Wayne State University Wayne State University Theses 1-1-2014 Proteomic Discovery Of Protein Phosphatase 1 Catalytic Subunit Protein Interaction Partners In Human Skeletal Muscle Zhao Yang Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_theses Part of the Medicinal Chemistry and Pharmaceutics Commons Recommended Citation Yang, Zhao, "Proteomic Discovery Of Protein Phosphatase 1 Catalytic Subunit Protein Interaction Partners In Human Skeletal Muscle" (2014). Wayne State University Theses. Paper 360. This Open Access Thesis is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Theses by an authorized administrator of DigitalCommons@WayneState. PROTEOMIC DISCOVERY OF PROTEIN PHOSPHATASE 1 CATALYTIC SUBUNIT PROTEIN INTERACTION PARTNERS IN HUMAN SKELETAL MUSCLE by Zhao Yang THESIS Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE YEAR 2014 MAJOR Pharmaceutical Sciences Approved By: Advisor Date © COPYRIGHT BY ZHAO YANG 2014 All Rights Reserved DEDICATION To the world beloved ii ACKNOWLEDGMENTS I would like to express my deep appreciation to Dr. Zhengping Yi, my research supervisor, for his patient guidance, enthusiastic encouragement and useful critiques of this research work. I would also like to thank my committee members, Dr. Fei Chen and Dr. Moh H. Malek, for their valuable and constructive advice in keeping my progress on schedule. My completion of this project could not have been accomplished without the generous help of Dr. Michael Caruso. I would also like to extend my thanks to Dr. Xiangmin Zhang and Dr. Danjun Ma, for their help in offering me the resources in running the program, and to other group members of the laboratory, Mr. Yue Qi, Ms. Divyasri Damacharla, Mr. Nishit Shah, and Mr. Majed Alharbi for their continuous support during the project. Finally, I wish to thank my parents for their support and encouragement throughout my study. iii TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... VI LIST OF TABLES ..................................................................................................................... VIII CHAPTER 1 INTRODUCTION .................................................................................................... 1 1.1 DIABETES MELLITUS AND INSULIN RESISTANCE .......................................................... 1 1.1.1 History of Diabetes .......................................................................................... 1 1.1.2 Insulin resistance ............................................................................................. 3 1.1.3 Signaling associated with Insulin Sensitivity .................................................... 4 1.2 PP1 C AND PHOSPHORYLATION , AND INSULIN RESISTANCE ......................................... 5 1.2.1 PI3k-AKT-mTOR Pathway ............................................................................... 5 1.2.2 Phosphatases: Function, Category and Families ............................................. 7 1.2.3 Introduction of Protein Phosphatase 1 Catalytic Subunit ................................. 8 1.3 HYPERINSULINEMIC -EUGLYCEMIC CLAMPS AND SKELETAL MUSCLE .......................... 11 1.3.1 Hyperinsulinemic-euglycemic Clamps ........................................................... 11 1.3.2 Tissues response to Insulin ........................................................................... 11 1.3.3 Studying skeletal muscle as a promising approach in T2D research ............. 12 1.4 MASS SPECTROMETRY IN DISCOVERY OF PROTEIN -PROTEIN INTERACTIONS .............. 13 CHAPTER 2 RESEARCH DESIGN AND METHODS ................................................... 15 2.1. MATERIALS ........................................................................................................... 15 2.1.1 Antibody ........................................................................................................ 15 2.1.2 Reagents ....................................................................................................... 16 2.2. SUBJECTS ............................................................................................................ 16 2.3 HYPERINSULINEMIC -EUGLYCEMIC CLAMP WITH MUSCLE BIOPSIES . ............................ 16 iv 2.4 PLASMA INSULIN CONCENTRATION DETERMINATIONS . ............................................... 17 2.5 PROTEOMICS SAMPLE PREPARATION AND ANALYSIS . ................................................ 17 2.5.1 Muscle biopsy processing ............................................................................. 