The effects of cholesterol lowering agents on the proteome of primary human hepatocytes [Elektronische Ressource] / von Martin Wörner
268 Pages
English

The effects of cholesterol lowering agents on the proteome of primary human hepatocytes [Elektronische Ressource] / von Martin Wörner

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The effects of cholesterol lowering agents on the proteome of primary human hepatocytes Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät III Chemie, Pharmazie, Bio- und Werkstoffwissenschaften der Universität des Saarlandes von Martin Wörner Saarbrücken 2009 Tag des Kolloquiums: 12.07.2010 Dekan: Prof. Dr. Stefan Diebels Berichterstatter: Prof. Dr. Rita Bernhardt Prof. Dr. Elmar Heinzle Vorsitzender: Prof. Dr. Uli Müller Akademischer Mitarbeiter: Dr. Klaus Hollemeyer Index IIIInnnnddddeeeexxxx ABBREVIATIONS I SSUUMMMMAARRYY IIVV SSUUMMMMAARRYY IIVVSUMMARY (GERMAN) V 1 INTRODUCTION 1 1.1 CHOLESTEROL 1 1.1.1 PHYSIOLOGICAL ROLE 1 1.1.2 TRANSPORT 2 1.1.3 BIOSYNTHESIS AND DEGRADATION 3 1.1.4 REGULATION OF BIOSYNTHESIS, TRANSPORT AND DEGRADATION 6 1.1.5 INHIBITORS OF CHOLESTEROL BIOSYNTHESIS 8 1.2 PRIMARY HUMAN HEPATOCYTES 11 11..33 PPRROOTTEEOOMMIICCSS 1122 11..33 PPRROOTTEEOOMMIICCSS 11221.3.1 SAMPLE PREPARATION 12 1.3.2 SAMPLE SEPARATION 13 1.3.3 PROTEIN IDENTIFICATION 17 1.3.4 QUANTITATION 19 1.4 AIM OF THE WORK 22 2 MATERIALS 23 2.1 CHEMICALS 23 2.2 INSTRUMENTS / MATERIALS 25 2.3 BUFFER / SOLUTIONS / WATER 26 33 MMEETTHHOODDSS 2277 33 MMEETTHHOODDSS 22773.1 CELL CULTURE 29 3.1.1 PRIMARY HUMAN HEPATOCYTES 29 3.1.

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Published 01 January 2009
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The effects of cholesterol lowering agents
on the proteome of primary human hepatocytes




Dissertation
zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät III
Chemie, Pharmazie, Bio- und Werkstoffwissenschaften
der Universität des Saarlandes



