整合蛋白是一種細胞表面黏著受器，其功能是影響細胞的黏著、遷移跟生長。纖維黏結蛋白是一種細胞外基質的糖蛋白，會與整合蛋白作用。纖維黏結蛋白的組成是由三種不同類別的重複性單元所組成，包括12種第一型、2種第二型、15到17種第三型。特別的是，第三型纖維黏結蛋白的第十個模組對於整合蛋白αvβ3具有低的親和力。我們若要利用第三型纖維黏結蛋白設計對於整合蛋白的藥物，必須使其具有高穩定性、選擇性以及活性。因此我們利用第三型纖維黏結蛋白的第十個模組作為鷹架蛋白，去設計對於整合蛋白αvβ3具有專一性的拮抗劑。在本實驗中，我們將：(1)套用實驗室對於去整合蛋白的研究出的整合蛋白特殊辨識序列或者是之前研究篩選出的對於整合蛋白的特殊辨識序列到第三型纖維黏結蛋白的第十個模組上，(2)並增加一對雙硫鍵於RGD loop上，並改變其雙硫鍵的鍵結模式，(3)接著進行對整合蛋白αvβ3具有專一性的第三型纖維黏結蛋白第十個模組蛋白突變株作特性的分析，(4)最後也將第三型纖維黏結蛋白第十個模組的蛋白突變株應用於癌症的研究上。現在我已利用大腸桿菌系統成功表達20種以上的第三型纖維黏結蛋白第十個模組蛋白突變株，並得到純的蛋白產物。在功能分析上顯示所有的蛋白突變株對於抑制血小板凝集能力是低的，顯示其較不會引起血小板缺乏症。在抑制細胞黏著試驗中，證明了套用去整合蛋白序列的FN-III10(PRGDMPD)突變株對於整合蛋白αvβ3具有高度的親和力。當加入一對雙硫鍵鍵結在RGD loop上，顯示整合蛋白αvβ3傾向於喜好CX7C的patterns，而非CX4C 及CX8C patterns。而且FN-III10(PRGDMPD)選擇性地抑制整合蛋白αvβ3，其IC50值為101 nM。利用量熱儀以及硫酸胺鹽沉澱法測試其熱穩定度及溶解度，分別顯示Tm質與溶解度：FN-III10(CPRGDMPDC)(85℃)(27.8 mg/ml) > FN-III10(81℃)(7.3 mg/ml) > FN-III10(PRGDMPD)(74℃)(5.3 mg/ml)，表示當加入一對雙硫鍵鍵結於RGD loop上，可以增加第三型纖維黏結蛋白第十個模組蛋白突變株的熱穩定性和溶解度。再來，利用NMR技術分析證明了FN-III10(CPRGDMPDC)具有正確的摺疊以及動力學分析指出其RGD loop具高穩定性，因此較能辨識整合蛋白αvβ3。此外，在癌症研究上，由體外試驗證明了FN-III10(CPRGDMPDC)可以抑制A375細胞的遷移能力以及HUVEC細胞的tube生成。在體內試驗中，FN-III10(CPRGDMPDC)在減少Lewis lung cancer cells在C57/B6老鼠身上的腫瘤生長上的結果沒有顯著的意義，但仍可以延長老鼠的壽命。總結來說，我們已經成功的將第三型纖維黏結蛋白第十個模組蛋白改變成對於整合蛋白αvβ3具有高度親和力、選擇性，並且具有高的熱穩定性和溶解度。我們的結果顯示我們對於整合蛋白αvβ3專一性的第三型纖維黏結蛋白第十個模組蛋白突變株，較先前所發表的FN-III10(PRGDWNEG)好。此外，如何應用這個專一性的蛋白突變株於整合蛋白αvβ3相關的疾病的研究，也正在進行中。

Integrins are cell-surface adhesion receptors that affect cell adhesion, migration and proliferation. Fibronectin (FN) is an extracellular matrix glycoprotein that binds to integrins. FN is composed of three different types of homologous repeating domains, including FN-I, FN-II, and FN-III. FN contains 12 of FN-I, 2 of FN-II, and 15-17 of FN-III repeats. In particular, FN-III10 can bind to integrin αvβ3 with low affinity. To use FN-III10 as integrin drugs, it is essential to engineer them to have high stability, selectivity, and potency. Therefore, we propose to use FN-III10 as the scaffold to design integrin αvβ3-specific antagonists. In this study I: (1) incorporate the integrin-specific recognition sequences into FN-III based on the results of our study on disintegrin or reported sequences; (2) modify disulfide bond pattern (CXnC) within RGD loop; (3) characterize integrin αvβ3-specific FN-III10 variants; and (4) study anti-tumor activity of integrin αvβ3-specific FN-III10 variants. Now I have expressed > 20 FN-III10 variants in E. coli and purified them to homogeneity. Functional analysis showed that all FN-III10 variants had lower activity in inhibiting platelet aggregation, suggesting that they exhibited low thrombocytopenia effect. Cell adhesion inhibition study demonstrated that FN-III10(PRGDMPD) mutant incorporating disintegrin sequence had higher affinity to integrin αvβ3. The incorporation of disulfide linkage into the RGD loop showed that integrin v3-specific FN-III10 variants preferred the CX7C but not CX4C and CX8C patterns, and FN-III10(CPRGDMPDC) selectively inhibited integrin αvβ3 with an IC50 value of 93 nM. Differential scanning calorimetry analysis and solubility measurements by ammonium sulfate precipitation showed that their Tm values and solubility FN-III10(CPRGDMPDC) (85 oC; 27.8 mg/ml) > FN-III10 (81oC; 7.3 mg/ml) > FN-III10(PRGDMPD) (74 oC; 5.3 mg/ml). These results indicate that the incorporation of a disulfide bond into the RGD loop of FN-III10 can increase its thermostability and solubility. NMR and dynamics analyses demonstrated that FN-III10(CPRGDMPDC) has the correct folding and the RGD loop of FN-III10(CPRGDMPDC) exhibited higher rigidity with preference for integrin v3 binding. Integrin αvβ3-specific FN-III10 (CPRGDMPDC) protein can inhibit A375 cell migration and HUVEC tube formation in vitro. The xenograft mouse model showed that it only slightly reduced the growth of Lewis lung cancer cells in C57/B6 mice; however, it can prolong their survival in vivo. In conclusions, we have successfully engineered FN-III10 scaffold to produce an integrin αvβ3-specific variant with high potency, selectivity, thermostability, and solubility. Our results also showed that this integrin αvβ3-specific FN-III10 variant is superior than the reported variant, FN-III10(PRGDWNEG). The further optimization of integrin v3-specific FN-III10 variant for the treatment of integrin αvβ3-related diseases is ongoing.

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