ABPP+分子对接+MST微量热泳动技术揭示FBP作用靶点

1. 探针制备：活性分子探针的设计合成，以及探针活性鉴定

2. 将探针与总蛋白孵育（或与细胞孵育后提取总蛋白）

3. 上述孵育完成后，先后进行UV照射（使活性分子与靶蛋白共价相连）、 点击化学反应（给活性分子引入报告基团，如生物素）

4. 通过报告基团对靶蛋白进行富集鉴定

ABPP实验基本流程图

文献案例解读

主要研究结果

图1 光亲和FBP探针（PA-FBP）的化学合成路线

ALDH2 (ab87451, Abcam) was labeled on lysine residues using the Protein Labeling   Kit RED-NHS 2nd Generation (NanoTemper Technologies). Measurements were carried   out in MST-buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.05% Tween-20). The labeled   protein was mixed with FBP and incubated for 5 min at room temperature before being   loaded into Monolith NT.115 Capillaries (NanoTemper Technologies). Samples were then   transferred into the Monolith NT.115 device and MST experiments were carried out at   Nano-red. MST was measured using a Monolith NT.115 instrument (NanoTemper   Technologies) at an ambient temperature of 23.5 oC. Instrument parameters were 20%   excitation power and Medium MST power. Error bars = SD. from two independent   experiments.

Fluorescence imaging

To determine the subcellular localization of the PA-FBP, HepG2 cells were plated in 8well coverslips (Cellvis, C8-1.5H-N) overnight to reach 70% confluence. The growth   medium was aspirated, and the cells were incubated with 10 mM PA-FBP at 37 °C for 90   min. The cells were washed with PBS for three times, and the cells was exposed to 365nm UV light for 5 min on ice, as described above. Cells were treated with 0.1% TritonX100/PBS and then were fixed with 4% Paraformaldehyde Fix Solution for 15 min. Fixed   cells were washed 3 × 5 min with PBS. The cells were reacted with 50 µM CuSO4/BTTAA (1:6), 50 µM TMARA-N3 (82561-66-9), and 2.5 mM sodium ascorbate for 15 min at room temperature in the dark. The reaction mixture was aspirated, and the cells were washed 3 × 5 min with PBS. The fixed cells were blocked at room temperature for 30 min, and the cells were washed 3 × 5 min with PBS. The cells incubated with primary antibodies at room temperature for 1 h followed by incubation with secondary antibodies, Hoechst and MitoTracker Deep Red at room temperature for 30 min. The cells were washed 3 × 5 min with PBST and the coverslips were then imaged on a Zeiss LSM880 confocal microscope.

图5 FBP与ALDH2的结合位点鉴定

分子对接方法

The crystal structure of human ALDH2 (in complex with NAD+ and PEG MME 550, PDB:   8DR9), was uploaded from the RCSB database at a resolution of 1.50 Å. Molecular docking   simulations were carried out using SYBYL to determine the interactions between FBP and   ALDH2 and identify the binding site of FBP on ALDH2. The docking complex was   demonstrated by PyMOL and Discovery Studio.

活细胞中FBP-ALDH2-ROS轴的确定

研究者通过流式细胞实验发现，FBP以剂量依赖性方式增加细胞内ROS水平（图6A）。在HepG2细胞中敲低ALDH2同样导致ROS水平上升（图6B），而FBP引起的ROS增加可以通过ALDH2激活剂Alda-1恢复（图6C）。这表明FBP通过抑制ALDH2增加细胞内ROS水平。荧光成像显示，FBP浓度增加导致线粒体明显碎片化（图6D），这是由于FBP引起的氧化应激相关的剂量依赖性线粒体碎片化（图6E，F）。在敲低ALDH2的HepG2细胞中，FBP诱导的线粒体碎片化程度与正常细胞相同（图6G）。综上，FBP抑制ALDH2活性导致ROS水平上升和线粒体形态改变。

图6 HepG2细胞中FBP-ALDH2-ROS轴的测定

研究总结

参考文献