von
Martin Wörner


Saarbrücken
2009















Tag des Kolloquiums: 12.07.2010

Dekan: Prof. Dr. Stefan Diebels

Berichterstatter: Prof. Dr. Rita Bernhardt
Prof. Dr. Elmar Heinzle

Vorsitzender: Prof. Dr. Uli Müller

Akademischer Mitarbeiter: Dr. Klaus Hollemeyer Index
IIIInnnnddddeeeexxxx
ABBREVIATIONS I
SSUUMMMMAARRYY IIVV SSUUMMMMAARRYY IIVV
SUMMARY (GERMAN) V
1 INTRODUCTION 1
1.1 CHOLESTEROL 1
1.1.1 PHYSIOLOGICAL ROLE 1
1.1.2 TRANSPORT 2
1.1.3 BIOSYNTHESIS AND DEGRADATION 3
1.1.4 REGULATION OF BIOSYNTHESIS, TRANSPORT AND DEGRADATION 6
1.1.5 INHIBITORS OF CHOLESTEROL BIOSYNTHESIS 8
1.2 PRIMARY HUMAN HEPATOCYTES 11
11..33 PPRROOTTEEOOMMIICCSS 1122 11..33 PPRROOTTEEOOMMIICCSS 1122
1.3.1 SAMPLE PREPARATION 12
1.3.2 SAMPLE SEPARATION 13
1.3.3 PROTEIN IDENTIFICATION 17
1.3.4 QUANTITATION 19
1.4 AIM OF THE WORK 22
2 MATERIALS 23
2.1 CHEMICALS 23
2.2 INSTRUMENTS / MATERIALS 25
2.3 BUFFER / SOLUTIONS / WATER 26
33 MMEETTHHOODDSS 2277 33 MMEETTHHOODDSS 2277
3.1 CELL CULTURE 29
3.1.1 PRIMARY HUMAN HEPATOCYTES 29
3.1.2 HUMAN COLON CARCINOMA CELL LINE 116 30 Index
3.1.3 SCHIZOSACCHAROMYCES POMBE 30
3.1.4 ECHERICHIA COLI 30
3.2 PROTEIN ISOLATION 31
3.2.1 CELL DISRUPTION 31
3.2.2 DIFFERENTIAL CENTRIFUGATION 32
3.2.3 CLEANING THE MICROSOMES 32
3.3 DETERMINING THE PROTEIN CONCENTRATION 33
3.3.1 PLUSONE™ 2D QUANT KIT 33
3.3.2 BICINCHONINIC ACID ASSAY 33
3333....4444 TTTTWWWWOOOO DDDDIIIIMMMMEEEENNNNSSSSIIIIOOOONNNNAAAALLLL GGGGEEEELLLL EEEELLLLEEEECCCCTTTTRRRROOOOPPPPHHHHOOOORRRREEEESSSSIIIISSSS ((((2222DDDD –––– PPPPAAAAGGGGEEEE)))) 33334444
3.4.1 ISOELECTRIC FOCUSSING 34
3.4.2 SODIUM DODECYLSULFATE–POLYACRYLAMIDE GEL ELECTROPHORESIS 36
3.4.3 STAINING 38
3.4.4 IMAGE DIGITALISATION 38
3.4.5 IN-GEL DIGESTION 39
3.4.6 SPOTTING 40
3.5 NANO HIGH-PRESSURE LIQUID CHROMATOGRAPHY (NHPLC) 41
3.5.1 SAMPLE PREPARATION 41
3.5.2 FIRST DIMENSION, BASIC ELUENT 42
3.5.3 SECOND DIMENSION, ACIDIC ELUENT, ION-PAIRING 42
3.5.4 MATRIX MIXING AND SPOTTING 43
33..66 MMAASSSS SSPPEECCTTRROOMMEETTRRYY BBYY MMAALLDDII--TTOOFF//TTOOFF 4444 33..66 MMAASSSS SSPPEECCTTRROOMMEETTRRYY BBYY MMAALLDDII--TTOOFF//TTOOFF 4444
3.6.1 IN-GEL DIGESTS 44
3.6.2 NLC-MS SAMPLES 47
3.7 REAL-TIME POLYMERASE CHAIN REACTION 49
3333....8888 BBBBIIIIOOOOIIIINNNNFFFFOOOORRRRMMMMAAAATTTTIIIICCCCSSSS 55550000
3.8.1 IMAGE ANALYSIS 50
3.8.2 PROTEIN IDENTIFICATION 55
3.8.3 PROTEIN QUANTITATION 56
3.8.4 DATA HANDLING 57
3.8.5 DATABASES 59
4 RESULTS 61
4.1 GENERAL REMARKS 61 Index
4.2 EVALUATION OF THE METHODICAL APPROACHES 62
4.2.1 TWO DIMENSIONAL GEL ELECTROPHORESIS 63
4.2.2 NANO HIGH PRESSURE LIQUID CHROMATOGRAPHY 71
4.2.3 SUMMARY 88
4.3 THE EFFECTS OF RSV AND LEK-935 89
4.3.1 CYTOSOLIC FRACTION / TWO-DIMENSIONAL GEL ELECTROPHORESIS 89
4.3.2 MICROSOMAL FRACTION / NANO HIGH PRESSURE LIQUID CHROMATOGRAPHY 99
4.3.3 VALIDATION OF THE RESULTS BY RT-PCR 103
4.3.4 SUMMARY OF REGULATIONS 103
4444....4444 BBBBIIIIOOOOIIIINNNNFFFFOOOORRRRMMMMAAAATTTTIIIICCCC AAAANNNNAAAALLLLYYYYSSSSIIIISSSS OOOOFFFF TTTTHHHHEEEE RRRREEEEGGGGUUUULLLLAAAATTTTEEEEDDDD PPPPRRRROOOOTTTTEEEEIIIINNNNSSSS 111100006666
4.4.1 ROSUVASTATIN TREATMENT 106
4.4.2 LEK-935 TREATMENT 112
5555 DDDDIIIISSSSCCCCUUUUSSSSSSSSIIIIOOOONNNN 111111117777
5.1 EXPERIMENTAL APPROACHES, THEIR QUIRKS AND RESULTS 117
5.1.1 PROTEOMICS 117
5.1.2 TWO-DIMENSIONAL GEL ELECTROPHORESIS 118
5.1.3 NANO LIQUID CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRY 127
5.1.4 SUMMARY 132
5.2 THE EFFECTS OF CHOLESTEROL LOWERING AGENTS 133
5.2.1 THE EFFECTS OF ROSUVASTATIN ON THE PROTEOME OF PRIMARY HUMAN HEPATOCYTES 133
5.2.2 THE EFFECTS OF LEK-935 ON THE PROTEOME OF PRIMARY HUMAN HEPATOCYTES 142
5.3 SUMMARY AND OUTLOOK 145
6666 RRRREEEEFFFFEEEERRRREEEENNNNCCCCEEEESSSS 111144447777
APPENDIX 165
APPENDIX I: PUBLICATIONS RESULTING FROM THIS WORK 165
CONTRIBUTIONS TO INTERNATIONAL MEETINGS 165
MANUSCRIPTS 165
AAPPPPEENNDDIIXX IIII:: DDEETTAAIILLSS OOFF TTHHEE PPRROOTTEEIINNSS FFOOUUNNDD TTOO BBEE RREEGGUULLAATTEEDD 116677 AAPPPPEENNDDIIXX IIII:: DDEETTAAIILLSS OOFF TTHHEE PPRROOTTEEIINNSS FFOOUUNNDD TTOO BBEE RREEGGUULLAATTEEDD 116677 Index
SAMPLE 1 2D SPOT DETAILS 167
SSAAMMPPLLEE 11 NNLLCC--MMSS RREESSUULLTTSS 118811 SSAAMMPPLLEE 11 NNLLCC--MMSS RREESSUULLTTSS 118811
SAMPLE 2 2D SPOT DETAILS 184
SAMPLE 2 NLC-MS DETAILS 209
AAPPPPEENNDDIIXX IIIIII:: SSPPOOTT IIDDEENNTTIIFFIICCAATTIIOONNSS FFRROOMM 22DD GGEELLSS 221111 AAPPPPEENNDDIIXX IIIIII:: SSPPOOTT IIDDEENNTTIIFFIICCAATTIIOONNSS FFRROOMM 22DD GGEELLSS 221111
SAMPLE 1 211
SAMPLE 2 216
APPENDIX IV: ITRAQ™ EVALUATION 219
APPENDIX V COMPLETE LIST OF PROTEINS IDENTIFIED BY NLC-MS 228
APPENDIX VI: KEGG PATHWAYS AFFECTED 248
SAMPLE 1 248
RSV TREATMENT 248
LEK-935 TREATMENT 249
SAMPLE 2 250
RSV TREATMENT 250
LEK-935 TREATMENT 252
ACKNOWLEDGEMENTS 255
Abbreviations I
AAAAbbbbbbbbrrrreeeevvvviiiiaaaattttiiiioooonnnnssss

