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Contents Slides
Proteomics of obesity and adipose tissue: overview 1 -11
Protein profiling methodologies 12 – 23
Protein secretion by adipocytes 24 – 47
Molecular mechanisms for obesity-related physiology 48 – 59
Proteomics in obesity: summary 60
Some literature references 61
Abbreviations used 62
Obesity in The Netherlands
BMI > 30 kg/m2
25 kg/m2 < BMI > 30 kg/m2
BMI = weightlength 2
Overgewicht = overweightErnstig overgewicht = obese
ASPECTS OF PROTEIN FUNCTION
• Protein identification• Protein quantification• Protein turnover• Posttranslational modifications• Protein interactions and complex formation• Cellular localization and translocation• Structural analysis
WHAT TYPE OF INFORMATION?
Standard:
- Cell fractionation
- Affinity chromatography
- 1D and 2D gel
electrophoresis
- Western blotting
- ELISA
- Immunocytochemistry
Moderately advanced:
- HPLC
- LC-MS
-MALDI-TOF MS
-Peptide fingerprinting
- SELDI-TOF MS
- ICAT
- MIDA
- Edman degradation
-(Recombinant) protein
production
- Yeast two-hybrid system
- Phage display
Advanced:
- Multi-dimensional LC-MS- Imaging MS- SPR-BIA- BIA-MS- Chemical protein synthesis- Antibody/protein arrays-Chip-based microprocessing- Crystallography- NMR- Protein modeling- Tandem MS- FT-MS
Methodologies
• “ Gene structure alone tells us very little about the physiological function of proteins since it ignores the co- and post- translational modifications to which they are subjected.”
(Edmund Fisher, 1997, Nobel Laureate Physiology & Medicine 1992)
• Proteins are the “working horses” in the cell
• Proteins are still preferred targets for drug- and nutritional intervention
• Body fluids contain proteins and usually not RNA
Proteomics, why?
extract proteins
separate them
determine relative quantity
identify differential proteins
changed genes/pathways
TechniquesTechniques
2D –gel electrophoresis
staining
mass spectrometry
pathway analysis
PROTEIN (GENE)-IDENTIFICATION
M/Z
PROTEIN FRAGMENTS FINGERPRINT
Bouwman et al., Rapid Comm Mass Spectrom 2005
HUGO PROTEINDATABASE
TRYPTIC PEPTIDES
Protein-identification via fingerprinting
M/Z
M/Z
PROTEIN FRAGMENTS FINGERPRINT
Excretion products from adipocytes
Cholesteryl ester transfer protein
Plasminogen activator inhibitor-1 (PAI-1)
Leptin
Lipoprotein lipase (LPL)
ApoE
Retinol-binding protein
Vasc. endoth. growth factor (VEGF)
TNF-
IL-6
Angiotensinogen
Hepatocyte growth factor
Insulin-like growth factor-1 (IGF-1)
Adiponectin
Bradley et al. Recent Prog Horm Res. 2001;56:329-58
IL-8
Phospholipid transfer protein
Cardio-vascular effectsInsulin signaling
Weight regulation
Lipid handling
Pro-inflamm. cytokines
Resistin
Growth factorsOthers
Excreted proteins
Research approach: secretome profiling
Fat storage
Model system: mouse 3T3-L1 (pre-)adipocytes
Excreted proteins during 3T3-L1 differentiation
subtotal
identified spots 161
unknown spots 32
total 193
41 different proteins as candidates of secreted proteins related with adipocyte differentiation
EXCRETED PROTEINS
Newly identified:
Macrophage migration inhibitory factor - infiltrationPigment epithelium derived factor - vascularisation Prohibitin - cell growth and differentiation
Collagens and modifiers:
Type I, III, IV, V, VIMatrix metalloproteinase 2Procollagen C-proteinase enhancer proteinProtein-lysine 6-oxidase
EXCRETED PROTEINS
Newly identified:
Macrophage migration inhibitory factor - infiltrationPigment epithelium derived factor - vascularisation Prohibitin - cell growth and differentiation
Collagens and modifiers:
Type I, III, IV, V, VIMatrix metalloproteinase 2Procollagen C-proteinase enhancer proteinProtein-lysine 6-oxidase
