A heat shock protein 90 binding domain in endothelial nitric-oxide synthase influences enzyme function.
Previous reports suggest heat shock protein 90 (hsp90) associates with endothelial nitric-oxide synthase (eNOS) to increase nitric oxide (*NO) generation. Ansamycin inhibition of chaperone-dependent activity increases eNOS generation of superoxide anion (O(2)(*)) 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 *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)(*) 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)(*) rather than *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 *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 *NO and O(2)(*) generation from eNOS and, therefore, vascular function.
Xu
Hao
H
Department of Surgery, Division of Pediatric Surgery, Medical College of Wisconsin, Children's Research Institute, Cardiovascular Research Center, Milwaukee, WI 53226, USA.
Shi
Yang
Y
Wang
Jingli
J
Jones
Deron
D
Weilrauch
Dorothee
D
Ying
Rong
R
Wakim
Basam
B
Pritchard
Kirkwood A
KA
D000818:Animals; D001011:Aorta / Q000378:metabolism; D002417:Cattle; D049109:Cell Proliferation; D004798:Enzymes / Q000737:chemistry; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000737:chemistry; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary
D004798:Enzymes; D018841:HSP90 Heat-Shock Proteins; D010455:Peptides; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester