Radical changes in the idea of the organization of intracellular space that occurred in the early 2010s made it possible to consider the formation and functioning of so-called membrane-less organelles (MLOs) based on a single physical principle: the liquid–liquid phase separation (LLPS) of biopolymers. Weak non-specific inter- and intramolecular interactions of disordered polymers, primarily intrinsically disordered proteins, and RNA, play a central role in the initiation and regulation of these processes. On the other hand, in some cases, the “maturation” of MLOs can be accompanied by a “liquid–gel” phase transition, where other types of interactions can play a significant role in the reorganization of their structure. In this work, we conducted a bioinformatics analysis of the propensity of the proteomes of two membrane-less organelles, formed in response to stress in the same compartment, for spontaneous phase separation and examined their intrinsic disorder predispositions. These MLOs, amyloid bodies (A-bodies) formed in the response to acidosis and heat shock and nuclear stress bodies (nSBs), are characterized by a partially overlapping composition, but show different functional activities and morphologies. We show that the proteomes of these biocondensates are differently enriched in proteins, and many have high potential for spontaneous LLPS that correlates with the different morphology and function of these organelles. The results of these analyses allowed us to evaluate the role of weak interactions in the formation and functioning of these important organelles.

The formation and function of membrane-less organelles (MLOs) is one of the main driving forces in the molecular life of the cell. These processes are based on the separation of biopolymers into phases regulated by multiple specific and nonspecific inter- and intramolecular interactions. Among the realm of MLOs, a special place is taken by the promyelocytic leukemia nuclear bodies (PML-NBs or PML bodies), which are the intranuclear compartments involved in the regulation of cellular metabolism, transcription, the maintenance of genome stability, responses to viral infection, apoptosis, and tumor suppression. According to the accepted models, specific interactions, such as SUMO/SIM, the formation of disulfide bonds, etc., play a decisive role in the biogenesis of PML bodies. In this work, a number of bioinformatics approaches were used to study proteins found in the proteome of PML bodies for their tendency for spontaneous liquid–liquid phase separation (LLPS), which is usually caused by weak nonspecific interactions. A total of 205 proteins found in PML bodies have been identified. It has been suggested that UBC9, P53, HIPK2, and SUMO1 can be considered as the scaffold proteins of PML bodies. It was shown that more than half of the proteins in the analyzed proteome are capable of spontaneous LLPS, with 85% of the analyzed proteins being intrinsically disordered proteins (IDPs) and the remaining 15% being proteins with intrinsically disordered protein regions (IDPRs). About 44% of all proteins analyzed in this study contain SUMO binding sites and can potentially be SUMOylated. These data suggest that weak nonspecific interactions play a significantly larger role in the formation and biogenesis of PML bodies than previously expected.

Radical changes in the idea of the organization of the intracellular space that occurred in the early 2010-s made it possible to consider the formation and functioning of the so-called membrane-less organelles (MLOs) based on a single physical principle: the liquid-liquid phase separation (LLPS) of biopolymers. Weak nonspecific inter- and intramolecular interactions of disordered polymers, primarily of intrinsically disordered proteins and RNA, play a central role in the initiation and regulation of these processes. On the other hand, in some cases, the "maturation" of MLOs can be accompanied by the "liquid-gel” phase transition, where other types of interactions can play a significant role in reorganization of their structure. In this work, we conducted a bioinformatics analysis of the propensity of the proteomes of two membrane-less organelles formed in response to stress in the same compartment, nucleolus, for spontaneous phase separation and looked at their intrinsic disorder predispositions. These nucleolar MLOs, amyloid bodies (A-bodies) formed in the response to acidosis and heat shock and nuclear stress bodies (nSBs), are characterized by the partially overlapping composition, but show different functional activities and morphologies. We show that the proteomes of these nucleolar biocondensates are differently enriched in proteins, many of with high potential for spontaneous LLPS that correlates with different morphology and function of these organelles. The results of these analyses allowed us to evaluate the role of weak interactions in the formation and functioning of these important organelles.

The formation and functioning of membrane-less organelles (MLOs) is one of the main driving forces in the molecular life of the cell. These processes are based on the separation of biopolymers into phases regulated by multiple specific and nonspecific inter- and intramolecular interactions. Among the realm of MLOs, a special place is taken by the promyelocytic leukemia nuclear bodies (PML-NBs or PML bodies), which are the intranuclear compartments involved in the regulation of cellular metabolism, transcription, maintenance of genome stability, response to viral infection, apoptosis, and tumor suppression. According to the accepted models, specific interactions, such as SUMO/SIM, formation of disulfide bonds, etc., play a decisive role in the biogenesis of PML bodies. In this work, a number of bioinformatics approaches were used to study proteins found in the proteome of PML-bodies for their tendency to spontaneous liquid-liquid phase separation (LLPS), which is usually caused by weak nonspecific interactions. 205 proteins found in PML bodies have been identified. It has been suggested that UBC9, P53, HIPK2, and SUMO1 can be considered as the scaffold proteins of PML bodies. It was shown that more than half of the proteins in the analyzed proteome are capable of spontaneous LLPS, with 85% of the analyzed proteins being intrinsically disordered proteins (IDPs), and remaining 15% being proteins with intrinsically disordered protein regions (IDPRs). About 44% of all proteins analyzed in this study contain SUMO binding sites and can potentially be SOMYylated. These data suggest that weak nonspecific interactions play a significantly larger role in the formation and biogenesis of PML bodies than previously expected.

Ubiquitin-specific protease 7 (USP7/HAUSP) is one of the most studied deubiquitinating enzymes and plays a crucial role in regulating numerous cellular processes, making it a promising therapeutic target. In the nucleus, USP7 partially colocalizes with PML nuclear bodies (PML-NB)—multifunctional membraneless organelles involved in post-translational modifications and protein complexes assembly. The molecular basis and functional significance of this association remain uncharacterized. In this study, comparison of USP7 and PML interactomes revealed a significant overlap of 166 shared proteins. Functional enrichment analysis showed that USP7 and PML may operate within a common molecular context related to transcriptional regulation, chromatin remodeling, and DNA damage responses. Furthermore, these processes are also linked to cellular senescence and human aging (CellAge and GenAge databases). Focused analysis of overlaps between the USP7 interactome and core PML-NB proteins identified 61 proteins forming a dense “small-world” network. Most are prone to liquid–liquid phase separation, are intrinsically disordered, and serve as substrates for SUMOylation or ubiquitination. These findings not only expand our understanding of the molecular functions of USP7 but also highlight PML-NB as an important cellular context for investigating mechanisms associated with USP7 activity.