Protein Modification and Catabolic Fates of Lipid

PROTEIN MODIFICATION AND CATABOLIC FATES OF LIPID

PEROXIDATION PRODUCTS

CHUAN SHI

Submitted in partial fulfillment of the requirements for

the Degree of Doctor of Philosophy

Dissertation Advisor: Gregory P. Tochtrop, Ph.D.

Department of Chemistry

CASE WESTERN RESERVE UNIVERSITY

January 2017 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

______Chuan Shi candidate for the Doctor of Philosophy degree *.

Rajesh Viswanathan (signed)______(chair of the committee)

Anthony Pearson ______

Michael Zagorski ______

Henri Brunengraber ______

Gregory Tochtrop ______

______

(date) ______Dec. 8, 2016

*We also certify that written approval has been obtained for any proprietary material contained therein.

This thesis is dedicated to my parents in the deepest appreciation and gratitude for their unconditional love, endless support and continuous encouragement throughout every step in my life

TABLE OF CONTENTS

Table of Contents ...... i

List of Figures ...... v

List of Schemes ...... vii

List of Tables ...... viii

Acknowledgement ...... ix

List of Abbreviations ...... xi

Abstract ...... xv

Chapter 1. General Introduction ...... 1

1.1 Oxidative stress ...... 2

1.2 Lipid peroxidation ...... 3

1.3 Fates of LPO products and their relevance on disease states ...... 5

1.3.1 Reactions with proteins and other endogenous nucleophiles ...... 5

1.3.2 Metabolic fate ...... 8

1.4 Research Strategy ...... 11

1.4.1 Characterization of the enzymes in catabolic pathway of 4-HNE ...... 11

1.4.2 Investigation of the relevance of EKODEs in diseases or aging ...... 13

1.5 References ...... 15

Chapter 2. Identification of Relevant Enzymes in the Catabolic Pathways of 4-

Hydroxyacids and Lipid Peroxidation Products ...... 23

2.1 Introduction ...... 24

2.2 Results and Discussions ...... 28

2.2.1 Kinase activity assay ...... 28

2.2.2 Kinase purification ...... 33

2.2.3 Verification of kinase candidates ...... 39

2.3 Conclusions ...... 43

2.4 Experimental Sections ...... 44

2.4.1 Materials and Methods ...... 44

2.4.2 GHP-CoA kinase activity assay ...... 46

2.4.3 Kinase purification ...... 53

2.4.3 Kinase purification ...... 48

2.4.4 Verification of kinase candidate ...... 50

2.5 Acknowledgement ...... 52

2.6 References ...... 53

Chapter 3. Mechanistic Study of Protein Modification by Epoxyketooctadecenoic Acids

(EKODEs) ...... 59

3.1 Introduction ...... 60

3.2 Results and Discussions ...... 63

3.2.1 Mechanistic study of the reactions of EKODE model compounds with

analogues of nucleophilic amino acids ...... 63

3.2.2 Reactions of EKODEs with cysteine ...... 68

3.2.3 Mass spectrometric characterization of the modification of human serum

albumin (HSA) by trans-EKODE-(E)-IIb ...... 73

3.3 Conclusions ...... 79

3.4 Experimental Sections ...... 80

3.4.1 Materials and Methods ...... 80

3.4.2 Mechanistic study of the reactions of EKODE model compounds with

analogues of nucleophilic amino acids ...... 81

3.4.3 Reactions of EKODEs with cysteine ...... 87

3.4.4 Mass spectrometric characterization of the modification of HSA by trans-

EKODE-(E)-IIb ...... 89

3.5 Acknowledgement ...... 92

3.6 References ...... 93

Chapter 4. Immunochemical Detection of EKODE-Cysteine Adducts in Oxidative Stress,

Aging, and Diseases ...... 99

4.1 Introduction ...... 100

4.2 Results and Discussions ...... 103

4.2.1 Strategies for antigen preparation ...... 103

4.2.2 Production and characterization of antibodies against EKODE-cysteine adduct

...... 108

4.2.3 Immunochemical detection of EKODE-cysteine adduct in nervous system...113

4.2.4 Immunochemical detection of EKODE-cysteine adduct in cardiovascular

disease and other tissues ...... 117

4.3 Conclusions ...... 121

4.4 Experimental Sections ...... 122

4.4.1 Materials and Methods ...... 122

4.4.2 Synthesis of antigens against EKODE-cysteine adduct ...... 123

4.4.3 Production and characterization of anti-EKODE-Cys antibodies ...... 126

iii

4.4.4 Immunochemical detection of endogenous EKODE-Cys adducts in biological

samples ...... 130

4.5 Acknowledgement ...... 133

4.6 References ...... 134

Chapter 5. Future Directions ...... 140

5.1 Identification of relevant kinase in the catabolic pathways of 4-hydroxyacids .....141

5.2 Protein modification of EKODEs ...... 143

5.3 References ...... 145

Appendix ...... 147

Bibliography ...... 148

LIST OF FIGURES

Figure 1.1 Production of lipid peroxidation products ...... 4

Figure 1.2 Fates of lipid peroxidation products ...... 5

Figure 1.3 Proposed metabolic pathway of EKODEs ...... 12

Figure 2.1 4-P-acyl-CoAs identified in extracts of rat livers perfused with C4 to C11 4- hydroxyacids ...... 25

Figure 2.2 SDS-PAGE analysis of the expression of GHB-CoA transferase ...... 29

Figure 2.3 Synthesis of GHP-CoA...... 31

Figure 2.4 Kinase activity assay using GHP-CoA as substrate ...... 33

Figure 2.5 Kinase activity test of ammonium sulfate precipitation of pig liver homogenate ...... 34

Figure 2.6 FPLC purification of 30-45% AS precipitate using IEX ...... 36

Figure 2.7 FPLC purification of IEX fractions 22-25 using Blue-sepharose ...... 37

Figure 2.8 SDS-PAGE analysis of Blue-sepharose fraction 9 ...... 38

Figure 2.9 SHPK activity test of recombinant SHPK ...... 41

Figure 2.10 GHP-CoA activity test ...... 42

Figure 3.1 Structures of EKODE isomers ...... 60

Figure 3.2 Proposed reactivity of EKODEs with biological nucleophiles ...... 62

Figure 3.3 Time course of the reactions of N-acetyl-cysteine-methyl ester with EKODEs and EKODE model compounds ...... 70

Figure 3.4 LC-MS analysis of the reaction of trans-EKODE-(E)-IIb with N-acetyl- cysteine-methyl ester ...... 72

Figure 3.5 LC-MS analysis of trypsin digestion of EKODE-modified HSA ...... 75

Figure 3.6 LC-MS analysis of EKODE-modified His-338 peptide ...... 76

Figure 3.7 MS/MS product ion scan of peptide containing EKODE-modified His-338

(m/z 593.3595, +3) ...... 77

Figure 4.1 Design of antigen ...... 103

Figure 4.2 MALDI-TOF analysis ...... 107

Figure 4.3 Serum antibody titer in rabbits immunized with EKODE-Cys-spacer-KLH

...... 108

Figure 4.4 Epitope characterization of anti-EKODE-Cys antibodies by competitive

ELISA ...... 109

Figure 4.5 Cross reactivity of LPO products modified proteins ...... 111

Figure 4.6 Detection of EKODE-Cys adducts in BSA treated with linoleic acid under nonenzymatic oxidation conditions ...... 112

Figure 4.7 Detection of EKODE-Cys adducts in M17 cells treated with H2O2 ...... 114

Figure 4.8 Immunochemical detection of EKODE-Cys adducts in human brain tissue during the aging process ...... 116

Figure 4.9 Distribution of (A) EKODE Ib-Cys and (B) EKODE IIb-Cys adducts in mouse tissues ...... 118

Figure 4.10 Immunochemical detection of EKODE-Cys adducts in ischemic mouse heart

...... 120

LIST OF SCHEMES

Scheme 2.1 Proposed mechanism of isomerization from 4-hydroxy-acyl-CoA to 3- hydroxy-acyl-CoA via 4-P-acyl-CoA ...... 26

Scheme 3.1 Preparation of EKODE model compounds ...... 63

Scheme 3.2 Proposed mechanism of the ring opening reaction of EKODE II model compound with butanethiol ...... 66

Scheme 3.3 Synthesis the EKODE II derivatives and their reaction with butanethiol .....67

Scheme 3.4 Synthesis of trans-EKODE-(E)-Ib and trans-EKODE-(E)-IIb ...... 68

Scheme 4.1 Synthesis of EKODE-Cys-spacer-KLH conjugate as the antigen for antibody production ...... 105

vii

LIST OF TABLES

Table 2.1 Kinase candidates identified in the purification of pig liver and the BLAST results of amino acid sequences in human protein database ...... 40

Table 3.1 Adducts of EKODE model compounds with amino acid surrogates ...... 64

Table 3.2 Modified Cys/His/Lys residues in HSA treated with 1 mM EKODE ...... 74

Table 3.3 EKODE-modified peptides identified in HSA treated with EKODE at various molar ratio ...... 78

Table 4.1 Characterization of antigens and coating agents using MALDI-TOF and

TNBSA assay ...... 106

viii

ACKNOWLEDGEMENTS

First I want to express my sincere gratitude to my advisor and mentor Dr. Gregory

P. Tochtrop for providing me with the inspiration, wisdom, and passion necessary for me to continue my doctoral program, for giving me the motivation and freedom to grow as an independent researcher, for being supportive for me to pursue my career in pharmaceutical industry, and for always being there during my darkest times after I was diagnosed with hepatocellular carcinoma. It was my good fortune to have Dr. Tochtrop as my advisor and to work with him in the past years. His support and encouragement were far beyond what

I have ever expected and asked for. I am sincerely and deeply grateful for the tremendous time he has spent mentoring me and for everything he has done for me.

I would like to thank Dr. Henri Brunengraber and Dr. Guofang Zhang for providing the opportunity to perform LC-MS/MS based experiments in their labs. The collaboration with them expanded my horizon and the scope of this thesis. Their lab members Dr.

Qingling Li, Sophie Roussel-Kochheiser and John Koshy offered great support for my research and are highly appreciated.

I would also like to thank Dr. Xiongwei Zhu for offering the opportunity to carry out immunoassays in his lab and for generously providing human neuroblastoma cells and human brain tissues to test our antibodies. I would like to express my thanks to his lab members, Dr. Li Li, Dr. Wenzhang Wang, and Sandra L Siedlak, for teaching me the techniques and knowledge of immunoassays.

Special thanks to Dr. Naidong Weng and Dr. Wenying Zhang at Janssen

Pharmaceuticals for allowing me to use their instruments to carry out peptide mapping work described in Chapter 3. The knowledge and experience of LC-MS they shared with me served as a solid foundation for not only my thesis work, but also my career in pharmaceutical industry. Their support for my doctoral work and professional career is truly invaluable.

Sincere thanks to all my colleagues and friends I have met at CWRU. They made my PhD candidacy an enjoyable and memorable journey. In particular, I would like to thank my labmate, Dr. Jianye Zhang, for his guidance and support in my first two years at

CWRU. The synthetic skills I learned from him are essential for the work described in

Chapter 3. Dr. Sushabhan Sadhukhan provided great support for the protein purification work in Chapter 2. Dr. Yong Han has always been very helpful for all the instrumental work in our lab. His technical support and enlightening discussion are greatly appreciated.

I would like to thank my thesis committee members Dr. Rajesh Viswanathan, Dr.

Anthony J. Pearson, Dr. Michael G. Zagorski, and Dr. Henri Brunengraber for their guidance, support and encouragement throughout my time as a graduate student.

My most heartfelt thanks must go out to my parents. It was their encouragement and support that gave me the tenacity to finish my graduate studies. It was their unconditional love that brought me the light of hope at my darkest and the strength at my weakest. There are no words to express my gratitude for their guidance and support throughout every step of my life, and for everything they have given me. All I can say is thank you and I love you.

LIST OF SYMBOLS AND ABBREVIATIONS

4-HNE 4-hydroxy-2-(E)-nonenal

4-P-acyl-CoA 4-phosphoacyl-CoA

4-P-GHP-CoA 4-phospho-pentanoyl-CoA

2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-

ABTS diammonium salt

Ac-CoA acetyl CoA

ADH alcohol dehydrogenase

AHT Anhydrotetracycline

ALD alcoholic liver diseases

ALDH aldehyde dehydrogenase

ALE advanced lipoxidation end-product

AMBIC ammonium bicarbonate

AOR alkenal/one oxidoreductase

ATP Adenosine triphosphate

BCA bicinchoninic acid

BLAST Basic Local Alignment Search Tool

BSA bovine serum albumin

CAT catalase

CDS calibration delivery system

CHD coronary heart disease

CoA coenzyme A

CV column volume

DHN 1,4-dihydroxynonene

DMSO dimethyl sulfoxide

DNP 1,4-dinitrophenylhydrazine

EDC 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride

EKODEs epoxyketooctadecenoic acids

ELISA enzyme-linked immunosorbent assay

FA formic acid

FPLC fast protein liquid chromatography

G6P glucose 6-phosphate

GHB γ-hydroxybutyrate

GHB-CoAT 4-hydroxybutyrate-CoA transferase

GHP γ-hydroxypentanoic acid

GPx glutathione peroxidase

GSH glutathione

GSSG glutathione disulfide

GST glutathione S-transferase

HAA 4-hydroxynonanal

HHCOSY 1H-1H correlation spectroscopy

HNA 4-hydroxy-2-nonenoic acid

HPLC high performance liquid chromatography

HRMS high-resolution mass spectrometry

xii

HRP horseradish peroxidase

HSA human serum albumin

IDA information dependent acquisition

IEX ion-exchange chromatography

IMAC immobilized metal affinity chromatography

IPTG Isopropyl β-D-1-thiogalactopyranoside

IS internal standard

KLH keyhole limpet hemocyanin

LOOHs lipid hydroperoxides

LPO lipid peroxidation

MA mercapturic acid

MALDI-TOF matrix-assisted laser desorption/ionization-time

MDA malondialdehyde

MRM multiple reaction monitoring

NADH reduced nicotinamide adenine dinucleotide

NADPH reduced nicotinamide adenine dinucleotide phosphate

NFT neurofibrillary tangles

NHS N-hydroxysuccinimide

ONE 4-oxo-2-nonenal

PBS phosphate buffered saline

PCC pyridinium chlorochromate

PE phosphatidylethanolamine

PMA phosphomolybdic acid

xiii

PMSF phenylmethanesulfonylfluoride

PPL porcine pancreatic lipase

PPP pentose phosphate pathway

PUFAs polyunsaturated fatty acids

RNS reactive nitrogen species

ROS reactive oxygen species

SDS-PAGE sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SHPK sedoheptulokinase

SODs superoxide dismutases

SPE solid phase extraction

TBST Tris-buffered saline with Tween-20

TFA trifluoroacetic acid

THF tetrahydrofuran

TIC total ion current

TLC thin-layer chromatography

TNBSA 2,4,6-Trinitrobenzene sulfonic acid

Trx/TrxR thioredoxin/thioredoxin reductase

xiv

Protein Modification and Catabolic Fates of Lipid Peroxidation Products

Abstract By

Chuan Shi

The imbalance between the production of reactive oxygen/nitrogen species

(ROS/RNS) and their consumption by antioxidants leads to excess free radicals and peroxides, which can attack various components of cell or the entire organism.

Polyunsaturated fatty acids (PUFAs) are especially vulnerable to free radical mediated oxidation. The resulting oxidative degradation of PUFAs gives reactive bifunctional aldehyde such as 4-hydroxy-2-nonenal (4-HNE) as well as polyoxygenated products with the full carbon chain of the fatty acid such as epoxyketooctadecenoic acids (EKODEs).

Many of lipid peroxidation (LPO) products are highly electrophilic and can subsequently react with biological nucleophiles including protein side chains and DNA bases, consequently leading to enzyme inactivation and gene mutation. A number of metabolic fates of LPO products have been discovered and are critical for counteracting the oxidative damage. In addition, parallel catabolic pathways of 4-HNE were identified while the mechanism was not fully elucidated yet.

The first goal of my work was to generate a better understanding of the transformations involved in the catabolic pathways of 4-HNE. An LC-MS/MS based assay was developed to probe the presence of 4-hydroxy-acyl-CoA kinase activity, which generates 4-phosphoacyl-CoA (4-P-acyl-CoA) as a key intermediate during the

xv isomerization of 4-hydroxy-acyl-CoA to 3-hydroxy-acyl-CoA. This assay was used to guide the purification of the kinase from liver tissues. A number of purification techniques including ammonium sulfate precipitation, fast protein liquid chromatography (FPLC) and

SDS-PAGE were utilized. Protein sequencing of the resulting protein fractions gave five kinase candidates. Sedoheptulose kinase, the most relevant candidate with oxidative stress was tested for 4-hydroxy-acyl-CoA kinase activity.

In addition to the catabolic fates of LPO products, we also investigated the chemical nature and biological consequences of the protein modification by these molecules. A series of EKODE model compounds were synthesized and applied in the reaction with model nucleophiles to identify the adducts. Michael adducts of cysteine and histidine were discovered as well as an unexpected ring-opening products in the reaction of EKODE II model compound with cysteine analogues. The mechanism was found to be an intra- molecular rearrangement of the Michael adduct, which was revealed by the reactions of

EKODE II derivatives with butanethiol and confirmed by our analytical tools including

LC-MS and UV spectrometer. The reactions of EKODEs with HSA were also explored using LC-MS based assays, where Cys-34, His-67, His-146, and His-440 were found to be the most reactive residues. Those findings led us to further investigate the relevance of

EKODE-Cys adducts in aging and disease state. Polyclonal antibodies were produced and characterized, and subsequently immunoassays were developed to detect and quantify

EKODE-Cys adducts in biological samples. An H2O2 concentration dependent increase of

EKODE-Cys adducts was identified in neuroblastoma cells. The accumulation was also identified in human brain tissues during aging process as well as ischemic rat heart tissues.

xvi

Chapter 1

General Introduction

1.1 Oxidative stress

Oxidative stress has been associated with a growing list of disease states including

Alzheimer’s disease1, cardiovascular disease2 and cancer3. The molecular basis of oxidative stress can be explained by the imbalance between the production of reactive oxygen/nitrogen species (ROS/RNS) and their consumption by antioxidants. Excess

ROS/RNS are present during oxidative stress and they can attack various components of cell or the entire organism eventually leading to cell death.

ROS and RNS are free radicals and peroxides including hydrogen peroxide (H2O2),

- superoxide anion (·O2 ), hydroxyl radical (·OH), peroxy radical (ROO·), nitric oxide

(NO·), peroxynitrite (ONOO-), organic hydroperoxide (ROOH), alkoxy radical (RO·), and so forth4. The endogenous sources for ROS/RNS production are derived from the intracellular metabolism in mitochondrial and peroxisomes, as well as different cellular

5 enzyme systems such as NADPH oxidases . The consumption of O2 by mitochondrial leads

- 6 - to the production of superoxide ·O2 via a one-electron reduction . ·O2 itself is relatively stable, but it can combine with nitrogen monoxide (NO) to generate peroxynitrite7, 8, or dismute non-enzymatically or with the catalysis of superoxide dismutases (SODs) to form

9 H2O2 . These secondary products are more aggressive and can be further converted into various free radicals and peroxide or react with biomacromolecules such as proteins10.

At normal physiological conditions, ROS/RNS are critical to maintain homeostasis.

They can act as essential elements of host defense mechanism against tissue injury and infection, or generated as a result of the stimulation by growth factors to regulate a proliferative response11. In heathy tissue, the production of ROS/RNS is largely

counteracted by an antioxidant defense system that neutralizes and removes ROS/RNS.

Antioxidants include enzymatic scavengers such as SOD, which catalyzes the conversion

12 from superoxide to H2O2 , as well as catalase (CAT) and glutathione peroxidase (GPx), which detoxify cellular peroxides13. A number of non-enzymatic small molecules including glutathione (GSH)14, ascorbate15, and acetylcysteine16 also play an important role in scavenging ROS. During oxidative stress, the antioxidant defense system is compromised or overwhelmed by ROS/RNS production. The resulting excess ROS/RNS attack all cellular components such as proteins, DNA and lipids17. Consequently these reactions lead to DNA damage, mitochondrial malfunction, cell membrane damage and eventually cell death18, 19.

1.2 Lipid peroxidation

Although ROS can certainly function stochastically on all biomacromolecules to cause cell damage, polyunsaturated fatty acids (PUFAs) are especially vulnerable to free radical mediated oxidation due to their bis-allylic structures (Figure 1.1). The resulting oxidative degradation of PUFAs is termed lipid peroxidation (LPO). These chain reactions are initiated by ROS/RNS via abstraction of an active bis-allylic hydrogen from PUFA to generate a conjugated pentadienyl fatty acid radical, which subsequently reacts with an oxygen molecule to form a dienyl peroxyl radical (LOO·). This unstable species abstract an active bis-allylic hydrogen from a nearby PUFA to give a second pentadienyl fatty acid radical and a lipid hydroperoxide (LOOH). The pentadienyl fatty acid radical repeats this cycle to propagate the chain reaction, while hydroperoxide can undergo second round of

oxidation at different site of unsaturation to give polyoxygenated intermediate. The resulting intermediate is unstable and can undergo chain-cleaving fragmentation to give reactive bifunctional aldehyde such as 4-hydroxy-2-nonenal (4-HNE), 4-oxo-2-nonenal

(ONE), and malondialdehyde (MDA), acrolein, and so forth. Meanwhile, polyoxygenated products such as epoxyketooctadecenoic acids (EKODEs), which retain the full carbon chain of the fatty acid, were also discovered.

O Linoleic Acid

Oxidative stress ROS/RNS

O2 OO OO

Mechanism not fully elucidated

Epoxyketooctadecenoic Acids (EKODEs) Alkenal Series O O

(CH2)7COOH C5H11 H O O trans-EKODE-(E)-IIb 4-ONE(4-OHE) O O

(CH2)7COOH C5H11 H O OH trans-EKODE-(E)-Ib 4-HNE(4-HHE)

Figure 1.1 Production of lipid peroxidation products

1.3 Fates of LPO products and their relevance on disease states

1.3.1 Reactions with proteins and other endogenous nucleophiles

Many of LPO products are good electrophiles that are highly reactive with biological nucleophiles such as nucleophilic protein side chains and DNA bases.

