Triacylglycerol Synthesis and Stress Response in Fission Yeast Schizosaccharomyces pombe ZHANG QIAN A THESIS SUBMITTED FOR THE DEGREE OF PHD DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS First of all, I would like to express my deepest gratitude to my mentor, Dr. Robert Yang Hongyuan, whose advise, guidance, encouragement and scientific excellence have made this thesis possible. It is an honor to be his graduate student and the learning experience under his guidance has been both challenging and rewarding. His continuous support and encouragement have given me strong confidence throughout my entire graduate training. Heartfelt thanks also go to his laboratory members Woo Wee Hong, Low Choon Pei, Zhang Shao Chong, Chieu Hai Kee, Li Hongzhe, Li Ou, Liew Li Phing, Wang Peng Hua, Alex Lim, Yvonne Tay, Xiao Han, Tan Eric, and Dr. Li Tianwei, for their invaluable, unreservedly generous technical help and kind words of encouragement. I am also grateful to Dr. Mohan Balasubramanian, Dr. Wang Hongyan and Volker Wachtler for kindly providing yeast strains and technical assistance. Sincere thanks to Dr. Naweed Naqvi, Dr. Matt Whiteman, Dr. Marie Clement, Dr. Tian Seng Teo, and Dr. Alan Munn for their help and advice on this project. Finally, special thanks are to my family, especially my husband, who has been with me all these while, for his unfailing support and love. TABLE OF CONTENTS Acknowledgement ……………………………………………………………………… Table of content …………………………………………………………………………3 Abstract …………………………………………………………………………………11 List of tables ……………………………………………………………………………12 List of figures ……………………………………………………………………………12 Abbreviation and symbols used …………………………………………………………18 1. Introduction……………………………………………………………………………22 1.1 Functions of TAG…………………………………………………………………23 1.2 Synthesis of TAG and its metabolic pathways……………………………………24 1.2.1 Biosynthesis of triacylglycerols(TAG) …………………………………… 24 1.2.1.1 Phosphaditic Acid Pathway………………………………………………24 1.2.1.2 Monoacylglycerol Pathway………………………………………………28 1.2.1.3 GPAT ……………………………………………………………………28 1.2.1.4 DAG acyltransferase ……………………………………………………30 1.2.1.4.1 DGAT ……………………………………………………………32 1.2.1.4.2 DAG transacylase …………………………………………………32 1.2.1.4.3 Lecithin-DAG transacylase ………………………………………32 1.2.1.4.4 Regulation of enzymes responsible for DAG esterification ………33 1.2.2 Hydrolysis of TAG …………………………………………………………33 1.3 Regulation of TAG metabolism …………………………………………………35 1.3.1 Nutritional regulation of TAG metabolism…………………………………36 1.3.2 Hormonal regulation and signaling pathways involved in TAG metabolism………………………………………………38 1.3.2.1 Hormonal regulation and signaling pathways in TAG lipogenesis………38 1.3.2.2 Hormonal regulation and signaling pathway in TAG lipolysis …………39 1.3.2.2.1 Catecholamines and glucagons ………………………………39 1.3.2.2.2 Leptin …………………………………………………………41 1.3.2.3 Transcritional regulation of TAG metabolism by SREBP1 ……………42 1.4 TAG and diseases …………………………………………………………………45 1.4.1 Congenital generalized lipoatrophy (CGL)…………………………………45 1.4.2 Diet induced obesity ………………………………………………………46 1.4.3 TAG and heart disease ……………………………………………………47 1.4.4 TAG and type diabetes……………………………………………………47 1.5 Relationship between TAG and lipotoxicity………………………………………49 1.6 TAG biosynthesis in yeast S. cerevisiae: acylation of DAG ……………………53 1.7 S. pombe as a good model and tool for lipid metabolism research. ………………58 1.8 Our specific aim …………………………………………………………………61 2. Materials and methods ………………………………………………………………63 2.1 Strain and media …………………………………………………………………63 2.2 Enzyme identification and characterization ………………………………………63 2.2.1 Disruption of plh1+ and dga1+ ………………………………………………63 2.2.2. In vivo DAG or sterol esterification assays …………………………………66 2.2.3. In vitro DAG esterification or sterol esterification assays …………………67 2.2.4. Site Directed Mutagenesis by PCR Overlap Extension ……………………73 2.2.5. Modification of active residue of Plh1p ……………………………………78 2.3 Phenotype characterization ………………………………………………………79 2.3.1 Growth curve analysis ……………………………………………………79 2.2 Viability in various growth phases…………………………………………79 2.3.3 Viability under various stress conditions …………………………………80 2.3.3.1 Viability in osmotic