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

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

2. 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

3. BMI > 30 kg/m2 25 kg/m2 < BMI > 30 kg/m2 Obesity in The Netherlands weight length BMI = 2 Overgewicht = overweight Ernstig overgewicht = obese

4. CHILD OBESITY

5. Obesity linked with diseases Reduced fertility Obesity Diabetes cardiovascular diseases Cancer

6. ADIPOSE TISSUE: adipocytes / stromal cells

7. GENE-EXPRESSION PROFILING mouse 3T3-L1 (pre)adipocytes

8. RNA PROTEIN METABOLITE GENE GENETICS PROTEOMICS METABOLOMICS TRANSCRIPTOMICS

9. 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

10. 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

11. 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

12. PROTEIN PROFILING

14. PROTEINS Cy3Cy5 MIX INCUBATE

15. ANTIBODY ARRAY PROTEINS Cy3Cy5 MIX INCUBATE

16. extract proteins separate them determine relative quantity identify differential proteins changed genes/pathways Techniques 2D –gel electrophoresis staining mass spectrometry pathway analysis

17. Iso-electric focussing Protein profiling: impression of identity and quantity Mass separation

19. PROTEIN (GENE)-IDENTIFICATION PROTEIN FRAGMENTS FINGERPRINT M/Z Bouwman et al., Rapid Comm Mass Spectrom 2005

20. MALDI-TOF MASS SPECTROMETRY + - flight tube laser detector proteins/peptides + matrix M/Z

21. Protein-identification via fingerprinting PROTEIN FRAGMENTS FINGERPRINT M/Z TRYPTIC PEPTIDES PROTEIN DATABASE HUGO M/Z

22. tandem MS (MS/MS)

23. PROTEINS MEMBRANE STRUCTURAL CYTOSOLIC 2D-GELS 2D-LC

24. ADIPOCYTE FUNCTIONING IN OBESITY Protein secretion by adipocytes

25. Obesity at cellular level THE SECRETOME

26. 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

27. Research approach: secretome profiling Model system: mouse 3T3-L1 (pre-)adipocytes Excreted proteins Fat storage

28. Inhibition of protein excretion from 3T3-L1 cells Day 12 37°C 37°C + BFA 20°C

29. 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

30. 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

31. 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

32. Novel adipokine in human plasmaPigment epithelium-derived factor(PEDF) r = 0.583

33. 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

34. THERAPY KILLER PEPTIDE ADIPOCYTE VASCULAR EPITHELIUM PROHIBITIN Kolonin et al., Nat. Med. 2004

35. 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

36. 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

37. BASAL LAMINA COLLAGEN IV STAINING Pierleoni et al., Eur, J. Histochem. 1998

38. 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

39. 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

40. 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

41. Comparison on transcriptome and proteome of secretory genes

42. Collagen post-translational modifications Alberts et al. Molecular Biology of The Cell. 3rd

43. Collagen processing enzymes – Ins – Ins + Ins + Ins

44. 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

45. SECRETOME: SECRETED INTERVENTION PROTEINS TARGETS ECM COMPONENTS INSULIN-REGULATED

46. MOLECULAR MECHANISMS FOR OBESITY-RELATED PHYSIOLOGY EC Framework Programme 5 project NUGENOB : Low Fat-Burners vs High Fat-Burners

48. Whole body nutrient oxidation: carbohydrates fatty acids LFO > HFO Different fatty acid flux visible in adipose proteome?

49. LFO HFO • Encountered problems: • Quality of the tissue (biopsy) • Contamination (blood) • Normalization 2D-differential proteins: plasma and blood cell derived

50. VASCULARISATION 50 µm 50 µm Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in VivoLarson DR et al. 2003 Science 300 ( 5624) ,1434-1436