Targeting the Nck/PERK interaction to improve β cell function and survival George Kefalas Division of Experimental Medicine Department of Medicine McGill University Montreal, Quebec, Canada December 2017 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © George Kefalas, 2017 Abstract The failure of pancreatic β cells to produce insulin is a central problem in both Type 1 and Type 2 diabetes, however the molecular mechanisms leading to β cell failure remain unclear. Consequently, investigating mechanisms that promote β cell function and survival is paramount in the development of therapies to prevent and treat both types of diabetes. PKR-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein involved in maintaining β cell homeostasis. Previously, our group identified the adaptor protein Nck1 as a negative regulator of PERK. Indeed, we demonstrated that Nck1, by directly binding PERK autophosphorylated on Tyr561, limits PERK activation and signaling. In accordance, we found that Nck1 depletion in β cells promotes PERK activity and signaling, increases insulin biosynthesis, and promotes cell viability in response to diabetes-related stresses. Herein, we explore the therapeutic potential of abrogating the interaction between Nck and PERK in order to improve β cell function and survival. To do so, we used a peptide containing the minimal sequence of PERK that is involved in binding Nck1 conjugated to the cell permeable protein transduction domain from the HIV protein TAT. In the current study, we confirm that the synthetic phosphopeptide TAT-pY561 sequesters Nck and prevents its interaction with PERK. Moreover, we demonstrate that TAT-pY561 penetrates the β cell membrane and promotes basal PERK activity. Furthermore, we report that pretreatment of β cells with TAT-pY561 inhibits apoptosis induced by glucolipotoxicity. Finally, we further characterize TAT-pY561 at the biochemical level by comparing its affinity for Nck with unphosphorylated and mutant peptide variants. Taken together, our data support the potential of sequestering Nck using a synthetic peptide to enhance basal PERK activity and create more resilient β cells. ii Résumé L’incapacité des cellules β du pancréas endocrine à produire suffisamment d'insuline est un problème majeur chez les diabétiques. Cependant, les mécanismes moléculaires menant à la défaillance des cellules β restent flous. Par conséquent, l’identification de mécanismes contrôlant la fonction des cellules β pancréatiques est d’importance majeure pour le design de thérapies efficaces visant à prévenir et traiter le diabète. Plusieurs études ont révélé le rôle essentiel de la protéine kinase du réticulum endoplasmique PERK dans le maintien fonctionnel des cellules β pancréatiques. Dans ce contexte, notre groupe a découvert que l’adaptateur Nck1 limite l’activation de PERK. En effet, nous avons démontré que la liaison directe de Nck1 au résidu Tyr561 phosphorylé de PERK réduit l’activité basale de PERK. Nous avons par ailleurs révélé que la déplétion de Nck1 dans les cellules β augmente l'activité basale de PERK et la biosynthèse de l'insuline, et améliore la viabilité des cellules β en réponse aux stress liés au diabète. Dans cette thèse, nous avons exploré le potentiel thérapeutique de prévenir l'interaction entre Nck et PERK afin d'améliorer la fonction et la survie des cellules β. Dans cette optique, nous avons utilisé le peptide TAT-pY561 composé de la séquence minimale de PERK impliquée dans la liaison avec Nck1 conjuguée au domaine de transduction protéique de la protéine TAT du VIH. Dans cette étude, nous confirmons que le phosphopeptide synthétique TAT-pY561 séquestre Nck et empêche son interaction avec PERK. En outre, nous démontrons que TAT-pY561 est capable de pénétrer la membrane des cellules β et d’augmenter l’activité basale de PERK. De plus, nous rapportons que le prétraitement des cellules β avec TAT-pY561 inhibe l'apoptose induite par la glucolipotoxicité. Finalement, nous avons plus amplement caractérisé le peptide TAT-pY561 en comparant son affinité pour Nck avec une variante non phosphorylée ou mutée du peptide iii TAT-pY561. Dans leur ensemble, ces données supportent le potentiel de séquestration de Nck à travers l’usage d’un peptide synthétique afin d’améliorer l'activité basale de PERK et de créer des cellules β plus robustes. iv Acknowledgments The authorship of this thesis will be attributed to only one person, however multiple people are responsible for its creation and for all of the efforts behind it. First and foremost, I’d like to express my gratitude to my supervisor, Dr. Louise Larose, for taking a chance on me and providing me with the opportunity to pursue my graduate studies in her laboratory. Thank you for always challenging me and for mentoring me in such a way that helped me grow as both a scientist and a person. The lessons I have learned from you will stick with me throughout my entire life as a scientist. Thanks also to all members of the Larose lab from the past few years, especially Dr. Lama Yamani, Dr. Hui Li, Dr. Julie Dusseault, Dr. Bing Li, Nida Haider, and Cindy Baldwin. I would not have enjoyed my time as a graduate student nearly as much if it were not for the pleasant environment you all helped to create. Throughout my graduate studies I had the privilege of being supported by many brilliant and caring researchers. Thanks to the members of my research advisory committee, Dr. Geoffrey Hendy, Dr. Stéphane Laporte, and Dr. Simon Wing, for the stimulating discussions that were held concerning my project. Thanks to our collaborators, Dr. Jennifer Estall and Dr. Nathalie Jouvet, for the passion they demonstrated towards this project. Finally, thanks to the Canadian Institutes of Health Research, the Fonds de recherche du Québec – Santé, and the McGill University Division of Experimental Medicine for providing me with the financial support to pursue these graduate studies. Next, I would like to thank the faculty, staff, and students of the Polypeptide Laboratories and the MeDiC program for all that they have done to make it so enjoyable to come to work every day. I never once dreaded coming to the lab simply because I knew the laughs we’d share would brighten up my day. Over the past few years, the MUHC-RI has served as more than just an v academic setting for me; it’s truly been a home away from home. While many people can claim to have met and worked with amazing colleagues here, I am honored to say that it is here that I built amazing friendships, ones that I am certain will last a lifetime. Finally, I’d like to acknowledge my family and friends, especially those who undertook this journey of graduate school with me over the past two years: Alexandra Lewis, Andrew Dixon, Joseph Szymborski, Arielle Leone, and Vivian Stavrakos. Through all the highs and lows of this era of our lives, it’s been a blast having you all by my side. Lastly, I’d like to thank my parents, Anna and Peter, and my late grandfather, George, for all that they have done for me over the past 25 years to make this entire experience a possibility for me. vi Preface & Author Contributions This thesis was written in accordance with the guidelines outlined by the Faculty of Graduate and Postdoctoral Studies of McGill University. The author wrote the entire thesis, accompanied by editorial comments by Dr. Louise Larose. Experimental design, laboratory work, and data analysis contributing to the final results shown in this thesis were performed by the author under the supervision and guidance of Dr. Louise Larose. Cindy Baldwin (research technician) assisted with several experimental protocols. vii Table of Contents Abstract ........................................................................................................................................... ii Résumé ........................................................................................................................................... iii Acknowledgments........................................................................................................................... v Preface & Author Contributions ................................................................................................... vii Table of Contents ......................................................................................................................... viii List of Figures ................................................................................................................................ xi List of Abbreviations .................................................................................................................... xii Chapter 1: Introduction ............................................................................................................... 1 1.1 Glucose Homeostasis & β Cell Physiology ..................................................................... 2 1.1.1 Anatomy of the Pancreas .......................................................................................... 3 1.1.2 Insulin Biogenesis ..................................................................................................... 4 1.1.3 Insulin Secretion ....................................................................................................... 6 1.1.4 Insulin Signaling ....................................................................................................... 8 1.1.5 Clinical Significance ..............................................................................................