Eva A. Shmidt

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.

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.