There are six actin isoforms encoded by different genes. Actin isoforms have similar sequences but different functions and are only partially interchangeable. Sequences of muscle and non-muscle actins (α- and β-isoforms) show largest differences. Spectral analysis revealed that α- and β-actins are rather similar. β-Actin was shown to form inactivated state, similar to that of α-actin. Inactivated actin is a unique stable state of muscle and non-muscle actins.

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.

Tetrapyrrole-binding proteins are promising near-infrared fluorescent markers. We recently showed that the near-infrared fluorescent protein iRFP713, derived from bacteriophytochrome from Rhodopseudomonas palustris (RpBphP2), when mutated (iRFP713/C15S/V254C; hereafter referred to as iRFP) and complexed with phycocyanobilin (PCB) as a chromophore (iRFP-PCB), has a significantly higher fluorescence quantum yield than that of iRFP complexed with biliverdin (iRFP-BV). Here, we show that iRFP-PCB can be used as a fluorescent biomarker in eukaryotic cells (HEK293T) and that the effective brightness of iRFP-PCB is more than four times higher than that of iRFP-BV. We elucidated the structural basis of iRFP interaction with PCB by determining its crystal structure. Interestingly, we observed both parallel and antiparallel arrangements of iRFP protomers in an asymmetric unit cell. We used molecular dynamics simulations to show that the mobility of the protein and chromophore covalently bound to Cys254 is similar in different assembly states and in complexes with BV or PCB. Overall, the results indicate that PCB is a promising chromophore for the development of new fluorescent biomarkers from bacterial phytochromes and provide a basis for the further engineering of biomarkers from iRFP and related proteins.

Actin is one of the most widespread and most conserved proteins. At the same time, six actin isoforms are known, encoded by different genes. These isoforms differ slightly in amino acid sequence and have similar structures, but differ in localization and functioning. During functioning, actin interacts with a large number of proteins, which are combined according to this feature into a pool of so-called actin-binding proteins. The question arises whether and how the proteins interacting with different actin isoforms differ. Since the pool of actin-binding proteins includes hundreds of proteins, it was logical to use bioinformatics analysis to solve the questions. In this work, it is shown that the functionality of the α-, β-, and γ-actin interactomes differ significantly, but their structural characteristics are close.

Cells are crowded entities, but the intracellular space represents an inhomogeneously crowded environment, where the concentrations of macromolecules (proteins, nucleic acids, etc.) are not uniformly distributed throughout the cell resulting in regions with different levels of crowding. Liquid-liquid phase separation (LLPS)-driven formation of various membrane-less organelles (MLOs) represents a means for the control, regulation, and redistribution of cellular crowded environment. Because MLOs contain the high concentrations of biological macromolecules (proteins and RNAs), often significantly exceeding those of the surrounding cytoplasm or nucleoplasm, their inside represents an overcrowded milieu. It is well-known that the appearance of the stress-induced MLOs represents a reaction to various types of stresses, enabling the protection of the genetic and protein material during hostile conditions. However, stress can also cause structural, functional, and compositional changes in the MLOs, which are constitutively present in the cells, thereby causing the reshuffling of the overcrowded environment. This chapter describes stress-induced changes in several MLOs (nucleolus, Cajal bodies, paraspeckles, nuclear speckles, NELF-Bodies, nucleolar stress bodies, PML-bodies, stress-granules, and Р-bodies) found in the eukaryotic cells.

Trans-activation response (TAR) DNA-binding protein 43 (TDP-43) is an RNA-binding protein involved in the processing, transport, and regulation of mRNA translation. It is distributed in many tissues, including the brain, where it is found mainly in hippocampal neurons. Abnormal localization, hyperphosphorylation, and aggregation of TDP-43 are pathological signs of a group of neurodegenerative diseases known as TDP-43 proteinopathies. Despite the growing understanding of the physiological role of TDP-43 in ensuring neuronal plasticity and the formation of long-term memory, to date, there is no comprehensive data on the molecular and cellular mechanisms of the transformation of functional membraneless organelles (MLOs) containing TDP-43 into toxic aggregates and the pathogenesis of associated diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This review is devoted to highlighting the role of MLOs in the formation of irreversible aggregates, the role of TDP-43 in the formation of MLOs and their relationship with pathological forms of TDP-43, most often found in people suffering from neurodegenerative diseases.

