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UNIVERSITY of CALIFORNIA RIVERSIDE Development of Quantitative FRET Technology for SENP Enzyme Kinetics Determinations and High UNIVERSITY OF CALIFORNIA RIVERSIDE Development of Quantitative FRET Technology for SENP Enzyme Kinetics Determinations and High-sensitive High-throughput Screening Assay for Protease Inhibitor Discovery A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Bioengineering by Yan Liu December 2012 Dissertation Committee: Dr. Jiayu Liao, Chairperson Dr. Jerome Schultz Dr. David Kisailus Dr. Dimitrios Morikis Copyright by Yan Liu 2012 ii The Dissertation of Yan Liu is approved: Committee Chairperson University of California, Riverside iii Acknowledgements I would like to thank Dr. Jiayu Liao for his great support in my Ph.D training, and Dr. Jerome Schultz, Dr. David Kisailus, Dr. Dimitrios Morikis for their helpful advice. Dr. Morikis and Dr. Chris Kieslich offered the computational method to determine the SUMO4 mutation sites. Dr. Victor Rodgers and Dr. Jerome Schultz provided valuable advice for calculation model optimization. Dr. David Carter helped a lot on compound screening instrumentation. I also have to thank all of the members in Dr. Liao’s group for very close collaborative work and helpful discussion, especially Dr. Yang Song and Dr. Yongfeng Zhao’s help for teaching and training and Ms. Ling Jiang’s technical help, Ms. Hong Xu’s help for instrument maintenance. This work is supported by funding from University of California-Riverside and the National Institutes of Health. The text of this dissertation, in part, is a reprint of the material as is appeared in Front. Biol. 2012, Vol 7 (1), p57-64; Anal. Biochem . 2012, 422, p14-21. Listed in the publication, Dr. Jiayu Liao supervised the research which forms the basis for this dissertation, Yang Song provided theoretical help for spectrum analysis, Vipul Madahar provided technical help during the research. iv Dedication I would like to dedicate this dissertation to my parents (Haicheng Liu and Qianting Ma) and my boyfriend (Jian Gong) for their constant support and encouragement throughout my Ph.D study. v Abstract of the Dissertation Development of Quantitative FRET Technology for SENP Enzyme Kinetics Determinations and High-sensitive High-throughput Screening Assay for Protease Inhibitor Discovery by Yan Liu Doctor of Philosophy, Graduate Program in Bioengineering University of California, Riverside, December 2012 Dr. Jiayu Liao, Chairperson SUMOylation is an important post-translational protein modification mechanism which plays important roles in a variety of biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. SENP, SUMO specific protease, can either mature pre-SUMO or deconjugate SUMO from its target proteins. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. FRET is an energy transfer phenomenon which occurs between two spectrum-overlapping fluorophores in close proximity. The efficiency of FRET is highly dependent on the distance between the donor and acceptor fluorophores, which makes FRET very powerful in the detection of molecular interactions and biomolecular conformational changes. A novel highly sensitive FRET-based protease assay was designed and developed to study the activities and specificities of SENP in SUMOylation vi signaling pathway and to identify bioactive chemical compounds which can specifically inhibit SENPs’ activities. The endopeptidase and isopeptidase activities of SENP1/2/5/6/7c toward SUMO1/2/3 were studied and verified by western-blot assay. A novel quantitative FRET analysis was performed to study the protease kinetics to compare the specificities to substrate. The direct emissions of donor and acceptor were considered in FRET signal analysis. The mathematical algorithm dependent on pre-established standard curves or later optimized internal calibration method related the hydrolyzed substrate concentration to detected fluorescent signals during the dynamic processes, and thus derived the kinetics constants. Then, the developed protease assay and protease kinetics analysis methods were applied to study the pre-SUMO4’s maturation and the product inhibition in pre-SUMO’s maturation, which indicated the potential application in biomechanism study and chemical compounds’ inhibition characterization. Finally, the FRET-based assay was developed to HTS assay to screen small chemical SENP inhibitors. The screening conditions were optimized to achieve satisfactory Z’ factor value and high signal-to-noise ratio. 55,000 compounds were screened by the developed HTS assay. The general principles of the quantitative FRET analysis in protease kinetics, including the characterization of inhibition, can be applied to other substrate-protease with inhibitors. The FRET-based protease assay as well as the HTS assay provided a powerful tool for large-scale and high-throughput applications. vii Table of Contents Introduction……………………………………………………………………………1 Chapter I Abstract……………………………………………………………………...