Stress-granules, P-bodies, and cell aging: A bioinformatics study.

At the molecular level, aging is often accompanied by dysfunction of stress-induced membrane-less organelles (MLOs) and changes in their physical state (or material properties). In this work, we analyzed the proteins included in the proteome of stress granules (SGs) and P-bodies for their tendency to transform the physical state of these MLOs. Particular attention was paid to the proteins whose gene expression changes during replicative aging. It was shown that the proteome of the studied MLOs consists of intrinsically disordered proteins, 30-40% of which are potentially capable of liquid-liquid phase separation (LLPS). Proteins whose gene expression changes during the transition of human cells to a senescent state make up about 20% of the studied proteomes. There is a statistically significant increase in the number of positively charged proteins in both datasets studied compared to the complete proteomes of these organelles. An increase in the relative content of DNA-, but not RNA-binding proteins, was also found in the SG dataset with senescence-related processes. Among SGs proteins potentially involved in senescent processes, there is an increase in the abundance of potentially amyloidogenic proteins compared to the whole proteome. Proteins common to SGs and P-bodies, potentially involved in processes associated with senescence, form clusters of interacting proteins. The largest cluster is represented by RNA-binding proteins involved in RNA processing and translation regulation. These data indicate that SG proteins, but not proteins of P-bodies, are more likely to transform the physical state of MLOs. Furthermore, these MLOs can participate in processes associated with aging in a coordinated manner.

Nucleolar- and Nuclear-Stress-Induced Membrane-Less Organelles: A Proteome Analysis through the Prism of Liquid-Liquid Phase Separation.

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.

Bioinformatics Analysis of Actin Interactome: Characterization of the Nuclear and Cytoplasmic Actin-Binding Proteins.

Actin is present in the cytoplasm and nucleus of every eukaryotic cell. In the cytoplasm, framework and motor functions of actin are associated with its ability to polymerize to form F-actin. In the nucleus, globular actin plays a significant functional role. For a globular protein, actin has a uniquely large number of proteins with which it interacts. Bioinformatics analysis of the actin interactome showed that only a part of actin-binding proteins are both cytoplasmic and nuclear. There are proteins that interact only with cytoplasmic, or only with nuclear actin. The first pool includes proteins associated with the formation, regulation, and functioning of the actin cytoskeleton predominate, while nuclear actin-binding proteins are involved in the majority of key nuclear processes, from regulation of transcription to DNA damage response. Bioinformatics analysis of the structure of actin-binding proteins showed that these are mainly intrinsically disordered proteins, many of which are part of membrane-less organelles. Interestingly, although the number of intrinsically disordered actin-binding proteins in the nucleus is greater than in the cytoplasm, the drivers for the formation of the membrane-less organelles in the cytoplasm are significantly (four times) greater than in the nucleus.

On the Prevalence and Roles of Proteins Undergoing Liquid-Liquid Phase Separation in the Biogenesis of PML-Bodies.

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.