Recent scientific findings highlight the crucial role of liquid-liquid phase separation (LLPS) in the compartmentalization of enzyme systems. A synthesis of the extant data indicates that lipid rafts and condensates formed by phase separation are also implicated in signal transduction, including participation in recognized receptor systems. The intrinsically disordered nature of many membrane-binding proteins, coupled with their propensity for LLPS, provides condensate formation, which can bind to or form on the membranes. Moreover, condensates can form simultaneously on both sides of the membrane at lipid raft regions facilitating signal transmission across the membrane. The finding that LLPS plays a direct role in cell signaling, especially in well-defined transmembrane signaling pathways, represents a substantial, yet largely unrecognized, advancement in understanding of intracellular signal transduction mechanisms.

Despite extensive research, the features associated with the aging phenotype are not all-inclusive and need to be updated on a regular basis to incorporate new findings. We propose to include the dysfunction of membrane-less organelle (MLO) as a new aging hallmark. Special scaffold proteins with a high degree of intrinsic disorder drive the formation of MLOs via the liquid-liquid phase separation (LLPS) process. Aberrant behavior of MLOs was shown to be associated with the pathogenesis of many neurodegenerative diseases. In this work, we challenge the aging through bidirectional bioinformatics analysis of human proteins found in Granulome consisting of 7,264 protein and Ageome containing 1,624 aging-related proteins. The analysis indicates the interconnectivity of MLOs and aging. Approximately 67% of the Ageome are presented in Granulome thereby constituting the Intersectome that include 1,084 proteins showing an enrichment significantly higher than for the random datasets of the same size. Furthermore, for proteins in 10 representative MLOs, we analyzed in detail molecular functions, association with the already known aging hallmarks, and the roles in MLO formation (scaffold, client, or regulator). Cumulatively, our results strengthen the hypothesis that the dysfunction of MLOs can serve as a potent new aging hallmark.

Besides the conventional monomeric globular (G-actin) and polymeric fibrillar (F-actin) forms, actin can adopt a thermodynamically stable inactivated oligomeric state (I-actin) when it loses bound nucleotide and coordinating divalent cation under mild denaturing or stress conditions. However, the supramolecular organization of these assemblies remains poorly understood. Here, using size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) and negative-stain transmission electron microscopy (NS-TEM), we show that heat-inactivated actin (heat-I-actin) forms flat, disk-like oligomers ("beads") that further assemble into linear, unbranched chains. SEC-SAXS reveals strongly elongated particles whose cross-sectional parameters closely match those previously established for I-actin oligomers by hydrodynamic measurements, while NS-TEM directly visualizes bead-like particles of ∼165 Å in diameter that occasionally align into unbranched chains, structures not seen for G- or F-actin. Together with previously published structural insights, these data support a multistage assembly pathway in which I-actin subunits first form dimers, then condense into flat oligomeric beads, which in turn connect into one-dimensional supramolecular chains. We discuss how such assemblies may relate to poorly characterized short oligomers of nuclear actin.

It became clear more than 20 years ago that the nucleolus not only performs the most important biological function of assembling ribonucleic particles but is also a key controller of many cellular processes, participating in cellular adaptation to stress. The nucleolus’s multifunctionality is due to the peculiarities of its biogenesis. The nucleolus is a multilayered biomolecular condensate formed by liquid–liquid phase separation (LLPS). In this review, we focus on changes occurring in the nucleolus during cellular stress, molecular features of the nucleolar response to abnormal and stressful conditions, and the role of long non-coding RNAs transcribed from the intergenic spacer region of ribosomal DNA (IGS rDNA).

In the last decade, much attention is given to study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in response to acidosis and heat shock, and disassemble after the end of stress. One of the proteins involved in the biogenesis of A-bodies, regardless of the type of stress, is Von-Hippel Lindau protein (VHL). Known also as a tumor suppressor, VHL is capable to form amyloid fibrils both in vitro and in vivo in response to environment acidification. As for majority of amyloidogenic proteins fusion with different tags is used for increase of VHL solubility. Here, we first performed AFM-study of fibrils formed by VHL protein and by VHL fused with GST-tag (GST-VHL) at acidic conditions. It was shown that formed by full-length VHL fibrils are short heterogenic structures with persistent length of 2.400 nm and average contour length of 409 nm. GST-tag catalyzes VHL amyloid fibril formation, superimpose chirality, increases length and level of hierarchy, but decreases rigidity of amyloid fibrils. The obtained data indicate that tagging can significantly affect the fibrillogenesis of the target protein.