2D Two dimensional
2D-PAGE Two dimensional gel electrophoresis
ACN Acetonitrile
ACTH Adrenocorticotropic hormone
APS Ammoniumpersulfate
BCA Bicinchoninic acid
CHAPS 3-[(3-cholamidopropyl)dimethylammonium]-1-propansulfonate
CHCA Alpha-cyano-4-hydroxy-cinnamic acid
CID Collision induced dissociation
Cl Chloride
CO Carbon dioxide 2
CoA Coenzyme A
CoA Coenzyme A
CYP Cytochrome P450
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DTT Dithiothreitol
DTT Dithiothreitol
E.coli Escherichia coli
EBSS Earl’s balanced salt solution
EDTA Ethylene diamine tetraacetic acid
EGTA Ethylene glycol tetraacetic acid
ER Endoplasmic reticulum
ESI Electrospray ionisation
1 1Glu-fib (Glu )-fibrinopeptide B
HCT-116 Human carcinoma cell line 116
HDL High density lipoprotein
HEPES 4-(2-hydroxyethyl)-1-piparazineethanesulfonic acid
HFBA Heptafluorobutyric acid
HMG-CoA 3-hydroxy-3-methyl-glutaryl-CoA
HPLC High pressure liquid chromatography II Abbreviations
IAA Iodacetamide
ICAT Isotope coded affinity tag
IDL Intermediate density lipoprotein
IEF Isoelectric focussing
IP Ion paired
IPGstrips Immobilised pH gradient gels
IPP Isopentenyl-pyrophosphate
iTRAQ™ Isotope tags for relative and absolute quantification
KCl Potassium chloride
LC Liquid chromatography
LDL Low density lipoprotein
LEK-935 2-((3,4-dichlorophenethyl)(propyl)amino)-1-(pyridin-3-yl)ethanol
MALDI Matrix assisted laser desorption/ionisation
mRNA Messenger RNA
MS Mass spectrometry / spectrometer
MS/MS Tandem mass spectrometry
Na HPO4 Disodium hydrogen phosphate 2
NaCl Sodiumchloride
NaH PO Sodium dihydrogen phosphate 2 4
NaHCO Sodium hydrogen carbonate 3
nLC-MS Nano liquid chromatography coupled to mass spectrometry
NO Nitric oxide
PAGE Polyacrylamide gel electrophoresis
PCR Polymerase chain reaction
PFF Peptide-fragment fingerprint
PMF Peptide-mass fingerprint
PMSF Phenylmethylsulfonylfluoride
PSD Post-source decay
PS-DVB Poly-(sterene-divenlybenzene)
RNA Ribonucleic acid
RP Reversed phase
RSV Rosuvastatin
RT Real time
S.pombe Schizosaccharomyces pombe Abbreviations III
S/N Signal to noise
SCX Strong cation exchange
SDS Sodiumdodecylsulfate
SILAC Stable isotope labelling by amino acids in cell culture
SREBP Sterol regulatory element binding protein
TEMED N,N,N',N'-tetraethylmethanediamine
TFA Trifluoro acetic acid
TFE Trifluoro ethanol
TOF Time of flight
Tris Tris(hydroxymethyl)aminomethane
UdS Saarland university
VLDL Very low density lipoprotein
ZChL Central chemical repository

