Vascular Biology Program and Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
Silicon Therapeutics, Boston, MA, USA.
Sci Rep. 2019 Mar 15;9(1):4632. doi: 10.1038/s41598-019-40929-4.
Antizyme inhibitor (AZIN) stimulates cell proliferation by binding to and sequestering the cell cycle suppressor antizyme. Despite the important role of the antizyme-AZIN protein-protein interaction (PPI) in cell cycle regulation, there are no assays for directly measuring the binding of AZIN to antizyme that are amenable to high throughput screening. To address this problem, we developed and validated a novel antizyme-AZIN intramolecular FRET sensor using clover and mRuby2 fluorescent proteins. By introducing alanine mutations in the AZIN protein, we used this sensor to probe the PPI for key residues governing the binding interaction. We found that like many PPIs, the energy of the antizyme-AZIN binding interaction is distributed across many amino acid residues; mutation of individual residues did not have a significant effect on disrupting the PPI. We also examined the interaction between Clover-AZIN and antizyme-mRuby2 in cells. Evidence of a direct interaction between Clover-AZIN and antizyme-mRuby2 was observed within cells, validating the use of this FRET sensor for probing intracellular antizyme-AZIN PPI. In conclusion, we have developed and optimized a FRET sensor which can be adapted for high throughput screening of either in vitro or intracellular activity.
抗酶抑制剂 (AZIN) 通过与细胞周期抑制因子抗酶结合并将其隔离来刺激细胞增殖。尽管抗酶-AZIN 蛋白-蛋白相互作用 (PPI) 在细胞周期调控中起着重要作用，但目前还没有直接测量 AZIN 与抗酶结合的适用于高通量筛选的测定方法。为了解决这个问题，我们使用报春花和 mRuby2 荧光蛋白开发并验证了一种新型的抗酶-AZIN 分子内 FRET 传感器。通过在 AZIN 蛋白中引入丙氨酸突变，我们使用该传感器来探测关键残基，以探测控制结合相互作用的 PPI。我们发现，与许多 PPI 一样，抗酶-AZIN 结合相互作用的能量分布在许多氨基酸残基上；单个残基的突变对破坏 PPI 没有显著影响。我们还在细胞中检查了 Clover-AZIN 和 antizyme-mRuby2 之间的相互作用。在细胞内观察到 Clover-AZIN 和 antizyme-mRuby2 之间的直接相互作用的证据，验证了该 FRET 传感器用于探测细胞内抗酶-AZIN PPI 的用途。总之，我们已经开发并优化了一种 FRET 传感器，可适用于体外或细胞内活性的高通量筛选。
