Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 86
Текст из файла (страница 86)
The putative CyPA-binding proteins were pulled down by glutathione agarose beads, and separated on a SDS-PAGE gel. The proteins on thegel were visualized by silver staining. The stained protein band that correspondedto the position of putative CyPA-binding proteins was excised and subjected totryptic hydrolysis.
Tryptic peptides were spotted with a-cyano-4-hydroxycinnamicacid as matrix and analyzed using a MALDI-TOF mass spectrometer (PE Biosystems VOYAGER System 4187) [31]. Search of MS Fit with the resulting peptidemasses in the NCB database, yielded several protein candidates. One group of proteins identified were cytoskeleton proteins, including b-spectrin and a-actin. Another group included proteins related to redox state such as AOP-2 (antioxidantprotein 2, also termed 1–cysteine peroxiredoxin).
AOP-2 belongs to a large familyof bacterial and eukaryotic enzymes thought to be part of the cellular defenseagainst oxidative stress [32, 33]. Several membrane proteins were also identified.The analysis appeared to be technically sound since proteins homologous to AOP2 such as antioxidant protein 1 (AOP-1) and peroxiredoxin VI (Prx VI) were previously identified as CyPA binding proteins [34, 35].
Using the yeast two hybridsystem Jaschke et al. found that human CyPA binds to AOP-1 and stimulates itsenzymatic activity [34]. Lee at al. identified a 20-kD binding protein for Prx VI asCyPA, and demonstrated that CyPA enhances Prx VI peroxidase activity as an immediate electron donor [35].18.3.2Confirmation of 43-kD CyPA-binding Protein as a a-ActinThe SOXF-associated proteins identified by SDS-PAGE and MALDI-TOF MS wereverified by GST-CyPA affinity pull-down assay and by immunoprecipitation withanti-CyPA antibodies followed by immunoblotting with antibodies against the coprecipitating proteins. As shown in Fig. 18.3, a-actin was detected in the pellets ofGST-CyPA but not GST (negative control) precipitated from VSMC lysates.
Of interest, a-actin did not bind to the GST-CyPA R55A mutant, which lacks isomeraseactivity (Fig. 18.3, lane 3), suggesting that isomerase activity is required for the interaction of CyPA with a-actin. CyPA binding to a-actin was further confirmed byimmunoprecipitation from VSMC lysates. These results demonstrate that CyPAinteracts with actin in VSMC. Since cytoskeletal proteins such as a-actin and b-18.4 ConclusionsFig. 18.3 Specific interactions of CyPA withactin.
GST-CyPA proteins were preparedand used to co-precipitate CyPA bindingproteins from VSMC lysates. Shown is anexperiment in which VSMC lysates were incubated for 16 hours with GST-CyPA constructs, the beads were subjected to SDSPAGE, and actin was detected by westernblot. Wild type CyPA (W) was mutated to(R55A = R) to inhibit isomerase activity.G = GST.spectrin are thought to be involved in protein trafficking in the early secretorypathway [36], we speculate that these interactions might be a component of themechanisms by which CyPA is secreted in response to oxidative stress.
Furtherstudies are needed to clarify the functional importance of these interactions forCyPA-mediated cellular effects.18.4ConclusionsSeveral general concepts arise from our study of SOXF in vascular smooth musclecells. First, our approach was facilitated by analyzing a sub-proteome. We havestudied two subproteomes to date. In our initial studies we analyzed conditionedmedium from cells exposed to oxidative stress. Subsequently we studied proteinsthat co-precipitated with cyclophilin A from intracellular sources.
By dramaticallyreducing the number of potential proteins that could interact with these proteinswe simplified the proteomics analysis. Second, we designed our experiments tocontain several controls that facilitated identification of important proteins.
For example, we compared medium from cells exposed to oxidative stress to mediumfrom untreated cells. For cyclophilin A we had access to proteins that lacked critical domains required for protein–protein interactions and enzyme activity.
Thesebiological controls significantly improved our ability to identify biologically important interacting proteins. Third, it is possible to use cross-linking reagents to increase the stability of protein-protein interactions which can increase the likelihood of finding transient interactions that are biologically important. Fourth we31331418 Vascular SOXF and associated proteinsfound that using early passage cultured cells yielded information that was relevantto the in vivo situation.
These results indicate the utility of cultured cells and theimportance of correlating in vitro results with in vivo experiments. Finally, andperhaps most important, a proteomics approach was excellent for discovery basedresearch as we identified biologically important molecules that could not havebeen predicted based on current literature and knowledge.18.5AcknowledgementsWe thank Drs. Ruedi Abersold and Steve Gygi for their invaluable expertise inmass spectrometry. We also thank Drs. Andreas Pahl and Holger Bang for theirgenerous gift of antibody CyPA mAb7F1. We thank Drs.
