High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells.
High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells.
复制标题
DOI：
10.1371/journal.pone.0289707
复制
发表时间：
2023
期刊：
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
我们以前已经证明，人肝型磷酸果糖激酶1（PFK 1）招募其他速率决定酶在葡萄糖代谢组织多酶代谢组件，称为glucosomes，在人体细胞。然而，葡萄糖体如何可逆地组装和分解以功能性地调节葡萄糖代谢，从而有助于人类细胞生物学，这在很大程度上仍然是难以捉摸的。我们开发了一种高含量的定量高通量筛选（qHTS）试验，以确定调控机制，控制PFK 1介导的葡萄糖体组件从稳定转染的HeLa Tet-On细胞。初始qHTS具有针对激酶抑制剂富集集合的激酶活性化合物库。因此，三种化合物，已知抑制细胞周期蛋白依赖性激酶2，核糖体蛋白S6激酶和极光激酶A，分别进行了鉴定，并在高分辨率荧光单细胞显微镜下进一步验证。随后使用小发夹RNA的敲除研究进一步证实了Aurora激酶A对HeLa细胞中PFK 1组装体形成的积极作用。重要的是，这里所有鉴定的蛋白激酶都被研究为控制人类细胞中细胞周期进程的一个特定级联的关键信号节点。总的来说，我们的qHTS方法解开了调节人类细胞中PFK 1介导的糖体组装形成的细胞周期相关信号网络。
We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells.
登录
查看更多内容
DOI：
10.1016/j.jbc.2022.101675
发表时间：
2022-03
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Jeon M;Chauhan KM;Szeto GL;Kyoung M;An S
通讯作者：
An S
影响因子：
7.7
作者：
Fuller, Gregory G.;Han, Ting;Kim, John K.
通讯作者：
Kim, John K.
影响因子：
11.2
作者：
Ali S;Heathcote DA;Kroll SH;Jogalekar AS;Scheiper B;Patel H;Brackow J;Siwicka A;Fuchter MJ;Periyasamy M;Tolhurst RS;Kanneganti SK;Snyder JP;Liotta DC;Aboagye EO;Barrett AG;Coombes RC
通讯作者：
Coombes RC
影响因子：
3.7
作者：
Dranchak P;MacArthur R;Guha R;Zuercher WJ;Drewry DH;Auld DS;Inglese J
通讯作者：
Inglese J
影响因子：
46.9
作者：
通讯作者：
--