| 摘要: | Grb2-SH2 區塊在致癌基因 Ras 的訊息傳遞路徑中，對於細胞的增生與分化，扮演很重要的角色。龍鳳娣博士之實驗室致力於研發Grb2 -SH2之胜肽拮抗物，發表一個抑制 Grb2 -SH2區塊效力非常好之前導胜肽 (Fmoc-Glu-Tyr-Aib-Asn-NH2)，但此胜肽對抗乳癌細胞 (MCF-7) 之增生的效力不佳，推測可能是此胜肽對細胞膜之穿透性不佳，無法進入細胞中發揮其抑制 Grb2-SH2之功效。因此本研究之設計為將穿膜胜肽RGD-4C (CDCRGDCFC) 與前導胜肽耦合，以期增加胜肽之穿膜效率，進而達到抑制細胞內Grb2-SH2 的效用。結果顯示設計之胜肽對乳癌細胞株 (MCF-7) 之增生具有抑制作用。因此，以分生方法構築 Grb2-SH2 質體，並大量表現及純化出 Grb2-SH2，以提供實驗室未來應用表面膜漿共振技術研發出對Grb2-SH2具有高親和力及對癌細胞具有抗增生功效之胜肽。本論文之另一個標的蛋白質是 Id (Inhibitor of DNA binding/Inhibitor of differentiation) 蛋白質。Id1 是 DNA 結合蛋白質的抑制物，屬於抑制 DNA 與轉錄因子結合的蛋白質家族，且包含了一個高度保留的 helix-loop-helix (HLH) 區塊，其過度表現會導致腫瘤的形成或腫瘤轉移，在訊息傳遞途徑中扮演很重要的角色，因此，可當作癌症治療重要標的。2010 年龍鳳娣博士之實驗室發表一個有效之前導胜肽 (peptide 3C) ，對 Id1 具有高親和力及對癌細胞具有抗增生功效 (IC50 =25 μM)。由於 Id1 蛋白質其結構仍未被發表，因此，本研究進行 Id1 HLH domain 之基因轉殖、表現及純化蛋白質的工作。未來將利用 X 光晶譜學技術解析出 Id1 蛋白質及 Id1與peptide 3C 複合體之蛋白質晶體結構，以期了解 peptide 3C與Id1蛋白質的結合活性部位，進一步修飾 peptide 3C以提高其抗癌功效。本論文之結果所提供之資訊有助於研發出更有潛力之抗癌藥物。
The growth factor receptor-bound protein-Src homology 2 (Grb2-SH2) plays an important role in the oncogenic Ras signaling pathway, which involves in cell proliferation. Lung’s laboratory has focused on developing antagonists of Grb2-SH2 and reported a lead peptide, Fmoc-Glu-Tyr-Aib-Asn-NH2, which exhibited high potent affinity for Grb2-SH2, but it showed poor bioactivity on breast cancer cell line (MCF-7). We speculated that the cell permeability of the lead peptide is too low to penetrate into the cell, limiting its function as inhibitor of Grb2-SH2. In this study, to enhance the cell permeability of peptides for inhibition of the intracellular Grb2-SH2, we designed the peptide analogs by incorporation of the cell-penetrating peptide (RGD-4C peptide；CDCRGDCFC ) into the lead peptide. Our results demonstrated that the designed peptides exhibited anti-proliferative effect on MCF-7 cancer cells. Therefore, we constructed the Grb2-SH2 gene, expressed and purified the Grb2-SH2 protein for further development and discovery of the high-affinity and potent Grb2-SH2 inhibitors by using surface plasmon resonance (SPR)-based method. Another target protein for this study is Id1 proteins, which are inhibitors of differentiation or DNA binding, act as dominant negative antagonists of the basic helix–loop–helix (bHLH) family of transcription factors, and contain a highly conserved dimerization motif known as the HLH domain. Id1 protein plays an important role in the cell growth, differentiation and tumorigenesis, which involved tumor metastasis of overexpression of Id1 protein in tumor cells. Thus, it has been the potential target for cancer intervention. In 2010, Lung’s laboratory reported a lead peptide, peptide 3C, which exhibited high potent inhibitory effects on breast cancer cell line (MCF-7) and colon cancer cell line (HT-29), the IC50 value of peptide 3C was determined as 25 μM in both cells. The crystal structure of the Id1 protein has been reported in the literature, therefore, we performed the construction, expression, purification and crystallization of human Id1 in order to determine the crystal structures of human Id1 and the complex of Id1 and peptide 3C for understanding their active site for binding and modification of peptide 3C to enhance the anticancer potency. Results of these studies should provide important information for development of anti-cancer drugs. |
