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ABC Transporters’ Interactome: Mapping the Human ABCG2 & ABCG1 Protein-Protein Interactions Kristen Lee Institute of Parasitology McGill University Montréal, Québec April 2021 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science ©Kristen Lee, 2021 TABLE OF CONTENTS ABSTRACT .................................................................................................................................... 2 ABRÉGÉ......................................................................................................................................... 2 ACKNOWLEDGEMENTS ............................................................................................................ 4 CONTRIBUTIONS TO ORIGINAL KNOWLEDGE ................................................................... 6 CONTRIBUTION OF AUTHORS................................................................................................. 6 LIST OF ABBREVIATIONS ......................................................................................................... 7 INTRODUCTION .......................................................................................................................... 9 METHODOLOGY ....................................................................................................................... 38 RESULTS ..................................................................................................................................... 46 DISCUSSION ............................................................................................................................... 72 SUMMARY AND CONCLUSION ............................................................................................. 81 REFERENCES ............................................................................................................................. 84 Page 1 of 110 ABSTRACT ABC transporters play essential roles of detoxification and selective transport of substances in humans. ABCG2 is a half-transporter mainly expressed at the plasma membrane in many healthy tissues including tissue barriers and the gastrointestinal tract. Determining protein-protein interactions of ABCG2 can elucidate a concrete mechanism of regulation (e.g., activation and inhibition) in diseased and healthy states. Moreover, differential tissue regulation of ABCG2 can fully exploit ABCG2’s native roles and broad substrate specificity. Mutations and alterations of expression have been linked to pathologies such as Alzheimer’s, diabetes, cancer, genetic disorders and many infections. Understanding signaling pathways through protein interactions can uncover new therapeutics and drug targets against such ailments. This project uses a proximity-dependent biotinylation approach (BirA) to identify two types of ABCG2 interacting proteins: direct interactors and those within a 10Å sphere. ABCG2 fused with BirA at its N-terminus was stably transfected into HEK293F (an embryonic cell line) and several clones were functionally characterized. Biotinylated proteins, using the latter HEK-BirA-huABCG2 transfectants, identified by mass spectrometry were compared to those interacting with ABCG1 (using HEK- BirA-ABCG1 transfectants), an endosomal transporter that is involved in lipid homeostasis. Proteins that interacted with ABCG2 and ABCG1 were analyzed and compared for differential subcellular localizations and cellular pathways. ABRÉGÉ Les transporteurs ABC sont essentiels pour la désintoxication et le transport sélectif des substances dans les cellules humaines. ABCG2 est un demi-transporteur exprimé principalement dans la membrane cytoplasmique de nombreux tissus sains, tels que les barrières tissulaires et le système digestif. En déterminant les interactions protéines-protéines de ABCG2, cela nous permet Page 2 of 110 d’élucider les mécanismes d’activation et d’inhibition dans les cas de maladies et dans les cas normaux. Les mutations et les altérations dans l’expression de génétiques sont liées aux maladies telles que la maladie d'Alzheimer, le diabète, le cancer, les anomalies génétiques et plusieurs infections. La découverte d’une nouvelle voie de signalisation peut dévoiler de nouvelles thérapeutiques et cibles contres ces maladies. Ce projet utilise l'approche BioID (avec BirA) pour identifier des interactions protéines-protéines avec ABCG2 dans une sphère d'interaction de 10Å. BirA fusionné au N-terminus de ABCG2 a été transfecté dans HEK293F (un ligne cellulaire embryonnaire) et plusieurs clones stables ont été caractérisés fonctionnellement. Les protéines, qui sont biotinylées par HEK-BirA-huABCG2 et identifiées par la spectrométrie de masse ont été comparés à ceux qui interagissent avec ABCG1 (en utilisant HEK-BirA-huABCG1), un