Effects of Naturally-Occurring Polymorphisms on the Recombinant Expression Levels and Enzymatic Activity of the Rhesus Monkey Arylamine N-Acetyltransferase 1 (NAT1)”

DEMOCRITUS UNIVERSITY OF THRACE

SCHOOL OF HEALTH SCIENCES

DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS

Undergraduate Research Project

titled:

“Effects of naturally-occurring polymorphisms on the recombinant expression levels and enzymatic activity of the Rhesus monkey arylamine N-acetyltransferase 1 (NAT1)”

By:

Ioanna Stefani

-1103-

Supervised by: Assistant Professor Giannoulis Fakis, D. Phil

Alexandroupolis

October, 2015

ΔΗΜΟΚΡΙΤΕΙΟ ΠΑΝΕΠΙΣΤΗΜΙΟ ΘΡΑΚΗΣ

ΣΧΟΛΗ ΕΠΙΣΤΗΜΩΝ ΥΓΕΙΑΣ

ΤΜΗΜΑ ΜΟΡΙΑΚΗΣ ΒΙΟΛΟΓΙΑΣ ΚΑΙ ΓΕΝΕΤΙΚΗΣ

Διπλωματική Εργασία

με τίτλο:

«Επιπτώσεις φυσικών πολυμορφισμών στα επίπεδα έκφρασης και στην ενζυμική ενεργότητα της Ν-ακετυλοτρανσφεράσης των αρυλαμινών 1 (ΝΑΤ1) του πιθήκου Ρέζους»

Της φοιτήτριας:

Ιωάννα Στεφανή

-1103-

Επιβλέπων: Επίκουρος Καθηγητής Γιαννούλης Φακής, D. Phil

Αλεξανδρούπολη

Οκτώβριος, 2015

Abstract

Arylamine N-acetyltransferases (NATs) are enzymes of the xenobiotic metabolism that play an important role in detoxifying chemicals. They catalyze the transfer of an acetyl group from the AcCoA to an acceptor, usually arylamines but also aryl hydrazines. The enzymes of the NAT family are present in many organisms and their function in detoxifying chemicals is linked with evolutionary advantage in overcoming the difficulties in the chemical environment. The human NAT1 enzymes have been widely studied and their polymorphic substitutions in the coding region have been linked to differences in the enzyme activity. In turn, this is linked to variable pharmacological response, e.g. different metabolizing rate of isoniazid a drug used for the treatment of tuberculosis. The polymorphisms of these enzymes and their effect on the activity have been also studied in model organisms, such as rodents The possible correlation with chemically induced endometriosis has led to the study of NAT enzymes in Non-Human Primate (NHPs) models. The NAT1 and NAT2 polymorphisms in a Rhesus macaque population have been identified and investigated for their impact on the enzymatic activity but also on the protein stability.

In the present study, two naturally occurring single nucleotide polymorphisms (SNPs) of the (MACMU)NAT1 that cause non-synonymous changes in the amino acid sequence [p.Leu89Phe (L89F) and p.Asp115Tyr (D115Y)] were inserted in the reference sequence (Wt). The four variants (reference protein wt, one carrying the L89F polymorphism, one carrying the D115Y polymorphism and one carrying both L89F and D11Y) were expressed in E. coli BL21 cells and purified through affinity chromatography (IMAC). The effects of the non-synonymous substitutions on the enzymatic activity were studied with DTNB enzymatic activity assays. In addition, a total of 9 NAT1 protein variants (7 carrying one SNP, 1 carrying two SNPs and reference (MACMU)NAT1 protein) have been investigated through immunoblotting for their presence in the soluble and the insoluble fraction of the bacterial whole cell lysates.

The study of these NAT1 variants showed that the alterations in the amino acid chain can affect the enzymatic activity. The presence of the D115Y or the L89F substitutions reduced the enzymatic activity, while the NAT1 variant that carries both D11Y and L89F substitutions demonstrated almost no activity. In addition, the immunoblotting experiments showed that the polymorphisms in the amino acid sequence may affect the recombinant expression of the protein variants from E. coli BL21 cells. In some cases both the enzymatic activity and the expression of the polymorphisms are reduced, where in other cases the enzymatic activity and expression may not be similarly affected.

There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved.

Charles Darwin, On the Origin of Species, First Edition, 24 November 1859

Acknowledgments

I will take advantage of this chance to express my gratitude to my Supervisor Assistant Professor Dr Giannoulis Fakis for the opportunity he gave me to be part of the Laboratory’s team and whose continuous advice and supervision the past two years have been priceless in the scientific, academic and career aspects of my life. In addition, I would also like to express my heartfelt thanks to Assistant Professor Dr Sotiria Boukouvala for her priceless scientific advice and problem solving help during my training at the Laboratory and my undergraduate thesis. Moreover, I would like to thank the students of the laboratory Theodora Tsirka, Marinakis Nikos and Rizou Sofia for their help and guidance during the early training of me at the laboratory. In addition, I would like to thank the students Kanellopoulou Ioanna, Karamanou Georgia, Kyrizaki Athina and Olbasalis Giannis for the special moments we have had together at the lab. Moreover, I would like to thank the PhD student Angelou Eftychia for her significant advice and help in casual laboratory arising problems. Furthermore, I would like to express my deepest thanks to the PhD student Garefallaki Vasiliki for her continuous and priceless guidance during my project, the patience she had during my training, the laboratory experience she passed me, the priceless scientific, career, life and even language advice she provided me and the opportunity she gave me to be a part of the SymBioSE 2015. Additionally, I would like to thank the undergraduate student Drakomathioulaki Nafsika and Giannouri Despina for their important scientific, laboratory and career advice. I also want to express my thanks to my classmate and laboratory companion Savvidou Olga for all her help, understanding and patience that taught me so much. Last, but not least, I would like to express my whole-hearted thanks to the MSc student Bouraki Genovefa and the undergraduate student Konstantopoulou Maria for their invaluable help and understanding in the laboratory and especially everyday life, from the moving to the paperwork. To conclude and taken the opportunity, I would like to thank my family for their continuous love and support, emotional and financial, during my undergraduate and graduate studies. Without their support and understanding, nothing would be possible.

Presentations arising from this undergraduate thesis

Part of the results of this undergraduate thesis was presented at:

A) 18th Symposium of Biology Students in Europe, Alexandroupolis, 23-31 of July 2015

Title: Effects of naturally-occurring polymorphisms on the function of NAT1 enzyme of the primate Macaca mulatta (Rhesus monkey).

Authors: Stefani Ioanna1, Marinakis Nikolaos1*, Giannouri Despoina1*, Rizou Sophia1*, Tsirka Theodora1, Sabbagh Audrey2, Boukouvala Sotiria1, Fakis Giannoulis1, 1Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece, 2Institut de Recherche pour le Développement (IRP-UMR216), Université Paris Descartes, France,*Equal contribution

B) 66th Congress of the Hellenic Society of Biochemistry and Molecular Biology, Athens, 11-13 of December 2015

Title: Effects of naturally-occurring polymorphisms on the recombinant expression levels and the function of NAT1 enzyme of the primate Macaca mulatta (Rhesus monkey).

Authors: Ioanna Stefani, Sotiria Boukouvala, Giannoulis Fakis, Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece

0. TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION……………………………………...1 1.1 Primates………………………………………………………………………...2 1.1.1 Rhesus Macaque (Macaca mulatta)…………………………...... 3 1.2 Xenobiotic Metabolism…………………………………………………………4 1.3 Arylamine N-acetyltransferase………………………………………………….5 1.3.1 Arylamine N-acetyltransferase enzymatic activity…………………6 1.3.2 Arylamine N-acetyltransferases (NATs) tertiary structure……………7 1.3.3 Arylamine N-acetyltransferase substrates……………………………..8 1.3.4 NAT inhibitors……………………………………………………10 1.4 NATs in prokaryotes…………………………………………………………11 1.5 NATs in eukaryotes………………………………………………………….14 1.5.1 NATs in fungi………………………………………………………14 1.5.2 NATs in animals……………………………………………………17 1.5.3 NATs in human………………………………………………………20 1.5.4 NAT polymorphisms and disease…………………………………21 1.6. Aims of the present study………………………………………………………..22

CHAPTER 2: MATERIALS AND METHODS…………………..…23

2.1. Quantification of samples using Spectrophotometer……………………………24 2.2. Quantification of total protein concentration in bacterial lysates with Bradford Assay…………………………………………………………………………………25 2.3. Isolation of plasmid DNA from Bacterial cell cultures…………………………26 2.4. Transformation of E. coli competent cells………………………………………27 2.5. Nucleic acid amplification with the Polymerase Chain Reaction………………28 2.6. Agarose gel electrophoresis……………………………………………………30 2.7. DNA Sequencing………………………………………………………………...31 2.8. Recombinant expression of (MACMU)NAT1 proteins………………………....31 2.9. Purification of recombinant (MACMU)NAT1 protein with affinity chromatography……………………………………………………………………....33 2.10. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).35 2.11. Enzymatic activity assays of the recombinant (MACMU)NAT1 proteins….....38 2.12. Protein Analysis by Immunoblotting (Western Blot)………………………….40

CHAPTER 3: RESULTS……………………………………………...45

3.1. PCR Screening of transformed E. coli JM109 cells for the presence of the (MACMU)NAT1 construct in the plasmid…………………………………………..46 3.2. Sequencing of the inserted construct in the pET28b+ plasmid isolated from the transformed E. coli JM109 cultures………………………………………………….46 3.3. PCR screening of transformed E. coli BL21 cells for the presence of the (MACMU)NAT1 construct in the plasmid………………………………………….47

iii

3.4. Recombinant expression and purification of the (MACMU)NAT1 Wt, L89F, D115Y and L89F+D115Y protein variants……………………………………….48 3.5. Enzymatic activity of the recombinant (MACMU)NAT1 protein variants (L89F, D115Y and L89F+D115Y) from Macaca mulatta…………………………………..50 3.5.1. Standard Curves…….…………………………………………………..50 3.5.2. Enzymatic activity assays………………………………………………53 3.6. Detection and study of the expression profile of the recombinant polymorphic NAT1 proteins from Macaca mulatta by immunoblotting…………………………..55

CHAPTER 4: CONCLUSIONS AND DISCUSSION……………….71

CHAPTER 5: BIBLIOGRAPHY……………………………………..75

CHAPTER 1 INTRODUCTION

[1]

1.1 Primates

The order Primates is a part of the class Mammalia of the Phylum Chordota of the Kingdom Animalia. The first major branch of the Order Primates is taxonomically divided into two Suborders: Prosimii and Anthropoidea. This distinction is made on a number of anatomical and behavioural features however the most obvious ones are found in the skull. There are 4 infaorders, Platyrrhini and Catarrhini of the Anthropoidea suborder and Lemuriformes, Lorisiformes and Tarsiiformes of the Prosimii suborder. The four infaorders are further classified in 13 families: Lemuridae, Cheirogaleidae, Indridae, Daubentoniidae, Galagonidae, Loridae, Megaladapidae, Tarsiidae, Cebidae, Cercopithecidae, Callitrichidae, Hylobatidae, Hominidae, that consist of 279 species (Ref.Text-1) (Figure 1.1). From another perspective, the order is split into Strepsirhini and Haplorhini with the Tarsiiformes moved from the other prosimians and grouped with the anthropoids (Sellers, 2000).

Figure 1.1: Traditional Primate Taxonomy. http://www.yorku.ca/kdenning/+2140%202005-6/2140-8Nov2005.htm

The primates share important characteristics, like fingers with the ability to grasp on all four limbs, flat nails instead of sharp claws and stereoscopic vision and distance perception. The primates are grouped in three main groups: (1) The New World

[2]

Primates inhabit mainly in Central and South America, (2) The Old World Primates and the (3) Anthropoids are native to Africa and Asia. (Figure 1.2)

Figure 1.2: Natural range of non-human primates http://anthro.palomar.edu/primate/prim_1.html

The old world apes of the Haplorrhini suborder belong to the Cercopithecidae family which consists of 96 species, including the Mandril (Mandrillus), Baboons (Papio), and Macaque (Macaca) and Surili (Presbytis). (RefText-1)

1.1.1 Rhesus Macaque (Macaca mulatta)

Although Rhesus Macaque (Macaca mulatta) is thought to have diverged from the common ancestor with human about 25 million years ago, it is the most widely used non-human primate in basic and applied biomedical research (The Rhesus Macaque Genome Sequencing and Analysis Consortium- Richard A. Gibbs, 2007). Their anatomical, genetic and physiological similarities with human set them among the most appropriate animal research models.

In nature, Rhesus monkeys have broad geographic distribution that reaches from Afghanistan to India and northern Thailand. According to McDonnel Genome Institute website (http://genome.wustl.edu/genomes/detail/macaca-mulatta/), the species was also abundant in southern China and Tibet, until 60 years ago when humans led to the extinction of these populations.

The rhesus macaque (Macaca mulatta) is a widely used small primate model of human disease, development, and behavior with the most studies to have principally used this primate as a NHP model for infectious diseases, pharmacology, and neuroscience research (Carlsson et al., 2004). They have also similar reproductive system with humans with cycle of 29 days making them suitable Non-Human-Primate (NHP) for studies of the disorders associated with the reproductive system, such as endometriosis (Fakis et al., 2007).

[3]

1.2 Xenobiotic Metabolism

The term xenobiotic metabolism describes as a whole the metabolic pathways that modify a compound which is foreign to the organism.It is important in Biotransformation and Drug metabolism. As described by Williams in 1959, the xenobiotic metabolism reactions can be classified in phase I and phase II reactions. Later, phase III was also suggested (Omiecinski et al., 2010).

Phase I includes all the reactions that introduce a –OH, -COOH, -NH2 or –SH group to the initial compound. These reactions include Hydrolysis, Oxidation and Reduction. Initially, the phase I reactions were characterized by the monooxygenation function which is catalyzed by the cytochrome P450s. Further studies of the CYP enzyme family have described a great amount of xenobiotic chemicals that are being detoxified with N- and O- dealkylation, aliphatic and aromatic hydroxylation, N- and S- oxidation and deamination reactions.

Phase II consists of all conjugation reactions that are related to the conjugation of a highly hydrophilic group (sulfate, glucuronic acid, mercapturic acid, methyl, acetyl) to the initial compound. Increasing the hydrophilicity promotes the xenobiotic’s excretion, typically through urine. These conjugation reactions are mostly catalyzed by transferases. Examples of enzyme families that catalyze Phase II reactions of xenobiotic metabolism are demonstrated in Table 1.

Table 1: Examples of enzyme families that catalyze Phase II reactions.

Enzyme family Symbol UDP glucuronosyltransferases UGTs sulfotransferases STs N-acetyltransferases (arylamine N- NATs acetytransferase) glutathione S-transferases GSTs methyltransferases (DNAMTs, OMTs, SMTs)

The last group and newly proposed term, Phase III reactions, consists of the reactions that are carried out by active membrane transporters for the transmission of drugs and other xenobiotics through the cellular membranes. The main enzyme described by thiw term is the P-glycoprotein (ABC: ATP- Binding cassette) that is of great interest for its function in the resistance of cancer cells to chemotherapeutics. Lately, organic anion transporters and organic cation transporters have been also included to the Phase III reactions (Omiecinski et al., 2010).

