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ABSTRACT: Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, Gα and Gβγ in the cytosol. In this study, we show novel nuclear functions of Gβγ through demonstrating interaction of Gβ(2) with integral components of chromatin and effects of Gβ(2) depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented Gβ(2) levels in the nucleus and Gβ(2) interacted with specific nucleosome core histones and transcriptional modulators. Depletion of Gβ(2) repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. Gβ(2) interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the Gβ(2)-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of Gβγ with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.
PLoS ONE 01/2013; 8(1):e52689. · 4.09 Impact Factor
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ABSTRACT: Abstract Aim: Loss of superoxide dismutase (SOD) activity is a defining biochemical feature of asthma. However, mechanisms for the reduced activity are unknown. We hypothesized that loss of asthmatic SOD activity is due to greater susceptibility to oxidative inactivation. Result: Activity assays of blood samples from asthmatics and healthy controls revealed impaired dismutase activity of copper-zinc SOD (CuZnSOD) in asthma. CuZnSOD purified from erythrocytes or airway epithelial cells from asthmatic was highly susceptible to oxidative inactivation. Proteomic analyses identified that inactivation was related to oxidation of cysteine 146 (C146), which is usually disulfide bonded to C57. The susceptibility of cysteines pointed to an alteration in protein structure, which is likely related to the loss of disulfide bond. We speculated that a shift to greater intracellular reducing potential might account for the change. Strikingly, measures of reduced and oxidized glutathione confirmed greater reducing intracellular state in asthma, compared with controls. Similarly, greater free thiol in CuZnSOD was confirmed by ∼2-fold greater N-ethylmaleimide binding to C146 in asthma as compared with controls. Innovation: Greater reducing potential under a chronic inflammatory state of asthma, thus, leads to susceptibility of CuZnSOD to oxidative inactivation due to cleavage of C57-C146 disulfide bond and exposure of usually unavailable cysteines. Conclusion: Vulnerability of CuZnSOD influenced by redox likely amplifies injury and inflammation during acute asthma attacks when reactive oxygen species are explosively generated. Overall, this study identifies a new paradigm for understanding the chemical basis of inflammation, in which redox regulation of thiol availability dictates protein susceptibility to environmental and endogenously generated reactive species. Antioxid. Redox Signal. 00, 000-000.
Antioxidants & Redox Signaling 08/2012; · 8.20 Impact Factor
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ABSTRACT: Gamma-carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively-charged residue. Gla is generated by a single enzyme, the gamma-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla-peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins.
Journal of Proteome Research 04/2012; · 5.11 Impact Factor
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ABSTRACT: Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and β are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIβ. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIβ activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.
Proteomics 03/2011; 11(5):829-42. · 4.43 Impact Factor
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ABSTRACT: Chronic activation of angiotensin II (AngII) type 1 receptor (AT(1)R), a prototypical G protein-coupled receptor (GPCR) induces gene regulatory stress which is responsible for phenotypic modulation of target cells. The AT(1)R-selective drugs reverse the gene regulatory stress in various cardiovascular diseases. However, the molecular mechanisms are not clear. We speculate that activation states of AT(1)R modify the composition of histone isoforms and post-translational modifications (PTM), thereby alter the structure-function dynamics of chromatin. We combined total histone isolation, FPLC separation, and mass spectrometry techniques to analyze histone H2A in HEK293 cells with and without AT(1)R activation. We have identified eight isoforms: H2AA, H2AG, H2AM, H2AO, H2AQ, Q96QV6, H2AC and H2AL. The isoforms, H2AA, H2AC and H2AQ were methylated and H2AC was phosphorylated. The relative abundance of specific H2A isoforms and PTMs were further analyzed in relationship to the activation states of AT(1)R by immunochemical studies. Within 2 hr, the isoforms, H2AA/O exchanged with H2AM. The monomethylated H2AC increased rapidly and the phosphorylated H2AC decreased, thus suggesting that enhanced H2AC methylation is coupled to Ser1p dephosphorylation. We show that H2A125Kme1 promotes interaction with the heterochromatin associated protein, HP1α. These specific changes in H2A are reversed by treatment with the AT(1)R specific inhibitor losartan. Our analysis provides a first step towards an awareness of histone code regulation by GPCRs.
