Brigham Young University BYU ScholarsArchive All Theses and Dissertations 2015-06-01 Studies on the Roles of Translationally Recoded Proteins from Cyclooxygenase-1 and Nucleobindin Genes in Autophagy Jonathan J. Lee Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Chemistry Commons BYU ScholarsArchive Citation Lee, Jonathan J., "Studies on the Roles of Translationally Recoded Proteins from Cyclooxygenase-1 and Nucleobindin Genes in Autophagy" (2015). All Theses and Dissertations. 6538. https://scholarsarchive.byu.edu/etd/6538 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in All Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Studies on the Roles of Translationally Recoded Proteins from Cyclooxygenase-1 and Nucleobindin Genes in Autophagy Jonathan J. Lee A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Daniel L. Simmons, Chair Richard K. Watt Joshua L. Andersen Barry M. Willardson Jeffery Barrow Department of Chemistry and Biochemistry Brigham Young University June 2015 Copyright © 2015 Jonathan J. Lee All Rights Reserved ABSTRACT Studies on the Roles of Translationally Recoded Proteins from Cyclooxygenase-1 and Nucleobindin Genes in Autophagy Jonathan J. Lee Department of Chemistry and Biochemistry, BYU Doctor of Philosophy Advances in next-generation sequencing and ribosomal profiling methods highlight that the proteome is likely orders of magnitude larger than previously thought. This expansion potentially occurs through translational recoding, a process that results in the expression of multiple variations of a protein from a single messenger RNA. Our laboratory demonstrated that cyclooxygenase-3/1b (COX-3/1b), a frameshifted, intron-1-retaining, alternative splice variant from the COX-1 gene, is multiply recoded, which results in the translation of at least seven different COX-3 proteins. Two of the recoded COX-3 proteins that we identified are active prostaglandin synthases and are inhibited by non-steroidal anti-inflammatory drugs (NSAIDs). Here we show that the other non-prostaglandin-generating recoded COX-3 proteins perform new roles in innate immunity, a process in which COX are known to generally function. Our analyses determined that these recoded COX-3 proteins bind at or near the amino-terminal region of ATG9a, a critical regulator of both canonical (i.e. digestive autophagy associated with mTORc inhibition and nutrient deprivation) and non-canonical (i.e. xenophagy involved in the innate immune response to invading organisms) autophagy. We further show that this process requires mTORc signaling activity, which opposes the digestive pathway. As a final confirmation of the biological relevance of these recoded COX-3 proteins and their central role in xenophagy, we demonstrate that expression of these COX-3 proteins in an encephalomyocarditis virus infection model system differentially affects infectious virion production. These COX-3 proteins also associate with recoded cytosolic nucleobindin around large, innate immune-related, large LC3-II positive structures (LLPSs). Through mutagenizing catalytic residues of recoded COX-3 proteins and drug assays, we determine LLPS formation is dependent on oxylipin generation. Keywords: Cyclooxygenase, COX-3, Oxylipin, Autophagy, Xenophagy, Non-canonical autophagy, Nucleobindin ACKNOWLEDGEMENTS I must share my most heartfelt appreciation to Dr. Simmons for his support, patience, friendship, and direction as his graduate student. His foresight and knowledge have aided me in my experimental design and analysis of data. Dr. Simmons’ sound advice has led me to better understand the process of science and discovery. His energy has been a boon in my graduate career and I hope to emulate his excitement in science in future projects. I would also like to thank both Dr. Simmons, Dr. Bradshaw, and Jacob Cuttler for editing this dissertation. I am also appreciative of the advice and counsel given me by my committee members Dr. Willardson, Dr. Andersen, Dr. Barrow, and Dr. Watt. Their expression of support and guidance has helped me solve difficult questions which enabled me to move forward in my projects. I must give special thanks to Dr. Andersen for loaning me material for my projects and giving me counsel in the area of autophagy. I am extremely grateful for the work provided by the undergraduate students Stephen Hill, Tim Visser, Matthew Lelegren, and especially Gideon Logan. Gideon has provided almost all of the confocal images in this dissertation and has given me insights that have propelled my work forward. I must also express gratitude to Dr. John Hunter who has given me advice on techniques and writing. It has been a great pleasure working with everyone involved in the work and writing of this dissertation. Finally, I must express my deepest love for my wife, Melissa, who has been patient and supportive of my endeavors in the Biochemistry Ph.D. program. Her care and love has sustained me through the many difficulties I faced as a graduate student. I must also say thanks to all those not listed here who have also shown kindness to me and given me support. TABLE OF CONTENTS Abstract……………………………………………………………………..