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The Role of the Chaperone CCT in Assembling Cell Signaling Complexes Brigham Young University BYU ScholarsArchive Theses and Dissertations 2020-07-21 The Role of the Chaperone CCT in Assembling Cell Signaling Complexes Nicole C. Tensmeyer Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Physical Sciences and Mathematics Commons BYU ScholarsArchive Citation Tensmeyer, Nicole C., "The Role of the Chaperone CCT in Assembling Cell Signaling Complexes" (2020). Theses and Dissertations. 9192. https://scholarsarchive.byu.edu/etd/9192 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected]. The Role of the Molecular Chaperone CCT in Assembling Cell Signaling Complexes Nicole C. Tensmeyer A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Barry M. Willardson, Chair Josh L. Andersen John C. Price Pam Van Ry Department of Chemistry and Biochemistry Brigham Young University Copyright © 2020 Nicole C. Tensmeyer All Rights Reserved ABSTRACT The Role of the Molecular Chaperone CCT in the Assembly of Signaling Complexes Nicole C. Tensmeyer Department of Chemistry and Biochemistry, BYU Doctor of Philosophy In order to function, proteins must be folded into their native shape. While this can sometimes occur spontaneously, the process can be hindered by thermodynamic barriers, trapped intermediates, and aggregation prone hydrophobic interactions. Molecular chaperones are proteins that help client proteins or substrates overcome these barriers so that they can be folded properly. One such chaperone is the chaperonin CCT, a large MDa protein made up of 16 paralogous subunits that form a double ring structure. CCT encapsulates its substrates in a central cavity, where they are sequestered and folded, using ATP binding and hydrolysis to drive conformational changes in the CCT-substrate complex. CCT mediates the folding of many substrates involved in a variety of cellular process, including the cytoskeletal proteins actin and tubulin, and the G protein subunit Gβ, which signals downstream of GPCRs in a variety of cellular processes. We showed that CCT is responsible for folding the β-propeller containing proteins, mLST8 and Raptor, which are subunits of the mTOR complexes. The mTOR complexes (mTORC1 and mTORC2) are master regulators of cell growth and survival by controlling processes such as protein synthesis, energy metabolism, cell survival pathways and autophagy. CCT folds mLST8 and Raptor and help them assemble into the mTOR complexes. As a result, CCT is required for functional mTOR signaling. Furthermore, we solved a 4.0 Ǻ resolution structure of mLST8 bound to CCT. Surprisingly, mLST8 is found in the center of the folding cavity, in between the rings, despite previous evidence suggesting that substrates bind only in the apical domains. Given its role in folding and assembling the mTOR complexes, G proteins, and many other proteins involved in cell survival pathways, CCT has been implicated in cancer. CCT upregulation often correlates with a worse prognosis, likely because uncontrolled growth requires increased chaperone capacity. The peptide CT20P has been shown to have cytotoxic effects in cancer cells, likely through its binding to CCT. We characterized CT20P, showing that it binds to CCT and inhibits its substrate-folding functions in cells. We specifically showed that a GFP-CT20P fusion protein inhibited the assembly of two important signaling complexes Gβγ and mTORC1. These results show that CT20P is a useful inhibitor for the study of CCT function. Keywords: chaperones, CCT, mTOR signaling, G protein signaling, CT20P ACKNOWLEDGEMENTS I would like to acknowledge my funding from the Simmons Center for Cancer Research, the Roland K. Robbins Research Fellowship, and the NIH. I would like to thank my committee for their support, and I am especially grateful to my advisor, Dr. Barry Willardson, for his help in every step of this process. The biggest thank you goes to my husband, Chris, for being my greatest supporter in this and every other endeavor in my life. TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x 1. INTRODUCTION ................................................................................................................... 1 1.1 Introduction ...................................................................................................................... 1 1.2 Chaperones ....................................................................................................................... 2 1.2.1 The role of chaperones in the cell ............................................................................. 2 1.2.2 Chaperonins .............................................................................................................. 6 1.2.3 The Chaperonin CCT ................................................................................................ 7 1.2.4 CCT substrates .......................................................................................................... 9 1.2.5 The folding mechanism of CCT ............................................................................. 10 1.2.6 The co-chaperones of CCT ..................................................................................... 14 1.2.7 CCT in disease ........................................................................................................ 15 1.3 mTOR: a master regulator of cell growth and metabolism ............................................ 17 1.3.1 The mTOR complexes ............................................................................................ 18 1.3.2 mTORC1 activation ................................................................................................ 20 1.3.3 Effects of mTORC1 activation ............................................................................... 22 1.3.4 mTORC2 activation ................................................................................................ 24 1.3.5 Effects of mTORC2 activation ............................................................................... 27 iv 1.3.6 mTOR signaling in disease ..................................................................................... 28 1.4 G protein signaling ......................................................................................................... 29 1.5 Conclusion ...................................................................................................................... 30 2. G proteins .............................................................................................................................. 31 2.1 Introduction .................................................................................................................... 31 2.2 G protein signaling ......................................................................................................... 32 2.3 The G protein gene family ............................................................................................. 34 2.4 G protein structure .......................................................................................................... 37 2.5 G protein biogenesis ....................................................................................................... 39 2.6 G-Protein activation ....................................................................................................... 41 2.7 Effector activation .......................................................................................................... 44 2.8 G protein inactivation ..................................................................................................... 46 2.9 Physiology of G protein signaling – vision .................................................................... 47 2.10 Physiology of G protein signaling – synaptic transmission ........................................... 48 2.11 Physiology of G protein signaling – insulin section ...................................................... 50 2.12 Acknowledgements ........................................................................................................ 51 3. Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly ........................................................................................................................................ 52 3.1 Introduction .................................................................................................................... 52 3.2 Results ............................................................................................................................ 56 v 3.2.1 mLST8 and Raptor β-propellers bind CCT ............................................................ 56 3.2.2 CCT contributes to mTORC assembly and signaling ............................................. 58 3.2.3 PhLP1 does not assist in mTORC assembly ........................................................... 61 3.2.4 cryo-EM structure of the mLST8-CCT assembly intermediate .............................
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