Abstract 222: Oxidative Modification of Extracellular Matrix Proteins Alters the Hsp90 Interactome in Human Umbilical Vein Endothelial Cells
Lipid peroxidation increases the production of thiol-reactive α, β-unsaturated aldehydes such as 4-hydroxy-2-nonenal (4-HNE). Exposure of endothelial cells (EC) to 4-HNE impairs cell function. Interestingly, heat shock protein 90 (hsp90), which interacts with a multitude of client proteins, is upregulated under conditions that promotes oxidative stress. However, to our knowledge no one has examined the effects of oxidative modification of matrix proteins on cell function. Here we hypothesize that oxidative modification of matrix perturbs EC function with respect to the hsp90 interactome. To test this hypothesis, we treated gelatin-coated culture dishes with 10 μM Linoleic Acid (LA) under conditions that promoted oxidation. Dishes were washed free of excess LA and seeded with human umbilical vein EC. Native dishes without LA treatment were used as control. The hsp90 client proteins were identified by immunoprecipitation of hsp90 and nanoLC-MS/MS analysis. Oxidative modification of gelatin caused a marked shift in the hsp90-interactome (n = 4). EC maintained on LA-treated gelatin expressed more hsp90 (1.5 X) than controls. Only ~33% of the proteins were present in both groups at similar levels. Hsp90 client proteins in EC maintained on LA-treated gelatin also had increased levels of DNA repair protein BRCA2, protein kinase Tec, protein phosphatase RPTP-rho, ubiquitin ligase Pellino 3, chaperone protein nucleophosmin, retinoic acid receptor, 2 histone deacetylases (e.g. Sin3b), 4 transcription factors (e.g. Prox1), 5 cytoskeleton protein and stress fibers (e.g. tubulin), 9 ribosomal proteins. GO analysis revealed that most of these proteins are involved in stress responses such as protein synthesis, degradation, stress fiber formation, transcription repression, DNA repair and senescence. These data show that EC enter into a stress-response status with marked alterations in the hsp90 interactome upon sensing oxidative modification of extracellular matrix proteins. Such changes may play a role in impaired anti-atherogenic responses in the vessel wall.