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PROTEOMICS OF OBESITY Part II Prof. Dr. E.C.M. Mariman PowerPoint PPT Presentation


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PROTEOMICS OF OBESITY Part II Prof. Dr. E.C.M. Mariman. Contents Slides Proteomics of obesity and adipose tissue: overview1 -11 Protein profiling methodologies12 – 23 Protein secretion by adipocytes24 – 47 Molecular mechanisms for obesity-related physiology48 – 59

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PROTEOMICS OF OBESITY Part II Prof. Dr. E.C.M. Mariman

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PROTEOMICS OF OBESITY

Part II

Prof. Dr. E.C.M. Mariman


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ContentsSlides

Proteomics of obesity and adipose tissue: overview1 -11

Protein profiling methodologies12 – 23

Protein secretion by adipocytes24 – 47

Molecular mechanisms for obesity-related physiology48 – 59

Proteomics in obesity: summary60

Some literature references61

Abbreviations used62


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BMI > 30 kg/m2

25 kg/m2 < BMI > 30 kg/m2

Obesity in The Netherlands

weight

length

BMI =

2

Overgewicht = overweight

Ernstig overgewicht = obese


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CHILD OBESITY


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Obesity linked with diseases

Reduced

fertility

Obesity

Diabetes

cardiovascular

diseases

Cancer


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ADIPOSE TISSUE:

adipocytes / stromal cells


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GENE-EXPRESSION PROFILING

mouse 3T3-L1 (pre)adipocytes


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RNA

PROTEIN

METABOLITE

GENE

GENETICS

PROTEOMICS

METABOLOMICS

TRANSCRIPTOMICS


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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


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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


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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


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PROTEIN PROFILING


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PROTEINS

Cy3Cy5

MIX

INCUBATE


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ANTIBODY ARRAY

PROTEINS

Cy3Cy5

MIX

INCUBATE


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extract proteins

separate them

determine relative quantity

identify differential proteins

changed genes/pathways

Techniques

2D –gel electrophoresis

staining

mass spectrometry

pathway analysis


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Iso-electric focussing

Protein profiling: impression of identity and quantity

Mass separation


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PROTEIN (GENE)-IDENTIFICATION

PROTEIN

FRAGMENTS

FINGERPRINT

M/Z

Bouwman et al., Rapid Comm Mass Spectrom 2005


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MALDI-TOF MASS SPECTROMETRY

+ -

flight tube

laser

detector

proteins/peptides + matrix

M/Z


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Protein-identification via fingerprinting

PROTEIN

FRAGMENTS

FINGERPRINT

M/Z

TRYPTIC

PEPTIDES

PROTEIN

DATABASE

HUGO

M/Z


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tandem MS (MS/MS)


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PROTEINS

MEMBRANE STRUCTURAL CYTOSOLIC

2D-GELS

2D-LC


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ADIPOCYTE FUNCTIONING IN OBESITY

Protein secretion by adipocytes


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Obesity at cellular level

THE SECRETOME


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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


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Research approach: secretome profiling

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

Excreted proteins

Fat storage


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Inhibition of protein excretion from 3T3-L1 cells

Day 12

37°C

37°C

+ BFA

20°C


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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


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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


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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


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Novel adipokine in human plasmaPigment epithelium-derived factor(PEDF)

r = 0.583


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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


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THERAPY

KILLER

PEPTIDE

ADIPOCYTE

VASCULAR

EPITHELIUM

PROHIBITIN

Kolonin et al., Nat. Med. 2004


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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


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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 1.9virtually none

  • 3T3-L1 9.6 3.2abundant

  • 9.9 3.3

  • Green & Meuth, Cell 3:127-133,1974


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    BASAL LAMINA

    COLLAGEN IV STAINING

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


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    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


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    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


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    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


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    Comparison on transcriptome and proteome

    of secretory genes


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    Collagen post-translational modifications

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


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    Collagen processing enzymes

    – Ins

    – Ins

    + Ins

    + Ins


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    • 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


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    SECRETOME:

    SECRETED INTERVENTION

    PROTEINS TARGETS

    ECM COMPONENTS INSULIN-REGULATED


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    MOLECULAR MECHANISMS FOR

    OBESITY-RELATED PHYSIOLOGY

    EC Framework Programme 5 project NUGENOB :

    Low Fat-Burners vsHigh Fat-Burners


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    Whole body nutrient oxidation:

    carbohydrates

    fatty acids

    LFO > HFO

    Different fatty acid flux visible in adipose proteome?


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    LFO

    HFO

    • Encountered problems:

      • Quality of the tissue (biopsy)

      • Contamination (blood)

      • Normalization

    2D-differential proteins: plasma and blood cell derived


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    VASCULARISATION

    50 µm

    50 µm

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


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    courtesy of Peter Arner/Gabby Hul


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    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


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    GenMAPP:


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    Protein descriptionLFO/HFO

    Methylmalonate-semialdehyde dehydrogenase2.41

    (MMSDH)

    Superoxide dismutase, Mn (Mn-SOD)1.90

    Prohibitin (PHB)1.83

    Annexin A21.48

    Fructose-bisphosphate aldolase A (ALDOA)-1.42

    p < 0.05


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    VALINE DEGRADATION PATHWAY

    MMSDH↑


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    • 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)


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    LFO/HFO

    glycolysis↓

    PHB1

    valine degradation

    Claessens et al., Proteomics, clinical applications 2007


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    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


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    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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    Abbreviations used


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