J. Abe and A. Baas, andmembers of the Berk laboratory for their valuable assistance. This work was supported by grants from National Institutes of Health (HL44721 and HL49192 toB. C. Berk ).18.6References123456Alexander RW. Theodore Cooper Memorial Lecture. Hypertension and thepathogenesis of atherosclerosis. Oxidativestress and the mediation of arterial inflammatory response: a new perspective.Hypertension. 1995; 25:155–161.Abe J, Berk BC. Reactive Oxygen speciesas mediators of signal transduction incardiovascular disease. Trends Cardiovasc.Med.
1998; 8:59–64.Omar HA, Cherry PD, Mortelliti MP,Burke-Wolin T, Wolin MS. Inhibitionof coronary artery superoxide dismutaseattenuates endothelium-dependent andindependent nitrovasodilator relaxation.Circ. Res. 1991; 69:601–608.Baas AS, Berk BC. Differential activationof mitogen-activated protein kinases byH2O2 and O–2 in vascular smooth musclecells.
Circ Res. 1995; 77:29–36.Mohazzab KM, Kaminski PM, WolinMS. NADH oxidoreductase is a majorsource of superoxide anion in bovine coronary artery endothelium. Am. J. Physiol.1994;266:H2568–2572.Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK,78910Harrison DG. Angiotensin II-mediatedhypertension in the rat increases vascularsuperoxide production via membraneNADH/NADPH oxidase activation.
Contribution to alterations of vasomotortone. J. Clin. Invest. 1996; 97:1916–1923.Wung BS, Cheng JJ, Hsieh HJ, ShyyYJ, Wang DL. Cyclic strain-inducedmonocyte chemotactic protein-1 gene expression in endothelial cells involves reactive oxygen species activation of activator protein 1. Circ. Res. 1997; 81:1–7.Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H2O2 for platelet-derived growthfactor signal transduction. Science. 1995;270:296–299.Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW.
Angiotensin IIstimulates NADH and NADPH oxidaseactivation in cultured vascular smoothmuscle cells. Circ. Res. 1994;7 4:1141–1148.Halliwell B. Free radicals, reactive oxygen species and human disease: a criticalevaluation with special reference toatherosclerosis. Br. J. Exp. Pathol. 1989;70:737–757.18.6 References111213141516171819Rao GN, Berk BC. Active oxygen speciesstimulate vascular smooth muscle cellgrowth and proto-oncogene expression.Circ. Res. 1992; 70:593–599.Liao D-F, Baas AS, Daum G, Berk BC.Purification of a secreted protein factorinduced by reactive oxygen species (ROS)in vascular smooth muscle cells (VSMC).Circulation.
1997; 96:I-901.Meloche S, Seuwen K, Pages G, Pouysségur J. Biphasic and synergistic activation of p44mapk (ERK1) by growth factors:correlation between late phase activationand mitogenicity. Mol. Endocrinol. 1992;6:845–854.York RD, Yao H, Dillon T, Ellig CL,Eckert SP, McCleskey EW, Stork PJ.Rap1 mediates sustained MAP kinase activation induced by nerve growth factor.Nature. 1998; 392:622–626.Liu G, Espinosa E, Oemar BS, LuscherTF. Bimodal effects of angiotensin II onmigration of human and rat smoothmuscle cells. Direct stimulation and indirect inhibition via transforming growthfactor-beta 1.
Arterioscler. Thromb. Vasc.Biol. 1997; 17:1251–1257.Sugo S, Minamino N, Shoji H, Kangawa K, Kitamura K, Eto T, Matsuo H.Production and secretion of adrenomedullin from vascular smooth musclecells: augmented production by tumornecrosis factor-alpha. Biochem. Biophys.Res. Commun. 1994; 203:719–726.Itoh H, Mukoyama M, Pratt RE, Gibbons GH, Dzau VJ. Multiple autocrinegrowth factors modulate vascular smoothmuscle cell growth response to angiotensin II. J. Clin. Invest. 1993; 91:2268–2274.Battegay EJ, Raines EW, Seifert RA,Bowen-Pope DF, Ross R. TGF-beta induces bimodal proliferation of connectivetissue cells via complex control of anautocrine PDGF loop. Cell. 1990; 63:515–524.Nakamura Y, Morishita R, Higaki J,Kida I, Aoki M, Moriguchi A, YamadaK, Hayashi S, Yo Y, Matsumoto K, et al.Expression of local hepatocyte growthfactor system in vascular tissues.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
