Interaction of Neuropeptide Y and Hsp90 through a Novel Peptide Binding Region

Abstract

Hsp90 is a molecular chaperone that binds and assists refolding of non-native and/or labile polypeptides and also bind various peptides. However, the rules of how Hsp90 recognizes substrates have not been well characterized. By surface plasmon resonance measurements, a physiologically active peptide, neuropeptide Y (NPY), with a strong binding property to Hsp90 was identified from screening of 38 randomly selected peptide candidates. We showed that the carboxy-terminal fragment of NPY (NPY13-36), which forms an amphipathic R-helix structure, preserved the strong binding to Hsp90. Immunoprecipitation and immunoblotting using HeLa cell extracts revealed that newly synthesized NPY precursors bound to Hsp90, suggesting that the in vitro binding experiments identified an interactive peptide in vivo. Proteolytic cleavage of the NPY13-36/Hsp90 complex, as well as binding site analysis using deletion mutants of Hsp90, revealed the NPY binding locus on Hsp90R as the 192 amino acid region following the N-terminal domain. By electron microscopic analysis using an anti-Hsp90 antibody against the sequence proximal to the highly charged region, we showed that the Hsp90 dimer bound to NPY13-36 at both ends. Mutation of arginine residues in NPY13-36 to alanine abrogated binding to Hsp90. Our studies indicate that the hinge region after the N-terminal domain of Hsp90 and the positive charges on NPY are important for this interaction.

The Region Adjacent to the Highly Immunogenic Site and Shielded by the Middle Domain Is Responsible for Self-Oligomerization/Client Binding of the HSP90 Molecular Chaperone

“…Notably, the present study demonstrated that residues 311-350 are sufficient for the oligomerization, and our results further demonstrated that residues 341-350 are critical for self-oligomerization of HSP90R. We recently demonstrated that residues 289-400 are also important for the binding to neuropeptide Y (26). Therefore, residues 311-350, being located adjacent to the highly immunogenic site (residues 291-304) of human HSP90R (14), seem to directly associate with client proteins, such as neuropeptide Y and Akt/PKB.…”

“…Therefore, residues 311-350, being located adjacent to the highly immunogenic site (residues 291-304) of human HSP90R (14), seem to directly associate with client proteins, such as neuropeptide Y and Akt/PKB. Interestingly, we recently reported that the electron microscopic image of the mAb K3720 (recognizing at residues 291-304) attached HSP90 dimer was indistinguishable to that of the HSP90 dimer bound to neuropeptide Y bridged to streptavidin (26). Moreover, it should be noted that residues 341-350, which are critical for the self-oligomerization of the N domain, completely overlap the region (residues 335-348) essential for the interaction with Akt/PKB (Figure 8).…”

Dysbalance in sympathetic neurotransmitter release and action in cirrhotic rats: Impact of exogenous neuropeptide Y

“…Previously, we showed that heat-shock protein-90 (HSP-90) acts as signaling mediator of nNOS-and eNOS dependent vasodilation in mesenteric arteries, and that increased nitrergic vasorelaxation observed in portal hypertension is mediated largely by HSP-90 [43,44]. However, the reported strong binding of processed NPY to HSP-90 might outline a mechanism of NPY-mediated NOS/NO regulation [45]. Furthermore, the proteolytic processing of NPY leads to selective activation of presynaptic Y2 receptors via NPY 3-36 [32].…”

Amelioration of portal hypertension and the hyperdynamic circulatory syndrome in cirrhotic rats by neuropeptide Y via pronounced splanchnic vasoaction

“…Since, in portal hypertension mesenteric arteries exhibit eNOS, iNOS and nNOS upregulation4–7 22 it is tempting to speculate that NPY may interfere with this vascular NO overproduction. Indeed, Ishiwatari-Hayasaka et al recently reported a novel peptide binding region for NPY close to the N-terminal domain of HSP90 a key regulator of NOS activity 47. In fact, among multiple biologically active peptides screened, NPY was found to exert the highest affinity and interaction with HSP90.…”