University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2008 The Development of a Dual-Tag Affinity Purification System and its Application to Elucidate the Interacting Protein Network Surrounding the Human Telomere Binding Proteins TRF1, TRF2, and POT1 Richard John Giannone University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation Giannone, Richard John, "The Development of a Dual-Tag Affinity Purification System and its Application to Elucidate the Interacting Protein Network Surrounding the Human Telomere Binding Proteins TRF1, TRF2, and POT1. " PhD diss., University of Tennessee, 2008. https://trace.tennessee.edu/utk_graddiss/523 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Richard John Giannone entitled "The Development of a Dual-Tag Affinity Purification System and its Applicationo t Elucidate the Interacting Protein Network Surrounding the Human Telomere Binding Proteins TRF1, TRF2, and POT1." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Life Sciences. Yisong Wang, Major Professor We have read this dissertation and recommend its acceptance: Yie Liu, W. Hayes McDonald, Brynn Voy, Sundar Venkatachalam Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Richard John Giannone entitled “The Development of a Dual-Tag Affinity Purification System and its Application to Elucidate the Interacting Protein Network Surrounding the Human Telomere Binding Proteins TRF1, TRF2, and POT1.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Yisong Wang Major Professor We have read this dissertation and recommend its acceptance: Yie Liu W. Hayes McDonald Brynn Voy Sundar Venkatachalam Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) The Development of a Dual-Tag Affinity Purification System and its Application to Elucidate the Interacting Protein Network Surrounding the Human Telomere Binding Proteins TRF1, TRF2, and POT1 A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Richard John Giannone December 2008 DEDICATION I dedicate this dissertation to my grandfather John Demeter; a man who encouraged me to think critically, always question, and never be afraid to challenge conventional wisdom. ii ACKNOWLEDGEMENTS I would first like to thank my mentors, Drs. Yisong Wang and Yie Liu for their guidance and support regarding my research endeavors. I would especially like to acknowledge Dr. Yisong Wang for his great personal sacrifice, allowing me to finish the research contained herein despite extreme adversity. I would like to acknowledge my committee members, Dr. W. Hayes McDonald, Dr. Brynn Voy, and Dr. Sundar Venkatachalam for their critical input, helpful suggestions, and time put into evaluating my course of study. A critical part of my research involved the use of mass spectrometry. With that, I would like to again thank Dr. Hayes McDonald as well as the entire Organic and Biological Mass Spectrometry (OBMS) group at Oak Ridge National Laboratory (ORNL) for both their helpful instruction and permission to use the facilities to generate the necessary data. I would also like to thank Dr. Elissa Chesler, Director of the Systems Genetics group at ORNL and Dr. Cynthia Peterson, Director of the Graduate Program for Genome Science and Technology at the University of Tennessee; both of you went above and beyond what was required and I truly appreciate the support you lent me. Of course, I would like to thank my fellow co-workers and friends, Dr. Hyekyung Plumley, Dr. Yonglong Zhou, Ying Huang, Marla Gomez, (Dr.) David Rhee, and especially (Dr.) Jun Wu and his support, camaraderie, and fellowship; trying at times, but fun while it lasted. Finally and most importantly, I would like to thank my family, Richard H. Giannone, Denyse Giannone, and Jessica Giannone for their love, support, guidance and motivation throughout my life and especially during these recent few years. iii ABSTRACT Protein-protein interactions (PPI) play a vital role in almost every cellular process. Although many methodologies exist to probe PPIs, one of the most successful and widely employed is tandem affinity purification coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS). Although best demonstrated in yeast, TAP has encountered significant hurdles in its application to mammalian systems, especially the observed low yield of bait protein and its interacting partners after two consecutive purifications. To address these issues, a novel dual-tag affinity purification (DAP) system was developed that not only enhances bait protein recovery, but also allows for rapid evaluation of dual-tag compatibility with a protein of interest based on its known subcellular localization. In addition, several tags of varying composition were constructed to allow for maximal bait protein compatibility. With this system, mammalian bait protein yield was improved by more than 200% relative to previously published results. Capitalizing on this success, DAP was applied to human telomere binding proteins TRF1, TRF2, and POT1 to garner a greater understanding of the protein networks that involve the telomere. Expectedly, all the members of the telosome complex were identified at frequencies that lend evidence towards the currently accepted architecture. Also identified were several other novel proteins and subcomplexes that may enhance our understanding of telomere maintenance / length regulation. For instance, members of the classical nuclear import system co-purified with both TRF1 and TRF2. Although previously documented for TRF1, TRF2’s association with importin alpha (KPNA2) and beta (KPNB1) has not been demonstrated till now. Interestingly, further study revealed that KPNA2 acts as a negative regulator of TRF2 nuclear localization. This observation could have far-reaching implications as TRF2 is thought to be also heavily involved in the DNA damage response. Along these lines, a more in- depth MS analysis revealed several putative phosphorylation sites along TRF2’s sequence. One site, pS380, seems to be phosphorylated by the DNA-damage kinase ATM and plays a role in a cell’s proliferative capacity, possibly affecting telomere length regulation. The studies contained here within demonstrate the efficacy of DAP-LC-MS/MS to provide useful leads with regards to the study of PPIs. iv TABLE OF CONTENTS Chapter 1: The Study of Protein-Protein Interactions Using Tandem Affinity Purification and Mass Spectrometry.………………………………1 Chapter 2: Introduction to the Human Telomere…………………………………..20 Chapter 3: A Dual-Tagging System for the Affinity Purification of Mammalian Protein Complexes……………………………………..40 Chapter 4: Exploring the Protein Network Surrounding the Human Telomere Binding Proteins TRF1, TRF2, and POT1…………………..…61 Chapter 5: Probing Novel Sites of TRF2 Phosphorylation by Mass Spectrometry………………………………………………………………...97 Chapter 6: TRF2’s Interacting with KPNA2, a Member of the Classical Nuclear Import Mechanism………………………………………………140 Work Cited …………………………………………………………………….……..183 Appendix A …………………………………………………………………………..202 Vita ………………………………………………………………………………….…217 v LIST OF TABLES Table 3.1: Peptide identifications of known TRF2 interacting proteins or complexes……………………………………………………59 Table 4.1: Bulk MudPIT MS/MS data for each dual-tag affinity purified telomere binding protein after searching with DBDigger and filtering with DTASelect………………………………………...77 Table 4.2: Spectral count data for all identified members of the telosome complex organized by bait protein……………………...………...79 Table A.1: Known and putative interacting partners of the telomere binding proteins TRF1, TRF2, and POT1, sorted by frequency of identification………………………………………………………..203 vi LIST OF FIGURES Figure 1.1: A depiction of the various post-translational modifications that can be applied to each amino acid residue……………………..5 Figure 1.2: Data-dependent tandem mass spectrometry…………………………13 Figure 1.3: Tandem Affinity Purification…………………………………………….16 Figure 2.1: The human telomere, structure and function…………………………21 Figure 2.2: The lengthening of the 3’ G-rich overhang by telomerase in human cells……………………………………………………………26 Figure 2.3: The telosome…………………………………………………………….28 Figure 3.1: Features and validation of the dual-tags……………………………...44 Figure 3.2: Efficient purification of dual-tagged TRF2…………………………….56 Figure 4.1: Proper telomeric localization of POT1 and TRF1 fusion proteins…………………………………………………………………..74