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View Showing the Overlay of C Trace of Gankyrin WT (Black) and Modeled I79D Mutant (Green) Study of the Structure and Function Relationship of Oncoprotein Gankyrin Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yi Guo, B.S. Ohio State Biochemistry Graduate Program The Ohio State University 2009 Dissertation Committee: Professor Ming-Daw Tsai, Advisor Professor Ross Dalbey, Co-advisor Professor Shang-Tian Yang Copyright by Yi Guo, B.S. 2009 ABSTRACT Gankyrin, a newly defined oncoprotein also known as PSMD10 and P28, functions as a dual-negative regulator of the two most prominent tumor suppressor pathways: the CDK/pRb and MDM2/P53 pathways. Its aberrant expression has been prevalently found in human hepatocellular carcinomas (HCC) and esophagus squamous cell carcinomas (ESCC), which indicates gankyrin is a potential diagnostic and therapeutic target in cancers. Gankyrin is an ankyrin repeat (AR) protein which is composed of seven ankyrin repeats. Each repeat of gankyrin exhibits a canonical helix- turn-helix conformation and these seven repeats are stacked together near linearly to form a helix bundle, in which the neighboring ankyrin repeats are linked by loops of varied size which orientate perpendicularly to the axes of the helices of ankyrin repeats. Our previous studies showed that while both specific CDK4 inhibitor p16INK4A (P16, exclusively consists of four AR repeats) and gankyrin bind to cyclin-dependent kinase 4 (CDK4) in similar fashion, only P16 inhibits the kinase activity of CDK4. While this could explain why P16 is a tumor suppressor and gankyrin is oncogenic, the structural basis of these contrasting properties was unknown. In this study we show that a double mutant of gankyrin, L62H/I79D, inhibits the kinase activity of CDK4, similar to P16, and such CDK4-inhibtory activity is associated with the I79D but not L62H mutation. In addition, mutations at I79 and L62 bring about a moderate decrease in the ii stability of gankyrin. Further structural and biophysical analyses suggest that the substitution of Ile79 with Asp leads to local conformational changes in loops I–III of gankyrin. Taken together, our results allow the dissection of the “protein–protein binding” and “CDK4 inhibition” functions of P16, show that the difference between tumor suppressing and oncogenic functions of P16 and gankyrin, respectively, mainly resides in a single residue, and provide structural insight to the contrasting biological functions of the two AR proteins. The second part of the research on gankyrin involves an important structural characteristic of AR proteins: the presence of TPLH tetrapeptide or a variant at the beginning of canonical helix-turn-helix motif. Hydrogen bonding involving the Thr and His residues in the same and between adjacent tetrapeptide motifs presumably contributes to the formation of a hydrogen-bonding network and consequently the stability of the molecule. Thus, we investigated the structural role of this TPLH network in AR proteins by studying gankyrin, an oncogenic protein composed of seven ARs and six TPLH tetrapeptides, and p16INK4a, a tumor suppressor with four ARs and no TPLH tetrapeptides. Our results show that disrupting the TPLH network in the middle by removing Thr or both Thr and His from AR4 and AR5 of gankyrin significantly decreases its stability in both chemical- and heat-induced unfolding. On the other hand, introducing the TPLH network in p16INK4a in the middle (on AR3) increases its conformational stability. Our results suggest that the hydrogen bonding between neighboring TPLHs stabilize the structure of AR proteins when the TPLH motifs are in the middle of a long stretch of ankyrin repeats. iii Dedicated to my family iv ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Ming-Daw Tsai, for his guidance during my Ph.D studies. Dr. Tsai has always been encouraging his students to think as a scientist, which has truly helped me in my scientific career. I would also like to thank Dr. Junan Li and my dissertation committee members, Dr. Ross Dalbey and Dr. Shang-Tian Yang, for their time and vested interest in my research. Dr. Junan Li has been guiding me with my research during the past two years. With his help on research, I was able to tackle problems and finish research projects. I would like to acknowledge my past lab members, including Hongyuan Li, Shngjiang Tu, Anjali Mahajan, Haiyan Song, Yu Wang, Sandeep Kumar, Hyun Lee, Brandon Lamarche, and Marina Bakhtina. This group of incredibly talented scientists has taught me so much not only about science, but also about being human. It is an honor to have worked with each of these individuals. Finally, I thank my family for the unconditional love and support. My mother has been extremely supportive of my scientific career even though she is thousands of miles away. My lovely wife, Jie, has been my most solid support during my Ph.D career. v VITA November 29, 1980. Born – Wuhan, Hubei, China. June 2002. Bachelor of Science in Biochemistry, Wuhan University. 