[4]

1.3 Arylamine N-acetyltransferase

Arylamine N-acetyltransferase is the enzymatic activity which was initially described by Fritz Lipmann in 1951 as the activity responsible for the N- acetylation of arylamines. In his work, he proposed that these enzymes, that were isolated from pigeon liver extracts, should be named “acetokinases” in similarity to with the phosphokinases (Lipmann et al., 1951).

The arylamine N-acetyltranferases (NATs) are phase II enzymes of the xenobiotic metabolism and have been classified by the International Union of Biochemistry and Molecular Biology (IUBMB) as the enzymes responsible for three distinct enzymatic activities. Primarily, they catalyze the conjugation of an acetyl group to the amino group of aromatic amines (EC 2.3.1.5) as demonstrated in Figure 1.3. Additionally, the NATs are also found to have N-hydroxyarylamine O-acetyltransferase activity (EC 2.3.1.118) as well as aromatic-hydroxylamine O-acetyltransferase (EC 2.3.1.56).

Figure 1.3: AcCoA dependent N-acetylation of an arylamine. (EC 2.3.1.5 Arylamine N- acetyltransferase). http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/enzymes/GetPage.pl?ec_number=2.3.1.5

The enzymes of the NAT family have been extensively studied for their role in the metabolism of drugs and environmental toxins. They are soluble proteins of similar sizes, around 30-34kDa. They possess an important role in the detoxification of potentially toxic exogenous compounds but they are also considered to be responsible for the bioactivation of chemicals to carcinogenic compounds. Their presence in a broad range of organisms, from prokaryotes to eukaryotes, demonstrates their important role in the interaction between the organisms and the chemicals in their environment.

Because of their function in metabolizing carcinogenic and toxic compounds, the NAT isoenzymes have been broadly studied for their association with cancer and several other diseases, including infectious (McDonagh et al., 2014). In addition, given their broad presence in nature, NATs their role has been examined through comparative and evolutionary studies (Karagianni et al., 2015; Sabbagh et al., 2013).

[5]

1.3.1 Arylamine N- acetyltransferase enzymatic activity

The arylamine N- acetyltransferase isoenzymes catalyze the transfer of an acetyl group from an acetyl donor –mainly AcCoA, although recent studies have characterized other coenzymes as acyl donors (Karagianni et al., 2015) - to an arylamine –acetyl acceptor. Site directed mutagenesis and enzyme kinetic studies of mammalian, avian and rabbit NATs with cysteine inhibitors and specific labeling have introduced the Cys68 to be the catalytic residue (Sim et al., 2012). Later studies, including the solving of the crystal structures, identified a cysteine-histidine-aspartate catalytic triad that is conserved in the N-terminus of the protein (Sinclair et al., 2000; Butcher et al. 2002; Wu et al., 2007; Sim et al., 2012). In human NAT isoenzymes, the catalytic triad consists of the residues Cys68, His107 and Asp122.

Figure 1.4: Illustration of the conserved catalytic residues of the active site (of the HUMAN NAT1 sequence) and the proposed catalytic mechanism with acetyl-CoA as acyl donor (Sim et al., 2012).

The catalytic reaction has been described to take place in two steps, through a Ping Pong Bi Bi mechanism. Like most of such partial exchange reactions, where the enzyme reacts with the substrate to form a product which dissociates before the second substrate enters to the active site of the enzyme (Lueck and Fromm, 1973), AcCoA and the arylamine enter the active site sequentially. The AcCoA binds to the side chain of the Cys68 in the active site, forming a cysteinyl thioester, an acetyl- enzyme intermediate. Following the release of CoA, the transfer of the acetyl group

[6] from the enzyme to the substrate takes place, forming the acetylated product that is then released from the enzyme (Boukouvala & Fakis, 2005; Minchin et al., 2007;).

As it has become apparent after solving the crystal structures of NAT enzymescrystal structures (Sinclair, et al.,, 2000; Wu et al., 2007), the acetyl acceptor (arylamine) binding site is overlapping with the CoA binging site and thus confirming the two step two reaction catalytic mechanism of the NAT isoenzymes (Zhou et al., 2013).

1.3.2 Arylamine N-acetyltransferases (NATs) tertiary structure

The bacterial N- acetyltransferase from Salmonella typhimurium was the first crystal structure of a NAT isoenzyme that was solved (Sinclair et al., 2000). The three dimensional structure of the folded peptide revealed a spherical molecule that is composed of three domains, around 80-90aa each, where the second and third are linked by an interdomain helix of 27aa. Domain I is a helical bundle and domain II a β- sheet barrel that connects through the helical interdomain to domain III, an α/β lid. The catalytic triad (Cys69, His107 and Asp122) is made up of the first two domains, together with a helical bundle and a beta-barrel (Sinclair et al., 2000; Boukouvala and Fakis, 2005). This structural motif was found to be similar to the cysteine proteases and transglutaminases suggesting that these enzyme families had a common protein ancestor (Sinclair et al., 2000).

Figure 1.5: Structural comparison of bacterial NAT (panel A) and human NAT (panel B). The structures of NAT from Salmonella typhimurium (PDB code: 1E2T) and human NAT1 (PDB code: 2PQT) are shown. The insertion in the human NAT is indicated in purple. The structures of the C-terminal residues in both enzymes are shown in red (Zhou et al., 2013). In 2007, Wu and his coworkers resolved the crystal structures of (HUMAN)NAT1 and (HUMAN)NAT2, a (HUMAN)NAT1 in complex with the irreversible inhibitor

[7]

2-bromoacetanilide, a (HUMAN)NAT1 mutant at the active site and a (HUMAN)NAT2 in complex with CoA. Α very significant finding was the existence of an insertion in the eukaryotic sequence which results to a different structure of the C-terminus of the enzyme. In addition, the substrate binding pocket of human NAT1 was found to be smaller (163Å) than that of NAT2 (257Å), as a result of two key residue substitutions, Arg127 and Tyr129 in NAT1 that have bulkier side chains compared to the serine residues of NAT2 at these positions. This finding can explain the difference in substrate specificity of the two isoenzymes (Wu et al., 2007; Zhou et al. 2013).

Likewise the prokaryotic structure, the crystal structure of the human NATs proposes a motiff, of three domains. The N-terminal domain (residues 1-83) consists mainly of five helices (α1-α5). The second domain consists of the residues 84-192 which form 9 β strands, interrupted by short helices and are connected through an α-helical interdomain to the third domain which consists of four anti-parallel β-strands and a helix. The 17-residue bracket (167-183) is created by the insertion and is only present in eukaryotic structures (Wu et al., 2007) –Figure 1.5.

Up to this date, more NAT crystal structures are available contributing to the understanding of the distinct substrate selectivity of the enzymes but also to the design of selective inhibitors for therapeutic purposes. Comparison of the structures has showed differences in the CoA-binding site between the prokaryotic and the eukaryotic NAT. This finding could lead to design of novel targets that could selectively bind to the prokaryotic NAT enzyme (Zhou et al., 2013).

1.3.3 Arylamine N-acetyltransferase substrates

The enzymes of the NAT family catalyze the acetylation of a variety of toxic compounds, mainly arylamines, arylhydrazines, as they are implicated in the detoxification of xenobiotics and therapeutic drugs whereas they are also responsible for the bioactivation of carcinogens.

Throught various studies over the past years, NATs have been associated with the metabolism of several drugs including: anti-microbial agents, e.g. isoniazid, sulfamethaxazole, dapsone; anti- arrhythmic and hematology agents, e.g. procainamide, hydralazine; anti-inflammatory, e.g. 5-aminosalicyl acid, sulfasalazine, caffeine (Figure 1.6.A). In addition, they also acetylate industrial and environmental carcinogens, for instance benzidine and 2- aminofluorene (Boukouvala and Fakis, 2005; McDonagh et al. 2014). Recent studies underline their role in the metabolism of toxic products of the secondary metabolism such as benzoxalinones, which are produced by the plants for defense (Karagianni et al. 2015).

[8]

Figure 5: A. Chemical structure of common NAT substrates. a) isoniazid, b) hydralazine, c) phenelzine, d) p-aminobenzoic acid, e) p-aminosalicylic acid, f) 5- aminosalicylic acid, g) procainamide, h) sulphamethazine, i) sulphamethoxazole, j) dapsone. k) 2- aminofluorene, l) benzidine, m) 2-naphthylamine, n) 4-aminobiphenyl, o) 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (Boukouvala and Fakis 2005). B. Chemical structures of arylamine and arylhydrazine compounds tested as substrates in a high throughput analysis of eukaryotic NATs (Kawamura et al., 2005).

Despite their high conservation between species and their high amino acid similarity, the different NAT isoenzymes show distinct substrate specificity (Kawamura et al. 2005). It was initially observed, after studies with 5 -aminosalicyl and sulphamethazine, that the human liver possessed two distinct catalytic activities (Jenne, 1965). Later studies found that the two human isoenzymes were both catalyzing the acetylation of arylamines, but with separate substrate specificities, where NAT1 selectively acetylates smaller substrates, such as 5-aminosalicyl, p- aminobenzoic acid and p-aminobenzoylglutamate while NAT2 acetylates bulkier arylamines and arylhydrazines, like sulphamethazine, isoniazid, dapsone and procainamide (reviewed by Sim et al. 2014). The substrate specificity profile of the eukaryotic enzymes has been extensively studied and it has been mainly used for their classification of the enzymes of the NAT family (Kawamura et al. 2005) (Figure 1.6.B). The difference in substrate specificity was explained after the solving of the crystal structure of the human isoenzymes by Wu and his colleagues in 2007, which revealed the differences of the volumes of the binding site that this substrate selectivity was explained (Figure 1.7).

[9]

Figure 1.7: Substrate binding pockets of NAT1 and NAT2. Schematic representation of pAS in the substrate binding pocket of NAT1 (D) and SMZ in NAT2 (F). pAS and SMZ are colored in red. The residues involved in substrate binding and specificity are labeled. Hydrogen bonds are drawn as dashed lines. The Van der Waals surfaces in the enzyme’s pocket are shown as green-cyan (NAT1) or cyan (NAT2) planes (Wu et al., 2007).

1.3.4 NAT inhibitors

The development of NAT inhibitors has been stated to be essential for many reasons. First, inhibitors were employed in the detection of the amino acids that form the active site, to provide information about the potential endogenous role of the NAT enzymes in the cell and also for the solving of the crystal structures. Newer studies are examining the potential inhibition of the enzyme by small molecules that could be utilized for therapeutic purposes, for instance the discovery of compounds that could selectively inhibit the prokaryotic NAT for the treatment of tuberculosis (Sim et al., 2012).

The screening of compounds is focused on small molecules that bind to the substrate binging site or the AcCoA binding site, although it must be noted that these sites are partially overlapping (Zhou et al., 2013). In addition, it has been suggested that besides the residues that form the active site, the development of inhibitors should be directed to the designing of molecules that bind with increased affinity to residues that stabilize the enzyme- inhibitor complex. Such example is the case of K100 for the human NAT1 that does not possess any catalytic role, but is found to be important for AcCoA binding to the enzyme and by extension to a possible inhibitor (Minchin et al., 2015).

[10]

To date, the screens have identified small molecules belonging to two chemical classes, the 2-aminoalcohols and 1,2,4-triazole classes, that could selectively inhibit bacterial NAT. Those compounds have been examined for potential anti-tubercular activity (Sim et al., 2012) and although they did not show strong inhibition activities, they lead to the discovery of analogs with more promising effects. Piperonidols have been investigated as potential antimycobacterial agents allowing the identification of the piperidinol scaffold as selective prokaryotic NAT inhibitor. The piperonidol derivatives demonstrated structural complementarity to the binding pocket of mycobacterial NAT that causes specific and covalent modification of the active site cysteine of NAT (Abuhammad et al., 2014).

Additionally, taking into consideration the association of NAT1 with breast cancer, screens for molecules that inhibit the human NAT1 have been carried out for the discovery of possible antitumor drugs. Among the molecules with inhibiting abilities rhodanine and naphthoquinone have been further examined. Rhodanine has showed strong inhibitory abilities as well as cytotoxicity suggesting its potential use as antitumor drug. On the other hand, naphthoquinone, which selectively binds to human NAT1, showed a promising potential application as biomarker because it possesses the property of changing color –from blue to red- after binding to human NAT1 (Sim et al., 2012).

The inhibition of NAT enzymes has been also examined in studies concerning the effects of nanomaterials on the human NATs. Stated the fact that several nanomaterials are present in a wide range of products for everyday use, the finding that an activity of detoxifying pollutants of NAT could be inhibited is very worrying. However, the nanoparticles have not so far shown a clear effect on the NAT activity, as findings suggest that different nanomaterials induce different responses ranging from substrate dependent inhibition to even enhancement of the activity (Deng et al., 2014).

1.4 NATs in prokaryotes

NAT enzyme homologues have been identified in many taxonomic groups, indicating the significant role they maintain for the adaptation of distinct organisms in the changing chemical environments. Beyond the Salmonella typhimurium NAT, which has contributed to significant discoveries in the field because of its application as a model for biochemical and structural studies (Sinclair et al., 2000), and mycobacterial NATs that have been widely studied for their clinical importance in tuberculosis (Abuhammad et al., 2014), these phylogenetic investigations are revealing the broad existence of NAT-like sequences in bacterial genomes (Vagena et al., 2008; Glenn et al., 2010).

Mining through public genome databases has identified NAT homologous sequences in a plethora of bacterial taxa. So far, NAT-like sequences have been recognized in

[11]

Actinobacteria, Bacteroidetes/ Chlorobi, Cyanobacteria, Firmicutes and Proteobacteria (Figure 1.8) (www.nat.mbg.duth.gr/Prokaryotic%20NATs_2013.htm). Noteworthy was the discovery of a NAT homologue in an archaeal species, the Halogeometricum borinquense (Glenn et al. 2010).

Figure 1.8: The phylogenetic tree from the analysis of 115 prokaryotic and eukaryotic NAT amino acid sequences. The rooted phylogram resulting from Bayesian Inference is presented along with associated posterior probabilities (P75%). Bolded branches indicate bootstrap support (P90%) from Neighbor-Joining and Maximum Parsimony analyses. The fungal and animal clades were condensed to conserve space. Protist NATs with phylogenetic affinity to the bacterial NATs are shown circled within the two boxed areas of bacteria (Glenn et al., 2010).

[12]

Figure 1.9: Reactions catalyzed by the enzymes RifF and NAT. The product of the RifF reaction (proansamycin X) is the direct precursor of the rifamycin W. The substrate used to illustrate the NAT reaction is the antitubercular drug isoniazid (INH).

Although these enzymes show sequence similarity and retain the NAT-like scaffold of a spherical protein with three domains and the same or similar catalytic triad, some studies have identified novel functions of the homologous enzymes, suggesting possible functional adaptivity cases (Sim et al., 2008). Of great interest is the occasion of the RifF enzymes that has the amide synthase activity and is responsible for the termination of the assembly of the polyketide backbone during the biosynthesis of Rifamycin. The sequence of the RifF protein from Amycolatopsis mediterranei, an actinomycete that produces the important antibiotic Rifamycin B, shows 26% identity and 40% homology with the proteins of the NAT family (Pompeo et al., 2002; Verma et al., 2011). Given the fact the RifF protein is structurally similar to the NATs, possesses highly conserved regions and catalyzes reactions that as chemically analogous (Figure 1.9), it is suggested that the NAT structure has evolved to permit a range of functions in different organisms (Pompeo et al., 2002; Sim et al., 2008).

Concerning the important application of isoniazid (INH), a typical NAT substrate, in the treatment of tuberculosis, the presence of NAT isoenzymes in Mycobacteria is of particular interest (Payton et al., 1999; Upton et al., 2001). This led to further investigations that produced the very interesting finding that mycobacterial NAT possesses an endogenous role in the cell. In-frame deletions of the Mycobacterium bovis BCG nat caused phenotypic changes on growth, cell morphology, cell wall lipid composition and also increased sensitivity of mycobacteria to intracellular killing by mouse macrophages. The NAT activity was associated with the biosynthesis of mycolic acids and their derivatives and proposed as a possible factor for maintaining the homeostasis of acetyl-CoA (Bhakta et al., 2004).

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1.5 NATs in eukaryotes

The enzymes of the NAT family are widely present and hold a very important role in the xenobiotic metabolism of eukaryotic organisms. Biochemical, genetic, and molecular approaches, and more recently surveys through genomic databases, have identified NAT enzymes in a range of single-cell and multicellular eukaryotes (Vagena et al., 2008; Glenn et al., 2010). Historically, the NAT enzymes of laboratory animals, such as the chicken, rabbit, mouse, rat and hamster, have been subjected to pharmacological and toxicological studies, because of their significant similarity with human NATs (Sim et al., 2012).

Comparative genomic studies in public databases have revealed the existence of NAT homologues in divergent taxonomic groups (Vagena et al., 2008; Glenn et al., 2010). NAT genes have been identified in Protista, Fungi and Animals, leading to the assumption that NATs must have originally appeared to an ancestral prokaryote and have evolved in the following prokaryotic and eukaryotic descendants (Vagena et al. 2008).

Interestingly, studies have not identified any NAT gene in the kingdom of plants, with two exceptions in the Citrus sinensis genome and the alga Chlamydomonas reinhardtii genomes that were divergent and therefore have been attributed to be possible bacterial contamination. These studies also confirmed the total absence of NAT genes in the genomes of the Canidae family members (Vagena et al., 2008; Glenn et al., 2010; Sabbagh et al. 2013). The absence of the NAT genes from these organisms is considered to have been caused by gene loss events early in the evolution in plants and after the separation of the canids from their ancestor (Glenn et al., 2010; Sabbagh et al. 2013).

NAT homologues are present, and usually more than one NAT gene per genome, in certain taxa of protists (Figure 1.10). A mixed phylogeny of NATs between bacteria and protists was indicated supporting the possibility of occurrence of horizontal gene transfer events (Glenn et al., 2010; Sim et al., 2012).

1.5.1 NATs in fungi

NAT genes have been so far found in three phyla of the Fungi Kingdom, the Microsporidia, Basidiomycota and Ascomycota (http://nat.mbg.duth.gr/ Eukaryotic%20NATs_2013.htm#_Table_2:_Distribution_and annotation). Of particular interest is the case of ascomycetes. In the species that infect animals or humans only one NAT gene was found whereas in the plant pathogenic fungi multiple NAT loci have been recognized. This may indicate a possible involvement of the NAT enzymes in the pathogenicity of plants (Glenn et al., 2010) – (Figure 1.10).

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The existence of several NAT isoenzymes in the plant pathogenic ascomyces is considered to be an evolutionary result that adds distinct functions in those fungi, rather than just a product of genetic redundancy (Karagianni et al., 2015). Initially, the role of NATs in plant pathogenic fungi was associated with the detoxification of benzoxanoids, toxic compounds that are secreted by the plants for defense against fungal infections. Besides the acetyl-dependent N- acetylation of arylamines, novel functions for detoxifying environmental pollutants have been revealed from recent investigations, proposing the possibility of that fungal NATs have evolved to perform diverse functions that add advantages to the pathogen fitness. They also suggest the categorization of the homologous isoenzymes of the plant associated fungi of the genera of Aspergillus and Fusarium, into three groups, depending on the function they are involved in. The first group consists of all the homologs that catalyze acetylation reactions using acetyl-CoA and propionyl-CoA as cofactors, the second group is formed by the isoenzymes of the plant pathogens that are active with malonyl-CoA and the third group contains the enzymes that have minimal activity with acetyl-CoA compounds.

The enzymes of the first and second group are of great agro-economic importance as they are responsible for the antimicrobials produced from cereal plants that are widely grown as crops. The resistant infection of crops by those microbes can cause major damage and therefore the detoxification mechanisms that add the survival advantage to the fungi are of significant interest. The tolerance of the pathogens from the Fusarium genus to an anti-fungal toxin produced by cereal plants, 2-benzoxazolinone (BOA), was discovered to be a result of an endogenous detoxification pathway implicating two genetic loci (FDB1 and FDB2) (Glenn et al., 2002, 2003). Later, the FDB2 locus was characterized as NAT (Glenn & Bacon, 2009).

Besides the agroeconomic importance of the NAT genes in fungi, the existence of multiple NAT genes identified per fungal genome generated the interest for investigation of their potential role, apart from the acetyl-dependent acetylation (Vagena et al., 2008; Glenn et al., 2010; Karagianni et al., 2015). The investigations have shown that the homologs of the third group retain the ability to non-selectively bind acetyl-, malonyl-, propionyl- and succinyl-CoA but their function is not well characterized yet. This, along with the presence of A. flavus NAT3 in a putative biosynthetic gene cluster, which is responsible for the production of a polyketide, and the occasion of the NAT-homolog RifF locus of the rifamycin biosynthetic gene cluster (Pompeo et al., 2002), strongly suggest a hypothesis that the multiple NAT- homologs per fungal genome may have a role in the secondary metabolism of the fungi (Karagianni et al., 2015).

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Figure 1.10: Phylogenetic analysis of 79 NAT amino acid sequences from fungi. The NATs of species typically pathogenic to humans or animals are circled (Glenn et al. 2010).

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1.5.2 NATs in animals

NAT sequences have been found in the genomes not only in the vertebrates, but also in the lower animal phyla, such as mollusks, worms and arthropods. Homologs of the NAT genes have been identified in the echinoderm Strongylocentrotus purpuratus, the hemichordate Saccoglossus kowalevskii, the urochordate Ciona intestinali and the cephalochordate Branchiostoma floridae (Glenn et al., 2010; Sim et al., 2012). However, no NAT-like sequence was found in the genomic sequence of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae (Boukouvala & Fakis, 2005). A recent study reports the existence of arylamine N- acetyltransferase activity in the blue secretion of the gastropod mollusk Telescopium telescopium, a “horn snail” that naturally inhabits in the Sundarban mangrove. An interesting finding was that the NAT like activity in the secretion was increasing during the monsoon and post-monsoon periods, suggesting its association with the decreased level of heavy metal contamination in sampling sites leading to the idea that this NAT-like protein could be used for bio-monitoring of the coastal pollution (Gorain et al., 2014).

The presence of NAT genes has been noted in species from all major vertebrate taxa, except the dog genome where no NAT sequence or NAT activity has been found (Vagena et al., 2008; Glenn et al., 2010; Sabbagh et al. 2013), with variable number of sequences present in each genome (Sim et al., 2012; Sabbagh et al., 2013).

The NAT genes in rodents have been extensively studied for the characterization of the role of NAT. In mouse, the existence of three genes (Nat1, Nat2, and Nat3) with 870bp intronless ORFs became evident in the murine NAT locus (Boukouvala and Fakis, 2005). The Nat1 was encodes a product that acetylates isoniazid (INH), while Nat2 encodes a product that acetylates p-aminobenzoic acid (pABA) and p-anisidine (pANS). Finally the Nat3 gene, unlike the human pseudogene (NATP), does not contain any nonsense mutations and is considered able to encode a functional polypeptide (Boukouvala et al., 2002). The Nat loci in mice are localized on chromosomal band 8 B3.1-3.3. Nat loci in rats are found in the 16p14 chromosomal region that is syntenic with human 8p22 and mouse 8 B3.1-3.3 (Boukouvala & Fakis, 2005).

In addition, rodents have been used for the study of the endogenous role of NAT. The ubiquitous expression of the murine NAT2 (which is homologous to the human NAT1) along with the ability of recombinant human NAT1 to N-acetylate p- aminomenzoylglutamate (p-ABG), a major folate catabolite and its expression in the embryo from the early developmental stages) suggest a possible implication of murine NAT2 and its homolog human NAT1 in the metabolism of folate (Boukouvala and Fakis, 2005).

The evolutionary history of the NAT gene family in vertebrates was subjected to phylogenetic analysis (Glenn et al., 2010; Sabbagh et al., 2013). Among other interesting findings, these studies have revealed events of positive selection and

[17] accelerated amino acid evolution in three Nat sequences in a bat species, showing strong evolutionary effect in the sequence of these xenobiotic metabolizing enzymes. Additionally, specific gene duplication events are suggested by the fact that in most species carrying more than one functional NAT gene the paralogs were more similar to each other than the corresponding orthologs. However, the three nat genes in the NAT locus of the mouse and rat genomes are considered to have been present before the divergence of the two rodent species. Although the investigations have proposed that NAT evolution is driven by xenobiotic exposure, no sign of adaptive selection during primate evolution was reported at any NAT coding site (Sabbagh et al., 2013). Above all, this phylogenetic analysis supports that the NAT gene family has evolved under a dynamic process called birth-and-death evolution, where duplication and deletion events have contributed to the genetic diversity of the NAT locus in the vertebrate genomes.

Comparative genomic studies have identified a highly conserved NAT locus in human and other primates (Sabbagh et al., 2013). Although the human NAT genes and their products have been thoroughly studied, little is known about NATs in non-human primates. Two functional NAT genes have been found in all 22 primate species studied so far, whereas a third NAT sequence similar to the human pseudogene NATP was found in a small set of them (Sabbagh et al., 2013). The Maximum Likelihood tree that has resulted from these studies clustered the NAT sequences in three groups that contained human NAT1, NAT2 and NAT3 respectively (Figure 1.11). In addition, the NATP sequences have been found only in the catarrhine species (Cercopithecidae, Hylobatidae and Hominidae) and the absence of the NATP sequence in the Hylobates lar is suggested to be due to gene loss. The evidence also propose that the three human genes have arisen from a first duplication event in the common ancestor of Simiiformes generating the NAT1 gene and a NAT ancestor which was later, in the ancestor of Catarrhini, duplicated generating the NAT2 gene and NATP sequence.

The significant interest of biochemical research in non-human primates (NHP) has been underlined in many pharmacological studies. NHPs have been characterized as the ideal model for such studies because of the anatomical and physiological similarities with humans. Rhesus macaque (Macaca mulatta) has been used for many years as a model for the study of endometriosis, as it menstruates and can develop endometriotic lesions similar to the human disease. For the study of chemically induced endometriosis and investigation of possible association between variants of the NAT genes and the disease, screening of four monkeys has been conducted and revealed two NAT2 alleles responsible for the coding of two alloenzymes with an amino acid (Val231Ile) substitution (Fakis et al., 2007). The discovery of this naturally occurring polymorphism with functional significance, revealed an alteration to the substrate selectivity of the enzyme. This amino acid switch caused lower activity of the NAT2 isoenzyme with NAT2 substrates, but surprisingly made it more active with NAT1 substrates. It was later confirmed that the change in the affinity towards the bulkier NAT2 substrates was due to changes in the spatial characteristics of the loop

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(125-129). The side chain of Ile231 is believed to push the loop towards the interior of the substrate binding pocket and thus limiting the space available to substrates in the binding cleft (Fakis et al., 2007; Wu et al., 2007; Tsirka et al., 2013).

Figure 1.11: Phylogenetic tree of the NAT gene family in primates from 19 distinct primate species. The clades of Strepsirrhini, Platyrrhini, Cercopithecidae, Hylobatidae and Hominidae are shown in turquoise, green, blue, pink and red, respectively (Sabbagh et al., 2013)

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1.5.3 NATs in human

In human, two NAT genes, NAT1 and NAT2, and one pseudogene, NATP, have been identified and mapped to the 8p22 chromosomal region. The two NAT genes differ in gene structure, pattern of developmental and tissue expression, while their products, NAT1 and NAT2 isoenzymes, have different substrate selectivity profile. The genes have intronless ORFs of 870bp encoding for proteins of 290aa (30-34kDa) in size (Boukouvala & Fakis, 2005).

Although the coding region of the NAT genes is intronless, their 5’-UTR has been found to contain one or more short upstream non-coding exons (NCEs) that are separated from each other and the coding exon by long introns (Boukouvala & Sim, 2005; Fakis et al., 2007). This conserved organization of the NAT genes is thought to be linked with transcriptional regulation, where alternative spicing of the NCEs in the 5’-UTR results in a transcript with different 5’-UTR and the same coding region (Boukouvala & Sim, 2005; Butcher et al., 2005). It has been suggested that differential utilization of upstream NCEs is linked with cell-specific gene expression and regulation of transcriptional and translational efficiency. Recent research suggests that the NAT1 and NAT2 transcripts contain diverseregulation elements that cause differences in the expression rates. (Boukouvala & Fakis, 2005).

The human NAT1 isoenzyme is ubiquitously expressed and studies suggest it has a possible endogenous role in folate metabolism. NAT1 mRNA and protein have been found in every foetal and adult tissue examined, from blastocyst stage, suggesting the possible involvement of NAT1 in cell homeostasis and development. Human NAT1 has substrate specificity for small arylamines and N-acetylation activity against a major folate catabolite (see also 1.5.2), p-aminobenzoylglutamate (p-ABG), has been also found (Boukouvala & Fakis, 2005; Sim et al., 2012).

Human NAT2 activity has been detected in liver and intestine suggesting its role for detoxifying chemical compounds that pass through the liver and the intestinal epithelium, whereas NAT1 is thought to be responsible for local detoxification and bioactivation (Boukouvala & Fakis, 2005). NAT2 isoenzyme selectively acetylates bulkier arylamines than the NAT1 substrates.

Human NAT Polymorphisms

Both NAT1 and NAT2 loci are polymorphic, with the NAT1*4 and NAT2*4 alleles considered as the wild type alleles, both responsible for rapid acetylator profile (Boukouvala & Fakis, 2005). To date, 28 NAT1 alleles and 88 NAT2 alleles have been described (according to NAT nomenclature database: nat.mbg.duth.gr). Of these alleles, some carry point mutations in the coding region that are either synonymous or cause amino acid substitutions which affect the acetylator phenotype, frameshift alterations or mutations in the untranslated regions that cause alterations in the transcript’s stability and translation rate.

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The NAT polymorphisms and the resulting acetylation phenotypes have been associated with predisposition to a number of diseases .The most typical of these is the association of slow NAT2 genotypes among bladder cancer patients exposed to arylamines. The NAT allelic frequencies show considerable inter-ethnic variance. In particular, for Caucasian populations, studies have reported frequencies of 75% NAT1*4 and 20% NAT1*10 for the NAT1 gene; and 25% NAT2*4 and 50% NAT2*5 for the NAT2 gene (Boukouvala & Fakis, 2005).

1.5.4 NAT polymorphisms and disease

The association between NAT gene polymorphisms and various diseases has been broadly studied for its clinical importance because of their significant role of in detoxifying pollutants as well as drug metabolizing effects on complex diseases). NAT polymorphisms have been studied in relation to their potential association with various disorders, for instance neurodegenerative diseases, autoimmune disorders. They are also implicated in infectious diseases, as NAT is responsible for the metabolism of therapeutic drugs, such as the acetylation of isoniazid (INH) used for the treatment of tuberculosis (Boukouvala & Fakis, 2005; Sim et al., 2012).

