UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA REDOX REGULATION OF NF-B ACTIVATION BY HYDROGEN PEROXIDE AND EFFECTS ON GENE EXPRESSION Virgínia Mylena de Oliveira Marques Tese orientada pelo Prof. Doutor Fernando Antunes e pela Prof. Doutora Luísa Cyrne DOUTORAMENTO EM BIOQUÍMICA (REGULAÇÃO BIOQUÍMICA) 2008 Contents AGRADECIMENTOS .......................................................................................................................................... v RESUMO ............................................................................................................................................................ vii SUMMARY ......................................................................................................................................................... xi ABBREVIATION LIST ................................................................................................................................... xiii CHAPTER I - GENERAL INTRODUCTION .................................................................................................. 1 PART 1 - THE NF-B PATHWAY ............................................................................................................................................ 1 1. Prologue ........................................................................................................................................................ 1 2. The Rel/NF-B proteins ................................................................................................................................ 2 3. The IB proteins ........................................................................................................................................... 4 4. The inducible activation of NF-B ............................................................................................................... 5 4.1 The classical pathway ........................................................................................................................... 6 4.1.1 The IB-Kinase (IKK) complex ...................................................................................................................... 8 4.1.2 IKK activation ............................................................................................................................................... 9 4.1.3 From cell receptors to IKK activation ........................................................................................................... 9 4.1.4 Down-regulation of IKK activation ............................................................................................................. 12 4.2 The alternative pathway of NF-B activation ..................................................................................... 13 5. Regulation of NF-B activation .................................................................................................................. 14 5.1 Regulation by the IB proteins ............................................................................................................ 14 5.1.1 The oscillatory response of NF-B ............................................................................................................. 18 5.2 Transcriptional regulation of NF-B .................................................................................................. 19 5.3 Phosphorylation of the Rel/NF-B proteins ........................................................................................ 20 5.4 Acetylation of the Rel/NF-B proteins ................................................................................................ 22 5.5 The nuclear role of IKK .................................................................................................................... 24 5.6 The B site ........................................................................................................................................... 24 PART 2 - HYDROGEN PEROXIDE ........................................................................................................................................... 26 1. Reactive oxygen species (ROS) – “Les enfants terribles” ......................................................................... 26 2. Hydrogen Peroxide metabolism .................................................................................................................. 27 2.1 Consumption of H2O2 .......................................................................................................................... 28 2.2 Production of H2O2.............................................................................................................................. 29 3. The intracellular redox state ........................................................................................................................ 31 4. Oxidative modifications of cellular proteins ............................................................................................... 32 5. H2O2 regulates cellular signaling systems ................................................................................................... 33 5.1 H2O2 regulates kinases and phosphatases ........................................................................................... 34 5.2 H2O2 regulates transcription factors ................................................................................................... 38 i PART 3 – NF-B AND H2O2 ................................................................................................................................................. 40 1. H2O2 as the universal mediator of NF-B activation .................................................................................. 40 2. Is NF-B regulated by H2O2?...................................................................................................................... 41 2.1 H2O2 and the IKK complex .................................................................................................................. 42 2.2 H2O2 and the IBs ............................................................................................................................... 43 2.3 H2O2 and the transactivation potential of NF-B ............................................................................... 44 PART 4 – SCOPE OF THE THESIS ........................................................................................................................................... 45 REFERENCES .......................................................................................................................................................................... 47 CHAPTER II ...................................................................................................................................................... 59 A QUANTITATIVE STUDY OF NF-B ACTIVATION BY H2O2: RELEVANCE IN INFLAMMATION AND SYNERGY WITH TNF- ................................................................................................................................................................................................ 59 1. Abstract ....................................................................................................................................................... 59 2. Introduction ................................................................................................................................................. 60 3. Materials and Methods ................................................................................................................................ 61 4. Results ......................................................................................................................................................... 65 4.1 Calibrated exposure of MCF-7 cells to H2O2 ...................................................................................... 65 4.2 Estimation of H2O2 cytosolic concentrations in MCF-7 cells ............................................................. 66 4.3 Titration of NF-B activation with steady-state concentrations of H2O2 and with ............................. 67 TNF- in MCF-7 cells ............................................................................................................................... 67 4.4 Modulation by H2O2 of NF-B activation by TNF- .......................................................................... 69 4.5 Modulation by H2O2 of gene expression induced by TNF- via NF-B ............................................. 71 5. Discussion ................................................................................................................................................... 74 6. References ................................................................................................................................................... 78 7. Supplementary information ......................................................................................................................... 84 CHAPTER III .................................................................................................................................................... 91 COOPERATIVE EFFECT OF H2O2 AND TNF- ON NF-B ACTIVATION: MODULATION OF IB-, IB- AND IB- ..... 91 1. Abstract ....................................................................................................................................................... 91 2. Introduction ................................................................................................................................................