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Substrate Specificity of Human N-Acetyltransferase 8 for Aromatic Cysteine Conjugates

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Substrate Specificity of Human N-Acetyltransferase 8 for Aromatic Cysteine Conjugates Substrate Specificity of Human N-Acetyltransferase 8 for Aromatic Cysteine Conjugates by Reema Deol A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology (Toxicology) Guelph, Ontario, Canada © Reema Deol, August, 2015 ABSTRACT SUBSTRATE SPECIFICITY OF HUMAN N-ACETYLTRANSFERASE 8 FOR AROMATIC CYSTEINE CONJUGATES Reema Deol Advisor: University of Guelph, 2015 Professor P. David Josephy The mercapturic acid pathway is a metabolic route for the detoxication of xenobiotics, processing drugs and toxicants to mercapturic acids (N-acetylcysteine conjugates). An N- acetyltransferase enzyme, NAT8, catalyzes the transfer of an acetyl group from acetyl-CoA to the cysteine amino group, producing a mercapturic acid, which is excreted in the urine. Human NAT8 was recently cloned and expressed but little is known concerning its activity and specificity with regard to substrates of toxicological importance. I expressed human NAT8 in HEK 293T cells and used colorimetric assays with DTNB and HPLC analysis (UV or MS detection) to measure the activity of NAT8 for various cysteine substrates. All of the S-aryl- substituted (benzyl, 4-nitrobenzyl, and menaphthyl) cysteine conjugates tested are substrates for NAT8-catalyzed acetylation, with the exception of S-trityl-cysteine. This information provides insight into the final step in the glutathione-dependent biotransformation of toxicologically significant xenobiotics, such as polycyclic aromatic hydrocarbons. STATEMENT OF WORK PERFORMED All of the work presented in this thesis was completed by me, except for the following: Dr. Maria Veiga-Da-Cunha, Universite de Louvain, Belgium, cloned NAT8 DNA into expression vector, pEF6-HisB. The pEF6-NAT8 expression plasmid was provided to us by Dr. Veiga-Da-Cunha and used to express NAT8 in HEK 293T cells. I performed all NAT8 reactions and prepared samples for analysis via LC-MS. The LC- MS method (see Chapter 2.8) was developed by Dr. Dyanne Brewer at the Advanced Analysis Centre, Mass Spectrometry facility. Samples were separated and the desired ions were detected using LC-MS by Dr. Dyanne Brewer and Dr. Armen Charchoglyan. They also determined the area under the curve for specific ions. I used the provided data (area of substrates) to perform all calculations for enzyme kinetics. Undergraduate research project students assisted with several tasks. Rachael McNeilly assisted with the initial expression of NAT8 in HEK 293T cells and preliminary detection of NAT8 using western blot analysis. Tena Watts helped test specificity of NAT8 for cysteine conjugates using a colorimetric DTNB assay. Lastly, I worked closely with Caio Gomes de Barros Martins to recrystallize and improve purity of both menaphthyl-cysteine and its corresponding mercapturic acid. iii ACKNOWLEDGMENTS I am very grateful to my advisor, Dr. P David Josephy for providing me with the opportunity to work in his lab. He offered invaluable knowledge, patience, and support throughout my graduate studies at the University of Guelph. I am extremely appreciative for being able to attend not only several conferences but also many lab fun-filled activities. I would also like to thank my advisory committee members, Dr. Gordon Kirby and Dr. Richard Manderville for their unwavering guidance. A special thanks to Dr. Armen Charchoglyan and Dr. Dyanne Brewer for their expert support with LC-MS. I wish to thank all MCB faculty, specifically, Dr. Marc Coppolino and Dr. Nina Jones for their advice and guidance regarding tissue culturing techniques. I am extremely thankful to all the current and past members of the Josephy Lab for making this a wonderful experience. I am indebted to Kajaparan Jeyanthan, not only for his friendship but also for being a mentor throughout my studies. I wish to thank Hilary Groom, Caio Martins, and Rachael McNeilly for their constant support and friendship. Pam Loughran, Joban Dhanoa, Tena Watts, Meghan Lambie, Naomi Dickson, and Quan Ly I thank for their amity and creating a joyful lab. I would also like to thank my fellow colleagues, Maria Anillo, David Martynowicz, Charles Wrobleski, Evan Mallete, Matiyo Ojehomon, Haidun Liu, and Brianna Guild for their support and comic relief throughout this journey. Lastly, to my parents, family, and friends, I could not have done this without you all. Thank you for being my rock of support and providing me with unfaltering encouragement and motivation to persevere. iv Table of Contents ABSTRACT .................................................................................................................................... ii STATEMENT OF WORK PERFORMED ................................................................................... iii ACKNOWLEDGMENTS ............................................................................................................. iv LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix ABBREVIATIONS ....................................................................................................................... xi Chapter 1: Introduction ................................................................................................................... 1 1.1 Bioactivation and Detoxication ........................................................................................ 1 1.2 Cysteine Conjugates ......................................................................................................... 2 1.2.1 Metabolism of Cysteine Conjugates .............................................................................. 2 1.2.2 In-Vivo Acetylation of Cysteine Conjugates .................................................................. 6 1.2.3 Bioactivation of Cysteine Conjugates ............................................................................ 7 1.3 N-Acetyltransferases ...................................................................................................... 11 1.3.1 Aspartate N-Acetyltransferase (NAT8L) ..................................................................... 16 1.3.2 Aspartate N-Acetyltransferase (NAT8L) Structure ..................................................... 18 1.3.3 NAT8L Mechanism ..................................................................................................... 21 1.3.4 NAT8L Kinetics........................................................................................................... 23 1.4 N-Acetyltransferase 8 (NAT8) ....................................................................................... 24 1.4.1 Cloning of NAT8 ......................................................................................................... 24 v 1.4.2 NAT8 Topology ........................................................................................................... 25 1.4.3 Purification of NAT8 ................................................................................................... 28 1.4.4 NAT8 Enzyme Activity ............................................................................................... 29 1.4.5 Identification of NAT8 ................................................................................................ 33 1.4.6 Single Nucleotide Polymorphisms and Genome-Wide Association Studies ............... 35 1.5 Research Objectives ....................................................................................................... 38 Chapter 2: Materials and Methods ................................................................................................ 41 2.1 Chemicals ............................................................................................................................ 41 2.2 Synthesis of Cysteine Conjugates and Mercapturic acids .................................................. 41 2.2.1 Menaphthyl-cysteine .................................................................................................... 41 2.2.2 N-Acetyl-(S-menaphthyl)-L-cysteine .......................................................................... 42 2.2.3. 4-S-Nitrobenzyl-L-cysteine and N-Acetyl-(4-nitro-S-benzyl)-L-cysteine .................. 42 2.3 Preparation of Stock Solutions............................................................................................ 42 2.4 Plasmid DNA Extraction .................................................................................................... 43 2.5 HEK 293T Expression ........................................................................................................ 44 2.6 Colorimetric Enzyme Assays .............................................................................................. 45 2.7 NAT8 HPLC Activity Assay .............................................................................................. 47 2.8 NAT8 Activity LC-MS Analysis ........................................................................................ 50 Chapter 3: Results ......................................................................................................................... 56 3.1 Synthesis of Menaphthyl-Cys and its Mercapturic Acid .................................................... 56 vi 3.2 Plasmid DNA Extraction ...................................................................................................
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