2004 – 2005. Ohio State Biochemistry Program Fellow, The Ohio State University. 2005 – present. Graduate Teaching and Research Associate, The Ohio State University. PUBLICATIONS 1. Guo, Y. , Yuan, C., Weghorst, C. M., Tsai, M.-D., and Li, J. (2009) Diversified Contributions of Conserved TPLH Tetrapeptides to the Conformational Stability of Ankyrin Repeat Proteins. J Biol Chem (submitted). 2. Li, J. and Guo, Y. (2009) Gankyrin Oncoprotein: Structure, Function, and Involvement in Cancer. Current Chemical Biology (In Press). 3. Guo, Y. , Mahajan, A., Yuan, C., Joo, S. H., Weghorst, C. M., Tsai, M.-D., and Li, J. (2009) Comparisons of the Conformational Stability of Cyclin-Dependent Kinase (CDK) 4-Interacting Ankyrin Repeat (AR) Proteins. Biochemistry 48, 4050-4062. 4. Mahajan, A. †, Guo, Y.† , Yuan, C., Weghorst, C. M., Tsai, M.-D., and Li, J. (2007) Dissection of protein-protein interaction and CDK4 inhibition in the oncogenic versus tumor suppressing functions of gankyrin and p16. J. Mol. Biol. 373, 990-1005. ( †A.M. and Y.G. contributed equally to this work) FIELDS OF STUDY Major Field: Ohio State Biochemistry Program vi TABLE OF CONTENTS Page Abstract . ii Dedication . iv Acknowledgments . v Vita . vi List of Tables. xi List of Figures . .xii List of Abbreviations . .xiv Chapters: 1. Introduction . 1 1.1 The discovery of oncoprotein gankyrin . 1 1.2 The structure of gankyrin . 3 1.3 Diverse functions of gankyrin . 5 1.3.1. Gankyrin and Rb, p53 . 6 1.3.2. Gankyrin and the proteasome . 8 1.3.3. Gankyrin and MAGE A4, RelA . 8 1.3.4. The structure basis for the function of gankyrin . .9 vii 1.4 Gankyrin and cancer . 12 1.4.1. Aberrant expression of gankyrin is prevalent in human cancers . 12 1.4.2. Aberrant expression of gankyrin could be an early event in the development of human cancers . 13 1.4.3. Potential mechanisms underlying the involvement of gankyrin in cancer . 14 1.5 Summary . 16 2. Dissection of protein-protein interaction and cdk4 inhibition in the oncogenic versus tumor suppressing functions of gankyrin and p16 . 18 2.1 Introduction . 18 2.2 Results . 23 2.2.1. Structure-based protein engineering . 23 2.2.2. I79D and L62H/I79D of gankyrin bind and inhibit CDK4 . 24 2.2.3. I79D and L62H/I79D mutations bring about substantial perturbation to the local conformation of gankyrin . 27 2.2.4. I79D and L62H/I79D destabilize the global structure of gankyrin 33 2.3 Discussion . 35 2.3.1. Possible conformational adjustments of local loops upon I79D mutation of gankyrin . 35 2.3.2. Structural basis for the decreased stability of the gankyrin mutants . 39 2.3.3. Structural basis for the functional diversity of the AR proteins . 40 2.3.4. Potential biological significance . 42 2.4 Materials and Methods . 43 viii 2.4.1. Cloning, expression, and purification of human gankyrin and its mutants . 43 2.4.2. Pull-down assay . 44 2.4.3. In Vitro CDK4 kinase assay . 45 2.4.4. Circular dichroism (CD) analyses of gankyrin proteins . 46 2.4.5. NMR analyses . 47 2.4.6. Bioinformatics analysis . 47 3. Contributions of conserved tplh tetrapeptides to the conformational stability of ankyrin repeat proteins . 48 3.1 Introduction . 48 3.2 Results . 54 3.2.1. Mutagenic effect on gankyrin-CDK4 association . 55 3.2.2. Mutagenic effect on the conformational stability of gankyrin . 57 3.2.3. Mutagenic effect on the structure of gankyrin . 61 3.2.4 Mutagenic effect on P16 . 68 3.3 Discussion . 70 3.4 Materials and Methods . 76 3.4.1. Database analysis. 76 3.4.2. Cloning, expression, and purification of human gankyrin, p16 INK4a and their mutants. 77 3.4.3. Pull-down assay . 78 3.4.4. In vitro CDK4 kinase assay . 78 3.4.5. Circular dichroism (CD) analyses . 79 ix 3.4.5. NMR analyses . 80 Bibliography . 81 x LIST OF TABLES Table Page 2.1 Biochemical and biophysical parameters of gankyrin and its mutant proteins . 35 3.1 Conformational stability of TPLH mutants of gankyrin and P16 . 54 xi LIST OF FIGURES Figure Page 1.1 Sequence alignments of human, wolf, mouse, rat, hamster, zebrafish, and yeast gankryin proteins . 3 1.2 Solution structure of human gankyrin . ..
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