Proteomics investigation into cardiac endothelial cells using the Orbitrap at the Proteomics...

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Proteomics investigation into cardiac endothelial cells using the Orbitrap at the Proteomics facility

of the University of Stellenbosch

Salome Smit

Central Analytical Facility

University of Stellenbosch

Overview

1. Proteomics analysis of cardiac endothelial cells2. SILAC experiment with HIV-1Tat protein3. Summary

• 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

CARDIAC MICROVASCULAR ENDOTHELIAL CELLS = 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-cardiomyocyte arrangement

• cardiomyocytes are regarded as the primary cellular recipients of paracrine messengers secreted by CMECs, such as NO and endothelin-1.

• CMECs: PIVOTAL ROLE IN BOTH MYOCARDIAL FUNCTION AND INJURY

• Optimal diffusion of oxygen and nutrients

• Reciprocal signalling with cardiomyocytes

• Regulate cardiomyocyte growth and development

• Regulation of cardiomyocyte contractile function & rhythmicity

• Therefore, CMECs are now recognized as important regulators of myocardial function.

TNF-α: RELEASE, BINDING AND EFFECTS

TNF-R1 TNF-R2

APOPTOSISINFLAMMATORY RESPONSE / PRO-SURVIVAL

Inflammation; Tissue Injury (eg Ischaemia); Aging; Cardiovascular Risk Factors (Obesity; DM);

Heart Failure

↑TNF-α Release

ANTI-APOPTOSIS / ANTI-NECROSIS

Vascular endothelial cells are the PRIMARY targets of circulating TNF-α (Pober 2004); Express both TNF-R1 and TNF-R2

(Madge 2001)

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

METHODS: LARGE-SCALE PROTEOMICS: LTQ Orbitrap Velos MS (Thermo Scientific): Central Analytical Facility, FHS, Univ Stellenbosch SDS-PAGE

IN-GEL TRYPSINISATION

NANO LIQUID CHROMATOGRAPHY

MASS SPECTOMETRY

PROTEIN ID

• 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

Control TNF-α

Control Down226

TNF-αUp

Shared: 1214

Sieve™: 1511 proteins

Control TNF-α

Control Down

TNF-α Up143

Shared: 1056

Maxquant™: 1102 proteins

TNF-α: 5ng / ml ; 24h

PROTEIN REGULATION:

UP REGULATED AND TNF-α ONLY:

DAVID Bioinformatics Resources®

16 proteins

MITOCHONDRIAL PROTEINS:

• ATP Synthase subunits (TNF only);• Acetyltransferase component of Pyruvate dehydrogenase (3-fold);• Carnitine-Acylcarnitine carrier protein (3-fold);• Acyl CoA dehydrogenase ( 5-fold);• Isocitrate dehydrogenase (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)

51 proteins

13 proteins

Function, pathway, process P-value

Nucleic Acid Metabolism 0.000004

Protein Synthesis 0.00006

Protein Trafficking 0.00009

EIF-2 Signalling 0.000015

Glutathione Metabolism 0.0004

Interleukin Signalling 0.0007

Oxidative Stress 4x10-7

Mitochondrial Dysfunction 0.008

Ingenuity® Systems

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)

DOWN REGULATED AND CONTROL ONLY:

Cellular Assembly & Organisation 1.7 x 10-9

Cellular Function & Maintenance 1.7 x 10-9

Cell Morphology 1.7 x 10-7

Cellular Growth & Proliferation 3.8 x 10-7

Integrin Signalling 2.4 x 10-10

Caveolar-mediated Endocytosis 4.5 x 10-9

Clathrin-mediated Endocytosis 3.3 x 10-8

Actin Cytoskeleton Signalling 2.7 x 10-7

Ingenuity® Systems

EVIDENCE OF TNF-α SIGNALLING:

• TNF-R1 and Death Associated Protein (TRADD): Expressed only in TNF-α stimulated cells • NF-κB: Expressed only in TNF-α stimulated cells

Proteomics:

• Complement C4 (2.2-fold); • ICAM-1 (only TNF-α); • MHC Class 1 (1.6-fold); • IL-1 (TNF-α only)

Inflammatory / Immune Protein Expression:

IκB-α

β-tubulin

Control 0.5ng/ml 5ng/ml 20ng/ml

IκB EXPRESSION

eNOS-NO Pathway:• eNOS: 27%• eNOS: 63%• NO: 44%• NO: 33%• NO: 23%

NOS-NO BIOSYNTHESIS PATHWAYS:

Control TNF-α 5ng/ml

PKB/Akt REGULATION AND ACTIVATION

PROTEOMICS:• Heat shock protein 90-α (5.8-fold)• Heat shock protein 90-β (42-fold)

HEAT SHOCK PROTEIN 90 EXPRESSION

β- tubulin

Control TNF 0.5ng TNF 0.5ng TNF 20ng

NO PRODUCTION

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%)

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)

Glutathione Metabolism 0.0004

Oxidative Stress 4x10-7

Ingenuity® Systems

Control 0.5ng/ml 5ng/ml

p22-phox

β-tubulin

P22-PHOX EXPRESSION

OXIDATIVE STRESS PARAMETERS:

20ng/ml

MITOCHONDRIAL ROS PRODUCTION:

Control: + MitoSoxTM20 µm

TNF-α: + MitoSoxTM

20 µm

FACS confirmed results

Thus proteomic results confirmed

Apoptosis / Cell Death:• Apoptosis: 50% of studies • Apoptosis: 38% of studies• Necrosis: 13% of studies

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)

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.

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.

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.

• 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

Recent successes with Orbitrap

Orbitrap Velos

Neuroblastoma cells 7539

M. smegmatis 3271

M. Bovis 2368

P. falciparum 1681

CMEC 1663

V. Cholera 1411

Urine biomarkers 1500

Sample from another MS unit 546 vs 24

Single human peptide in Arabidopsis

Acknowledgements

Thanks to:Prof Hans Strijdom – US – CMECAnd students Amanda, Mashudu and Corli

Dr Putuma Gqamana – UCT – SILAC proteomics, neuroblastoma cellsDr Brandy Gqamana-Young – UCT – Urine proteomics

Mae Newton-Foot and Zhou Fang – US – M. SmegmatisLouise Vos – US – M. Bovis

Dr Martella du Preez and Lisa Schaeffer – CSIR – V. Cholera

Dr Cobus Zwiegelaar – Azargen – human peptide

Proteomics LaboratorySenior Analyst: Dr Salome SmitOffice: 021 938 9632Fax nr : 086 690 7602email: salomiesmit@sun.ac.zaUniversiteit van StellenboschBesoek / Visit: www.sun.ac.za/saf

www.facebook.com/pages/CAF-Proteomics-lab-University-of-Stellenbosch/278646975539969

Proteomics investigation into cardiac endothelial cells using the Orbitrap at the Proteomics...

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