Standard abbreviations for amino acids
A Ala Alanine L Leu Leucine
R Arg Arginine K Lys Lysine
N Asn Asparagine M Met Methionine
D Asp Aspartic acid F Phe Phenylalanine
C Cys Cysteine P Pro Proline
Q Gln Glutamine S Ser Serine
E Glu Glutamic acid T Thr Threonine
G Gly Glycine V Val Valine
H His Histidine W Trp Tryptophan
I Ile Isoleucine Y Tyr Tyrosine
IV Summary
SSSSuuuummmmmmmmaaaarrrryyyy
Cholesterol plays a crucial role for human life. It is a part of eukaryotic lipid bilayers,
necessary for cell division and serves as a precursor for steroid hormones. The effects
of cholesterol lowering agents are not yet fully understood. This study describes, for
the first time, the effects of the HMG-CoA reductase inhibitor rosuvastatin and the
new CYP51A1 inhibitor LEK-935 on the proteome of primary human hepatocytes.
Samples derived from two different human donors were analysed. They were sub-
fractionated prior to the proteome analysis to enhance the resolution of the analysis.
The cytosolic and microsomal fractions were analysed in a semi-quantitative manner
by 2D-PAGE and nLC-MS respectively. A final set of 44 proteins was found to be
differentially expressed. This set contains proteins already known to be affected and
involved in the cholesterol biosynthesis. It also contains proteins that cannot be
directly related to cholesterol metabolism and that have not yet been described to be
affected by cholesterol lowering agents. The finding of the already known proteins
validates the chosen experimental design while the other proteins provide new
information and represent targets for further investigations. RT-PCR measurements
performed at a chosen set of proteins validate the results. They furthermore underline
the huge inter-individual differences observed during the proteome analysis.