The Role of the S. cerevisiae Sco2p and Its Homologues in Antioxidant Defense Mechanisms

The Sco proteins, present in all kind of organisms, are regarded as one of the key players in the cytochrome c oxidase (COX) assembly. However, experimental and structural data, such as the presence of a thioredoxin-like fold, suggest that Sco proteins may also play a role in redox homeostasis. Our current studies in S. cerevisiae have strongly suggested an antioxidant role to Sco2 protein (ySco2p). While the single deletion of SCO2 does not result in a distinctive phenotype, the concomitant deletion of superoxide dismutase 1 (SOD1) leads to an increased sensitivity to oxidative stress generating agents (paraquat, menadione, plumbagin) compared to the respective single mutants. Since S. cerevisiae is a good model to functionally characterize genes from more complex organisms, identification of such a phenotype has paved the way to test whether the Sco2 homologues from other organisms are able to substitute for the function of ySco2p. The Sco homologues from Homo sapiens, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster and Kluyveromyces lactis were integrated into the genome of the double deletion mutant. The functional complementation was tested by both growth and biochemical ROS assays. All homologues except for K. lactis K07152 and A. thaliana HCC1 were able to complement the phenotype, indicating their role in antioxidant defense. Interestingly, pathogenic human SCO2 point mutations failed to restore this function. The observation of non-functional homologues despite of the high sequence similarity to ySco2p strengthened our hypothesis on the importance of conserved aminoacid(s) for the defensive role. For this purpose, selected homologues were aligned and the conservation was judged not only based on identity but also similarity (e.g. charge, hydrophobicity). Interestingly, alignment results have pointed out an aminoacid site (located 15 aminoacids downstream of CxxxC motif) that a positively charged lysine is found only in the non-functional homologues. Subsequent mutagenesis analyses verified the functional importance of this aminoacid site (gain and loss of functions) and revealed the detrimental effect of positive charge on antioxidant function. In order to explain the observed functional change, further effort will be put into the calculations of the electrostatic potential and identifications of protein-protein interactions.:Contents List of figures x List of tables xii Abbreviations xiii 1 Introduction 1 1.1 ROS production 1 1.2 Oxidative stress 2 1.3 Antioxidant response 3 1.4 The thioredoxin fold: From structure to function 6 1.5 Sco proteins 7 1.5.1 Structural similarity of Sco proteins to antioxidant enzymes 8 1.5.2 Current knowledge about Sco proteins of S. cerevisiae 9 1.6 Background studies 10 1.7 Using yeast as a model 11 1.7.1 Cross-species complementation studies 11 1.7.2 Yeast model for human mitochondria studies 12 1.8 Aim of the study 12 2 Materials & Methods 14 2.1 Materials 14 2.1.1 Chemicals and Reagents 14 2.1.2 Equipments 16 2.1.3 Kits 17 2.1.4 Antibodies 18 2.1.5 Plasmid 18 2.1.6 Primers 19 2.1.7 S. cerevisiae strains 22 2.1.8 Media 22 2.2 Methods 24 2.2.1 Cultivation of S. cerevisiae cells 24 2.2.1.1 Culture conditions 24 2.2.1.2 Preparation of glycerol stocks 24 2.2.2 Molecular Biology Methods 24 2.2.2.1 S. cerevisiae genomic DNA isolation 24 2.2.2.2 RNA isolation 25 2.2.2.2a Cultured mammalian cells (HEK293) 25 2.2.2.2b Drosophila melanogaster 25 2.2.2.3 RNA purity and concentration determination 25 2.2.2.4 Reverse transcription 25 2.2.2.5 Polymerase chain reaction 25 2.2.2.5a Standard PCR 25 2.2.2.5b Overhang PCR 26 2.2.2.5c Overlap extension PCR 27 2.2.2.5d Site-directed mutagenesis by overlap extension PCR 27 2.2.2.6 DNA agarose gel electrophoresis 28 2.2.2.7 DNA gel extraction and clean-up 29 2.2.2.8 DNA sequencing 29 2.2.2.9 Southern blotting 29 2.2.2.9a DNA preparation 29 2.2.2.9b Blotting 30 2.2.2.9c Preparation of a DIG-labelled probe 30 2.2.2.9d Hybridization of the DIG-labelled probe to DNA 30 2.2.2.9e Detection of hybridized DIG-labelled URA3 probe 31 2.2.2.10 Yeast transformation 32 2.2.2.11 Growth assay 32 2.2.3 Protein methods 33 2.2.3.1 Isolation of crude mitochondria from yeast 33 2.2.3.2 SDS-PAGE 33 2.2.3.3 Protein transfer 34 2.2.3.4 Colloidal Coomassie gel staining 34 2.2.3.5 Protein detection 35 2.2.3.6 Stripping the membrane and reprobing 35 2.2.4 Biochemical methods 35 2.2.4.1 Methylene Blue staining 36 2.2.4.2 Quantification of ROS 36 2.2.4.2a Amplex Red staining 36 2.2.4.2b Lipid peroxidation assay 36 2.2.5 Bioinformatics 37 2.2.6 Statistical Analysis 37 3 Results 40 3.1 Selection of homologues by bioinformatic analysis 40 3.2 Generation of recombinant strains 42 3.3 Confirmation of site-specific integration by check PCR 44 3.4 Verification of single site integration by Southern Blotting 44 3.5 Analysis of the functional homology between selected homologues and ySCO2 45 3.5.1 Complementation assay in solid media 45 3.5.2 Complementation assay in liquid media 47 3.6 Determination of cell viability 48 3.7 Quantification of ROS 51 3.7.1 Quantification of extracellular H2O2 51 3.7.2 Quantification of lipid peroxidation 53 3.8 Investigation of the expression and subcellular localization of homologues 55 3.9 Investigation of the impact of pathogenic hSCO2 mutations on its antioxidant role 58 3.10 Mutational analysis of ySCO2 60 3.11 Identification of functionally important residues 61 3.12 Prediction of salt bridges 65 3.13 Alanine mutagenesis 66 4 Discussion 68 4.1 Functional homology between the selected homologues and ySCO2 68 4.1.1 A. thaliana homologues, HCC1 & HCC2 68 4.1.2 H. sapiens homologues, hSCO1 & hSCO2 69 4.1.3 D. melanogaster homologue, SCOX 70 4.1.4 Yeast homologues, K07152 & SpSCO1 70 4.2 The localization and expression pattern of homologues 71 4.3 The impact of pathogenic hSCO2 mutations on its antioxidant role 72 4.4 Mutational analysis of ySCO2 73 4.5 Attempts to understand the underlying reason(s) behind charge-related functional change 74 4.6 Potential mechanisms associated with the antioxidant action of ySco2p 78 5 Summary 81 6 References 8