利用NMR研究Stip1蛋白C端部分與熱休克蛋白90和熱休克蛋白70之結合

NMR Study of Stip1 C-terminal portion in binding Hsp90 and Hsp70

曹凱勛(Tsao, Kai Hsun)

指導教授 ： 蘇士哲

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2015年)

https://doi.org/10.6843/NTHU.2015.00260

摘要

Stip1 (STress-Inducible Protein 1) 是一個60kD的佐監護蛋白，它含有兩個DP功能區塊及三個TPR功能區塊，Stip1的主要功能是幫助熱休克蛋白90與熱休克蛋白70連結，TPR功能區塊對於佐監護蛋白（如：Stip1、HIP、CHIP）在與熱休克蛋白結合十分重要，Stip1-Hsp90-Hsp70的三元複合體主要功用在於客戶蛋白的折疊，有趣的是，雖然在Stip1之C端的DP2在之前研究中發現似乎不與熱休克蛋白結合，但它於客戶蛋白的折疊扮演關鍵的角色，相關機制尚未明瞭，我們的合作者發現，利用pull-down assay可證明Stip1在少了DP2這段功能區後，熱休克蛋白90及熱休克蛋白70可被偵測的量均減少，他們也發現在Stip1最C端的24個胺基酸 (Peptide 520)可減少卵巢癌腫瘤的生長，在本研究中，我們試著利用核磁共振光譜解析DP2和Peptide520的作用，並將之結果應用在癌症治療上，我們利用蛋白質骨架鑑定得知DP2的二級結構，且NOESY也可提供我們關於Peptide520的二級結構資訊，我們證明了Stip1之TPR2A-2B會與熱休克蛋白90之middle功能區與C端功能區有強交互作用，但DP2仍然未被鑑定出與熱休克蛋白90或熱休克蛋白70的連結，我們也證明了熱休克蛋白70的ATP水解酶功能區與熱休克蛋白90之middle功能區與C端功能區有直接的交互作用，從實驗得知，Peptide520不會影響Stip1與熱休克蛋白90跟熱休克蛋白70與熱休克蛋白90的連結，DP2與Peptide520在Stip1-Hsp90-Hsp的三元複合體中所扮演的角色需要更進一步解析。

關鍵字

核磁共振光譜 ； 熱休克蛋白

並列摘要

Stip1 (STress-Inducible Protein 1) is a 60 kD co-chaperone containing two DP (aspartate-proline) domains and three TPR (tetratricopeptide repeat) domains, and the main function of Stip1 is to assist the connection between Hsp90 (Heak shock protein 90) and Hsp70 (Heat shock protein 70). TPR domains are important for co-chaperones, such as Stip1, HIP and CHIP, to bind to Hsps. The Stip1-Hsp90-Hsp70 ternary complex is responsible for client protein folding. Interestingly, one of the DP domains in Stip1, DP2, plays a central role in client protein folding process, while the mechanism is less understood. There is no report to define the interaction between DP2 and Hsp90 or Hsp70. However, we noticed the deletion of DP2 in Stip1 decreases the ability in binding Hsp90 and Hsp70 in the pull-down assay. In addition, the last C-terminal fragment (Peptide 520) of Stip1 reduced the growth of ovarian tumor cell. In this study, we try to define the roles of DP2 and Peptide520 by NMR titration experiments. The structure information of DP2 and Peptide520 were established by NMR backbone assignment. Based on the NMR titration experiments, Stip1 TPR2A-2B showed strong interaction with Hsp90 middle and C-terminal domain, whereas DP2 remains no interaction with any domain of Hsp90 or Hsp70. We noticed the direct interaction between Hsp70 ATPase domain and Hsp90 middle and C-terminal domain. However, Peptide 520 has no effect in modulating the binding of Stip1-Hsp90 and Hsp90-Hsp70. The roles of DP2 and Peptide 520 in the Stip1-Hsp90-Hsp70 ternary complex still need to be further elucidated.

並列關鍵字

NMR ； heat shock protein ； stress inducible protein 1 ； chaperone

參考文獻

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