to sterols, sphingolipids, and biochemical problems.

This research program concerns multiple ongoing projects dealing with the biochemistry, chemistry, and actions of sterols, sphingolipid bases (and their derivatives), and isoprenoid alcohols and acids as well as potential interrelationships in their metabolism and actions. One major project concerns the chemistry, actions, and metabolism of certain 4,4-dimethylsterols very recently found to affect meiosis in mammalian oocytes. Another major focus involves the chemical nature and metabolism of C27 sterols found to accumulate in tissues of subjects with the Smith-Lemli-Opitz syndrome, an hereditary disorder of development associated with a defect in the late stage of cholesterol biosynthesis. Another hereditary disorder of development, suggested to be a defect in squalene epoxidase, is also being studied. Several projects involve studies of the biochemistry and actions of different oxysterols as potential inducers of calcification in specialized cells of arteries (calcifying vascular cells), a subject of importance in the pathogenesis and detection of atherosclerotic lesions. Also included are fundamental studies of the actions of a variety of oxysterols on cholesterol esterification and their actions on acyl coenzyme A-cholesterol acyltransferase, studies of relevance to the intestinal absorption of cholesterol and the pathogenesis of atherosclerosis. Other studies concern the effects of selected oxysterols on the nuclear orphan receptor LXRalpha, recently shown to be important in the in vivo regulation of cholesterol metabolism. Another exciting project concerns studies directed towards determination of the chemical nature of the endogenous inducer(s) of cytochrome P450BM-3 in Bacillus megaterium, with potential for transfer of knowledge from this system to the control of the induction of drug-metabolizing enzymes in humans. Isoprenoid alcohols and acids, regulators of cell growth and apoptosis, will be studied with regard to their syntheses, methods for their separation, and studies of their actions. Sphingolipid bases and their 1-phosphate derivatives, of considerable current interest in biology and medicine, will be studied with regard to their chemistry, methods for their quantitation, and selected actions.

Protists cause numerous human diseases, including malaria, African sleeping sickness, Chagas' disease, leishmaniasis, giardiasis, and amebic meningitis. Treatments for each of these is inadequate, as few pharmaceutically exploitable metabolic differences have been found between humans and protists. Sterol biosynthesis is a promising but underexplored area for finding vital protist enzymes that lack human homologs, and the primary goal of this proposal is identifying novel drug targets in protist sterol metabolism. Animals and fungi synthesize the sterol ring system in one step using lanosterol synthase, which cyclizes oxidosqualene to lanosterol. The overwhelming majority of protists in which sterol biosynthesis has been examined synthesize that ring system in two steps: cycloartenol synthase cyclizes oxidosqualene to cycloartenol, which is then converted to lanosterol by cyclopropyl isomerase. These organisms should be susceptible to specific inhibitors of either cycloartenol synthase or cyclopropyl isomerase. Presented in this proposal are experiments to characterize these enzymes from several protists. A series of compounds designed to specifically inhibit cycloartenol synthase is described. Recombinant organisms that express the protist cycloartenol synthases and cyclopropyl isomerases will be constructed to provide enzymes with which to test the drug candidates. Metabolic research in pathogenic protists is hindered by difficulties in obtaining sufficient biomass for classical experiments with radiolabeled tracers. The approach described here uses molecular biological techniques to clone and express protist genes responsible for forming the sterol ring and to determine which pathway various protists use. This laboratory has constructed several metabolically engineered yeast strains in which the normal sterol pathway (which goes through lanosterol) is supplemented with components of the plant sterol biosynthetic pathway (which goes through cycloartenol). These strains can utilize either lanosterol or cycloartenol as sterol precursors, and will serve as hosts to clone cycloartenol synthase, cyclopropyl isomerase, or lanosterol synthase by genetic complementation.

