School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong, China.
Int J Mol Sci. 2018 Mar 7;19(3):763. doi: 10.3390/ijms19030763.
Nucleic acid aptamers hold promise as therapeutic tools for specific, tailored inhibition of protein targets with several advantages when compared to small molecules or antibodies. Nuclear WW domain containing E3 ubiquitin ligase 1 (WWP1) ubiquitin ligase poly-ubiquitinates Runt-related transcription factor 2 (Runx2), a key transcription factor associated with osteoblast differentiation. Since WWP1 and an adapter known as Schnurri-3 are negative regulators of osteoblast function, the disruption of this complex has the potential to increase bone deposition for osteoporosis therapy. Here, we develop new DNA aptamers that bind and inhibit WWP1 then investigate efficacy in an osteoblastic cell culture. DNA aptamers were selected against three different truncations of the HECT domain of WWP1. Aptamers which bind specifically to a C-lobe HECT domain truncation were observed to enrich during the selection procedure. One particular DNA aptamer termed C3A was further evaluated for its ability to bind WWP1 and inhibit its ubiquitination activity. C3A showed a low µM binding affinity to WWP1 and was observed to be a non-competitive inhibitor of WWP1 HECT ubiquitin ligase activity. When SaOS-2 osteoblastic cells were treated with C3A, partial localization to the nucleus was observed. The C3A aptamer was also demonstrated to specifically promote extracellular mineralization in cell culture experiments. The C3A aptamer has potential for further development as a novel osteoporosis therapeutic strategy. Our results demonstrate that aptamer-mediated inhibition of protein ubiquitination can be a novel therapeutic strategy.
核酸适体作为治疗工具，具有特异性、靶向性抑制蛋白质靶标，与小分子或抗体相比具有几个优势。核 WW 结构域包含 E3 泛素连接酶 1（WWP1）泛素连接酶多泛素化 Runt 相关转录因子 2（Runx2），Runx2 是与成骨细胞分化相关的关键转录因子。由于 WWP1 和一种称为 Schnurri-3 的衔接蛋白是成骨细胞功能的负调节剂，因此破坏这种复合物有可能增加骨质疏松症治疗的骨沉积。在这里，我们开发了新的 DNA 适体，它们可以结合并抑制 WWP1，然后在成骨细胞培养物中研究其疗效。针对 WWP1 的 HECT 结构域的三种不同截断物选择了 DNA 适体。在选择过程中观察到结合到 C lobe HECT 结构域截断物的适体富集。一种称为 C3A 的特定 DNA 适体进一步评估了其结合 WWP1 和抑制其泛素化活性的能力。C3A 对 WWP1 表现出低 µM 的结合亲和力，并且被观察为 WWP1 HECT 泛素连接酶活性的非竞争性抑制剂。当 SaOS-2 成骨细胞用 C3A 处理时，观察到部分核定位。在细胞培养实验中，C3A 适体也被证明能够特异性促进细胞外矿化。C3A 适体具有作为新型骨质疏松症治疗策略进一步开发的潜力。我们的结果表明，适体介导的蛋白质泛素化抑制可能是一种新的治疗策略。
