Proteomics Department

PREVIOUS PROJECTS

-Endothelial cell receptors; Transport of macromolecules in vascular cells. (Antohe et al., Microcirculation Endothelium and Lymphatics, 1986;1988; Antohe and Poznansky, Pharmaceutical Enzymes, 1997; Borvac et al., Int Immunol.,1998; Antohe et al.,Endothelium.,1997;Dobrila et al.,Int Immunol.,1992; Antohe et al., Hum. Immunol., 2001; Antohe et al.,Cell Tissue Res., 2005).
-Albumin binding proteins function in receptor mediated binding and transcytosis of albumin across endothelial cells. (Antohe et al. Eur J Cell Biol.,1991,1993; Heltianu et al., Eur. J Cell Biol. 1994 and Microvasc Res.,1989; Antohe et al., Eur J Cell Biol.,1998).
-Endothelial heart-type fatty acid binding proteins (FABP) are the main carriers for fatty acids. (Antohe et al., Eur J Cell Biol.,1991;1998; Antohe et al., J Liposome Res., 2004).
-Human placental endothelial cells express neonatal receptors (FcRn) which discriminate and monitor the intracellular pathway of IgG. (Radulescu et al., Hum Immunol., 2004; Antohe et al., Hum Immunol., 2001,2004).
-Low density lipoprotein (LDL) binding induces asymmetric redistribution of LDL receptors in endothelial cells. (Antohe et al., Endothelium: J of Endoth. Cell Res., 1997; Antohe et al., Eur J Cell Biol., 1999).
-Monoclonal antibodies as therapeutic tools. (Antohe et al., J Liposome Res.,2004; Radulescu et al.,Med Sci Monit., 2004 and Hum Immunol.,2004).
-Role of the folic acid receptors in the pathobiology of cardiovascular diseases. (Antohe et al., Cell and Tissue Research, 2005; Antohe F., Archives of Physiology and Biochemistry, 2006).
-Hyperlipidemia induces endothelial cell dysfunction. (Antohe et al., Atherosclerosis Suppl., 2006, 2008; Ivan et al., J of Receptors and Signal Transduction, 2010; Uyy et al., Microvasc. Res., 2010; Haraba et al., Int. J of Cardiology, 2011; Haraba et al., Cell and Tissue Research, 2011; Haraba and Antohe, Digest J of Nanomaterials and Biostructures, 2011).
-Eye dysfunction associated with inflammatory systemic disease. (Cojocaru et al., Annals of the Rheumatic Diseases, 2004 and 2006; Oftalmologia, 2006; Cojocaru et al., Digest J of Nanomaterials and Biostructures, 2011).

CURRENT PROJECTS

Cellular and molecular mechanisms that govern the molecular traffic in response to pathological stimuli in vascular cells

Goal:
To extend the studies beyond the classical methods (biochemical, immunological, microscopic, et al.) to top level proteomic approach using high performance qualitative and quantitative mass spectrometry equipments, namely LTQ Orbitrap VelosPro with the highest power of resolution and sensitivity of targeted proteomics and the extremely selective triple quadrupol mass spectrometer TSQ Vantage recently acquired by the European Community Structural Funds Grant, CARDIOPRO (2009-2012),(Fig. 1).
Main objectives
I.Analytical proteomics of membrane proteins isolated from normal and activated cells/tissue fragments under various risk factors (hyperlipidemic diet, hyperglycemia, reactive oxygen species) for cardiovascular diseases and their complications (atherosclerosis, diabetes). The benefits could be new biomarkers (or set of markers) related to certain cellular dysfunctions, relevant receptors and/or components of the signaling pathways altered under stress factors, and important post-translational modifications of proteins responsible for vascular pathologies that could become the targets in personalized therapies.
It was reported that sub-endothelial accumulation of lipids leads to the formation of foam cells primarily derived from macrophages and in the late stages of atherosclerosis even from endothelial cells (EC). We have reproduce the formation of EC- derived foam cells in culture (Fig. 2)(Radulescu et al., 2004; Antohe F., 2006, Ivan L et al. 2010) and plan to uncover the signaling pathways and key factors responsible for the phenotypic changes induced by high lipid diet insults in EC.
Biological membranes contain a mosaic of micro-domains (rafts, caveolae) with unique molecular composition that play an important role in the membrane organization and function (Fig. 3). The specialized micro-domains are often the target of intra- or extracellular
stimuli affecting their dynamics or signal transduction. The role and the expression of caveolins (markers for caveolae) both at protein and gene level in normal and pathological conditions (diabetes, hyperlipidemia) will be examined.
The current proteomic project (Fig. 4) aims to define and characterize the protein pattern alterations in experimental hyperlipidemic conditions and under statin therapy. Using high resolution 2D-DIGE technology we have recently found significant changes in the protein profile of lipid rafts in the two conditions as opposed to control. The proteins showing significant difference in the expression levels were selected as candidates to be identified by mass spectrometry.
II. Functional proteomics aiming to study the molecular structure and the functional interaction between proteins that will allow the possibility to generate theories that could lead to a therapeutic strategy in cardiovascular pathology. Previously unknown proteins may be discovered by their association with one or more proteins that are already known. Protein interaction analysis may also uncover unique, unforeseen functional roles for well-known proteins. The discovery or verification of an interaction is the first step on the road to understanding where, how and under what conditions these proteins interact in vivo and the functional implications of these interactions. The new PHERAstar FS, could perform complex assays, such as FRET and BRET, AlphaLISA and Alpha Screen, due to the unique modular optical system, sequential dual excitation, simultaneous dual emission, and ratio-metric calculations (Fig. 5).
Our objectives are to evidence the specific interactions between the lipid-rafts co-localized proteins (receptors, signal transductions components, particular enzymes or structural caveolae proteins) involved in cellular metabolic and regulatory processes associated with vascular dysfunctions.