Their pharmacogenetic role is also important in cardiovascular disorders for their ability to acetylate various arylamines that are used for treatment of patients with circulatory problems (McDonagh et al., 2014).

Of particular interest is the relationship of the NAT polymorphism with many types of cancers, as NAT enzymes have been linked with the risk for carcinogenesis, response or resistance to treatment, and also characterized as possible drug targets (McDonagh et al., 2014). Many studies have identified the association between the slow NAT2 phenotype and bladder cancer and have agreed slow NAT2 genotype to be significant risk factor for bladder cancer exposure to arylamines (Boukouvala & Fakis, 2005; Sim et al, 2012).

In contrast to bladder cancer, fast NAT2 phenotype has been linked to increased predisposition for colorectal carcinogenesis, as it is responsible for the bioactivation of pro-carcinogens that are present in food (Boukouvala & Fakis, 2005). Deregulation of expression of the NAT1 has been implicated in breast cancer. Association of NAT1 overexpression and estrogen/progesterone receptor positive breast tumor has been reported (Sim et al., 2012).

Further interest arises from the effects of NAT1 in developmental disorders, because of the implication of NAT1 in folate catabolism (Boukouvala & Fakis, 2005). The detection of N-acetylated p-aminobenzoylglutamate in urine of Nat2 (homologous to human NAT1) wild type mice and its absence in Nat2 null mice strongly suggest the endogenous role of NAT1 in early development.

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1.6. Aims of the present study

The aim of this undergraduate thesis was to determine the effects of the naturally occurring polymorphisms p.Leu89Phe (L89F), p.Asp115Tyr (D115Y) and p.Leu89Phe+p.Asp115Tyr (L89F+D115Y) on the function of the NAT1 enzyme of the primate Macaca mulatta (Rhesus monkey).

In addition, continuing previous work from our group on the naturally occurring polymorphisms of the NAT1 enzyme of the Rhesus monkey, the effects of all nine polymorphisms on the recombinant expression have been also studied.

More specifically, our aim was to employ recombinant protein expression and purification of the NAT1 polymorphic variants (L89F, D115Y and L89F+D115Y) and wild type reference NAT1 protein. We aimed to determine the enzyme activity of these protein variants by applying DTNB enzyme assays that measure the amount of released CoA from the acetyltransferase reaction Furthermore, the localization of the recombinant products of all nine NAT1 variants in the whole bacterial lysate was to be investigated through immunoblotting with a commercial anti-His antibody and our polyclonal Ab-183 (specific for the C terminus of the mammalian NAT1).

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CHAPTER 2 MATERIALS & METHODS

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2.1. Quantification of samples using Spectrophotometer

In order to quantify the concentration of proteins or nucleic acids without losing a significant amount of the sample, quantification is performed with the use of the NanoDropTM Spectrophotometer. Using fiber optic technology and the inherent surface tension properties of the sample of liquid samples, the microsamples are retained during the measurement without the use of cuvettes (Desjardens et al., 2009). (Figure 2.1)

Figure 2.1: The Nanodrop 1000 sample retention system. (A) A sample volume of 2μL is dispensed onto the lower of the optical pedestal. (B) Once the instrument lever arm is lowered, the upper optical pedestal engages the sample, forming a liquid column with the path length defined by the gap between the two optical surfaces (Desjardens et al., 2009)

The intensity of the blank solution and the sample are both used (Equation 2.1.a.) for the calculation of the Absorbance. The value of the Absorbance is then applied on the Beer-Lambert equation for the calculation of the sample concentration (Desjardens et al., 2009) (Equation 2.1.b.). Nucleic acid sample concentrations are quantified using the absorbance value at 260nm and protein sample concentrations are quantified at 280nm. Maximum absorbance is measured for nucleic acids at 260nm and for

proteins at 280nm. Pure DNA samples show A260/A280 purity ratio near ~1.8.

Equation 2.1.a.: Calculation of the sample Equation 2.1.b.: The Beer-Lambert absorbance value equation for the calculation of sample concentration

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2.1.1. Materials

 Spectrophotometer NanoDropTM 2000, Thermo Fischer Scientific  Pipette (P2, Gilson)  Sample (DNA, Protein)  Blank Solution (Reference solution, where the sample is dissolved)  Tips (Kisker)

 ddH2O  Gloves  Absorbent paper

2.1.2. Method

For the quantification of the sample concentration via the NanoDrop Spectrophotometer, the appropriate updated software is needed. At first, the arm is raised and the pedestals are being washed with ddH2O and wiped to prevent previous sample carry-over. 1μL of the blank solution is pipetted onto the lower measurement pedestal and the software is told to blank this sample. The remaining blank solution is rewiped and 1μL of the sample for quantification is loaded. The measure choice is selected and the result of quantification after is processed by the software is then observed on the screen.

2.2. Quantification of total protein concentration in bacterial lysates with Bradford Assay

The total protein concentration of bacterial lysate solutions may be quantified with the Bradford Assay spectrophotometer method. The calculation relies on the color change and thus the absorbance value change of a dye after its interaction with the protein molecule. The Coomassie Brilliant Blue G-250 dye, also known as Bradford Reagent, binds to specific amino acids that are found in most of the proteins. When the dye is attached and under specific temperature and pH conditions, the color of the dye changes from brownish to blueish. The intensity of the blue color is linearly proportional to the quantity of the protein in the sample. This color change can be calculated by measuring the absorbance of the sample and the reference (blank) sample at the 595nm (Ref-Text-3).

In order to perform Bradford Assay, a standard curve from samples of known protein concentrations must be created in advance. The absorbance of the unknown sample is then subjected to the formula, which has resulted from the standard curve, and its concentration can be calculated (Ref.Text-4).2.1.1. Materials

 Eppendorf Biophotometer® Plus  Bradford Reagent (Sigma-Aldrich)  Bacterial lysate solutions (serial dilutions in 20mM Tris HCl pH 7.5)  BSA standards of appropriate concentration ( 0, 0.25, 0.5, 1.0, 1.4 mg/mL)

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 3mL disposable cuvettes (BRAND semi micro)  Pipette (P200, P1000 Gilson)  Tips (Kisker)  Gloves 2.1.2. Method

The Bradford Reagent is gently mixed and brought to room temperature. 1.5mL of the Bradford Reagent is mixed in a cuvette with 50μL of the bacterial lysate solution. The reference (blank) sample is created by mixing 1.5mL of Bradford Reagent with 50μL of 20mM Tris HCl pH 7.5. The samples are incubated for 10min at room temperature. The absorbance of each sample is measured at 595nm. The concentration of each sample is calculated from the standard curve’s formula and the value of the sample’s absorbance measured at 595nm.

The BSA standards are created by serially diluting 2mg/mL BSA solution. Each sample is prepared by mixing 50μL of BSA solution and 1.5mL of Bradford Reagent. For each concentration two samples are prepared. They are 10min for incubation in room temperature and then measured at 595nm. The average absorbance is related to the concentration and the appropriate standard curve resulting from these samples’ concentration and amount is created. The appropriate formula for that describes the standard curve is being used to determine the concentration of the unknown samples.

2.3. Isolation of plasmid DNA from Bacterial cell cultures

The extraction of plasmid DNA from E. coli cells may be performed with different approaches either depending on the physicochemical properties of the molecules or even by binding on special substrates. Isolation can be performed by centrifugation in gradient of CsCl, isolation with polyethylene glycol-PEG or purification by column chromatography. For this study, QΙAprep Spin Miniprep kit for Plasmid Purification (QIAgen) was used. The isolation is performed with alkaline lysis of the cells, neutralizing and adjustment to high-salt binding conditions followed by absorption of the DNA onto a silica membrane. After washing the column to remove the salts, the DNA is eluted (Ref.Text-5)

2.3.1. Materials

 E. coli BL21 (DE3) pLys cells/ E. coli JM109 cells (stored at -80°C with 25% v/v glycerol) with the desired NAT1 sequence in pET28b+ expression vector  Microcentrifuge (Eppendorf 5415c)  Centrifuge (Eppendorf 5810)  Incubator (Velp Scientifica FOC 225i)  Dry Block (Thermmolyne: type 17600 Dri-Bath)  QiAprep Spin Miniprep kit (250) for Plasmid Purification(QIAgen)  Luria Bertani (LB) Medium [Tryptone (pancreatic digest of casein) 100g/L, yeast extract 5g/L, NaCl 5g/L] (sterilized at 121°C for 1h)

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 Antibiotic kanamycin (50mg/ml, sterilized via filtration, stored at -20°C)  Disposable inoculating loops  Pipettes (P2, P20, P200, P1000, Gilson)  Tubes (1.5mL)  Tips (Kisker)

2.3.2. Method

2μL of E. coli cells from the glycerol stock that is stored at -80°C with 25% v/v glycerol with the desired recombinant sequence in the expression plasmid pET28b(+), are being inoculated in 10mL of LB medium with kanamycin (50 μg/ml). The bacterial cell cultures are being left to incubate overnight at 37°C with continuous agitation (225rpm). After the overnight incubation, the cultures are centrifuged at 3000rpm for 10min and the supernatant is removed.

Figure 2.2: Plasmid DNA isolation procedure. (QIAprep® Miniprep Handbook)

Then the QΙAprep protocol is followed. (Figure 2.2). The bacterial pellet is resuspended in 500μL buffer P1. 500μL buffer P2 are added and mixed thoroughly by inverting the tube 4-6 times followed by the adding and mixing of 700μL buffer N3. The suspension is centrifuged at 13000rpm (17900rcf) for 10min. The supernatant is transferred to a QIAprep spin column. The columns are centrifuged for 60s and the flow-through is discarded. The column is washed by adding 500μL buffer PB and centrifugation at 13000rpm for 60s. The flow through is discarded and the column is rewashed with 750μL buffer PE and 60s centrifugation at 13000rpm. The flow- through is discarded and the column is centrifuged again for 60s. The column is transferred to a clean tube. The elution is performed by adding 30μL EB buffer preheated at 70°C, 60s at room temperature and 60s centrifugation at 13000rpm.

2.4. Transformation of E. coli competent cells

Bacterial cells can be easily transformed with naked DNA under appropriate conditions. In order to insert a DNA molecule in bacterial cells, the cells must be suitably prepared. The transformation efficiency is increased when the cells are being processed with salts, e.g. CaCl2, and exposed to a short heat shock before adding of the plasmid DNA. The Ca2+ cations neutralize the negative charge of the membrane and the application of high temperature creates small openings in the membrane who allow the supercoiled plasmid to enter the bacterium. The small openings close after

[27] the sudden drop of the temperature. The cells are left to grow and express the plasmid with the resistance factor in medium without antibiotic and then are left to grow in a solid growth medium which contains antibiotic, in order to select the colonies that have accepted the plasmid. Injection of DNA molecules in bacterial cells could also be achieved by electroporation (Ref.Text-6).

2.4.1 Materials

 E. coli JM109 (for plasmid DNA extraction) and BL21 (for protein expression) strain competent cells (stored at -80°C)  Recombinant DNA with the desired sequence (pET28b+ plasmid mutagenesis product with Kanamycin resistance gene)  Kanamycin solution 50mg/mL (sterile through filtration-stored in -20°C)  Sterile L shaped disposable loops  Incubator (Velp Scientifica FOC 225i)  Waterbath  Growth medium for solid culture sterile at 121°C for 20min(LB Agar)  Luria Bertani (LB) Medium [Tryptone (pancreatic digest of casein) 100g/L , yeast extract 5g/L, NaCl 5g/L] (sterilized at 121°C for 20min)  Petri dishes (polystyrene disposable, sterile, size 100x15mm)  Tubes 0.5mL-1.5mL  Pipettes (P2, P20, P200, P1000 Gilson)  Tips (Kisker, Axygen)  Absorbent paper  Disposable Latex gloves

2.4.2. Method

1μL of plasmid DNA (mutagenesis product) is added in 20μL JM109 or 100μL BL21 E. coli competent cells. The cells are left for 20min (JM109) or 30min (BL21) with regular shaking. The cells are subjected to heat shock for 30s (JM109) or 45s (BL21) at 42°C. The tubes are left in ice for 2min (JM109) or 5min (BL21). Follows addition of 180μL (JM109) or 900μL (BL21) warm LB medium and the cells are left for 1h at 37°C to incubate with continuous agitation (225rpm). After the incubation 100μL (JM109) or 150μL (BL21) of each culture are spread on LB agar petri dish with kanamycin (50μg/mL). The petri dishes are left for overnight incubation at 37°C.

The next day, the petri dishes are observed for the presence of colonies. A colony is then picked with a sterile inoculation loop and is used to inoculate 10mL of LB medium with kanamycin (50μg/mL). This is incubated overnight at 37°C with continuous agitation (225rpm), in order to obtain a transformed culture.

2.5. Nucleic acid amplification with the Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) was invented at the middle of the 1980’s by Kary Mullis and since then has brought a revolution in molecular genetics. PCR gives

[28] the potential to easily, rapidly and cheaply replicate large quantities of nucleic acids from samples containing very limited starting material. The technique is based on the use of a DNA polymerase, nearly always a thermostable polymerase e.g. Taq, for the amplification of a DNA template in repetitive replication cycles. The enzyme is guided to the desired sequence by a pair of short oligonucleotides (primers) that hybridize on either side of the target sequence. The PCR constitutes a very powerful technique and as a result the product can be then detected through electrophoresis in a gel (Ref.Text-7).

In order to confirm whether the appropriate insert was cloned into thepET28b+vector, a number of colonies were screened by PCR. The primers (T7 promoter primer #69348-3, T7 terminator primer #69337-3) hybridize on either sides of the cloning site. (Appendix 1)

2.5.1. Materials

 Thermocycler (Gradient Cycler PTC-225, MJ Research)  Laminar flow hood (Telstar Mini V/PCR)  E. coli BL21 and JM109 liquid cultures with pET28b+ for screening (DNA template)  DNA Polymerase [LA Taq(TaKaRa), GO Taq(Promega): 5u/μL]  Polymerase Buffer (TaKaRa, Promega: 5x)  dNTPs mixture (2.5mM of each)

 MgCl2 (25mM)  Primers (T7 promoter primer #69348-3, T7 terminator primer #69337-3 5pmol/μl)  PCR strips (Axygen)  PCR caps (Axygen Scientific)  Tubes (0.5- 1.5 ml)  Pipettes (2, 20, 200 μl, Gilson, Eppendorf)  Tips (Kisker, Axygen)

 ddH2O  Absorbent paper  Disposable Latex gloves

2.5.2. Method

A reaction mastermix, which contains all of the components except the template, is prepared in the laminar flow hood (Table 2.5.2.a). The Taq polymerase is the last component that is added to the mastermix. The mastermix is distributed to the PCR tubes. 2μL of each liquid culture and 2μL of ddH2O for the non-template control (NTC) are then pipetted to the strip tubes, outside the laminar flow hood, at the bench.