17 2.5.2 Immunoprecipitation, 1D SDS-PAGE, in-gel digestion and mass spectrometry ............................................................................................................................... 18 2.5.3 Sample data analysis .................................................................................... 19 2.5.4 Statistical analysis ......................................................................................... 21 2.5.5 Bioinformatics analysis .................................................................................. 21 CHAPTER 3 RESULTS ................................................................................................ 22 CHAPTER 4 DISCUSSION ........................................................................................... 24 4.1 PP1 C INTERACTION PARTNERS AS PP1 REGULATORY PROTEINS .............................. 25 4.2 PP1 C INTERACTION PARTNERS INVOLVED IN CYTOSKELETON DYNAMICS ................... 28 4.3 PP1 C INTERACTION PARTNERS IN MITOCHONDRIAL FUNCTION .................................. 30 4.4 INTERACTION PARTNERS ACT IN METABOLISM PATHWAYS ......................................... 31 4.5 INTERACTION PARTNERS RELATE TO PROTEIN SYNTHESIS AND DEGRADATION , ......... 33 4.6 OTHER PP1 C INTERACTION PARTNERS ................................................................... 37 4.7 INFLUENCE OF MUSCLE FIBER -TYPE COMPOSITION .................................................. 38 4.8 SUMMARY ............................................................................................................. 39 REFERENCES ............................................................................................................. 60 ABSTRACT .................................................................................................................. 80 AUTOBIOGRAPHICAL STATEMENT .......................................................................... 82 v LIST OF FIGURES Figure 1. The causal relationship and tissue involvement of Type 2 Diabetes development…………………………………………………………………..............40 Figure 2. Overview of signal molecules involved in insulin signaling pathway….41 Figure 3. The sequence of three major isoforms of PP1c: PPP1CA isoform 1, PPP1CB, PPP1CC isoform 1………………………………………………………...42 Figure 4. Structure information of PP1c ...………...……………………………….43 Figure 5. Interactions between PP1c and its interaction partner, MYPT1 to form a PP1 holoenzyme ………………………………………………………………………45 Figure 6. Regulation patterns of interaction partners on PP1c …………………..46 Figure 7. Summary of glucose metabolism change after Type 2 diabetes among different tissues during euglycemic insulin clamp studies…………………………47 Figure 8. Outline of clinical and proteomics data acquisition and analysis. A). General flow chart of clinical and proteomics data acquisition and analysis; B). Detailed illustration of clinical visit 2 ...………………………………………………48 Figure 9. Detailed Proteomic Analysis workflow……………………………………50 Figure 10. Network analysis of enriched PP1c interaction partners in human skeletal muscle revealed by proteomics and Ingenuity Pathway Analysis ……..51 vi Figure 11. Significantly enriched pathways for the PP1c interaction partners identified in this study revealed by Ingenuity Pathways Analysis. A). Significantly enriched pathways. The total number of identified PP1c interaction partners for a given pathway in this study is denoted beside each bar. B) PP1c interaction partners in EIF2 Signaling……………………………………………………………52 vii LIST OF TABLES Table 1. Clinical characteristics participants in the PP1c interaction partner study …………………………………………..……………………………………………… 54 Table 2. The 46 proteins/ protein groups met the 2 rigorous criteria (See Methods for details) for classification as PP1c interaction partners in human skeletal muscle ..………………………………………………………………………………. 55 Table 3. Significantly enriched pathways for the PP1c interaction partners identified in the study revealed by Ingenuity Pathways Analysis……………….. 59 viii 1 CHAPTER 1 INTRODUCTION 1.1 Diabetes mellitus and Insulin resistance Diabetes mellitus, or simply diabetes, is a metabolic disorder that is marked by hyperglycemia, high blood glucose. In 2014, 29.1 million individuals in the United States, which is about 9.3% of the population, have diabetes [1]. Among all cases of diabetes, about 90% to 95% are Type 2 Diabetes (T2D), the diabetes form generally results from defects in insulin action [1]. Furthermore, other related complications of diabetes will afflict patients with their cardinal, ocular, renal, and nervous system dysfunction, mainly resulting from hyperglycemia [2]. 1.1.1 History of Diabetes Unfortunately, though recognition of diabetes