Bassam T. Wakim

Comparative proteomic analysis of PAI-1 and TNF-alpha-derived endothelial microparticles

Endothelium‐derived microparticles (EMPs) are small vesicles released from endothelial cells in response to cell injury, apoptosis, or activation. Elevated concentrations of EMPs have been associated with many inflammatory and vascular diseases. EMPs also mediate long range signaling and alter downstream cell function. Unfortunately, the molecular and cellular basis of microparticle production and downstream cell function is poorly understood. We hypothesize that EMPs generated by different agonists will produce distinct populations of EMPs with unique protein compositions. To test this hypothesis, different EMP populations were generated from human umbilical vein endothelial cells by stimulation with plasminogen activator inhibitor type 1 (PAI‐1) or tumor necrosis factor‐alpha (TNF‐α) and subjected to proteomic analysis by LC/MS. We identified 432 common proteins in all EMP populations studied. Also identified were 231 proteins unique to control EMPs, 104 proteins unique to PAI‐1 EMPs and 70 proteins unique to TNF‐α EMPs. Interestingly, variations in protein abundance were found among many of the common EMP proteins, suggesting that differences exist between EMPs on a relative scale. Finally, gene ontology (GO) and KEGG pathway analysis revealed many functional similarities and few differences between the EMP populations studied. In summary, our results clearly indicate that EMPs generated by PAI‐1 and TNF‐α produce EMPs with overlapping but distinct protein compositions. These observations provide fundamental insight into the mechanisms regulating the production of these particles and their physiological role in numerous diseases.

STAT1-dependent expression of energy metabolic pathways links tumour growth and radioresistance to the Warburg effect.

BackgroundThe Signal Transducer and Activator of Transcription 1 (STAT1) has traditionally been regarded as a transmitter of interferon signaling and a pro-apoptotic tumour suppressor. Recent data have identified new functions of STAT1 associated with tumourigenesis and resistance to genotoxic stress, including ionizing radiation (IR) and chemotherapy. To investigate the mechanisms contributing to the tumourigenic functions of STAT1, we performed a combined transcriptomic-proteomic expressional analysis and found that STAT1 is associated with regulation of energy metabolism with potential implication in the Warburg effect.MethodsWe generated a stable knockdown of STAT1 in the SCC61 human squamous cell carcinoma cell line, established tumour xenografts in athymic mice, and compared transcriptomic and proteomic profiles of STAT1 wild-type (WT) and knockdown (KD) untreated or irradiated (IR) tumours. Transcriptional profiling was based on Affymetrix Human GeneChip® Gene 1.0 ST microarrays. Proteomes were determined from the tandem mass spectrometry (MS/MS) data by searching against the human subset of the UniProt database. Data were analysed using Significance Analysis of Microarrays for ribonucleic acid and Visualize software for proteins. Functional analysis was performed with Ingenuity Pathway Analysis with statistical significance measured by Fishers exact test.ResultsKnockdown of STAT1 led to significant growth suppression in untreated tumours and radio sensitization of irradiated tumours. These changes were accompanied by alterations in the expression of genes and proteins of glycolysis/gluconeogenesis (GG), the citrate cycle (CC) and oxidative phosphorylation (OP). Of these pathways, GG had the most concordant changes in gene and protein expression and demonstrated a STAT1-dependent expression of genes and proteins consistent with tumour-specific glycolysis. In addition, IR drastically suppressed the GG pathway in STAT1 KD tumours without significant change in STAT1 WT tumours.ConclusionOur results identify a previously uncharacterized function of STAT1 in tumours: expressional regulation of genes encoding proteins involved in glycolysis, the citrate cycle and mitochondrial oxidative phosphorylation, with predominant regulation of glycolytic genes. STAT1-dependent expressional regulation of glycolysis suggests a potential role for STAT1 as a transcriptional modulator of genes responsible for the Warburg effect.

PROTEOMIC ANALYSIS OF ARTICULAR CARTILAGE VESICLES FROM NORMAL AND OSTEOARTHRITIC CARTILAGE

OBJECTIVE Articular cartilage vesicles (ACVs) are extracellular organelles found in normal articular cartilage. While they were initially defined by their ability to generate pathologic calcium crystals in cartilage of osteoarthritis (OA) patients, they can also alter the phenotype of normal chondrocytes through the transfer of RNA and protein. The purpose of this study was to analyze the proteome of ACVs from normal and OA human cartilage. METHODS ACVs were isolated from cartilage samples from 10 normal controls and 10 OA patients. We identified the ACV proteomes using in-gel trypsin digestion, nanospray liquid chromatography tandem mass spectrometry analysis of tryptic peptides, followed by searching an appropriate subset of the Uniprot database. We further differentiated between normal and OA ACVs by Holm-Sidak analysis for multiple comparison testing. RESULTS More than 1,700 proteins were identified in ACVs. Approximately 170 proteins satisfied our stringent criteria of having >1 representative peptide per protein present, and a false discovery rate of ≤5%. These proteins included extracellular matrix components, phospholipid binding proteins, enzymes, and cytoskeletal components, including actin. While few proteins were seen exclusively in normal or OA ACVs, immunoglobulins and complement components were present only in OA ACVs. Compared to normal ACVs, OA ACVs displayed decreases in matrix proteoglycans and increases in transforming growth factor β-induced protein βig-H3, DEL-1, vitronectin, and serine protease HtrA1 (P < 0.01). CONCLUSION These findings lend support to the concept of ACVs as physiologic structures in articular cartilage. Changes in OA ACVs are largely quantitative and reflect an altered matrix and the presence of inflammation, rather than revealing fundamental changes in composition.