PLoS ONE 01/2010; 5(9):e12552. · 4.09 Impact Factor
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ABSTRACT: Hyperhomocysteinemia has long been associated with atherosclerosis and thrombosis and is an independent risk factor for cardiovascular disease. Its causes include both genetic and environmental factors. Although homocysteine is produced in every cell as an intermediate of the methionine cycle, the liver contributes the major portion found in circulation, and fatty liver is a common finding in homocystinuric patients. To understand the spectrum of proteins and associated pathways affected by hyperhomocysteinemia, we analyzed the mouse liver proteome of gene-induced (cystathionine beta-synthase (CBS)) and diet-induced (high methionine) hyperhomocysteinemic mice using two-dimensional difference gel electrophoresis and Ingenuity Pathway Analysis. Nine proteins were identified whose expression was significantly changed by 2-fold (p < or = 0.05) as a result of genotype, 27 proteins were changed as a result of diet, and 14 proteins were changed in response to genotype and diet. Importantly, three enzymes of the methionine cycle were up-regulated. S-Adenosylhomocysteine hydrolase increased in response to genotype and/or diet, whereas glycine N-methyltransferase and betaine-homocysteine methyltransferase only increased in response to diet. The antioxidant proteins peroxiredoxins 1 and 2 increased in wild-type mice fed the high methionine diet but not in the CBS mutants, suggesting a dysregulation in the antioxidant capacity of those animals. Furthermore, thioredoxin 1 decreased in both wild-type and CBS mutants on the diet but not in the mutants fed a control diet. Several urea cycle proteins increased in both diet groups; however, arginase 1 decreased in the CBS(+/-) mice fed the control diet. Pathway analysis identified the retinoid X receptor signaling pathway as the top ranked network associated with the CBS(+/-) genotype, whereas xenobiotic metabolism and the NRF2-mediated oxidative stress response were associated with the high methionine diet. Our results show that hyperhomocysteinemia, whether caused by a genetic mutation or diet, alters the abundance of several liver proteins involved in homocysteine/methionine metabolism, the urea cycle, and antioxidant defense.
Molecular & Cellular Proteomics 12/2009; 9(3):471-85. · 7.40 Impact Factor
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ABSTRACT: Glutamyl-prolyl tRNA synthetase (EPRS) is a component of the heterotetrameric gamma-interferon-activated inhibitor of translation (GAIT) complex that binds 3'UTR GAIT elements in multiple interferon-gamma (IFN-gamma)-inducible mRNAs and suppresses their translation. Here, we elucidate the specific EPRS phosphorylation events that regulate GAIT-mediated gene silencing. IFN-gamma induces sequential phosphorylation of Ser(886) and Ser(999) in the noncatalytic linker connecting the synthetase cores. Phosphorylation of both sites is essential for EPRS release from the parent tRNA multisynthetase complex. Ser(886) phosphorylation is required for the interaction of NSAP1, which blocks EPRS binding to target mRNAs. The same phosphorylation event induces subsequent binding of ribosomal protein L13a and GAPDH and restores mRNA binding. Finally, Ser(999) phosphorylation directs the formation of a functional GAIT complex that binds initiation factor eIF4G and represses translation. Thus, two-site phosphorylation provides structural and functional pliability to EPRS and choreographs the repertoire of activities that regulates inflammatory gene expression.
Molecular cell 08/2009; 35(2):164-80. · 14.61 Impact Factor
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ABSTRACT: The recent introduction of electrospray ionization techniques that are suitable for peptides and whole proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for proteins. The advantages gained by these approaches over traditional Edman Degradation sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the protein. This powerful technique has been instrumental for the study of proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of protein expression in cystic fibrosis as an example.
Methods in molecular biology (Clifton, N.J.) 02/2009; 544:325-41.
