A heat shock protein 90 binding domain in endothelial nitric-oxide synthase influences enzyme function

作者：Xu Hao; Shi Yang^*; Wang Jingli; Jones Deron; Weilrauch Dorothee; Ying Rong; Wakim Basam; Pritchard Kirkwood A Jr

来源：Journal of Biological Chemistry, 2007, 282(52): 37567-37574.

DOI：10.1074/jbc.M706464200

Previous reports suggest heat shock protein 90 (hsp90) associates with endothelial nitric-oxide synthase (eNOS) to increase nitric oxide (center dot NO) generation. Ansamycin inhibition of chaperone-dependent activity increases eNOS generation of superoxide anion (O-2 radical anion) upon enzyme activation. In the present study we identify where hsp90 binds to eNOS using overlapping decoy peptides based on the amino acid (aa) sequence of eNOS (291-420). B1, B2, and B3 peptides inhibited hsp90 association with eNOS in cell lysates from proliferating bovine aortic endothelial cells. B2 (aa 301-320), common to both B1 and B3, decreased stimulated center dot NO production and hsp90 association in bovine aortic endothelial cells. The B2/ B3 peptide was redesigned to TSB2 that includes a TAT protein transduction domain and shortened to 14 aa. TSB2 impaired vasodilation of isolated facialis arteries in vitro and in vivo and increased eNOS-dependent O-2 radical anion radical anion generation in native endothelial cells on mouse aortas, whereas a control peptide, TSB(Ctr), which has the four glutamic acids in TSB2 substituted with alanine, showed no such effects. Site-directed mutagenesis of eNOS at 310, 314, 318, and 323 Glu to Ala yields an eNOS mutant that exhibited reduced hsp90 association and generated O-2 radical anion rather than center dot NO upon activation. Together, these data demonstrate that hsp90 associates with eNOS at aa 310 -323. Moreover, a decoy peptide based on this sequence is sufficient to displace hsp90 from eNOS and uncouple eNOS activity from center dot NO generation. Thus, Glu-310, Glu-314, Glu-318, and Glu-323 in eNOS, although each does not do much by itself, synergistically they increase " cooperativity" in the association step that is critical for maintaining hsp90-eNOS interactions and promoting coupled eNOS activity. Such chaperone-dependent signaling may play an important role in modulating the balance of center dot NOandO(2) radical anion generation from eNOS and, therefore, vascular function.

• 出版日期2007-12-28