[29]

Table 2.5.2.a: Reaction Components for PCR Screening Component (final concentration) NTC PCR Reaction Template - 2μL Forward Primer(0.5μΜ) 2.5μL 2.5μL Reverse Primer(0.5μΜ) 2.5μL 2.5μL Taq DNA Polymerase (1U/reaction) 0.13μL 0.13μL Buffer(1x) 5μL 5μL MgCl2(2mM) 2μL 2μL dNTPs(0.2mM) 2μL 2μL ddH2O 10.87μL 8.87μL

The strips are centrifuged (spin down) and are placed in the thermocycler. The applied thermal profile is shown at the Table 2.5.2.b.

Table 2.5.2.b: Thermal Profile for the PCR Screening STEP1 95°C for 10min Denaturation STEP2 95°C for 30s Denaturation STEP3 55°C for 30s Annealing STEP4 72°C for 1min Extending STEP5 Repeat STEP2-STEP4 Denaturation, Annealing, for 34 more times Extending x34 times STEP6 72°C for 2min Final extending STEP7 End

2.6. Agarose gel electrophoresis

Agarose gel electrophoresis is the most broadly applied technique in molecular biology. The most common application of the technique is the separation, on the basis of their length, and the observation of nucleic acids after dyeing with appropriate stain. Agarose is needed at suitable concentration for the creation of a gel in which the electrophoresis of the molecules is performed. The samples are loaded into wells and electric field is applied in the tank. The negatively charged DNA molecules migrate from the cathode to the anode at speeds that depend on their molecular weight (length in base pairs). The separated DNA may be visualized with Ethidium Bromide (EtBr) fluorescent dye under UV light. The DNA fragments may also be extracted from the agarose gel, if necessary for other applications (Ref.Text-3). 2.6.1. Materials

 TBE Electrophoresis buffer 0.5x [to make 1000mL of 5xTBE solution: 54g Tris Base, 27.5g Boric acid, 20mL of 0.5M EDTA pH8.0]  Agarose powder (BioRad)  Gel Electrophoresis Systems (Owl™ EasyCast™: Gel plate, combs)  Microwave (Matsui MS106 0.6 FT)

[30]

 1Kb Ladder (Sigma)  Samples (Amplified PCR product) with dye  Conical flask (100mL)  Ethidium Bromide fluorescent dye- EtBr 10mg/mL  Power supply (PowerPac™ Basic Power Supply -BioRad)  UV trans illuminator and digital camera (Molecular Imager® Chemi Doc XRS+ Imaging System Gel Doc™ XR System)  Pipettes (P20, Gilson)  Tips (Kisker)  Gloves  Absorbent paper

2.6.2. Method

0.3g of agarose powder is dissolved in a conical flask in 30mL of 0.5x TBE buffer. The mixture is boiled in the microwave oven and then slightly cooled before the adding of EtBr (final concentration: 0.5μg/mL). The melted solution is poured into the specially prepared mold with the appropriate comb. Once the agarose solidifies, the combs are removed from the gel and 0.5x TBE buffer is added in the electrophoresis tank. 5μL of DNA molecular weight marker (DNA ladder) is loaded for the estimation of fragments’ size and then 6μL of each sample (5μL product and 1μL loading buffer) are loaded into the wells. Constant electric field (60V) is applied to the gel chamber. The DNA bands are visualized under illumination with UV light and photographed.

2.7. DNA Sequencing

In order to verify that the plasmid contains the NAT1 sequence with the desired SNPs, the plasmid DNA was subjected to sequencing. For the sequencing reaction, sample of the purified plasmid DNA (concentration between 30-100ng/μL) was sent to the GATC Biotech Company. The sequencing was performed using the ABI 3730xl system that applies the Sanger technology (Webpage-2). The resulting chromatograph is then evaluated at the BioEdit Sequence Alignment Editor v7.2.5.

2.8. Recombinant expression of (MACMU)NAT1 proteins

Eschericia coli is a frequently used host for the expression of recombinant proteins. It allows the expression of pure, soluble and functional proteins in large quantities. For the recombinant expression of proteins in E. coli, a suitable expression E. coli strain and a suitable expression vector with the desired sequence are required (Ref.Text- 7).For the expression of the recombinant (MACMU)NAT1 proteins from the E. coli BL21 (DE3)pLysS strain, the appropriate (MACMU)NAT1 is inserted in pET28b(+) expression vector (Appendix 1). The pET28b(+) vector carries a cloning/expression region that is being transcribed by the T7 RNA polymerase (Appendix-1). The E. coli BL21 (DE3)pLysS strain is lysogenic for λ-DE3, which contains the T7 bacteriophage

[31] gene I, encoding T7 RNA polymerase under the control of the lac UV5 promoter (Webpage-1). T7 RNA polymerase is expressed upon addition of isopropyl-1-thio-β- D-galactopyranoside (IPTG) and consequently the recombinant expression of the pET28b(+) is then induced. 2.8.1. Materials

 E. coli BL21(DE3)pLysS glycerol stocks (25% v/v glycerol, stored at-80°C) that contain the pET28b(+) with the appropriate (MACMU)NAT1 sequence  Refrigerated centrifuge (Sorvall Evolution RC by Kendro Laboratory Products)  Incubator (Velp Scientifica FOC 225i)Eppendorf Biophotometer® Plus  Luria Bertani (LB) Medium [Tryptone (pancreatic digest of casein) 100g/L , yeast extract 5g/L, NaCl 5g/L] (sterilized at 121°C for 20min)  Terrific Broth (TB) Growth Medium (Sigma- Aldrich) [47.6g medium powder

(12g/L tryptone, 24g/L yeast extract, 9.4g/L K2HPO4, 2.2g/L KH2PO4) and 8mL glycerol in 1L ddH2O – sterilized for 1h at 121°C]  Antibiotic kanamycin (50mg/ml, sterilized via filtration, stored at -20°C)  1M IPTG solution (Isopropyl β-D-1-thiogalactopyranoside) stored at -20°C  Conical flasks (500mL, 200mL)  Tubes (50mL)  Pipettes (P200, P1000, Gilson)  Tips (Kisker)  Disposable inoculating loops  Disposable Latex gloves  Absorbent paper

2.8.2. Method

Frozen glycerol stocks of E. coli BL21 (DE3)pLysS with the appropriate (MACMU)NAT1 construct in pET28b(+) vector are used for the inoculation of 10mL LB medium with kanamycin (final concentration 5μg/mL) and incubated overnight at 37°C with continuous agitation (225rpm).

The next day 1mL/0.8mL are injected in conical flasks that contain 200mL/50mL TB medium with kanamycin (final concentration 5μg/mL) followed by incubation at 37°C with continuous agitation (225rpm) until the OD(600nm) of the medium reaches 0.8. The induction of the expression is performed by adding Isopropyl β-D-1- thiogalactopyranoside (IPTG) in final concentration of 1mM and overnight incubation at 16°C with continuous agitation (150rpm).

The next day the liquid cultures are centrifuged for 20min at 3000rpm (4°C). The supernatant is discarded and the bacterial pellet is stored at -80°C for a minimum of one day.

[32]

2.9. Purification of recombinant (MACMU)NAT1 protein with affinity chromatography

Affinity chromatography is one of the most broadly applied techniques for the purification of many biological molecules and especially proteins. The technique is based on the affinity of the protein molecules for special chemical groups. The recognition and binding of the protein molecules at the column may be performed with various ways. Either by covalent bonding of the chemical group in the column, passing of the protein mixture through the column and elution of the desired protein (that has attached in the column) by passing a solution which contains in high concentration a chemical group that binds with greater affinity at the column (Ref. Text-13).

Figure 2.3: Illustration of purification through affinity chromatography column. The protein of interest is bound to the ligand attached to polymer bead and then eluted after the washing of the column with solution of the ligand. (http://affinity-chromatography.tripod.com/ac.html)

[33]

Immobilized metal affinity chromatography (IMAC) is technique that relies on a molecule’s affinity for certain metals immobilized onto a chelating surface. His- tagged proteins can be purified by IMAC. The Ni2+ charged resins have the ability to selectively bind the poly-His tail of the produced recombinant proteins (Figure 2.4) that are found in protein mixture (Ref.Text-14). The elution can be achieved by washing the column with imidazole solution of increasing concentrations since the imidazole competes with the binding of the imidazole ring of histidine at the column.

Figure 2.4: Partial structure of Profinity IMAC Resin. Illustration of UNOsphere

Bead with coupled IDA functional ligand, shown charged with Ni2+. Wavy lines indicate available binding sites (Ref.Text-14).

The in pET28b(+) expression vector carries upstream the cloning site a 6xHis tag (Appendix 1). The (MACMU)NAT1 proteins that are expressed from the pET28b(+) can then be purified from the protein mixture through IMAC. 2.9.1. Materials

 Bacterial pellet of E. coli BL21(DE3) pLysS culture [centrifuged and separated from the medium after the overexpression of the (MACMU)NAT1 protein and stored at -80°C]  Incubator [cooled at 4°C (Velp Scientifica FOC 225i)]  Refrigerated centrifuge (Sorvall Evolution RC by Kendro Laboratory Products)  Sonicator (Heat systems misonix Sonicator, Ultrasonic processor XL, XL2015)  Lysis Buffer [20mM Tris-HCl pH 7.5, 300mM NaCl, 5mM Imidazole, 5% v/v glycerol, 0.1% CHAPS, 1μL (per 10mL Lysis Buffer) 2-Mercaptoethanol, Protease inhibitor mixture (50μL per 10mL Lysis Buffer)]  Profinity IMAC Ni-Charged Resin BioRad (stored at 4°C)  Chromatography columns (Polyprep Chromatography columns, BioRad)  Binding Buffer (20mM Tris-HCl pH 7.5, 20mM NaCl)  Imidazole wash solutions in binding buffer (25mM, 50mM, 100mM, 250mM, 500mM)

[34]

 1M Imidazole solution  Tubes (1,5mL, 5mL, 15mL)  Pipettes (P20, P200, P1000, Gilson)  Tips (Kisker)  Gloves  Absorbent paper

2.9.2. Method

The tubes containing bacterial pellets from the 200mL cultures are removed from - 80°C freezer and are left horizontally on ice to thaw slowly. 7.5mL lysis buffer is added into each tube and the pellet is resuspended and vortexed to achieve cell lysis. Next, the lysate is submitted to 5-6 cycles of 15s sonication at 20-30Hz with interim 30s pause on ice, until it gets milky and then centrifuged for 40min at 20000rcf (4°C). The supernatant is mixed with 400μL resin and allowed to bind for 1.5h with continuous agitation at 4°C. The mixture is transferred in chromatography columns followed by subsequent elutions of the column with 1mL binding buffer fractions with increasing concentration (25mM, 50mM, 100mM, 250mM and 500mM) of imidazole. The elution fractions are quantified and analyzed with SDS PAGE so that the proper fraction can be selected for further investigation.

2.10. Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Electrophoresis is the ability of a charged molecule to move through an electric field and is usually performed in gels that work as molecular filters and therefore reinforce the separation. Small molecules travel easily through the gel, whereas large molecules are more difficult to move. Electrophoresis of proteins is usually performed in a small vertical polyacrylamide gel. The acrylamide is polymerized through the linkage of the acrylamide with the methylene-bis-acrylamide (Figure 2.5).

Figure 2.5: Demonstration of the acrylamide polymerization. (http://www.sci.sdsu.edu/TFrey/Chem365/Proteins/ProteinsChem365.html)

[35]

The protein mixture is initially dissolved in SDS (Sodium Dodecyl Sulphate) which is an anionic detergent and consequently destroys all the non-covalent interactions of a protein. The reduction of disulfide bonds is mediated by the addition of 2- mercaptoethanol or dithiothreitol (DTT). The SDS anions are bound in the main peptide chain in ratio of 1 SDS anion per 2 amino acids. The denatured protein-SDS complex carries large negative electric charge, proportional to the protein’s molecular weight, and therefore travels through the gel (from the cathode to the anode) in dependence of the protein’s size (Figure 2.6).

Figure 2.6: Emulsification by sodium dodecyl sulfate gives proteins a net negative charge. (https://www.nationaldiagnostics.com/electrophoresis/article/biological-macromolecules- proteins)

The proteins in the gel can be visualized by dyeing the gel with silver or Coomassie Blue (Ref.Text-13). In addition, in order to identify the size of the protein bands of the gel, an amount of molecular weight marker is also loaded in the gel and electrophoresed among with the protein samples.

Figure 2.7: Demonstration of the SDS PAGE vertical cast. http://www.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg

[36]

For the SDS PAGE, the gel should be prepared by the mixing and polymerization of two polyacrylamide solutions: the separating gel solution and the stacking gel solution, between a cast of two glass plates (Figure 2.7). The separating solution is the part of the gel where the separation of the protein mixture is performed. The composition of the stacking solution (lower pH, increased SDS concentration) provides a denaturing environment for further denaturation of the protein molecules, but also glycerol assists the stacking of the molecules into the wells of the gel.

The acrylamide concentration depends on the molecular weight of the proteins we wish to separate. For the (MACMU)NAT1 proteins the appropriate polyacrylamide concentration is between 10%-12%.

2.10.1. Materials

 Vertical gel electrophoresis system (BioRad Mini PROTEAN Tetra System)  Glass Plates and combs (Mini PROTEAN Tetra Cell Plates and Combs)  Power supply (PowerPac™ Basic Power Supply -BioRad)  Dry Block (Thermmolyne: type 17600 Dri-Bath)  Documentation System (Molecular Imager® Chemi Doc XRS+ Imaging System Gel Doc™ XR System)  Molecular Weight Marker (PageRuler™ Plus Prestained Protein Ladder, 10 to 180 kDa, stored at -20°C)  Separating gel solution [0.37Μ Tris-HCl pH 8.8, 1% w/v SDS, 11% w/v of 30% (w/v) acrylamide/bis-acrylamide solution, 1% Ammonium Persulfate

(APS), 0.1% Ν,Ν,Ν,Ν-tetramethylethylenediamine (TEMED), ddH2O]  Stacking gel solution [1Μ Tris-HCl pH 6.8, 10% w/v SDS, 30% w/v 30% (w/v) acrylamide/bis-acrylamide solution, 50% glycerol, 1% Ammonium Persulfate (APS), 0.1% Ν,Ν,Ν,Ν-tetramethylethylenediamine (TEMED),

ddH2O]  1x SDS Running Buffer (25 mM Tris base, 192 mM Glycine, 0.1% SDS)  SDS Loading Buffer: [70mM Tris-HCl pH 6.8, 5% 2-mercaptoethanol, 40% glycerol, 3% w/v SDS, 0.05% (w/v) bromophenol blue]  Stain solution [1g Coomassie Brilliant Blue in 1L Destain solution(30% Methanol, 10%Acetic Acid)]  Destain solution (30% Methanol, 10% Acetic Acid)  Pipettess (P2, P20, P200, P1000, Gilson)  Tips (Kisker)  Absorbent paper  Gloves

2.10.2. Method

For preparation of the polyacrylamide gel, separating gel solution is poured between the two glass plates. After the separating gel is solidified, the stacking gel solution is poured between the plates, on top of the separating, and the combs are also inserted at

[37] the top, to create the wells. Meanwhile, the samples are prepared by adding loading buffer, in the equal volume as the protein mixture, and heating at 90°C for 5min.

After polymerization, the gel cast is placed in the electrophoresis tank. The tank is filled with 1x SDS Running buffer and the combs are removed from the gel. The samples and the protein ladder (5μL) are loaded in the wells (Figure 2.8). Electric field of 120V is applied to the tank until all the samples reach same point of the gel and then it is increased to 180V until the loading dye reaches the bottom of the cast.