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ABSTRACT: Phosphorylation of ribosomal protein L13a is essential for translational repression of inflammatory genes by the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex. Here we show that IFN-gamma activates a kinase cascade in which death-associated protein kinase-1 (DAPK) activates zipper-interacting protein kinase (ZIPK), culminating in L13a phosphorylation on Ser(77), L13a release from the ribosome, and translational silencing of GAIT element-bearing target mRNAs. Remarkably, both kinase mRNAs contain functional 3'UTR GAIT elements, and thus the same inhibitory pathway activated by the kinases is co-opted to suppress their expression. Inhibition of DAPK and ZIPK facilitates cell restoration to the basal state and allows renewed induction of GAIT target transcripts by repeated stimulation. Thus, the DAPK-ZIPK-L13a axis forms a unique regulatory module that first represses, then repermits inflammatory gene expression. We propose that the module presents an important checkpoint in the macrophage "resolution of inflammation" program, and that pathway defects may contribute to chronic inflammatory disorders.
Molecular cell 12/2008; 32(3):371-82. · 14.61 Impact Factor
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ABSTRACT: We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.
Nucleic Acids Research 12/2008; 37(2):382-92. · 8.03 Impact Factor
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ABSTRACT: A proteomic approach was taken to study how fish respond to changes in calorie availability, with the longer-term goal of understanding the evolution of lipid metabolism in vertebrates. Zebrafish (Danio rerio) were fed either high (3 rations/day) or low (1 ration/7 days) calorie diets for 5 weeks and liver proteins extracted for proteomic analyses. Proteins were separated on two-dimensional electrophoresis gels and homologous spots compared between treatments to determine which proteins were up-regulated with high-calorie diet. Fifty-five spots were excised from the gel and analyzed via LC-ESI MS/MS, which resulted in the identification of 69 unique proteins (via multiple peptides). Twenty-nine of these proteins were differentially expressed between treatments. Differentially expressed proteins were mapped to Gene Ontology (GO) terms, and these terms compared to the entire zebrafish GO annotation set by Fisher's exact test. The most significant GO terms associated with high-calorie diet are related to a decrease in oxygen-binding activity in the high-calorie treatment. This response is consistent with a well-characterized response in obese humans, indicating there may be a link between lipid storage and hypoxia sensitivity in vertebrates.
Comparative Biochemistry and Physiology Part D Genomics and Proteomics 12/2008; 3(4):275-82. · 1.72 Impact Factor
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ABSTRACT: Apolipoprotein A-I (apoAI) acts as an ABCA1-dependent acceptor of cellular phospholipids and cholesterol during the biogenesis of HDL, but this activity is susceptible to oxidative inactivation by myeloperoxidase. We tried to determine which residues mediated this inactivation and create an oxidant-resistant apoAI variant.
Mass spectrometry detected the presence of tryptophan, methionine, tyrosine, and lysine oxidation in apoAI recovered from human atheroma. We investigated the role of these residues in the myeloperoxidase-mediated loss of apoAI activity. Site-directed mutagenesis and chemical modification were used to create variants of apoAI which were tested for ABCA1-dependent cholesterol acceptor activity and oxidative inactivation. We previously reported that tyrosine modification is not required for myeloperoxidase-induced loss of apoAI function. Lysine methylation did not alter the sensitivity of apoAI to myeloperoxidase, whereas site-specific substitution of apoAI methionine to valine increased the sensitivity of apoAI to myeloperoxidase. ApoAI tryptophan residues were identified as essential in apoAI function and oxidant sensitivity as substitution of all four apoAI tryptophan residues to leucine led to loss of function, but the conservative substitution to phenylalanine retained full function and was resistant to oxidative inactivation.
Tryptophan modification of apoAI is primarily responsible for the myeloperoxidase-mediated loss of the cholesterol acceptor activity of apoAI.
