Protein interactome of 3′,5′‐cAMP reveals its role in regulating the actin cytoskeleton
Protein interactome of 3′,5′‐cAMP reveals its role in regulating the actin cytoskeleton
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3-,5-2-cAMP蛋白相互作用组揭示其调节肌动蛋白细胞骨架的作用
DOI：
10.1111/tpj.16313
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Tsiavaliaris, Georgios
中科院分区：
文献类型：
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作者：
Figueroa, Nicolás E.;Franz, Peter;Luzarowski, Marcin;Martinez‐Seidel, Federico;Moreno, Juan C.;Childs, Dorothee;Ziemblicka, Aleksandra;Sampathkumar, Arun;Andersen, Tonni Grube;Tsiavaliaris, Georgios
蛋白质相互作用物的鉴定非常适合小分子的功能表征。 3',5'-cAMP 是一种进化上古老的信号代谢物，在植物中基本上未被表征。为了深入了解 3',5'-cAMP 的生理作用，我们使用化学蛋白质组学方法，即热蛋白质组分析 (TPP)，来公正地鉴定 3',5'-cAMP 蛋白靶标。 TPP 测量配体结合后蛋白质热稳定性的变化。全面的蛋白质组学分析得出了 51 种蛋白质的列表，这些蛋白质在与 3',5'-cAMP 一起孵育后，其热稳定性发生了显着改变。该列表包含代谢酶、核糖体亚基、翻译起始因子以及与植物生长调节相关的蛋白质，例如细胞分裂周期 48。为了在功能上验证所获得的结果，我们重点关注 3',5'-cAMP 在调节肌动蛋白细胞骨架中的作用，这表明 51 种已识别蛋白质中存在肌动蛋白。 3',5'-cAMP 补充通过诱导肌动蛋白成束影响肌动蛋白组织。与这些结果一致，通过喂养或通过 3',5'-cAMP 代谢的化学调节获得的 3',5'-cAMP 水平的增加足以部分挽救肌动蛋白 2 actin7 突变体的短下胚轴表型，肌动蛋白水平严重受损。观察到的拯救是针对 3',5'-cAMP 的，正如使用位置异构体 2',3'-cAMP 所证明的那样，对于植物细胞报告的纳摩尔 3',5'-cAMP 浓度也是如此。3',5'-cAMP-肌动蛋白配对的体外表征反对肌动蛋白和 3',5'-cAMP 之间的直接相互作用。讨论了 3',5'-cAMP 影响肌动蛋白动力学的替代机制，例如通过干扰钙信号传导。总之，我们的工作提供了特定的资源，3',5'-cAMP 相互作用组，以及对植物中 3',5'-cAMP 介导的调节的功能洞察。
Identification of protein interactors is ideally suited for the functional characterization of small molecules. 3′,5′‐cAMP is an evolutionary ancient signaling metabolite largely uncharacterized in plants. To tap into the physiological roles of 3′,5′‐cAMP, we used a chemo‐proteomics approach, thermal proteome profiling (TPP), for the unbiased identification of 3′,5′‐cAMP protein targets. TPP measures shifts in the protein thermal stability upon ligand binding. Comprehensive proteomics analysis yielded a list of 51 proteins significantly altered in their thermal stability upon incubation with 3′,5′‐cAMP. The list contained metabolic enzymes, ribosomal subunits, translation initiation factors, and proteins associated with the regulation of plant growth such as CELL DIVISION CYCLE 48. To functionally validate obtained results, we focused on the role of 3′,5′‐cAMP in regulating the actin cytoskeleton suggested by the presence of actin among the 51 identified proteins. 3′,5′‐cAMP supplementation affected actin organization by inducing actin‐bundling. Consistent with these results, the increase in 3′,5′‐cAMP levels, obtained either by feeding or by chemical modulation of 3′,5′‐cAMP metabolism, was sufficient to partially rescue the short hypocotyl phenotype of theactin2 actin7mutant, severely compromised in actin level. The observed rescue was specific to 3′,5′‐cAMP, as demonstrated using a positional isomer 2′,3′‐cAMP, and true for the nanomolar 3′,5′‐cAMP concentrations reported for plant cells.In vitrocharacterization of the 3′,5′‐cAMP–actin pairing argues against a direct interaction between actin and 3′,5′‐cAMP. Alternative mechanisms by which 3′,5′‐cAMP would affect actin dynamics, such as by interfering with calcium signaling, are discussed. In summary, our work provides a specific resource, 3′,5′‐cAMP interactome, as well as functional insight into 3′,5′‐cAMP‐mediated regulation in plants.