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J. Biol. Chem., Vol. 282, Issue 52, 37567-37574, December 28, 2007

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

Hao Xu, Yang Shi¹, Jingli Wang, Deron Jones, Dorothee Weilrauch, Rong Ying, Basam Wakim^¶, and Kirkwood A. Pritchard, Jr.²

From the Department of Surgery, Division of Pediatric Surgery, and the Departments of Anesthesiology and ^¶Biochemistry, Medical College of Wisconsin, Children's Research Institute, Cardiovascular Research Center, Milwaukee, Wisconsin 53226

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 () 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 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 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 generation from eNOS and, therefore, vascular function.

Received for publication, August 6, 2007 , and in revised form, October 29, 2007.

^* This work was supported by National Institutes of Health Grants HL71214 and HL48251 (to K. A. P.), American Heart Association Postdoctoral Fellowship Award 0520103Z (to H. X.), and National Institutes of Health Grant HL080468 (to Y. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence may be addressed: Medical College of Wisconsin, Children's Research Institute, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-955-4634; E-mail: yangshi{at}mcw.edu. ² To whom correspondence may be addressed: Medical College of Wisconsin, Children's Research Institute, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-955-5615; E-mail: kpritch{at}mcw.edu.

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