Substantial efforts have been made by various groups during the past decades to understand the chemical nature and biological consequences of the reactions of LPO products with biological nucleophiles.

Figure 1.2 Fates of lipid peroxidation products

The most widely studied LPO-derived reactions are the formation of protein adducts. Given the alkenal structure of many LPO products, the high reactivity towards thiol and amino groups are responsible for the formation of most LPO-derived protein adducts (Figure 1.2). One of the most common reactions is Michael addition, where a nucleophilic protein residue such as cysteine, lysine, and histidine is added to a conjugated carbon-carbon double bond (C=C-C=O). Another common reaction is Schiff-base formation of carbonyl with primary amine of lysine, which often accounts for LPO-derived crosslinking of proteins. Once Michael adducts and Schiff base are formed initially, they can often undergo tautomerization, cyclization, oxidation, or dehydration to produce advanced lipoxidation end-products (ALE). For example, once the initial Michael adduct of 4-HNE is generated, it further undergo cyclization through the hydroxyl group at carbon

4 to form stabilized hemiacetal adducts20. In addition, an irreversible adduct, HNE-derived

2-pentylpyrrole, was identified in the reaction of 4-HNE and lysine residue. It is generated via Paal-Knorr condensation of the initial Schiff base adduct21, 22. This stable end product has been immunochemically detected in human serum samples using polyclonal antibodies at elevated levels in patients with cardiovascular disease or in end-stage renal disease23.

Furthermore, due to the bifunctional aspect of many LPO products such as 4-HNE

(electrophilic C-3 nad carbonyl group), they can form intra- or inter-molecular protein crosslinks via a combination of Michael addition at the conjugated carbon-carbon double bond and Schiff base formation at the carbonyl. Another protein crosslinking adduct of 4-

HNE was also identified as a lysine derived dihydro-pyrole derivative. This fluorophore is generated by two lysine residues and a 4-HNE molecule involving a Schiff base formation, a Michael addition, a series of oxidation, and finally a cyclization to give 2-hydroxy-2-

pentyl-1,2-dihydropyrrol-3-one iminium24, 25.

Although a conclusive definition of the role of LPO products in human pathology is yet to be fulfilled, direct correlations of LPO-derived protein modifications and many disease states have been identified26. It was estimated that 1-8% endogenously generated

4-HNE will modify proteins27. In most cases, these LPO-derived reactions impair the functions of protein by generating adducts at the active site to inhibit the enzymatic activity or initiating protein aggregation by forming crosslinks.

4-HNE has been widely reported to influence the activity of many enzymes due to its ability to rapidly form adducts with cysteine, histidine and lysine residue, as summarized in a review by Poli et al28. For examples, 4-HNE and 4-ONE covalently modify LKB1, a unique serine/threonine kinase tumor suppressor, at Cys210 residue, thereby inhibiting its

AMP-kinase activity and may facilitate tumor progression29. As in the case of the thioredoxin/thioredoxin reductase (Trx/TrxR) system, Cys32/Cys35 of Trx and

Cys496/Sec497 of TrxR are the primary targets of 4-HNE modification30. Because of the crucial role of Trx/TrxR system in redox regulation and many signaling pathways, the inactivation of its enzymatic activity by 4-HNE implies the potential impact of LPO products in fundamental cell functions.

LPO derived protein modification may also interfere normal physiology by mediating conformational changes of protein. Tau proteins are expressed predominately in neurons and interact with tubulin to form axonal microtubules. The formation of tau- containing neurofibrillary tangles (NFT) is considered as the key mediator of the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease, Parkinson

disease and Down syndrome31. The presence of HNE-lysine adducts of tau in the NFT region of brain tissues from AD patients is documented by different groups using immunohistochemical detection32-34. In addition, increased level of HNE-derived modification of tau is observed after treatment of normal tau with 4-HNE, but only when tau was in the phosphorylated state35. These findings indicate that the phosphorylation dependent modification of tau by LPO products are major conformational changes that contribute to NFT formation and consequent progression of neurodegenerative diseases.

Other endogenous nucleophiles such DNA bases can also react with LPO products.

A number of LPO-derived DNA adducts have been identified and they are considered to be responsible for DNA mutation and consequent disease state36, 37. 4-HNE, for example, can react with all four DNA bases with different reactivity: G>C>A>T38. The Michael adduct generated from the reaction of 4-HNE and the deoxyguanine moiety of DNA can undergo cyclization to give 1,N-2-propane adducts in the form of four diastereomers39, 40

(Figure 1.2) and the resulting mutagenicity of these adducts are dictated by their stereochemistry41. Another group of biological nucleophiles, phospholipids phosphatidylethanolamine (PE), which contain a primary amino group, can also produce adducts with HNE through Michael addition or Schiff base formation42. The subsequent cyclization of the Schiff base adduct gives a pyrrole derivative that is stable at pH from 6.5 to 8.543.

1.3.2 Metabolic fate

Due to the cytotoxicity of LPO products, and to the fact that these molecules have

a longer half-life compared with ROS/RNS, understanding the capability of organisms to inactivate or eliminate LPO products is critical, as it may give key insights into the pathogenesis of these molecules. Clear links have been made between increasing levels of

LPO products or LPO-derived protein adducts and various disease states such as

Alzheimer’s disease44, 45 and cardiovascular disease46. However, the physiological relevance is not necessarily originated from elevated production of LPO products. Rather, the cellular damage by LPO products can be rationalized by a diminished capability of tissues to catabolize and scavenge LPO products, resulting in accumulation of these reactive molecules and subsequent protein adducts47, 48.

The most dogmatic LPO product is 4-HNE and the largest portion of previous reports focuses on how this molecule react and modify the functions of various proteins/enzymes, while the knowledge of its metabolic fate is largely limited to the biotransformation with its intact nine carbon framework49. The major metabolic pathways involve the elimination of 4-HNE via glutathionylation by glutathione S-transferase (GST) and/or subsequent mercapturic acid (MA) formation, followed by excretion through kidney50, 51 (Figure 1.2). Other major metabolites include 4-hydroxy-2-nonenoic acid

(HNA) and 1,4-dihydroxynonene (DHN), which are the corresponding carboxylic acid and alcohol that are formed with the enzymatic catalysis by aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) respectively. The formation of glutathione or mercapturic acid conjugates can often be combined with oxidation/reduction of the 4-HNE moiety as well as the lactone formation of HNA to give urinary metabolites such as DHN-

GSH, HNA-GSH, and HNA-lactone-GSH52-54. Furthermore, the carbon-carbon double bond can be saturated by a NAD(P)H dependent alkenal/one oxidoreductase (AOR) to give

4-hydroxynonanal (4-HAA)55.

In addition to the metabolism studies, which dealt with the intact nine carbon framework, our group and our collaborators discovered the catabolic fate of 4-HNE47, 48. It was found that 4-HNE catabolism can proceed via two parallel pathways (Figure 1.2).

While they can be processed through a β-oxidation/α-oxidation sequence (Pathway A), the predominant pathway involves a phosphorylation and an isomerization of 4-hydroxy-acyl-

CoA to 3-hydroxy-CoA (Pathway B). Phosphorylated intermediates, 4-phosphoacyl-CoA

(4-P-acyl-CoAs), were also identified in extracts of rat livers perfused with C4 to C11 4- hydroxyacids. This observation indicates that 4-hydroxy-nonanoic acid, which is endogenously derived from the oxidation and saturation of 4-HNE, can be catabolically processed through a phosphorylation mediated pathway in addition to the common β- oxidation/α-oxidation pathways.

Parallel metabolic pathways are found in many fundamental pathways that are critical for normal physiology. For example, glycolysis is the process that converts glucose to pyruvate and it is one of the most dogmatic metabolic pathways that occurs in nearly all organisms. The fate of pyruvate is dictated by physiological conditions via different parallel pathways. In aerobic organism under normal conditions, pyruvate is catabolized to carbon dioxide through citric acid cycle. On the other hand, once the organism is transferred to anaerobic conditions, pyruvate can proceed down a parallel pathway to be catabolized to lactate. Other examples include bile acid biosynthesis via either neutral or acidic pathways56 and β-oxidation in either peroxisome or mitochondria. These examples further illustrate the importance of this parallelism as a fundamental process in normal physiology. In the case of 4-hydroxyacids catabolism, the catabolic pathway is so robust

that liver can utilize the liberated acetyl-CoA, propionyl-CoA, and formate as its primary carbon source47. Given the robust nature and the carefully evolved parallelism, this catabolic pathway may play an important role in either normal physiology or disease pathogenesis. A highly plausible hypothesis is that loss of capability to catabolically scavenge LPO products lead to their accumulation and subsequently contribute to disease progression. Considering the existence of parallel pathways, it is also possible that different pathways may dominate in different physiological conditions or in different tissues.

1.4 Research Strategy

1.4.1 Characterization of the enzymes in catabolic pathway of 4-HNE

Although the parallel catabolic pathways of 4-HNE have been discovered by our lab and our collaborators, the mechanism of the transformation of 4-hydroxyacyl-CoA to

3-hydroxyacyl-CoA is not fully elucidated yet. The presence of the phosphorylated intermediate 4-P-acyl-CoA indicates that a kinase-driven reaction is a key step in the novel catabolic pathway. Characterization of this kinase will lead to a better understanding of the mechanism as well as the pathological implication of 4-HNE catabolism. Furthermore, when we carefully exam the structures of EKODEs, the most intuitive route by which they would potentially be catabolized is via initial rounds of β-oxidation until the internal α,β- unsaturated carbonyl epoxides are encountered. The resulting functionality, especially for

EKODE-Ib series, would be quite similar to 4-hydroxyakenal series (Figure 1.3). It is possible that after four cycles of β-oxidation, trans-EKODE-(E)-Ib can undergo similar pathways as 4-HNE to produce catabolic end products. Therefore, a better understanding

of the catabolism of 4-HNE may provide insights into the catabolic fates of other LPO products.

O O

OH O trans-EKODE-(E)-Ib

-Oxidation (4 Cycles)

SCoA O

Similar catabolic pathways as 4-HNE?

Metabolic/catabolic end products

Figure 1.3 Proposed metabolic pathway of EKODEs

Although we simultaneously discovered the parallel catabolic pathways of many 4- hydroxy-n-acids (C4-C11), we started the identification of the kinase from γ- hydroxypentanoic acid (GHP). The first and foremost reason behind this decision is that 4- hydroxy-4-phospho-pentanoyl-CoA (4-P-GHP-CoA) was the most abundant 4-P-acyl-

CoAs that was identified in extracts of rat livers perfused with C4 to C11 4-hydroxy-n- acids. Therefore, this molecule represents our best chance of probing the presence of the kinase activity. Furthermore, GHP is readily available and it can be utilized by γ- hydroxybutyrate-CoA transferase (GHB-CoAT) to incorporate CoA moiety. It allows

synthesis of large quantity of GHP-CoA, which was used as substrate in our kinase assay.

An LC-MS/MS based assay was developed to detect the production of 4-P-GHP-

CoA as a result of kinase activity. This assay was then used to guide the purification of liver homogenates, which involved ammonium precipitation, ion-exchange chromatography, affinity chromatography, and gel electrophoresis. The resulting protein bands were subjected to protein sequencing to identify kinase candidates.

1.4.2 Investigation of the relevance of EKODEs in diseases or aging

Despite the fact that a large number of protein adducts generated by LPO products have been discovered, the knowledge of the reactivity of many poly-oxygenated products such as EKODEs is still limited. The study of EKODE-derived protein modifications will lead to a more comprehensive understanding of the toxicity and biological relevance of

LPO products.

In an effort to investigate the detailed mechanism of the formation of EKODE- derived protein modification, we started our project from model studies using individual amino acids since the products of their reactions with EKODEs can be isolated by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), and their structures can be determined by UV/Vis, NMR and LC-MS. In addition, instead of

EKODEs and amino acids with full structure, EKODE model compounds and surrogates of nucleophilic amino acids were first used in model studies to simplify the synthesis of

EKODEs and the characterization of adducts. After the mechanisms of adducts formation were revealed, the reactivity of EKODEs with full structure was confirmed by monitoring

their reactions with amino acid analogues using LC-MS. EKODEs were then incubated with model protein at various molar ratio in physiological buffer. The resulting mixture was subjected to enzymatic digestion and subsequent peptide mapping by high-resolution mass spectrometry (HRMS) to reveal modification sites.

Once the reactions of EKODEs with model protein were elucidated, we proceeded to investigate the relevance of EKODE-derived protein modification in aging and disease states. An immunochemical approach was carried out since antibodies are able to bind various proteins that are modified by the same LPO product of interest, and therefore, it defines the overall impacts of protein modification derived from specific LPO product.

Since the cysteine thiol was found to be the most reactive amino acid toward EKODEs and stable cysteine adducts were discovered, we synthesized conjugates of EKODE-Cys adducts and carrier protein, which were used to generate anti-EKODE-Cys polyclonal antibodies. The antibodies were purified and characterized by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Immunoassays were then performed to evaluate the relative level of EKODE-Cys adducts in oxidative stress, cardiovascular diseases, and aging.

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Chapter 2

Identification of Relevant Enzymes in the Catabolic

Pathways of 4-Hydroxyacids and Lipid Peroxidation

Products

2.1 Introduction

Many of the lipid peroxidation (LPO) products generated from oxidative stress are highly electrophilic and can subsequently react with nucleophilic protein side chains and

DNA bases via Michael addition or formation of Schiff bases1, 2. The resulting adducts may undergo a series of secondary reactions to form stable end products. Substantial efforts have been made to ascertain the chemical nature of protein modification by LPO products and how the modification modulates the activities of proteins and enzymes3-5.

Considering the cytotoxicity of LPO products and the fact that they are usually much more stable than ROS, the capability of organisms to inactivate or eliminate LPO products is critical for counteracting the oxidative damage caused by protein/DNA modification of LPO products. The metabolic fates of the most dogmatic LPO products, 4- hydroxy-2-(E)-nonenal (4-HNE), have been extensively studied6-8. The major transformations include the oxidation or reduction of the aldehyde functional groups to generate 4-hydroxy nonanoic acid or 1,4-dihydroxy-2-nonene, saturation of the C=C double bond, and glutathionylation catalyzed by glutathione S-transferases (GSTs)9.

In addition to the metabolism studies, which dealt with the intact nine-carbon framework, our group and our collaborators discovered the catabolic fate of 4-HNE10, 11. It was found that 4-HNE catabolism can proceed via two parallel pathways. While they can be processed through a β-oxidation/α-oxidation sequence, the predominant pathway involves a phosphorylation and an isomerization of 4-hydroxy-acyl-CoA to 3-hydroxy-

CoA. Phosphorylated intermediates, 4-phosphoacyl-CoA (4-P-acyl-CoAs), were also identified in extracts of rat livers perfused with C4 to C11 4-hydroxyacids (Figure 2.1). This

observation indicates that 4-hydroxyacids, which are either exogenously introduced through drugs of abuse including γ-hydroxybutyrate (GHB) or endogenously derived from the oxidation and saturation of 4-HNE, can be catabolically processed through a phosphorylation mediated pathway in addition to the common β-oxidation/α-oxidation pathways.

Figure 2.1 4-P-acyl-CoAs identified in extracts of rat livers perfused with C4 to C11 4- hydroxyacids. Each peak denotes the 4-P-acyl-CoAs derived from corresponding 4- hydroxyacid.

The proposed mechanism of isomerization from 4-hydroxy-acyl-CoA to 3- hydroxy-acyl-CoA via 4-P-acyl-CoA is shown in Scheme 2.1. The key step is the phosphorylation of 4-hydroxy-acyl-CoA, which is likely driven by a kinase action. The resulting 4-P-acyl-CoA undergoes dehydrogenation, hydration and dephosphorylation to yield the enol form of 3-ketoacyl-CoA, which is then processed through cycles of β- oxidation to give short chain acyl-CoAs.

Scheme 2.1 Proposed mechanism of isomerization from 4-hydroxy-acyl-CoA to 3- hydroxy-acyl-CoA via 4-P-acyl-CoA

Parallel metabolic pathways are found in many fundamental pathways that are critical for normal physiology. For example, glycolysis is the process that converts glucose to pyruvate and it is one of the most dogmatic metabolic pathways that occurs in nearly all organisms. The first step in glycolysis is the phosphorylation of glucose facilitated by hexokinases including glucokinase. The resulting glucose 6-phosphate (G6P) can be processed through the glycolysis pathway to produce pyruvate and release energy in the form of reduced nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP). G6P can also enter pentose phosphate pathway (PPP), which is a metabolic pathway parallel to glycolysis, to generate pentoses and reduced nicotinamide adenine dinucleotide phosphate (NADPH). In addition to the two major metabolic pathways, G6P may also be converted to glycogen to store excess glucose. Due to the prominent position and multiple fates of G6P, the activity of glucokinase plays an important role in the regulation of carbohydrate metabolism. Clear links have been made between the mutations of glucokinase gene and metabolic diseases such as type 2 diabetes12, 13 and hyperinsulinemic hypoglycemia14. The research of glucokinase has led to the discovery of glucokinase activators as new pharmacotherapy for diabetes15-17.

Therefore, identification of the kinase involved in the catabolic pathway of 4-hydroxyacids as well as 4-HNE and other LPO products may lead to important discovery in the biological relevance of LPO products in normal physiology and diseases.

In this chapter, we developed a LC-MS/MS based assay to probe the presence of 4- hydroxy-acyl-CoA kinase activity. This assay was used to guide the purification of the kinase where crude liver homogenates were purified via ammonium sulfate precipitation and various chromatography techniques. Given the prominent role of kinases in metabolic

pathways, identification of the kinase may give key insights into the metabolic fates of 4- hydroxyacids and 4-HNE. In addition, many of other LPO products such as EKODEs have a similar core structure as 4-HNE. Consequently, understanding the metabolism and relevant enzymes for 4-HNE may facilitate the metabolic study of other LPO products.

2.2 Results and Discussions

2.2.1 Kinase activity assay

To guide the purification of potential kinase involved in the metabolism of 4- hydroxyacids including 4-hydroxynonenal, a kinase activity assay needs to be established to probe the presence of kinase in the extraction from crude biological materials. The most reliable methods for detection of kinase reactions are radiometric-based assays, which are performed in the presence of 32P-γ-ATP or 33P-γ-ATP18. The use of radioisotopes, however, presents a major limitation for its application in large scale of enzyme purification. Another widely used approach for kinase screening is a fluorescence-based assay to detect the production of ADP or consumption of ATP from kinase reactions19. But this universal kinase activity assay is limited by the selectivity and may be not suitable for the purpose of kinase purification. Therefore, a LC-MS/MS based kinase activity assay was developed to identify the production of 4-P-acyl-CoAs in the kinase-catalyzed reaction. A multiple reaction monitoring (MRM) method targeting at the product of the kinase reaction provides a selective and sensitive approach to probe the kinase activity.

4-Hydroxy-4-phospho-pentanoyl-CoA (4-P-GHP-CoA) was the most abundant 4-

P-acyl-CoA that was identified under identical LC-MS/MS conditions in extracts of rat

livers perfused with 2 mM of C4 to C11 4-hydroxy-n-acids11. It has also been well characterized by 31P-NMR and LC-MS/MS. Therefore, γ-hydroxypentanoyl-CoA (GHP-

CoA) was prepared to be used as the substrate of the kinase activity assay. The most common approach for the synthesis of acyl-CoA is the reaction of acid anhydride with

CoA20, 21. However, this method was not successful for the synthesis of GHP-CoA due to the facile formation of γ-hydroxypentanoic lactone22.

Figure 2.2 SDS-PAGE analysis of the expression of GHB-CoA transferase

An enzymatic approach, therefore, was developed using recombinant γ- hydroxybutyrate-CoA transferase (GHB-CoAT)23, which has been reported to be able to

use GHP as substrate24. The expression cell strain, E. coli BL21 CodonPlus(DE3)-RIL transformed with pASK-IBA(3+) vector containing GHB-CoAT gene, was grown aerobically at 37 °C in standard-I-medium with carbenicillin and chloramphenicol. At an

OD578 of 0.5-0.6, the culture was induced with anhydrotetracycline (AHT) and incubated for 5 hours. The heterologous expression resulted in the production of recombinant GHB-

CoAT with C-terminal fused to a Strep-tag for affinity purification. After cell lysis with sonication, the protein purification of the resulting supernatant was performed based on the highly effective interaction between the Strep-tag and the Strep-Tactin resin column. GHB-

CoAT was eluted with desthiobiotin and subjected to SDS-PAGE analysis. As shown

Figure 2.2, GHB-CoAT with high purity appeared at 49 kDa after multiple wash and elution steps. The purified GHB-CoAT can be stored at -80 °C for years without loss of activity.

GHP was synthesized by the base-catalyzed hydrolysis of GHP lactone in 0.1 M

NaOH (Figure 2.3 A). Meanwhile, acetyl CoA (Ac-CoA) was prepared by the reaction of free CoA with acetic anhydride, which subsequently served as a CoA donor to convert

GHP to GHP-CoA in the presence of GHB-CoAT. The reaction mixture was characterized by LC-MS/MS using the typical transitions of CoA esters, which contain daughter ions generated from the loss of a non-variable fragment (m/z 507) from phosphate backbone of

CoA25. As shown in Figure 2.3 B, the majority of free CoA was converted to Ac-CoA

(peak height ~ 7.3E5) with only a small amount remained (peak height ~ 4.5E4). The yield of the conversion of GHP-CoA from Ac-CoA was estimated to be around 30% based on the ratio of the peak height assuming the MS response of both compounds were similar.

XIC of +MRM (5 pairs): 768.000/261.000 Da ID: CoA from Sample 37 (GHP-CoA 5uM_Zorbax C8_2.1X100mm_0.4mL/min) of CoA std.wiff... Max. 4.5e4 cps.

