Institute of Biophysics, The Czech Academy of Sciences, v.v.i., Královopolská 135, 61265 Brno, Czech Republic.
Masaryk Memorial Cancer Institute, Research Centre for Applied Molecular Oncology, Zluty kopec 7, 65653 Brno, Czech Republic.
Bioelectrochemistry. 2021 Aug;140:107808. doi: 10.1016/j.bioelechem.2021.107808. Epub 2021 Mar 26.
In this paper we compare electrochemical behavior of two homolog proteins, namely anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), playing an important role in cancer cell biology. The slight variation in their protein structures has an impact on protein adsorption and orientation at charged surface and also enables AGR2 and AGR3 to form heterocomplexes. We confirm interaction between AGR2 and AGR3 (i) in vitro by immunochemical and constant current chronopotentiometric stripping (CPS) analysis and (ii) in vivo by bioluminescence resonance energy transfer (BRET) assay. Mutation of AGR2 in dimerization domain (E60A) prevents development of wild type AGR2 dimers and also negatively affects interaction with wild type AGR3 as shown by CPS analysis. Beside new information about AGR2 and AGR3 protein including their joint interaction, our work introduces possible applications of CPS in bioanalysis of protein complexes, including those relatively unstable, but important in the cancer research.
在本文中，我们比较了两种同源蛋白（AGR2 和 AGR3）的电化学行为，它们在癌细胞生物学中起着重要作用。它们的蛋白质结构略有差异，这影响了在带电表面上的蛋白质吸附和取向，也使 AGR2 和 AGR3 能够形成异源复合物。我们通过免疫化学和恒流计时库仑 stripping（CPS）分析在体外证实了 AGR2 和 AGR3 之间的相互作用（i），并通过生物发光共振能量转移（BRET）测定在体内证实了相互作用（ii）。二聚化结构域（E60A）突变的 AGR2 阻止了野生型 AGR2 二聚体的形成，并且如 CPS 分析所示，也对与野生型 AGR3 的相互作用产生负面影响。除了有关 AGR2 和 AGR3 蛋白的新信息，包括它们的联合相互作用外，我们的工作还介绍了 CPS 在蛋白质复合物生物分析中的可能应用，包括那些相对不稳定但在癌症研究中很重要的复合物。
