Project description:ChIP-Sequencing using antibody to eNOS in cells from a primary prostate cancer with poor outcome and metastatic LNCaP cells, in basal condition and after E2 treatment
Project description:Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of L-arginine and molecular oxygen into L-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell-derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist stimulated NO synthesis and decreased phosphorylation of eNOS at Ser1177, a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser1177 in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells.
Project description:Extensive reports in the past decade have shown the importance of eNOS expression in atherogenesis. However, both the eNOS knockout and overexpression mouse models exhibit paradoxical results of increased atherosclerotic lesions. Our genetic approach herein utilizes endogenous point-mutations at the eNOS S1176 site to effectively manipulate eNOS activity rather than total eNOS protein expression levels. Our efforts definitively show that eNOS S1176 phosphorylation is sufficient to mitigate atherosclerotic lesion progression.