and oxidative Stress …………………………80 2.3.3.2. Heat shock stress treatment ………………………………………80 2.3.4 Fluorescence microscopy …………………………………………………81 2.3.4.1 Nile Red staining …………………………………………………81 2.3.4.2 DNA Staining ……………………………………………………81 2.3.4.3 GFP fluorescence …………………………………………………82 2.3.4.4 TUNEL assay………………………………………………………82 2.3.4.5. Annexin V staining ………………………………………………83 2.3.4.6. ROS staining………………………………………………………83 2.3.4.7. TMRE staining ……………………………………………………84 2.3.5. Measurement of fatty acid biosynthesis by C14-acetate incorporation …84 2.3.6. Fatty Acid Analysis by using GCMS ……………………………………85 2.3.7 Analysis of DAG Accumulation by Steady-state Labeling ………………87 3. Characterization of Plh1p and Dga1p in S. pombe……………………………………89 3.1 Introduction ………………………………………………………………………89 3.2 Identification of plh1+ and dga1+ in S. pombe …………………………………90 3.3 Deletion of plh1+ and dga1+ in S. pombe ………………………………………91 3.4 Characterization of Deletion Mutants ……………………………………………91 3.4.1 Deletion of plh1+ and dga1+ resulted in viable yeast ……………………91 3.4.2 TAG synthesis in cells with deletion of plh1+ and dga1+ …………………91 3.4.3 Analysis of plh1+ or dga1+ by overexpression ……………………………93 3.4.4 In vitro and in vivo esterification assays ……………………………………93 3.4.4.1 In vitro microsomal assays of DAG esterification …………………93 3.4.4.2 Assays of sterol esterification ………………………………………94 3.4.4.2.1 Identification of candidate genes for sterol esterification in S. pombe………………………………………………………… 95 3.4.4.2.2 In vitro microsomal assays of sterol esterification ………96 3.4.5 Substrate specificities of Plh1p and Dga1p …………………………………97 3.5 Characterization of Plh1p …………………………………………………………97 3.5.1 Introduction …………………………………………………………………97 3.5.2 Conserved structure elements in Plh1p ……………………………………99 3.5.3 Chemical modification of serine, histidine and cysterine …………………99 3.5.4 Site-directed mutagenesis of the acid residue of the catalytic triad ………100 3.6. Localization of Plh1p and Dga1p ………………………………………………101 3.7 Summary …………………………………………………………………………101 4. Phenotype characterization …………………………………………………………117 4.1 Growth property of cells deficient in TAG biosynthesis under various conditions ………………………………………………………………118 4.1.1 Growth of cells deficient in TAG biosynthesis in stationary phase and log-phase ………………………………………………………118 4.1.2 Detection of cell death in cells upon entering stationary phase ……………119 4.1.3 Growth of cells deficient in TAG biosynthesis in stress conditions ………121 4.2 Mating Behavior of cells deficient in TAG biosynthesis…………………………122 4.2.1 Growth property of h90 DKO in rich medium ……………………………123 4.2.2 Mating behavior in late stationary phase in YES medium ……………… 123 4.2.3 Mating ability and growth property in ME…………………………………124 4.2.4 Growth property of DKO of 266 and h90 in ME …………………………124 4.3 Lipid profiles under deficiency of DAG esterification …………………………125 4.3.1 Fatty acid metabolism ……………………………………………………126 4.3.1.1 Fatty acid biosynthesis ……………………………………………126 4.3.1.2 Total fatty acid level ………………………………………………126 4.3.2 DAG biosynthesis at steady state increased markedly in DKO mutants upon entry into stationary phase …………………………………………………127 4.3.3 Assay for [3H] oleate incorporation into phospholipids and ergesterol ester in DKO and wild type cells……………………………………………………127 4.3.4 DAG level under high salt stress …………………………………………128 4.4 Summary …………………………………………………………………………128 Role of DAG and sphingolipids in cell death of DKO ………………………………151 5.1 Introduction ………………………………………………………………………151 5.2 Role of DAG accumulation in the cell death of DKO……………………………151 5.2.1 Detection of cell death induced by DiC8 DAG ……………………………152 5.2.1.1 Growth of cells on YES plate containing high concentration of DAG…………………………………………152 5.2.1.2 Viability test under high concentration DAG treatment……………152 5.2.1.3 Cell morphology under DAG treatment……………………………153 5.2.2 Role of DAG in high concentration fatty acid treatment …………………153 5.2.2.1 Viability under high concentration fatty acid treatment……………154 5.2.2 Cell death under high concentration of fatty acid exposure ………154 5.2.2.3 DAG level under fatty acid treatment………………………………154 5.2.2.4 Viability test of DKO with overexpression