This multidisciplinary collaborative research is aimed at determining how and why plants synthesize diverse triterpenoids. This project will integrate reverse genetics in Arabidopsis, heterologous expression of cDNAs in yeast, spectroscopic and chromatographic structural determination, and gene expression analysis to elucidate the function of Arabidopsis triterpenoid biosynthetic genes and thereby triterpenoid biosynthetic pathways, control mechanisms, and biological function. Three of the characterized triterpenoid biosynthetic enzymes convert a shared substrate to different compounds; other enzymes to be studied may similarly provide structural diversity, metabolize different substrates, or provide means to spatially or temporally control expression and thus product formation. The effects of the mutations on plant growth, development, and triterpenoid composition will facilitate linking each triterpenoid biosynthetic gene with an enzyme of known catalytic activity and a specific biological role. Genes to be studied include apparent oxidosqualene cyclases, farnesyl pyrophosphate synthases, squalene synthases, squalene epoxidases, and cycloeucalenol isomerase. This work will provide a comprehensive accounting of triterpenoid skeletons synthesized by Arabidopsis, establish which compounds are derived from each gene product, and determine the spatial and temporal expression patterns of the various biosynthetic genes. These experiments will not only elucidate the biological importance of triterpenoid diversity, facilitating future modifications for agricultural benefit, but will provide students with broad interdisciplinary training that bridges modern chemistry and biology. The data from these studies will be publicly available on the project web site (http://www.bioc.rice.edu/~bartel/At_triterpenoids.html), and the knockout seeds will be deposited in the ABRC at Ohio State University for distribution to the community.

Plants possess a variety of mechanisms to protect themselves from attack by herbivores. One system that is induced upon attack by herbivorous insects is the production of terpenoids that can serve in either direct or indirect defense against the herbivore. It has previously been shown that the maize sesquiterpene cyclase gene stc1, located in 9S, is induced in corn seedlings being foraged by beet armyworm (BAW) larvae. The product of this gene is a volatile terpenoid that most likely serves to attract wasps that parasitize the BAW larvae. Preliminary work leading to this proposal has now shown that stc2, the ortholog of stc1 in 6L, is induced by a different insect pest, the southwestern corn borer (SWCB). The P.I.s propose a genetic, molecular, and biochemical dissection of the two orthologous maize genes involved in this novel type of inducible plant defense system, as follows:

1. Characterization of the induction of the stc2 gene by the SWCB and other related corn borers. These experiments will establish the pattern of stc2 induction in response to regurgitant from the SWCB and whether the plant response is specific to that insect species or not.

2. Determination of the complete structure of the stc2 gene by isolating a full-length stc2 cDNA from seedling sheaths that have been foraged by SWCB. This will enable identification of the N-terminus of the protein, confirmation of the predicted intron-exon structure of the transcript, and expression of the STC2 enzyme in a heterologous system for biochemical characterization of its activity.

3. Assessment of the possible role of stc2 as an insect resistance gene by isolating and sequencing the stc2 gene from the non-inducible, SWCB-susceptible Ki3 inbred line. This would identify the basis of the noninducibility of stc2 in Ki3 and provide further evidence that stc2 corresponds to a quantitative trait locus (QTL) for SWCB resistance that maps very close to the location of stc2 in 6L.

4. Determination of the nature of the terpenoid product catalyzed by the heterologously expressed STC1 and STC2 enzymes. These experiments will identify the nature of the terpenoids made by these enzymes and confirm whether the product of STC1 is a sesquiterpenoid, as suggested by the earlier biochemical genetics data.

5. Identification of the most likely subcellular localization of the STC proteins with constructs that fuse their putative chloroplast transit peptides to GFP. This will confirm whether the predicted N-terminal targeting sequence in each of the three sequenced stc1 alleles behaves as a chloroplast transit peptide in vivo.

The proposed activity will have clear broader impacts. The work combines genetics, molecular biology, and biochemistry to analyze the response of maize plants to insect pests. It will serve as excellent training ground for student and postgraduate researchers interested in applying biochemistry and molecular genetics to address practical problems in plant biology. Both the P.I and co-P.I. are members of minorities and their labs are heavily populated by under-represented groups. The project is relevant to long-term improvement in U.S. agriculture in that it addresses mechanisms by which maize defends itself from insect attack. The identification of genes involved in insect resistance will aid breeders in developing naturally resistant crop species. Understanding natural defense mechanisms in major crops will allow alternative insect control strategies and reduce our dependence on potentially toxic pesticides. Thus, the problem to be investigated is of scientific, economic and environmental significance.

