22/07/54 Oral Pre se ntatio n CRDC 5 The 5 th Tropical and Sub-tropical Crops The 5th Tropical and Sub ‐ tropical Crops Research Symposium (CRDC5) Research Symposium (CRDC5) Responses of Transcription Factor to Oxidative Stress in Saccharomyces cerevisiae Piyasuda Thepnok Biochemical Technology School of Bioresources and Technology King Mongkut’s University of Technology Thonburi Introduction Collection of proteins called transcription factors mediate binding of RNA polymerase Initiation of transcription Transcription Elongation of RNA chain g Termination of transcription 2 2 1
22/07/54 Oral Pre se ntatio n CRDC 5 Introduction T F Transcription Factor (TF) � Proteins that bind to specific sequence of DNA DNA � Control transcription � Regulate transcription of gene either � Regulate transcription of gene either positively or negatively Transcription regulator 3 3 � In S. cerevisiae , Major class of transcriptional regulators is composed of sub-family of zinc finger proteins “Finger–like” shape zinc cluster proteins Play important role in transcriptional processes Crystal structures of Gal4p o rs 4 4 2
22/07/54 Oral Pre se ntatio n CRDC 5 Zinc cluster proteins in S. cerevisiae � Complete sequencing of S. cerevisiae genome � Identification of 55 members � Based on well-conserved consensus amino acid sequence Cys-X 2 -Cys-X 6 -Cys-X 5–12 -Cys-X 2 -Cys-X 6–8 -Cys Function of Zinc cluster proteins � Act as transcription regulators in wide range of cellular processes � A mino acid and vitamin synthesis � Carbon and nitrogen metabolism � Meiosis and morphogenesis � Stress response and pleiotropic drug resistance (PDR) 6 6 3
22/07/54 Oral Pre se ntatio n CRDC 5 Role of Zinc cluster protein Role Gene name Phenotype EDS1 ( YBR033W ) Expression is dependent on Rpb2p Δ ybr150c is sensitive to thiabendazole TAS1 (YBR150C) Interacts with Rds2p in yeast two - hybrid system YBR239C YDR520C Δ y dr520c is slightly sensitive to caffeine YER184C Unknown YFL052W Δ yfl052w is hypersensitive to heat shock at 37 o C YJL103C May be involved in oxidative phosphorylation YJL206C Δ ykl222c is sensitive to caffeine YKL222C YKR064W YLL054C YLR278C Δ ylr278c is sensitive to caffeine YNR063W Previous study � Akache, B., Wu, K. and Turcotte, B., 2001 � Examined phenotypes of strains carrying deletion of genes encoding zinc cluster proteins on non-fermentable carbon source : Lactate, Glycerol 8 4
22/07/54 Oral Pre se ntatio n CRDC 5 � Akache, B., Wu, K. and Turcotte, B., 2001 9 4 My previous study . Growth of S. cerevisiae strains with deleted zinc cluster genes on media containing glucose, oleic or linoleic acids as a sole carbon source Carbon source Strain Synthetic media YP media Glucose Oleic Linoleic Glucose Oleic Linoleic acid acid acid acid Wild-type ++++ ++++ ++++ ++++ ++++ ++++ ∆ yor380w ++++ ++ ++ ++++ ++ ++ ( ∆ FZP ) ++++ normal growth +++ moderate growth ++ impaired growth + severly impaired growth - no growth 10 5
22/07/54 Oral Pre se ntatio n CRDC 5 Phenotypic analysis of wild-type and deletion strains Wild-type ∆ FZP SD + 2 % Glucose YP + 2 % Glucose Wild-type ∆ FZP SD + 0.125 % Oleic acid YP + 0.125% Oleic acid Wild-type ∆ FZP SD + 0.125% Linoleic acid YP + 0.125 % Linoleic acid : Deletion of FZP which encodes for zinc cluster proteins resulted in impaired growth on oleic and linoleic acids 11 Oxidative stress Oxidative agents � Oxidative stress : Caused by reactive oxygen species (ROS) species (ROS) - hydrogen peroxide (H 2 O 2 ) � ROS generated during respiration and oxidation of nutrients Reduce growth rate and affect cell viability � Generated ROS may damage cellular macromolecules : nucleic acids, proteins and lipids 6
22/07/54 Oral Pre se ntatio n CRDC 5 � Oxidative agent : H 2 O 2 � Produced during dehydrogenation in β -oxidation � O � One major target of H 2 O 2 attack is unsaturated j t t f H O tt k i t t d lipids, leading to autocatalytic lipid peroxidation (Gunstone, 1996) Membrane damage and may impaired growth of g y p g unsaturated fatty acid � To overcome oxidative stress Essential for yeast cells to be able to respond and adapt rapidly to stress condition for survival � Cells possess variety of defenses Defenses systems : Enzymatic defense systems - Catalase : Used to maintain cellular redox state - Catalase : Used to maintain cellular redox state : Found in peroxisomes : Represent sole site for fatty acid β -oxidation 7
