Intrinsically disordered proteins and proteins containing intrinsically disordered regions are highly abundant in the proteome of eukaryotes and are extensively involved in essential biological functions. More recently, their role in the organization of biomolecular condensates has become evident and along with their misregulation in several neurologic disorders. Currently, most studies involving these proteins are carried out in vitro and using purified proteins. Given that in cells, condensate-forming proteins are exposed to high, millimolar concentrations of cellular metabolites, we aimed to reveal the interactions of cellular metabolites and a representative condensate-forming protein. Here, using the arginine–glycine/arginine–glycine–glycine (RG/RGG)-rich cold inducible RNA binding protein (CIRBP) as paradigm, we studied binding of the cellular metabolome to CIRBP. We found that most of the highly abundant cellular metabolites, except nucleotides, do not directly bind to CIRBP. ATP, ADP, and AMP as well as NAD⁺, NADH, NADP⁺, and NADPH directly interact with CIRBP, involving both the folded RNA-recognition motif and the disordered RG/RGG region. ATP binding inhibited RNA-driven phase separation of CIRBP. Thus, it might be beneficial to include cellular metabolites in in vitro liquid–liquid phase separation studies of RG/RGG and other condensate-forming proteins in order to better mimic the cellular environment in the future.

中文翻译：

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ATP调节RNA驱动的冷诱导RNA结合蛋白相分离

本质上无序的蛋白质和含有本质上无序区域的蛋白质在真核生物的蛋白质组中非常丰富，并且广泛参与基本的生物学功能。最近，它们在生物分子凝聚物组织中的作用已经变得明显，并且它们在几种神经系统疾病中的调节不当。目前，大多数涉及这些蛋白质的研究都是在体外进行的，并使用纯化的蛋白质。鉴于在细胞中，冷凝物形成蛋白暴露于高浓度的细胞代谢物，我们旨在揭示细胞代谢物与代表性的冷凝物形成蛋白的相互作用。在这里，使用富含精氨酸-甘氨酸/精氨酸-甘氨酸-甘氨酸 (RG/RGG) 的冷诱导 RNA 结合蛋白 (CIRBP) 作为范例，我们研究了细胞代谢组与 CIRBP 的结合。我们发现大多数高度丰富的细胞代谢物，除了核苷酸，不直接与 CIRBP 结合。ATP、ADP 和 AMP 以及 NAD⁺、NADH、NADP ⁺和 NADPH 直接与 CIRBP 相互作用，涉及折叠的 RNA 识别基序和无序的 RG/RGG 区域。ATP 结合抑制了 RNA 驱动的 CIRBP 相分离。因此，将细胞代谢物纳入 RG/RGG 和其他冷凝物形成蛋白的体外液 - 液相分离研究可能是有益的，以便在未来更好地模拟细胞环境。