5.42 4.5e4 4.0e4 Free CoA 3.0e4 m/z = 768/261 1.88 2.0e4

Intensity, cps 1.04 1.0e4 2.23 9.28 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min XIC of +MRM (5 pairs): 810.000/303.000 Da ID: Acetyl CoA from Sample 37 (GHP-CoA 5uM_Zorbax C8_2.1X100mm_0.4mL/min) of CoA ... Max. 7.3e5 cps.

3.16 7.3e5

6.0e5 Acetyl-CoA m/z = 810/303 4.0e5

1.04 3.43

Intensity, cps Intensity, 2.0e5 2.89 3.86 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min XIC of +MRM (5 pairs): 868.000/361.000 Da ID: GHP-CoA from Sample 37 (GHP-CoA 5uM_Zorbax C8_2.1X100mm_0.4mL/min) of CoA s... Max. 3.5e5 cps.

5.40 3.5e5 3.0e5 GHP-CoA

2.0e5 m/z = 868/361

1.04

Intensity, cps 1.0e5

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min

Figure 2.3 Synthesis of GHP-CoA. (A) Synthetic route; (B) LC-MS/MS analysis of the reaction catalyzed by GHB-CoAT showing XIC of free-CoA (top panel), acetyl-CoA

(middle panel), and GHP-CoA (bottom panel)

To test the kinase activity assay, fresh pig liver was homogenized in Kbrebs buffer and incubated with GHP-CoA and ATP in Tris buffer containing magnesium chloride and phosphatase inhibitor. The reaction was conducted at 37 °C for 30 minutes and the mixture was partially purified by solid phase extraction (SPE) based on anion exchange. d9-

Pentanoyl-CoA, an isotopic labeled acyl-CoA, was added prior to sample cleanup as internal standard (IS) to ensure assay accuracy, and therefore, the variability in SPE and

LC-MS/MS could be compensated by IS. The production of 4-P-GHP-CoA as a result of kinase activity was monitored by the MRM transition of m/z 948>441. In the reaction using liver homogenate, 4-P-GHP-CoA was successfully identified (Figure 2.4). Meanwhile, a negative control was also performed using the same reaction condition without adding

ATP, which is an essential component of the kinase reaction. As a result, no significant amount of 4-P-GHP-CoA was detected. Zhang et al. reported that the concentration of 4- phosphobutyryl-CoA was at low pmol/g in the liver of wild-type mice. Since only small amount (≤ 4 mg) of liver homogenate was used for kinase reaction, it was likely that the endogenous level of 4-P-GHP-CoA in pig liver was negligible and did not have interference with the kinase activity assay.

Figure 2.4 Kinase activity assay using GHP-CoA as substrate. Top panel: negative control using liver homogenate without adding ATP; Bottom panel: positive control using liver homogenate.

2.2.2 Kinase purification

The GHP-CoA kinase activity has been identified in extracts of rat livers perfused with 4-hydroxy-n-acids. However, the cost and availability of rat liver present the major limitations for its use as the source of GHP-CoA kinase. Therefore, pig liver was selected as the starting material due to the availability of large quantities of fresh tissues, and because it showed comparable activity in our initial kinase assay test (data not shown).

Freshly obtained pig liver was homogenized with Krebs buffer in a blender and the soluble fraction was subjected to ammonium sulfate precipitation. It was found that the kinase activity appeared in both fractions obtained from 30% and 45% saturated ammonium sulfate (Figure 2.5). However, the kinase activity in 45% fraction was 73% higher than that was in 30% fraction and a much larger quantity of protein was obtained from 45% saturated ammonium sulfate. Therefore, only the 45% fraction was subjected to desalting by dialysis in 10 mM Tris buffer with 10 mM NaCl and subsequently purified by fast protein liquid chromatography (FPLC) based on ion-exchange (IEX).

2.0E‐02 1.8E‐02 1.6E‐02 1.4E‐02 1.2E‐02 1.0E‐02 8.0E‐03 6.0E‐03 4.0E‐03

Peak Area Ratio (P‐GHP‐CoA/IS) 2.0E‐03 0.0E+00 15‐30 30‐45 45‐60 Homogenate Ammonium sulfate saturation (%)

Figure 2.5 Kinase activity test of ammonium sulfate precipitation of pig liver homogenate

The IEX chromatography was performed by loading the dialyzed ammonium sulfate fraction to a SP-Sepharose fast-flow column (cation-exchange) and eluting using a

10 mM - 1 M NaCl gradient (Figure 2.6 A). The unbound proteins were washed out quickly as indicated by one major protein peak after 5 minute (fraction 3 and 4), which showed noticeable GHP-CoA kinase activity (Figure 2.6 B). The presence of kinase in fraction 3 and 4 was likely due to the fast flow rate (20 mL/min) applied in IEX purification that washed out a small amount of the kinase. The majority of kinase activity was found to be located in fractions 22-25 and it appeared relatively unstable in the eluting buffer.

Therefore, the fractions were pooled and immediately subjected to desalting by dialysis with molecular weight cutoff of 3500 Da.

The fractions from IEX were then applied to Blue-Sepharose fast flow column for affinity purification. The blue dye in the Blue-Sepharose column binds several enzymes including kinases and dehydrogenases, and the interaction can be disrupted by high concentration of salts. After washing out unbound proteins, the resin-binding proteins were eluted using a multiple-step gradient of NaCl (Figure 2.7). Fraction 9, which contained the majority of the kinase activity, was eluted in the early stage of the gradient indicating that

GHP-CoA kinase had only weak binding interaction with the resin.

The fraction from a Blue-Sepharose column was then concentrated by centrifugal filter (10 kDa cutoff), resolved by 15% SDS-PAGE, and visualized by Coomassie Blue staining (Figure 2.8). Several individual protein bands were identified, indicating that most of the hepatic proteins from the crude homogenate of pig liver were removed by previous purification steps, whereas some co-eluted proteins were still present. The selected protein bands or sections were then excised and sent to Taplin Mass Spectrometry Facility

(Harvard Medical School, MA, USA) for protein sequencing via peptide mass fingerprinting.

B 9 8 7 6 5 4 3 2 Activity (pM/mg/min) 1 0 3 18192021222324252627282930313335 Fractions

Figure 2.6 FPLC purification of 30-45% AS precipitate using IEX. (A) FPLC chromatography; (B) Kinase activity assay of FPLC fractions

B 30

Activity (pM/mg/min) 5

0 2 9 10 11 15 16 Fractions

Figure 2.7 FPLC purification of IEX fractions 22-25 using Blue-sepharose. (A) FPLC chromatography; (B) Kinase activity assay of FPLC fractions

Blue-Sepharose Protein marker Fraction 9

66 kDa

45 kDa

18.4 kDa

Figure 2.8 SDS-PAGE analysis of Blue-sepharose fraction 9. Left lane: concentrated Blue-

Sepharose fraction 9, selected gel regions were excised for protein sequencing; Right lane: protein marker.

2.2.3 Verification of kinase candidates

The identified peptides were searched in the protein database of pig (Sus scrofa) and proteins with three or more unique peptide matches was considered confidently identified from the sample. The identified protein candidates were manually examined to confirm the experimental molecular weight based on SDS-PAGE matched the theoretical value. Since many proteins in pig database are listed as uncharacterized, a homology search of amino acid sequences in human protein database was performed for the identified pig protein candidates using Basic Local Alignment Search Tool (BLAST) and kinase domain was found in 5 protein candidates (Table 2.1). Since an identity of 25% or higher indicates similarity of function, the proteins identified from the BLAST search in human protein database, which have identity scores of 87% - 100%, are considered as the human counterparts of the pig kinases.

KAD3 belongs to the well-characterized adenlylate kinase family that catalyzes the interconversion of three adenine nucleotides (ATP/ADP/AMP) in the cell26. The protein kinases CSK2127 and CPNE328 phosphorylate the hydroxyl group of serine or threonine of protein, while PNKP is involved in the 5'-phosoharylation of nucleic acids29. Among the five kinase candidates, sedoheptulokinase (SHPK) presents the highest relevance with oxidative stress and the metabolism of LPO products. SHPK converts the sedoheptulose to sedoheptulose-7-phosphate and plays an important role in the pentose phosphate pathway

(PPP)30. Since PPP generates NADPH and is part of the major cellular antioxidative

31 machinery , SHPK has been found to promote yeast’s tolerance of H2O2 induced oxidative stress32 and has also been reported by Haschemi et al. to act as a regulator of cellular metabolism and the redox state33.

Table 2.1 Kinase candidates identified in the purification of pig liver and the BLAST results of amino acid sequences in human protein database

Protein sequencing result BLAST result in human protein database

Peptides mass Entry name Matched protein (gene name) Substrate Identity matched (Da)

GTP:AMP phosphotransferase Adenosine F1SK45_PIG 8 25,594 92% (KAD3) monophosphate Casein kinase II subunit alpha Protein F2Z5I5_PIG 4 45,144 100% (CSK21) serine/threonine Sedoheptulokinase F1RLI9_PIG 7 51,491 Sedoheptulose 87% (SHPK) Polynucleotide 5'-dephospho- F1RHU1_PIG 6 57,532 88% phosphatase/kinase (PNKP) DNA Copine-3 Protein F1RXD5_PIG 3 59,644 95% (CPNE3) serine/threonine

Therefore, to ascertain if SHPK presents the kinase activity for the phosphorylation of GHP-CoA, mouse SHPK with a polyhistidine-tag was overexpressed in E. coli at 30 °C for 4 hours in the presence of isopropyl β-D-1-thiogalactopyranoside (IPTG) as the inducer34. After cell lysis by freeze-thaw, the extract was purified by Ni2+ based immobilized metal affinity chromatography (IMAC). The protein was then tested for the sedoheptulokinase activity using a LC-MS/MS based assay. Sedoheptulose was incubated with recombinant SHPK in the presence of ATP and Mg2+ and sample cleanup was performed by protein precipitation using organic solvents. The production of sedoheptulose-7-phosphate was monitored by MRM transition of m/z 289>97 as reported by Vas et al35. The reaction of the recombinant SHPK with sedoheptulose resulted in a distinctive peak after 6.28 minutes, which was consistent with the sedoheptulose-7-

phosphate standard (Figure 2.9). Meanwhile, the negative control, which was performed using the same conditions without adding ATP, did not show a detectable amount of phosphorylated products. These results suggest that protein expression was successful and the recombinant protein was active toward sedoheptulose, its known substrate.

6 1.8e6 6.28 6 Activity test of 6 1.2e66 recombinant SHPK 6 m/z = 289/97 5 6.0e55 5

Intensity, cps 5

0 1.8e66

6 6 Negative control 1.2e66 6 without adding ATP 5 m/z = 289/97 6.0e55 5

Intensity, cps 5

2.08 3.12 3.49 6.32 7.32 0 1.8e66

6 6.63 Sedoheptulose‐7‐ 1.2e66 6 phosphate standard 5 m/z = 289/97 6.0e55 5

Intensity, cps 5

0 10 20 3.030 40 50 6.060 70 80 9.090 10 0 11 0 12.012 0 Time, min

Figure 2.9 SHPK activity test of recombinant SHPK.

Recombinant SHPK was then tested for the GHP-CoA kinase activity using the

LC-MS/MS based assay described above. Unfortunately, 4-P-GHP-CoA was not observed in the reaction of SHPK with GHP-CoA (Figure 2.10), and therefore, SHPK is not likely the kinase involved in the metabolism of 4-hydroxy acids. Ongoing efforts are being made to identify GHP-CoA kinase from the other four kinase candidates obtained in our liver purification, as well as other unknown proteins.

Figure 2.10 GHP-CoA activity test of (A) recombinant SHPK; (B) positive control using liver homogenate

2.3 Conclusions

In this chapter, a LC-MS/MS based kinase activity assay was developed to monitor the presence of GHP-CoA kinase activity. The assay was used to guide the purification of

GHP-CoA kinase from pig liver, which started from ammonium sulfate precipitation of liver homogenate and was followed by FPLC purification using IEX chromatography and affinity chromatography. The FPLC fraction was resolved by SDS-PAGE and the protein bands were subjected to protein sequencing. Five kinase candidates were identified and sedoheptulose kinase, the most relevant candidate with oxidative stress, was successfully expressed. While it demonstrated sedoheptulose kinase activity, the enzyme did not show activity in the GHP-CoA kinase assay. Therefore, the remaining kinase candidates, as well as other potential enzyme candidates, will be tested to identify the kinase involved in the catabolism of 4-HNE and 4-hydroxyacids.

2.4 Experimental Sections

2.4.1 Materials and Methods

General chemicals, organic solvents, D-sedoheptulose, D-sedoheptulose-7- phosphate lithium salt, SPE cartridge (2-(2-Pyridyl) ethyl Silica Gel, Anion exchange, bed wt 100mg, vol 1 mL), EZBlue gel staining reagent were purchased from Sigma-Aldrich

(St. Louis, MO, USA). 5 × Kinase buffer I was purchased from Alfa Aesar (Ward Hill,

MA, USA). Protease inhibitor cocktail tablets (cOmplete, EDTA-free) were purchased from Roche (Indianapolis, IN, USA). Fresh whole pig livers were obtained from a local farm. Dialysis cassettes (3.5K MW cutoff, 12 mL) were purchased from Pierce (Waltham,

MA, USA). Amicon Ultra-15 Centrifugal Filter Units (10 kDa MW cutoff) were purchased from EMD Millipore (Billerica, MA, USA). SP-Sepharose Fast Flow, Blue-Sepharose 6

Fast Flow, Ni-Sepharose 6 fast flow resin, and PD-10 desalting columns were purchased from GE Healthcare (Pittsburgh, PA, USA). 2 × Laemmli Sample Buffer was from Bio-

Rad (Hercules, CA, USA). E. coli BL21 CodonPlus(DE3)-RIL with pASK-IBA(3+) vector containing GHB-CoAT gene was obtained from Dr. Wolfgang Buckel (Philipps-

Universitat, Germany). E. coli BL21(DE3) pLysS with pET-15b expression vector containing the open-reading-frame of mouse SHPK was obtained from Dr. Emile Van

Schaftingen (Universite´ Catholique de Louvain, Belgium).

SDS-PAGE. SDS Polyacrylamide gel and SDS running buffer was prepared according to standard protocols. 100 μL of protein solution or protein marker was mixed with 100 μL of 2 × loading buffer containing 2-mercaptoethanol and heated in boiling water for 10 minutes. The samples were then loaded to the wells of the gel and separated

at 100 V. The gel was then transferred to a plastic container and covered with EZBlue gel staining reagent. After shaking at r.t. for 2 hours, the gel was washed in 10% acetic acid

(v/v) for destaining until clear bands were observed.

LC-MS/MS analysis. LC-MS/MS employed an Ultimate 3000 HPLC system

(Sunnyvale, CA, USA) with autosampler that maintains samples at 4 °C. A Zorbax 300SB-

C8 column (2.1 mm × 100 mm, 3.5 m, Agilent, Santa Clara, CA) was used for LC separation at a flow rate of 0.2 mL/min. Mobile phase A was 100 mM ammonium formate in 95% water 5% acetonitrile pH 5, and mobile phase B was 95% acetonitrile 5% water with 5 mM ammonium formate.

For monitoring acyl-CoAs in the synthesis of GHP-CoA and 4-P-GHP-CoA in the kinase activity assay, a multiple-step gradient was as following: 0-1 min (2% B), 1-4 min

(2%-90% B), 4-6 min (90% B), 6-6.5 min (90%-2% B), and 6.5-15 min (2% B). The divert valve was set as: 0-1 A, 1-8 B, 8-15 A. 10 L of purified reaction mixture was separated by LC and subjected to MS analysis on a QTRAP 4000 mass spectrometer (AB Sciex,

Foster City, CA, USA). The ion source parameters in positive turbo ionspray mode were as follows: curtain gas 30 psi, collision gas high, GAS1 55 psi, GAS2 55 psi, ionspray voltage 5500 V, and source temperature 500 °C. For monitoring acyl-CoAs, the MS parameters were set as following:

Q1 Q3 Dwell Time (ms) ID DP EP CXP CE 768 261 50 CoA 90 10 10 50 810 303 50 Acetyl CoA 90 10 10 50 868 361 50 GHP-CoA 90 10 10 51 948 441 50 4-P-GHP-CoA 90 10 10 51 861 354 50 d9-Pentanoyl-CoA 90 10 10 51

For monitoring the production of sedoheptulose-7-phosphate in the SHPK activity test, an Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 m, Agilent) were used for LC separation at a flow rate of 0.2 mL/min. Mobile phase A was 750 mg/L ammonium formate in 90% water 10% acetonitrile pH 7.5, and mobile phase B was 90% acetonitrile 10% water with 750 mg/L ammonium formate. A multiple-step gradient was as following: 0-2 min

(2% B), 2-10 min (2%-60% B), 10-11 min (60-90% B), 11-16 min (90% B), 16-17 min

(90-2% B), 17-26 min (2% B). The divert valve was set as: 0-2 A, 2-10 B, 10-26 A. 10 L of purified reaction mixture was separated by LC and subjected to MS analysis on a

QTRAP 4000 mass spectrometer. The ion source parameters in negative turbo ionspray mode were as follows: curtain gas 30 psi, collision gas high, GAS1 50 psi, GAS2 50 psi, ionspray voltage -4500 V, and source temperature 500 °C. Sedoheptulose-7-phosphate was monitored using MRM transitions of -289/97. The MS parameters were set as following:

DP -47, EP -10, CE -29, and CXP 4.

2.4.2 GHP-CoA kinase activity assay

Expression of GHB-CoAT. E. coli BL21 CodonPlus(DE3)-RIL containing GHB-

CoAT gene was grown aerobically at 37 °C in standard-I medium (1.5 % Pepton, 0.3% yeast extract, 100 mM NaCl and 6 mM D-Glucose) with carbenicillin 50 mg/L and chloramphenicol 50 mg/L. When OD578 reached 0.5-0.6, AHT (400 μg/L) was added to the culture and induction was performed at r.t. for 4 hours. The harvested cells were resuspended in PBS and disrupted by sonication (3 × 5 minutes). The resulting supernatant from centrifugation was applied to Strep-tag column (5ml, IBA GmbH, Germany), which was previously equilibrated with washing buffer (100 mM Tris-HCl, 150 mM NaCl and 2 mM DTT). The column was then washed with 50 mL washing buffer and eluted in 1 mL

fractions by washing buffer containing 2.5 mM desthiobiotin. The purification steps including cell lysis, washing and eluting were evaluated by 15% SDS-PAGE and the gel was visualized by Coomassie blue. The fractions containing GHB-CoAT were stored at -

80 °C.

Synthesis of GHP-CoA. GHP was synthesized by the hydrolysis of valerolactone in basic condition. 1.1 mL of 1M NaOH was added to 8.9 mL of water, followed by addition of valerolactone (95 μL, 0.879 mmol). The mixture was incubated at 60 °C for 3 hours and cooled to room temperature. CoA trilithium salt (20 mg, 25.5 μmol) was dissolved in 2 mL of 0.1 M KHCO3 buffer and 40 μL of acetic anhydride was added. The reaction was performed at 4 °C to allow the formation of acetyl-CoA. 2 mL of 125 mM

KH2PO4 (pH 7.0) and 2 mL of GHP mixture were added to the acetyl-CoA solution. After adjusting the pH to 7, 400 μL of GHB-CoAT solution was added to the above mixture and the reaction was incubated at r.t. for 45 minutes. 10 μL of the mixture of injected was subjected to LC-MS/MS analysis to identify the formation of GHP-CoA. The final concentration of GHP-CoA was estimated to be around 0.85 mM based on the LC-MS/MS signal ratio to acetyl-CoA.

Kinase activity assay. 20 μL of protein fractions, 20 μL of GHP-CoA, 10 μL of

100 mM ATP and 15 μL of kinase buffer (100 mM Tris-HCl, 50 mM magnesium chloride,

5 mM dithiothreitol, and 5 mM sodium orthovanadate, pH 7.4) were added in an Eppendorf tube and mixed by gentle vortex. After incubating at 37 °C for 30 minutes, 10 μL of 0.02 mM d9-pentanoyl-CoA was added to the reaction and mixed. The SPE cartridge was activated by adding 1 mL of methanol followed by 1 mL of loading buffer (methanol/5% acetic acid 1:1). The reaction mixture was then added to the 925 μL of loading buffer and

mixed by vortexing. After being centrifuged at 4 °C for 5 minutes, 800 μL of the supernatant was loaded to the cartridge, which was then washed with 1 mL of loading buffer. The acyl-CoAs were then eluted by 1 mL of the mixture of ammonium formate and methanol in a 1:1 ratio, followed by 1 mL of the mixture in 1:3 ratio and 1 mL of methanol.

The combined elutes were dried under nitrogen and reconstituted in 100 μL of LC mobile phase A. 10 μL was injected for LC-MS/MS analysis. The concentration of 4-P-GHP-CoA was calculated based on the peak area ratio of analyte to IS.

2.4.3 Kinase purification

Liver homogenization. All of the protein purification steps were conducted in a cold room or a refrigerator to maintain the working temperature at 4 °C. Two of the freshly obtained whole pig livers were placed in blender and homogenized at low speed setting for

5 × 30 seconds with 30 seconds interval. 100 g of the crude homogenate was added to 450 mL of pre-chilled extraction buffer (50 mM HEPES, 100 mM KCl, 1 mM EDTA, 0.05 mM

DTT, 9 protease inhibitor tablets added, pH 7.4) and homogenized for 15 minutes with pause to prevent heating up. The resulting homogenate was centrifuged at 8000 RPM for

30 minutes at 4 °C to remove insoluble material. 10 mL of the supernatant was snap frozen in liquid nitrogen and stored at -80 °C until analysis.

