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.

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg 2+ -dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg 2+ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidinepurine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

The discovery of intrinsically disordered proteins (IDPs) that do not have an ordered structure and nevertheless perform essential functions has opened a new era in the understanding of cellular compartmentalization. It threw the bridge from the mostly mechanistic model of the organization of the living matter to the idea of highly dynamic and functional "soft matter". This paradigm is based on the notion of the major role of liquid-liquid phase separation (LLPS) of biopolymers in the spatial-temporal organization of intracellular space. The LLPS leads to the formation of self-assembled membrane-less organelles (MLOs). MLOs are multicomponent and multifunctional biological condensates, highly dynamic in structure and composition, that allow them to fine-tune the regulation of various intracellular processes. IDPs play a central role in the assembly and functioning of MLOs. The LLPS importance for the regulation of chemical reactions inside the cell is clearly illustrated by the reorganization of the intracellular space during stress response. As a reaction to various types of stresses, stress-induced MLOs appear in the cell, enabling the preservation of the genetic and protein material during unfavourable conditions. In addition, stress causes structural, functional, and compositional changes in the MLOs permanently present inside the cells. In this review, we describe the assembly of stress-induced MLOs and the stress-induced modification of existing MLOs in eukaryotes, yeasts, and prokaryotes in response to various stress factors.