研究人類肝癌衍生生長因子之HATH區域的蛋白質骨架動態在單體和雙體間之異同

Probing the Protein Backbone Dynamics of the HATH Domain of Human Hepatoma-Derived Growth Factor in Monomer and Dimer

田世齊(Shih-Chi Tien)

指導教授 ： 黃太煌 ； 呂平江

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(2006年)

https://doi.org/10.6843/NTHU.2006.00559

摘要

人類肝癌衍生生長因子(hHDGF)是屬於一個新發現的蛋白質家族，命名為人類肝癌衍生生長因子相關蛋白質家族(HDGF-related protein)，簡稱為HRP蛋白質家族。目前此蛋白質家族已知包含六個蛋白質，其功能目前包括：血管、腎臟新生、腫瘤生長、細胞增生和活化核酸轉錄等。所有HRP蛋白質家族的蛋白質在N端的序列上有很高的相似度(homologous to the amino terminal of HDGF)，此段序列簡稱為HATH domain。但這六個蛋白質在C端序列有很大的差異性。人類肝癌衍生生長因子是一含有240個胺基酸的蛋白質，可分做兩部份：第一段是從N端的前100個胺基酸序列，有完整結構稱之HATH domain；第二段是從胺基酸序列101至240，其結構還未知。
人類肝癌衍生生長因子的HATH domain會與細胞膜外的肝素(heparin)產生鍵結，會讓外生性的人類肝癌衍生生長因子更容易進入細胞。而C端可能可以幫助蛋白質由細胞質進入細胞核中，讓此因子在細胞核中發生細胞增生或活化的反應。之前的實驗發現，HATH domain的單體間，會相互分享部分的結構，而形成雙體(dimer)此一特別的雙體，稱為domain-swapped dimer。這個特殊的雙體相較於單體，跟肝素有更高鍵結力。
我的論文題目主要是在利用核磁共振去研究人類肝癌衍生生長因子的HATH domain的單體及雙體在分子動態上的差異性。我們利用異核核磁共振脈衝序列，在14.7 Tesla的靜磁場下，量測遲緩速率(relaxation rate)和15N-H異核交互作用增異參數(NOE)。並使用無模型法則(Model-free formulism)去決定分子動態參數：次序參數(S2)、有效相關時間(τe)、化學交換參數(Rex)；另外利用進一步的簡化光譜密度(reduced spectral density mapping)的對應得到了光譜密度函數去分析比較單體及雙體在分子動態的差異性。

關鍵字

蛋白質 ； 核磁共振

並列摘要

Human hepatoma-derived growth factor (hHDGF) is a member of HRPs (HDGF-related proteins) family of proteins. HRPs belong to a new protein family that has been known in nephrogenesis, tumorigenesis, vascular development, cell proliferation and transcriptional activation. All the HRPs have the conserved N-terminal homologous to the amino terminal of HDGF (HATH) domain, but vary in the C-terminal domain. hHDGF is a 240-amino acid protein, which can be divided into two parts: the first is the well-structured HATH domain, from residues 1-100 and the structure has been determined by NMR; the second is the C-terminal domain, from residues 101-240 , which is disorder.
HATH domain has been shown to bind to heparin and heparin sulfate located outside the surface of cell membrane and facilitated internalization of the protein into cell. The C-terminal domain may help translocate the protein from cytoplasm to nucleus, and serve as a signal to stimulate cell growth. Previous studies showed that HATH domain monomer shares a portion of structure with each other to form the particular dimer called the domain-swapped dimer. Dimer has much higher heparin-binding affinity to heparin than that of the monomer.
My thesis work is aimed at determining the difference in dynamics by NMR relaxation method between monomer and the homo-dimer of the HATH domain. 15N spin relaxation rates and heteronuclear NOE determined at 600 MHz field were analyzed by Modelfree approach to extract the dynamic parameter: S2 (order parameter), τe (effective correlation time) and Rex (chemical exchange rate). I also use Reduced Spectral Density Mapping to determine the spectral density functions: J(ω0.87H), J(0) and J(ωN). The results showed that the domain swapped semi-dimer packs similar in spatial and orientation compares to the monomer by order parameters and spectral density function, J(0.87H). Besides fast local fluctuation loop motions act similar in both monomer and dimer, there were much more micro- to millisecond motions in monomer. Both in monomer and dimer, the L2 loop region acts more complicated dynamic motions with fitting to effective correlation times and exchange rates. We demonstrated the dynamic similarity and differences in monomer and dimer of HATH domain in wide-ranged time scale by using NMR relaxation experiments.

並列關鍵字

NMR ； HATH domain ； protein ； monomer ； dimer

參考文獻

2. Izumoto, Y.; Kuroda, T.; Harada, H.; Kishimoto, T.; Nakamura, H., Hepatoma-derived growth factor belongs to a gene family in mice showing significant homology in the amino terminus. Biochem Biophys Res Commun 1997, 238, (1), 26-32.

3. Ikegame, K.; Yamamoto, M.; Kishima, Y.; Enomoto, H.; Yoshida, K.; Suemura, M.; Kishimoto, T.; Nakamura, H., A new member of a hepatoma-derived growth factor gene family can translocate to the nucleus. Biochem Biophys Res Commun 1999, 266, (1), 81-7.

4. Dietz, F.; Franken, S.; Yoshida, K.; Nakamura, H.; Kappler, J.; Gieselmann, V., The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies. Biochem J 2002, 366, (Pt 2), 491-500.

5. Ge, H.; Si, Y.; Roeder, R. G., Isolation of cDNAs encoding novel transcription coactivators p52 and p75 reveals an alternate regulatory mechanism of transcriptional activation. Embo J 1998, 17, (22), 6723-9.

6. Sue, S. C.; Chen, J. Y.; Lee, S. C.; Wu, W. G.; Huang, T. H., Solution structure and heparin interaction of human hepatoma-derived growth factor. J Mol Biol 2004, 343, (5), 1365-77.