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Abstract：
Plakophilin 1 (PKP1), a member of the p120ctn subfamily of the armadillo (ARM)-repeat-containing proteins, is an important structural component of cell-cell adhesion scaffolds although it can also be ubiquitously found in the cytoplasm and the nucleus. RYBP (RING 1A and YY1 binding protein) is a multifunctional intrinsically disordered protein (IDP) best described as a transcriptional regulator. Both proteins are involved in the development and metastasis of several types of tumors. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with RYBP by using in cellulo methods, namely immunofluorescence (IF) and proximity ligation assay (PLA), and in vitro biophysical techniques, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), and isothermal titration calorimetry (ITC). We also characterized the binding of the two proteins by using in silico experiments. Our results showed that there was binding in tumor and non-tumoral cell lines. Binding in vitro between the two proteins was also monitored and found to occur with a dissociation constant in the low micromolar range (~10 μM). Finally, in silico experiments provided additional information on the possible structure of the binding complex, especially on the binding ARM-PKP1 hot-spot. Our findings suggest that RYBP might be a rescuer of the high expression of PKP1 in tumors, where it could decrease the epithelial-mesenchymal transition in some cancer cells.
摘要：
斑素1(PKP1),Armadillo（ARM）含重复蛋白的p120ctn亚家族的成员，是细胞-细胞粘附支架的重要结构成分，尽管它也可以在细胞质和细胞核中普遍存在。RYBP（RING1A和YY1结合蛋白）是一种多功能的内在无序蛋白（IDP），最好被描述为转录调节因子。两种蛋白质都参与几种类型肿瘤的发展和转移。我们研究了PKP1的Armadillo结构域（ARM-PKP1）与RYBP的结合，即免疫荧光(IF)和邻近连接测定(PLA)，和体外生物物理技术，即荧光，远紫外（远UV）圆二色性（CD），和等温滴定量热法(ITC)。我们还通过使用计算机模拟实验表征了两种蛋白质的结合。我们的结果表明，在肿瘤和非肿瘤细胞系中存在结合。还监测了两种蛋白质之间的体外结合，发现其解离常数在低微摩尔范围（〜10μM）。最后,计算机模拟实验提供了有关结合复合物可能结构的其他信息，特别是绑定ARM-PKP1的热点。我们的研究结果表明，RYBP可能是PKP1在肿瘤中高表达的拯救者。在那里它可以减少一些癌细胞的上皮间质转化。
