Interaction of Neuropeptide Y and Hsp90 through a Novel Peptide Binding Region
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- 作者：Haruko Ishiwatari-Hayasaka ; Mikako Maruya ; Amere Subbarao Sreedhar ; Takayuki K. Nemoto ; Peter Csermely ; and Ichiro Yahara
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：November 11, 2003
- 年：2003
- 卷：42
- 期：44
- 页码：12972 - 12980
- 全文大小：150K
- 年卷期：v.42,no.44(November 11, 2003)
- ISSN：1520-4995
文摘
Hsp90 is a molecular chaperone that binds and assists refolding of non-native and/or labilepolypeptides and also bind various peptides. However, the rules of how Hsp90 recognizes substrateshave not been well characterized. By surface plasmon resonance measurements, a physiologically activepeptide, neuropeptide Y (NPY), with a strong binding property to Hsp90 was identified from screeningof 38 randomly selected peptide candidates. We showed that the carboxy-terminal fragment of NPY(NPY13-36), which forms an amphipathic 
-helix structure, preserved the strong binding to Hsp90.Immunoprecipitation and immunoblotting using HeLa cell extracts revealed that newly synthesized NPYprecursors bound to Hsp90, suggesting that the in vitro binding experiments identified an interactivepeptide in vivo. Proteolytic cleavage of the NPY13-36/Hsp90 complex, as well as binding site analysisusing deletion mutants of Hsp90, revealed the NPY binding locus on Hsp90 as the 192 amino acidregion following the N-terminal domain. By electron microscopic analysis using an anti-Hsp90 antibodyagainst the sequence proximal to the highly charged region, we showed that the Hsp90 dimer bound toNPY13-36 at both ends. Mutation of arginine residues in NPY13-36 to alanine abrogated binding toHsp90. Our studies indicate that the hinge region after the N-terminal domain of Hsp90 and the positivecharges on NPY are important for this interaction.
-helix structure, preserved the strong binding to Hsp90.Immunoprecipitation and immunoblotting using HeLa cell extracts revealed that newly synthesized NPYprecursors bound to Hsp90, suggesting that the in vitro binding experiments identified an interactivepeptide in vivo. Proteolytic cleavage of the NPY13-36/Hsp90 complex, as well as binding site analysisusing deletion mutants of Hsp90, revealed the NPY binding locus on Hsp90 as the 192 amino acidregion following the N-terminal domain. By electron microscopic analysis using an anti-Hsp90 antibodyagainst the sequence proximal to the highly charged region, we showed that the Hsp90 dimer bound toNPY13-36 at both ends. Mutation of arginine residues in NPY13-36 to alanine abrogated binding toHsp90. Our studies indicate that the hinge region after the N-terminal domain of Hsp90 and the positivecharges on NPY are important for this interaction.