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WANG-DISSERTATION-2014.Pdf Copyright by Feng Wang 2014 The Dissertation Committee for Feng Wang Certifies that this is the approved version of the following dissertation: A Co-translational Ubiquitination Pathway for Quality Control of Newly Synthesized Proteins Committee: Jon Huibregtse, Supervisor Jaquelin Dudley Arlen Johnson Tanya Paull Rick Russell A Co-translational Ubiquitination Pathway for Quality Control of Newly Synthesized Proteins by Feng Wang, B.S.; M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August, 2014 Dedication To my family (Fuqiang, Jinyu and Gang) and Jingjie for their love and support. Acknowledgements I would like to express my deepest gratitude to Dr. Jon Huibregtse, for his encouragement, guidance, patience, and enthusiasm for science. I could not ask for a better mentor. I am also grateful to the members of my committee, Drs. Jaquelin Dudley, Arlen Johnson, Tanya Paull, and Rick Russell, for their constructive criticism, great suggestions, and helpful advice. I also would like to thank all the past and present members of the Huibregtse lab: Dr. Larissa Canadeo, Dr. Hazel O'Connor, Nancy Lyon, Nicholas Maskalenko, Caleb Swaim, Dr. Hyung Cheol Kim, Aaron Charlson, Amanda Vaughn and Kyungwoon Seo, for their friendship and support throughout the years. Finally, I would like to thank my family, Jingjie, and my friends, for their love and support during graduate school. v A Co-translational Ubiquitination Pathway for Quality Control of Newly Synthesized Proteins Feng Wang, Ph.D. The University of Texas at Austin, 2014 Supervisor: Jon M. Huibregtse Previous studies indicated that 6%-30% of newly synthesized proteins are rapidly degraded by the ubiquitin-proteasome system. This has generally been assumed to occur post-translationally, following failure of chaperone-assisted folding mechanisms. However, the extent and significance of co-translational quality control remains largely unknown. In investigations of ISG15, an interferon-induced ubiquitin-like protein, our lab found that ISG15 is conjugated to a very broad spectrum of newly synthesized proteins. The major ligase for ISG15, Herc5, co-fractionated with polysomes, and further studies indicated that the processes of translation and ISGylation were closely coupled. Here, I employ an in vitro run-off translation system and puromycin labeling experiments to demonstrate that nascent polypeptides are ISGylated within active translation complexes, providing direct support for the co-translational mechanism for ISG15 conjugation. Approaches developed for studying co-translational ISGylation were subsequently used to examine co-translational ubiquitination (CTU), which we hypothesized might be vi important in quality control of newly synthesized proteins. Consistent with this, I found that the pathway for degradation of newly synthesized proteins was initiated while proteins were being translated, with ubiquitination of actively translating nascent polypeptides. CTU is a conserved and robust pathway from yeast to mammals, with 5-6% of total nascent polypeptides being ubiquitinated in S. cerevisiae, and 12-15% in human cells. CTU products contained primarily K48-linked polyubiquitin chains, consistent with a proteasomal targeting function. Although nascent chains previously have been shown to be ubiquitinated within stalled and defective translation complexes (referred to here as CTUS), nascent chain ubiquitination also occurred within active translation complexes (CTUA). CTUA accounted for approximately two-thirds of total CTU (CTUT) in human cells and approximately half of CTUT in yeast cells. CTUA was increased in response to agents that induce protein misfolding, whereas CTUS was increased in response to agents that led to translational misreading or stalling. These results indicate that ubiquitination of nascent chains occurs in two contexts and define CTUA as a component of a quality control system that marks proteins for destruction before their synthesis is complete. Finally, decreased translation fidelity is thought to lead to the accumulation of misfolded proteins and hasten the aging process. As CTU is a pathway for quality control of newly synthesized proteins, I explored whether CTU plays a protective role during the replicative aging process in budding yeast. Consistent with previous reports using human cells, I found that newly synthesized proteins are a major source of proteasome substrates under non-stressed conditions. Transient proteasome inhibition (using MG132) led to a decrease of yeast replicative life span (RLS), whereas simultaneous treatment with vii cycloheximide, a translation inhibitor, suppressed this effect. Deletion of Ltn1, the major E3 ligase of the CTUS pathway, also shortened the RLS of yeast. Together, these results provide a preliminary set of evidence supporting the hypothesis that the quality of newly synthesized proteins is an important determinant of aging. viii Table of Contents List of Tables ................................................................................................... xii List of Figures ................................................................................................. xiii Chapter 1: Introduction .................................................................................... 1 1.1 A brief overview of the ubiquitin-proteasome system (UPS) .......................... 1 1.1.1 Ubiquitin............................................................................................... 2 1.1.2 Ubiquitin activating enzymes (E1s) ....................................................... 6 1.1.3 Ubiquitin conjugating enzymes (E2s) .................................................... 7 1.1.4 Ubiquitin ligases (E3s) .......................................................................... 8 1.1.5 Deubiquitinating enzymes (DUBs) ...................................................... 12 1.1.6 The proteasome ................................................................................... 13 1.2 ISG15: An anti-viral ubiquitin-like protein .................................................. 14 1.2.1 Ubiquitin-like proteins ........................................................................ 14 1.2.2 ISG15 and its pathway enzymes .......................................................... 17 1.2.3 Anti-viral function of ISG15 ............................................................... 18 1.2.4 ISG15 targets a broad range of newly synthesized proteins ................. 21 1.3 Cellular proteostasis and its linkage with aging ............................................ 23 1.3.1 Challenges of protein folding in vivo .................................................. 24 1.3.2 Strategies to facilitate protein folding in vivo ...................................... 25 1.3.3 Strategies for clearance of misfolded proteins ..................................... 28 1.3.4 Classic model for quality control of newly synthesized proteins .......... 32 1.3.5 Aging is linked to a gradual loss of cellular proteostasis ...................... 34 1.4 Goals of my doctoral work .......................................................................... 41 Chapter 2: ISG15 is co-translationally conjugated to ribosome-associated nascent polypeptides ............................................................................... 43 2.1 Introduction ................................................................................................. 43 2.2 Materials and methods ................................................................................. 45 2.3 Results ......................................................................................................... 49 ix 2.3.1 Establishment of in vitro run-off translation and biotin-puromycin conjugation reactions .......................................................................... 49 2.3.2 Polysome-associated nascent chains are ISGylated.............................. 54 2.3.3 ISGylation of nascent polypeptides of individual target proteins ......... 60 2.4 Discussion ................................................................................................... 62 Chapter 3: A co-translational ubiquitination pathway for quality control of newly synthesized proteins ................................................................... 64 3.1 Introduction ................................................................................................. 64 3.2 Materials and methods ................................................................................. 67 3.3 Results ......................................................................................................... 72 3.3.1 CTU is a robust process in mammalian cells ....................................... 72 3.3.2 CTU predominantly occurs on cytosolic rather than ER-associated polysomes ........................................................................................... 77 3.3.3 CTU targets are polyubiquitinated with K48 and K11 chains .............. 79 3.3.4 CTU occurs within both stalled and active translation complexes ........ 85 3.3.5 CTU is enhanced under conditions that promote protein misfolding or translational errors .............................................................................. 90 3.4 Discussion ................................................................................................... 95 Chapter
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