Candidate Urinary Biomarker Discovery in Ureteropelvic Junction Obstruction: A Proteomic Approach

PURPOSE Ureteropelvic junction obstruction may either worsen and require surgery, improve or remain stable. It may take upward of 3 years for the natural history to unfold. Urinary proteome analysis using capillary electrophoresis mass spectrometry has been shown to differentiate between normal infants and those with ureteropelvic junction obstruction. We sought to confirm these findings using liquid chromatography/nano-spray mass spectrometry to examine the urinary proteome in patients with unilateral grade IV ureteropelvic junction obstruction compared to age matched healthy infants. MATERIALS AND METHODS Urine specimens were obtained from 21 healthy infants with normal maternal/fetal ultrasound and 25 infants with grade IV unilateral ureteropelvic junction obstruction. Specimens were prepared using standard methods and subjected to liquid chromatography/tandem mass spectrometry analysis. Normalized data were annotated using the IPA(R) knowledge platform. RESULTS There were 31 proteins significantly different in their level of abundance at 1 to 6 months, and 18 at 7 to 12 months compared to age matched controls. These proteins clustered into major functional networks. All of the biomarkers previously reported in clinical studies of ureteropelvic junction obstruction were observed with the notable exception of transforming growth factor-beta1. CONCLUSIONS These results confirm the presence of significant differences in the urinary proteome in unilateral ureteropelvic junction obstruction compared to age matched normal individuals. This study adds new information about levels of abundance of specific proteins and peptides in ureteropelvic junction obstruction, which may allow for better classification of disease subgroups and help to establish improved indications for the early selection of surgical candidates based on urinary protein biomarkers.

Pseudomonas aeruginosa ExoS ADP‐ribosyltransferase inhibits ERM phosphorylation

Pseudomonas aeruginosa causes life‐threatening infections in compromised and cystic fibrosis patients. Pathogenesis stems from a number of virulence factors, including four type III translocated cytotoxins: ExoS, ExoT, ExoY and ExoU. ExoS is a bifunctional toxin: the N terminus (amino acids 96–219) encodes a Rho GTPase Activating Protein (GAP) domain. The C terminus (amino acids 234–453) encodes a 14‐3‐3‐dependent ADP‐ribosyltransferase domain which transfers ADP‐ribose from NAD onto substrates such as the Ras GTPases and vimentin. Ezrin/radixin/moesin (ERM) proteins have recently been identified as high‐affinity substrates for ADP‐ribosylation by ExoS. Expression of ExoS in HeLa cells led to a loss of phosphorylation of ERM proteins that was dependent upon the expression of ADP‐ribosyltransferase activity. MALDI‐MS and site‐directed mutagenesis studies determined that ExoS ADP‐ribosylated moesin at three C‐terminal arginines (Arg553, Arg560 and Arg563), which cluster Thr558, the site of phosphorylation by protein kinase C and Rho kinase. ADP‐ribosylated‐moesin was a poor target for phosphorylation by protein kinase C and Rho kinase, which showed that ADP‐ribosylation directly inhibited ERM phosphorylation. Expression of dominant active‐moesin inhibited cell rounding elicited by ExoS, indicating that moesin is a physiological target in cultured cells. This is the first demonstration that a bacterial toxin inhibits the phosphorylation of a mammalian protein through ADP‐ribosylation. These data explain how the expression of the ADP‐ribosylation of ExoS modifies the actin cytoskeleton and indicate that ExoS possesses redundant enzymatic activities to depolymerize the actin cytoskeleton.

Nitration of PECAM-1 ITIM tyrosines abrogates phosphorylation and SHP-2 binding

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule with a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when phosphorylated, binds Src homology 2 domain-containing protein-tyrosine phosphatase (SHP-2). PECAM-1 is expressed at endothelial cell junctions where exposure to inflammatory intermediates may result in post-translational amino acid modifications that affect protein structure and function. Reactive nitrogen species (RNS), which are produced at sites of inflammation, nitrate tyrosine residues, and several proteins modified by tyrosine nitration have been found in diseased tissue. We show here that the RNS, peroxynitrite, induced nitration of both full-length cellular PECAM-1 and a purified recombinant PECAM-1 cytoplasmic domain. Mass spectrometric analysis of tryptic fragments revealed quantitative nitration of ITIM tyrosine 686. A synthetic peptide containing 3-nitrotyrosine at position 686 could not be phosphorylated nor bind SHP-2. These data suggest that ITIM tyrosine nitration may represent a mechanism for modulating phosphotyrosine-dependent signal transduction pathways.

Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging

Excess superoxide (O(2)(-)) and nitric oxide (NO) forms peroxynitrite (ONOO(-)) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO(-). Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O(2)(-)/ONOO(-) during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC-MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO(-). We also found that ONOO(-) directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO(-) formed oligomers. Resveratrol (RES), a scavenger of O(2)(-)/ONOO(-), reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O(2)(-) and ONOO(-) levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO(-) during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function.

The urine proteome for radiation biodosimetry: effect of total body vs. local kidney irradiation.

Victims of nuclear accidents or radiological terrorism are likely to receive varying doses of ionizing radiation inhomogeneously distributed over the body. Early biomarkers may be useful in determining organ-specific doses due to total body irradiation (TBI) or partial body irradiation. The authors used liquid chromatography and mass spectrometry to compare the effect of TBI and local kidney irradiation (LKI) on the rat urine proteome using a single 10-Gy dose of x-rays. Both TBI and LKI altered the urinary protein profile within 24 h with noticeable differences in gene ontology categories. Some proteins, including fetuin-B, tissue kallikrein, beta-glucuronidase, vitamin D-dependent calcium binding protein and chondroitin sulfate proteoglycan NG2, were detected only in the TBI group. Some other proteins, including major urinary protein-1, RNA binding protein 19, neuron navigator, Dapper homolog 3, WD repeat and FYVE domain containing protein 3, sorting nexin-8, ankycorbin and aquaporin were detected only in the LKI group. Protease inhibitors and kidney proteins were more abundant (fraction of total scans) in the LKI group. Urine protein (Up) and creatinine (Uc) (Up/Uc) ratios and urinary albumin abundance decreased in both TBI and LKI groups. Several markers of acute kidney injury were not detectable in either irradiated group. Present data indicate that abundance and number of proteins may follow opposite trends. These novel findings demonstrate intriguing differences between TBI and LKI, and suggest that urine proteome may be useful in determining organ-specific changes caused by partial body irradiation.

Identification of Proteins Interacting with GTP Cyclohydrolase I

GTP cyclohydrolase I (GCH-1) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, an essential cofactor for nitric oxide synthase and aromatic amino acid hydroxylase. To explore the interactome of GCH-1, we established a HEK 293 cell line stably expressing tetracycline-inducible FLAG-GCH-1. FLAG-GCH-1 and associated proteins were immunoprecipitated and analyzed by liquid chromatography/tandem mass spectrometry. Twenty-nine proteins, derived from different subcellular components such as cytosol, membranes, nucleus and mitochondria were identified to interact with GCH-1. Cell fractionation studies also showed that GCH-1 was present in the cytosol, membranes and nucleus. Gene ontology analysis revealed that GCH-1 interactome was involved in a variety of biological processes such as signal transduction, apoptosis, metabolism, transport and cell organization. To our knowledge, this study is the first to provide a comprehensive analysis of the GCH-1 interactome. Findings expand the number and diversity of proteins that are known to associate with GCH-1.

A Synthetic Polyvalent Ligand for α5β1 Integrin Activates Components of the Urothelial Carcinoma Cell Response to Bacillus Calmette-Guérin

PURPOSE Prior study has shown that bacillus Calmette-Guérin binds to and cross-links α5β1 integrins present on the surface of urothelial carcinoma cells. Antibody mediated cross-linking of α5β1 integrins can reproduce signal transduction, gene transactivation and phenotypic changes, similar to those observed in response to bacillus Calmette-Guérin. We evaluated the effect of a synthetic polyvalent ligand for α5β1 on these elements of the tumor response to bacillus Calmette-Guérin. MATERIALS AND METHODS The consensus α5β1 integrin binding tripeptide RGD was linked to a MAP8 backbone to result in an octavalent construct targeting α5β1 integrin. RGD-MAP8 was used to determine its effect on signaling pathway activation (nuclear factor-κB, NRF2 and CEBP), gene expression (p21, interleukin-6 and 8, CXCL1, CXCL2 and CCL20) and cytotoxicity (trypan blue exclusion and HMGB1 release) in human urothelial carcinoma cells. Results were compared to those of treatment with bacillus Calmette-Guérin or the missense peptide GRD-MAP8. RESULTS The RDG-MAP8 construct significantly increased nuclear factor-κB signaling and p21 expression relative to controls. Compared to bacillus Calmette-Guérin treatment, only p21 expression was comparable for cells treated with RGD-MAP8, averaging 70% of bacillus Calmette-Guérin induced expression. RGD-MAP8 failed to have a significant effect on CEBP or NRF2 activation, gene expression or cell viability. CONCLUSIONS Intracellular signaling, gene transactivation and phenotypic changes in response to RGD-MAP8 were qualitatively and quantitatively different than those observed in response to bacillus Calmette-Guérin. Results suggest that while α5β1 integrin cross-linking contributes to the bacillus Calmette-Guérin response, it alone is insufficient to duplicate the full spectrum of bacillus Calmette-Guérin induced changes in urothelial carcinoma cell biology.