Abstract 17910: A Novel Mechanism for Regulating Proteasome Function in Vascular Endothelial Cells.
The proteasome plays important roles in the maintenance of proteomic homeostasis through destruction of abnormal, aged and oxidized proteins. Proteasomal malfunction has been implicated in many age-related diseases including cardiovascular diseases, such as heart failure. We recently reported that the proteolytic activity of the proteasome is under metabolic control, by O-GlcNAcylation and PKA phosphorylation. O-GlcNAcylation, which is elevated by hyperglycemia and implicated in diabetes and its complications, is the first endogenous proteasome inhibitor ever described. In this study, we report a novel mechanism modulating proteasomal function, namely through the reduction of O-GlcNAcylation by heat shock protein 90 (hsp90) inhibitors. Primary cultures of bovine aortic endothelial (BAE) cells and human lung microvascular endothelial (HLMVE) cells were exposed to hsp90 inhibitors radicicol (1.7μM) or 17-AAG (2.7μM) for 16 hr. Hsp90 inhibition resulted in proteasome-dependent degradation of the enzyme O-GlcNAc transferase (OGT), and consequently, in the reduction of O-GlcNAcylation, in both BAE and HLMVE cells. More importantly, hsp90 inhibition attenuated the increase in global O-GlcNAcylation in cells exposed to high glucose (30mM), glucosamine (5mM), or to the O-GlcNAcase inhibitor, PUGNAc (100μM). Treating the BAE and HLMVE cells with proteasome inhibitors MG132 (10μM), epoxomycin (1μM) or Bortezomib (1μM), in the absence or presence of hsp90 inhibitors revealed that hsp90 inhibition dramatically increased protein ubiquitination. Concomitantly, hsp90 inhibition accelerated the disposal of the ubiquitinated proteins, and prevented their cellular accumulation. Hsp90 inhibition stimulated the chymotrypsin-like peptidase activity in cell lysate as demonstrated by the increase in the cleavage of the fluorogenic peptide substrate suc-LLVY-AMC. These results strongly suggest that hsp90 inhibitors may be useful in improving protein quality control and cellular function by promoting protein ubiquitination and degradation in the cell. This would be the first pharmacological approach to stimulate the function of the ubiquitin-proteasome system and might have important therapeutical applications in age-related diseases.
- © 2010 by American Heart Association, Inc.