Copyright 2015 by Jessica L. Friedman ii Abstract ATP-binding cassette (ABC) transporters are a diverse family of transmembrane proteins that facilitate the ATP powered translocation of substrates across cellular membranes. Multidrug resistance-associated protein 4 (MRP4) is a highly polymorphic, human ABC transporter with a wide array of substrates, including, antiviral drugs and prostaglandins. Previously, altered antiviral drug transport capacities of nonsynonymous single nucleotide polymorphic MRP4 variants, relative to reference MRP4 transport activity, were reported. This project focused on characterizing the ATP hydrolysis capabilities of the G487E MRP4 variant (rs11568668); G487 is located in an ATP binding cassette, also known as the nucleotide binding domain, of MRP4. Previously published works report a 0.008 minor allele frequency in Asian American individuals (n = 120) and show decreased transport activity with G487E. Novel data on G487E MRP4 activity from this study corroborate the decreased drug transport findings. This study characterized prostaglandin E2 – stimulated ATP hydrolysis by human MRP4 reference and G487E variant expressed in insect cells. MRP4 specific activity was determined by its selective inhibition with beryllium fluoride. Reference MRP4 reached a maximal ATP hydrolysis velocity of 27.4 ± 1.07 nmoles Pi/mg protein/minute; for comparison, ATP hydrolysis in Sf21 membranes absent of exogenous transporters reached a maximum velocity of 18.4 ± 0.55 nmoles Pi/mg protein/minute. The G487E MRP4 variant had an intermediate Vmax value of 24.4 ± 0.93 nmoles Pi/mg protein/minute. As measured by an ANOVA, Vmax values for the Sf21, MRP4 reference and G487E MRP4 membranes were significantly different (p < 0.0001). In contrast, Km values were similar for the three sample types and ranged from 0.34 ± 0.16 µM for reference MRP4 to 0.44 ± 0.18 µM for G487E MRP4. Structural rationales for these data iii were explored using novel homology models of the first nucleotide binding domain of MRP4. The G487E mutation was determined to only affect the MRP4 binding affinity of ATP; prostaglandin E2 binding was not impacted. These results suggest that the reduced transport phenotype of G487E MRP4 relative to reference MRP4 is due, at least in part, to impaired ATP hydrolysis. Acknowledgements This document marks the end of an incredible journey in academic research and the beginning of an exciting new career in pharmaceutical consulting. I want to thank my friends, family, and mentors who enhanced this experience and helped make this transition possible. To my thesis committee, thank you for your support in my decision to leave UCSF a few years earlier than originally planned. Deanna Kroetz, thank you for being an incredibly supportive PI and friend. Despite my career interests being far outside of your expertise, you facilitated the series of informational interviews that led me to health care consulting and gave me the time to explore and enter this career path. Thank you for your encouragement through this entire project; I am thrilled that I could leave the lab with detailed instructions for the successful execution of MRP4 ATPase assays and preliminary MRP4 variant ATPase activity data. Jaime Fraser, thank you for your continual support through my UCSF journey, from my recruitment in 2013 to my Masters thesis submission in 2015. Kathy Giacomini, thank you for your insight into career transitions in non-research science careers and your feedback on my Masters thesis. To my PSPG cohort, thank you for being involved in every step of this journey – becoming a California resident, balancing fellowship applications, rotations, project proposals, with journal clubs and 220 presentations, surviving TAing pharmacy students, and solidifying our next career steps. iv To my friends and family, thank you for always being a phone call, text, or video call away – moving 3,000 miles did not feel so far because you were always so close. Grandpa, thank you for inspiring my resilience and determination. Dad, thank you for trying to give advice even when my questions were beyond your expertise and your unwavering confidence in me. The work described in Chapter 2 was performed with guidance from published work: Sauna ZE, Nandigama K, Ambudkar S V. Multidrug resistance protein 4 (ABCC4)-mediated ATP hydrolysis: Effect of transport substrates and characterization of the post-hydrolysis transition state. J Biol Chem. 2004;279(47):48855-48864. doi:10.1074/jbc.M408849200. I appreciate the helpful advice of the Ambudkar laboratory at the National Cancer Institute while setting up this assay. Finally, I would like to thank everyone in the Kroetz and Giacomini labs for their support in and out of the laboratory. Without you, I would still be looking for the MilliQ water (hidden in the fish room). v Table of Contents Abstract ......................................................................................................................................... iii Acknowledgements ...................................................................................................................... iv List of Tables .............................................................................................................................. viii List of Figures ............................................................................................................................... ix Chapter 1 : Introduction .............................................................................................................. 1 ABC Transporters .................................................................................................................................. 1 Human ABC Transporters .................................................................................................................... 1 Functional Characteristics .................................................................................................................... 5 ABC Transporter Associated Diseases................................................................................................. 6 Multidrug Resistance-Associated Protein 4 (MRP4) ........................................................................... 8 Tissue Distribution ............................................................................................................................... 8 Interaction with Drugs .......................................................................................................................... 8 ATP Binding and Hydrolysis ............................................................................................................. 11 MRP4 Homology Modeling ............................................................................................................... 13 ABCC4 Genetic Variants .................................................................................................................... 15 Goals and Rationale for Project.......................................................................................................... 19 References ............................................................................................................................................. 21 Chapter 2 : Effect of MRP4 G487E Variant on ATPase Activity .......................................... 26 Materials and Methods ........................................................................................................................ 26 Sf21 Cell Culture ................................................................................................................................ 26 Sf21 Cell Infection and Harvest ......................................................................................................... 27 Membrane Isolation............................................................................................................................ 27 vi Western Blotting ................................................................................................................................ 29 ATPase Activity Assay ...................................................................................................................... 30 Homology Template Search and Selection. ....................................................................................... 32 Model Building and Quality Assessment ........................................................................................... 32 Results .................................................................................................................................................... 33 Assay Development............................................................................................................................ 33 MRP4 Specific ATPase Activity ........................................................................................................ 35 Substrate-Dependent MRP4 ATPase Activity ................................................................................... 37 Proposed Reaction Mechanism .......................................................................................................... 39 Structural Observations ...................................................................................................................... 40 Discussion .............................................................................................................................................. 46 Assay Optimization