Figure 2.8: Preparation of the polyacrylamide gel and assembling of the electrophoresis cell. (http://www.bio-rad.com/en-gr/applications-technologies/performing-protein-electrophoresis)

At the end of the electrophoresis, the gel is removed from the glass plates and incubated for 1h in stain solution and for 2h in destain solution. The appropriate photographs of the gels are taken and archived with the help of the Molecular Imager System. Alternatively, the separated bands can be detected with immunoblotting, and thus the gel may be incubated in transfer buffer without staining.

2.11. Enzymatic activity assays of the recombinant (MACMU)NAT1 proteins

The acetyltransferase enzymatic activity of the NAT enzymes is often measured by the measurement of the acetyl coenzyme-A hydrolysis. The acetylation of the substrate can be determined through the DTNB Assay (Ellman’s Reagent), which enables the measuring of CoA produced from the hydrolysis of acetyl-coenzyme-A (Figure 2.9).

Figure 2.9: Reaction of DTNB with a R-SH https://en.wikipedia.org/wiki/Ellman%27s_reagent DTNB [5,5'-dithio-bis-(2-nitrobenzoic acid)] was first introduced by Ellman in 1959 as a compound for quantitating free sulfhydryl groups in solution. DTNB reacts with a free sulfhydryl group and produces a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB2-) which is yellow colored and has maximum absorbance at 405nm. The reaction is rapid and stoichiometric and with the addition of one mole of thiol releasing one mole of TNB. The sulfhydryl groups from the produced CoA may be

[38] estimated by comparison to a standard curve composed of known concentrations of CoA.

The enzymatic activity assays are performed at four time points for each substrate and each (MACMU)NAT1. The amount of the produced CoA is determined by the comparison of the average of the absorption values (A405nm) at each time point with the standard curve of known concentrations of CoA for every substrate. After the analysis of the diagrams that resulted from the Microsoft Excel program, the enzymatic activity is determined, defined as nmoles of N-acetylated substrate / time (min) / μg of protein.

2.11.1. Materials

 Microplate Reader (TECAN Infinite® M1000 PRO)  96 –well plate (optical clear, flat bottom)  100mM Arylamine solutions in DMSO (Dimethyl Sulfoxide) [ p- aminobenzoic acid (pABA), 5- aminosalicylic acid (5- AS), p- anisidine (pANS), sulfamethazine (SMZ), Sigma- Aldrich, (stored at -20°C)]  20 mM Acetyl- CoA solution (Sigma- Aldrich, stored at -20°C)  20 mM CoA solution (Sigma- Aldrich, stored at -20°C)  20 mM Tris- HCl pH 7.5  DTNB solution (5mM DTNB, 6.4M Gu-HCl in 20mM Tris-HCl pH 7.5)  Working aliquots of arylamines 1mM, 2 mM in 20mM Tris- HCl pH 7.5

 Working aliquots of 2mM Ac-CoA in ddH2O  Working aliquots of 2mM CoA in ddH2O  Automatic pipettes (Eppendorf multipipette plus)  Tips (Eppendorf combtips 0.5, 1.25 ml)  Pipettes (2, 20, 200, 1000 μl, Gilson)  Tips (Kisker)  Tubes (50mL, 1.5mL, 0.5mL)  Absorbent paper  Gloves

2.11.2. Method

Prior to the enzymatic activity assays, standard curves of the absorption (A405nm) of mixtures that contain arylamine and known concentrations of CoA are created. The reactions, for each different concentration of CoA (0, 1, 2, 3, 5, 7, 9, 10, 15, 20 nmol), are performed in triplicates (Figure 2.10). The mixture for each reaction contains the corresponding concentration of CoA and 0.5mM arylamine-substrate in 20mM Tris- HCl pH 7.5. Before the measurement at 405nm, 12.5μL DTNB solution is also added. The average measurements for each triplicate are used for the designing of a standard curve, for each arylamine-substrate, of the absorption related to the concentration of CoA.

[39]

The enzymatic activity assays are performed in 96 well plates in the volume of 50μL per reaction per well. The enzymatic assays are performed in duplicates and contain 1μg of purified (MACMU)NAT1 protein mixed with 500μM of arylamine substrate (either pABA, or 5-AS, or pANS, or SMZ) in 20mM Tris-HCl pH 7.5. The reaction is initiated by adding 400μM AcCoA and is terminated by adding 12.5μL DTNB solution. Finally, the measurement of the absorbance of each well at 405nm is performed using the microplate reader.

Figure 2.10: 96 well plate of the reactions for the standard curve.

The amount of produced CoA is calculated by the transformation of the average absorbance for each duplicate through the standard curve’s equation and the acetylation profile of each enzyme for each substrate are presented on a diagram of the produced CoA values in relation to time.

2.12. Protein Analysis by Immunoblotting (Western Blot)

Western blot is a very sensitive technique that enables the detection of proteins, even in very small quantities. The technique relies on the ability of the antibodies to bind to their epitope strongly and with specificity. Furthermore it allows the quantitative and qualitative identification of protein molecules from a protein mixture.

The method requires the immobilization of the protein molecules on a membrane (e.g. nitrocellulose or PolyVinylidene DiFluoride-PVDF) through dry or liquid transfer (Figure 2.11). The protein mixture has been previously separated by SDS-PAGE and then they are transferred to the membrane, where the interaction of the proteins with the antibodies will take place. The transfer of the peptides from the polyacrylamide gel (after electrophoresis) to the membrane is performed in an electric field with the negatively charged protein molecules of the gel travelling from the negative pole to the positive pole and become attached to the membrane through hydrophobic interactions (electrotransfer) (Ref.Text-13).

[40]

Figure 2.11: Electrotransfer of proteins from a gel to a solid support membrane (e.g. nitrocellulose or PVDF). http://www.gelifesciences.com/webapp/wcs/stores/servlet/CategoryDisplay?categoryId =1179764&catalogId=10102&productId=&top=Y&storeId=11251&langId=-1 Prior to detection, the membrane is incubated in blocking solution, which contains BSA or milk casein. The BSA or the casein proteins block the uncovered places of the membrane so that the antibody will not recognize nonspecific places.

The protein-antibody complex, at the membrane surface, can be detected by adding a secondary antibody that specifically recognizes the first antibody (Figure 2.12). Usually a reporter enzyme [Alkaline Phosphatase (AP) or Horseradish Peroxidase (HRP)], linked to the primary or the secondary antibody, drives a colorimetric or chemiluminescence reaction after exposure to the appropriate substrate.

Figure 2.12: Immunodetection work-flow: Blocking, primary antibody incubation, secondary antibody incubation. (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf)

[41]

More specifically, the probe (reporter enzyme) may be conjugated either to the primary antibody (monoclonal commercial antibody) or conjugated to the secondary antibody that recognizes the heavy chain of the primary antibody. For example, the reporter enzyme HRP is able to oxidize luminol in the presence of hydrogen peroxide. When the oxidized luminol returns to its original state, light is produced. The produced light may be detected on a film or by an automated documentation system (Ref.Text-18).

Figure 2.13: Illustration of a secondary antibody conjugated to HRP that recognizes the heavy chain of a primary antibody which is bound to a peptide. http://www.perkinelmer.com/CMSResources/Images/44-148631tsa_ihc_direct_460.jpg

For the purposes of this study, the software (Image LabTM) of the automated documentation system (Chemidoc XRS+) was also used for the analysis of the detected bands.

2.12.1. Materials

 Polyacrylamide gel after electrophoresis and separation of the desired protein mixture.  Western Blot apparatus including gel holder cassettes, foam pads and tank (Criterion™ Blotter -BioRad)  Documentation System (Molecular Imager® Chemi Doc XRS+ Imaging System Gel Doc™ XR System)  PVDF membrane for protein Blotting (Immun-Blot™ - BioRad)  Power supply (PowerPac™ Basic Power Supply -BioRad)  Filter paper(Chromatography paper 3mm CHR Whatman)  Tris-Buffered Saline-Tween 20 (TBST) buffer [20mM Tris-HCl pH 7.5, 100mM NaCl, 0.1% v/v Tween 20]  Anti-His mouse monoclonal antibody (HRP conjugate, Sigma)  Anti-rabbit secondary antibody (HRP conjugate, Sigma)  Ab-183 primary antibody from rabbit antiserum (against CVPKHGDRFFTI of human NAT1 C-terminal peptide – BSA conjugate)

[42]

 Transfer Buffer (25mM Tris base, 192mM glycine, 10% v/v Methanol)  Blocking buffer (5% skimmed milk in TBST)  Chemiluminescence Kit (Immun-Star™, WesternC™ kit, BioRad)  Methanol  Polystyrene box  Ice packs  Tubes (50mL)  Pipettes (P20, P1000 Gilson)  Tips (Kisker)  Absorbent paper

2.12.2. Method

After the electrophoresis, the polyacrylamide gel is soaked into transfer buffer for 15min. In addition, the foam pads and the filter papers are also soaked into transfer buffer. The PVDF membrane is presoaked in methanol to activate and then into transfer buffer. The Blot sandwich is then assembled in the following order (from the cathode to the anode): foam pad, filter paper, gel, membrane, filter paper, foam pad (Figure 2.14). The cassette is positioned in the appropriate slot in the western blot tank, which is filled with transfer buffer and is placed in a polystyrene box with ice packs. Electric current 400mA is applied to the apparatus for 1.5h. Upon completion of transfer, the membrane is incubated in the blocking buffer overnight at 4°C with shaking. Next, the primary antibody (diluted in blocking buffer) is incubated for 1.5h followed by 3 washings of 15min with TBST while agitating. The secondary antibody is incubated after the washings for 1h followed by 3 washings of 15min with agitation.

Figure 2.14: Assembly of the transfer cassette-sandwich http://stanxterm.aecom.yu.edu/wiki/index.php?page=Western_blotting

[43]

The detection is carried out by mixing 700μL ECL and 700μL hydrogen peroxide (Chemiluminescence Kit) on the membrane, waiting for 3min on the bench at room temperature and subsequent visualization with the help of the CCD camera and the analysis software.

[44]

CHAPTER 3 RESULTS

[45]

3.1. PCR Screening of transformed E. coli JM109 cells for the presence of the (MACMU)NAT1 construct in the plasmid

Liquid cultures (15mL) were obtained from selected colonies transformed with pET28b+ constructs. They were screened by PCR for the presence of the inserted (MACMU)NAT1 sequence (Figure 3.1). The primers used for PCR screening hybridize on either side of the cloning region. Plasmid DNA was also isolated from both cultures and sequenced.

Figure 3.1: Detection of the amplified by PCR insert of the pET28b+ vector that has been used to transform E. coli JM109 cells. Both selected cultures (C1 and C2) have been found positive for the presence of an insert with molecular weight (MW) between 1000bp and 500bp. The region was amplified with T7 forward and T7 reverse primers and the products have been separated through electrophoresis in 2% w/v agarose gel. (L: Sigma 1Kb Ladder, C1: Colony1, Colony2, N: Negative template control).

3.2. Sequencing of the inserted construct in the pET28b+ plasmid isolated from the transformed E. coli JM109 cultures

Plasmid DNA was isolated from the C1 liquid culture originating from transformed E. coli JM109 colonies. Sequencing was performed to evaluate the insert’s presence and orientation. Sequence analysis was conducted with the use of BioEdit software and it is demonstrated in Figure 3.2.

The sequence of the cloned insert was further analyzed through alignment with the (MACMU)NAT1 wild type sequence. The existence of the desired substitutions, p.Leu89Phe and p.Asp117Tyr resulting through site directed mutagenesis were identified (Figure 3.3). (Note: The site-directed mutagenesis experiments were performed by Doctorate student T. Tsirka, but the sequence analysis was conducted by the author).

[46]

Figure 3.2: A . Analysis using the ORF finding tool confirmed the existence of an ATG (initiation) codon upstream the 6xHis Tag region within a sequence of 932 nucleotides (138-1070). B. This region also contained the NAT1 initiation codon (Figure 2A) and is terminated by the TAG termination codon that is also contained downstream (Figure 2B). C. The nucleotide sequences were converted to the corresponding peptide sequence, with the use of the virtual translation tool, demonstrating the first Met residue, the six His residues and the initiation Met of the NAT1 sequence. D. The translation is terminated 311 residues downstream.

Figure 3.3: Alignment of the sequencing result with the (MACMU)NAT1 wild type (reference) sequence. The mutant sequence was found positive in the virtual translation for the existence of the two amino acid substitutions, p.Leu89Phe (L89F) and p.Asp115Tyr (D115Y).

In summary, the plasmids were found suitable for transformation of E. coli BL21 cells (expression strain), and can be used further for expression of the desired mutant protein.

3.3. PCR screening of transformed E. coli BL21 cells for the presence of the (MACMU)NAT1 construct in the plasmid

Plasmid DNA was isolated from liquid cultures (15 ml) of E. coli JM109 cells containing pET28b+ with the (MACMU)NAT1 sequence with the identified amino acid substitutions. This DNA was then used for the transformation of E. coli BL21 cells. Two transformed colonies from each dish were picked and grown in LB medium. The liquid cultures were then screened through PCR for the presence of the insert in the plasmid, using PCR primers that bind on either side of the cloning region. The result of the electrophoresis of the PCR products is demonstrated in Figure 3.4. Although all four of them were found positive for the existence of the insert in the plasmid, two of them (1B and 1A) were stored in -80°C with glycerol.

[47]

Figure 3.4: Agarose gel (2%w/v) electrophoresis of the PCR amplification products (primers: T7 forward and T7 reverse). All four cultures (2 from each dish) have been found positive for the existence of an insert with molecular weight (MW) between 1000bp and 500bp (L: 1Kb Sigma Ladder, N: Negative template control, -: Empty well, 1A: Dish 1- Colony A, 1B: Dish 1-Colony B, 2A: Dish 2-Colony A, 2B: Dish 2-Colony B).

3.4. Recombinant expression and purification of the (MACMU)NAT1 Wt, L89F, D115Y and L89F+D115Y protein variants

The recombinant (MACMU)NAT1 variant proteins containing the selected polymorphisms were expressed in 200mL TB medium cultures after induction with 1mM IPTG. The soluble cell fraction that contained all the soluble protein was then passed through the BioRad IMAC column. The recombinant proteins that bound to the nickel ions were eluted through increasing imidazole concentrations (25, 50, 100, 250 mM) in binding buffer. 15μL from each fraction were mixed with 5μL loading buffer and then 15μL from the mixture were loaded on a SDS-polyacrylamide gel. In addition 4μL of protein ladder were loaded on the gel and the electrophoresis result has been observed after staining with Coomassie Blue.

[48]

Figure 3.5: SDS PAGE of the purified recombinant NAT1 proteins along with non-purified bacterial samples after staining with Coomassie Blue.

[49]

Figure3.5: A. SDS PAGE of the recombinant NAT1 reference protein (Wt). The lanes contain: BI- Bacterial pellet sample before induction, La- Protein ladder as molecular weight marker, AI-Bacterial pellet after induction, Pe- Bacterial lysate pellet, Sup: Bacterial lysate supernatant, FT: the elution flow through, and the NAT1 purified fractions in 25, 50, 100, 250 and 500mM imidazole.