Natural aging and age-related diseases involve the acceleration of replicative aging, or senescence. Multiple proteins are known to participate in these processes, including the promyelocytic leukemia (PML) protein, which serves as a core component of nuclear-membrane-less organelles known as PML nuclear bodies (PML-NBs). In this work, morphological changes in PML-NBs and alterations in PML protein localization at the transition of primary fibroblasts to a replicative senescent state were studied by immunofluorescence. The fibroblasts were obtained from both healthy donors and donors with premature aging syndromes (ataxia-telangiectasia and Cockayne syndrome). Our data showed an increase in both the size and the number of PML-NBs, along with nuclear enlargement in senescent cells, suggesting these changes could serve as potential cellular aging markers. Bioinformatic analysis demonstrated that 30% of the proteins in the PML interactome and ~45% of the proteins in the PML-NB predicted proteome are directly associated with senescence and aging processes. These proteins are hypothesized to participate in post-translational modifications and protein sequestration within PML-NBs, thereby influencing transcription factor regulation, DNA damage response, and negative regulation of apoptosis. The findings confirm the significant role of PML-NBs in cellular aging processes and open new avenues for investigating senescence mechanisms and age-associated diseases.

The analysis of cryo-electron tomography images of human and rat mitochondria revealed that the mitochondrial matrix is at least as crowded as the cytosol. To mitigate the crowding effects, metabolite transport in the mitochondria primarily occurs through the intermembrane space, which is significantly less crowded. The scientific literature largely ignores how enzyme systems and metabolite transport are organized in the crowded environment of the mitochondrial matrix. Under crowded conditions, multivalent interactions carried out by disordered protein regions (IDRs), may become extremely important. We analyzed the human mitochondrial proteome to determine the presence and physiological significance of IDRs. Despite mitochondrial proteins being generally more ordered than cytosolic or overall proteome proteins, disordered regions plays a significant role in certain mitochondrial compartments and processes. Even in highly ordered enzyme systems, there are proteins with long IDRs. Some IDRs act as binding elements between highly ordered subunits, while the roles of others are not yet established. Mitochondrial systems, like their bacterial ancestors, rely less on IDRs and more on RNA for LLPS compartmentalization. More evolutionarily advanced subsystems that enable mitochondria-cell interactions contain more IDRs. The study highlights the crucial and often overlooked role played by IDRs and noncoding RNAs in mitochondrial organization.

Actin is a key protein of the muscle contraction system [...]

The multifunctional promyelocytic leukemia protein (PML) is involved in the regulation of various cellular processes in both physiological and pathological conditions. Specifically, PML is one of the inositol-1,4,5-trisphosphate receptors (IP3Rs) activity regulators and can influence Ca2+ transport from the endoplasmic reticulum (ER) to mitochondria. In this work, the effects of PML knockout on calcium homeostasis in the cytosol, ER, and mitochondria of HeLa cells were studied upon stimulation with histamine, which induces Ca2+ mobilization from the ER via IP3Rs. We utilized calcium indicators with different subcellular localizations, including synthetic dyes Fura-2 (cytosolic), Xrhod-5F (mitochondrial), and protein sensor R-CEPIAer (ER), as well as mitochondrial potential-sensitive probes Rh123 and TMRM. Our results show that PML knockout induced changes in HeLa cell and mitochondrial morphology, slightly decreased basal and integral Ca2+ levels, enhanced mitochondrial Ca2+ uptake from the cytoplasm, and maintained residual mitochondrial potential after depolarization. Additionally, it reduced the Ca2+ pool in ER membranes not associated with histamine receptor activation and, consequently, IP3Rs. These findings suggest that changes in calcium ion transport due to PML knockout in HeLa cells affect mitochondrial activity.

In addition to the well-known monomeric globular (G-actin) and polymeric fibrillar (F-actin) forms, actin can exist in the so-called inactivated form (I-actin). Hsp70 chaperon, prefoldin, and CCT chaperonin are required to obtain native globular state. In contrast, I-actin is spontaneously formed in the absence of intracellular folding machinery. I-actin can be obtained from G-actin by elimination of divalent ion, incubation in presence of small concentrations of denaturants, and by heat exposure. Since G-actin is a quasi-stationary, thermodynamically unstable form, it can gradually transform into inactivated state in the absence of chelating/denaturating agents or heat exposure, but the transition is much slower. I-actin was shown to associate into oligomers up to the molecular weight of 14-16 G-actin monomers, though the structure of these oligomers remains uncharacterized. This study employs small-angle X-ray scattering to reveal novel insights into the oligomerization process of such spontaneously formed inactivated actin. These oligomers are differentiated from F-actin through comparative analysis, highlighting a unique oligomerization pathway.