臺灣博碩士論文加值系統

Growth factor receptor-bound protein 2 (簡稱Grb2)是一個轉接蛋白質被發現可以和非常多種控制細胞分裂、成長的蛋白質相結合。這些蛋白質包括嵌合蛋白質Shc、erbB等生長激素受體，FAK，以及細胞內和癌症有關的蛋白質BCR-abl。Grb2 SH2和這些蛋白質的結合是透過和蛋白質上的含磷酸化酪氨酸如pYXNX序列相互作用。Grb2和這些蛋白質結合後再利用其SH3區塊和另一蛋白質Sos作用，然後Sos再利用其功能活化Ras這個蛋白質。而Ras接著調控一系列的kinase作用，控制細胞的分裂及生長。最近Grb2被發現和某些癌症的生成有緊密關係，而對Grb2 SH2區塊設計出的新藥被認為在癌症治療上扮演一個重要角色。我們試著將Grb2 SH2區塊以分生方法大量表現並將之純化，純化後的樣品使用旋光儀、核磁共振做結構上及其穩定度的分析；由旋光儀的實驗可得知Tm值為55℃、Cm大約為2M、△G(H2O)=1.19 kcal/mole；將Grb2 SH2 domain及不含磷酸化酪胺酸的環狀胜G1TE做一維及二維核磁共振光譜，根據核磁共振實驗結果G1TE是一圓環結構，比較G1TE 及Bcl-Abl磷酸化酪胺酸胜與Grb2 SH2 domain結合的結構圖形，推測G1TE上的Tyr3、Val6和Gly7三個殘基可能扮演與Grb2 SH2 domain結合的重要角色；未來將利用Grb2 SH2 domain和G1TE結合的複合體結構分析及最新的Grb2 SH2 domain研究報告來設計與Grb2 SH2 domain結合更好的胜、小分子，發展可抑制Grb2的藥物。

The SH2 domain of Grb2 binds phophotyrosyl peptides with the consensus sequence pYXNX within several proteins including the adapter proteins Shc, FRS-2, growth factor receptors such as the members of the erbB family, morphology-determining proteins such as FAK, and cellular oncogenes such as Bcr-Abl. Binding of the Grb2 SH2 domain to the receptors relocates the Grb2 SH3 domain binding proteins, i.e. Sos, close to the plasma membrane. Then, Sos due to its guanine nucleotide exchange activity, converts the GDP-bound inactive form of Ras to its GTP-bound active form. Activated Ras triggers the kinase cascade which is essential for cell growth and differentiation. A particularly important role for Grb2 in human cancer has been proposed for cells transformed by high levels of erbB2 (HER-2 or neu) expression. Recent studies have indicated that Grb2 function is required for cell transformation by the neu and Bcr-Abl oncogenes. Thus , the design of specific inhibitors to Grb2 SH2 domain holds the promise of targeted treatment of breast cancer and cancer. Based on circular dichroism measurements, its melting temperature was found to be 55℃, at pH7.4. The Gibbs free energy (△GH2O) of the intrinsic stability and thermodynamic spontaneity of unfolding were found to be low, 1.19 kcal/mole by Gdn×HCl denaturation experiments, as compared to 3.96 kcal/mole (BTK SH3 domain) and 3.03 kcal/mole (Itk SH3 domain)[1], indicating poor stability of Grb2 SH2 domain. Until now , most of the inhibitors designed for the SH2 domain of Grb2 are focused on the phosphotyrosine containing peptides and peptidomimetics. However, these compounds all face the stability (chemical and/or biological) of the pTyr side chain phosphate ester and the poor cellular penetrability problems. Recently, nonphosphorylated cyclic peptide ligands were discovered for the Grb2 SH2 domain using phage display library. G1TE, One of these cyclic peptides, was determined using two-dimensional NMR and simulated annealing methods. The average RMS-deviations were found to be 0.41±0.11 A for the backbone heavy atoms C, Cα and N, and 1.03±0.14 A for all heavy atoms in a family of 10 structures. Structural comparison between G1TE and Bcr-Abl phosphopeptide bound to Grb2 SH2 domain revealed that G1TE may form a larger circle-like binding surface than the Bcr-Abl phosphopeptide. Since these cyclic peptides do not contain pTyr, they define a new type of SH2 domain binding motif that may advance the design of Grb2 inhibitors.

目錄
誌謝 I
中文摘要 II
英文摘要 III
序論 1
材料與方法 4
結果與討論 17
參考文獻 20
圖 26

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