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Determining HDAC8 Substrate Specificity by Noah Ariel Wolfson A

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Determining HDAC8 Substrate Specificity by Noah Ariel Wolfson A Determining HDAC8 substrate specificity by Noah Ariel Wolfson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biological Chemistry) in the University of Michigan 2014 Doctoral Committee: Professor Carol A. Fierke, Chair Professor Robert S. Fuller Professor Anna K. Mapp Associate Professor Patrick J. O’Brien Associate Professor Raymond C. Trievel Dedication My thesis is dedicated to all my family, mentors, and friends who made getting to this point possible. ii Table of Contents Dedication ....................................................................................................................................... ii List of Figures .............................................................................................................................. viii List of Tables .................................................................................................................................. x List of Appendices ......................................................................................................................... xi Abstract ......................................................................................................................................... xii Chapter 1 HDAC8 substrates: Histones and beyond ...................................................................... 1 Overview ..................................................................................................................................... 1 HDAC introduction ..................................................................................................................... 2 Known HDAC8 substrates .......................................................................................................... 4 Candidate non-histone HDAC8 Substrates ................................................................................. 8 HDAC8 complex formation ...................................................................................................... 11 Enzyme structure affects substrate specificity .......................................................................... 15 Catalytic Mechanism and Regulation of HDAC8 activity ........................................................ 22 HDAC8 localization .................................................................................................................. 26 Post-translational modification of HDAC8 ............................................................................... 28 Concluding remarks .................................................................................................................. 32 Acknowledgements ................................................................................................................... 33 Bibliography .............................................................................................................................. 33 Chapter 2 An enzyme-coupled assay measuring acetate production for profiling histone deacetylase specificity .................................................................................................................. 45 Introduction ............................................................................................................................... 45 Materials and Methods .............................................................................................................. 47 Reagents................................................................................................................................. 47 Acetyl-CoA synthetase preparation ....................................................................................... 47 HDAC8 expression and purification ..................................................................................... 49 iii Fluor de Lys assay ................................................................................................................. 49 Acetate assay kit .................................................................................................................... 50 Optimized stopped coupled acetate assay ............................................................................. 50 Optimized continuous coupled acetate assay ........................................................................ 52 Fluorescamine assay .............................................................................................................. 52 Results ....................................................................................................................................... 53 Assay ..................................................................................................................................... 53 Optimization of Assay for Measuring HDAC Activity ......................................................... 56 Stopped Assay ....................................................................................................................... 58 Continuous Assay .................................................................................................................. 62 Reactivity of HDAC8 with peptides ...................................................................................... 66 Discussion ................................................................................................................................. 71 Acknowledgements ................................................................................................................... 76 Bibliography .............................................................................................................................. 76 Chapter 3 Profiling small peptide substrates to identify HDAC8 recognition motif(s) ............... 81 Introduction ............................................................................................................................... 81 Materials and Methods .............................................................................................................. 82 Reagents................................................................................................................................. 82 HDAC8 expression and assay ............................................................................................... 82 Improving the assay conditions ............................................................................................. 83 FlexPepBind protocol ............................................................................................................ 84 Modeling the structures of peptide-protein complexes ......................................................... 84 Step 1: Preparation of a coarse starting model of a peptide-HDAC8 complex ..................... 85 Step 2: Optimization of the peptide-HDAC8 complex structure .......................................... 86 Step 2(a) Extensive optimization using Rosetta FlexPepDock ............................................. 86 Step 2(b) Short minimization of starting structure ................................................................ 87 Parameters optimized for HDAC8 FlexPepBind ................................................................... 87 iv Definition of axes for rigid body movements ........................................................................ 89 Addition of an electrostatic term to default scoring function score12 .................................. 90 Step 3: Ranking of the peptide substrates based on the optimized peptide-receptor complex structures ................................................................................................................................ 90 Dataset 1: Training and test set: 361 GXKacZGC peptides .................................................. 91 Initial calibration set: 5 substrates & 5 non-substrates .......................................................... 92 Dataset 2: Validation set: 361 GRKacXZC peptides ............................................................ 92 Statistical tests ....................................................................................................................... 92 Binary distinction between substrates and non-substrates .................................................... 93 Prediction of relative substrate activity ................................................................................. 93 Results ....................................................................................................................................... 93 First round of calibration ....................................................................................................... 93 Analysis of performance in Training and Test sets ............................................................... 94 Searching for novel, non-histone substrates .......................................................................... 95 Experimental validation of de-novo predictions ................................................................... 96 Second round of calibration ................................................................................................. 100 Improved starting structure and score function ................................................................... 101 Second round of Phosphosite screen ................................................................................... 103 Assessing the second set of peptides ..................................................................................
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