Wang Xin, Cui Jin, Li Wei, Zeng Xianglu, Zhao Jian, Pei Gang
State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Graduate School, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
J Alzheimers Dis. 2015;47(4):927-37. doi: 10.3233/JAD-150313.
Elucidation of γ-secretase structure and dynamic conformational changes is of importance to drug discovery targeting this enzyme. Electron microscopy analyses provided important structural information, but the dynamic changes of γ-secretase in cells need to be explored further. We found that PS1 internal fluorescence resonance energy transfer (FRET) probes can incorporate into γ-secretase complex and possess secretase activity. Our results from fluorescence lifetime image microscopy (FLIM) and acceptor photobleaching FRET show different PS1 internal FRET when PS1 fluorescent probes expressed alone or with other secretase subunits Aph1aL, Nicastrin, and Pen2, indicating that PS1 internal FRET could be applied for probing conformational change of γ-secretase complex. Further, we accessed whether γ-secretase activity interfering compounds induced different conformational changes of PS1. Our results show that both γ-secretase modulators and inhibitors affect PS1 internal FRET but in different manners. These results demonstrate that FLIM and acceptor photobleaching FRET could be applied to monitor different PS1 conformational changes in γ-secretase.
阐明γ-分泌酶的结构和动态构象变化对于以该酶为靶点的药物发现至关重要。电子显微镜分析提供了重要的结构信息，但γ-分泌酶在细胞中的动态变化仍需进一步探索。我们发现PS1内部荧光共振能量转移（FRET）探针可以整合到γ-分泌酶复合物中并具有分泌酶活性。我们通过荧光寿命成像显微镜（FLIM）和受体光漂白FRET得到的结果显示，当PS1荧光探针单独表达或与其他分泌酶亚基Aph1aL、Nicastrin和Pen2一起表达时，PS1内部FRET不同，这表明PS1内部FRET可用于探测γ-分泌酶复合物的构象变化。此外，我们研究了γ-分泌酶活性干扰化合物是否会诱导PS1的不同构象变化。我们的结果表明，γ-分泌酶调节剂和抑制剂均会影响PS1内部FRET，但方式不同。这些结果表明，FLIM和受体光漂白FRET可用于监测γ-分泌酶中PS1的不同构象变化。
