Introduction Collection of proteins called transcription factors - - PDF document

Oral Pre se ntatio n CRDC 5 22/07/54 1

The 5th Tropical and Sub‐tropical Crops Research Symposium (CRDC5) The 5th Tropical and Sub-tropical Crops 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 Elongation of RNA chain

Transcription

g Termination of transcription

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Transcription Factor (TF)

Introduction

T F DNA

Proteins that bind to specific sequence of DNA Control transcription Regulate transcription of gene either Regulate transcription of gene either positively or negatively Transcription regulator

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In S. cerevisiae, Major class of transcriptional regulators is composed of sub-family of zinc finger proteins zinc cluster proteins “Finger–like” shape

Crystal structures of Gal4po rs

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Play important role in transcriptional processes

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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-X2-Cys-X6-Cys-X5–12-Cys-X2-Cys-X6–8-Cys Act as transcription regulators in wide range

Function of Zinc cluster proteins

• f cellular processes

Amino acid and vitamin synthesis

Carbon and nitrogen metabolism Meiosis and morphogenesis

Stress response and pleiotropic drug

resistance (PDR)

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Role Gene name Phenotype

Role of Zinc cluster protein

Unknown

EDS1(YBR033W) TAS1 (YBR150C) YBR239C YDR520C YER184C YFL052W YJL103C Expression is dependent on Rpb2p Δybr150c is sensitive to thiabendazole Interacts with Rds2p in yeast two-hybrid system Δydr520c is slightly sensitive to caffeine Δyfl052w is hypersensitive to heat shock at 37oC May be involved in oxidative phosphorylation YJL206C YKL222C YKR064W YLL054C YLR278C YNR063W Δykl222c is sensitive to caffeine Δylr278c is sensitive to caffeine

Akache, B., Wu, K. and Turcotte, B., 2001

Previous study

Examined phenotypes of strains carrying deletion of genes encoding zinc cluster proteins

• n non-fermentable carbon source

: Lactate, Glycerol

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Oral Pre se ntatio n CRDC 5 22/07/54 5 Akache, B., Wu, K. and Turcotte, B., 2001

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. Growth of S. cerevisiae strains with deleted zinc cluster genes

• n media containing glucose, oleic or linoleic acids as a sole

carbon source

My previous study

Strain Carbon source Synthetic media YP media Glucose Oleic acid Linoleic acid Glucose Oleic acid Linoleic acid Wild-type ++++ ++++ ++++ ++++ ++++ ++++ ∆yor380w (∆FZP) ++++ ++ ++ ++++ ++ ++ ++++ normal growth +++ moderate growth ++ impaired growth + severly impaired growth - no growth

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∆FZP Wild-type

Phenotypic analysis of wild-type and deletion strains

YP + 2 % Glucose SD + 0.125 % Oleic acid SD + 2 % Glucose YP + 0.125% Oleic acid

∆FZP Wild-type

SD + 0.125% Linoleic acid YP + 0.125 % Linoleic acid

∆FZP Wild-type

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: Deletion of FZP which encodes for zinc cluster proteins resulted in impaired growth on oleic and linoleic acids

Oxidative stress Oxidative agents Oxidative stress : Caused by reactive oxygen species (ROS) species (ROS)

• hydrogen peroxide (H2O2)

ROS generated during respiration and

• xidation of nutrients

Reduce growth rate and affect cell viability Generated ROS may damage cellular macromolecules : nucleic acids, proteins and lipids

Oral Pre se ntatio n CRDC 5 22/07/54 7 Oxidative agent : H2O2

Produced during dehydrogenation in β-oxidation

O j t t f H O tt k i t t d One major target of H2O2 attack is unsaturated 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

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Peroxisomes

Main functions of peroxisomes is degradation of

Organelles and compartments in yeast cell

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

• f plasma membrane
• f plasma membrane

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Objective :

Present study

This study aims to understand roles of uncharacterized zinc cluster proteins Examined sensitivity of deletion strain to

• xidative stress,

: Process commonly occur during non-fermentation : Fatty acid metabolism normally generates

• xidative stress

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Methodology

Phenotypic analysis Growth assay of wild-type and deleted zinc cluster strains

• n oxidative agent : H2O2 , diamide

Spot assay

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Se r ial dilution

Phenotypic analysis

Cultur e of S.c e r e visiae wild- type and de le tion str ains on YPD me dia

Spot assay

Results

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Oral Pre se ntatio n CRDC 5 22/07/54 11 cell cell

∆FZP

Wild‐type ∆FZP ∆FZI YP + 3 mM H2O2 ∆FZP Wild‐type ∆FZI SD + 3 mM H2O2 YP + 2 % Glucose SD + 2 % Glucose YP + 0.4 mM diamide ∆FZP Wild‐type ∆FZI

Figure 1. Growth assay of zinc cluster ΔFZP and ΔFZI strains on glucose in presence of 3 mM H2O2 and 0.4 mM Diamide

SD + 0.4 mM diamide

Fatty acid metabolism normally generates

• xidative stress

Discussion

H2O2 produced during dehydrogenation in β-oxidation can induce such stress response Lead to membrane damage Causing impaired growth on fatty acids

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Oral Pre se ntatio n CRDC 5 22/07/54 12 Yeast cells must adapt to promptly response to such stress

Discussion

such stress Suggests that FZP plays important role to ensure survival under this condition

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FZP

?

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Putative target genes of zinc cluster protein FZP in pathway of fatty acid β-oxidation and peroxisomal biogenesis.

Oral Pre se ntatio n CRDC 5 22/07/54 13 PEX13 is required for appressorium-mediated plant infection by Anthracnose fungus Colletotrichum orbiculare Fujihara et al., 2009 Colletotrichum orbiculare Appressorium melanization is required for

pathogenicity

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p g y

Howard et al. 1991; Wang et al. 2005.

• Appressorium : Initial host penetration structure

Darkly melanized infection structure

Ap Ap S Ap S

Kubo and Furusawa 1991; Tsuji et al. 1997

Ap : Appressorium S : Spore

Ap

Oral Pre se ntatio n CRDC 5 22/07/54 14 Melanin functions to trap osmolytes within cell, generating high turgor pressure necessary for mechanical penetration of plant cuticle and cell wall cell wall

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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

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Expected benefits

May be beneficial for development of new antifungal agents against plant-pathogens antifungal agents against plant pathogens

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FZP

?

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Putative target genes of zinc cluster protein FZP in pathway of fatty acid β-oxidation and peroxisomal biogenesis.

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Conclusion

Deletion of zinc cluster FZP impairs utilization

• f oleic acid or linoleic acid for growth
• f oleic acid or linoleic acid for growth

Sensitivity to oxidative agents such as H2O2 and diamide is also affected in ΔFZP strain, when compared to wild-type strain

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Putative zinc cluster regulator FZP may function to regulate genes encoding for protein necessary for fatty acid utilization and tolerance to oxidative stress

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Oral Pre se ntatio n CRDC 5 22/07/54 17

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 .

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Thank you

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