Scratching below the surface: wound healing and alanine mutagenesis provide unique insights into interactions between eristostatin, platelets and melanoma cells.
Scratching below the surface: wound healing and alanine mutagenesis provide unique insights into interactions between eristostatin, platelets and melanoma cells.
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深入探究：伤口愈合和丙氨酸突变为了解毛立抑素、血小板和黑色素瘤细胞之间的相互作用提供了独特的见解。
DOI：
10.1159/000092417
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发表时间：
2005
影响因子：
--
通讯作者：
Paquette-Straub,Carrie
中科院分区：
文献类型：
--
作者：
McLane,MaryAnn;Zhang,Xiaoming;Tian,Jing;Zelinskas,Claire;Srivastava,Apoorva;Hensley,Brett;Paquette-Straub,Carrie
为了研究去整合素eristostatin的分子机制，利用10个重组丙氨酸突变体进行了细胞功能研究。ADP诱导的血小板聚集揭示了这种去整合素的RGD环(R24、R27、G28、N31)和C末端(W47、N48、G49)中的七个残基的关键作用。使用体外划痕伤口愈合试验，四个人黑色素瘤细胞株在暴露于野生型eristostatin时产生了类似的结果。与对照组相比，所有Eristostatin处理的细胞对损伤区域的愈合都较少。当使用纤维连接蛋白作为基质时，这种现象被重现。C8161细胞在N端突变体P4A作用下伤口愈合明显延迟，而在R24A或G28A作用下则无明显延迟。来自我们实验室和其他实验室的证据表明，αIIb、α4和α5整合素都不直接参与eristostatin的相互作用。Eristostatin不影响培养24 h后黑色素瘤细胞的数量，也不影响细胞的凋亡。然而，在这些黑色素瘤细胞暴露于eristostatin后进行的磷酸化研究显示，几个酪氨酸磷酸化分子发生了变化。
To study the molecular mechanism of the disintegrin eristostatin, cellular functional studies were performed using ten recombinant alanine mutants. ADP-induced platelet aggregation revealed critical contributions of seven residues within the ‘RGD loop’ (R24, R27, G28, N31) and C-terminus (W47, N48, G49) of this disintegrin. Using an in vitro scratch wound healing assay, four human melanoma cell lines yielded similar results when exposed to wildtype eristostatin. All eristostatin-treated cells healed less of the wounded area than control conditions. This phenomenon was reproduced when using fibronectin as the matrix. C8161 cells showed significant delay in wound closure with the N-terminal mutant P4A but not with R24A or G28A. Evidence from our laboratory and others suggests neither alpha IIb, alpha 4 nor alpha 5 integrins are directly involved in eristostatin’s interactions. Eristostatin did not affect the number of melanoma cells in culture after 24 h or the development of apoptosis. However, phosphorylation studies performed after these melanoma cells were exposed to eristostatin revealed changes in several tyrosine phosphorylated molecules.