Ammonium sulfate precipitation. Saturated ammonium sulfate solution was prepared by dissolving 541 g ammonium sulfate in 707 mL of water. The supernatant of the liver homogenate was transferred to a beaker and saturated ammonium sulfate solution was added drop wise with gentle stirring. At 15%, 30%, 45%, and 60% of ammonium sulfate saturation, the homogenate was incubated for additional 20 minutes, followed by

centrifuging at 8000 RPM for 30 minutes at 4 °C to obtain protein precipitate. The resulting protein pellet was snap frozen in liquid nitrogen and stored at -80 °C until analysis.

IEX chromatography. 3 g of 45% ammonium sulfate protein precipitate was dissolved in 10 mL of FPLC buffer A (10 mM Tris, 10 mM NaCl, pH=7.4) and loaded into dialysis cassette (3.5k molecular cutoff). The protein fraction was dialyzed in 2 L of FPLC buffer A (10 mM Tris, 10 mM NaCl, pH=7.4) at 4 C overnight and was subjected to FPLC purification. The FPLC system, GE AKTA purifier (GE healthcare, Pittsburgh, PA, USA), is consisted with a UPC-900 UV monitor, P900 pumps and a Frac920 fraction collector.

The FPLC was connected to a SP Sepharose column (110 × 26 mm, column volume (CV)

58.4 mL), which was operated in a 4 C refrigerator. The sample was loaded to the column through an injection loop. The LC buffer A was 10 mM Tris, 10 mM NaCl, pH=7.4 and

LC buffer B was 10 mM Tris, 1M NaCl, pH=7.4. The FPLC was performed at a flow rate of 5 mL/min using a multiple-step gradient: 0-2 CV (0%B), 2-4.5 CV (0%-100%B), and

4.5-6.5 CV (100%B). 20 mL fractions were collected using automatic fraction collector.

The protein concentrations of the fractions were monitored by UV absorbance at 280 nm and the salt concentration was monitored by the conductance. 20 μL of the fractions with significant UV absorbance were subjected to kinase activity assay and the rest of the eluates were quick frozen and stored at -80 °C until further purification.

Blue-Sepharose column chromatography. The fractions 22-25 from IEX were pooled and dialyzed in FPLC buffer A at 4 C overnight. The fraction was then loaded to the FPLC system, which was connected to a Blue-Sepharose column (100 × 16 mm, CV

20.1 mL). The same FPLC buffer as IEX was used and a multiple-step gradient was: 0-3

CV (0%B), 3-5 CV (50%B), and 5-10 CV (100%B). The flow rate was 2.5 mL/min and

fraction size was 10 mL. The protein concentrations of the fractions were monitored by

UV absorbance at 280 nm and the salt concentration was monitored by the conductance.

20 μL of the fractions with significant UV absorbance were subjected to kinase activity assay and the rest of the eluates were quick frozen and stored at -80 °C until further purification.

SDS-PAGE and protein sequencing. 15 g of 45% ammonium sulfate precipitate was purified by SP-Sepharose and Blue-Sepharose columns using the procedures described above. The fraction 9 from Blue-Sepharose column were combined and concentrated to

300 μL using a centrifugal filter (10 kDa cutoff). 100 μL of the fraction was separated by

10% SDS-PAGE and visualized by EZBlue gel staining reagent. Five protein bands and sections were excised and sent to Taplin Mass Spectrometry Facility (Harvard Medical

School, MA, USA) for protein sequencing.

2.4.4 Verification of kinase candidate

Protein sequencing data mining. The identified peptides were searched in the protein database of pig (Sus scrofa) and proteins with three or more unique peptide matches was considered confidently identified from the sample. The identified protein candidates were manually examined to confirm the experimental molecular weight based on SDS-

PAGE matched the theoretical value. Homology search of the pig protein candidates in human protein database was performed using BLAST provided by the Universal Protein

Resource (UniProt, http://www.uniprot.org/blast/).

Expression of SHPK. E. coli BL21(DE3)pLysS harbouring pET15-mCARKL was grown aerobically at 37 °C in LB medium (10 g Tryptone, 5 g yeast extract, 10 g NaCl in

1 L of water) with 100 mg/L ampicillin and 25 mg/L chloramphenicol. When OD578 reached 0.5-0.6, the culture was cooled on ice for 20 min and IPTG (1 mM) was added to the culture. After induction at 30 °C for 4 hours, the cells were harvested by centrifuging at 6000 RPM at 4 °C for 10 minutes. The cell pellet was resuspended in lysis buffer (25 mM HEPES, 300 mM NaCl, 1 mg/mL lysozyme, 20 μL of protease inhibitor cocktail, pH

7.4) and disrupted by 3 cycles of freeze/thaw. The sample was centrifuged to remove insoluble material and the supernatant was loaded to a Ni-Sepharose 6 fast flow column, which was pre-equilibrated with 10 mL of buffer A (25 mM HEPES, 300 mM NaCl, 20 mM imidazole, pH7.4). The column was washed with 10 mL of buffer A and eluted with

8 mL of 50% buffer A and 50% buffer B (25 mM HEPES, 300 mM NaCl, 280 mM imidazole, pH7.4), followed by 5 mL of buffer B. 0.8 mL fractions were collected and the protein concentration was measured by UV absorbance at 280 nm. The eluted recombinant protein fractions were pooled, desalted by PD-10 column, and stored at -80 °C until analysis.

SHPK activity tests. 20 μL of protein fractions, 7 μL of 10 mM sedoheptulose, 10

μL of 100 mM ATP and 50 μL of 2 × kinase buffer were added in an Eppendorf tube and mixed by gentle vortex. After incubating the reaction mixture at 37 °C for 30 minutes, 800

μL of chilled acetonitrile was added to precipitate proteins. The samples were vortexed vigorously and then incubate at 4 °C for 30 minutes. The protein precipitate was removed by centrifugation and the supernatant was dried by nitrogen and reconstituted in 100 μL of

LC mobile phase A. 10 μL was injected for LC-MS/MS analysis and the D-sedoheptulose-

7-phosphate lithium salt was used as the standard.

2.5 Acknowledgement

All the LC-MS/MS based kinase activity tests were conducted in Dr. Henri

Brunengraber’s and Dr. Guofang Zhang’s labs. The bacterial strains for expressing GHB-

CoAT and mouse SHPK were generously provided by Dr. Wolfgang Buckel (Philipps-

Universitat, Germany) and Dr. Emile Van Schaftingen (Universite´ Catholique de Louvain,

Belgium) respectively.

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Chapter 3

Mechanistic Study of Protein Modification by

Epoxyketooctadecenoic Acids (EKODEs)

3.1 Introduction

The progress of oxidative stress can lead to oxidative damage in all biomacromolecules, but polyunsaturated fatty acids (PUFAs) are particularly susceptible to free radical mediated peroxidation1. The resulting lipid hydroperoxides (LOOHs) can subsequently undergo a variety of secondary reactions and some lead to chain cleavage to form reactive bifunctional aldehydes such as malondialdehyde (MDA), acrolein and 4- hydroxy-2-(E)-nonenal (4-HNE)2. Stable polyoxygenated products like epoxyketooctadecenoic acids (EKODEs) (Figure 3.1), which retain the intact chain of the fatty acids, have also been discovered3.

Figure 3.1 Structures of EKODE isomers

EKODEs, the most abundant oxidation products of linoleic acid generated under in vitro oxidation conditions4, have been shown to display prominent biological activities including stimulating corticosterone production, aldosterone secretion, and adrenal

steroidogenesis in rat adrenal cells5-7. EKODEs are also found to effectively activate pathways controlled by the antioxidant response element promoter8. EKODEs have been detected in vivo5, 9 and the concentration in human plasma was found to be 1-500 nM with a mean value of 50 nM from 24 human subjects7.

The biological relevance of LPO products is largely rationalized by their ability to covalently modify proteins10 and DNA11, which may consequently lead to enzyme inactivation12, 13 and gene mutation14, 15. Many of the LPO products contain α, β- unsaturated carbonyl as the major functional groups, which can react with biological nucleophiles to undergo Michael addition to the C=C double bond or form a Schiff-base to the carbonyl (Figure 3.2)10. The resulting adducts may subsequently undergo intra- molecular rearrangement to form stable advanced lipoxidation end products (ALE) including HNE-derived 2-pentylpyrroles16, 17. Some of the bifunctional aldehydes such as

4-HNE are able to crosslink proteins via simultaneous conjugation with nucleophilic protein residues (Cys, His, Lys) on the C=C bond and Schiff base condensation with Lys on carbonyl18. In addition, the epoxy group of EKODEs is susceptible to nucleophilic attack, which may lead to ring opening products.

Lin et al.4 reported that EKODEs can form adducts with histidine and the reaction mechanism of EKODE I adduct formation was confirmed to be Michael addition.

However, the mechanism of formation of EKODE II adduct was not confirmed yet and the reactions with other amino acid residues such as lysine and cysteine have not been elucidated. In this chapter, we continued to use model compounds as a convenient tool to reveal the mechanism of EKODE modification on model nucleophiles. The proposed mechanism was confirmed by using EKODE derivatives and EKODEs with full structure.

A peptide mapping experiment was then performed by using model protein to extend the acquired knowledge to EKODE modification of proteins.

Figure 3.2 Proposed reactivity of EKODEs with biological nucleophiles

3.2 Results and Discussions

3.2.1 Mechanistic study of the reactions of EKODE model compounds with analogues of nucleophilic amino acids

The mechanistic investigation of the reactions of EKODEs with amino acids is extremely challenging due to the facts that 1) the synthesis of EKODE is complicated and the yield is not satisfactory, which limits the availability of EKODEs as starting materials for studying their reactivity; and 2) EKODEs and amino acids contain multiple chiral centers that complicates the characterization of the reactions. Therefore, EKODEs were simplified into two model compounds trans-5,6-epoxy-3-(E)-undecen-2-one (EKODE I model compound) (3.3) and trans-5,6-epoxy-2-(E)-undecen-4-one (EKODE II model compound) (3.5) representing EKODE I and EKODE II series respectively (Scheme 3.1).

3.2 3.1 3.3

3.4 3.5

Scheme 3.1 Preparation of EKODE model compounds

EKODE model compounds contain the same core reactive groups, the epoxide and

α, β-unsaturated ketone, as the original EKODEs. Meanwhile, they have simplified structures allowing straightforward synthesis and reactivity characterization. EKODE I

model compound was synthesized through modified methods based on our previous studies4. EKODE II model compound was obtained by a Grignard reaction of an epoxy aldehyde (3.2) and then oxidized by pyridinium chlorochromate (PCC). In the meantime, three amino acids surrogates n-butylamine, imidazole and butanethiol were used in the model study to mimic the residues of nucleophilic amino acids, lysine, histidine and cysteine. Since these surrogates lack chiral centers and have minimized carbon chain, the characterization of the reaction products is expected to be less complicated.

The EKODE model compounds were incubated with amino acid surrogates in 50 mM PBS buffer (pH 7.4) containing 20% acetonitrile to mimic physiological conditions.

The resulting products were isolated, characterized and summarized in Table 3.1. The reaction of EKODE model compounds with 10 equivalents of n-butylamine at 37 °C for

24 hours resulted in no significant amount of Michael adducts or Schiff base adducts. This result is consistent with our previous proteomics study that no lysine adducts were observed when a model protein, apomyoglobin, was treated with EKODEs19.

Table 3.1 Adducts of EKODE model compounds with amino acid surrogates

3.6 3.7 3.8

In the reaction of EKODE II model compounds with imidazole, two diastereomers of Michael adducts were successfully identified. This is consistent with a previous report that EKODE I was able to form Michael adduct with imidazole4. The Michael adducts of histidine that generated from nucleophilic attack at α, β-unsaturated carbonyl have been extensively studied20. A number of LPO products, such as HNE21 and ONE22, have been reported to be capable of modifying histidine residues on protein and the resulting adducts were developed as biomarkers for oxidative stress23-25. Our results from the model study revealed the high possibility that EKODEs can modify proteins through covalent binding to histidine residues via Michael addition. Therefore, EKODEs have the potential to become indicators of oxidative damage to proteins during LPO reactions.

The sulfhydryl group of the cysteine residue is the most reactive nucleophile in proteins13. It has been broadly reported that thiol groups can attack the epoxy rings26 and the ring opening reaction normally occurs at the carbon near electron-deficient groups27.

In our study, the reaction of EKODE I model compound with butanethiol produced a ring opening product (3.6) at α position of the alkene group in 72 hours while the Michael adduct was not stable enough for isolation. The reaction of EKODE II model compound led to a stable Michael adduct (3.7) in 1 hour and interestingly, it produced a ring opening product (3.8) at β position of the carbonyl group in the 48 hours incubation. The structure of this unexpected ring opening product was further confirmed by 1H-1H correlation spectroscopy (HHCOSY). (Appendix)

Advanced reaction products originating from Michael adducts of LPO products on nucleophilic protein side chains have been reported28. The predominant initial reaction involves Michael addition to the α, β-unsaturated carbonyl. The resulting adducts are

relatively unstable and may be further converted to advanced end products. It is, therefore, reasonable to postulate that EKODE II compounds were nucleophilically attacked by sulfhydryl leading to the Michael adduct, which then underwent an intramolecular rearrangement through a six-member ring transition state to form the ring opening product containing the sulfur substituent at β position of carbonyl group (Scheme 3.2).

Scheme 3.2 Proposed mechanism of the ring opening reaction of EKODE II model compound with butanethiol

To test our hypothesis, three derivatives of EKODE II model compounds were synthesized (Scheme 3.3). Derivative A (3.10) and B (3.14) were prepared via a similar synthesis route as that for EKODE II model compound. Derivative C (3.19) was synthesized by using a previously reported method29. The derivatives were subsequently incubated with butanethiol at 1:1 ratio at r.t. The reaction with derivative A resulted in a

Michael adduct (3.11) in 4 hours which was stable at 37 °C for more than 2 days and no advanced products were observed. This can be explained by the fact that the geometry of the alkene Michael adducts does not favor the formation of six-member ring transition state. This observation supports our hypothesis that the intramolecular rearrangement from the six-member ring transition state is the key step for the ring opening reaction of epoxy ring at β position. Meanwhile, derivatives B (3.14) and C (3.19) cannot undergo Michael addition reaction and therefore, were directly attacked by free thiol group at α position.

3.1 3.1 3.16

3.17

3.2 3.12

3.18

3.9 3.13

A 3.10 B 3.14 C 3.19

3.11 3.15 3.20

Scheme 3.3 Synthesis the EKODE II derivatives and their reaction with butanethiol

3.2.2 Reactions of EKODEs with cysteine

Since trans-EKODE-(E)-Ib (3.22) and trans-EKODE-(E)-IIb (3.25) are the most abundant isomers of EKODE I and EKODE II family generated from nonenzymatic autoxidation of linoleic acid, they were synthesized and tested in the reaction with cysteine to confirm the reaction mechanisms that were established using EKODE model compounds and cysteine analogues. For the synthesis of trans-EKODE-(E)-Ib, the methyl ester of the final product was prepared via the method reported by Lin et al.4 (Scheme 3.4)

3.21 3.22

3.23 3.24

3.25 Scheme 3.4 Synthesis of trans-EKODE-(E)-Ib and trans-EKODE-(E)-IIb

The hydrolysis of the ester (3.21) was previously conducted by lithium hydroxide and the yield was not satisfactory due to the base-catalyzed hydrolysis of epoxide ring. The reaction was optimized by using porcine pancreatic lipase (PPL) as a biological catalyst in neutral conditions30 and the yield was improved to 62%. The synthesis of trans-EKODE-

(E)-IIb started with ozonolysis of methyl oleate (3.23) 31, which eliminated the tedious

multiple steps to obtain methyl 9-oxononanoate (3.24) from azelaic acid monomethyl ester.

The rest of the synthesis route followed previously reported methods4 except that the last step was catalyzed by lipase.

In an effort to investigate the mechanism of the generation of two contradictory ring opening products, the reactions of EKODEs and EKODE model compounds with N- acetyl-cysteine-methyl ester were monitored by UV spectroscopy at 37 °C (Figure 3.3).

The absorption at 234 nm, which corresponds to α, β unsaturated ketone, dropped rapidly in the first 5 hours, indicating that Michael addition dominated the early stage of the reaction. Subsequently the ring opening products began to form, resulting in the recovery of the conjugation and thus the increase of absorption at 234 nm. When EKODE I model compound was replaced with trans-EKODE-(E)-Ib, there was no significant impact on the reaction rate of Michael addition. Meanwhile, the ring opening reaction was remarkably slower and it can be explained by the higher steric hindrance in the full length EKODE.

On the contrary, the absorption recovery rates of trans-EKODE-IIb and EKODE II model compound were very close. This observation can be rationalized by our hypothesis that the ring opening reaction of EKODE II is an intra-molecular rearrangement of the Michael adduct so that the reaction rate was not significantly affected by steric effects. It is worth mentioning that ring opening products of EKODE II were relatively stable compared with the intact EKODEs, which degrade faster in solution (data not shown). This observation suggests that the EKODE-Cys adducts may accumulate once generated and they have the potential to become biomarkers for oxidative stress in chronic diseases.

Figure 3.3 Time course of the reactions of N-acetyl-cysteine-methyl ester with EKODEs and EKODE model compounds

The LC-MS analysis of the reaction of full length EKODE II with N-acetyl- cysteine-methyl ester was consistent with our UV study (Figure 3.4). After three hours, two of the four Michael adduct diastereomers (m/z 488) were generated and all of the four diastereomers were detected after 20 hours. This is consistent with the UV study that

Michael addition dominated the early stage of the reaction and also indicated that the

Michael addition was stereo-selective and kinetically controlled. This is in agreement with previous results by Lin et al. that the Michael addition of EKODE I model compound with imidazole showed stereo-selectivity (unpublished data). The anti-adducts, which are defined by the position of imidazole relative to the expoxy-ring, were favored comparing with syn-adducts. Michael adducts disappeared after incubating for 3 days and a new broad peak was detected. It was tentatively assigned as a mixture of diastereomers of the ring opening products, which was not resolved well in our short LC separation. This is consistent with our hypothesis that the ring opening products of EKODE II were derived from the initial Michael addition products. It is noteworthy that trace amount of EKODE adduct with two additional cysteines was detected (data not shown). It indicates that the ring opening products of EKODE may undergo Michael addition with cysteine or other nucleophiles due to the recovery of α, β-unsaturated carbonyl, but the reaction may be hindered by the increased steric effect in the ring opening products. This suggests that

EKODE may induce protein cross-linking, which is a common mechanism for the cytotoxicity of LPO products32.

Figure 3.4 LC-MS analysis of the reaction of trans-EKODE-(E)-IIb with N-acetyl- cysteine-methyl ester

3.2.3 Mass spectrometric characterization of the modification of human serum albumin (HSA) by trans-EKODE-(E)-IIb

It has been reported that HSA, the most abundant protein in human blood, can be extensively modified by LPO products through multiple nucleophilic residues including cysteine, histidine, and lysine33. Therefore, HSA was selected as a model protein to ascertain the EKODE-derived modification on biological nucleophiles at protein level. 10

μM HSA was incubated with 1000 μM EKODE overnight at 37 °C in PBS containing 20% acetonitrile to mimic physiological conditions. After overnight incubation, the modified protein was precipitated with organic solvents and resolubilized in ammonium bicarbonate

(AMBIC) buffer, followed by proteolytic digestion using trypsin. The resulting tryptic peptides were analyzed by using information dependent acquisition (IDA) by high- resolution mass spectrometry (HRMS) to reveal modification sites.

HSA peptides were subjected to peptide mapping by BioPharmaView software for the presence of +310.2144 mass shift, which corresponds to the addition of EKODE via

Michael addition or +292.4131 for the formation of Schiff base with lysine residue. The matching tolerance for peptide precursor and product ions were set at 10 ppm and 0.10 Da respectively. The peptide coverage was found to be 89.7%. When HSA was exposed to 1 mM EKODE, 7 modified peptides were identified and the modification sites were Cys-34,

His-67, His-146, His-338, His-440, His-464, and His-535 (Table 3.2). The observed monoisotopic m/z of the peptide precursors were in good agreement with the theoretical value with error less than 10 ppm. In addition, multiple charge states for the peptide containing Cys-34 (+3 and +4 charge) and His-338 (+3 and +4 charge) were identified. No

EKODE-modified lysine residue was identified and it was consistent with the results in our

model study that EKODEs were not reactive with lysine or the model compound of lysine.

Table 3.2 Modified Cys/His/Lys residues in HSA treated with 1 mM EKODE

RT Theoretical Observed Error Residue Peptide Charges (min) Mono m/z Mono m/z (PPM)

Cys-34 ALVLIAFAQYLQQCPFEDHVK 6.41 3 915.1642 915.1683 4.54

Cys-34 ALVLIAFAQYLQQCPFEDHVK 6.41 4 686.6249 686.6285 5.15

His-67 SLHTLFGDK 5.73 3 443.2551 443.2575 5.32

His-146 HPYFYAPELLFFAK 6.26 3 685.0410 685.0440 4.38

His-338 RHPDYSVVLLLR 5.64 3 593.3573 593.3597 3.95

His-338 RHPDYSVVLLLR 5.64 4 445.2698 445.2723 5.65

His-440 HPEAK 4.80 2 446.2629 446.2651 4.82

His-464 MPCAEDYLSVVLNQLCVLHEK 6.44 3 905.4666 905.4707 4.53

His-535 HKPK 4.32 2 410.2706 410.2736 7.45

The mass spectrum was also manually examined to confirm the peptide matching of software. Figure 3.5 A shows the total ion current (TIC) of the TOF scan of precursors and a large number of peaks were identified for the proteolytic peptides. When an extraction window of 12.5 mDa was used to extract the peptide containing EKODE- modified His-338 (m/z 593.3595, +3), a distinctive peak was obtained at retention time of

5.64 minutes (Figure 3.5 B). The high specificity was achieved by the high resolution of the mass spectrometer, which allowed the use of narrow extraction window. The HRMS scan at 5.64 minutes was then extracted by using an extraction window of 0.12 minutes to display the mass spectrum (Figure 3.6 A). The highlighted peaks correspond to the multiple charge states of His-338-modified peptide and the zoomed-in spectra are shown in Figure 3.6 B-D. The charge states can be confirmed by the isotopic distribution and

Figure 3.6 B-D match charge states +4, +3, and +2 respectively. To validate the site of

modification, the product ions of the matched peptide were examined. The MS/MS product ion scan of the peptide with EKODE-modified His-338 is shown in Figure 3.7, and

+ matched y- and b-product ion series are listed. The presence of matched product ions y10

+ and b2 indicates that the modification site is the histidine residue.

