Ulke-Lemée Annegret, Turner Sara R, MacDonald Justin A
Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4Z6, Canada.
J Cell Biochem. 2015 Nov;116(11):2667-75. doi: 10.1002/jcb.25215.
The smoothelin-like 1 (SMTNL1) protein is the newest member of the smoothelin family of muscle proteins. Two calmodulin (CaM)-binding domains (CBD1 for Ca-CaM; CBD2 for apo-CaM) have been described for the SMTNL1 protein using in vitro assays. We now demonstrate in situ associations of SMTNL1 and CaM in A7r5 smooth muscle cells using the proximity ligation assay (PLA). We quantified CaM-SMTNL1 proximity events accurately after taking into account variations in protein expression levels. The refined method allows quantification of in situ proximity after transient transfection with an associated error of <10%. The proximity of SMTNL1 and CaM in A7r5 cells could be reduced by scrambling the amino acid sequence and mutation of large hydrophobic amino acids of CBD1. The truncation of CBD2 did not influence SMTNL1 proximity to CaM. Ultimately, we conclude that SMTNL1 forms complex interactions with CaM in smooth muscle cells, with a role for CBD1 and possibly the intrinsically disordered region.
平滑肌样蛋白1（SMTNL1）是肌肉蛋白平滑肌样蛋白家族的最新成员。通过体外实验，已确定SMTNL1蛋白具有两个钙调蛋白（CaM）结合结构域（与Ca-CaM结合的CBD1；与脱钙CaM结合的CBD2）。我们现在使用邻近连接分析（PLA）在A7r5平滑肌细胞中证明了SMTNL1和CaM的原位结合。在考虑蛋白质表达水平的变化后，我们准确地量化了CaM-SMTNL1邻近事件。这种改进的方法允许在瞬时转染后对原位邻近度进行量化，相关误差<10%。通过打乱CBD1的氨基酸序列和突变大的疏水氨基酸，可以降低A7r5细胞中SMTNL1和CaM的邻近度。CBD2的截断不影响SMTNL1与CaM的邻近度。最终，我们得出结论，SMTNL1在平滑肌细胞中与CaM形成复杂的相互作用，CBD1以及可能的内在无序区域发挥作用。
