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Salome Smit

Proteomics investigation into cardiac endothelial cells using the Orbitrap at the Proteomics facility of the University of Stellenbosch. Central Analytical Facility University of Stellenbosch. Salome Smit. Overview Proteomics analysis of cardiac endothelial cells

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Salome Smit

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  1. Proteomics investigation into cardiac endothelial cells using the Orbitrap at the Proteomics facility of the University of Stellenbosch Central Analytical Facility University of Stellenbosch Salome Smit

  2. Overview Proteomics analysis of cardiac endothelial cells SILAC experiment with HIV-1Tat protein Summary

  3. CARDIAC MICROVASCULAR ENDOTHELIAL CELLS = CMECs • Vascular endothelium long thought to be a mere selectively permeable barrier between the circulation and sub-endothelial tissues, is now known to be a master regulator of vascular homeostasis, • Controlling functions such as vasomotor activity, thrombosis, inflammation and redox balance • When endothelial function becomes compromised as observed in cardiovascular risk conditions such as diabetes mellitus, vascular homeostasis is lost resulting in increased oxidative stress, a loss of nitric oxide (NO) bioavailability, increased endothelial cell expression of pro-inflammatory vascular adhesion molecules and increased endothelial permeability. • These pathophysiological changes underlie the phenomena of endothelial activation and endothelial dysfunction, of which the latter in particular is regarded as the early forerunner of atherosclerosis. All slides for CMEC work courtesy of Prof Hans Strijdom, University of Stellenbosch

  4. CMECs • In the heart, the myocardial capillaries (leading to ischaemic heart disease) are made up of cardiac microvascular endothelial cells (CMECs). • CMECs show distinct structural and functional adaptations compared to other endothelial cell phenotypes in view of their location in the myocardium where they are closely associated with surrounding cardiomyocytes. • There is intimate CMEC-cardiomyocytearrangement • cardiomyocytesare regarded as the primary cellular recipients of paracrine messengers secreted by CMECs, such as NO and endothelin-1.

  5. CMECs • CMECs: PIVOTAL ROLE IN BOTH MYOCARDIAL FUNCTION AND INJURY • Optimal diffusion of oxygenand nutrients • Reciprocal signalling with cardiomyocytes • Regulate cardiomyocyte growth and development • Regulation of cardiomyocytecontractile function & rhythmicity • Therefore, CMECs are now recognized as important regulators of myocardial function.

  6. TNF-α: RELEASE, BINDING AND EFFECTS Inflammation; Tissue Injury (egIschaemia); Aging; Cardiovascular Risk Factors (Obesity; DM); Heart Failure ↑TNF-α Release TNF-R1 TNF-R2 INFLAMMATORY RESPONSE / PRO-SURVIVAL Vascular endothelial cells are the PRIMARY targets of circulating TNF-α (Pober 2004); Express both TNF-R1 and TNF-R2 (Madge 2001) ANTI-APOPTOSIS / ANTI-NECROSIS APOPTOSIS

  7. What is the effect of TNF-α on CMEC? What would be a novel and optimal method to study these cells? To gain the most information

  8. METHODS: LARGE-SCALE PROTEOMICS: LTQ Orbitrap Velos MS (Thermo Scientific): Central Analytical Facility, FHS, Univ Stellenbosch SDS-PAGE IN-GEL TRYPSINISATION NANO LIQUID CHROMATOGRAPHY • Relatively few papers measure large-scale protein expression and regulation in vascular endothelial cells of any type (“only” 350 since 2001): Surprising! (Richardson 2010) • Pubmed search: <5 papers reported on any form of proteomic analysis performed on CMECs MASS SPECTOMETRY PROTEIN ID

  9. PROTEIN REGULATION: Control Down 226 Control Down 77 TNF-α Up 143 TNF-α Up 269 Shared: 1214 Shared: 1056 Control Control TNF-α TNF-α Maxquant™: 1102 proteins Sieve™: 1511 proteins TNF-α: 5ng / ml ; 24h

  10. UP REGULATED AND TNF-α ONLY: 16 proteins DAVID Bioinformatics Resources®

  11. UP REGULATED AND TNF-α ONLY: 51 proteins • MITOCHONDRIAL PROTEINS: • ATP Synthase subunits (TNF only); • Acetyltransferase component ofPyruvatedehydrogenase (3-fold); • Carnitine-Acylcarnitine carrier protein (3-fold); • AcylCoAdehydrogenase( 5-fold); • Isocitratedehydrogenase(only TNF); • ADP/ATP translocase 2 ( 7.6-fold); • Cytochrome C1 ( 2-fold); • Electron transfer flavoprotein( 2-fold); • VDAC-1 ( 2-fold); • Cytochrome C1 ( 2-fold); • Cytochrome C oxidase (TNF only); • Glycerol-3-phosphate dehydrogenase(TNF only) DAVID Bioinformatics Resources®

  12. UP REGULATED AND TNF-α ONLY: 13 proteins DAVID Bioinformatics Resources®

  13. UP REGULATED AND TNF-α ONLY: Ingenuity® Systems

  14. DOWN REGULATED AND CONTROL ONLY: 27 proteins • CYTOSKELETON PROTEINS: • ADP Ribosylation Factor ( 5-fold); • Actin, alpha-1 ( 4-fold); • Actin, gamma-1 (control only); • Alpha actinin-4 ( 4-fold); • Cofilin-1 (5.5-fold); • Gelsolin( 43-fold); • Tubulin, beta 2 ( 31-fold); • R-ras( 52-fold) DAVID Bioinformatics Resources®