Figure 3.5 LC-MS analysis of trypsin digestion of EKODE-modified HSA. (A) TIC of full

HRMS scan; (B) XIC of peptide containing EKODE-modified His-338 at m/z 593.3595 using extraction window of 12.5 mDa

Figure 3.6 LC-MS analysis of EKODE-modified His-338 peptide. (A) Extracted HRMS scan spectrum at retention time of at 5.64 min; (B) (C) (D) Zoomed-in HRMS scan of highlighted area in (A)

Figure 3.7 MS/MS product ion scan of peptide containing EKODE-modified His-338

(m/z 593.3595, +3)

In an effort to identify the most reactive residues, lower concentrations of EKODE

(100 and 500 μM) was incubated with 10 μM HSA (Table 3.3) and the sequence coverage was 90.4% and 87.7% respectively. When HSA was exposed to lower concentration of

EKODE (500 μM), 1 cysteine and only 4 histidine residues were found to be modified, including Cys-34, His-67, His-146, His-338, and His-440. The relative level of EKODE to protein was then reduced to a ratio of 10:1, which was the lowest ratio where EKODE modification could be detected. As a result, the modification on His-338 was not detectable, whereas the other 4 residues including Cys-34 were still identified. Therefore,

Cys-34, His-67, His-146, and His-440 represent the most reactive residues of HSA toward

EKODE modification. Aldini et al. reported the covalent modification of HSA by HNE and it was found that the most reactive HNE-adduction site was Cys-34 (Michael adduct), followed by Lys-199 (Schiff base) and His-146 (Michael adduct)34. In another study reported by Liu et al., His-67 was found to be one of the 4 most reactive histidines, which

was readily modified by HNE through Michael addition35. Since EKODEs and HNE contain similar functional groups, it is highly possible that both LPO products display comparable selectivity in the reaction with nucleophilic residues of proteins. Cys-34 provides the largest fraction of free thiol in the blood serum and it works as a scavenger for ROS as well as an endogenous detoxifying agent for reactive carbonyl species36. The covalent modification of Cys-34 by EKODEs may dampen its ability to serve as antioxidants. In addition, our model study demonstrated that the reactions of EKODEs with cysteine could generate irreversible ring opening products, which were relatively stable in physiological conditions. These results indicate that EKODEs, after entering the general circulation, could rapidly modify serum albumin and the resulting adducts might accumulate over the course of oxidative stress. Therefore, the EKODE-derived adducts of

HSA Cys-34 may be suitable as biomarkers for oxidative damage in human diseases.

Table 3.3 EKODE-modified peptides identified in HSA treated with EKODE at various molar ratio

Molar ratio EKODE : HSA Residue Peptide

10:1 50:1 100:1

Cys-34 ALVLIAFAQYLQQCPFEDHVK ● ● ● His-67 SLHTLFGDK ● ● ● His-146 HPYFYAPELLFFAK ● ● ● His-338 RHPDYSVVLLLR ● ● His-440 HPEAK ● ● ● His-464 MPCAEDYLSVVLNQLCVLHEK ● His-535 HKPK ●

3.3 Conclusions

In this chapter, EKODE model compounds were synthesized and applied in the reaction with model nucleophiles to identify the adducts. It was found that EKODE can form Michael adducts with cysteine and histidine. In addition, ring opening products were identified in the reaction with cysteine analogues. The mechanism of formation of an unexpected product from the thiolysis of EKODE II model compound was revealed by the reactions of EKODE derivatives with butanethiol and it was found to be the intra-molecular rearrangement of the initial Michael adduct. trans-EKODE-(E)-Ib and trans-EKODE-(E)-

IIb were then synthesized using modified methods. The reactions of EKODEs with cysteine analogues were monitored by LC-MS and UV spectroscopy, and the observation was consistent with our proposed mechanism for the reaction of EKODE II model compound.

The reactions of EKODEs with HSA were also explored, where Cys-34, His-67, His-146, and His-440 were the most reactive residues. It is likely that EKODEs are important biological electrophiles that are involved in protein damage as a biological consequence of oxidative stress. EKODE-derived adducts of cysteine residues of proteins may be suitable as biomarkers for oxidative stress in diseases and aging.

3.4 Experimental Sections

3.4.1 Materials and Methods

General chemicals, organic solvents, HSA, and lipase from porcine pancreas were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sequencing Grade Modified

Trypsin was purchased from Promega (Madison, WI, USA).

Column chromatography was conducted by using silica gel (particle size 32-63

μM). Analytical thin-layer chromatography (TLC) was performed by glass-coated silica gel plates with fluorescent indicator (0.25 mm), and visualized by UV lamp (254 nm) or

TLC stains including phosphomolybdic acid (PMA) and 1,4-dinitrophenylhydrazine

(DNP). 1H and 13C- NMR spectra were recorded on a Varian Gemini spectrometer operating at 400 MHz and 100 MHz for 1H and 13C respectively (at the Department of

Chemistry, Case Western Reserve University). The residual solvent peak was used as the

1 13 internal references (CDCl3, 7.26 ppm for H NMR and 77.2 ppm for C NMR).

In addition to TLC, the reactions were also monitored by LC-MS. HPLC system is consisted with Shimadzu LC-20AB pumps and SIL-20A HT autosampler (Columbia, MD).

An Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 m, Agilent, Santa Clara, CA) was used for LC separation at a flow rate of 0.2 mL/min. Mobile phase A was 0.05% formic acid (FA) in water, and mobile phase B was 0.05% FA in methanol. A multiple-step gradient was as following: 0-2 min (60% B), 2-25 min (60%-95% B), 25-30 min (95% B),

30-31 min (95%-60% B), and 35 min (60% B). 10 L of reaction mixture was separated by LC and subjected to MS analysis on a Shimadzu LCMS-2020 mass spectrometer. The

DUIS interface setting was: interface temperature 350 °C, DL temperature 250 °C,

nebulizing gas flow 1.5 L/min, heating block 400 °C, interface voltage 3.5 kV, and detector voltage 0.95 kV. The m/z range was 100-1000.

3.4.2 Mechanistic study of the reactions of EKODE model compounds with analogues of nucleophilic amino acids

Synthesis of EKODE I model compound (trans-5,6-Epoxy-3-(E)-undecen-2- one) (3.3). trans-2,3-epoxyoctanal (3.2) was prepared by the epoxidation of trans-2- octenal (3.1) (10 g, 79.3 mmol) in methanol using 30% hydrogen peroxide solution (30 mL, 264 mmol) in the presence of NaHCO3 (5 g, 59.5 mmol). After 1 hour of reaction at r.t., 70 mL of brine was added to the mixture, followed by extraction with CH2Cl2 (3 × 200 mL). The combined organic layer was washed with brine (3 × 50 mL) and dried over

Na2SO4. The solvent was removed in vacuo and the residual oil was purified by column chromatography (hexane/ethyl acetate, 5:1) to give trans-2,3-epoxyoctanal (3.2) as a transparent oil (8.41 g, 74.6% yield). 1-triphenylphosphoranylidene-2-propanone (6.4 g,

20.1 mmol) in 50 mL of CH2Cl2 was then added dropwise to trans-2,3-epoxyoctanal (3.2)

(2.6 g, 18.3 mmol) in ice bath and the reaction was performed with vigorous stirring for 2 hours. The resulting reaction mixture was washed with brine (3 × 10 mL), dried over

Na2SO4, and concentrated with rotovap. The purification was conducted by column chromatography (hexane/ethyl acetate, 10:1) to yield trans-5,6-Epoxy-3-(E)-undecen-2-

1 one (3.3) as a yellow oil (2.87 g, 86.1%). H NMR (400 MHz, CDCl3) δ 0.90 (t, 3H, J =

7.0 Hz, CH3), 1.30-1.34 (4H, 2×CH2), 1.40-1.52 (2H, CH2), 1.62 (m, 2H, CH2), 2.26 (s,

3H, CH3), 2.91 (td, 1H, J = 5.6 Hz and 2.0 Hz, CHOC), 3.21 (dd, 1H, J = 6.8 Hz and 1.6

Hz, OCHC=C), 6.35 (d, 1H, J = 16.0 Hz, CHC=O), 6.47 (dd, 1H, J = 16.0 Hz and 6.8 Hz,

13 CHCO); C NMR (100 MHz, CDCl3) δ 14.5 (CH3), 22.7, 25.7, 27.6, 31.6, 31.9, 56.5

(CHO), 61.2 (CHO), 133.0 (C-2, C=C), 145.3 (C-3, C=C), 198.1 (C=O).

Synthesis of EKODE II model compound (trans-5,6-Epoxy-2-(E)-undecen-4- one) (3.5). 0.5 M 1-propenylmagnesium bromide solution in tetrahydrofuran (THF) (20 mL, 10 mmol) was added dropwise in a solution of trans-2,3-epoxyoctanal (3.2) (2g, 14.1 mmol) in 10 mL of anhydrous diethyl ether at -20 °C (brine ice bath). After stirring at r.t. for 1 hour, the reaction was quenched by saturated NH4Cl solution (40 mL) and extracted with diethyl ether (3 × 40 mL). The organic layers were combined, dried, concentrated in vacuo, and followed by purification over silica (hexane/ethyl acetate, 5:1) to give (E)-1-(3-

Pentyloxiran-2-yl)but-2-en-1-ol (3.4) as a transparent oil (1.4g, 77% yield). trans-5,6-

Epoxy-2-(E)-undecen-4-one (3.5) was prepared by the oxidation of (E)-1-(3-Pentyloxiran-

2-yl)but-2-en-1-ol (3.4) (1 g, 5.43 mmol) using PCC (1.9 g, 8.81 mmol) in 100 mL of anhydrous CH2Cl2 in the presence of pyridine (1 g, 12.6 mmol). The reaction mixture was stirred vigorously at r.t. for 1 h, filtrated by celite, and concentrated. The resulting mixture was subjected to column chromatography (hexane/ethyl acetate, 10:1) to give trans-5,6-

Epoxy-2-(E)-undecen-4-one as a transparent oil (3.5) (426 mg, 43.1%). 1H NMR (400

MHz, CDCl3) δ 0.88 (t, 3H, J = 7.2 Hz, CH3), 1.30-1.36 (4H, 2×CH2), 1.45-1.50 (2H, CH2),

1.57-1.68 (2H, CH2), 1.92 (dd, 3H, J = 7.0 Hz and 1.6 Hz, CH3), 3.05 (ddd, 1H, J = 6.0 Hz,

4.8 Hz and 2.0 Hz, CHOCH2), 3.34 (d, 1H, J = 2.0 Hz, CHOC=O), 6.25 (dq, 1H, J = 15.6

13 Hz and 1.6 Hz, O=CCH=C), 7.09 (dq, 1H, J = 15.6 Hz, 7.0 Hz, C=CHCH3); C NMR (100

MHz, CDCl3) δ 14.2 (C-2, CH3), 18.8 (C-11, CH3), 22.7, 25.7, 31.6, 32.0, 58.6 (CHO),

59.2 (CHO), 125.9 (C-3, C=C), 145.8 (C-2, C=C), 195.8 (C=O).

Reactions of EKODE model compounds with amino acid analogues. 100 μM

EKODE model compounds was incubated with 1 mM of amino acid analogues in 100 mL

of PBS containing 20% acetonitrile and the reactions were monitored by TLC and LC-MS.

After 24 hours of reaction with n-butylamine at 37 °C, no significant amount of adducts were identified. The reactions of EKODE model compound with imidazole afforded diastereomers of Michael adducts, which were characterized via previously reported procedures by Lin et al.4 and Zhang37. EKODE I model compound was incubated with butanethiol at 37 °C for 72 hours and the reaction was extracted by ethyl acetate (3 × 200 mL). The combined organic layer was washed with brine (3 × 50 mL), dried over Na2SO4

, and concentrated in vacuo. The residual oil was purified by column chromatography

(hexane/ethyl acetate, 10:1) to afford the ring opening product (3.6) of EKODE I model

1 compound ((E)-5-(butylthio)-6-hydroxyundec-3-en-2-one). H NMR (400 MHz, CDCl3) δ

0.78-0.88 (6H, 2×CH3), 1.20-1.60 (12H, 6× CH2), 2.24 (s, 3H, CH3), 2.38 (t, 2H, J = 7.6

Hz, CH2), 3.27 (dd, 1H, J = 6.0 Hz and 4.0 Hz, CHC=C), 3.82 (m, 1H, CHO), 5.96 (dd,

1H, J = 15.6Hz, CHC=O), 6.67 (dd, 1H, J = 16.0 Hz and 10.0 Hz, CHCS). To capture the

Michael adduct of EKODE II model compound with butanethiol, the reaction was performed at r.t. for 1 hour and immediately extracted and purified by using the same procedure that was used for the reaction of EKODE I model compound and butanethiol.

The resulting Michael adducts (3-(butylthio)-1-(3-pentyloxiran-2-yl)butan-1-one) (3.7)

1 contained two diastereomers. H NMR (400 MHz, CDCl3) δ 0.82-0.88 (6H, 2×CH3),

1.21(d, 3H, J = 6.8 Hz, CH3), 1.24-1.61 (12H, 6× CH2), 2.31-2.49 (m, 1H, CHO), 2.47 (t,

2H, J = 7.2 Hz, CH2S), 2.51-2.72 (m, 1H, CHOC=O), 2.97-3.07(m, 1H, CHS), 3.15-3.23

(m, 2H, CH2C=O). The ring opening products ((E)-6-(butylthio)-5-hydroxyundec-2-en-4- one) (3.8) was obtained by stirring the mixture EKODE II model compound with butanethiol at r.t. for 48 hours. The mixture was extracted and purified using the same

procedure to give the ring opening product of EKODE II model compound (3.8). 1H NMR

(400 MHz, CDCl3) δ 0.86 (t, 3H, J = 6.8 Hz, CH3), 0.93 (t, 3H, J = 7.2 Hz, CH3), 1.21-1.33

(4H, 2×CH2), 1.45 (m, 2H, CH2), 1.57 (m, 2H, CH2), 1.97 (dd, 3H, J = 7.2 Hz and 1.6 Hz,

C=CCH3), 2.6 (m, 2H, CH2S), 2.94 (ddd, 1H, J = 10.0 Hz, 3.2 Hz and 3.2 Hz, CHS), 3.52

(d, 1H, J = 5.2 Hz, OH), 4.48 (dd, 1H, J = 5.2 Hz and 3.2 Hz, CHC=O), 6.39 (dq, 1H, J =

15.6 Hz and 1.6 Hz, O=CCH=C), 7.08(dq, 1H, J = 15.6 Hz and 7.2 Hz, C=CHC); 13C NMR

(100 MHz, CDCl3) δ 13.9 (CH3), 14.2 (CH3), 18.9 (CH3), 22.3, 22.7, 27.2, 29.3, 31.3, 31.7,

31.9, 49.6 (CHS), 78.2 (COH), 127.1 (C-3, C=C), 145.8 (C-2, C=C), 199.2 (C=O).

Synthesis of EKODE derivative A (1-(3-Pentyloxiran-2-yl)but-2-yn-1-one)

(3.10). trans-2,3-epoxyoctanal (2g, 14.1 mmol) (3.2) was dissolved in 50 mL of anhydrous ethyl ether at -20 °C, followed by dropwise addition of 0.5 M 1-propynylmagnesium bromide in THF (20 mL, 10 mmol). After stirring at r.t. for 1 hour, the reaction was quenched by saturated NH4Cl solution (40 mL) and extracted with diethyl ether (3 × 40 mL). The organic layers were combined, dried over Na2SO4, and concentrated in vacuo.

The resulting oil was directly used without further purification for the synthesis of EKODE derivative A. The residual oil was reconstituted in anhydrous 20 mL of CH2Cl2, followed by addition of 0.3 M Dess-Martin periodinane solution in CH2Cl2 (30 mL, 9 mmol) and

NaHCO3 (2g, 23.8 mmol). After stirring at r.t. for 1 hour, the reaction was quenched with

20 mL of saturated Na2S2O3 solution. The aqueous layer was subsequently extracted with

CH2Cl2 (3 × 50 mL) and the combined organic layer was washed with brine (3 × 50 mL), dried over Na2SO4 and concentrated with rotovap. The purification was conducted by column chromatography (hexane/ethyl acetate, 10:1) to yield EKODE derivative A (3.10)

1 as a yellow oil (810 mg, 31.9% yield). H NMR (400 MHz, CDCl3) δ 0.83 (t, 3H, J = 7.0

Hz, CH3), 1.27-1.42 (6H, 2×CH2), 1.5-1.6 (m, 2H, COCH2), 1.99 (s, 3H, C≡CH3), 3.19 (dt,

13 1H, J = 5.2 Hz, and 0.8 Hz, COHCH2), 3.23 (m, 1H, COHC=O); C NMR (100 MHz,

CDCl3) δ 4.5 (C-1, CH3), 14.1 (C-11, CH3), 22.7, 25.7, 31.6, 31.7, 59.3 (C-6, CHO), 60.2

(C-5, CHO), 77.5 (C-2, C≡C), 93.9 (C-3, C≡C), 184.6 (C=O).

Synthesis of EKODE derivative B (trans-5,6-Epoxy-undecan-4-one) (3.14).

Grignard reagent was prepared by adding a solution of 1-bromopropane (300 mg, 2.5 mmol) in 5 mL of ether to a stirring mixture of Mg turnings and 5 mL of ether to maintain a gentle reflux. The reaction was stirred for additional 2 hours, followed by addition of a solution of trans-2-octenal (260 mg, 2.06 mmol) (3.1) in 10 mL of ether. After stirring for

1 hour at r.t. the reaction was quenched by 20 mL of saturate NH4Cl solution and washed with brine (3 × 20 mL). The organic layer was dried with Na2SO4 and concentrated. The residual oil was dissolved in 10 mL of anhydrous, followed by addition of Dess-Martin reagent (3 mL, 0.9 mmol). After stirring at r.t. for 30 minutes, the reaction mixture was then subjected to the same work-up and purification procedures described above to give

EKODE derivative B (3.14) as a transparent oil (175 mg, 52% yield). 1H NMR (400 MHz,

CDCl3) δ 0.90 (t, 6H, J = 7.0 Hz, 2×CH3), 1.29-1.34 (4H, 2×CH2), 1.42-1.47 (m, 2H, CH2),

1.54-1.65 (4H, 2×CH2), 2.43 (m, 2H, O=CCH2), 3.03 (td, 1H, J = 5.4 Hz and 0.8 Hz, CHO),

13 3.20 (d, 1H, J = 0.8 Hz, CHOC=O); C NMR δ (100 MHz, CDCl3) δ 13.9 (CH3), 14.2

(CH3), 16.8, 22.7, 25.7, 31.6, 32.0, 39.3 (C-3, CH2), 58.6 (C-6. CHO), 59.9 (C-5, CHO),

208.2 (C=O).

Synthesis of EKODE derivative C ((3-Pentyloxiran-2-yl)(phenyl)methanone)

(3.19). Phenylacetylene (3.17) (300 mg, 2.94 mmol) and hexanal (3.16) (300 mg, 3.00 mmol) were dissolved in anhydrous CH2Cl2, followed by addition of amberlyst-15 (300

mg). After stirring the reaction mixture at r.t. for 4 hours, the reaction was filtrated, concentrated, and purified over silica gel (hexane/ethyl acetate, 10:1) to give 1-phenyloct-

1 2en-1-one (3.18) as a brown oil (260 mg, 43.8%). H NMR (400 MHz, CDCl3) δ 0.89 (t,

3H, J = 7.2 Hz, CH3), 1.29-1.34 (4H, 2×CH2), 1.48-1.52 (m, 2H, CH2), 2.30 (m, 2H,

CH2C=C), 6.86 (dt, 1H, J = 15.2 Hz and 1.6 Hz, C=CH), 7.05 (dt, 1H, J = 15.2 Hz and 7.0

13 Hz, C=CHC=O), 7.43 (m, 2H, 2×C6H5), 7.51 (m, 1H, C6H5), 7.91 (m, 2H, 2×C6H5); C

NMR (100 MHz, CDCl3) δ 14.2 (CH3), 22.7, 28.1, 31.6, 33.0, 126.0 (C-8, C=C), 128.7

(C6H5), 128.7 (C6H5), 132.8 (C6H5), 138.2 (C6H5), 150.3 (C-9, C=C), 191.1 (C=O). The resulting compound (3.18) (260 mg, 1.29 mmol) was dissolved in 10 mL of methanol and the epoxidation was performed using 30% hydrogen peroxide solution (0.5 mL, 4.41 mmol) in the presence of NaHCO3 (300 mg, 3.57 mmol). After 2 hours of reaction at r.t.

90 mL of brine was added to the mixture, followed by extraction with CH2Cl2 (3 × 200 mL). The combined organic layer was washed with brine (3 × 50 mL) and dried over

Na2SO4. The solvent was removed in vacuo and the residual oil was purified by column chromatography (hexane/ethyl acetate, 5:1) to give EKODE derivative C (3.19) as a brown

1 oil (210 mg, 74.7% yield). H NMR (400 MHz, CDCl3) δ 0.89 (t, 3H, J = 7.2 Hz, CH3),

1.31-1.36 (4H, 2×CH2), 1.45 (m, 2H, CH2), 1.72 (m, 2H, CH2), 3.13 (ddd, 1H, J = 6.0 Hz,

4.8 Hz and 2.0 Hz, CHO), 4.01 (d, 1H, J = 2.0 Hz, CHOC=O), 7.49 (m, 2H, 2×C6H5), 7.61

13 (ddt, 1H, J = 8.0 Hz, 6.8 Hz, 1.2 Hz, C6H5), 8.01(m, 2H, 2×C6H5); C NMR (100 MHz,

CDCl3) δ 14.2 (CH3), 22.7, 25.8, 31.7, 32.2, 57.6 (C-9, CHO), 60.4 (C-8, CHO), 128.5

(C6H5), 129.0 (C6H5), 134.0 (C6H5), 135.8 (C6H5), 195.0 (C=O).