Arteriosclerosis Thrombosis and Vascular Biology 09/2008; 28(11):2063-70. · 6.37 Impact Factor
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ABSTRACT: Lipid transfer inhibitor protein (LTIP) is a physiologic regulator of cholesteryl ester transfer protein (CETP) function. We previously reported that LTIP activity is localized to LDL, consistent with its greater inhibitory activity on this lipoprotein. With a recently described immunoassay for LTIP, we investigated whether LTIP mass is similarly distributed. Plasma fractionated by gel filtration chromatography revealed two LTIP protein peaks, one coeluting with LDL, and another of approximately 470 kDa. The 470 kDa LTIP complex had a density of 1.134 g/ml, indicating approximately 50% lipid content, and contained apolipoprotein A-I. By mass spectrometry, partially purified 470 kDa LTIP also contains apolipoproteins C-II, D, E, J, and paraoxonase 1. Unlike LDL-associated LTIP, the 470 kDa LTIP complex does not inhibit CETP activity. In normolipidemic subjects, approximately 25% of LTIP is in the LDL-associated, active form. In hypercholesterolemia,this increases to 50%, suggesting that lipoprotein composition may influence the status of LTIP activity. Incubation (37 degrees C) of normolipidemic plasma increased active, LDL-associated LTIP up to 3-fold at the expense of the inactive pool. Paraoxon inhibited this shift by 50%. Overall, these studies show that LTIP activity is controlled by its reversible incorporation into an inactive complex. This may provide for short-term fine-tuning of lipoprotein remodeling mediated by CETP.
The Journal of Lipid Research 08/2008; 49(7):1529-37. · 5.56 Impact Factor
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ABSTRACT: Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1beta mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by > or =2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses.
PLoS ONE 01/2008; 3(10):e3367. · 4.09 Impact Factor
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ABSTRACT: The cyclin E/Cdk2 complex plays an essential role in the G(1)/S cell cycle transition and DNA replication. Earlier we showed that in hematopoietic tumor cells, caspase-mediated cleavage of cyclin E generates p18-cyclin E, which is unable to interact with Cdk2 and therefore plays a role independent of the cell cycle. The expression of a cleavage-resistant cyclin E mutant greatly diminishes apoptosis, indicating the critical role of cyclin E cleavage. p18-cyclin E expression can induce apoptosis or sensitization to apoptotic stimuli in many cell types. Here we identify Ku70 as a specific p18-cyclin E-interacting partner. In hematopoietic tumor cell lines, the association of p18-cyclin E with Ku70 induces the dissociation of Bax from Ku70, followed by Bax activation. This mechanism of Bax activation leads to the amplification of the apoptosis signal in all tumor cell lines examined. N-terminal Ku70 deletion mutants are unable to bind to p18-cyclin E to regulate its apoptotic effect. p18-cyclin E-mediated amplification of apoptosis is dependent on Bax and Ku70 being greatly diminished in Ku70(-/-) and Bax(-/-) mouse embryo fibroblasts and in hematopoietic cells where Bax knockdown was achieved by short interfering RNA. The p18-cyclin E/Ku70 and Bax/Ku70 interactions provide a balance between apoptosis and the survival of cells exposed to genotoxic stress.
Molecular and Cellular Biology 06/2007; 27(9):3511-20. · 5.53 Impact Factor
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ABSTRACT: We and others have shown that foam cell formation initiated by exposing macrophages to oxidized low density lipoprotein (oxLDL) triggers the differential expression of a number of proteins. Specifically, our experiments have identified peroxiredoxin I (Prx I) as one of these up-regulated proteins. The peroxiredoxins, a family of peroxidases initially described for their antioxidant capability, have generated recent interest for their potential to regulate signaling pathways. Those studies, however, have not examined peroxiredoxin for a potential dual functionality as both cytoprotective antioxidant and signal modulator in a single, oxidant-stressed system. In this report, we examine the up-regulation of Prx I in macrophages in response to oxLDL exposure and its ability to function as both antioxidant enzyme and regulator of p38 MAPK activation. As an antioxidant, induction of Prx I expression led to improved cell survival following treatment with oxLDL or tert-butyl hydroperoxide. The improved survival coincided with a decrease in measurable reactive oxygen species (ROS), and both the increased survival and reduced ROS were reversed by Prx I small interfering RNA transfection. Additionally, our data show that activation of p38 MAPK in oxLDL-treated macrophages was dependent on the up-regulation of Prx I. Reduction of Prx I expression by small interfering RNA transfection resulted in a significant decrease in p38 MAPK activation, whereas the up-regulation of Prx I expression with either oxLDL or ethoxyquin led to increased p38 MAPK activation. These results are consistent with multiple roles for Prx I in macrophage-derived foam cells that include functionality as both an antioxidant and a regulator of oxidant-sensitive signal transduction.
