i Front line antioxidant defenses in the freeze tolerant wood frog, Rana sylvatica: An in-depth analysis of mechanisms of enzyme regulation. Neal J. Dawson B.Sc. S.Sp. Honours Biochemistry Queen’s University, 2006 M.Sc. Biology Carleton University, 2009 A Thesis Submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology Carleton University Ottawa, Ontario, Canada © Copyright 2014 Neal J. Dawson ii The undersigned hereby recommend to the Faculty of Graduate Studies and Research acceptance of this thesis Front line antioxidant defenses in the freeze tolerant wood frog, Rana sylvatica: An in-depth analysis of mechanisms of enzyme regulation. submitted by Neal J. Dawson, B.Sc., M.Sc. in partial fulfillment of the requirements for the degree of Doctor of Philosophy ____________________________________ Chair, Department of Biology ___________________________________ Thesis Supervisor ___________________________________ External Examiner Carleton University iii ABSTRACT The wood frog, Rana sylvatica, is one of few species that can survive whole-body freezing during overwintering. Frogs endure freezing of up to 70% of their total body water, and demonstrate a complete lack of respiration, heart beat and brain activity. Freezing imposes multiple stresses including anoxia/ischemia, cellular dehydration when water is lost to extracellular ice masses, wide temperature changes, and potential physical damage by ice. One crucial adaptation for freezing survival is well-developed antioxidant defenses to protect tissues from abiotic stress while frozen and deal with rapid changes in the generation of reactive oxygen species associated with anoxia and reoxygenation over freeze/thaw cycles. This thesis explores the properties and regulation of key antioxidant enzymes, purified via novel schemes, from frog muscle – both Cu/Zn- and Mn-dependent isoforms of superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT). The studies show that changes in activity, stability, and substrate affinity of antioxidant enzymes during the frozen state may be significant preparatory mechanisms employed by R. sylvatica to support the transition from frozen to thawed states and deal effectively with oxidative stress accompanying reperfusion. Moreover, reversible protein phosphorylation plays a central role in regulating the activity of these enzymes to suit physiological needs throughout freeze-thaw cycles. For example, CuZnSOD from muscle of frozen frogs showed a significantly higher V compared to the control enzyme. max Muscle MnSOD from frozen frogs showed a significantly lower K for O -, higher m 2 phosphorylation, and increased enzyme stability compared to control MnSOD. GR from iv frog muscle showed a significantly lower K for GSSG in the face of physiological levels m of glucose encountered during freezing, as well as the potential for phosphorylation via endogenous kinases. CAT from muscle of frozen frogs showed a significantly lower K m for H O and a higher level of phosphorylation; furthermore, stimulation of endogenous 2 2 kinases decreased K H O similar to what occurred in muscle of frozen animals. This m 2 2 thesis provides compelling evidence for regulation of antioxidant enzymes via reversible protein phosphorylation and augmentation of key antioxidant enzymes during freezing of the frog, likely in preparation to endure oxidative stress encountered during reperfusion over winter freeze-thaw cycles. v ACKNOWLEDGEMENTS I have lived a truly full life up to this point, and I cannot possibly thank all those who have helped shape me into the person I am today. I apologize in advance for leaving out those who deserve my thanks. To my friends, you are all incredible people that I have had the pleasure of knowing and learning from. I would specifically like to thank Ken Storey for taking a chance on a student interested in “functional proteins.” I thank you for the opportunity you afforded me, the enthusiasm you instilled in me, and for introducing me to the wonders of the comparative world of science. I cannot thank you enough for my time under your guidance. I offer a special thanks to Jan Storey for her fascinating discussions and endless editorial support, not only for myself, but the entire Storey lab. Unfortunately I have been in the Storey lab for far too long to list all those that have guided, assisted, and simply kept me sane throughout it all. However, I would like to give a special thanks to Kyle Bigger, Ryan Bell and Ben Lant. You have had an immeasurable impact on my scientific career thus far, and I hope to continue to learn, wonder, and explore with you for years to come. Lastly I would like to thank my family. To my mother and father, Joan and Donald Dawson, you provided a home life so incredibly loving, that I am only now starting to understand how truly lucky I was to have you as parents. Jennifer, Debbie, Dan and Olivia, I thank you for the love and laugher you shared with me, and for always supporting me throughout my life. Finally, Dominique, I can’t thank you enough for your patience and support throughout my studies. You are my rock, my love, and I look forward to embarking together on the adventures life has yet to throw our way. vi TABLE OF CONTENTS Title Page i Acceptance Sheet ii Abstract iii Acknowledgements v Table of Contents vi List of Abbreviations vii List of Figures ix List of Tables xii List of Appendices xiii Chapter 1 General Introduction 1 Chapter 2 Free-radical first responders: The characterization 21 of CuZnSOD and MnSOD regulation during freezing of the freeze tolerant North American wood frog, Rana sylvatica. Chapter 3 Passive regeneration of glutathione: Purification and 69 properties of glutathione reductase regulation from the freeze tolerant North American wood frog, Rana sylvatica. Chapter 4 A hydrogen peroxide safety valve: The