Search

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. Furthermore, GdnHCl can disrupt these clusters and directly affect the ANS spectral characteristics. A model for the interaction between GdnHCl, crowders, and ANS is proposed. Using bovine serum albumin (BSA) as a model protein, the limitations of using ANS for studying conformational transitions induced by GdnHCl in the presence of crowding agents are demonstrated. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this work, two new biscyclometalated Ir(III) complexes with diimine ligands were synthesized. The compounds were characterized by 1D and 2D NMR spectroscopy and HR ESI mass spectrometry. All complexes exhibit efficient phosphorescence with pronounced sensitivity to the presence of oxygen. The photophysical properties of the obtained compounds, including absorption, emission spectra, lifetimes and quantum yields of phosphorescence were measured in various aqueous and model biological media with variations in pH, temperature and at various O2 concentrations. The complexes exhibit a very good sensory response to the changes in oxygen concentration, resulting in an increase in quantum yields and lifetimes by 2.8–5.5 times upon oxygen removal from aerated aqueous solutions. Biological tests with CHO‐K1 cell line have shown that these compounds display low toxicity, rapid internalization into cells and localization predominantly in lysosomes. For the most promising complex the phosphorescence lifetime imaging experiments were conducted, revealing that this sensor markedly changes the phosphorescence lifetime values in cells from 1.8 to 4.1 μs upon transition from normoxia to simulated hypoxia. The obtained results indicate that this type of iridium chromophores can be effectively used for assessing the oxygen status of living objects by using time‐resolved luminescent microscopy. [ABSTRACT FROM AUTHOR]

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. In this study, we characterized the intracellular localization and exchange dynamics with intracellular contents of TSPYL5 and its parts, utilizing TSPYL5 fusion proteins with EGFP. Our findings reveal that TSPYL5 can be localized in both the cytoplasm and nucleoplasm, including the nucleolus. The nuclear (nucleolar) localization of TSPYL5 is mediated by the nuclear/nucleolar localization sequences (NLS/NoLS) identified in the N-terminal intrinsically disordered region (4–27 aa), while its cytoplasmic localization is regulated by the ordered NAP-like domain (198–382 aa). Furthermore, our results underscore the significant role of the TSPYL5 N-terminal disordered region (1–198 aa) in the exchange dynamics with the nucleoplasm and its potential ability for phase separation. Bioinformatics analysis of the TSPYL5 interactome indicates its potential function as a histone and ribosomal protein chaperone. Taken together, these findings suggest a significant contribution of liquid–liquid phase separation to the processes involving TSPYL5, providing new insights into the role of this protein in the cell's molecular life. [ABSTRACT FROM AUTHOR]