Journal of Biological Chemistry 10/2006; 281(38):27991-8001. · 4.77 Impact Factor
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Sudakshina Ghosh,
Allison J Janocha,
Mark A Aronica,
Shadi Swaidani,
Suzy A A Comhair,
Weiling Xu,
Lemin Zheng,
Suma Kaveti, Michael Kinter,
Stanley L Hazen,
Serpil C Erzurum
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ABSTRACT: Reactive oxygen species and reactive nitrogen species produced by epithelial and inflammatory cells are key mediators of the chronic airway inflammation of asthma. Detection of 3-nitrotyrosine in the asthmatic lung confirms the presence of increased reactive oxygen and nitrogen species, but the lack of identification of modified proteins has hindered an understanding of the potential mechanistic contributions of nitration/oxidation to airway inflammation. In this study, we applied a proteomic approach, using nitrotyrosine as a marker, to evaluate the oxidation of proteins in the allergen-induced murine model of asthma. Over 30 different proteins were targets of nitration following allergen challenge, including the antioxidant enzyme catalase. Oxidative modification and loss of catalase enzyme function were seen in this model. Subsequent investigation of human bronchoalveolar lavage fluid revealed that catalase activity was reduced in asthma by up to 50% relative to healthy controls. Analysis of catalase isolated from asthmatic airway epithelial cells revealed increased amounts of several protein oxidation markers, including chloro- and nitrotyrosine, linking oxidative modification to the reduced activity in vivo. Parallel in vitro studies using reactive chlorinating species revealed that catalase inactivation is accompanied by the oxidation of a specific cysteine (Cys(377)). Taken together, these studies provide evidence of multiple ongoing and profound oxidative reactions in asthmatic airways, with one early downstream consequence being catalase inactivation. Loss of catalase activity likely amplifies oxidative stress, contributing to the chronic inflammatory state of the asthmatic airway.
The Journal of Immunology 06/2006; 176(9):5587-97. · 5.79 Impact Factor
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ABSTRACT: Apolipoprotein A-I (apoAI), the major protein of high density lipoprotein, plays an important role in reverse cholesterol transport via its activity as an ABCA1-dependent acceptor of cellular cholesterol. We reported recently that myeloperoxidase (MPO) modification of apoAI inhibits its ABCA1-dependent cholesterol acceptor activity (Zheng, L., Nukuna, B., Brennan, M. L., Sun, M., Goormastic, M., Settle, M., Schmitt, D., Fu, X., Thomson, L., Fox, P. L., Ischiropoulos, H., Smith, J. D., Kinter, M., and Hazen, S. L. (2004) J. Clin. Invest. 114, 529-541). We also reported that MPO-mediated chlorination preferentially modifies two of the seven tyrosines in apoAI, and loss of parent peptides containing these residues dose-dependently correlates with loss in ABCA1-mediated cholesterol acceptor activity (Zheng, L., Settle, M., Brubaker, G., Schmitt, D., Hazen, S. L., Smith, J. D., and Kinter, M. (2005) J. Biol. Chem. 280, 38-47). To determine whether oxidative modification of apoA-I tyrosine residues was responsible for the MPO-mediated inactivation of cholesterol acceptor activity, we made recombinant apoAI with site-specific substitutions of all seven tyrosine residues to phenylalanine. ApoAI and the tyrosine-free apoAI were equally susceptible to dose-dependent MPO-mediated loss of ABCA1-dependent cholesterol acceptor activity, as well as lipid binding activity. MPO modification altered the migration of apoAI on SDS gels and decreased its alpha-helix content. MPO-induced modification also targeted apoAI tryptophan and lysine residues. Specifically, we detected apoAI tryptophan oxidation to mono- and dihydroxytryptophan and apoAI lysine modification to chlorolysine and 2-aminoadipic acid. Thus, tyrosine modification of apoAI is not required for its MPO-mediated inhibition of cholesterol acceptor activity.
