Kitamura Akira, Shibasaki Ai, Takeda Kayo, Suno Ryoji, Kinjo Masataka
Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 605-8501, Japan.
Biochem Biophys Rep. 2018 Apr 12;14:58-63. doi: 10.1016/j.bbrep.2018.03.009. eCollection 2018 Jul.
Normal function and abnormal aggregation of transactivation response (TAR) DNA/RNA-binding protein 43 kDa (TDP-43) are directly associated with the lethal genetic diseases: cystic fibrosis, amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). The binding of TDP-43 to single-stranded DNA (ssDNA) or RNA is involved in transcriptional repression, regulation of RNA splicing, and RNA stabilization. Equilibrium dissociation constants () of TDP-43 and ssDNA or RNA have been determined using various methods; however, methods that can measure with high sensitivity in a short time using a small amount of TDP-43 in solution would be advantageous. Here, in order to determine the of TDP-43 and fluorescence-labeled ssDNA as well as the binding stoichiometry, we use fluorescence correlation spectroscopy (FCS), which detects the slowed diffusion of molecular interactions in solution with single-molecule sensitivity, in addition to electrophoretic mobility shift assay (EMSA). Using tandem affinity chromatography of TDP-43 dually tagged with glutathione-S-transferase and poly-histidine tags, highly purified protein was obtained. FCS successfully detected specific interaction between purified TDP-43 and TG ssDNA repeats, with a in the nanomolar range. The of the TDP-43 mutant was not different from the wild type, although mutant oligomers, which did not bind ssDNA, were observed. Analysis of the fluorescence brightness per dimerized TDP-43/ssDNA complex was used to evaluate their binding stoichiometry. The results suggest that an assay combining FCS and EMSA can precisely analyze ssDNA recognition mechanisms, and that FCS may be applied for the rapid and quantitative determination of the interaction strength between TDP-43 and ssDNA or RNA. These methods will aid in the elucidation of the substrate recognition mechanism of ALS- and FTLD-associated variants of TDP-43.
反式激活应答（TAR）DNA/RNA结合蛋白43千道尔顿（TDP-43）的正常功能与异常聚集直接关联到致命性遗传疾病：囊性纤维化、肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTLD）。TDP-43与单链DNA（ssDNA）或RNA的结合参与转录抑制、RNA剪接调控及RNA稳定化。已使用多种方法测定了TDP-43与ssDNA或RNA的平衡解离常数（）；然而，能够在短时间内使用溶液中少量的TDP-43进行高灵敏度测量的方法将更具优势。在此，为了测定TDP-43与荧光标记的ssDNA的以及结合化学计量比，我们除了采用电泳迁移率变动分析（EMSA）外，还使用了荧光相关光谱法（FCS），它能以单分子灵敏度检测溶液中分子相互作用的扩散减慢情况。通过对谷胱甘肽-S-转移酶和多组氨酸标签双标记的TDP-43进行串联亲和层析，获得了高度纯化的蛋白。FCS成功检测到纯化的TDP-43与TG ssDNA重复序列之间的特异性相互作用，其在纳摩尔范围内。尽管观察到不结合ssDNA的突变体寡聚体，但TDP-43突变体的与野生型并无差异。对每个二聚化的TDP-43/ssDNA复合物的荧光亮度进行分析，以评估它们的结合化学计量比。结果表明，结合FCS和EMSA的分析方法能够精确分析ssDNA识别机制，并且FCS可用于快速定量测定TDP-43与ssDNA或RNA之间的相互作用强度。这些方法将有助于阐明与ALS和FTLD相关的TDP-43变体的底物识别机制。