Reactions of EKODE derivatives with butanethiol. 100 μM EKODE derivative

A/B/C was incubated with 1 mM of butanethiol in 100 mL of PBS containing 20%

acetonitrile at 37 °C for 24 hours and the reactions were monitored by TLC and LC-MS.

The adducts were extracted and purified using the procedures described in the reactions of

EKODE model compounds with butanethiol. Adduct of derivative A ((E)-3-(butylthio)-1-

1 (3-pentyloxiran-2-yl)but-2-en-1-one) (3.11): H NMR (400 MHz, CDCl3) δ 0.82-0.88 (6H,

2×CH3), 1.28-1.61 (8H, 4×CH2), 2.26 (s, 3H, C=CCH3), 2.84 (t, 2H, J = 7.2 Hz, SCH2),

2.98 (m, 1H, CHO), 3.19 (d, 1H, J = 1.6 Hz, CHOC=O), 6.25 (s, 1H, CH=C); 13C NMR

(100 MHz, CDCl3) δ 13.9 (CH3), 14.2 (CH3), 22.1, 22.7, 24.7, 25.7, 30.7, 31.3, 31.7, 32.1,

59.1 (C-6, CHO), 59.9 (C-5, CHO), 114.1 (C-3, C=C), 164.1 (C-2, C=C), 194.0 (C=O).

Adduct of derivative B (5-(butylthio)-6-hydroxyundecan-4-one) (3.15): 1H NMR (400

MHz, CDCl3) δ 0.83 (t, 3H, J = 7.2 Hz, CH3), 0.87 (t, 3H, J = 7.6 Hz, CH3), 1.28-1.42

(11H, CH3, 4×CH2), 1.57 (m, 2H, CH2), 1.85 (m, 2H, CH2), 2.46 (m, 2H, CH2), 2.70 (m,

13 2H, CH2), 3.10 (d, 1H, J = 8.8 Hz, CHS), 3.91 (m, 1H, CHO); C NMR (100 MHz, CDCl3)

δ 13.8 (CH3), 13.9 (CH3), 14.3 (CH3), 17.7, 22.2, 22.9, 25.4, 30.1, 31.7, 31.9, 33.9, 42.9,

57.5 (CHS), 70.9 (CHOH), 208.2 (CHO). Adduct of derivative C (2-(butylthio)-3-hydroxy-

1 1-phenyloctan-1-one) (3.20): H NMR (400 MHz, CDCl3) δ 0.83 (t, 3H, J = 7.4 Hz, CH3),

0.89 (t, 3H, J = 7.0 Hz, CH3), 1.20-1.70 (m, 12H, 6×CH2), 2.53 (m, 2H, CH2), 4.00 (d, 1H,

J = 8.8 Hz, CHS), 4.19 (ddd, 1H, J = 8.8 Hz, 8.4 Hz and 2.8 Hz, CHO), 7.47 (m, 2H,

13 2×C6H5), 7.58 (ddt, 1H, J = 8.4 Hz, 8.0 Hz and 1.2 Hz, C6H5), 7.99 (m, 2H, 2×C6H5); C

NMR (100 MHz, CDCl3) δ 13.8 (CH3), 14.3 (CH3), 22.1, 22.9, 25.6, 29.6, 31.5, 32.0, 33.7,

51.7 (CHS), 71.2 (CHOH), 128.8 (C6H5), 128.9 (C6H5), 133.6 (C6H5), 136.4 (C6H5), 197.1

(C=O).

3.4.3 Reactions of EKODEs with cysteine

Synthesis of trans-EKODE-(E)-Ib (9-oxo-trans-12,13-epoxy-10(E)-octadecenoic acid)

(3.22). The methyl ester of trans-EKODE-(E)-Ib (methyl 13-oxo-trans-9,10-epoxy-11(E)- octadecenoate) (3.21) was synthesized using previously published method4. 232 mg of the methyl ester (3.21) (0.716 mmol) was dissolve in 10 mL of PBS containing 20% acetonitrile, followed by addition of PPL (1.4 g, 100-500 unites/mg protein). After stirring at r.t. for 2 hours, the reaction mixture was extracted with ethyl acetate (3× 40 mL). The combined organic layer was dried over Na2SO4, concentrated, and purified by column chromatography (hexane/ethyl acetate/methanol, 4:1:1) to afford trans-EKODE-(E)-Ib as a white powder (3.22) (137 mg, 62% yield).

Synthesis of trans-EKODE-(E)-IIb (trans-12,13-epoxy-11-oxo-9(E)- octadecenoic acid) (3.25). The ozonolysis of methyl oleate followed previously published method31. Briefly, ozone was bubbled through the solution of methyl oleate (3.23) in methanol/CH2Cl2 at -78 °C until a persistent purple color appeared. After addition of glacial acetic acid and Zn powder, the reaction was quenched by NaHCO3 and purified via distillation to give methyl 9-oxononanoate (3.24) (95% yield). The methyl ester of trans-

EKODE-(E)-IIb (methyl trans-12,13-epoxy-11-oxo-9(E)-octadecenoate) was then prepared through the synthesis route published by Lin et al.4 The enzymatic hydrolysis of the methyl ester was then performed using the same procedures that was used in the synthesis trans-EKODE-(E)-Ib.

Kinetic studies of the reaction of EKODEs and EKODE model compounds with N-acetyl-cysteine-methyl ester. 10 μM EKODEs/EKODE model compounds and

100 μM N-acetyl-cysteine-methyl ester were dissolved in PBS containing 20% acetonitrile.

The reaction mixture was added to UV quartz cuvettes with 10 mm path length and capped.

UV analysis was conducted on a Lambda 25 UV/VIS Spectrometer (PerkinElmer,

Waltham, MA) equipped with cell changer and peltier temperature controller. The spectrometer was programed to maintain the reactions at 37 °C and scan the UV absorbance from 250 nm to 210 nm every 30 min at a scan speed of 480 nm/min. The absorbance at

234 nm was used to monitor the disruption and recovery of the α, β-unsaturated carbonyl in the reactions.

LC-MS analysis of the reaction of trans-EKODE-(E)-IIb with N-acetyl- cysteine-methyl ester. 40 μL of 1 mM trans-EKODE-(E)-IIb stock solution in acetonitrile was mixed with 8 μL of 50 mM N-acetyl-cysteine-methyl ester solution in water, followed by addition of 752 μL of acetonitrile, and 3.2 mL of PBS. Final concentrations of trans-

EKODE-(E)-IIb and N-acetyl-cysteine-methyl ester were 10 μM and 100 μM respectively.

The reaction mixture was then incubated at 37 °C and 500 μL of the mixture were collected at 0 minute, 3 hours, 20 hours, and 3 days. The aliquots were stored at -80 °C until analysis by LC-MS. 50 L of the reaction mixture from each time point were separated by LC and subjected to MS analysis on a Shimadzu LCMS-2020 mass spectrometer. The reactions were monitored at an m/z range of 100 - 1000. The selected ion current of trans-EKODE-

(E)-IIb and EKODE-Cys adduct were extracted from the total ion current using an extraction window of 1.0 Da (m/z 310 ± 0.5 for trans-EKODE-(E)-IIb, and m/z 488 ± 0.5 for EKODE-Cys).

3.4.4 Mass spectrometric characterization of the modification of HSA by trans-

EKODE-(E)-IIb

The reaction of HSA with by trans-EKODE-(E)-IIb. 10 μM of HSA was incubated with various concentration of trans-EKODE-(E)-IIb (100/500/1000 μM) in PBS

containing 20% acetonitrile overnight. 400 μL of pre-chilled ethanol/ethyl acetate (1:1) was then added to the centrifuge tube containing 100 μL of reaction mixture and the tube was placed on ice for 30 minutes. After centrifugation for 15 minutes at 14800 g, the supernatant was discarded and the precipitate was washed with cold acetonitrile (3 × 400

μL). The protein pellet was reconstituted in 100 μL of 50 mM AMBIC, followed by addition of 9 μL of trypsin solution (0.5 mg/mL in AMBIC). The trypsin digestion mixture was then incubated overnight at 37 °C and quenched by addition of 10 μL of 10% TFA

(v/v).

LC-HRMS analysis using IDA method. LC-HRMS analysis employed a HPLC system consisted of Shimadzu LC20AD pumps and a SIL-HTC autosampler (Columbia,

MD, USA). A Kinetex XB-C18 column (2.1 mm × 50 mm, 2.6 m, 100 Å, Phenomenex,

Torrance, CA, USA) was used for LC separation at a flow rate of 0.4 mL/min. Mobile phase A was 0.2% formic acid (FA) in water, and mobile phase B was 0.2% FA in acetonitrile. The needle rinse solvent was 0.1% TFA in 50% acetonitrile in water (v/v/v).

A multiple-step gradient was as following: 0-1 min (5% B), 1-10 min (5%-90% B), 10-12 min (90% B), 12-12.5 min (90%-5% B), and 15 min (5% B). 5 L of the reaction mixture from each time point were resolved by LC and subjected to MS analysis on an API 5600

TripleTOF mass spectrometer (AB Sciex, Foster City, CA, USA) by IDA method. The ion source parameters in positive turbo ionspray mode were as follows: curtain gas 30 psi,

GAS1 45 psi, GAS2 45 psi, ionspray voltage 5500 V, and source temperature 500 °C. The

TOF scan of peptide precursors was conducted at a MS range of m/z 100-1600 and the product ion spectrum acquisition was triggered for precursors with intensity above 150 cps.

The TripleTOF instrument was auto-tuned on a daily basis using tuning solution (AB

Sciex) to ensure MS accuracy. APCI positive calibration standard (AB Sciex) was delivered at speed of 500 μL/min for 2 min by the calibration delivery system (CDS) every

5 sample injections for mass calibration. The acquired data was processed in

BioPharmaView software (AB Sciex) for peptide mapping. Variable modifications of

C18H30O4 (+310.2144 Da) on Cys, His, or Cys were included for peptide identification.

The matching tolerance for peptide precursor and product ion were set at 10 ppm and 0.10

Da respectively.

3.5 Acknowledgement

Some of the data from the experiments using EKODE model compounds were obtained by Dr. Jianye Zhang (Case Western Reserve University) and recorded in his PhD dissertation37. The peptide mapping experiments were performed in the laboratories at

Janssen Pharmaceuticals, Inc.

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17. Sayre, L. M.; Sha, W.; Xu, G.; Kaur, K.; Nadkarni, D.; Subbanagounder, G.;

Salomon, R. G., Immunochemical evidence supporting 2-pentylpyrrole formation on proteins exposed to 4-hydroxy-2-nonenal. Chemical Research in Toxicology 1996, 9,

1194-1201.

18. Sayre, L. M.; Lin, D.; Yuan, Q.; Zhu, X.; Tang, X., Protein Adducts Generated from Products of Lipid Oxidation: Focus on HNE and ONE. Drug Metabolism Reviews

2006, 38, 651-675.

19. Lin, D. Modification and Cross-Linking of Proteins by Lipoxidation Derived

Electrophiles: Chemical and Biological Consequences. Case Western Reserve University

2006.

20. Doorn, J. A.; Petersen, D. R., Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal. Chemical

Research in Toxicology 2002, 15, 1445-1450.

21. Uchida, K.; Stadtman, E. R., Modification of Histidine-Residues in Proteins by

Reaction with 4-Hydroxynonenal. Proceedings of the National Academy of Sciences of the

United States of America 1992, 89, 4544-4548.

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2003, 16, 512-523.

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2007, 79, 9174-9184.

24. Orioli, M.; Aldini, G.; Beretta, G.; Facino, R. M.; Carini, M., LC-ESI-MS/MS determination of 4-hydroxy-trans-2-nonenal Michael adducts with cysteine and histidine- containing peptides as early markers of oxidative stress in excitable tissues. Journal of

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Receptor Inhibitors. The Journal of Organic Chemistry 2008, 73, 7764-7767.

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Reaction Products Originating from the Initial 4-Oxo-2-nonenal-cysteine Michael Adducts.

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Modifications of Proteins. Case Western Reserve University 2011.

Chapter 4

Immunochemical Detection of EKODE-Cysteine

Adducts in Oxidative Stress, Aging, and Diseases

4.1 Introduction

Oxidative stress has been associated with a growing list of disease states including

Alzheimer’s disease1-4, cardiovascular disease5, 6 and cancer7-9. The progress of oxidative stress can lead to damage in all biomacromolecules but polyunsaturated fatty acids

(PUFAs) are particularly susceptible to the free radical mediated peroxidation, which results in the formation of a variety of reactive species, including bifunctional aldehydes and ketones10. It is established that many of those LPO products are highly electrophilic and subsequently cytotoxic and genotoxic through their ability to covalently modify protein11, phospholipids12, and DNA13, 14. In the past few decades, one of the major topics in this field has been directed at investigating the chemical natural of protein modification by LPO products. Reversible protein modification products, including Michael adducts and

Schiff base adducts, can be generated from the covalent reactions with nucleophilic protein side chains including lysine, histidine, and cysteine15. Subsequent cyclization reactions of lysine adduct can lead to stable products such as the HNE-derived 2-pentylpyrrole, which was termed the first example of an “advanced lipoxidation end-product” (ALE)16.

Despite the fact that some of the deleterious effects of the LPO products may arise from modification of DNA bases and consequent mutations17, 18, the pathogenicity of LPO products in disease progression are widely rationalized by the modification and crosslinking of proteins to modify or abrogate the proteins’ ability to function normally.

Since the side chains of cysteine, lysine and histidine are often involved in enzymatic catalysis, the most common outcome of protein modification by LPO products is enzyme inactivation19. The formation of cysteine and selenocysteine adducts with 4-HNE, for example, has been reported to contribute to the inactivation of thioredoxin and thioredoxin

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reductase, which subsequently leads to the dysregulation of cellular redox state20, 21. In addition, LPO product-modified proteins are resistant to degradation by inhibiting proteasome machinery. These modifications may contribute to the accumulation of modified proteins, which represent the cumulative oxidative damage during times of oxidative stress22.

Markers of protein modification by LPO products have been developed and utilized as valuable tools to ascertain the relevance of LPO products in various disease states and pathogenesis of aging23. The most extensively studied oxidative stress related diseases are neurological diseases such as Alzheimer’s disease and Parkinson’s disease24. Increased

HNE derived protein modifications in Alzheimer’s disease have been reported by a number of groups25-27. In addition, acrolein28 and MDA29-modified proteins have been identified as biomarkers to investigate the potential contribution of LPO to neurofibrillary pathology and neuronal demise. The biological relevance of MDA-modified proteins is also identified in atherosclerosis, which is a major cause of coronary heart disease (CHD) and strokes.

The detection of MDA derived protein adducts in atherosclerotic plaques was first reported by Haberland et al. using antibodies against MDA modified proteins30. MDA-LDL adduct was found to regulate pro-inflammatory and pro-atherogenic processes, which results in the production of foam cells31.

Although mass spectrometry based methods have been used for detection of protein adducts in oxidative stress, and they can usually reveal more structural information, immunoassays are the most widely used approach for the characterization and quantitation of protein adducts derived from LPO products. ELISA kits measuring HNE protein adduct levels are commercially available and have been used to study biological relevance of

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protein modification of LPO products32. The antibodies bind not only to the specific antigen that is used to produce antibodies, but also to various other proteins that contain the same epitope. Therefore, the antibodies can recognize a variety of proteins that are modified by the same LPO product of interest and the immunoassay can be used to determine the overall impact of protein modification originating from specific LPO products.

In the previous chapter, we reported a mechanistic study of the protein modification by EKODEs using model amino acids and model proteins. A key finding of this work was that EKODEs can form irreversible ring opening products with cysteine analogues in addition to the Michael adducts and the thiolysis of EKODE II model compounds was at the β position of the carbonyl. The mechanism of formation of this unexpected ring opening product was studied using EKODE derivatives and was established to be an intra-molecular rearrangement of the cysteine Michael adduct. The cysteine adducts were also identified in the reaction of EKODEs with HSA, while Cys-34 was found to be one of the most reactive residues of HSA toward EKODEs. Those findings led us to further investigate the relevance of EKODE-Cys adducts in aging and disease states. In an effort to detect and quantify EKODE-Cys adducts in biological samples, rabbit polyclonal antibodies were produced and characterized. Immunoassays were then performed to detect the presence of

EKODE-Cys adducts in oxidative stress, cardiovascular diseases, and aging.

4.2 Results and Discussions

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4.2.1 Strategies for antigen preparation

Small molecule antigens, usually referred to as haptens, are normally not able to initiate an immune response. Therefore, in order to produce antibodies that are specific to

EKODE-cysteine adducts, the haptens need to be attached to a large carrier protein to obtain immunogenicity (Figure 4.1). Due to its large size and complexity, keyhole limpet hemocyanin (KLH) stimulates a strong immune response and is the most widely used carrier protein to prepare antigens for antibody production. In addition to the carrier, an aminocaproyl spacer was also incorporated into the design of antigen. It can distance the hapten from the surface of carrier protein and therefore, improve the immunogenicity of the hapten, and reduce the immune response stimulated by carrier proteins33. Another advantage of incorporating a spacer is that the hapten-spacer conjugate can be separated and characterized by NMR and LCMS to validate the conjugation reaction, which was subsequently used for conjugating the hapten-spacer to the carrier protein.

Figure 4.1 Design of antigen

trans-EKODE-(E)-Ib and trans-EKODE-(E)-IIb were selected for antibody production since they are the most abundant isomers of EKODE I and EKODE II family

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identified from nonenzymatic autoxidation of linoleic acid34. EKODEs were incubated with N-acetyl-cysteine-methyl ester overnight in phosphate buffered saline (PBS) containing 20% ethanol. The reaction was briefly purified by column chromatography over silica to remove excess reagent to afford a mixture of EKODE-cysteine Michael adduct and ring-opening product. Carbodiimide compounds, such as 1-ethyl-3-(-3- dimethylaminopropyl) carbodiimide hydrochloride (EDC), provide an efficient and versatile method for crosslinking carboxylic acid to free amine on protein side chains. The carboxylic acid terminal of EKODE-cysteine adducts were activated by EDC to form an active O-acylisourea intermediate, which then reacted with N-hydroxysuccinimide (NHS) to produce a stable amine-reactive NHS ester (Scheme 4.1). The addition of NHS improved conjugation efficiency and the stability of the intermediate. The resulting NHS ester was significantly more stable than the O-acylisourea intermediate and it was purified by silica gel chromatography to remove excess crosslinking reagents which might cause random polymerization of the free spacer. The amine reactive NHS ester was then mixed with aminocaproic acid, an analogue of lysine, to generate an EKODE adduct-spacer conjugate.

This reaction mimics the conjugation with lysine residues on carrier protein and therefore, the same reaction conditions were used for the conjugation of EKODE-adduct-spacer conjugates with KLH. The conjugate with bovine serum albumin (BSA) was also produced as the coating agent for immunoassays.

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O Cys O O O O O O 20% ethanol OH + HS O OH Cys O O HN PBS EKODE O OH N-acetyl-cysteine-methyl ester OH

Cl EKODE-Cys adduct NNH C N O Cys O HN Cl EDC O H O N N DMSO unstable reactive O-acylisourea ester

DMSO N HO O NHS

O O Cys O O N O O stable amine-reactive NHS ester

OH H2N DMSO/PBS O

O Cys O O OH N H O EKODE-Cys-spacer conjugate

NH2

EDC/NHS KLH/ BSA

O Cys O O H N N KLH/ H BSA O EKODE-Cys-spacer-KLH conjugate

Scheme 4.1 Synthesis of EKODE-Cys-spacer-KLH conjugate as the antigen for antibody production

The resulting EKODE-adduct-spacer-BSA conjugate was characterized by matrix- assisted laser desorption/ionization-time of flight (MALDI-TOF) (Figure 4.2). Intact BSA showed a peak at around 66343 Da and it shifted to ~76848 Da in EKODE Ib-Cys-spacer-

BSA conjugate and ~78525 Da in EKODE IIb-Cys-spacer-conjugate due to the conjugation of hapten and spacer. The protein conjugates were further evaluated by a 2,4,6-

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trinitrobenzene sulfonic acid (TNBSA) assay to determine the loss of free ε-amino groups on lysine. The conjugation efficiency was estimated as the ratio of conjugated lysine residues to total lysine residues by comparing MALDI-TOF mass and TNBSA reading of protein conjugates with that of intact protein. As shown in Table 4.1, at least 29.2% of lysine residues were attached to hapten based on both MALDI-TOF and TNBSA assay.

Considering the fact that only 30-35 out of the 59 lysine residues are available for conjugation reactions35, the coupling efficiency was satisfactory. KLH conjugates were only tested by TNBSA because the large molecular weight of KLH was out of MS range of MALDI-TOF.

Table 4.1 Characterization of antigens and coating agents using MALDI-TOF and TNBSA assay

% Lys modified

MALDI-TOF TNBSA

EKODE Ib-Cys-spacer-BSA 29.7% 64.6%

EKODE Ib-Cys-spacer-KLH N/A 45.8%

EKODE IIb-Cys-spacer-BSA 34.4% 58.0%

EKODE IIb-Cys-spacer-KLH N/A 29.2%

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]

u. x104 66343.831 a. [

ens. 5 nt I m/z 66343.831 BSA

4 33123.492

10000 20000 30000 40000 50000 60000 70000 80000 90000 m/z ]

u. x104 a. [

ens.

nt EKODE Ib‐Cys‐spacer‐BSA I m/z 76848.195

76848.195

37971.977 2

20000 40000 60000 80000 100000 m/z ]

u. x104 a.

