A cell-based high-content screen identifies isocotoin as a small molecule inhibitor of the meiosis-specific MEIOB-SPATA22 complex†.
A cell-based high-content screen identifies isocotoin as a small molecule inhibitor of the meiosis-specific MEIOB-SPATA22 complex†.
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DOI：
10.1093/biolre/ioaa062
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发表时间：
2020-08-04
影响因子：
3.6
通讯作者：
Wang PJ
中科院分区：
文献类型：
--
作者：
Xu Y;Liu R;Leu NA;Zhang L;Ibragmova I;Schultz DC;Wang PJ
MEIOB和SPATA22是减数分裂特异性蛋白，它们相互作用，对减数分裂重组和生育至关重要。MEIOB中的天冬氨酸383 （D383）在生物化学研究中与SPATA22相互作用至关重要。在这里，我们报告了遗传学研究证实了D383对小鼠MEIOB功能的要求。由于MEIOB和SPATA22蛋白在睾丸中的耗竭，MeiobD383A/D383A小鼠表现出减数分裂停滞。我们开发了一种基于细胞的双分子荧光互补（BiFC）实验，其中MEIOB和SPATA22融合分裂YFP片段，它们在培养细胞中的共表达导致MEIOB - SPATA22二聚化和荧光团重建。正如预期的那样，破坏相互作用的D383A取代导致在BiFC检测中没有YFP荧光。小分子文库的高通量筛选以0.7%的速率确定候选命中化合物。Isocotoin是一种天然产物库中的hit化合物，可以抑制MEIOB-SPATA22相互作用，并以剂量依赖的方式促进其在HEK293细胞中的降解。因此，BiFC检测可以用于筛选破坏蛋白-蛋白相互作用或促进减数分裂特异性蛋白降解的小分子抑制剂。一种基于细胞的双分子荧光互补筛选试验确定了促进MEIOB和SPATA22降解的异戊二醇。
MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The MeiobD383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB–SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB–SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein–protein interactions or promote degradation of meiosis-specific proteins. A cell-based bimolecular fluorescence complementation screening assay identifies isocotoin that promotes degradation of MEIOB and SPATA22.
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影响因子：
64.5
作者：
Matzuk MM;McKeown MR;Filippakopoulos P;Li Q;Ma L;Agno JE;Lemieux ME;Picaud S;Yu RN;Qi J;Knapp S;Bradner JE
通讯作者：
Bradner JE
DOI：
10.1073/pnas.1635054100
发表时间：
2003-10-14
影响因子：
11.1
作者：
Schultz, N;Hamra, FK;Garbers, DL
通讯作者：
Garbers, DL
影响因子：
21.3
作者：
Connell, P;Ballinger, CA;Patterson, C
通讯作者：
Patterson, C
影响因子：
4.6
作者：
Ishishita S;Matsuda Y;Kitada K
通讯作者：
Kitada K
影响因子：
4.8
作者：
Paik, Jisun;Haenisch, Michael;Amory, John K.
通讯作者：
Amory, John K.