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Elischolar – a Digital Platform for Scholarly Publishing at Yale Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine Spring 5-2007 Biochemical and functional characterization of the tapasin/ERp57 conjugate David Ryan Peaper Yale University. Follow this and additional works at: http://elischolar.library.yale.edu/ymtdl Part of the Medicine and Health Sciences Commons Recommended Citation Peaper, David Ryan, "Biochemical and functional characterization of the tapasin/ERp57 conjugate" (2007). Yale Medicine Thesis Digital Library. 2230. http://elischolar.library.yale.edu/ymtdl/2230 This Open Access Dissertation is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale. For more information, please contact [email protected]. Biochemical and functional characterization of the tapasin/ERp57 conjugate A Dissertation Presented to the Faculty of the Graduate School of Yale University in Candidacy for the Degree of Doctor of Philosophy By David Ryan Peaper Dissertation Director: Peter Cresswell May 2007 iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract Biochemical and functional characterization of the tapasin/ERp57 conjugate David Ryan Peaper 2007 Recognition of MHC class I/peptide complexes is required for the generation of CD8+ T cell responses. Peptide loading onto MHC class I/p2m dimers occurs in the ER and involves both specific proteins and cellular chaperones. Tapasin is essential for peptide loading onto most MHC class I alleles, and it forms a mixed disulfide with the glycoprotein specific oxidoreductase ERp57. I have characterized the biochemical requirements for tapasin/ERp57 conjugate formation and addressed potential functions for ERp57 in peptide loading. Tapasin specifically recruits ERp57 into a mixed disulfide at the expense of free ERp57 in the ER. Other components of the MHC class I peptide loading complex are not required for conjugate formation, and, in contrast to models of glycoprotein folding, conjugate formation does not require the generation of monoglucosylated glycans. Once associated, tapasin has evolved to inhibit the reductase activity of the ERp57 a domain leading to the retention of ERp57 in the loading complex over the course of normal peptide loading. In contrast, calreticulin undergoes cycles of binding and release, and its presence in the loading complex is dependent upon MHC class I. ERp57 is a core structural component of the MHC class I loading complex, and it is permanently sequestered there by tapasin. i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Finally, I have further characterized cells expressing a tapasin mutant unable to form the conjugate. Loading complex assembly is impaired in these cells, and peptide loading is inefficiently catalyzed by this mutant. Additionally, ERp57 binding stabilizes the structural integrity of tapasin. When endogenous ERp57 expression was suppressed using RNAi, all residual ERp57 was bound to tapasin, and both protein disulfide isomerase and ERp72, other ER resident oxidoreductases, formed mixed disulfides with tapasin. When redox mutant ERp57 proteins were over-expressed in these cells, only slight changes were seen in MFIC class I trafficking. My data indicate that ERp57 is a key structural component of the MFIC class I loading complex and likely does not exhibit any redox activity in this context. The key features of ERp57 responsible for its function remain unknown. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgements My time in the Cresswell lab has been extremely enjoyable, and that is largely due to the atmosphere that Pete has fostered in the lab. His door is always open, and he is always excited to hear your most recent result no matter how mundane it may seem later. He gives his students great freedom to explore their interests and develop their own experiments, but is also there to bounce around ideas. His mentorship style has allowed me to grow and become a much better scientist than I think I could have been in any other lab. I would like to thank my committee of Jim, Sankar, and Ruslan. They have provided excellent feedback and support throughout graduate school. I would also like to thank the Section of Immunobiology in general and the Medzhitov and Schatz labs in particular for the use of reagents and equipment. Additionally, most of the data in Chapter 5 of this work would not have been possible were it not for Tom Taylor and the cell sorting facility. Finally, the MD/PhD office, Jim, Sue, Cheryl, and Marybeth, has been extremely supportive during all phases of medical and graduate school. I hope that continues until I graduate. During my five years in the lab, a number of people have come and gone, and they have all had some impact on the work presented here. I must emphasize the roles of Naveen, Tobi, Suk-Jo, KC, Anne, Weiming, Xiuyan, Allesandra, Randy, and Ella for their camaraderie, experimental ideas, and/or guidance during my earlier days. Additionally, Nancy is the rock that holds the lab together, especially while Pete is away. Finally, my bay-mates Pam and Rebecca have provided an invaluable buffering capacity v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to the sometimes loud and chaotic nature of the lab in addition to scientific feedback and ideas. While in New Haven I have had to opportunity to make some life-long friends, and their presence throughout this process has made life a lot more enjoyable. I would like to thank my parents, David and Patricia, and family for their support over the years. Their willingness to encourage my curiosity is largely responsible for where I am today. Thomas and Constance Perry have also acted as surrogate parents when I am not able to make it to Indiana for long stretches of time, and for that I am grateful. Finally, I dedicate this work to Kate for her love and support. vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents Abstract.............................................................................................................................................i Title Page....................................................................................................................................... iii Copyright Notice........................................................................................................................... iv Acknowledgements........................................................................................................................v Table of Contents.........................................................................................................................vii Table of Figures............................................................................................................................x Chapter 1: Introduction.............................................................................................................. 1 1.1: General function of MHC class I complexes.......................................................1 1.2: The structure of MHC class 1.................................................................................2 1.3: The generation and translocation of MHC class I peptide ligands ...............6 1.4: Overview of MHC class I assembly .......................................................................7 1.5: Protein folding in the endoplasmic reticulum.................................................... 8 1.6: Glycoprotein folding.................................................................................................9 1.7: Oxidative protein folding.......................................................................................14 1.8: ERp57: a glycoprotein specific oxidoreductase...............................................21 1.9: MHC class I: Early folding events ......................................................................23 1.10: Peptide loading in the ER......................................................................................24 1.11: Tapasin is an MHC class I specific chaperone ................................................. 26 1.12: Tapasin and ERp57 form a mixed disulfide within the MHC class I loading complex....................................................................................................... 28 1.13: Outstanding questions........................................................................................... 29 Chapter 2: Specific recruitment of ERp57 into the MHC class I loading complex by tapasin............................................................................................................................................31 2.1: Tapasin expression determines free ERp57 levels ...........................................32 2.2: Conjugate formation is independent of {32m, MHC class I HC, TAPI, and T A P2...........................................................................................................................34 vii Reproduced with permission of the copyright owner. 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