reversible 109 phosphorylation of catalase from the freeze tolerant North American wood frog, Rana sylvatica. Chapter 5 General Discussion 149 Appendices 167 vii LIST OF ABBREVIATIONS AMP adenosine monophosphate AMPK 5’ adenosine monophosphate-activated protein kinase BSA bovine serum albumin CAT catalase cDNA complementary deoxyribonucleic acid C concentration at which half of the protein is unfolded m CuZnSOD copper and zinc containing SOD DEAE+ diethylaminoethyl cellulose DEPC diethylpyrocarbonate dNTP deoxyribonucleotide triphosphate DSF differential scanning fluorimetry DTT dithiothreitol E activation energy a EDTA ethylenediaminetetraacetic acid EGTA ethylene glycol tetraacetic acid ER endoplasmic reticulum ETS electron transport system FeSOD iron containing superoxide dismutase GMP guanosine monophosphate GPx glutathione peroxidase GR glutathione reductase GSH reduced glutathione GSSG oxidized glutathione GST glutathione-s-transferase I half maximal inhibitory concentration 50 IMAC immobilized metal ion affinity chromatography IP inositol trisphosphate 3 K half maximal activation concentration a viii K Michaelis-Menten constant m KPi potassium phosphate MAPK mitogen activated protein kinase MnSOD manganese containing superoxide dismutase mRNA messenger ribonucleic acid NAD(P)H reduced form of nicotinamide adenine dinucleotide (phosphate) NBT nitrotetrazolium blue NiSOD nickel containing superoxide dismutase PCR polymerase chain reaction PKA protein kinase A PKC protein kinase C PKG protein kinase G PMSF phenylmethylsulfonyl fluoride PP1 protein phosphatase 1 PP2A protein phosphatase 2A PP2C protein phosphatase 2C PVDF polyvinylidene difluoride RNA ribonucleic acid ROS reactive oxygen species rRNA ribosomal ribonucleic acid RT room temperature SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis TBARS thiobarbituric acid reactive substances TBST tris buffered saline with tween T the melting point m V maximal velocity max XO xanthine oxidase XDH xanthine dehydrogenase ix LIST OF FIGURES Figure 1.1. Single electron reduction of oxygen and the ROS Page 3 intermediates that arise as a result. Figure 1.2. A simple overview of the primary antioxidant pathways and Page 7 free radical producing reactions. Figure 1.3. Composite freezing curve for R. sylvatica. Page 11 Figure 2.1. (A) Typical elution profile for control SOD on a DEAE+ Page 61 column. (B) Typical elution profile for control SOD on a copper chelate column. Figure 2.2. Copper chelate column elution profile for SOD superimposed Page 62 with western blot analysis showing the cross-reactivity of anti-CuZnSOD (dotted line) and anti-MnSOD (dashed line) with individual fractions. Figure 2.3. Immunoblot analysis of (A) purified CuZnSOD from the Page 63 muscle of control R. sylvatica and (B) purified MnSOD from the muscle of control R. sylvatica. Figure 2.4. (A) RT-PCR analysis of sod transcript levels in frog muscle. Page 64 (B) Western blotting analysis of the amount of SOD protein in frog muscle. Figure 2.5. Relative phosphorylation levels of MnSOD from muscle of Page 65 control and 24 h frozen frogs using Pro-Q diamond phospho- protein staining to analyze relative total phosphorylation of the enzyme, and dot-blot analysis of residue-specific phosphorylation. Figure 2.6. Predicted phosphorylation sites for CuZnSOD and MnSOD. Page 66 (A) Partial amino sequence for CuZnSOD, highlighting predicted phosphorylation sites (bold, underlined). (B) Kinases predicted to phosphorylate CuZnSOD at the amino acid sites highlighted in A. (C) Partial amino sequence for MnSOD, highlighting predicted phosphorylation sites (bold, underlined). (D) Kinases predicted to phosphorylate MnSOD at the amino acid sites highlighted in C. Figure 2.7. Stability of CuZnSOD from crude extracts of frog muscle. Page 67 Samples were incubated for 24 h at 4°C with varying concentrations of urea then treated with pulse proteolysis to digest denatured CuZnSOD. x Figure 2.8. Stability of MnSOD from crude extracts of frog muscle. Page 68 Samples were incubated for 24 h at 4°C with varying concentrations of urea then treated with pulse proteolysis to digest denatured MnSOD. Figure 3.1. (A) Typical elution profile for GR on a hydroxyapatite Page 101 column. (B) Typical elution profile for GR on a Cibacron blue column. Figure 3.2. DEAE+ elution profiles for purified GR from control and 24 h Page 102 frozen muscle after incubation to stimulate protein kinases. Figure 3.3. Purified GR from the muscle of control R. sylvatica. Page 103 Figure 3.4. Absence of bands indicates no apparent phosphorylation of Page 104 muscle GR from either control or 24 h frozen frogs as assessed by Pro-Q diamond phosphoprotein staining. Figure 3.5. Relative phosphorylation levels of GR from control and 24 h Page 104 frozen frog muscle after total kinase incubations as assessed by Pro-Q diamond phosphoprotein staining. Figure 3.6. Relative phosphorylation levels of wood frog muscle GR after Page 105 incubations to stimulate total kinases; control and 24 h frozen conditions are compared using Western-blot analysis of site- specific phosphorylation. Figure 3.7. A comparison of the predicted serine (S), threonine (T) and Page 106 tyrosine (Y) phosphorylation sites on GR that are shared between X. tropicalis and X. laevis. Figure 3.8. Arrhenius plot for purified GR from the muscle of control and Page 107 24 h frozen R. sylvatica. Figure 3.9. Differential scanning fluorimetry analysis of the thermal Page 108 stability of purified GR from control and 24 h frozen frog muscle. Figure 4.1. (A) Typical elution profile for CAT on a hydroxyapatite Page 141 column. (B) Typical elution profile for CAT on a Cibacron blue column. (C) Typical elution profile for CAT from control muscle on a DEAE+ column. (D) Typical elution profile for CAT from frozen muscle on a DEAE+ column.