Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology & Biochemistry, Medical University of Graz, Graz, Austria.
BioTechMed-Graz, Graz, Austria.
Protein Sci. 2021 Jul;30(7):1438-1453. doi: 10.1002/pro.4123. Epub 2021 May 22.
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.
天然无序蛋白质和含有天然无序区域的蛋白质在真核生物的蛋白质组中高度丰富，广泛参与重要的生物学功能。最近，它们在生物分子凝聚物的组织中的作用变得明显，并且在几种神经疾病中存在失调。目前，大多数涉及这些蛋白质的研究都是在体外进行的，并且使用纯化的蛋白质。鉴于在细胞中，形成凝聚物的蛋白质暴露于高、毫摩尔浓度的细胞代谢物中，我们旨在揭示细胞代谢物与代表性凝聚物形成蛋白之间的相互作用。在这里，我们使用富含精氨酸-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，研究了细胞代谢组与 CIRBP 的结合。我们发现，除核苷酸外，大多数高丰度的细胞代谢物并不直接与 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 CIRBP 驱动的 RNA 相分离。因此，在未来的 RG/RGG 和其他凝聚物形成蛋白的体外液-液相分离研究中，包括细胞代谢物可能是有益的，以便更好地模拟细胞环境。
