Design Genetic Fluorescent Probes to Detect Protease Activity and Calcium-Dependent Protein-Protein Interactions in Living Cells
Total Page：16
File Type：pdf, Size：1020Kb
Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry 8-25-2008 Design Genetic Fluorescent Probes to Detect Protease Activity and Calcium-Dependent Protein-Protein Interactions in Living Cells Ning Chen Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Part of the Chemistry Commons Recommended Citation Chen, Ning, "Design Genetic Fluorescent Probes to Detect Protease Activity and Calcium-Dependent Protein-Protein Interactions in Living Cells." Dissertation, Georgia State University, 2008. https://scholarworks.gsu.edu/chemistry_diss/43 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. DESIGN GENETIC FLUORESCENT PROBES TO DETECT PROTEASE ACTIVITY AND CALCIUM-DEPENDENT PROTEIN-PROTEIN INTERACTIONS IN LIVING CELLS by NING CHEN Under the Direction of Professor Jenny J. Yang ABSTRACT Proteases are essential for regulating a wide range of physiological and pathological processes. The imbalance of protease activation and inhibition will result in a number of major diseases including cancers, atherosclerosis, and neurodegenerative diseases. Although fluorescence resonance energy transfer (FRET)-based protease probes, a small molecular dye and other methods are powerful, they still have drawbacks or limitations for providing significant information about the dynamics and pattern of endogenous protease activation and inhibition in a single living cell or in vivo. Currently protease sensors capable of quantitatively measuring specific protease activity in real time and monitoring activation and inhibition of enzymatic activity in various cellular compartments are highly desired. In this dissertation, we report a novel strategy to create protease sensors by grafting an enzymatic cleavage linker into a sensitive location for changing chromophore properties of enhanced green fluorescent protein (EGFP) following protease cleavage, which can be used to determine protease activity and track protease activation and inhibition with a ratiometric measurement mode in living cells. Our designed protease sensors exhibit large relative ratiometric optical signal change in both absorbance and fluorescence, and fast response to proteases. Meanwhile, these protease sensors exhibiting high enzymatic selectivity and kinetic responses are comparable or better than current small peptide probes and FRET-based protease probes. Additionally, our protease sensors can be utilized for real-time monitoring of cellular enzymogen activation and effects of inhibitors in living cells. This novel strategy opens a new avenue for developing specific protease sensors to investigate enzymatic activity in real time, to probe disease mechanisms corresponding to proteases in vitro and in vivo, and to screen protease inhibitors with therapeutic effects. Strong fluorescence was still retained in the cleaved EGFP-based protease sensors, which stimulated us to identify the EGFP fragment with fluorescence properties for further understanding chromophore formation mechanisms and investigating protein-protein interactions through fluorescence complementation of split EGFP fragments. Through fusing EF-hand motifs from calbindin D9k to split EGFP fragments, a novel molecular probe was developed to simultaneously track the calcium change or calcium signaling pathways and calcium-dependent protein-protein interaction in living cells in real time. INDEX WORDS: Protease sensor, Ratiometeric measurement, EGFP, Calcium-dependent, Protein-protein interactions, Fluorescence complementation DESIGN GENETIC FLUORESCENT PROBES TO DETECT PROTEASE ACTIVITY AND CALCIUM-DEPENDENT PROTEIN-PROTEIN INTERACTIONS IN LIVING CELLS by NING CHEN A Dissertation Submitted in Partial Fulfillment of the Requirement of the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2008 Copyright by Ning Chen 2008 DESIGN GENETIC FLUORESCENT PROBES TO DETECT PROTEASE ACTIVITY AND CALCIUM-DEPENDENT PROTEIN-PROTEIN INTERACTIONS IN LIVING CELLS by NING CHEN Committee Chair: Jenny J. Yang Committee: Giovanni Gadda Zhi-ren Liu Yujun George Zheng Robert Wohlhueter Electronic Version Approved: Office of Graduate Studies Colleges of Arts and Sciences Georgia State University December 2008 iv ACKNOWLEGEMENTS All of the work in this dissertation is carried out under the direction of Dr. Jenny J. Yang. I would like to express my high appreciation to Dr. Jenny J. Yang for her kindly providing this research opportunity, demonstrative guidance, valuable suggestion, discussion and support throughout this research. I would like to thank Dr. Giovanni Gadda for the helpful guidance of catalytic kinetic studies on protease sensors and valuable discussion. I would like to thank Dr. Zhi-Ren Liu for the helpful guidance of mammalian cell culture and transfection. I would like to thank Dr. Lily Yang from Emory University for kindly providing MIA PaCa-2 cell lines, valuable guidance and discussion on caspase activation and inhibition. I would like to thank Dr. Rihe Liu from The University of North Carolina at Chapel Hill for kindly offering active caspase-3 and caspase-8 enzyme. I would like to thank Dr. Jin Zou, Yun Huang, Shen Tang, and Mike Kirberger from biosensor group for much research technical guidance, and lots of good suggestion and discussion. I would like to specially thank Mike Kirberger for his proofreading and suggestion. I would like to thank Dr. Shunyi Li for helpful guidance to protein purification, mass spectroscopy, amino acid analysis and discussion. I would like to thank Dr. Yiming Ye from the Centers for Disease Control and Prevention (CDC) for providing the graft approach on the design of EGFP-based protease sensor variants and protein sequence analysis. v I would like to thank Dr. Robert Wohlhueter from the Centers for Disease Control and Prevention (CDC) for providing a lot of comments on my dissertation and editing. I would like to thank Dr. Siming Wang and Sarah Cepada at Georgia State University for mass spectrometry analysis and discussion on protease sensors. I would like to thank Dr. Wei Yang for the help of NMR data analysis, much valuable guidance and discussion. I would like to thank Dr. Yang’s group members for their helpful discussion. I would like to thank my dissertation committee members, Drs. Jenny J. Yang, Giovanni Gadda, Zhi-Ren Liu, Yujun George Zheng and Robert Wohlhueter. I would like to thank my qualify examination committee, Drs. Giovanni Gadda, Binghe Wang and Kathryn B. Grant. I would also like to thank the Department of Chemistry at Georgia State University for providing the opportunity to complete my doctorate degree study and research. I would like to thank the Molecular Basis of Disease program in Georgia State University for supporting my study and research. I would like to specially thank my wife, Suqin Yao, for her help and support to my study. I would like to specially thank her for taking care of my son although she is busy in her work. The special appreciation also would give my parents for their support. vi TABLE OF CONTENTS ACKNOWLEGEMENTS ..........................................................................................................IV LIST OF TABLES ...................................................................................................................XIII LIST OF FIGURES .................................................................................................................XIV LIST OF ABBREVIATIONS .................................................................................................XIX Chapter 1 Background of monitoring intracellular processes related to protease activity and calcium signaling in living cells...................................................................... 1 1. 1 Intracellular processes....................................................................................................... 1 1. 2 Proteases............................................................................................................................ 2 1.2.1 Protease functions ........................................................................................................ 2 1.2.2 Significance of protease investigations........................................................................ 4 1.2.3 Protease classifications ................................................................................................ 6 1.2.4 Substrate specificity of proteases................................................................................. 8 1.2.5 Current methods for protease detection ....................................................................... 8 1.2.6 Criteria for an ideal protease sensors......................................................................... 13 1. 3 Calcium signaling............................................................................................................ 14 1.3.1 Regulation of intracellular calcium............................................................................ 16 1.3.2 Major players to mediate intracellular calcium accumulation................................... 17 1.3.3 Calcium signaling and related diseases.....................................................................