Novel adipokine in human plasma Pigment epithelium-derived factor
(PEDF)
0
2500
5000
7500
10000
12500
15000
15.0 20.0 25.0 30.0 35.0
BMI
PE
DF
, n
g/m
l
r = 0.583
EXCRETED PROTEINS
Newly identified:
Macrophage migration inhibitory factor - infiltrationPigment epithelium derived factor - vascularisation Prohibitin - cell growth and differentiation
Collagens and modifiers:
Type I, III, IV, V, VIMatrix metalloproteinase 2Procollagen C-proteinase enhancer proteinProtein-lysine 6-oxidase
EXCRETED PROTEINS
Newly identified:
Macrophage migration inhibitory factor - infiltrationPigment epithelium derived factor - vascularisation Prohibitin - cell growth and differentiation
Collagens and modifiers:
Type I, III, IV, V, VIMatrix metalloproteinase 2Procollagen C-proteinase enhancer proteinProtein-lysine 6-oxidase
Collagen synthesis by Preadipose and Non-Preadipose 3T3 cells
HyPro (%) Collagen / synthesized Formation of Adipose
total cell protein (%) cells in resting
3T3-M2 5.5 1.9 virtually none
3T3-L1 9.6 3.2 abundant 9.9 3.3
Green & Meuth, Cell 3:127-133,1974
- glucose
- insulin1. culture medium proteins
2. cellular total RNA
2 days treatment with
Modulation of the secretion of adipocytes
1. medium proteins: 2D and 1D gel analysis2. mRNA: RT-PCR and microarray analysis
Characterize:Characterize:
Major effect on 2D gel
collagen I alpha1 C-peptide
collagen III alpha1 C-peptide
collagen I alpha 2 C-peptide
high insulinbasal
high insulin + glucosehigh glucose
Microarray analysis Cy5 sample Cy3 reference
extra set : genes encoding mitochondrial proteins and secretory proteins
MWG mouse 10 K array with extra gene sets50-mer mouse non-redundant oligos: 10060 in house printed
protein description gene overall max FC overall max FC FC t-test P FC t-test P
up-regulated proteinscollagen type I alpha 1 (fragment and C-terminal peptide)
Col1a1 + 31.9 + 51.0 1.11 0.442 1.23 0.160
collagen type I alpha 2 (fragment and C-terminal peptide)
Col1a2 + 14.7 + 14.2 1.05 0.596 1.20 0.132
collagen type III alpha 1 (fragment and C-terminal peptide)
Col3a1 + 3.4 + 7.0 1.17 0.170 1.16 0.134
adipsin Adn + 6.0 + 3.6 -1.20 0.204 -1.11 0.397collagen type V alpha 1 (fragment) Col5a1 + 3.5 + 4.8 1.11 0.290 1.32 0.018matrix metalloproteinase-2 Mmp2 + 11.7 ± 2.3 1.17 0.502 1.31 0.102procollagen C-proteinase enhancer protein
Pcolce + 4.2 + 7.3 1.22 0.082 1.36 0.017
pigment epithelium-derived factor (fragment)
Serpinf1 + 3.2 + 1.9 1.19 0.334 1.14 0.400
extracellular SOD Sod3 ± -1.1 + 2.2 1.07 0.473 1.38 0.040SPARC, osteonectin Sparc + 7.0 + 4.2 1.03 0.623 1.18 0.076
metalloproteinase inhibitor 2 Timp2 ± -1.1 + newly appeared
-1.04 0.825 1.23 0.065
complement C3 beta chain C3 + 3.7 + 4.0 1.10 0.191 1.13 0.223
secretion level on 2D gel mRNA expression level on arrayIns/Basal InsGlc/Glc Ins/Basal InsGlc/Glc
Comparison on transcriptome and proteome of secretory genes
Collagen processing enzymes
P4ha1
0.00
0.50
1.00
1.50
2.00
2.50
Basal Glc Ins Ins+Glc
no
rmalized
sig
nal / R
ps15
Pcolce
0.00
0.50
1.00
1.50
Basal Glc Ins Ins+Glc n
orm
alized
sig
nal / R
ps15
fold t-test P fold t-test Parray 1.27 0.038 1.32 0.000PCR 1.46 0.008 1.48 0.016array 1.22 0.082 1.36 0.017PCR 1.47 0.045 1.34 0.066
proline 4-hydroxylase
procollagen C-proteinase enhancer protein
Name SymbolIns/Basal
P4ha1
Pcolce
Ins+Glc/Glc
– Ins + Ins – Ins + Ins
• from a proteomics viewpoint, insulin has a significant impact on protein secretion of 3T3-L1 adipocytes
• transcription is not the major regulation point for these secreted proteins
• the discrepancy of the insulin effect between transcript and secretion level of collagens, may be partly explained by the transcriptional regulation of processing enzymes.
