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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

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slide1

PROTEOMICS OF OBESITY

Part II

Prof. Dr. E.C.M. Mariman

slide2

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

slide3

BMI > 30 kg/m2

25 kg/m2 < BMI > 30 kg/m2

Obesity in The Netherlands

weight

length

BMI =

2

Overgewicht = overweight

Ernstig overgewicht = obese

obesity linked with diseases
Obesity linked with diseases

Reduced

fertility

Obesity

Diabetes

cardiovascular

diseases

Cancer

slide6

ADIPOSE TISSUE:

adipocytes / stromal cells

slide7

GENE-EXPRESSION PROFILING

mouse 3T3-L1 (pre)adipocytes

slide8

RNA

PROTEIN

METABOLITE

GENE

GENETICS

PROTEOMICS

METABOLOMICS

TRANSCRIPTOMICS

slide9

WHAT TYPE OF INFORMATION?

ASPECTS OF PROTEIN FUNCTION

  • Protein identification
  • Protein quantification
  • Protein turnover
  • Posttranslational modifications
  • Protein interactions and complex formation
  • Cellular localization and translocation
  • Structural analysis
slide10

Methodologies

  • 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

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

Standard:

- Cell fractionation

- Affinity chromatography

- 1D and 2D gel

electrophoresis

- Western blotting

- ELISA

- Immunocytochemistry

slide11

Proteomics, why?

  • “ 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
slide14

PROTEINS

Cy3Cy5

MIX

INCUBATE

slide15

ANTIBODY ARRAY

PROTEINS

Cy3Cy5

MIX

INCUBATE

slide16

extract proteins

separate them

determine relative quantity

identify differential proteins

changed genes/pathways

Techniques

2D –gel electrophoresis

staining

mass spectrometry

pathway analysis

slide17

Iso-electric focussing

Protein profiling: impression of identity and quantity

Mass separation

slide19

PROTEIN (GENE)-IDENTIFICATION

PROTEIN

FRAGMENTS

FINGERPRINT

M/Z

Bouwman et al., Rapid Comm Mass Spectrom 2005

slide20

MALDI-TOF MASS SPECTROMETRY

+ -

flight tube

laser

detector

proteins/peptides + matrix

M/Z

slide21

Protein-identification via fingerprinting

PROTEIN

FRAGMENTS

FINGERPRINT

M/Z

TRYPTIC

PEPTIDES

PROTEIN

DATABASE

HUGO

M/Z

slide23

PROTEINS

MEMBRANE STRUCTURAL CYTOSOLIC

2D-GELS

2D-LC

slide24

ADIPOCYTE FUNCTIONING IN OBESITY

Protein secretion by adipocytes

slide26

Excretion products from adipocytes

Cardio-vascular effects

Insulin signaling

ApoE

TNF-

Angiotensinogen

Resistin

Plasminogen activator

inhibitor-1 (PAI-1)

Adiponectin

Weight

regulation

Vasc. endoth. growth factor (VEGF)

Leptin

IL-6

Pro-inflamm.

cytokines

Retinol-binding protein

IL-8

Phospholipid transfer protein

Hepatocyte growth factor

Lipoprotein lipase (LPL)

Insulin-like growth factor-1 (IGF-1)

Cholesteryl ester

transfer protein

Lipid handling

Growth factors

Others

Bradley et al. Recent Prog Horm Res. 2001;56:329-58

slide27

Research approach: secretome profiling

Model system: mouse 3T3-L1 (pre-)adipocytes

Excreted proteins

Fat storage

excreted proteins during 3t3 l1 differentiation

41 different proteins as candidates of secreted proteins related with adipocyte differentiation

Excreted proteins during 3T3-L1 differentiation

subtotal

identified spots 161

unknown spots 32

total 193

slide30

EXCRETED PROTEINS

Newly identified:

Macrophage migration inhibitory factor - infiltration

Pigment epithelium derived factor - vascularisation

Prohibitin - cell growth and differentiation

Collagens and modifiers:

Type I, III, IV, V, VI

Matrix metalloproteinase 2

Procollagen C-proteinase enhancer protein

Protein-lysine 6-oxidase

slide31

EXCRETED PROTEINS

Newly identified:

Macrophage migration inhibitory factor - infiltration

Pigment epithelium derived factor - vascularisation

Prohibitin - cell growth and differentiation

Collagens and modifiers:

Type I, III, IV, V, VI

Matrix metalloproteinase 2

Procollagen C-proteinase enhancer protein

Protein-lysine 6-oxidase

slide33

EXCRETED PROTEINS

Newly identified:

Macrophage migration inhibitory factor - infiltration

Pigment epithelium derived factor - vascularisation

Prohibitin - cell growth and differentiation

Collagens and modifiers:

Type I, III, IV, V, VI

Matrix metalloproteinase 2

Procollagen C-proteinase enhancer protein

Protein-lysine 6-oxidase

slide34

THERAPY

KILLER

PEPTIDE

ADIPOCYTE

VASCULAR

EPITHELIUM

PROHIBITIN

Kolonin et al., Nat. Med. 2004

slide35

EXCRETED PROTEINS

Newly identified:

Macrophage migration inhibitory factor - infiltration

Pigment epithelium derived factor - vascularisation

Prohibitin - cell growth and differentiation

Collagens and modifiers:

Type I, III, IV, V, VI

Matrix metalloproteinase 2

Procollagen C-proteinase enhancer protein

Protein-lysine 6-oxidase

slide36

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

slide37

BASAL LAMINA

COLLAGEN IV STAINING

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

modulation of the secretion of adipocytes
Modulation of the secretion of adipocytes

Characterize:

2 days treatment with

- glucose

- insulin

1. culture medium proteins

2. cellular total RNA

1. medium proteins: 2D and 1D gel analysis

2. mRNA: RT-PCR and microarray analysis

slide39

high insulin

basal

high glucose

high insulin + glucose

collagen I alpha1 C-peptide

collagen III alpha1 C-peptide

collagen I alpha 2 C-peptide

Major effect on 2D gel

microarray analysis
Microarray analysis

Cy5 sample

Cy3 reference

extra set : genes encoding mitochondrial proteins and secretory proteins

MWG mouse 10 K array with extra gene sets

50-mer mouse non-redundant oligos: 10060 in house printed

slide42

Collagen post-translational modifications

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

collagen processing enzymes
Collagen processing enzymes

– Ins

– Ins

+ Ins

+ Ins

slide44
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

slide45

SECRETOME:

SECRETED INTERVENTION

PROTEINS TARGETS

ECM COMPONENTS INSULIN-REGULATED

slide46

MOLECULAR MECHANISMS FOR

OBESITY-RELATED PHYSIOLOGY

EC Framework Programme 5 project NUGENOB :

Low Fat-Burners vs High Fat-Burners

slide48

Whole body nutrient oxidation:

carbohydrates

fatty acids

LFO > HFO

Different fatty acid flux visible in adipose proteome?

slide49

LFO

HFO

  • Encountered problems:
        • Quality of the tissue (biopsy)
        • Contamination (blood)
        • Normalization

2D-differential proteins: plasma and blood cell derived

slide50

VASCULARISATION

50 µm

50 µm

Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in VivoLarson DR et al. 2003 Science 300 ( 5624) ,1434-1436

slide52

LOOK ONLY AT ADIPOSE-DERIVED PROTEINS:

- generate a human adipocyte protein profile

- generate a blood cell protein profile

- overlay with biopsy protein profile

Fat biopsy

Adipo’s

slide54

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

slide56

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, Vessalet al., FEBS J. 2006)
slide57

LFO/HFO

glycolysis↓

PHB1

valine degradation

Claessens et al., Proteomics, clinical applications 2007

slide58

PROTEOMICS IN OBESITY

  • 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
slide59

Recommended literature:

Nutritional Proteomics: methods and concepts for research in

nutritional science. Schweigert FJ. Ann Nutr Metab 51:99-

107, 2007.

Nutrigenomics and nutrigenetics: the \'omics\' revolution

in nutritional science. Mariman E. Biotechnol Appl

Biochem 44:119-28, 2006.

Proteomics in nutrition research: principles, technologies and

applications. 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 nutritive

And non-nutritive components of the diet. Barnes S, Kim H.

J Biochem Mol Biol 37:59-74, 2004.

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