DESCRIPTION (provided by applicant): Lactonamycin was isolated by Matsumoto and coworkers in 1996 from Streptomyces rishiriensis in a screen for new antibiotics active against drug resistant bacterial strains. Lactonamycin exhibits potent antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). Structurally related polyketides including the tetracenomycins and elloramycin display potent antitumor activity. The goals of this research program are as follows: 1) Development of general synthetic methods to access lactonamycin, elloramycin, saintopin E, and the numerous tetracenomycin structures. In particular the construction of the ABCD-ring systems through a strategy of tandem 1,4-addition-Dieckmann ring closure will provide access to the natural product targets as well as analogs. 2) Construct advanced synthetic intermediates postulated as biosynthetic precursors of lactonamycin as part of elucidating the biosynthetic pathway to lactonamycin. 3) Construct an appropriate form of the carbohydrate L-rhodinose for completion of a chemical synthesis of lactonamycin. 4) Expand and define the scope of the tandem 1,4-addition-Dieckmann ring closure for applications to related type II polyketide natural products. Further expansion of the methodology to the synthesis of condensed polycyclic heterocycles. 5) Expand the [3+2] cycloaddition reaction of quinones with nitrile oxides to gain access to diverse type II polyketide structures such as DNA helicase inhibitor heliquinomycin.

This project is awarding scholarships to academically talented but financially-disadvantaged graduate students pursuing interdisciplinary research in the Departments of Biochemistry and Cell Biology, Chemistry, Chemical Engineering, and Physics at Rice University. The scholarships are supporting 26 students through the first two years of study, at which point students are transitioning to faculty research funding. Recruiting efforts are targeting women and underrepresented minorities. Students are recruited at conferences sponsored by the Society for Advancement of Chicanos and Native Americans in Science, the National Society of Black Engineers, the American Indian Science and Engineering Society, and the Society of Hispanic Professional Engineers, as well as at the Annual Biomedical Research Conference for Minority Students. Support structures include strong faculty mentoring for students in their first two years of graduate study, the use of senior graduate student ambassadors to mentor entering students, and seminar courses on communication and on interdisciplinary research for each cohort of eight to nine students. The goals of the program include increasing the proportion of minorities and women enrolled in the four graduate departments, supporting students through their first two years of graduate study, and building students' confidence in their research abilities.

With this award from the Chemistry Research Instrumentation and Facilities: Departmental Multi-User Instrumentation program (CRIF:MU), Professors Seiichi P. Matsuda, Ronald J. Parry and colleagues from the Department of Chemistry at William Marsh Rice University will upgrade a 200 and a 500 MHz NMR spectrometers, both of which will have solid state capabilities. The instrument will be used to support research activities such as: 1) studies of the structure and reactivity of transition metals in organic chemistry; 2) analysis of the structures of complex nano and composite materials; 3) use of NMR techniques to understand and manage the Earth's carbon cycle; 4) biosynthetic studies of biologically active natural products; 5) investigations of graphene nanoribbons and derivatives; and 6) analysis of organic-inorganic hybrid nanoparticles.

Nuclear Magnetic Resonance (NMR) spectroscopy is one of the most powerful tools available to chemists for the elucidation of the structure of molecules. It is used to identify unknown substances, to characterize specific arrangements of atoms within molecules, and to study the dynamics of interactions between molecules in solids and in solution. Access to state-of-the-art NMR spectrometers is essential to chemists who are carrying out frontier research. The results from these NMR studies will have an impact in synthetic organic/inorganic chemistry, materials and biochemistry.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce in the United States by recognizing and supporting outstanding graduate students who are pursuing research-based master's and doctoral degrees in fields within NSF's mission. GRFP provides three years of support for the graduate education of individuals who have demonstrated their potential for significant achievements in science and engineering research. The award to this GRFP institution supports NSF Graduate Fellows pursuing graduate education at the institution.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science, technology, engineering, and mathematics (STEM) and in STEM education. The GRFP provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in STEM and STEM education. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).  <br/><br/>The Computer and Information Science and Engineering Graduate Fellowships (CSGrad4US) is intended for individuals who have some practical experience following their bachelor’s degree and are now interested in pursuing a research-based doctoral degree. CSGrad4US Fellowships are a part of an overall strategy by NSF's CISE directorate to develop the workforce necessary to ensure the Nation's leadership in advancing CISE research and innovation. The CSGrad4US fellowship provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in CISE disciplines. This award supports the CSGrad4US Fellows pursuing graduate education at this CSGrad4US institution.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.