A Heat Shock Protein 90 Binding Domain in Endothelial Nitric-oxide Synthase Influences Enzyme Function

Hao Xu,Yang Shi,Jingli Wang,Deron Jones,Dorothee Weilrauch,Rong Ying,Basam Wakim,Kirkwood A Pritchard

doi:10.1074/jbc.m706464200

Abstract

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.

Highlights

An earlier report by Sessa and co-workers [5] showed, using a yeast two-hybrid system, that hsp90 interacted with endothelial nitric-oxide synthase (eNOS) at amino acid 300–400 of eNOS
Effects of B2 on Basal and Stimulated eNOS-dependent 1⁄7NO Generation and hsp90 Association with eNOS—Because B2 contained aa sequences that were common to B1 and B3, we examined the effects of B2 on stimulated endothelial cell (EC) 1⁄7NO generation using the protein transduction domain (PTD) peptide PEP1 to facilitate B2 uptake
Immunoprecipitates of eNOS from these test groups reveal the B2:PEP1 mixture markedly decreased hsp90 association under basal conditions and when the cultures were stimulated with A23187 (Fig. 2B, p Ͻ 0.05, n ϭ 3)

Summary

EXPERIMENTAL PROCEDURES

Peptide Synthesis—Twelve overlapping peptides (B1–B12, 20-mers) were designed to span the entire region where hsp was reported to associate with eNOS (aa 291– 420, bovine) [5]. The eNOS-derived peptide B2, which possesses aa sequences that are common to B1 and B3, was incubated for 30 min with PEP1, a protein transduction domain peptide [7] (mole:mole ϭ 1:20) and added to the BAEC cultures at a final peptide concentration of 5 nM. WT-eNOS and eNOS-4A HEK-293 cells were lysed in Nonidet P-40 lysis buffer (50 mM Tris1⁄7Cl, 0.2 mM EDTA, 0.2 mM EGTA, 1% Nonidet P-40, 0.1 mM dithiothreitol, 5 nM BH4, 1ϫ protease inhibitors, 1ϫ phosphatase inhibitors, pH 7.5) by incubating on ice for 30 min followed with sonicating at 10% power for 30 s twice. HEK-293 cell proteins were lysed in 400 ␮l of MOPS lysis buffer and subjected to eNOS immunoprecipitation as described above.

RESULTS

DISCUSSION

FIGURE erates