通过光亲和标记分析活细胞中多胺-蛋白相互作用。

IF 3.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Maciej Zakrzewski, Zuzanna Sas, Benjamin Cocom-Chan, Moh Egy Rahman Firdaus, Marcin Kałek, Karolina Szczepanowska, Piotr Gerlach, Anna Marusiak, Remigiusz A Serwa
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引用次数: 0

摘要

多胺是人体必需的代谢物,在调节关键细胞过程中起着至关重要的作用。虽然先前的研究表明多胺通过非共价相互作用调节蛋白质功能,但缺乏可靠的分析方法限制了对活细胞中这些相互作用的系统鉴定。为了解决这一挑战,我们合成了一系列新的光亲和探针,并将它们应用于模型细胞系,鉴定了400多种具有显著多胺类似物结构依赖性特异性的推定蛋白质相互作用物。对探针修饰肽的分析揭示了数十种蛋白质结合物的光交联位点,并证明除了一种探针(精胺类似物)外,所有探针都是细胞内稳定的。通过凝胶内荧光扫描显示了这些探针的相互作用谱,并用荧光显微镜检查了它们的亚细胞定位。亚精胺类似物与核质中的蛋白质相互作用,与核仁和核斑点蛋白共定位,以及在细胞质中。相比之下,二胺类似物定位于高尔基体附近的囊泡状结构,这意味着不同类型的多胺表现出对特定细胞区室的倾向。值得注意的是,亚精胺类似物优先结合含有酸性延伸的蛋白质,通常位于内在无序的区域。针对这样的一个案例,我们提供了G3BP1/2和亚精胺类似物之间直接相互作用的细胞内证据,并提出了这种相互作用影响应力-颗粒动力学的假设。总的来说,本研究提供了活细胞中多胺类似物-蛋白质相互作用的全面概况,为其在细胞生理学中的作用提供了有价值的见解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Profiling polyamine-protein interactions in live cells through photoaffinity labeling.

Polyamines are essential metabolites that play a crucial role in regulating key cellular processes. While previous studies have shown that polyamines modulate protein function through non-covalent interactions, the lack of robust analytical methods has limited the systematic identification of these interactions in living cells. To address this challenge, we synthesized a series of novel photoaffinity probes and applied them to a model cell line, identifying over 400 putative protein interactors with remarkable polyamine analog structure-dependent specificity. Analysis of probe-modified peptides revealed photocrosslinking sites for dozens of protein binders and demonstrated that all but one of the probes, the spermine analog, were intracellularly stable. The interaction profiles of these probes were visualized through in-gel fluorescence scanning, and their subcellular localization was examined using fluorescence microscopy. Spermidine analogs interacted with proteins in the nucleoplasm, colocalizing with nucleolar and nuclear-speckle proteins, as well as in the cytoplasm. By contrast, diamine analogs localized to vesicle-like structures near the Golgi apparatus, implying that different polyamine types exhibit a proclivity for specific cellular compartments. Notably, spermidine analogs bound preferentially to proteins containing acidic stretches, often located within intrinsically disordered regions. Focusing on one such case, we provide in-cellulo evidence of direct interactions between G3BP1/2 and spermidine analogs and advance the hypothesis that such interactions influence stress-granule dynamics. Overall, this study provides a comprehensive profile of polyamine analogs-protein interactions in live cells, offering valuable insights into their roles in cellular physiology.

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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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