B. SDS PAGE of the recombinant NAT1 L89F protein. The lanes contain: 500-the NAT1 purified with 500mM imidazole fraction, La, BI, AI, Pe, Sup, FT, 25, 50, 100, two empty wells and the 250mM fraction.

C. SDS PAGE of the recombinant NAT1 D115Y protein. The lanes contain: La, BI, AI, Pe, Sup, FT, 25, 50, 100, 250, 500.

D. SDS PAGE of the recombinant NAT1 L89F+D115Y protein. The lanes contain:, BI, AI, Pe, La, Sup, FT, 25, 50, 100, 250, 500.

After examining these electrophoresis results, the fraction of each protein that was selected for the enzymatic activity assays was the fraction purified with 100mM imidazole. These fractions were found appropriate because they presented a balance between the protein concentration and protein purity.

3.5. Enzymatic activity of the recombinant (MACMU)NAT1 protein variants (L89F, D115Y and L89F+D115Y) from Macaca mulatta

The enzymatic activity of the recombinant (MACMU)NAT1 variant proteins p.Leu89Phe (L89F), p.Asp115Tyr (D115Y) and p.Leu89Phe + p.Asp115Tyr (L89F+D115Y) was studied through DTNB assays. The assays were performed in 96 well microplates, in final volume of 50μL. Each reaction was performed with 1μg NAT protein and 0.5mM arylamine substrate in 20mM Tris-HCl pH 7.5 buffer and it was initiated by adding 0.4mM AcCoA. The reactions were terminated by the addition of 12.5μL of DTNB solution at four different time points (0, 0.5, 1, 3min) and the absorbance at the 405nm has been determined by the microplate reader TECAN infinite M1000.

3.5.1. Standard Curves

The concentration of the produced CoA-SH was determined through the use of standard curves that were created in advance for each substrate. The standard curve for each substrate was created with at least 6 points of the determined absorption that was measured by 11 samples of different CoA-SH concentrations (0,1,2,3,4,5,7,9,10,15,20nmol) with 0.5mM substrate in 20mM Tris-HCl pH 7.5 (Figure 3.6). The reactions were performed in triplicates.

[50]

[51]

Figure 3.6: Standard curves for each of the used NAT substrates using known CoA-SH concentrations. A. Standard curve with p- aminobenzoic acid (pABA). B. Standard curve with 5-aminosalicylic acid (5AS). C. Standard curve with p-anisidine (pANS). D. Standard curve with sulfamethazine (SMZ).

[52]

3.5.2. Enzymatic activity assays

The investigation of the enzymatic activity of the NAT1 polymorphic proteins was performed with four substrates. The four different recombinant proteins, L89F, D115Y, L89F+D115Y as well as the reference protein Wild type (Wt) were examined for their activity with each substrate. Specifically, the substrates used were p- aminobenzoic acid (pABA) and 5-aminosalicylic acid (5AS) that are NAT1 selective; in addition the non-selective p-anisidine (pANS) and the NAT2 selective sulfamethazine (SMZ) substrates were used.

[53]

Figure 3.7: Results of the enzymatic activity assays of the recombinant NAT1 variants (Wt, L89F, D115Y, L89F+D115Y) as performed with 1μg of protein with AcCoA and four different substrates at four time points (0, 0.5, 1, 3min). A. Grouped enzymatic activity

results of all the NAT1 protein variants with pABA as substrate. B. Grouped enzymatic activity results of all the NAT1 protein variants with 5AS as substrate. C. Grouped enzymatic activity results of all the NAT1 protein variants with pANS as substrate. D. Grouped enzymatic activity results of all the NAT1 protein variants with SMZ as substrate.

[54]

Using pABA substrate, the measured specific enzymatic activity of the (MACMU)NAT1 L89F protein is 7,717 nmol /min /mg of purified protein, of the (MACMU)NAT1 D115Y protein is 11,3018 nmol /min /mg of purified protein and the specific enzymatic activity of the (MACMU)NAT1 L89F+D115Y protein is 0,7796 nmol /min /mg of purified protein.

Using 5AS substrate, the measured specific enzymatic activity of the (MACMU)NAT1 L89F protein is 5,2124 nmol /min /mg of purified protein, of the (MACMU)NAT1 D115Y protein is 6.6062 nmol /min /mg of purified protein and the specific enzymatic activity of the (MACMU)NAT1 L89F+D115Y protein is 0,6102 nmol /min /mg of purified protein.

Using pANS substrate, the measured specific enzymatic activity of the (MACMU)NAT1 L89F protein is 7,2528 nmol /min /mg of purified protein, of the (MACMU)NAT1 D115Y protein is 10,3026 nmol /min /mg of purified protein and the specific enzymatic activity of the (MACMU)NAT1 L89F+D115Y protein is 0,1072 nmol /min /mg of purified protein.

However, the specific enzymatic activity of the four NAT1 protein variants with the SMZ substrate was not possible to determine, due to very low levels of activity of the NAT1 variants with this substrate.

3.6. Detection and study of the expression profile of the recombinant polymorphic NAT1 proteins from Macaca mulatta by immunoblotting

Due to the diverse enzymatic activity of the NAT1 variants, a study of the expression profile with the immunoblotting technique was designed. At first, a trial western blot experiment was performed with protein samples that were previously purified (see Figure 3.5) and stored with 20% of glycerol at -80°C and they were detected with Anti-His antibody (Figure 3.8).

Figure 3.8: Depiction of trial western blot experiment with anti-His (1:20,000) at 62.9s exposure. From left to right the lanes contain: L: Ladder, 1: Wt pellet, 2: Wt supernatant, 3: Wt in 100mM imidazole, 4: L89F pellet, 5: L89F supernatant, 6: L89F in 100mM imidazole, 7: D115Y pellet, 8: D115Y supernatant, 9: D115Yin 100mM imidazole, 10: L89F+D115Y pellet, 11: L89F+D115Y supernatant, 12: L89F+D115Y in 100mM imidazole. [55]

Following the successful results of this trial experiment, it was decided to extend it to the eight (MACMU)NAT1 protein variants (G51A, M82V, L89F, D115Y, E155Q, F175L, R187Q and L89F+D115Y). All these variants were expressed as described previously (Section 2.8). For every protein variant, two whole cell lysate fractions (soluble supernatant and insoluble pellet) were quantified with Bradford Assay after suitable dilutions in 20mM Tris-HCl pH 7.5 (Figure 3.9). Additionally, they were analyzed on SDS PAGE gel and detected with immunoblotting using either Anti-His or an enzyme-specific antibody. Specifically, the antibodies used were a commercially available Anti-His antibody that binds to the 6xHis tag of the recombinant protein, and Ab-183, a polyclonal antibody that binds to the C-terminal region of the mammalian NAT1 proteins (Figure 3.10) but also recognizes the BSA protein. Due to this ability, BSA was added to the samples for the detection of differences in sample loading to the gel. Furthermore, stripping of the antibody from the membrane following incubation with the Ab-183 has been attempted. The same membrane was then re-incubated with Anti-His antibody.

Figure 3.9: The standard curve for BSA solutions of known concentrations (from 0 to 1,4 μg/μL) that has been used for the quantification through Bradford Assay of the whole cell lysate solutions.

[56]

D E

Figure 3.10: Immunoblotting detection of NAT1 protein variants (Wt, G51A, M82V) from whole cell lysate solutions with A.Ab-183 (diluted by mistake in 1:500 instead at 1:5000) antibodies and B Anti- His (1:20000). The lanes have been loaded with 10mg/per lane of total protein quantified with Bradford Assay. BSA protein in concentration 1μg/μL has been loaded in each sample. (M1/M2: 1μg/μL (5μL + Loading buffer), 1: Wt pellet, 2: G51A pellet, 3: M82V pellet, 4: Wt soluble, 5: G51A soluble, 6: M82V soluble, E: Empty well, L: Molecular weight marker).

[57]

Figure 3.10: Immunoblotting detection of NAT1 protein variants (Wt, G51A, M82V) from whole cell lysate solutions.

A. Depiction of the western blot experiment of 10mg/lane total protein detected with Ab-183 (diluted by mistake in 1:500 instead at 1:5000) antibody, 11.1s exposure.

B. Depiction of the western blot experiment of 10mg/lane total protein detected with Anti-His (1:20000 diluted) antibody, 34.2s exposure.

C. Depiction of the western blot experiment of 10mg/lane total protein detected with Anti-His antibody, 16.3s exposure, after stripping of the membrane that has previously incubated with Ab- 183.

D. SDS PAGE gel stained gel after transfer to the membrane that has later incubated with Ab- 183.

E. SDS PAGE gel stained gel after transfer to the membrane that has later incubated with anti- His.

The western blot experiment presented above (Figure 3.10) showed that the amount of 10mg of total protein is excessive and thus for the following experiments the amount of protein loaded per lane has been reduced to 0.2mg of total protein per lane. In addition, the evaluation of the stripping result showed differences of the signal in the membrane that has been incubated with the anti-His antibody in contrast with the membrane that has been originally incubated with the anti-His antibody, subjected to antibody stripping and incubated again with the Ab-183. Furthermore, the amount of BSA loaded in each lane was the same (2μL from 0.1μg/μL BSA solution.

[58]

Figure 3.11: Immunoblotting detection of NAT1 protein variants (Wt, E155Q, F175L, R187Q) from whole cell lysate solutions with A.Ab-183 (1:5000) antibodies and B Anti-His (1:20000). The lanes have been loaded with 0.2mg/per lane of total protein quantified with Bradford Assay. BSA protein in concentration 1μg/μL has been loaded in each sample. (M1/M2: 1μg/μL, 1: Wt pellet, 2: E155Q pellet, 3: F175L pellet, 4: R187Q pellet, 5: Wt soluble, 6:E155Q soluble, 7: F175L soluble, 8: R187Q soluble, E: empty well, L: Molecular weight marker).

[59]

Figure 3.11: Immunoblotting detection of NAT1 protein variants (Wt, E155Q, F175L, R187Q) from whole cell lysate solutions.

A. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Ab-183

(1:5000) antibody, 176.2s exposure.

B. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Anti-His (1:20 000 diluted) antibody, 77.0s exposure.

C. SDS PAGE gel stained gel replica gel of the gel that has been transferred to the membrane that has been incubated with the Ab-183.

D. SDS PAGE gel stained gel replica gel of the gel that has been transferred to the membrane that ha s been incubated with the Anti-His.

In order to minimize any potential differences between sample loadings of each protein (pipetting errors) it was decided to load each lane with the same volume (5μL) of total protein solution from samples that had the same concentration (0.04μg/μL). Equal volume of BSA (2μL from 0.1μg/μL BSA solution) was added to each sample before loading. BSA was loaded to all gels.

[60]

Figure 3.12: Immunoblotting detection of NAT1 protein variants (Wt, L89F, D115Y, L89F+D115Y) from whole cell lysate solutions with A. Ab-183 (1:5000) antibodies and B. Anti-His (1:20000). The lanes have been loaded with 5μL of 0.04μg/μL of total protein solution protein quantified with Bradford Assay. 2μL of 0.1μg/μL BSA protein in concentration has been added in each sample. (1: Wt pellet, 2: L89F pellet, 3: D115Y pellet, 4: L89F + D115Y pellet, 5: Wt soluble, 6: L89Fsoluble, 7: D115Y soluble, 8: L89F+D115Y soluble, E: empty well, L: Molecular weight marker).

[61]

Figure 3.12: Immunoblotting detection of NAT1 protein variants (Wt, L89F, D115Y, L89F+D115Y)

A. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Ab-183 (1:5000) antibody, 7.0s exposure.

B. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Anti-His (1:20000 diluted) antibody, 13.1s exposure.

C. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Ab-183 (1:5000) antibody, 297.0s exposure.

D. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Anti-His (1:20000 diluted) antibody, 164.1s exposure.

E. SDSAfter PAGE evaluation stained ofreplica the results gel of the and both the the development gels that have of a been suitable transferred experimental to both the membranes.procedure for the study of the expression profile of the polymorphisms, it was decided to extend the immunoblotting experiments to the variants in bacterial cell lysates. These new western blot experiments have been performed with a fixed and standardized procedure. Further evaluation of the differences in the detection of the reference protein (Wt) from the bacterial lysate pellet and the soluble bacterial lysate fraction has been performed by comparison of the band intensity as measured by the Chemidoc Imager and the ImageLab software (BioRad).

A B D

C E

[62]

Figure 3.13: Immunoblotting detection of NAT1 protein variants (Wt, E155Q, F175L, R187Q) from whole cell lysates (OR: whole lysate mixtures, Ioanna) with A. Polyclonal antibody Ab-183 (1:5000) and B Anti-His antibody (1:20000). The lanes were loaded with 5μL of 0.04μg/μL of total protein solution protein quantified with Bradford Assay. 2μL of 0.1μg/μL BSA protein was added to each sample. (1: Wt pellet, 2: E155Q pellet, 3: F175L pellet, 4: R187Q pellet, 5: Wt soluble, 6:E155Q soluble, 7: F175L soluble, 8: R187Q soluble. E: empty well, L: Molecular weight marker).

A. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Ab-183 (1:5000) antibody, 58.4s exposure.

B. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Anti-His (1:20000 diluted) antibody, 300.0s exposure.

C. Depiction of the repeat of the same western blot experiment of 0.2mg/lane total protein detected with Anti-His (1:20000 diluted) antibody, 7.0s exposure (Performed by the undergraduate student Maria Konstantopoulou).

D. SDS PAGE stained replica gel of the both the gels that was transferred to both membranes.

E. SDS PAGE stained replica gel of the gel that was loaded and transferred to the membrane that was incubated with anti-His antibody by Maria Konstantopoulou.

F. Demonstration of the intensity measurements of the membrane that was incubated with the Ab-183(1:5000) antibody, 34.2s exposure (Lane 4: Wt pellet, Lane 5: E155Q pellet, Lane 6: F175L pellet, Lane 8: R187Q pellet, Lane 12: Wt soluble, Lane13: E155Q soluble, Lane 14: F175L soluble, Lane 15: R187Q soluble. The graph that resulted from the saturated bands shows a round peak indicating that the amount quantified is lower than the original amount that would be measured from this amount of protein if it did not exceed the quantification limits.

[63]

Table 3.1: The intensity of each band from the immunoblotting detection of the NAT1 protein from whole cell lysate solutions (demonstrated in Figure 3.13 F), as measured by the ChemiDoc Imager (BioRad)

Lanes Sample Volume(Int) Saturated

4 Wt pellet 11459888 √

5 E155Q pellet 373312 -

6 F175L pellet 290122 -

7 R187Q pellet 8038294 √

12 Wt soluble 9502658 √

13 E155Q soluble 658278 -

14 F175L soluble 295960 -

15 R187Q soluble 5815370 √

From the above measurements, Table 3.1, and taking into account that some of the bands are saturated, we can observe that in the culture of the reference protein (Wt) we detected more than 30-fold the amount of the NAT1 protein that was detected in the pellet from the lysate of the culture with the E155Q NAT1 variant. Also the NAT1 in the R187Q culture is 21-fold higher than the amount of the NAT1 protein that is detected in the pellet of the culture with the E155Q NAT1. In addition, 14-fold NAT1 was found in the Wt soluble fraction than in the soluble fraction of the E155Q NAT1 culture.

