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Research in Biochemistry, Bioorganic, and Analytical Chemistry Our research group is focused on understanding the roles of specific lipid molecular species and phospholipases in cellular functions and human disease. The laboratory is highly interdisciplinary with research faculty, postdoctoral fellows, staff and graduate students coming from a variety of backgrounds including chemistry, biochemistry, pharmacology, and mathematics. We combine systems biology approaches with mass spectrometry to profile changes in cellular lipid species (lipidomics). Much of our work is focused on understanding the role of phospholipase D (PLD) in growth factor and G protein coupled receptor (GPCR) signaling networks. With our collaborators we are developing novel chemical inhibitors of PLD and ancillary proteins in the signaling network to better understand the role of PLD in cancers and tumorigenesis, as well as to explore its therapeutic potential. Areas of current research include: structure and enzymology of PLD; defining the signal transduction networks from the receptor tyrosine kinases and GPCRs to PLD; mechanisms by which phosphatidic acid (PA) regulates cell cycle, growth, and proliferation; roles of PLD in zebrafish; developing novel probes for defining specific lipid binding targets; precursor-product relationships of lipid enzymes using mass spectrometry; lipid perioxidation and antioxidant mechanisms; and advanced methods in lipid mass spectrometry. RESEARCH INTERESTS AND PROJECTS Signal Transduction Pathways of Phospholipase D. Our laboratory has a long-standing interest in enzymes involved in cellular signaling and particularly the dynamic balance between PA and diacylglycerol (DAG). One of the major cellular pathways for direct production of PA is PLD. This enzyme is expressed in certain bacteria, viruses, yeast, and ubiquitously in mammalian cells. Defining the functions of PLD along with the pathways by which PLD is activated by cell surface receptors has been a challenging undertaking. We are particularly interested in defining the roles of PLD-generated PA in EGF receptor functions and have recently developed a system to define both protein and lipid participants in this pathway using a strategic combination of proteomic and lipidomic approaches. PLD has been implicated in a number of human diseases including diabetes, myocardial disease, neurodegenerative disorders, infectious diseases, and cancers. We have developed a platform to screen small molecule libraries for inhibitors of PLD and other lipid signaling pathways using both enzymatic- and cell-based assays. In collaboration with Craig Lindsley’s group candidate small molecules are identified and compounds optimized to improve potency, isoform selectivity, and pharmacodynamic properties. Effective inhibitors are being used to define mechanisms of PLD action on cell growth, proliferation, and a variety of processes in cancer cells. Structure and Enzymology of Phospholipase D. The structure of a mammalian PLD phosphodiesterase has been elusive. Our goal is to understand how G proteins and kinases modulate the structure and catalytic activity of the enzyme in order to fully understand its molecular mechanism. We will use this information to further refine our cadre of inhibitors to be more specific and isoform selective. As a result of our detailed analysis of the lipid composition of macrophages, we have identified several atypical or novel species of glycerophospholipids. This includes plasmanyl and plasmenyl species of phosphatidic acid and phosphatidylinositols. The roles of these species in metabolism and cell signaling are currently not defined, but we are using state-of-the-art mass spectrometry to establish precursor-product relationships. This includes the perioxidation products of polyunsaturated fatty esters and determining mechanisms by which these species contribute to human disease. The focus of this project includes the study of products formed by perioxidation of different lipid classes, including electrophiles generated. In collaboration with the groups of Ned Porter and Larry Marnett in the Chemistry Program at Vanderbilt, we have developed a new class of lipid probe that is being used to track metabolic and signaling pathways through cells. These probes are being developed for both lipidomic and proteomic applications. Selected Publications Rappley I, Gitler AD, Selvy PE, LaVoie MJ, Levy BD, Brown HA, Lindquist S, Selkoe DJ. Evidence That alpha-Synuclein Does Not Inhibit Phospholipase D. Biochemistry. 