….40 Introduction…………………………………………………………………….41 Material and Methods………………………………...……………………….49 Results………………………………………………………………………….56 Discussion……..........................................................................................77 Chapter II Abstract……………………………………………………………………...….81 Introduction……………………………………………………………………..82 Material and Methods……………………………………………...………….88 Results………………………………………………………………………….94 Discussion…………………………………………………………...………..128 viii Chapter III Abstract……………………….…………………………………………...….137 Introduction………………………………………………………………….138 Material and Methods……………….……………………………………….148 Results………………………………………………………………...………155 Discussion…………………………………………………………………….170 Chapter IV Abstract…………………………………………………….……………...….177 Introduction………………………………………..………………………….178 Material and Methods……………………………………..…………………182 Results…………………………………….…………………………………..187 Discussion…………………………………………………………………….195 Conclusions………………………...…………………………………………………200 References………………...………………………………………………………….202 ix List of Figures Page 3. Figure 1 Comparison of SUMO and ubiquitin. Page 7, Figure 2 The SUMO conjugation cycle. Page 9, Figure 3 SENPs’ function at multiple points in the SUMO pathway. Page 11. Figure 4 Human SENP structure and localization. Page 27. Figure 5 Synthesized or discovered small chemical SENP inhibitors. Page 31. Figure 6 Basic concepts of FRET. Page 46. Figure 7 Examples of previous studies about SUMO-SENP pair. Page 50. Figure 8 Map Map of the bacterial expressional plasmids encoding interested proteins. Page 57. Figure 9 Design of FRET-based protease assay. Page 59. Figure 10 Comparison of FRET pair in the developed protease assay. Page 61-62. Figure 11 Characterization of pre-SUMO1/2/3’s maturation processing by SENP1/2/5/6/7C in developed FRET-based protease assay and confirmed in biochemistry western-blot analysis. Page 63. Figure 12 Emission spectrum of CyPet-(pre-SUMO2)-YPet’s maturation by SENP2C in real time monitoring. Page 67. Figure 13 Quantitative analysis of fluorescent signals. x Page 68. Figure 14 Standard curves of fluorescent signal versus related protein concentration. Page 73. Figure 15 Quantitative analysis of CyPet–(pre-SUMO1)–YPet digested by different ratio of SENP1C. Page 76. Figure 16 Michaelis–Menten graphical analysis of CyPet–(pre- SUMO1)–YPet’s processing by SENP1. Page 84. Figure 17 Examples of energy transfer-based assay to study SENP or DUBs. Page 95. Figure 18 Pre-established standard curves of fluorescent emission versus related protein concentration. Page 97-98. Figure 19 Time-course of fluorescence component changes during pre-SUMO maturation by SENP1/2C. Page 100-101. Figure 20 The concentration of digested substrate during pre- SUMO maturation process analyzed by internal calibration. Page 106-107. Figure 21 Michaelis–Menten graphical analysis of pre-SUMO’s maturation by SENP. Page 112. Figure 22. Velocities in time range 70-205 sec or 145-325 sec were plotted versus remaining substrate concentration at the medium point (137.5 sec in 70-205 sec time range or 235 sec in 145-325 sec time range) in Michaelis- Menten equation. xi Page 118. Figure 23. FRET-based protein assay to monitor the SUMO- RanGAP1C conjugation and deconjugation in vitro . Page 120. Figure 24. Coomassie staining of purified proteins in SUMO conjugation assay Page 121. Figure 25. Characterization of SUMO1/2 deconjugation from RanGAP1C by SENP1/2/5/6/7C in developed FRET-based protease assay. Page 123. Figure 26. Quantitative FRET analysis to compare SENP1’s endo- and iso-peptidase activities. Page 125. Figure 27. Quantitative FRET analysis in study the protease kinetics of SUMO1-RanGAP1C deconjugation by SENP1C. Page 133. Figure 28. Structure of the complex of SENP1 C603A bound to SUMO1–modified RanGAP1 and full-length SUMO-1. Page 136. Figure 29 Structure of the SENP2–SUMO precursor complexes. Page 141. Figure 30. Sequence alignment for pre-SUMO2 and pre-SUMO4. Page 143. Figure 31. Equilibrium scheme for enzyme turnover in the presence and absence of an inhibitor. Page 144. Figure 32. Representations of the three major forms of inhibitor interactions with enzymes. Page 146. Figure 33. Dose-response plot of enzyme fractional activity as a function of inhibitor concentration. xii Page 155. Figure. 34 Crystal structure of (pre-SUM2)-SENP2 and generated (pre-SUMO4)-SENP2. Page 157. Figure 35 Electrostatic clustering and free energies of association for pre-SUMO2 based mutations of pre-SUMO4 and pre-SUMO4 alanine-scan. Page 159. Figure 36 Characterization of wild type pre-SUMO4 as well as 3 pre-
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