of DGK+ ……………155 5.2.2.4.1 Detection of DAG Kinase activity ………………………155 5.2.2.4.2 Viability test of DKO with overexpression of dgk ……………………………………156 5.2.2.4.3 Cell morphology identification of DKO with dgk overexpression under fatty acids treatment………………156 5.2.3 Viability of DKO with dgk overexpression in ME medium ………………156 5.2.4 C1 treatment ………………………………………………………………157 5.3 Role of sphigolipids in the cell death of DKO ……………………………………158 5.3.1 Viability in ceramide treatment………………………………………………159 5.3.2 Viability in DHS treatment …………………………………………………159 5.3.3 Cell morphology in ceramide and DHS treatment……………………………159 5.3.4 Viability fumonisin B1 and myriocin treatment ……………………………161 5.3.4.1 fumonisin B1…………………………………………………………161 5.3.4.2 myriocin………………………………………………………………161 5.4 Summary……………………………………………………………………………161 6. Mechanism of cell death caused by TAG deficiency ………………………………179 6.1 Introduction ……………………………………………………………………179 6.2 Role of ROS in cell death of DKO under various stress conditions……………179 6.2.1 ROS accumulation under DAG treatment ………………………………180 6.2.2 ROS accumulation under fatty acid, and high salt treatment ……………180 6.2.3 ROS accumulation in ME medium ………………………………………181 6.2.4 Recovery of viability through TMPO treatment…………………………181 6.3 Role of caspase in the death of DKO cells ……………………………………182 6.3.1 Viability in caspase deletion strains………………………………………182 6.3.2 Viability of DKO under zVAD-fmk treatment …………………………183 6.4 Role of mitochondria …………………………………………………………184 6.4.1 TMRE staining……………………………………………………………184 6.4.2 Cyclosporin A treatment …………………………………………………185 6.5 cAMP and MAP kinase inhibitor ………………………………………………185 6.5.1 cAMP ……………………………………………………………………186 6.5.2 MAP kinase inhibitor ……………………………………………………186 6.6 Summary ………………………………………………………………………186 7. Discussion……………………………………………………………………………200 7.1 Identification of two enzymes responsible for DAG esterification in S. pombe…200 7.2 Altered lipid profiles under TAG absence ………………………………………201 7.3 DAG, ROS, lipotoxicity and lipoapoptosis ………………………………………203 7.4 Programmed cell death/Apoptosis or Necrosis? …………………………………208 7.4.1 Selection of cell death under different nutritional profiles…………………211 7.4.2 Mitochondria ………………………………………………………………219 7.4.3 Caspase ……………………………………………………………………221 7.5 Apoptosis in yeast: suicide or murder? …………………………………………222 7.6 Difference between fission yeast and budding yeast ……………………………226 7.7 Future work ……………………………………………………………………228 Reference ………………………………………………………………………………230 10 Campfield LA, et al, Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks, Science, 1995, 269(5223): 546-9 Cases, S., et al, Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis, Proc. 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Isolation and biochemical characterization of organelles from the yeast, Saccharomyces cerevisiae, Yeast, 1995, 11: 493-536 246 Bibliography Name: Zhang Qian Gender: Female Date of Birth: October 1971 Nationality: P.R.China Permanent Residency: Singapore Current position: Regulatory Scientist, Health Science Authority, Singapore Education Experience 1999-2005 PhD, Department of Biochemistry, Medical School, National University of Singapore, Singapore 1993-1996 Master of Science, Beijing University of Chinese Medicine, China 1989-1993 Bachelor of Medicine, Beijing University of Chinese Medicine, China Working Experience 1996-1999 Research Assistant, Beijing University of Chinese Medicine, China Publication Schizosaccharomyces pombe Cells Deficient in Triacylglycerols Synthesis Undergo Apoptosis upon Entry into the Stationary Phase, J. Biol. Chem., Vol. 278, Issue 47, 47145-47155, November 21, 2003 Patent Triacylglycerol-Deficient FissionYeast and its Uses (US 2004) 247 [...]