2009, 48(5): 1077-1083. Marlo JE, Niswender CM, Days EL, Bridges TM, Xiang Y, Rodriguez AL, Shirey JK, Brady AE, Nalywajko T, Luo Q, Austin CA, Williams MB, Kim K, Williams R, Orton D, Brown HA, Lindsley CW, Weaver CD, Conn PJ. Discovery and Characterization of Novel Allosteric Potentiators of M-1 Muscarinic Receptors Reveals Multiple Modes of Activity. Molecular Pharmacology. 2009, 75(3): 577-588. Lewis JA, Scott SA, Lavieri R, Buck JR, Selvy PE, Stoops SL, Armstrong MD, Brown HA, Lindsley CW. Design and synthesis of isoform-selective phospholipase D (PLD) inhibitors. Part I: Impact of alternative halogenated privileged structures for PLD1 specificity. Bioorganic & Medicinal Chemistry Letters. 2009, 19(7): 1916-1920. Lavieri R, Scott SA, Lewis JA, Selvy PE, Armstrong M, Brown HA, Lindsley CW. Design and synthesis of isoform-selective phospholipase D (PLD) inhibitors. Part II. Identification of the 1,3,8-triazaspiro[4,5]decan-4-one privileged structure that engenders PLD2 selectivity. Bioorganic & Medicinal Chemistry Letters. 2009, 19(8): 2240-2243. Milne SB, Ivanova PT, Armstrong MD, Myers DS, Lubarda J, Shulga YV, Topham MK, Brown HA, Epand RM. Dramatic differences in the roles in lipid metabolism of two isoforms of diacylglycerol kinase. Biochemistry. 2008, 47(36): 9372-9379. Garcia A, Zheng Y, Zhao C, Toschi A, Fan J, Shraibman N, Brown HA, Bar-Sagi D, Foster DA, Arbiser JL. Honokiol suppresses survival signals mediated by Ras-dependent phospholipase D activity in human cancer cells. Clinical Cancer Research. 2008, 14(13): 4267-4274. Brown HA. New mechanism for a classic folate inhibitor. Nature Chemical Biology. 2008, 4(10): 581-582. Sun Y, Fang Y, Yoon MS, Zhang C, Roccio M, Zwartkruis FJ, Armstrong M, Brown HA, Chen J. Phospholipase D1 is an effector of Rheb in the mTOR pathway. Proceedings of the National Academy of Sciences. 2008, 105(24): 8286-8291. Murphy, SC, Fernandez-Pol, S, Chung, PH, Murthy, SN, Milne, SB, Salomao, M, Brown, HA, Lomasney, JW, Mohandas, N, and Haldar, K. Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum. Blood. 2007, 15: 2132-2139. Ivanova, PT, Milne, SB, Byrne, MO, Xiang, Y, and Brown, HA. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry. Methods in Enzymoology. 2007, 432: 21-57. Brown, HA, Henage, LG, Preininger, AM, Xiang, Y, and Exton, JH. Biochemical Analysis of Phospholipase D. Methods in Enzymology. 2007, 434: 49-87. Callender, HL, Forrester, JS, Ivanova, P, Preininger, A, Milne, S, Brown, HA. Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm. Analytical Chemistry. 2007, 79(1): 263-72. Rouzer, CA, Ivanova, PT, Byrne, MO, Brown, HA, Marnett, LJ. Lipid profiling reveals glycerophospholipid remodeling in zymosan-stimulated macrophages. Biochemistry. 2007, 46(20): 6026-6042. Sud, M, Fahy, E, Cotter, D, Brown, A, Dennis, EA, Glass, CK, Merrill, AH, Murphy, RC, Raetz, CR, Russell, DW, Subramaniam, S. LMSD: LIPID MAPS structure database. Nucleic Acids Research. 2007, 35: D527-32. Milne, SB, Forrester, JS, Ivanova, PT, Armstrong, MD, and Brown, HA. Multiplexed Lipid Arrays of Antiimmunoglobulin M-Induced Changes in the Glycerophospholipid Composition of WEHI-231 Cells. Functional Lipidomics. 2006 Milne, S, Ivanova, P, Forrester, J, Alex Brown, H. Lipidomics: an analysis of cellular lipids by ESI-MS. Methods. 2006, 39(2): 92-103. Rouzer, CA, Ivanova, PT, Byrne, MO, Milne, SB, Marnett, LJ, Brown, HA. Lipid profiling reveals arachidonate deficiency in RAW264.7 cells: Structural and functional implications. Biochemistry. 2006, 45(49): 14795-808. Henage, LG, Exton, JH, Brown, HA. Kinetic analysis of a mammalian phospholipase D: allosteric modulation by monomeric GTPases, protein kinase C, and polyphosphoinositides. Journal of Biological Chemistry. 2006, 281(6): 3408-17. Preininger, AM, Henage, LG, Oldham, WM, Yoon, EJ, Hamm, HE, Brown, HA. Direct modulation of phospholipase D activity by Gbetagamma. Molecular Pharmacology. 2006, 70(1): 311-318. Fahy, E, Subramaniam, S, Brown, HA, Glass, CK, Merrill, AH, Murphy, RC, Raetz, CR, Russell, DW, Seyama, Y, Shaw, W, Shimizu, T, Spener, F, Van Meer, G, VanNieuwenhze, MS, White, SH, Witztum, J, and Dennis, EA. A Comprehensive Classification System for Lipids. Journal of Lipid Research. 2005, 46: 839-862. Milne, SB, Ivanova, PT, DeCamp, D, Hsueh, RC, Brown, HA. A targeted mass spectrometric analysis of phosphatidylinositol phosphate species. Journal of Lipid Researsh. 2005, 46(8): 1796-802. Specialties
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Vanderbilt University Department of Chemistry |
Professor of Pharmacology at Vanderbilt University School of Medicine