22/07/54 Oral Pre se ntatio n CRDC 5 Peroxisomes Organelles and compartments in yeast cell � Main functions of peroxisomes is degradation of Main functions of peroxisomes is degradation of fatty acids. � In S. cerevisiae , fatty acid β -oxidation is restricted to peroxisomes (Tanaka et al., 1982: Kunau et al., 1988) http://biochemie.web.med.uni-muenchen.de � Oxidative agent : Diamide : Can rapidly cross biological membrane and altered cellular physiology such as change in composition of plasma membrane of plasma membrane 8
22/07/54 Oral Pre se ntatio n CRDC 5 Present study Objective : � This study aims to understand roles of uncharacterized zinc cluster proteins � Examined sensitivity of deletion strain to oxidative stress, : Process commonly occur during non-fermentation : Fatty acid metabolism normally generates oxidative stress 17 Methodology Phenotypic analysis Growth assay of wild-type and deleted zinc cluster strains on oxidative agent : H 2 O 2 , diamide � Spot assay 18 11 9
22/07/54 Oral Pre se ntatio n CRDC 5 Phenotypic analysis Se r ial dilution e of S.c e r e visiae Cultur wild- type and de le tion str ains on YPD me dia Spot assay Results 20 10
22/07/54 Oral Pre se ntatio n CRDC 5 cell cell Wild ‐ type ∆ FZP ∆ FZP ∆ FZI SD + 2 % Glucose YP + 2 % Glucose Wild ‐ type ∆ FZP ∆ FZI SD + 3 mM H 2 O 2 YP + 3 mM H 2 O 2 Wild ‐ type ∆ FZP ∆ FZI SD + 0.4 mM diamide YP + 0.4 mM diamide Figure 1. Growth assay of zinc cluster Δ FZP and Δ FZI strains on glucose in presence of 3 mM H 2 O 2 and 0.4 mM Diamide Discussion � Fatty acid metabolism normally generates oxidative stress � H 2 O 2 produced during dehydrogenation in β -oxidation can induce such stress response Lead to membrane damage Causing impaired growth on fatty acids 22 11
22/07/54 Oral Pre se ntatio n CRDC 5 Discussion � Yeast cells must adapt to promptly response to such stress such stress � Suggests that FZP plays important role to ensure survival under this condition 23 ? ? FZP Putative target genes of zinc cluster protein FZP in pathway of fatty acid β -oxidation and peroxisomal biogenesis. 24 12
22/07/54 Oral Pre se ntatio n CRDC 5 � Fujihara et al., 2009 � PEX13 is required for appressorium-mediated plant infection by Anthracnose fungus Colletotrichum orbiculare Colletotrichum orbiculare � Appressorium melanization is required for p pathogenicity g y � Howard et al. 1991; Wang et al. 2005. 25 � � Appressorium : Initial host penetration structure Darkly melanized infection structure Ap Ap S S Ap Ap Ap : Appressorium S : Spore Kubo and Furusawa 1991; Tsuji et al. 1997 13
22/07/54 Oral Pre se ntatio n CRDC 5 � Melanin functions to trap osmolytes within cell, generating high turgor pressure necessary for mechanical penetration of plant cuticle and cell wall cell wall 27 � Plant-pathogenic fungi � Metabolic processes catalyzed by peroxisomal enzymes enzymes Essential for appressorium function in Magnaporthe spp. and Colletotrichum spp. � Asakura et al. 2006; Bhambra et al. 2006; Ramos-Pamplona and Naqvi, 2006 14
22/07/54 Oral Pre se ntatio n CRDC 5 Expected benefits � May be beneficial for development of new antifungal agents against plant-pathogens antifungal agents against plant pathogens ? ? FZP Putative target genes of zinc cluster protein FZP in pathway of fatty acid β -oxidation and peroxisomal biogenesis. 30 15
22/07/54 Oral Pre se ntatio n CRDC 5 Conclusion � Deletion of zinc cluster FZP impairs utilization of oleic acid or linoleic acid for growth of oleic acid or linoleic acid for growth � Sensitivity to oxidative agents such as H 2 O 2 and diamide is also affected in Δ FZP strain, when compared to wild-type strain 31 � Putative zinc cluster regulator FZP may function to regulate genes encoding for protein necessary for fatty acid utilization and tolerance to oxidative stress 32 16
22/07/54 Oral Pre se ntatio n CRDC 5 Acknowledgements � Assoc.Prof.Dr. Kanok Rattanakanokchai � Dr. Nitnipa Soontorngun � Assoc.Prof.Dr. Bernard Turcotte � Thai Government Science and Technology Scholarship � National Research Council of Thailand � Organizer of CRDC conferrence 33 . Thank you 34 17
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