整合蛋白參與許多生物體的重要機制，例如：血管新生、血栓形成、免疫反應、及骨質疏鬆，並且在許多人類重大疾病中扮演重要角色。許多重要的胞外基質蛋白中皆含有由arginine-glycine-aspartate三個氨基酸所組成的 RGD motif ，並且可以利用此序列與整合蛋白作用。然而也有研究指出，有些蛋白可以以非RGD序列與特定整合蛋白作用，例如：阿茲海莫症所引起的amyloid beta胜肽被發現可以利用RHD序列與整合蛋白alpha5beta1作用;而單株抗體OPG2則可利用RYD序列專一性地與整合蛋白alphaIIbbeta3結合。在本篇論文中，我們利用含有68個氨基酸及六對雙硫鍵，且擁有48PRGDMP53的序列的去整合蛋白:馬來腹蛇蛋白（簡稱Rho）作為模版，用以探討X50於RG50D motif之中對於去整合蛋白與整合蛋白之間辨識的影響。根據細胞黏著以及血小板凝集實驗結果，顯示RGD motif中的Gly的位置對於Rho與整合蛋白的辨識扮演重要角色。比較Rho與RXD突變株之間活性差異，發現Rho針對整合蛋白alphaVbeta3、alpha5beta1及alphaIIbbeta3皆擁有最好的抑制活性。相反的，RXD突變株對整合蛋白抑制活性較野生型差，其對整合蛋白的alphaVbeta33、alpha5beta31 及alphaIIbbeta33抑制能力分別下降2.8-35.4, 5.1-695.3及 8.0-1989.1倍，然而我們卻發現當Gly突變為Leu、Val、Ile、Asn及Asp時，RXD突變株會對提升對整合蛋白alphaVbeta3的特異性。在之前的研究中，當黑色素細胞瘤發展為較惡性、容易轉移的時期，其整合蛋白alphaVbeta3的表現量高於良性黑色素細胞瘤50-100倍。由流式細胞儀偵測細胞表面整合蛋白表現量，發現人類黑色素細胞瘤A375細胞的確會表現整合蛋白alphaVbeta3，並且也同時表現整合蛋白alpha5beta1及alphaVbeta5。因此延續以上實驗結果，我們進一步探討針對整合蛋白alphaVbeta3具有特異性的RXD突變株對於A375黑色素細胞瘤細胞黏著、移動及生長的影響。從細胞黏著實驗結果發現Rho與RLD及ARLDDL重組蛋白可以抑制細胞黏著於fibrinogen（IC50分別為 4.0, 13.1, 13.1 nM）以及vitronectin(IC50分別為135.5, 1556, 1768 nM)，但卻無法抑制細胞黏著於fibronectin。從細胞移動實驗實驗結果發現Rho與RLD及ARLDDL重組蛋白可以有效的抑制A375細胞的移動，其IC50分別為9.2, 37.4, 45.1 nM，而由細胞侵犯實驗結果顯示，Rho與RLD及ARLDDL重組蛋白也可有效抑制細胞侵犯，並其IC50分別為18.0, 50.5, 63.2 nM。此外也發現Rho與RLD及ARLDDL重組蛋白會透過改變細胞週期進而抑制A375細胞anchorage-depentdent的生長，並且改變細胞的型態。然而RLD及ARLDDL卻不會引起A375細胞凋亡及抑制細胞anchorage-indepentdent的生長。整體而言，我們利用一種去整合蛋白—馬來腹蛇蛋白，藉由改變RXD motif的序列，設計出針對整合蛋白alphaVbeta3具有特異性的突變株，並且發現針對整合蛋白alphaVbeta3具有特異性的突變株可以有效的抑制人類黑色素細胞瘤的黏著、移動、侵犯及生長。由本篇實驗結果顯示由我們所設計出的針對整合蛋白alphaVbeta3具有特異性的突變株有潛力可做為治療黑色素細胞瘤的藥物。

Integrins are involved in many biological processes such as angiogenesis, thrombosis, inflammation, osteoporosis, and thus play a key role in many severe human diseases. The arginine-glycine-aspartate (RGD) motif is found in several important extracellular matrix proteins, which serve as adhesive integrin ligands. It was also reported that the motifs other than RGD can be recognized by integrins. For example, an RHD motif found in the Alzheimer's-disease causing beta peptides can interact with integrin alpha5beta1, and an RYD motif is found in an integrin alphaIIbbeta3-binding antibody. In this study I used rhodostomin, a disintegrin containing 68 amino acid residues with a 48PRGDMP53 motif, as the scaffold to study the role of the middle residue in the RXD motif in recognizing integrins. According to the results of cell adhesion and platelet aggregation assays, I found that the middle residues in the RXD motif of Rho play an important role in recognizing integrins. Rho containing the RGD motif has the highest activity toward integrins alphaIIbbeta3, alphaVbeta3, and alpha5beta1. In contrast, mutations on the middle residue in the RXD motif caused 2.8-35.4-, 5.1-695.3-, and 8.0-1989.1-folds deceases in inhibiting integrins alphaVbeta3, alpha5beta1, and alphaIIbbeta3. We also found that the Rho mutants containing L, V, I, E, and D in the middle residue of the RXD motif can selectively inhibit integrin alphaVbeta3. It is reported that integrinalphaVbeta3 increases 50- to 100-fold as human melanoma cells progress to a more metastatic phenotype. It was consistent with our flow cytometric analysis that integrin alphaVbeta3 was highly expressed on the surface of A375 cells. Besides, A375 cells also expressed integrin alphaVbeta5 andalpha5beta1. I then used integrin alphaVbeta3-specific Rho mutants to study the activities of integrin alphaVbeta3-specific Rho mutants in inhibiting the proliferation, migration, invasion, and apoptosis of human melanoma cells. The cell adhesion analysis showed that Rho, RLD and ARLDDL mutants inhibited the adhesion of A375 to fibrinogen with the IC50 values of 4.0, 13.1, and 13.1 nM, to vitronectin with the IC50 values of 135.5, 1556, 1768 nM, but not to fibronectin. The migration analysis showed that Rho, RLD and ARLDDL mutants effectively inhibited cell migration with IC50 values of 9.2, 37.4, and 45.1 nM, respectively. The result of matrigel invasion assay showed that Rho, RLD and ARLDDL mutants effectively inhibited cell invasion with IC50 values of 18.0, 50.5, and 63.2 nM, respectively. They also reduced cell growth by changing cell cycle and changed cell morphology of A375 melanoma cell. In contrast, RLD and ARLDDL mutants cannot induce cell apoptosis and inhibit the formation of colonies in soft agar. In conclusion, we designed integrin alphaVbeta3-specific disintegrins by mutating the RXD motif of Rho, and found they can inhibit the adhesion, migration, invasion, and growth of human melanoma cells in the nanomolar concentration range. This study demonstrated that integrin alphaVbeta3-specific Rho mutants designed by this study are potential drug candidates for melanoma treatment.

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