The work flow of the New PROTEOMIC facility
Fig.1.Typical shotgun proteomic approach. Proteins are extracted; denaturized, digested and the resulting peptides are separated by HPLC, prior to the MS analysis. The later consists of ionization of the peptides, acquisition of a full spectrum (survey scan) and selection of the precursor ions to be fragmented, followed by acquisition of MS/MS spectra. The data can be processed to either quantify the different species or determine the peptide amino acid sequence through database search.

Fig.2. Generation of EC-derived foam cells in culture. Human EC grown in standard culture medium with 10% human serum (a) and activated cells (b, c) grown in hyperlipidemic human serum. The later accumulates lipid droplets as revealed by phase contrast (b) and fluorescence microscopy (c). The free cholesterol content of the lipid droplets was evidenced with filipin (c inset). (Ivan L et al 2010) .

Fig.3. Ultrastructural changes of lung capillary endothelial cells (EC) in double transgenic diabetic mice (left) compared with wild type control mice (right). The former, exhibit a highly convoluted apical plasmalemma, increased number of caveolae (pv) and ribosomes (r) and a hyperplasic basal lamina (bl). l: capillary lumen; Bars, 200 nm (Uyy E et al. 2010).



Fig.4. 2D-DIGE experiment showing up-regulation of specific proteins (rectangular domain containing MW: 155kDa-170kDa and pI: 4,3-4,7) in the hyperlipidemic condition (green Cy3 stained proteins) as opposed to the control state (red Cy5 stained proteins), (Suica V. et al. 2011).

Fig.5. BRET2 (Bioluminescence Resonance Energy Transfer) is an advanced, non-destructive, cell-based assay generation that is perfectly suited for proteomics applications, including receptor research and the mapping of signal transduction pathways light source.

Fig. 7. Identification of stress proteins in endothelial cells. SDS-PAGE separation (15 μg/lane) of protein extracted from non-activated (N) and activated (A) human endothelial cells showed different expression (arrows) of some peptides (a). A representative Western blot for heat shock protein Hsp 27 (b), and Hsp 90 (c), in cytosolic fraction of non-activated (N) and activated (A) endothelial cells. Densitometry analysis of immunoblots normalized to β- actin for three independent experiments with similar results. b: p<0.01; c: p= 0.003 (Ivan and Antohe, 2010).


Fig. 6. (a) mRNA level of HMGB1 normalized to β-actin (real time PCR); Melt-curve analysis was used to identify nonspecific products. (b) HMGB1 signaling pathway in heart tissue revealed by immunoblotting using HMGB1, RAGE, AKT1, phospho-AKT1 (Thr-308) , -Tubulin and Lamin B antibodies followed by the appropriate HRP coupled secondary antibodies; control group : C, hyperlipidemic group: H, fluvastatin treated hyperlipidemic group: Ht (R. Haraba et al., 2011). (c) Schematic representation of HMGB1 signaling events in the heart tissue of animals fed a lipid rich diet (Boteanu R., 2012).

III.Alarmins and membrane proteins in health and disease. Alarmins (HMGB1, HSP proteins, annexin family) are endogenous chemotactic activators released under stress condition that induce specific cellular responses by interactions with receptors located in the membrane lipid bilayer.In the hyperlipidemic animal model, we demonstrated that the enhanced HMGB1 is linked to the up-regulation of RAGE, in association with AKT phosphorylation. Fluvastatin treatment resulted in significant reduction of HMGB1, RAGE and AKT phosphorylation expression in the investigated tissue. PI3K/AKT pathway can regulate the activity of Sp1 or NF-kB transcription factors, which induce the production of HMGB1, RAGE and other cytokines. Thus, a feedback loop action of HMGB1 that amplifies the initial signal and further enhances inflammation in hyperlipidemic conditions, which led to the development of atherosclerotic lesions, was proposed (R. Haraba et al., International J of Cardiology, and Cell and Tissue Research 2011). Future studies aiming to identify the transcription factors activated by HMGB1-RAGE signaling are necessary to be done for a better understanding the inflammatory process at all stages of the disease (Fig. 6).
Under the high lipid stress endothelial cells become activated and express high amount of heat shock proteins (HSP 27, 70 and 90) demonstrating the stimulated response to severe insults (Fig. 7).New CARDIOPRO equipments
- LTQ (linear trap quadrupole) Orbitrap Velos Pro
high performance mass spectrometer, Thermo Scientific
- TSQ (triple stage quadrupole) Vantage, Thermo Scientific
- Pherastar FS high performance spectrophotometer, BMG Labtech
- Typhoon FLA 9500 high performance variable mode laser scanner, GE Healthcare
- Ettan DALT twelve high-throughput 2-D electrophoresis system, GE Healthcare
- Micro Rotofor preparative isoelectric focusing system, Bio-Rad
- EXQuest Spot Cutter, Bio-Rad
- Concentrator Plus evaporation system, Eppendorf
-Fluorescence inverted microscop AxioVert A1.FL Zeiss