[

ens. nt I m/z 78525.085 EKODE IIb‐Cys‐spacer‐BSA 78525.085

1.5

1.0

0.5

0.0 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 m/z

Figure 4.2 MALDI-TOF analysis of (A) intact BSA, (B) EKODE Ib-Cys-spacer-BSA conjugate, (C) EKODE IIb-Cys-spacer-BSA conjugate

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4.2.2 Production and characterization of antibodies against EKODE-cysteine adduct

The KLH conjugates were used to immunize rabbits (Cocalico Biologicals) and the antibody titer was monitored by enzyme-linked immunosorbent assay (ELISA) using the

BSA-derived conjugates as coating agent, BSA as ELISA plate blocking agent, goat anti- rabbit IgG (horseradish peroxidase (HRP) conjugated) as secondary antibody. Antibody titer increased steadily over a period of 91 days and reached a plateau around day 70

(Figure 4.3). In order to purify and enrich the specific IgG fractions, the BSA conjugates of EKODE-Cys adducts were immobilized on aldehyde-activated beaded agarose resin through Schiff base bond formed with primary amines. The resulting affinity resin was then used for immunocapture of specific antibodies from the final bleed. Irrelevant endogenous antibodies were removed by washing the resin-bound complex and desired

IgG fraction was dissociated at low pH.

0.50 EKODE Ib-Cys 0.45 EKODE IIb-Cys 0.40

0.35

0.30

405 0.25 A 0.20

0.15

0.10

0.05 0 20406080100 Time (days)

Figure 4.3 Serum antibody titer in rabbits immunized with EKODE-Cys-spacer-KLH

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Epitope characterization of the purified antibodies was performed by competitive

ELISA. Negative competitors were synthesized by incubating EKODEs with N-acetyl- histidine-methyl ester or 4-HNE with N-acetyl-cysteine-methyl ester (Figure 4.4 C).

A B

1.2 1.2

1.0 1.1

0.8 1.0 0.9 0 0.6 0 0.8 B/B B/B 0.4 EKODE IIb-Cys 0.7 EKODE Ib-Cys EKODE IIb-His EKODE Ib-His HNE-Cys 0.2 0.6 HNE-Cys EKODE Ib-Cys EKODE IIb-Cys 0.5 0.0 1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 competitors (nmol/well) competitors (nmol/well)

Figure 4.4 Epitope characterization of anti-EKODE-Cys antibodies by competitive ELISA. (A) anti-EKODE IIb-Cys antibody competitive ELISA; (B) anti-EKODE Ib-Cys antibody competitive ELISA; (C) Structures of competitors for anti‐EKODE IIb antibody

The anti-EKODE-Cys antibodies were pre-adsorbed with competitors at various levels and the mixture was applied in ELISA with the same coating agents and same

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secondary antibody system used for measurement of antibody titer. As shown in Figure

4.4 A and 4.4 B, EKODE-His adducts and HNE-Cys adducts did not display significant inhibition of antibody binding, while the corresponding EKODE-Cys adducts, as expected, are more potent inhibitors. These results suggest that antibody recognition involves the full structure segment of EKODE-Cys adduct, including both the EKODE backbone and cysteine moiety. Therefore, the anti-EKODE-Cys antibodies have the potential to differentiate EKODE-Cys adduct from protein modification by other LPO products as well as modification by EKODE at protein residues other than cysteine. It is worth mentioning that while anti-EKODE IIb-Cys antibody exhibited excellent specificity toward the corresponding EKODE IIb isomers, both EKODE-Ib-Cys and EKODE-IIb-Cys adducts showed similar levels of inhibition toward anti-EKODE-Ib-Cys antibody, indicating that anti-EKODE-Ib-Cys antibody cannot differentiate the two EKODE isomers.

Lipid peroxidation leads to the formation of a large number of reactive aldehydes including HNE and ONE, which can react with nucleophilic residues on proteins. The cross-reactivity of aldehyde-protein adducts with anti-EKODE-Cys antibodies can introduce significant interference into the immunoassay. Therefore, the specificity of anti-

EKODE-Cys antibodies was further evaluated by western blot analysis with BSA modified by various LPO products. BSA was incubated with EKODEs as well as other common

LPO products including HNE, ONE, acrolein and octenal. The resulting BSA adducts were resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to membrane for western blot analysis. As shown in Figure 4.5, aldehyde- modified BSA was not recognized by the antibody, whereas EKODE adducts displayed a concentration-dependent reactivity. In addition, anti-EKODE-IIb-Cys antibody was able

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to differentiate the two EKODE isomers, which was consistent with competitive ELISA results. The excellent specificity of the antibodies might be attributed to the antibody purification step where specific BSA conjugates of EKODE-Cys adducts rather than generic protein G were used.

10uM 100uM 10uM 100uM 100uM 100uM 100uM 100uM EI EI EII EII HNE ONE Acrolein Octenal

A. anti-EKODE Ib-Cys antibody

B. anti-EKODE IIb-Cys antibody

Figure 4.5 Cross reactivity of LPO products modified proteins with (A) anti-EKODE Ib-

Cys antibody and (B) anti-EKODE IIb-Cys antibody.

The antibodies were then applied in the immunochemical detection of EKODE-Cys adducts that are generated when proteins are exposed to PUFAs under nonenzymatic oxidation conditions. BSA, a model protein containing a free cysteine residue Cys-34, was incubated with linoleic acid and an iron/ascorbate-mediated free radical-generating system at 37 °C for a period of 24 h (Figure 4.6). The negative control (0 h) consisted of native

BSA and did not show cross-reactivity with anti-EKODE-Cys antibodies. Meanwhile, the formation of EKODE-Cys adducts during the incubation was identified by immunoblot analysis. The incubation of BSA with linoleic acid under non-enzymatic oxidation conditions resulted in a time-dependent increase in the EKODE derived modification on

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cysteine residue. The results suggest that EKODE-Cys adducts have the potential to become biomarkers for oxidative stress. It is worth mentioning that a new protein band between 100-150 kDa was generated after 2 h incubation. This was tentatively assigned as

BSA dimer which was generated presumably from the protein cross-linking by EKODEs or other lipid peroxidation products36.

0h 0.5h 1h 2h 4h 8h 24h

KDa

150 100 75

A. anti-EKODE Ib-Cys antibody

150 100 75

B. anti-EKODE IIb-Cys antibody

Figure 4.6 Detection of EKODE-Cys adducts in BSA treated with linoleic acid under nonenzymatic oxidation conditions using (A) anti-EKODE Ib-Cys antibody and (B) anti-

EKODE IIb-Cys antibody

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4.2.3 Immunochemical detection of EKODE-cysteine adduct in nervous system

The central nervous system is particularly vulnerable to oxidative damage due to its high PUFA level, high rate of oxygen consumption, and comparatively limited endogenous antioxidant defense mechanisms. The reactive LPO products derived from oxidative damage of polyunsaturated lipids readily modify proteins and DNA. One of the major topics in this field is to identify the relevance of these adducts in neurodegenerative disease and aging3, 32, 37. In an effort to ascertain the nature of EKODE adducts, the anti-

EKODE-Cys antibodies were applied to identify EKODE adducts in nervous systems.

M17, a human neuroblastoma cell line widely used for in vitro research of the central nervous system, was maintained in serum-free Opti-MEM media. M17 cells were then treated with increasing concentrations of hydrogen peroxide for 24 hours to mimic the oxidative damage derived from oxidative stress. Treatment of high concentrations of H2O2

(0.5 mM) resulted in a significant loss of cell viability (data not shown), whereas lower concentration did not induce notable cell death and the cell lysate was analyzed by western blot. As shown in Figure 4.7, endogenous cysteine adducts of both EKODE-Ib and

EKODE-IIb were identified in control groups which were not treated with H2O2. β-Actin was used as an internal control and the levels of EKODE-Cys adduct considerably increased when the cells were exposed to hydrogen peroxide. 24 Hours exposure of 0.1 mM H2O2 resulted in 50% increase in EKODE-Ib-Cys adduct and over 100% increase in

EKODE-IIb-Cys adduct. This observation was possibly due to the relatively higher activity of EKODE-IIb compared with EKODE-Ib34. Under mild oxidative conditions, which were induced by a low concentration of hydrogen peroxide, the higher levels of EKODE-Cys adduct reflected the elevated production of LPO due to oxidative damage of PUFAs. When

113

M17 cells were treated with 0.25 mM of hydrogen peroxide, the level of EKODE-Ib-Cys slightly increased, while EKODE-IIb-Cys showed a small decrease. This can be explained by the fact that EKODEs are polyoxygenated products that retain the full carbon chain of

PUFAs and they may undergo chain cleavage reactions to convert to other LPO products under severe oxidative conditions. Therefore, the formation of EKODE-Cys adducts can be impacted by the equilibrium of EKODE production and EKODE elimination via oxidative cleavage under severe oxidative stress.

Figure 4.7 Detection of EKODE-Cys adducts in M17 cells treated with H2O2

In an effort to identify the correlation of EKODE-Cys adducts with aging process, the anti-EKODE-Cys antibodies were tested with human brain tissues from different age

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groups. To assess if the western blot results can reflect the level of EKODE adducts, the homogenate of human brain tissue was incubated with trans-EKODE-(E)-Ib and analyzed using anti-EKODE-Ib-Cys antibody. The intensity of the two major protein bands at 25-37 kDa showed a concentration-dependent increase (Figure 4.8A). This result also confirmed that the major proteins detected by western blot were derived from EKODE modification.

Brain tissues from control patients at different ages were then tested with anti-EKODE-

Cys antibodies. Significant changes in the levels of both EKODE-Cys adducts were observed as a function of age. As shown in Figure 4.8B, the identities of EKODE-modified proteins were consistent across different age groups, while the concentrations of EKODE adducts in middle-aged (30-40 years) subjects were over 300% higher than they were in young subjects (Figure 4.8C). The elevated level of EKODE-Cys adducts was consistent with the oxidative stress theory of aging, which attributes the phenomenon of aging to endogenously generated oxidants and the subsequent oxidative damage of biomolecules.

Since the ring-opening reaction of EKODE with cysteine is irreversible and the resulting adduct is relatively stable, it is possible that EKODE-Cys adducts, once generated as a result of LPO, are able to accumulate through the aging process. The results indicate that

EKODE-Cys adducts may serve as biomarkers for aging and aging related diseases such as Alzheimer’s disease and Parkinson’s disease. It is worth mentioning that studies using antibodies against HNE-derived protein adducts showed no correlation between the degree of HNE modification and age25. This may be explained by the fact that EKODEs are polyoxygenated products that are generated under mild oxidative conditions, whereas HNE is a chain cleavage product. Consistent therewith, EKODEs are more sensitive to the mild oxidative damage induced by normal aging conditions. Although anti-EKODE-Cys

115

antibodies have the potential to become a useful tool for quantitation of oxidative stress, more detailed studies are needed for elucidating the biological relevance of EKODE-Cys adducts with aging since many factors such as PUFAs intake may have impacts on the production of EKODEs and EKODEs derived protein adducts.

A B

Ctrl 10uM 100uM

Figure 4.8 Immunochemical detection of EKODE-Cys adducts in human brain tissue during the aging process. A. Human brain tissue incubated with increasing concentrations of trans-EKODE-(E)-Ib; B. Detection of EKODE-Cys adducts in human brain tissue in aging process; C. Quantitation of EKODE-Cys adducts

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4.2.4 Immunochemical detection of EKODE-cysteine adduct in cardiovascular disease and other tissues

The development of anti-EKODE-Cys antibodies provided a useful approach for identifying the production of EKODE-Cys adducts and showing the tissue distribution.

Tissues from eight different mouse organs were homogenized in Krebs buffer. Protein concentrations of the tissue homogenates and blood samples were measured by bicinchoninic acid (BCA) assay. The tissue homogenates and blood samples were then resolved on SDS-PAGE and subjected to western blot analysis. As shown in Figure 4.9, both EKODE-Ib-Cys and EKODE-IIb-Cys were heavily accumulated in liver and heart, whereas no traceable level of immunogenic molecules were detected in intestine and spleen. Since liver is the major organ for detoxification, one of the major topics in this field has been directed to the identification of LPO products generated in liver and hepatic proteins modified by LPO products. Substantial studies appeared using immunochemical approaches to detect hepatic protein adducts and use them as biomarkers for oxidative stress in liver diseases such as alcoholic liver diseases (ALD)38-40. This is consistent with our finding that liver was one of the major organs where EKODE-Cys adducts accumulated. Meanwhile, high level EKODE-Cys adducts were also found in heart tissues.

Heart is vulnerable to oxidative damage due to the high consumption of oxygen and nutrition for energy production41. Meanwhile, the cell turnover in this highly oxidative organ is extremely low. Therefore, LPO products generated as a result of oxidative damage in cardiomyocytes accumulate over time and some of the LPO products have been directly associated with heart diseases including CHD. It is worth mentioning that EKODE-Cys adducts were present in blood, although the abundance was relatively low compared with

117

that in liver and heart. It indicates that anti-EKODE-Cys antibodies may be used for immunochemical detection of EKODE-modified proteins in blood. Therefore, it may provide a less invasive approach that is more practical for clinical application.

Figure 4.9 Distribution of (A) EKODE Ib-Cys and (B) EKODE IIb-Cys adducts in mouse tissues

In an effort to investigate the relevance of EKODE-Cys adducts in cardiovascular diseases, isolated heart perfusion experiments were performed to model cardiac ischemia42.

Rat hearts were perfused for baseline equilibration, followed by global no-flow ischemia for a period of time. Hearts were then quick-frozen and homogenized for western blot analysis. The majority of EKODE modified proteins in control groups (0 min) were identified at 37-50 kDa, which was consistent with the result in tissue distribution

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experiment (Figure 4.10). There was no significant change in the abundance of both

EKODE-Cys adducts after 5 minutes of heart ischemia, whereas the level of EKODE-Ib-

Cys increased to over 160% at 15 minutes and 399% at 30 minutes. The accumulation of

EKODE-IIb-Cys was even more remarkable and it was 274% at 15 minutes and 517% at

30 minutes. The results suggest that there was a clear link between the levels of EKODE- modified proteins with the ischemic state in heart. It is possible that the accumulation of

EKODE-Cys adducts with ischemia time is a result of elevated production of ROS that, in turn, lead to EKODE-modified cardiac proteins. However, since the ischemic heart lacks oxygen supply, the accumulation of EKODE-Cys adduct is more likely due to the reduced rate of the disposal of LPO products43. Particularly, glutathione (GSH) is depleted rapidly during ischemia due to the diminished biochemical pathway to regenerate GSH from glutathione disulfide (GSSG)44, 45. GSH is the major endogenous antioxidant that can scavenge ROS as well as form covalent conjugates with LPO products such as HNE.

Although the GSH adduct of EKODE has not been reported, it is highly possible that GSH can react with EKODE via similar mechanisms as EKODE-Cys adducts formation, which was elaborated in Chapter 3. Therefore, the loss of GSH during heart ischemia may compromise the detoxification mechanism that effectively scavenges EKODEs and leads to accumulation of EKODE derived protein adducts. In addition, since EKODE may be eliminated through enzymatic catabolism, ischemia-induced malfunction of the relevant enzymes can also result in the accumulation of EKODEs. During heart ischemia, ATP is depleted within minutes. Subsequently ATP-dependent enzymes such as kinases lose the ability to process and eliminate cytotoxic LPO products including EKODEs that can initiate modification of cardiac proteins.

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Figure 4.10 Immunochemical detection of EKODE-Cys adducts in ischemic rat heart using (A) anti-EKODE Ib-Cys antibody and (B) anti-EKODE IIb-Cys antibody, and (C) quantitation results

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4.3 Conclusions

In this chapter we synthesized an antigen by conjugating EKODE-Cys adducts to

KLH, which was used for producing antibodies. The resulting polyclonal antibodies were characterized by competitive ELISA and immunoblot. It was found that the anti-EKODE-

Cys antibodies does not have significant cross-reactivity with proteins modified by other

LPO products and antibody recognition of epitope involves both the EKODE backbone and cysteine moiety. The antibodies were then applied in the detection of EKODE-Cys adducts in neuroblastoma cells under oxidative stress induced by H2O2. A H2O2 concentration dependent increase in EKODE-Cys adducts was identified, which indicates the accumulation of EKODE-Cys adducts in oxidative stress. The accumulation was also identified in human brain tissues during aging process and this observation is consistent with the free radical theory of aging. The tissue distribution of EKODE-Cys adducts was assessed and it was found that adducts were heavily accumulated in liver and heart. A heart perfusion experiment was then performed to mimic the heart ischemia conditions and elevated levels of EKODE-Cys adducts with ischemia time were observed. All of the above results suggest that there is a clear link between the levels of EKODE-Cys adducts and oxidative stress as well as the related disease states. The EKODE-Cys adducts may be used as a biomarker for oxidative stress and the anti-EKODE-Cys antibodies provide a valuable tool for evaluating this biomarker.

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4.4 Experimental Sections

4.4.1 Materials and Methods

General chemicals, organic solvents, N-acetyl-L-cysteine methyl ester (≥90%

HPLC), BSA (essentially fatty acid free), acrolein, trans-2-octenal were purchased from

Sigma-Aldrich (St. Louis, MO, USA). NHS, EDC, Imject mcKLH, Slide-A-Lyzer Dialysis

Cassettes (10K MWCO, 3 mL), TNBSA (5% w/v in methanol), Sinapinic acid MALDI matrices, Goat anti-Rabbit IgG (H+L) Secondary Antibody (HRP conjugate), and 1-Step

ABTS Substrate Solution (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]- diammonium salt), AminoLink Plus Micro Immobilization Kit (0.1 mL), BCA Protein

Assay Kit, Restore Western Blot Stripping Buffer were purchased from Fisher Scientific

(Waltham, MA, USA). 6-Aminocaproic acid was obtained from Alfa Aesar (Ward Hill,

MA, USA). 2 × Laemmli Sample Buffer was from Bio-Rad (Hercules, CA, USA).

Immobilon PVDF membrane and ECL detection reagent were purchased from Millipore

(Billerica, MA, USA). 10 × cell lysis buffer was purchased from Cell signaling technology

(Danvers, MA, USA). Complete Protease Inhibitor Cocktail Tablets were purchased from

Roche (Basel, Switzerland).

Column chromatography was conducted using silica gel (particle size 32-63 μM)

Analytical thin-layer chromatography (TLC) was performed by glass-coated silica gel plates with fluorescent indicator (0.25 mm), and visualized by UV lamp (254 nm) or TLC stains including phosphomolybdic acid (PMA) and 1,4-dinitrophenylhydrazine (DNP).

In addition to TLC, the reactions were also monitored by LC-MS. HPLC system is consisted with Shimadzu LC-20AB pumps and SIL-20A HT autosampler (Columbia, MD).

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30-31 min (95%-60% B), and 35 min (60% B). 10 L of reaction mixture was separated by LC and subjected to MS analysis on a Shimadzu LCMS-2020 mass spectrometer. The

DUIS interface setting was: interface temperature 350 °C, DL temperature 250 °C, nebulizing gas flow 1.5 L/min, heating block 400 °C, interface voltage 3.5 kV, and detector voltage 0.95 kV. The m/z range was 100- 1000.

4.4.2 Synthesis of antigens against EKODE-cysteine adduct

Preparation of EKODE-Cys adducts. 140 mg (0.452 mmol) of trans-EKODE-

(E)-Ib or trans-EKODE-(E)-IIb was dissolved with 1 mL of 20% acetonitrile in PBS buffer

(pH 7.4). N-acetyl-L-cysteine methyl ester (85.6 mg, 0.484 mmol) was then added and the reaction was stirred at r.t. overnight, at which time the reaction mixture was extracted with

5 mL of ethyl acetate for 3 times. The organic layer was combined, dried over sodium sulfate, and filtered. The filtrate was concentrated and briefly purified by silica gel chromatography using hexanes/ethyl acetate (1:1) to give a mixture of ring opening product and Michael adduct as a transparent oil (163 mg, 74.1% yield). The mixture of EKODE-

Cys adducts was characterized by LC-MS, which showed a major peak at m/z 488.20

([M+H]+) and 510.15 ([M+Na]+).

Preparation of EKODE-Cys-NHS ester. 163 mg (0.335 mmol) of EKODE-Cys adducts were dissolved with 4 mL of dimethyl sulfoxide (DMSO) in a round-bottom flask,

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followed by the addition of EDC (642 mg, 3.34 mmol) of and NHS (963 mg, 8.37 mmol).

After stirring at r.t. overnight, 25 mL of water was added to the reaction and extracted with

25 mL of ethyl acetate for 3 times. The combined organic layer was dried on sodium sulfate and filtered. After removal of solvent by rotavap, column chromatography with hexanes/ethyl acetate/methanol (5:5:1) yielded EKODE-Cys-NHS ester as a transparent oil

(143 mg, 74.2% yield). The product was subjected to LC-MS analysis and m/z was found at 607.15 ([M+Na]+).

Preparation of EKODE-Cys-spacer. EKODE-Cys-NHS ester (143 mg, 0.245 mmol) was dissolved in 1.5 mL of DMSO. A solution of aminocaproic acid (321 mg, 2.45 mmol) in 3.5 mL PBS was then added. The reaction mixture was stirred vigorously at r.t. for 4 hours, followed by addition of 5 mL of water and extraction with ethyl acetate (5 ×

25 mL). The combined organic layer was dried over Na2SO4 and the solvent was removed in vacuo. The residual oil was purified by column chromatography over silica

(hexanes/ethyl acetate/methanol, 5:5:1) to give EKODE-Cys-spacer as a transparent oil

(62.6 mg, 42.6% yield). The [M+H]+ of the product was found at m/z 601.30 and the

[M+Na]+ was found at m/z 623.30.

