Proteasome activator PA28 and major histocompatability complex class I processing in vitro and in vivo.

Proteasomes are large (~700kD) intracellular multicatalytic proteases found in eukaryotic cells and some archaebacteria and eubacteria. These proteases are responsible for degrading intracellular proteins for multiple cellular functions. In vertebrates, proteasomes also generate most of the peptide epitopes that are presented to cytotoxic T lymphocytes via major histocompatability complex (MHC) class I molecules on the cell surface. Proteasomes are important in both maintaining immunologic selftolerance as well as facilitating the immune response towards intracellular pathogens. It is believed that the core proteasome particle is latent in cells and therefore requires activator protein complexes to enhance its activity in vivo. The PA28 family of proteasome activators contains three members: alpha, beta, and gamma. PA28 α and β are ubiquitously distributed in the cell and expression is enhanced in vitro following exposure to interferon-γ (IFNγ). PA28γ is localized to the nucleus and its expression is unaffected by IFNγ. We demonstrate that the expression of PA28 α and β along with other proteasome genes are enhanced by IFNγ in vivo. These IFNγ-inducible genes demonstrate true constitutive expression that is only partially dependent on components of the IFNγ signaling cascade, and are completely independent of an IFNγ stimulus. Furthermore, it has been demonstrated in vitro that PA28 may affect the specificity of proteasomes, and work in conjunction with specific catalytic sites to enhance the peptide repertoire generated by proteasomes. To investigate the functions of PA28γ, we generated a PA28γ-deficient mouse strain. These mice display defects in multiple proteasomemediated pathways including MHC I antigen presentation. The effect of PA28γ on antigen processing and presentation is antigen-specific, not global. To better understand the antigen specificity of the PA28 complexes, recombinant protein was generated and the cleavage specificities of the mouse PA28α/β-proteasome complex were examined in vitro using natural peptide substrates. Acknowledgements: I need to extend so many thanks to all the people who have helped me during my tenure at U.C. In particular I must admit that I am terribly grateful to have been mentored by John Monaco. He has been an excellent mentor and friend. He provided me with the freedom and resources to pursue anything I wanted professionally both in and out of the lab, yet he was always there when I need him to keep me focused on the task at hand. Even as I start the next step in my career, I know that I will be able to count on John any time I am in need of his assistance. I would like to thank my committee members both past and present: George Babcock, Edmund Choi, George Deepe, Tom Doetschman, and Ken Blumenthal. My entire committee has also provided me with excellent guidance and great support. The members of the Monaco lab have also made my time here at U.C. very memorable, and I couldn’t have accomplished what I have without your help. I would like to thank Jill Beyette, Laura Elenich, David Ginsburg, Jeanna Guenther, Sherry Haley, Timothy Hubbell, Scott McCluskey, Herbert Runnels, Helena Russell, Jay Slack, Scott Wenderfer, Diane Wiginton, and Hiroko Yamazaki. Especially Herb who was also vital in my graduate training for taking me under his wing, training me as a researcher, and being a great friend I need to thank my collaborators, without whom I could not have accomplished the volume of work presented here. Todd Schell and Satvir Tevethia at the Penn State University College of Medicine for the SV40 studies in the PA28γ-deficient mice. Jack Bennink, Yunjung Cho, and John Yewdell at NIAID/NIH for the influenza studies in the PA28γ-deficient mice. Reta Gibbons, George Deepe, and the entire Deepe lab, here at U.C. , for helping me perform the Histoplasma capsulatum infection studies. Miguel Cruz and Reshma Rangwalla for initiating the immunoproteasome expression project and providing data for the manuscript. Lastly, but certainly not least, I need to thank Herbert Runnels for generating the constructs and developing the PA28γ-deficient mice, which I was able to characterize and use in a number of studies. I need to thank Carl Huether, Annette Muckerheide, Linda Finke, Tracy Reed, Richard Davis and the entire Preparing Future Faculty program for training me to not only be a researcher, but also a teacher and a mentor. I owe a great debt of gratitude to Annette Muckerheide for taking me under her wing and really advising me on how to balance my teaching, my research, and my personal life. She is an exceptional person and has touched my heart with her kindness and wisdom. I would also like to thank the students, faculty, and staff members of the department of Molecular Genetics, Biochemistry, and Microbiology for their assistance and support throughout the years. Also I would like to thank the Bob Colbert, Tom Griffin, and their lab members for discussions, constructive criticisms, and support. I also need to thank my friends and family, who have stood by my side for the last five years, supported my decisions, listened when I needed them to, advised me on many occasions, and provided pleasant distractions when appropriate. I especially owe gratitude to my parents, who have always supported and encouraged me. I also thank Pamela Buelterman who has provided me with perspective, advice, and who has always been there when I needed her. Her love has made me stronger and more determined to succeed. Dedication: I dedicate this dissertation and all my work over the past five years in the loving memory of my grandfather, Glenn J. Barton. He always supported me, loved me, and always believed in me that I would accomplish amazing things. It is his encouragement and stern support that echoes in my mind and memories and drives me to make him proud. I know that he was, is, and always will be proud of me. His presence is always felt and I know that he is beside me guiding and protecting me even today. Thank you, and I miss you.