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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


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


BMI > 30 kg/m2

25 kg/m2 < BMI > 30 kg/m2

Obesity in The Netherlands

weight

length

BMI =

2

Overgewicht = overweight

Ernstig overgewicht = obese


CHILD OBESITY


Obesity linked with diseases

Reduced

fertility

Obesity

Diabetes

cardiovascular

diseases

Cancer


ADIPOSE TISSUE:

adipocytes / stromal cells


GENE-EXPRESSION PROFILING

mouse 3T3-L1 (pre)adipocytes


RNA

PROTEIN

METABOLITE

GENE

GENETICS

PROTEOMICS

METABOLOMICS

TRANSCRIPTOMICS


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


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


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


PROTEIN PROFILING


PROTEINS

Cy3Cy5

MIX

INCUBATE


ANTIBODY ARRAY

PROTEINS

Cy3Cy5

MIX

INCUBATE


extract proteins

separate them

determine relative quantity

identify differential proteins

changed genes/pathways

Techniques

2D –gel electrophoresis

staining

mass spectrometry

pathway analysis


Iso-electric focussing

Protein profiling: impression of identity and quantity

Mass separation


PROTEIN (GENE)-IDENTIFICATION

PROTEIN

FRAGMENTS

FINGERPRINT

M/Z

Bouwman et al., Rapid Comm Mass Spectrom 2005


MALDI-TOF MASS SPECTROMETRY

+ -

flight tube

laser

detector

proteins/peptides + matrix

M/Z


Protein-identification via fingerprinting

PROTEIN

FRAGMENTS

FINGERPRINT

M/Z

TRYPTIC

PEPTIDES

PROTEIN

DATABASE

HUGO

M/Z


tandem MS (MS/MS)


PROTEINS

MEMBRANE STRUCTURAL CYTOSOLIC

2D-GELS

2D-LC


ADIPOCYTE FUNCTIONING IN OBESITY

Protein secretion by adipocytes


Obesity at cellular level

THE SECRETOME


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


Research approach: secretome profiling

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

Excreted proteins

Fat storage


Inhibition of protein excretion from 3T3-L1 cells

Day 12

37°C

37°C

+ BFA

20°C


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


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


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


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

r = 0.583


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


THERAPY

KILLER

PEPTIDE

ADIPOCYTE

VASCULAR

EPITHELIUM

PROHIBITIN

Kolonin et al., Nat. Med. 2004


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


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


    BASAL LAMINA

    COLLAGEN IV STAINING

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


    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


    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

    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


    Comparison on transcriptome and proteome

    of secretory genes


    Collagen post-translational modifications

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


    Collagen processing enzymes

    – 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 vsHigh Fat-Burners


    Whole body nutrient oxidation:

    carbohydrates

    fatty acids

    LFO > HFO

    Different fatty acid flux visible in adipose proteome?


    LFO

    HFO

    • Encountered problems:

      • Quality of the tissue (biopsy)

      • Contamination (blood)

      • Normalization

    2D-differential proteins: plasma and blood cell derived


    VASCULARISATION

    50 µm

    50 µm

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


    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

    Fat biopsy

    Adipo’s


    GenMAPP:


    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


    VALINE DEGRADATION PATHWAY

    MMSDH↑


    • 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)


    LFO/HFO

    glycolysis↓

    PHB1

    valine degradation

    Claessens et al., Proteomics, clinical applications 2007


    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


    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.


    Abbreviations used


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