Preparation of EKODE-Cys-spacer-NHS ester. Same procedures as for the synthesis of EKODE-Cys-NHS ester was used. 31 mg (51.6 μmol) of EKODE-Cys-spacer was dissolved in 1 mL of DMSO, followed by addition of EDC (90 mg, 516 μmol) and

NHS (148 mg, 1.29 mmol). The reaction mixture was stirred at r.t. overnight and 5 mL of water was added and subsequently extracted with ethyl acetate (5 × 25 mL). The dried organic layer was concentrated and purified by column chromatography to yield EKODE- ys-spacer-NHS ester as a transparent oil (22mg, 61.0% yield). The product was subjected

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to LC-MS analysis and [M+H]+ was found at m/z 698.35 and [M+Na]+ was found at m/z

720.35.

Preparation of EKODE-Cys-spacer-protein conjugates. 200 μL of EKODE-

Cys-spacer-NHS ester solution in DMSO (10 mg/mL, 14.3 mM) was added to 466 μL of protein solution (BSA or KLH 4.35 mg/mL in PBS). After stirring at r.t. for 4 hours, the reaction mixture was dialyzed in dialysis cassettes (10K molecular weight cut off) against

2 L of 30% DMSO in PBS with gentle stirring at 4 °C. The buffer was changed to 2L of

20% DMSO in PBS after 2 hours of dialysis and subjected to 4 hours of additional dialysis.

The reaction mixture was then dialyzed in PBS overnight.

Characterization of EKODE-Cys-spacer-protein conjugates by TNBSA assay.

KLH, BSA and the dialyzed solutions of EKODE-Cys-spacer-BSA and EKODE-Cys- spacer-KLH (~ 1 mg/mL in PBS) were diluted to 200 μg/mL with PBS. 0.5 mL of the

0.01% (w/v) solution of TNBSA in 0.1 M sodium bicarbonate (pH 8.5) was then added to

1 mL of each protein solution in Eppendorf tubes and mixed by vortex. The reaction mixture was then incubated at 37 °C for 2 hours and quenched by addition of 0.5 mL of

10% SDS and 0.25 mL of 1 N HCl. The samples were transferred to UV quartz cuvettes and the concentration of unmodified lysine was determined by measuring the absorbance of the solution at 335 nm on LAMBDA 25 instruments (Perkin Elmer, Waltham, MA) while the protein concentration was measured by the absorbance at 280 nm. The extent of modification of lysine was then calculated based on the following equation:

A335unmodified lysine in protein conjugates/A280protein conjugates Modified lysin% 1 A335unmodified lysine in BSA/KLH/A280BSA/KLH

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Characterization of EKODE-Cys-spacer-BSA conjugates by MALDI-TOF.

The MALDI matrix solution was prepared by dissolving 1 tube of sinapinic acid (1 mg) in

100 μL of 30% acetonitrile in water with 0.1% trifluoroacetic acid (TFA). 1 μL of EKODE-

Cys-spacer-BSA conjugate was applied onto a stainless steel MALDI sample block, followed by addition of 1 μL of MALDI matrix solution and mixed by pipette tips. The samples were dried at r.t. and subjected to MALDI-MS analysis, which was performed using a Bruker BiFlex III MALDI-TOF equipped with a pulsed nitrogen laser source (λ =

337 nm) and used at an acceleration voltage of 28 kV operated in the reflection mode

(Voyager Biospectrometry Workstation, PerSeptive Biosystems, Framingham, MA, USA).

The number of modified lysine was determined based on the addition of molecular weight due to the conjugation of EKODE-Cys-spacer and the extent of lysine modification was calculated based on the fact that BSA contains 59 lysine residues.

4.4.3 Production and characterization of anti-EKODE-Cys antibodies

Immunization and ELISA analysis of antibody titers. The EKODE-Cys-spacer-

KLH conjugates were sent to Cocalico Biologicals (Reamston, PA) for antiserum production. The rabbits were immunized on days 14, 21, 49, 77. Test bleeds were collected on days 14, 35, 56, 70, 84 from the time of initial inoculation and exsanguination was performed on day 91 for production bleed. In order to determine anti-EKODE-Cys antibody levels in rabbit blood serum, the corresponding EKODE-Cys-spacer-BSA conjugates were used as coating agents. 50 μL of BSA conjugate solution (20 μg/mL in

PBS) was added to each well of sterilized Baxter ELISA plate and incubated at 4 °C overnight. The coating solution was then discarded and the plate was washed 3 times using

300 μL of wash buffer (50 mM pH 7.4 PBS with 0.05% v/v Tween-20). The remaining

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active sites on the plate were blocked by addition of 300 μL of 3% (w/v) BSA in PBS to each well and followed by incubation at 37 °C for 2 hours. The plate was then washed with washing buffer (3 × 300 μL), followed by addition of 50 μL of rabbit serum from each test bleed or prebleed (before injection of antigen as negative response control), which were pre-diluted 1:10,000 with 1% BSA in PBS (w/v). 1% BSA in PBS without serum was used as blank and each sample was analyzed in duplicate. The plate was then incubated at 37 °C for 2 hours and washed with washing buffer (3 × 300 μL). The stock solution of HRP conjugated goat anti-rabbit IgG secondary antibody (0.4 mg/mL in water/glycerol, 1:1) was diluted 1:5000 with PBS and 100 μL of the diluted solution was added to each well. The plate was incubated at r.t. for 1 hour and again washed 3 times. 1-step ABTS substrate solution (150 μL) was added to each well to reveal the immunocomplex bound to the plate.

After incubating at r.t. for 30 minutes, the reaction was quenched by 100 μL of 1% SDS solution in water and the absorbance of the samples at 405 nm was measured by Bio-Rad

450 Microplate reader (Hercules, CA, USA).

Purification of antibody via immunoaffinity capture. In an effort to obtain the specific anti-EKODE-Cys antibodies from the crude antiserum, EKODE-BSA conjugates were prepared by mixing 100 μL of 0.5 mM trans-EKODE-(E)-Ib or trans-EKODE-(E)-

IIb solution in ethanol with 400 uL of 1mg/mL BSA solution in PBS and incubating at 37

°C overnight. The resulting EKODE-BSA conjugates were immobilized on MicroLink protein coupling column using the following procedures: The columns were centrifuged at

1000 × g for 2 minutes to remove the storing buffer. 300 μL of coupling buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) was added to suspend the resin, followed by centrifugation at 1000 × g for 1 minutes to remove the buffer. This step was repeated two

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more times to equilibrate the column. Subsequently 150 uL of the EKODE-BSA conjugates and 150 uL of coupling buffer were added to the column and mixed by gentle swirling. 2 uL of sodium cyanoborohydride solution (5 M in 0.01 M NaOH) was then added and the column was incubated overnight at 4 °C with gentle end-over-end mixing. After washing the column with coupling buffer (3 × 300 uL), the reaction was stopped by adding 2 × 300 uL of quenching buffer (1 M Tris, 0.05% NaN3, pH 7.4). 200 uL of quenching buffer and

4 uL of sodium cyanoborohydride solution were then applied to the column and incubated at r.t. for 30 minutes to block the active binding sites on the resin. After washing the resin containing immobilized EKODE-BSA conjugates 3 times with 300 uL of washing buffer

(1 M NaCl, 0.05% NaN3), 300 uL of antiserum was added to the column and incubated overnight at 4 °C with gentle end-over-end mixing. The resin-bound immunocomplex was then washed 3 times with 300 uL of washing buffer containing 0.05% Tween-20, followed by washing with coupling buffer (3 × 300 uL). 100 uL of elution buffer (pH 2.8, contains primary amine) was then slowly added and incubated at r.t. for 10 minutes. The dissociated antibodies were collected by centrifugation and the resulting solution was neutralized immediately by adding 5 uL of 1M Tris, pH 9.0.

Characterization of anti-EKODE-Cys antibodies by competitive ELISA. The competitor EKODE-His adducts were synthesized by mixing tran-EKODE-(E)-Ib or tran-

EKODE-(E)-IIb (10 mg, 32.3 μmmol) with N-acetyl-L-histidine methyl ester (34 mg, 161 mmol) overnight at r.t. in 1 mL of 20% acetonitrile in PBS. HNE-Cys adducts were obtained via the reaction of HNE (20 mg, 128 mmol) with N-acetyl-L-cysteine methyl ester

(45 mg, 254 mmol) in 1 mL of chloroform with overnight stirring at r.t. Both negative competitors and EKODE-Cys adducts were subjected to eight serial dilutions with PBS.

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50 μL of the diluted competitors and a positive control (PBS with no competitor) were incubated with 50 μL of purified antiserum (1:1000 diluted with 1% BSA in PBS) overnight at 4 °C. The resulting antibody-competitor mixture was analyzed by ELISA with the same conditions used to measure antibody titers. The measured absorbance values for duplicate samples were averaged and the relative values to positive control (B/B0) were plotted against the log of concentration.

Characterization of anti-EKODE-Cys antibodies by western blot. To evaluate the cross-reactivity of anti-EKODE-Cys antibodies with the protein adducts of other LPO products, the BSA adducts were prepared by incubating 90 μL of 1 mg/mL BSA solution in PBS with 10 μL of 1mM or 100 μM LPO products (EKODEs, HNE, ONE, acrolein, octenal) in ethanol at 37 °C for 24 hours. 1 μL of modified BSA was mixed with 9 μL of water and 10 μL of 2 × loading buffer containing 2-mercaptoethanol, and 8 μL of the mixture was separated by 15% SDS-PAGE at 100 V (gel was prepared according to common protocols). The resolved proteins were then electro-transferred onto Immobilon

PVDF membrane at 30 V overnight at 4 °C and incubated with blocking agent (10% non- fat milk in 0.1% Tween-20 in Tris-buffered saline (TBST)) with gentle shaking for 1 hours at r.t. After washing with TBST for 3 × 5 minutes, the anti-EKODE-Cys antibodies (diluted

1:1000 in 1% milk in TBST) were then applied to the membrane and incubated overnight at 4 °C. The blots were then washed again with TBST for 3 × 10 minutes, followed by addition of secondary antibody (HRP linked goat anti-rabbit IgG, 1:10000 diluted in

TBST). After incubating at r.t. for 1.5 hours and washing for 3 × 10 minutes, the blots were developed by ECL detection reagent for 5 minutes and visualized by exposing to X-ray film.

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Immunochemical detection of EKODE-Cys adducts in BSA treated with oxidized linoleic acid. 1 mg/mL BSA was incubated with 10 mM linoleic acid in PBS containing 10% ethanol at 37 °C in the presence of 0.5 mM FeSO4 and 1 mM ascorbic acid. At each time point of 30 min, 1h, 2h, 4h, 8h and 24h, 100 μL of aliquot was collected and stored at -20 until analysis. The modified BSA was resolved by SDS-PAGE and subjected to western blot analysis using anti-EKODE-Cys antibodies by the procedures described above.

4.4.4 Immunochemical detection of endogenous EKODE-Cys adducts in biological samples

Immunochemical detection in human neuroblastoma cells. Human neuroblastoma cell line M17 was maintained in serum-free Opti-MEM media (Invitrogen,

Gaithersburg, MD, USA) with 5% donor calf serum and 1% penicillin/streptomycin.

Hydrogen peroxide solution 30% (w/w) in water was added to a final concentration of 0.25 mM when cellular confluence was around 80% - 90%. The cells were collected after 24 hours of H2O2 treatment and cell lysis was performed in lysis buffer with protease inhibitor phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitor NaF. The protein concentration was determined using BCA protein assay and 20 μg of protein was resolved in duplicate by SDS-PAGE and analyzed by western blot using anti-EKODE-Cys antibodies. The membrane was then incubated in stripping buffer at r.t. for 30 minutes to remove the primary and secondary antibodies. After washing with TNBST for 3 × 10 minutes, the blots were then probed by β-actin as an internal control. Quantitation of the blots was conducted by a computer-assisted scanning system (Quantity One 4.3, Bio-Rad).

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Immunochemical detection in human brain tissues. Frozen frontal cortex samples were homogenized in lysis buffer with protease inhibitors cocktail added. After determining protein concentration with BCA assay, 30 μg of protein were resolved using

SDS-PAGE. Proteins were then transferred to Immobilon PVDF membrane for western blot analysis using anti-EKODE-Cys as the primary antibody and β-actin as an internal control. The quantitation result generated from Quantity One was plotted against the age of the subjects. In order to assess if the western blot results can reflect the level of EKODE adducts, 20 μL of 1 mg/mL homogenate of human brain tissue was incubated with 10 μM or 100 μM of trans-EKODE-(E)-Ib overnight at 37 °C and analyzed by western blot using anti-EKODE-Ib-Cys antibody.

Immunochemical detection in various mouse tissues. C57BL/6 mouse (2 month- old) was anesthetized with avertin and organs were quickly excised. All experiments were performed in accordance with the Institutional Animal Care and Use Committee at Case

Western Reserve University. The tissues were homogenized in lysis buffer with protease inhibitors cocktail added. After determining protein concentration with BCA assay, 30 μg of protein were resolved using SDS-PAGE and subjected to immunoblot using anti-

EKODE-Cys antibodies.

Immunochemical detection in rat heart ischemia. Adult, male Sprague–Dawley rats (300–350 g) were fed ad libitum for 8–12 days with standard laboratory chow before experiments. The heart perfusion experiments were performed as previously described42.

The control group was a 15-minutes perfusion with no ischemia. The different ischemic perfusion groups were based on 15-minutes perfusion to allow for baseline equilibration, followed by various time (5 min, 15 min, and 30 min) of global no-flow ischemia. Each

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group consisted of 3 rats (n= 3 × 4) and hearts were quick-frozen at the end of the protocol for each group. Powdered frozen organs were homogenized in lysis buffer with protease inhibitors. Protein concentration was measured by BCA assay and 30 μg of protein were separated using SDS-PAGE. Proteins were then transferred to Immobilon PVDF membrane for western blot analysis using anti-EKODE-Cys as the primary antibody and

β-actin as an internal control. Quantitation of the blots was conducted by a computer- assisted scanning system (Quantity One 4.3, Bio-Rad).

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4.5 Acknowledgement

All the western blot experiments were performed in Dr. Xiongwei Zhu’s lab

(Department of Pathology, Case Western Reserve University). Human brain tissues, M17 cells (treated with H2O2), and mouse organs were obtained from Sandra Siedlak, Dr.

Wenzhang Wang and Dr. Li Li in Dr. Zhu’s lab. The perfused rat hearts (with or without ischemia) were obtained from Dr. Guofang Zhang and Dr. Henri Brunengraber.

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Chapter 5

Future Directions

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5.1 Identification of relevant kinase in the catabolic pathways of 4-hydroxyacids

In Chapter 2, we reported our preliminary work on the identification of relevant kinase involved in the isomerization of 4-hydroxyacyl-CoA to 3-hydroxyacyl-CoA via 4- phophoacyl-CoA. An LC-MS/MS based kinase activity assay was developed to guide our purification of kinase from pig liver. A number of purification techniques were employed in the purification workflow including ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, and SDS-PAGE. Five kinase candidates were identified from sequencing of purified protein fractions and sedoheptulose kinase, the most relevant candidate with oxidative stress, was successfully expressed and tested for kinase activity. Although sedoheptulose kinase did not show GHP-CoA kinase activity in our test, the activity of the other four kinase candidates was not ascertained yet and these candidates will be tested in the near future. We also would like to search for potential kinase candidates in the literature and we have identified two interesting candidates. ACAD10 and ACAD11 belong to the acyl-CoA dehydrogenase family which is a group of mitochondrial enzymes that catalyze the dehydrogenation of acyl-CoA esters. He et al.1 reported that in addition to the acyl-CoA dehydrogenases domain, there is a predicted aminoglycoside phosphotransferase domain in both ACAD10 and ACAD11, and there is also a hydrolase domain in ACAD10. Since the enzymatic functions of ACAD10 and ACAD11 are closely related to the transformations involved in the isomerization pathway, we will express both enzymes and test their GHP-CoA kinase activity.

In addition, the kinase purification workflow may be further optimized to reveal more kinase candidates. Since the protein fractions from FPLC showed low activity at pM/mg/min level, it raised the possibility that a large portion of kinase activity may be lost

141

during or before the FPLC purification. Most of the physicochemical properties of GHP-

CoA kinase were not revealed yet, so it was possible that the kinase was not stable under our current FPLC or ammonium sulfate precipitation conditions. Several factors including buffer type, salt concentration, pH, temperature, and additives may have significant impacts on kinase stability2. Therefore, a comprehensive stability test can be performed to determine the optimal conditions for the purification workflow.

Ammonium sulfate precipitation and ion exchange chromatography were employed at the beginning of our purification workflow because they were general approaches applicable to most proteins. They offer great sample capacity and are readily accessible for quick purification, but at the same time they may sacrifice selectivity. Blue-Sepharose- based affinity chromatography provided additional level of selectivity by using NAD- similar ligand to bind dehydrogenase and kinase. However, it also binds a number of highly abundant proteins such as albumin, which may have considerable interference with the binding of kinase. Therefore, a more specific FPLC technique may be used to improve the selectivity of the purification. ATP-Sepharose-based affinity chromatography has been reported by different groups in the purification of kinases3-5. The ATP-Sepharose column is commercially available and provides excellent selectivity for kinase through non- covalent interaction between protein and ATP immobilized to column. ATP affinity chromatography may serve as a polishing stage of the purification workflow to concentrate kinase fraction and to remove trace contaminants that may interfere with protein sequencing.

Other questions abound. Are the catabolic pathways of 4-HNE universal processes applicable to other lipid peroxidation products? 4-HNE shares similar functional groups

142

with a number of other lipid peroxidation products including EKODEs. We can investigate the catabolism of EKODE from the synthesis of isotopically labeled EKODEs. The resulting isotopomers can then be used in liver perfusion experiments to illustrate the catabolism pathways using a combination of metabolomics and mass isotopomer analysis.

5.2 Protein modification of EKODEs

In Chapter 3 and Chapter 4, we reported model studies to reveal the mechanistic details of the reactions of EKODEs and nucleophilic protein side chains. We also developed specific antibodies to detect and quantify the protein adducts of EKODEs in biological samples. Using immunochemical assays, we found correlations between the level of EKODE cysteine adducts and several oxidative stress related processes such as aging and heart ischemia.

Although we have developed immunoassays using western blots to detect EKODE- modified proteins, they are not suitable for biomarker validation due to their low throughput and incapability of quantitative analysis. The next step in this work is to develop high-throughput and quantitative assays for bioanalysis of protein modification of

EKODEs. In our tissue distribution experiments we detected considerable amount of

EKODE-modified proteins in mouse blood samples using anti-EKODE-Cys antibodies.

Therefore, we can develop ELISA assays for quantitation of EKODE adducts in blood, which can be a less invasive, high-throughput, and quantitative approach that is more suitable for clinical application. A sandwich ELISA method can be developed by coating the plate with anti-EKODE-Cys antibodies. This assay will be a valuable tool that enables

143

us to validate EKODE adducts as potential biomarkers of oxidative stress related diseases for clinical application.

In addition to nucleophilic protein side chains, it is very likely that EKODEs can also form adducts with glutathione through Michael addition at carbon-carbon double bond. Glutathionylation presents one of the major antioxidant defenses against oxidative damage and it plays a key role in metabolizing highly reactive electrophiles such as 4-HNE and 4-ONE6. We are interested in understanding the interplay between the glutathionylation and protein adducts formation of EKODEs. Therefore, a quantitative assay will be developed to measure endogenous GSH conjugates of EKODEs and free

EKODEs. A variety of derivatization methods have been developed to improve the sensitivity of LC-MS/MS analysis of fatty acids7-9. For example, a picolylamine derivatization method was developed by Li et al.10, and an excellent sensitivity with a limit of detection in the low femtomole range was reported. We will continue our study on

EKODEs toward the evaluation of their glutathionylation pathway as well as the understanding of the interplay between different fates of EKODEs as they enable us to gain insights into the biological relevance of EKODEs in normal physiology and disease states.

144

5.3 References

1. He, M. A.; Pei, Z. T.; Mohsen, A. W.; Watkins, P.; Murdoch, G.; Van Veldhoven,

P. P.; Ensenauer, R.; Vockley, J., Identification and characterization of new long chain

Acyl-CoA dehydrogenases. Molecular Genetics and Metabolism 2011, 102, 418-429.

2. Nealon, D. A.; Henderson, A. R., Stability of commonly used thiols and of human creatine kinase isoenzymes during storage at various temperature in various media.

Clinical Chemistry 1977, 23, 816-829.

3. Davies, S. P.; Hawley, S. A.; Woods, A.; Carling, D.; Haystead, T. A. J.; Hardie,

D. G., Purification of the AMP-activated protein kinase on ATP-γ-Sepharose and analysis of its subunit structure. European Journal of Biochemistry 1994, 223, 351-357.

4. Hawley, S. A.; Davison, M.; Woods, A.; Davies, S. P.; Beri, R. K.; Carling, D.;

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5. Jeansonne, D. P.; Bordes, T. J.; Bennett, C. A.; Kothandaraman, G.; Bush, J. G.;

Vaccaro, J. A., A rapid ATP affinity-based purification for the human non-receptor tyrosine kinase c-Src. Protein Expression and Purification 2006, 46, 240-247.

6. Esterbauer, H.; Schaur, R. J.; Zollner, H., Chemistry and Biochemistry of 4-

Hydroxynonenal, Malonaldehyde and Related Aldehydes. Free Radical Biology and

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7. Yang, W.-C.; Adamec, J.; Regnier, F. E., Enhancement of the LC/MS analysis of fatty acids through derivatization and stable isotope coding. Analytical Chemistry 2007, 79,

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145

8. Johnson, D. W., Alkyldimethylaminoethyl ester iodides for improved analysis of fatty acids by electrospray ionization tandem mass spectrometry. Rapid Communications in Mass Spectrometry 2000, 14, 2019-2024.

9. Pettinella, C.; Lee, S. H.; Cipollone, F.; Blair, I. A., Targeted quantitative analysis of fatty acids in atherosclerotic plaques by high sensitivity liquid chromatography/tandem mass spectrometry. Journal of Chromatography B 2007, 850, 168-176.

10. Li, X. N.; Franke, A. A., Improved LC-MS Method for the Determination of Fatty

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146

Appendix 1H-1H correlation spectroscopy (HHCOSY) of the ring opening product of

EKODE II model compounds

147

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