| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 103 === Integrins are a family of α/β heterodimeric receptors and modulate many cellular processes. These integrin ligands employ a variant of the RGD, NGR, or LDV motifs as a key element of their major recognition site. RGD-containing ligands are shown to collaborate with specific flanking residues, synergistic site, and C-terminal region to control their integrin binding affinity and specificity. The NGR motif in the 5th and 7th type I repeats of fibronectin is known to bind integrin α5β1, and the spontaneous deamidation of NGR to isoDGR, an integrin αvβ3-binding motif, plays an important role in fibronectin fibril assembly and in regulating endothelial cell adhesion and proliferation. In my study I used rhodostomin (Rho), a disintegrin containing a 48PRGDMP53 motif, as a protein scaffold to investigate the regions of disintegrins involved in integrin interactions. The cell adhesion assay, NMR technique, X-ray crystallography, and molecular docking were used to study structure and functional relationships of disintegrin and integrins. To date, we have successfully used Rho to design potent and selective integrin-specific antagonists, including 48ARLDDL, an integrin αvβ3-specific mutant, and KG, an integrin αvβx-specific mutant. In my study I found the N-terminal region (residues 39-47) adjacent to the RGD motif that plays an important role in interacting with integrins αvβ3, αIIbβ3 and α5β1. The cell adhesion analysis of N-terminal mutants showed that Rho mutant with a 39KKKRT sequence exhibited the highest inhibitory activity with 3.6-, 4.6-, and 6.2-fold increases in inhibiting integrins αIIbβ3, αvβ3, and α5β1 in comparison with those of Rho 48ARGDNP-67NGLYG mutant. We also showed that Rho N-terminal mutant with a 46RR sequence exhibited 3.7- and 3.8-fold increases in inhibiting integrins αvβ3 and α5β1. In contrast, Rho mutant with a 46DD sequence exhibited 7.2-, 124.5-, and 〉582.0-fold decreases in inhibiting integrins αIIbβ3, αvβ3, and α5β1. The C-terminal mutations of 54DD into YY caused the increases 2.7- and 3.8-fold increases in inhibiting integrins αvβ3 and α5β1. 3D structures of Rho, KKKRT, and R46E mutants were determined by X-ray crystallography. Structural analysis showed that R42 of Rho KKKRT mutant interacted with C-terminal Y67 and H68 residues, which was not found from Rho with a SRAGK sequence. The docking of Rho and its KKKRT mutant into integrins showed that the sidechain of the R42 residue of KKKRT mutant interacted with the residue D216 of β1 and β3. In contrast, these interactions were absent in the Rho-integrin complexes. The sidechain orientation of the R46 residue in Rho and the E46 residue in R46E mutant were different. The docking analysis showed that the sidechain of R46 of Rho, but not E46 of R46E mutant, formed a salt bridge with D159 of αIIb subunit. These results demonstrate that the regions adjacent to the RGD motif in disintegrins affect their function and interactions with integrins. The incorporation of GNGRG amino acid sequence into Rho was used to study its role in integrin recognition. Mass and NMR analyses found that it can be converted into isoDGR and DRG isomers. The cell adhesion analysis showed that two isomers exhibited similar integrin αvβ3 inhibitory activity with the IC50 value of ~250 μM. In contrast, two isomers exhibited higher integrin α5β1 inhibitory activity. The DGR isomer had the IC50 value of 4.5 μM that is 6-fold more active than that of isoDGR isomer, suggesting that integrin ligands with an isoDGR motif are better ligands for integrin α5β1. Flow cytometry and western blot analysis showed that integrin β3-knockdown A375 cells were established. The adhesion analysis showed that the β3-knockdown cells cannot adhere on fibrinogen. The migration analysis showed that Rho, RLD and ALRDDL mutants inhibited A375 cell with the IC50 values of 9.3, 37.4, and 45.1 nM. In contrast, Rho, RLD and ALRDDL mutants inhibited the migration of β3-knockdown cells with the IC50 values of 0.5, 3, and 〉 100 μM. This result demonstrated that integrin αvβ3-specific mutant inhibited migration of human melanoma cells A375 cells via an integrin β3-dependent pathway. The use of Rho, ARLDDL and KG mutants in inhibiting angiogenesis, pancreatic cancer, and melanoma were evaluated. We found that they effectively inhibited tube formation, migration, and cell growth of endothelial cells with the IC50 values of 5-50 nM. They also inhibited the adhesion of pancreatic cancer cells (AsPC-1, Bx-PC-3, PANC-1, and Mia Paca-2) to vitronectin and fibronectin. In contrast, they cannot inhibit the adhesion of human melanoma A375 cell to fibronectin. We found that Rho and KG inhibited the migration of pancreatic cancer and melanoma cells with the IC50 values of 40-250 nM. The cell viability analysis showed that they also inhibited cell growth and induced cell apoptosis via AKT/caspase-3 pathway. Xenograft animal model showed that KG can inhibit the growth of pancreatic tumor. The results of this will provide new insight into the design of integrin-specific drugs for cancer therapy.
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