Pitfalls of Using ANS Dye Under Molecular Crowding Conditions.

The 1-anilino-8-naphthalenesulfonate (ANS) fluorescent dye is widely used in protein folding studies due to the significant increase in its fluorescence quantum yield upon binding to protein hydrophobic regions that become accessible during protein unfolding. However, when modeling cellular macromolecular crowding conditions in protein folding experiments in vitro using crowding agents with guanidine hydrochloride (GdnHCl) as the denaturant, the observed changes in ANS spectral characteristics require careful consideration. This study demonstrates that crowding agents can form clusters that interact differently with ANS.

PML Nuclear Bodies and Cellular Senescence: A Comparative Study of Healthy and Premature Aging Syndrome Donors' Cells.

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.

Bioinformatics analysis of proteins interacting with different actin isoforms.

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.

Time-resolved fluorescence of ANS dye as a sensor of proteins LLPS.

The explosive growth in the number of works addressing the phase separation of intrinsically disordered proteins has driven both the development of new approaches and the optimization of existing methods for biomolecular condensate visualization. In this work, we studied the potential use of the fluorescent dye ANS as a sensor for liquid-liquid phase separation (LLPS), focusing on visualizing condensates formed by the stress-granules scaffold protein G3BP1. Using fluorescence lifetime imaging microscopy (FLIM), we demonstrated that ANS can accumulate in RNA-induced G3BP1 condensates in aqueous solutions, but not in G3BP1 condensates formed under macromolecular crowding conditions in highly concentrated PEG solutions.

Ageing drop by drop: Disturbance of the membrane-less organelle biogenesis as an aging hallmark.

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.

New strokes to the portrait of inactivated actin.

In addition to the well-known monomeric and polymeric forms of actin there is another unique thermodynamically stable state of this protein, called "inactivated actin" (I-actin). I-actin is formed at moderate concentration of a denaturant, release of Ca ions and/or ATP, or after heating. This state is a monodisperse associate and it has the same spectral characteristics regardless of the method of preparation.

Promyelocytic leukemia protein (PML) knockout increases mitochondrial Ca uptake in HeLa cells.

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 (IPRs) activity regulators and can influence Ca 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 Ca mobilization from the ER via IPRs.

Stress-Induced Evolution of the Nucleolus: The Role of Ribosomal Intergenic Spacer (rIGS) Transcripts.

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).

Correction: Sulatskaya et al. Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T. 2018, , 2762.

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Correction: Stepanenko et al. Trypsin Induced Degradation of Amyloid Fibrils. , , 4828.

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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.

Von-Hippel Lindau protein amyloid formation. The role of GST-tag.

In the last decade, much attention was given to the study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in the 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).

Broken but not beaten: Challenge of reducing the amyloids pathogenicity by degradation.

Background: The accumulation of ordered protein aggregates, amyloid fibrils, accompanies various neurodegenerative diseases (such as Parkinson's, Huntington's, Alzheimer's, etc.) and causes a wide range of systemic and local amyloidoses (such as insulin, hemodialysis amyloidosis, etc.).

Order wrapped in chaos: On the roles of intrinsically disordered proteins and RNAs in the arrangement of the mitochondrial enzymatic machines.

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.

Structural determinants of odorant-binding proteins affecting their ability to form amyloid fibrils.

The formation of amyloid fibrils is associated with many severe pathologies as well as the execution of essential physiological functions by proteins. Despite the diversity, all amyloids share a similar morphology and consist of stacked β-strands, suggesting high amyloidogenicity of native proteins enriched with β-structure. Such proteins include those with a β-barrel-like structure with β-strands arranged into a cylindrical β-sheet.

Prediction of the Feasibility of Using the ≪Gold Standard≫ Thioflavin T to Detect Amyloid Fibril in Acidic Media.

Ordered protein aggregates, amyloid fibrils, form toxic plaques in the human body in amyloidosis and neurodegenerative diseases and provide adaptive benefits to pathogens and to reduce the nutritional value of legumes. To identify the amyloidogenic properties of proteins and study the processes of amyloid fibril formation and degradation, the cationic dye thioflavin T (ThT) is the most commonly used. However, its use in acidic environments that induce amyloid formation in vitro can sometimes lead to misinterpretation of experimental results due to electrostatic repulsion.

Insights into the Cellular Localization and Functional Properties of TSPYL5 Protein.

In recent years, the role of liquid-liquid phase separation (LLPS) and intrinsically disordered proteins (IDPs) in cellular molecular processes has received increasing attention from researchers. One such intrinsically disordered protein is TSPYL5, considered both as a marker and a potential therapeutic target for various oncological diseases. However, the role of TSPYL5 in intracellular processes remains unknown, and there is no clarity even in its intracellular localization.

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.

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.

PML Body Biogenesis: A Delicate Balance of Interactions.

PML bodies are subnuclear protein complexes that play a crucial role in various physiological and pathological cellular processes. One of the general structural proteins of PML bodies is a member of the tripartite motif (TRIM) family-promyelocytic leukemia protein (PML). It is known that PML interacts with over a hundred partners, and the protein itself is represented by several major isoforms, differing in their variable and disordered C-terminal end due to alternative splicing.

Mammalian odorant-binding proteins are prone to form amorphous aggregates and amyloid fibrils.

Odorant-binding proteins are involved in perceiving smell by capturing odorants within the protein's β-barrel. On the example of bovine odorant-binding protein (bOBP), the structural organization of such proteins and their ability to bind ligands under various conditions in vitro were examined. We found a tendency of bOBP to form oligomers and small amorphous aggregates without disturbing the integrity of protein monomers at physiological conditions.

Chaotic aging: intrinsically disordered proteins in aging-related processes.

The development of aging is associated with the disruption of key cellular processes manifested as well-established hallmarks of aging. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) have no stable tertiary structure that provide them a power to be configurable hubs in signaling cascades and regulate many processes, potentially including those related to aging. There is a need to clarify the roles of IDPs/IDRs in aging.