Wang et al., Diabetologia 2006
MOLECULAR MECHANISMS FOR OBESITY-RELATED PHYSIOLOGY
EC Framework Programme 5 project NUGENOB :
Low Fat-Burners vs High Fat-Burners
Whole body nutrient oxidation:
carbohydrates fatty acids
LFO > HFO
Different fatty acid flux visible in adipose proteome?
Encountered problems:
• Quality of the tissue (biopsy)• Contamination (blood)• Normalization
2D-differential proteins: plasma and blood cell derived
LFO HFO
50 µm 50 µm
Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo Larson DR et al. 2003 Science 300 ( 5624) ,1434-1436
VASCULARISATION
LOOK ONLY AT ADIPOSE-DERIVED PROTEINS:
- generate a human adipocyte protein profile - generate a blood cell protein profile - overlay with biopsy protein profile
Adipo’s Fat biopsy
Protein description LFO/HFO
Methylmalonate-semialdehyde dehydrogenase 2.41(MMSDH)
Superoxide dismutase, Mn (Mn-SOD) 1.90
Prohibitin (PHB) 1.83
Annexin A2 1.48
Fructose-bisphosphate aldolase A (ALDOA) -1.42
p < 0.05
Adipose proteome data indicate that LFO persons have:
-Decreased glycolysis/gluconeogenesis capacity (ALDOA)
-Increased valine catabolism (MMSDH)
-Increased ROS scavenging in the mitochondria (Mn SOD)
-Inhibition of pyruvate carboxylase (PHB1, Vessal et al., FEBS J. 2006)
•Proteomics can be used to identify proteins related to human obesity using in vitro or animal models
•The influence of nutrition and nutritional hormones on cell function can be studied
•Novel targets for intervention can be found
•Mechanistic models are provided for physiological differences in humans
PROTEOMICS IN OBESITY
Recommended literature:
Nutritional Proteomics: methods and concepts for research innutritional science. Schweigert FJ. Ann Nutr Metab 51:99-107, 2007.
Nutrigenomics and nutrigenetics: the 'omics' revolution in nutritional science. Mariman E. Biotechnol ApplBiochem 44:119-28, 2006.
Proteomics in nutrition research: principles, technologies andapplications. Fuchs D, Winkelmann I, Johnson IT, Mariman E,Wenzel U, Daniel H. Br J Nutr 94:302-14, 2005.
Nutriproteomics: identifying the molecular targets of nutritiveAnd non-nutritive components of the diet. Barnes S, Kim H.J Biochem Mol Biol 37:59-74, 2004.
Abbreviations usedAbbreviations used
BFA Brefeldin-A
BIA-MS Biomolecular interaction analysis – mass spectrometry
Cy-3, Cy-5 Fluorescent cyanine dyes
ECM Extra-cellular matrix
ELISA Enzyme-Linked immunosorbent assay
FC Fold change
FT-MS Fourier transform mass spectrometry
HPLC High performance liquid chromatography
HUGO Human Genome Organisation
ICAT Isotope-coded affinity tags
IL- 6 (etc) Interleukin -6 (etc)
M/Z Mass /charge ratio
MALDI-TOF Matrix-assisted laser desorptio/ionisation – time of flight
MIDA Mass isotopomer distribution analysis
MWG MWG Biotech AG
PHB Prohibitin
ROS Reactive oxygen species
SELDI-TOF Surface enhanced laser desorption ionization – time of flight
SPR-BIA Surface plasmon resonance – biomolecular interaction analysis