A C

[64]

B D

Figure 3.14: Immunoblotting detection of NAT1 protein variants (Wt, L89F, D115Y,L89F+D115Y) from whole cell lysate solutions with A.Ab-183 (1:5000) antibodies and B Anti-His (1:20000). The lanes have been loaded with 5μL of 0.04μg/μL of total protein solution protein quantified with Bradford Assay. 2μL of 0.1μg/μL BSA protein in concentration has been added in each sample. (1: Wt pellet, 2: L89F pellet, 3: D115Y pellet, 4:L89F+D115Y pellet, 5: Wt soluble, 6: L89F soluble, 7: D115Y soluble, 8: L89F+D115Y soluble. E: empty well, L: Molecular weight marker).

[65]

Figure 3.14: Immunoblotting detection of NAT1 protein variants (Wt, L89F, D115Y, L89F+D115Y) from whole cell lysate solutions

A. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Ab-183 (1:5000) antibody, 79.5s exposure.

B. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Anti-His (1:20000 diluted) antibody, 221.5s exposure.

C. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Ab-183 (1:5000) antibody, 31.2s exposure.

D. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Anti-His (1:20000 diluted) antibody, 46.3s exposure.

E. SDS PAGE stained replica gel of both gels that was transferred to both membranes.

F. Demonstration of the intensity measurements of the membrane that has been incubated with the Ab-183 (1:5000) antibody, 46.3s exposure (Lane 4: Wt pellet, Lane 5: L89F pellet, Lane 6: D115Y pellet, Lane 8: L89F+D115Y pellet, Lane 12: Wt soluble, Lane13: L89F soluble, Lane 14: D115Y soluble, Lane 15: L89F+D115Y soluble. The graph that has resulted from the saturated bands shows a round peak indicating that the amount quantified is lower than the original amount that would be measured from this amount of protein if it did not exceed the quantification limits.

Table 3.2: The intensity of each band from the immunoblotting detection of the NAT1 protein from whole cell lysate solutions (shown in Figure 3.14 F), as measured by the ChemiDoc Imager (BioRad)

Lanes Sample Volume(Int) Saturated 4 Wt pellet 10298211 √

5 L89F pellet 419418 - 6 D115Y pellet 211842 - 7 L89F+D115Y 140940 - pellet 12 Wt soluble 7595532 √

13 L89F soluble 251604 - 14 D115Y soluble 197820 - 15 L89F+D115Y 141840 - soluble

[66]

From the above measurements, Table 3.2, and taking into account that some of the bands are saturated, we can observe that in the culture of the reference protein (Wt) detected more than 24-fold the amount of the NAT1 protein that is detected in the pellet from the lysate of the culture with the L89F NAT1 variant . In addition, 30-fold NAT1 was found in the Wt soluble fraction than in the soluble fraction of the L89F NAT1 culture.

A B

[67]

Figure 3.15: Immunoblotting detection of NAT1 protein variants (Wti: Wt induced, Wtu: Wt uninduced, G51A, M82V) from whole cell lysate solutions with A.Ab-183 (1:5000) antibodies and B Anti-His (1:20000). The lanes have been loaded with 5μL of 0.04μg/μL of total protein solution protein quantified with Bradford Assay. 2μL of 0.1μg/μL BSA protein in concentration has been added in each sample. (1: Wti pellet, 2: Wtu pellet, 3: G51A pellet, 4: M28V pellet, 5: Wti soluble, 6: Wtu soluble, 7:G51A soluble, 8:M82V soluble. E: empty well, L: Molecular weight marker).

A. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Ab-183 (1:5000) antibody, 61.4s exposure.

B. Depiction of the western blot experiment of 0.2mg/lane total protein detected with Anti-His (1:20000 diluted) antibody, 7.0s exposure.

C. SDS PAGE stained replica gel of both the gels that was transferred to both membranes.

D. Demonstration of the intensity measurements of the membrane that has been incubated with the Ab-183(1:5000) antibody, 10.1s exposure (Lane 4: Wti pellet, Lane 5: Wtu pellet, Lane 6: G51A pellet, Lane 8:M82V pellet, Lane 12: Wti soluble, Lane 13: Wtu soluble, Lane 14: G51A soluble, Lane 15: M82V soluble. The graph that has resulted from the saturated bands shows a round peak indicating that the amount quantified is lower than the original amount that would be measured from this amount of protein if it did not exceed the quantification limits.

Table 3.3: The intensity of each band from the immunoblotting detection of the NAT1 protein from whole cell lysate solutions (shown in Figure 3.15 D), as measured by the ChemiDoc Imager (BioRad)

Lanes Sample Volume(Int) Saturated 4 Wti pellet 6579474 √

5 Wtu pellet 1592713 - 6 G51A pellet 105963 - 7 M82V pellet 186249 - 12 Wti soluble 3105850 -

13 Wtu soluble 680380 - 14 G51A soluble 272137 - 15 M82V soluble 83580 -

[68]

From the above measurements, Table 3.3, and taking into account that some of the bands are saturated, we can observe that in the culture of the reference protein (Wt) detected more than 15-fold the amount of the NAT1 protein that is detected in the pellet from the lysate of the culture with the G51A NAT1 variant . In addition, 2.5- fold NAT1 was found in the Wt soluble fraction than in the soluble fraction of the G51A NAT1 culture.

An unexpected finding was detected while studying the NAT1 protein variants (Wti, Wtu, G51A, M82V) demonstrated in Figure 3.15, where expression was noted from the Wt without induction. More specifically, it was decided for this investigation to load samples from the two fractions of the bacterial cell culture (Wtu) grown at the same conditions (16°C, overnight incubation, continuous agitation) but with the only difference from the other Wt culture, Wti, to be the absence of the IPTG. Surprisingly though, we detected NAT protein in this culture.

For that reason, further bioinformatics and immunoblotting investigations were conducted.

B C

[69]

Figure 3.16: Results of the investigation for the detection of NAT1 protein in the lysate from the uninduced culture. A. Alignment of the (HUMAN)NAT1 protein and an ECOLX protein of the N- acetyltransferase family. The C-terminal region of the (HUMAN)NAT1, CVPHGDRFFTI, that is recognized by Ab-183 is absent from the bacterial protein. B,C,D. Immunoblotting detection of NAT1 protein variants (Wti, Wtu, G51A, M82V) from bacterial cell pellet samples before and after the overnight growth at 16°C that was previously collected from the cultures that have been then lysed for the detection of the NAT protein with western have been loaded on SDS PAGE and then detected with immunoblotting (Figure 3.15). B. Depiction of the western blot experiment of 12μL of resolubilized in 20mM Tris-HCl pH 7.5 bacterial pellet detected with Ab-183 (1:5000) antibody, 182.2s exposure. C. Depiction of the western blot experiment of 12μL of resolubilized in 20mM Tris- HCl pH 7.5 bacterial pellet detected with Anti-His (1:20000 diluted) antibody, 212.4s exposure. D. SDS PAGE stained replica gel of the both the gels that have been transferred to both the membranes.

From the study demonstrated above (Figure 3.16), it became evident that the bacterial culture that expressed the reference protein (Wt) showed leaky expression profile. Through literature research, we have conducted a trial to see if the leaky expression stops in presence of glucose. Four different cultures of the Wt cultures were grown at the same conditions, but with different growth media and the recombinant proteins expressed were purified through affinity chromatography. It was then demonstrated that the expression in absence of IPTG, referred as “leaky expression” was eliminated in presence of 0.5% glucose in the growth medium.

Figure 3.17: Investigation of the expression profile of the (MACMU)NAT1 protein in presence of glucose and/or IPTG. The proteins have been purified from the bacterial lysates through IMAC and the purified products have been loaded in gel and electrophoresed. L: Molecular Weight Protein Marker , BI: Before induction (referring to the overnight incubation at 16°C), AI: After induction (referring to the overnight incubation at 16°C), Pe: Pellet, Sup: Supernatant, FT: Flow through, 25, 50, 100, 250, 500mM imidazole). –Expression and Purification of proteins from these four cultures has been performed by Maria Konstantopoulou with the help of Olga Savvidou.

[70]

CHAPTER 4 CONCLUSIONS AND DISCUSSION

[71]

4.1 Conclusions

This study aimed to investigate the effects of the naturally occurring polymorphisms of the NAT1 enzyme of the primate Macaca mulatta. The polymorphic protein variants of the NAT1 enzyme were studied in enzymatic activity assays for the characterization of their substrate profile. Furthermore, immunoblotting experiments were conducted for the characterization of the stability and the expression profile in the recombinant system of the protein.

Enzymatic activity assays were performed for the three variants (L89F, D115Y and L89F+D115Y) where 1μg of purified NAT1 enzyme was assayed for acetylation activity with four substrates (pABA, 5AS, pANS and SMZ). For the western blot experiments, all nine (MACMU)NAT1 variants were expressed in E. coli BL21 expression strain and a total of 0.2μg of total protein from the soluble and the insoluble part of the bacterial total lysate has been used for the detection of each variant.

The enzymatic activity assays showed that the presence of the L89F or the D115Y amino acid substitution lowers the enzymatic activity, whilst the presence of both amino acid substitutions (L89F+D115Y) nullifies the activity with every substrate tested. The western blot experiments showed that the amount of each variant present in the lysate was proportional to the soluble and insoluble fraction. In addition, the western blot experiments showed differences in the expression of each variant from the bacterial expression system. More specifically, a pattern of respective amounts of NAT1 protein variant was detected in the soluble and its respective insoluble part. Furthermore, the amount of NAT1 protein detected was always lower in the polymorphic variants than the reference (Wt) protein.

Noteworthy was the puzzling detection of NAT1 protein in the fractions originating from bacterial cultures where IPTG was not added. This finding led to a new series of experiments to test the reason for the detection of recombinant NAT1 reference protein (Wt) without induction of the expression of this culture. For that reason, we had to test the possibility that a bacterial protein was detected. The BLAST results showed that the NAT homologous proteins of the E. coli do not possess the polypeptide region detected by the Ab-183. Next, a new immunoblotting test was designed and bacterial cell pellet samples collected before and after the overnight incubation of the 50mL cultures were used for the detection of the NAT1 protein. These experiments confirmed that the (MACMU)NAT1 protein was expressed before and without the addition of IPTG, therefore it constituted “leaky” expression. Additional experiments examining the leaky expression of the (MACMU)NAT1 protein using different culture media demonstrated that the leaky expression is eliminated by adding 0.5% glucose in the growth medium.

[72]

4.2. Discussion

The western blot experiments were designed to investigate the stability of the recombinant enzyme in the bacterial expression system. It was hypothesized that the polymorphisms in the amino acid sequence could cause lowering of the protein stability in the E. coli cell and thus the protein should be localized in inclusion bodies that would be found in the insoluble part. What we observed was that the amount of the protein detected in the soluble part was proportional to the amount of protein present in the insoluble part and both were less than the amount of the reference protein detected in the respective parts. It was then considered that the presence of the amino acid substitution may affect the recombinant expression of the protein. The expression of all the variants, except the one carrying the R187Q substitution was shown to be decreased, compared to the expression of the Wt (reference protein). In addition, the protein variant that carried two amino acid substitutions (L89F+D115Y) demonstrated the lowest expression of all variants.

Although the expression of the enzymes was shown to have a pattern similar to their activity profile, for instance variants that showed reduction in acetylation activity (enzymatic activity assays performed by Giannouri 2014; Marinakis, 2014; Rizou 2014 and Zaliou 2013) were also found to have lower expression; the amount of protein produced should not be related to the reduced activity. This is resulting from the fact that the enzymatic activity assays have been performed for all substrates with equal amount of protein (1μg of protein for each reaction).

Of particular interest is the case of the variant carrying the R187Q amino acid substitution. This variant was shown to have a slow acetylation activity (Zaliou 2013), although the immunoblotting experiments showed that its expression is almost as high as the reference protein. This supports the hypothesis that the amount of protein expressed is not linked to the activity of the enzyme.

The effect of these polymorphisms to the acetylating activity, even though none of them is affecting the catalytic triad (Sinclair et al., 2000; Wu et al., 2007), generates interest for further investigation of the causes for lowering the activity. Taking into account the results of the study on the (MACMU)NAT2 V231I (Tsirka et al., 2014), where the amino acid substitution led to structural alterations that did not directly affect the active site, a structural in silico modeling study could give clear evidence of the reasons for the effects of the amino acid substitutions on the enzymatic activity.

The dramatic effect of the amino acid substitutions on the expression of the protein variants remains unclear. It would be useful to measure the optical density (at 600nm) of the culture after the overnight induction of protein expression in order to examine whether the bacterial cultures are affected by the expression of particular variants. Additional control tests that would examine possible leaky expression from the promoter should be also carried out prior to the expression of proteins in order to avoid differences of the expression of the recombinant proteins.

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To conclude, we can clearly observe that these naturally occurring non-synonymous polymorphisms of the (MACMU)NAT1 enzyme affect the acetylation activity of the variant enzymes, but they also lower the expression of the variants, indicating possible alterations in the tertiary structure of the enzyme caused by the substitutions. The results from the present study contribute to our knowledge on acetylation phenotypes that result from genetic polymorphisms. The combination of different NAT polymorphisms that result in different acetylation phenotypes which may then result to different pharmacological responses and distinct susceptibility in various diseased has not been yet well characterized. Thus any additional knowledge on the effects of gene polymorphisms on the protein product could help to improve understanding of the genotype-phenotype correlation.

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5. BIBLIOGRAPHΥ

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Undergraduate theses

o Giannouri, D. (2014). “Functional in- vitro study of polymorphisms p.Leu89Phe and p.Glu155Gln of the NAT1 enzyme in the primate Macaca mulatta (Rhesus)” BSc Thesis, Democritus University of Thrace, Department of Molecular Biology and Genetics, Alexandroupolis, Greece. o Marinakis, N. (2014). “Functional study of polymorphisms p.Asp115Tyr and p.Phe175Leu of the NAT1 enzyme in the primate Macaca mulatta (Rhesus)" BSc Thesis, Democritus University of Thrace, Department of Molecular Biology and Genetics, Alexandroupolis, Greece. o Rizou, S. (2014). “Functional study of polymorphisms p.Met82Val and p.Gly51Ala of the NAT1 enzyme in the primate Macaca mulatta (Rhesus)”. BSc Thesis, Democritus University of Thrace, Department of Molecular Biology and Genetics, Alexandroupolis, Greece. o Zaliou, S. (2013). “A comparative functional study of the enzyme family pf Arylamine N- Acetyltransferases (NAT) in lower and higher eukaryotes”. BSc Thesis, Democritus University of Thrace, Department of Molecular Biology and Genetics, Alexandroupolis, Greece.

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Internet sources:

- http://animaldiversity.org/accounts/Macaca_mulatta/#geographic_range, - http://genome.wustl.edu/genomes/detail/macaca-mulatta/ - http://www.issg.org/database/species/ecology.asp?si=1205&lang=EN\ - http://nat.mbg.duth.gr - www.iubmb.org - www.ncbi.nlm.nih.gov/ - www.scopus.com - (Webpage-1) https://www.promega.co.uk/products/cloning-and-dna-markers/cloning-tools- and-competent-cells/bacterial-strains-and-competent-cells/bl21_de3_plyss-competent-cells/ - (Webpage-2) http://www.gatc-biotech.com/en/index.html

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Appendix:

 Appendix 1: Novagen pET-28b(+) map (Retrieved from: https://www.staff.ncl.ac.uk/p.dean/pET28.pdf)