Abstract 1154: E310, 314, 318 and 323 in eNOS Play a Critical Role in Maintaining eNOS-hsp90 Interactions and Coupled eNOS Activity
Endothelial nitric oxide synthase (eNOS) generates nitric oxide (NO) in blood vessels to promote vasodilation. Typically, eNOS activity utilizes NADPH, O2 and L-arginine (L-Arg) to synthesize NO. However, eNOS activity becomes uncoupled from NO production whenever the activated O2 is released from the Fe-HEME of eNOS to generate superoxide ion (O2·−) rather than reacting with L-Arg to produce NO. Previously we showed using selective hsp90 inhibitors that disrupting hsp90 association with eNOS uncouples eNOS. Recently we observed that a decoy peptide based on the amino acid sequence in eNOS (a.a. #310 – 323, bovine) disrupts hsp90-eNOS interactions, while a control peptide that substitutes all four glutamic acids (E) with alanines (A) loses this ability. On the basis of this observation we hypothesized that the four E in eNOS, E310, E314, E318 and E323 play a critical role in promoting hsp90 association with eNOS and accordingly, coupled eNOS activity. To test this hypothesis, we used site directed mutagenesis to mutate eNOS cDNA into eNOS-4A, which generates the mutant eNOS protein that contains the four substitutions E310A, E314A, E318A and E323A when expressed. eNOS-4A and wild type (wt) eNOS pcDNA3 plasmids were transfected into HEK293 cells and stable cell lines generated with G418. Characterization of these two enzymes reveals that eNOS-4A consumes NADPH at the same rate as wt eNOS. Pulldown assays reveal that although eNOS-4A bound the same level of CaM as wt eNOS in A23187 stimulated HEK293 cells, eNOS-4A lost the ability to interact with hsp90. A23187-stimulated wt eNOS HEK293 cells generate high levels of NO and low levels of O2 ·− but, A23187-stimulated eNOS-4A HEK293 generate only O2 ·− by an L-NAME-inhibitable mechanism. These data clearly show that substituting A for E at 310, 314, 318 and 323 in eNOS disrupts eNOS-hsp90 interactions and uncouples eNOS activity from NO generation. As these substitutions do not interfere with O2·− generation by eNOS-4A, observations here provide NADPH consumption or CaM binding or definitive proof that E310, E314, E318 and E323 play a critical role in facilitating/directing hsp90 association with eNOS to increase coupled activity and NO generation.