PROTEOMICS OF OBESITY

Part II

Prof. Dr. E.C.M. Mariman

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

CHILD OBESITY

Obesity linked with diseases

ObesityDiabetes

Cancercardiovascular diseases

Reducedfertility

ADIPOSE TISSUE: adipocytes / stromal cells

GENE-EXPRESSION PROFILING

mouse 3T3-L1 (pre)adipocytes

GENE RNA PROTEIN METABOLITE

GENETICS

TRANSCRIPTOMICS

PROTEOMICS

METABOLOM

ICS

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?

PROTEIN PROFILING

PROTEINS

Cy3 Cy5

MIX

INCUBATE

PROTEINS

Cy3 Cy5

MIX

INCUBATE

ANTIBODY ARRAY

extract proteins

separate them

determine relative quantity

identify differential proteins

changed genes/pathways

TechniquesTechniques

2D –gel electrophoresis

staining

mass spectrometry

pathway analysis

Protein profiling: impression of identity and quantity

Iso-electric focussing

Mass separation

PROTEIN (GENE)-IDENTIFICATION

M/Z

PROTEIN FRAGMENTS FINGERPRINT

Bouwman et al., Rapid Comm Mass Spectrom 2005

MALDI-TOF MASS SPECTROMETRY

+ -

laser

proteins/peptides + matrix

detector

flight tube

M/Z

HUGO PROTEINDATABASE

TRYPTIC PEPTIDES

Protein-identification via fingerprinting

M/Z

M/Z

PROTEIN FRAGMENTS FINGERPRINT

tandem MS (MS/MS)

PROTEINSMEMBRANE STRUCTURAL CYTOSOLIC

2D-GELS

2D-LC

ADIPOCYTE FUNCTIONING IN OBESITY

Protein secretion by adipocytesProtein secretion by adipocytes

Obesity at cellular level

THE SECRETOME

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

Inhibition of protein excretion from 3T3-L1 cells

Day 12

20°C

37°C+ BFA

37°C

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

THERAPY

ADIPOCYTE

VASCULAREPITHELIUM

PROHIBITIN

KILLERPEPTIDE

Kolonin et al., Nat. Med. 2004

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

BASAL LAMINA

COLLAGEN IV STAINING

Pierleoni et al., Eur, J. Histochem. 1998

- 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

Alberts et al. Molecular Biology of The Cell. 3rd

Collagen post-translational modifications

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

SECRETOME:

SECRETED INTERVENTION PROTEINS TARGETS

ECM COMPONENTS INSULIN-REGULATED

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

courtesy of Peter Arner/Gabby Hul

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

GenMAPP:

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

MMSDH↑

VALINE DEGRADATION PATHWAY

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)

glycolysis↓LFO/HFO

PHB1

valine degradation

Claessens et al., Proteomics, clinical applications 2007

•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