Journal of Biological Chemistry 11/2005; 280(40):33775-84. · 4.77 Impact Factor
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ABSTRACT: The uptake of oxidized low density lipoprotein (oxLDL) by macrophages leads to foam cell formation and fatty streaks, which represent early sites of potential atheroma development. We developed a cell culture model of chronic oxLDL exposure to determine whether hallmark parameters of oxLDL uptake and cytotoxicity are altered during foam cell formation and to determine changes in protein and mRNA expression that distinguish acute and chronic oxLDL exposure. Although the extent of oxLDL uptake did not change, a resistance to oxLDL-induced cytotoxicity was observed in the chronically exposed cells. Macrophages that have been chronically exposed to oxLDL required a 40% higher concentration of oxLDL to achieve 50% survival in a 48-h treatment relative to macrophages subjected to a single oxLDL exposure. A main feature of the differentially expressed proteome was a series of significantly overexpressed antioxidant and antioxidant-related proteins in the oxLDL-exposed cells. A large proportion of these proteins (45%) was overexpressed in the chronically exposed cells prior to the oxLDL treatment, indicative of the unique phenotype produced by the chronic treatment. Analysis of the transcriptome also revealed a broad increase in the expression of antioxidant and antioxidant-related proteins. In addition, the transcriptome experiments found an increased inflammatory response under conditions of both acute and chronic oxLDL exposure. Overall the combined functional, proteomic, and transcriptomic experiments show that macrophages respond to oxLDL by developing an oxidative stress resistance that increases and stabilizes with chronic exposure. Furthermore this protective response and the increased foam cell survival that it supports amplifies their proatherogenic role by promoting a continued inflammatory state.
Molecular & Cellular Proteomics 11/2005; 4(10):1522-40. · 7.40 Impact Factor
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ABSTRACT: Mammalian transient receptor potential canonical (TRPC) genes encode a family of nonselective cation channels that are activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. In Drosophila photoreceptor cells, TRP channels are found in large, multimolecular signaling complexes in association with the PDZ-containing scaffolding protein, INAD. A similar mammalian TRPC "signalplex" has been proposed, but has yet to be defined. In the present study, affinity-purified polyclonal antibodies against TRPC5 and TRPC6 were used to immunoprecipitate signalplex components from rat brain lysates. Immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and sequenced by mass spectrometry. Proteins identified in the immunoprecipitates included cytoskeletal proteins spectrin, myosin, actin, drebrin, tubulin, and neurabin; endocytic vesicle-associated proteins clathrin, dynamin and AP-2; and the plasmalemmal Na(+)/K(+)-ATPase (NKA) pump. Several of these interactions were confirmed by reciprocal immunoprecipitation followed by Western blot analysis. In lysates from rat kidney, TRPC6, but not TRPC3, was found to coimmunoprecipitate with the NKA pump. Likewise, TRPC6, stably expressed in human embryonic kidney (HEK) cells, coimmunoprecipitated with endogenous NKA and colocalized with the pump to the plasmalemma when examined by immunofluorescence microscopy. Cell surface biotinylation experiments in intact HEK cells, confirmed that both the Na(+) pump and TRPC6 were present in the surface membrane and appeared to interact. Lastly, TRPC6 coimmunoprecipitated with the NKA pump when the proteins were coexpressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. These observations suggest that TRPC6 and the Na(+) pump are part of a functional complex that may be involved in ion transport and homeostasis in both the brain and kidney.
Pflügers Archiv - European Journal of Physiology 11/2005; 451(1):87-98. · 4.46 Impact Factor