  15. DOWN REGULATED AND CONTROL ONLY: Ingenuity® Systems

  16. EVIDENCE OF TNF-α SIGNALLING: Proteomics: • TNF-R1 and Death Associated Protein (TRADD): Expressed only in TNF-α stimulated cells • NF-κB: Expressed only in TNF-α stimulated cells TRADD Control 0.5ng/ml 5ng/ml 20ng/ml IκB-α β-tubulin Inflammatory / Immune Protein Expression: • Complement C4 (2.2-fold); • ICAM-1 (only TNF-α); • MHC Class 1 (1.6-fold); • IL-1 (TNF-α only) IκB EXPRESSION

  17. eNOS-NO Pathway: • eNOS: 27% • eNOS: 63% • NO: 44% • NO: 33% •  NO: 23%

  18. NOS-NO BIOSYNTHESIS PATHWAYS: Control TNF 0.5ng TNF 0.5ng TNF 20ng HSP90 β- tubulin PKB/Akt REGULATION AND ACTIVATION HEAT SHOCK PROTEIN 90 EXPRESSION Control TNF-α 5ng/ml NO PRODUCTION p-PKB t-PKB • PROTEOMICS: • Heat shock protein 90-α(5.8-fold) • Heat shock protein 90-β(42-fold)

  19. Oxidative Stress: • ROS: 63% of studies • Nitrosative stress: 25% of studies • NADPH-oxidase: 25% of studies • ROS included: Superoxide (50%), mito- ROS (25%) and H2O2 (13%)

  20. PROTEOMIC DATA SUGGEST ANTI-OXIDANT PROTEINS AND OXIDATVE STRESS RESPONSE: • PROTEOMICS: • Park-7 ( 2-fold ) • SOD [Mn], mitochondrial (2-fold) • Thioredoxin ( 3-fold) • Glutathione-s-transferase (only in TNF) • Glutathione peroxidase, GPX4 (only TNF) • Peroxiredoxin (2-fold) DAVID Bioinformatics Resources® Ingenuity® Systems

  21. OXIDATIVE STRESS PARAMETERS: Control 0.5ng/ml 5ng/ml 20ng/ml p22-phox β-tubulin P22-PHOX EXPRESSION

  22. MITOCHONDRIAL ROS PRODUCTION: FACS confirmed results 20 µm 20 µm Thus proteomic results confirmed Control: + MitoSoxTM TNF-α: + MitoSoxTM

  23. Apoptosis / Cell Death: • Apoptosis: 50% of studies •  Apoptosis: 38% of studies • Necrosis: 13% of studies

  24. APOPTOSIS / CELL DEATH: • PROTEOMICS: • Bid (TNF only) • RACK-1 (2.7-fold) • PEA-15 (inhibits TNF-R1-mediated Caspase 8 activity) (6.3-fold) • VDAC-1 (1.6-fold) • BOK (TNF only) • Metadherin(anti-apoptotic) (TNF only) • Gelsolin (anti-apoptotic) ( 43-fold)

  25. Take home message???? • Vascular endothelial cells neglected in proteomics • CMEC basically no thorough study • This is novel and important study to gain knowledge into cardiovascular disease • Increase in oxidative stress due to TNF-α – proteins increase to counteract • eNOS – decreased HSP90 • Cells undergo apoptosis – increase in apoptotic proteins and increase in some anti-apoptotic proteins – therefore cells are fighting back • Due to increase in apoptotic proteins and hence increase in cell death the proteins involved in may be the result of cytoskeleton organisation which is decreased.

  26. Quantitative proteomic analysis of HIV-1 Tat apoptosis in SH-SY5Y neuroblastoma cells Putuma P. Gqamana1ǂ, Tariq Ganief1ǂ, Salome Smit2, Shaun Garnett1,Andrew Nel1, and Jonathan Blackburn1┴. Quantitative proteomic analysis of HIV-1 Tat induced apoptosis in SH-SY5Y neuroblastoma cells. Manuscript in preparation.

  27. Tat associated with neural cell death and probable agent of HIV associated dementia • 2849 proteins were identified from SILAC treated cells which were either phospho-enriched or phospho-depleted (therefore reduced complexity of sample) • 17 up regulated and 72 down regulated proteins identified from SILAC • Dysregulation of proteins identified associated with several neurodegenerative disorders • Cell adhesion proteins down regulated – associated with apoptosis • Proteins identified may also have role in weakening of immune response • From results: • Tat neurotoxicity may activate early signalling via tyrosine phosphorylation receptors and cause mitochondrial and oxidative stress leading to apoptosis. This will form basis of future biomarker discovery for HIV associated dementia Putuma P. Gqamana1ǂ, Tariq Ganief1ǂ, Salome Smit2, Shaun Garnett1,Andrew Nel1, and Jonathan Blackburn1┴. Quantitative proteomic analysis of HIV-1 Tat induced apoptosis in SH-SY5Y neuroblastoma cells. Manuscript in preparation.

  28. Recent successes with Orbitrap

  29. Acknowledgements Thanks to: Prof Hans Strijdom – US – CMEC And students Amanda, Mashudu and Corli Dr Putuma Gqamana – UCT – SILAC proteomics, neuroblastoma cells Dr Brandy Gqamana-Young – UCT – Urine proteomics Mae Newton-Foot and Zhou Fang – US – M. Smegmatis Louise Vos – US – M. Bovis Dr Martella du Preez and Lisa Schaeffer – CSIR – V. Cholera Dr Cobus Zwiegelaar – Azargen – human peptide

  30. Proteomics Laboratory Senior Analyst: Dr Salome Smit Office: 021 938 9632 Fax nr : 086 690 7602 email: salomiesmit@sun.ac.za Universiteit van Stellenbosch Besoek / Visit: www.sun.ac.za/saf www.facebook.com/pages/CAF-Proteomics-lab-University-of-Stellenbosch/278646975539969

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