Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave., 194064 St. Petersburg, Russia.
Almazov National Medical Research Centre, Institute of Hematology, 197341 St. Petersburg, Russia.
Int J Mol Sci. 2022 Jan 30;23(3):1613. doi: 10.3390/ijms23031613.
In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4-6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid-liquid phase separation (LLPS) leading to the formation of PML bodies.
在这项工作中，我们进行了全长 PML 异构体及其 C 末端结构域在有或没有内源性 PML 存在的情况下形成 PML 体的比较研究。基于对 PML 异构体氨基酸序列中固有无序倾向分布的分析，从氨基酸残基 395 开始的区域（即由外显子 4-6 编码的序列）被指定为这些蛋白质的 C 末端结构域。我们证明，每个全长核 PML 异构体在没有其他 PML 异构体的情况下都能够形成核液滴隔间。这些液滴与野生型细胞中观察到的核质交换具有接近的动态特征。只有 PML-II 和 PML-V 异构体的 C 末端结构域能够被包含在内源性 PML 体的组成中，同时部分分布在核质中。PML-II 异构体的 C 末端结构域形成的液滴是动态的液滴隔间，无论是否存在内源性 PML。PML-V 的 C 末端结构域在 PML 敲除细胞中形成动态的液滴隔间，但当 PML-V 异构体的 C 末端插入现有的内源性 PML 体时，该蛋白的分子不再与核质交换。实验表明，破坏 PML 泛素化的 K490R 取代促进 PML-II 和 PML-V 异构体的 C 末端结构域在内源性 PML 敲除 HeLa 细胞中的弥散分布，但不在野生型细胞中。这些数据表明，PML-II 和 PML-V 异构体的 C 末端结构域具有形成动态液滴样隔间的能力，而与 PML 的有序 N 端 RBCC 基序无关。这表明 PML 异构体的 C 末端结构域之间的非特异性相互作用在引发液-液相分离（LLPS）从而形成 PML 体方面起着重要作用。
