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 cells
2. SILAC experiment with HIV-1Tat protein
3. Summary
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
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
• 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
Inflammation; Tissue Injury (eg Ischaemia); Aging;
Cardiovascular Risk Factors (Obesity; DM);
Heart Failure
↑TNF-α Release
TNF-R1
APOPTOSIS
INFLAMMATORY
RESPONSE /
PRO-SURVIVAL
TNF-R2
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:
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
PROTEIN REGULATION:
Control
Down
77
Control
Shared:
1056
TNF-α
Up
143
TNF-α
Maxquant™: 1102 proteins
Control
Down
226
TNF-α
Up
269
Shared:
1214
Control
TNF-α
Sieve™: 1511 proteins
TNF-α: 5ng / ml ; 24h
UP REGULATED AND TNF-α ONLY:
16 proteins
DAVID Bioinformatics Resources®
UP REGULATED AND TNF-α ONLY:
51 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)
DAVID Bioinformatics Resources®
UP REGULATED AND TNF-α ONLY:
13 proteins
DAVID Bioinformatics Resources®
UP REGULATED AND TNF-α ONLY:
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)
DAVID
Bioinformatics
Resources®
DOWN REGULATED AND CONTROL ONLY:
Function, pathway, process
P-value
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:
Proteomics:
• TNF-R1 and Death Associated Protein (TRADD):
Expressed only in TNF-α stimulated cells
• NF-κB: Expressed only in TNF-α stimulated cells
Control 0.5ng/ml
5ng/ml
TRADD
20ng/ml
IκB-α
β-tubulin
Inflammatory / Immune Protein
Expression:
IκB EXPRESSION
• Complement C4 (2.2-fold);
• ICAM-1 (only TNF-α);
• MHC Class 1 (1.6-fold);
• IL-1 (TNF-α only)
eNOS-NO Pathway:
•
•
•
•
•
eNOS: 27%
eNOS: 63%
NO: 44%
NO: 33%
 NO: 23%
NOS-NO BIOSYNTHESIS PATHWAYS:
NO
PRODUCTION
Control TNF 0.5ng TNF 0.5ng TNF 20ng
HSP90
β- tubulin
Control
TNF-α 5ng/ml
p-PKB
HEAT SHOCK
PROTEIN 90
EXPRESSION
t-PKB
PKB/Akt
REGULATION
AND
ACTIVATION
PROTEOMICS:
• Heat shock protein 90-α (5.8-fold)
• Heat shock protein 90-β (42-fold)
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)
DAVID Bioinformatics Resources® • Peroxiredoxin (2-fold)
Function, pathway, process
P-value
Glutathione Metabolism
0.0004
Oxidative Stress
4x10-7
Ingenuity® Systems
OXIDATIVE STRESS PARAMETERS:
Control
0.5ng/ml
5ng/ml
20ng/ml
p22-phox
β-tubulin
P22-PHOX EXPRESSION
MITOCHONDRIAL ROS PRODUCTION:
20 µm
Control: +
MitoSoxTM
20 µm
TNF-α: + MitoSoxTM
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.
• Tat associated with neural cell death and probable agent of HIV associated dementia
• 2849 proteins were identified from SILAC treated cells which were either phosphoenriched 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.
Recent successes with Orbitrap
Orbitrap Velos
MS
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
Single human peptide in Arabidopsis
546 vs 24

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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
29
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-ofStellenbosch/278646975539969