... cerevisiae S pombe Schizosaccharomyces pombe SAPK stress- activated protein kinase SREBP sterol regulatory element-binding protein Sty1p suppressor of tyrosine kinase 1 protein TAG triacylglycerols TGH TAG hydrolase 20 TKO Δdga1Δplh1Δpca1 of S pombe TLC thin layer chromatography TMRE tetramethylrhodamine ethyl ester TUNEL terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labelling VLDL very... lipogenesis in both liver and adipose tissues, resulting in elevated postprandial plasma TAG levels On the other hand, fasting could induce net loss of TAG from fat cells through reducing lipogenesis in adipose tissue and increasing rate of lipolysis (Kerstan S, 2001) Glucose is another critical factor to initiate TAG biosynthesis Plasma glucose is an indicator of reduced or excess food intake and could... type and DKO under hydrogen peroxide treatment ….…………… 137 Figure 4.11 DAPI staining and TUNEL assay in cells under high concentration of KCl …………………138 Figure 4.12 DAPI staining and TUNEL assay in cells under H2O2 treatment…………139 Figure 4.13 Viability of h90 WT and DKO in YES medium upon entry into stationary phase…………………………………………………………………… 140 Figure 4.14 DAPI staining and TUNEL assay of h90 WT and. .. absence upon stationary phase and salt stress …………………………………………………………….203 Figure 7.2 The crosstalk of stress activated MAP kinase pathway and cAMP dependent kinase pathway in S pombe …………………………………………… 217 Figure 7.3 Upstream signaling events determine final modes of cell death………… 218 Figure 7.4 Program Cell Death in monocellular organism yeast ……………………226 17 ABBREVIATIONS AND SYMBOLS USED ACAT acyl-CoA:... evidence suggesting that DAG, not sphingolipids, mediates fatty acid-induced lipoapoptosis in yeast Lastly, we show that generation of reactive oxygen species is essential to lipoapoptosis Therefore, we suggest that the TAG biosynthesis in stressful conditions provides a buffering form for highly reactive or toxic molecules such as DAG or ROS The inhibition of TAG synthesis in fission yeast may generate... Colony forming under DHS treatment……………………………………174 Figure 5.16 DAPI staining and TUNEL assay of cells under ceramide treatment…….175 Figure 5.17 DAPI staining and TUNEL assay of cells under DHS treatment…………176 Figure 5.18 Colony forming assay under fumunisin B1 treatment…………………….177 Figure 5.19 DAPI staining under fumonisin treatment……………………………… 177 Figure 5.20 Colony forming assay under myriocin B1 treatment……………………... treatment…………………… 178 Figure 5.21 DAPI staining under myriocin treatment…………………………………178 Chapter VI Figure 6.1 ROS staining under DAG treatment……………………………………… 187 Figure 6.2 ROS staining under palmitic acid treatment……………………………… 187 Figure 6.3 ROS staining under KCl treatment…………………………………………188 Figure 6.4 ROS staining in ME medium………………………………………………188 Figure 6.5 Colony forming assay of cells with or without... et al, 2000) Another case is related to hibernating grizzly bears They store enormous amount of body fats (most of which are TAG) in preparation for their long sleep Using body fat as their sole fuel, bears can survive the whole winter without eating, drinking, urinating or defecating (Nelson, D L and Cox, M M., 2000) Indeed, the appearance of TAG is again a victory of nature to show how a specific molecule... lipase D 27 the pathway of TAG and phospholipid biosynthesis Within the cell, there appear to be separate lipid pools, and these isoenzymes may participate in distinct biological pathways (Rustow B and Kunze D., 1985; Binaglia, L., et al ,1982) 1.2.1.2 Monoacylglycerol Pathway In mammals, the monoglycerol pathway primarily takes place in the intestinal mucosal cells, hepatocytes and adipocytes After pancreatic... as acyl donors Gat2p, on the other hand, prefers G-3-P and 16 carbon fatty acids A reduced PA pool and an increased PS/ PI ratio were observed in both gat1 and gat2 single deletion 29 strains Deletion of GAT1 resulted in a 50% increase in the rate of TAG synthesis whereas deletion of GAT2 reduced the rate of TAG synthesis by 50% (Zaremberg, V., and McMaster, C.R 2002) These results suggest that acylation . cerevisiae S. pombe Schizosaccharomyces pombe SAPK stress- activated protein kinase SREBP sterol regulatory element-binding protein Sty1p suppressor of tyrosine kinase 1 protein TAG triacylglycerols. 1 Triacylglycerol Synthesis and Stress Response in Fission Yeast Schizosaccharomyces pombe ZHANG QIAN . in S. pombe …………………………………89 3.1 Introduction ………………………………………………………………………89 3.2 Identification of plh1 + and dga1 + in S. pombe …………………………………90 3.3 Deletion of plh1 + and dga1 + in S. pombe