……………………………………….. ii Acknowledgments……………………………………………………………………..……………………….. iii List of Tables……………………………………………………………………..………………………………… vii List of Figures……………………………………………………………………..……………………………….. viii Abbreviations……………………………………………………………………..………………………………. xi Chapter 1: Autophagy, recoded cyclooxygenase and oxylipins………...................... 1 Abstract……………………………………………………………………..…………………………….. 1 Background………………………………………………………………………………………………. 2 Macroautophagy…………………………………………………………………………… 2 Non‐canonical autophagy and innate immunity...………………………….4 Cargo selection and lipid’s role in autophagy……………….…...…………. 6 References……………………………………………………………………..………………........... 9 Chapter 2: Recoded COXs complex with ATG9a, a vital component of autophagy……………………………………………………………….…………………………………………… 12 Abstract……………………………………………………………………..…………………………….. 12 Background……………………………………………………………………..………………………..12 ATG9a is a transmembrane protein that shuttles between Golgi and autophagic structures…………………………………………………….12 Regulation of ATG9a is dependent on both protein binding and post‐translational modifications…………………………………………….. 13 ATG9a is involved in innate immunity ………………………………………….. 14 Materials and Methods……………………………………………………………………………..16 Materials………………………………………………………………………………………. 16 Cell Culture……………………………………………………………………..……………. 16 Plasmid DNA purification……….…………………………………..………………… 16 DC protein assay………..…………………………………………………………………. 17 Transient transfection………………..………………………………………………… 17 Site‐directed mutagenesis………………………….…………………………......... 18 Co‐immunoprecipitation of ectopic ATG9a and rc57…………………….. 19 Immunoblot analysis…………………………………………………………………….. 20 Collagenizing glass slides………………………………………………………………. 20 Confocal microscopy…..………………………………………………………………… 21 Acceptor photobleach FRET analysis…………………………………………….. 21 EMCV infection…………………….………………………………………………………..22 Plaque forming assay……………………………………………………………………..22 iv Results……………………………………………………………………..………………………………. 23 Confocal microscopy of rcCOXs identifies a role in autophagy………. 23 rcCOXs do not localize to amphisomes but rc50 associates with autolysosomes……………………………………………………………………….25 Permeabilization of cells shows rcCOXs are not intraluminal autophagosomal cargo……………………………………………… 25 rcCOXs co‐localize with ATG9a vesicles…………………………………………. 29 rcCOXs binds near or at the N‐terminus of ATG9a………………………… 31 rcCOXs do not co‐localize with macroautophagic omegasomes……. 34 The rcCOX/ATG9a complex does not require COX catalytic residues………………………………………………………….……….. 36 mTOR inhibitor blocks rcCOX/ATG9a complex………………………………. 38 rcCOXs differential effect on EMCV replication…………………………….. 39 Discussion…………………………………….……………………………………………………………40 References…………………………………….…………………………………….…………………….44 Chapter 3: Recoded COXs and nucleobindin induce large LC3‐II positive structures (LLPSs)……………………………….………………………………… 48 Abstract……………………………………………………………………..…………………………….. 48 Background……………………………………………………………………..………………………..48 Nucleobindin is a multi‐domain protein with many physiological roles and two subcellular locations..…………………….…..49 Cytosolic nucleobindin occurs through translational recoding…………………………………………………………………… 50 Materials and Methods……………………………………………………………………………..51 Sample preparation for p62 immunoblot analysis.……………………….. 51 Results……………………………………………………………………..………………………………. 51 Site‐directed mutagenesis demonstrates that cNuc is a recoded form of Nuc…………………………………………………….………… 51 Recoded COXs translocate with cNuc to LLPSs and act synergistically to drive their formation…………………………………………. 54 Co‐transfection of cNuc abolishes interaction between rcCOX and ATG9a………………………………………………………………….......... 56 cNuc blocks autophagic flux before amphisome formation……………………………………………………………………………………… 57 Discussion…………………………………….………………………………………………………….. 60 References…………………………………….…………………………………….…………………… 63 Chapter 4: Large LC3‐II positive structure (LLPS) formation is affected by rcCOX redox activity and requires oxylipin metabolism……………….………………. 68 Abstract……………………………………………………………………..…………………………….. 68 Background……………………………………………………………………..………………………..69