demi- transporteur dans la même famille de ABCG2 qui reste à l’endosome et qui transporte le cholestérol. Ces protéines qui interagissent avec ABCG2 et ABCG1 ont été analysées et comparées pour déterminer leurs emplacements dans la cellule et leurs voies cellulaires. Page 3 of 110 ACKNOWLEDGEMENTS I would like to offer my absolute gratitude to Professor Elias Georges, my project investigator, for granting me this amazing opportunity in being able to contribute to the growing field of biochemistry and health sciences. Each experience in his lab has challenged me to think critically and independently as well as contribute to my maturity as a person. I thank every one of my lab members, namely: Dr. Fadi Baakdah, Dr. Georgia Limniatis, and Zahra Sahaf for training me upon my arrival at the lab as well as offering me advice during stressful times. I will never forget laughing till my stomach hurt because of Fadi’s dad jokes (and constantly reminding him my name is Kristen with an E) or discussing key issues with Georgia (ie. stress shopping and eating cheese because a result made no sense). I thank Haritha Menon for supporting me as we worked through the stress of our M.Sc. projects together and pushed each other to stay consistent at the gym. My lab mates have been like family to me and I am immensely grateful to have shared these past few years with them and call them friends. Special thanks to my committee member Professor Sébastien Faucher not only for being an active member of my committee but also training me during my undergraduate degree as one of his interns. Additional thanks to the Fonds de recherche du Quebec – Nature et technologies (FRQNT) for providing me with the scholarship to financially support me through my degree. I would also like to thank the entirety of the Institute of Parasitology at McGill, namely fellow students: Nisha Ramamurthy, Rishi Rajesh, Dr. Mark Kaji, Jennifer Noonan, Maude Dagenais, Jysiane Cardot, Moti Sobat, Elizabeth Siciliani, Cathy Shang Kuan and Eyre Nomi. I will always have nightmares from the horror movie Rishi, Nisha and Mark dragged me to see but will still cherish the time we spent together. Thank you to Dr. Norma Bautista-Lopez, Victoria Muise, Kathy Keller, Linhua Zhang, Mike Massé, Shirley Mongeau and Serghei Dernovici. Page 4 of 110 I offer my thanks to Kyle Roux for graciously providing us with the pcDNA3.1 mycBioID and pcDNA3.1 MCS-BirA(R118G)-HA vectors. I also would like to thank Chris Hicks for providing me with technical support and ensuring I did not lose my data when my computer crashed several times. My deepest gratitude to my best friend, Caroline Estrada (M.Sc.), for illustrating the diagrams of our fusion protein. She has been my rock throughout my academic career, and I am honoured to have her in my life. I want to thank the rest of my amazing friends outside of the university who provided stability and encouragement throughout my degree. I extend my thanks to my college biology teachers: Dr. Shireef Darwish for helping me realize my passion for molecular biology, and Dr. J. P. Parkhill for offering the advice of never giving up in the pursuit of higher education despite the hurdles you may face. Finally, I would like to offer my most sincere gratitude to my amazing parents, Maria Scordilis and Kenny Lee, who have influenced the woman and scientist I am today. Without them I would have not been encouraged to push myself to my limits to strive for success. They have taught me to fight against all odds, never give up on my dreams and stay true to myself. Page 5 of 110 CONTRIBUTIONS TO ORIGINAL KNOWLEDGE The current knowledge on these transporters is limited to studies demonstrating knocking down or amplifying expression of proteins and pathways to alter expression of ABCG1 or ABCG2. Many studies try to explore resolution techniques involving using inhibitors of these ABCG transporters that have low toxicity, however, most have not been approved for use in human trials. Understanding possible differences in the interactions between these transporters in healthy versus diseased states, such as cancer, could better isolate therapies that target solely the transporters involved in disease and not hinder their native functions in healthy tissues. In this thesis we provide a list of proteins hypothesized to interact and therefore take part in a mechanism or pathway involved in mitigating ABCG1 and ABCG2 expression and activity. CONTRIBUTION OF AUTHORS The experiments conducted for the project of this thesis were carried out under the oversight of Professor Elias Georges, who supervised the experimental design and is involved in the editing of this thesis and any subsequent manuscripts. The construction of vectors with the N- terminal
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