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. 2023 Dec 19;25(1):39.
doi: 10.3390/ijms25010039.

Insights into the Cellular Localization and Functional Properties of TSPYL5 Protein

Sergey A Silonov  1 , Eugene Y Smirnov  1 , Eva A Shmidt  1 , Irina M Kuznetsova  1 , Konstantin K Turoverov  1 , Alexander V Fonin  1
Affiliations

Insights into the Cellular Localization and Functional Properties of TSPYL5 Protein

Sergey A Silonov et al. Int J Mol Sci. .

Abstract

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.

Keywords: TSPYL5; fluorescence recovery after photobleaching (FRAP); intrinsically disordered proteins (IDPs); intrinsically disordered regions (IDRs); liquid–liquid phase separation (LLPS); protein–protein interactions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TSPYL5-EGFP localization. (A) Localization of the chimeric protein TSPYL5-EGFP in the cytoplasm and nucleolus of HeLa cells. The fluorescent signal intensity in the nucleolus may vary among different cells in the same cell line (a1–a4). (B) Presence of nucleolar localization at different stages of the HeLa cell cycle using the Fucci system. (C) Simplified structure of the nucleolus, showing the fibrillar center, dense fibrillar component, and granular component. Green color highlights the data from fluorescent microscopy showing the localization of TSPYL5-EGFP in the granular component of the nucleolus. (D) Model illustrating the working principles of the Fucci system for cell cycle analysis [18]: Cdt1-mCherry expression is observed during the G1 phase, while Geminin-TagBFP expression is suppressed; both proteins are present during the S phase; Geminin-TagBFP is expressed and Cdt1-mCherry expression is suppressed during the G2 and M phases. Yellow dashed line indicates the cell nucleus; white arrow indicates the nucleolus. Scale bar 10 μm. The illustrations in (C,D) were created with BioRender.com (accessed on 10 September 2023).
Figure 2
Figure 2
TSPYL5 structure and fragment analysis. (A) Schematic representation of the TSPYL5 amino acid sequence and its fragments with the results of bioinformatics analysis (details in the Materials and Methods section). DNA-binding regions (orange), RNA-binding regions (pink), NAP-like domain (red), droplet-promoting regions (DPRs, cyan), and nuclear localization signal (NLS, blue) are shown. (B) Enlarged image of the TSPYL5-EGFP fragment 1–198 aa localization in the putative nucleolus granular component. (C) TSPYL5-EGFP fragment localization in HeLa cell line. The presence of nuclear and nucleolar localization is shown for TSPYL5 fragments 1–50 aa, 1–198 aa, and 1–382 aa. The numbers represent the positions of amino acid residues (aa). The yellow dashed line indicates the cell nucleus. Scale bar 10 µm.
Figure 3
Figure 3
Fluorescence recovery after photobleaching (FRAP) of full-length TSPYL5-EGFP and its fragments. (A) FRAP recovery curves. The red curve representing the recovery of the 1–198 aa fragment closely overlaps with the green curve representing full-length TSPYL5. (B) Three-dimensional structure of TSPYL5 according to AlphaFold2 prediction (AF-Q86VY4-F1) [30]. Regions of TSPYL5 are highlighted: 1–50 aa (yellow), 50–198 aa (red), and 198–417 aa (green).
Figure 4
Figure 4
Analysis of human TSPYL5 interactome. (A) Three major blocks of the TSPYL5 interactome based on their functions and frequent occurrence in the database (histones, RNA helicases, ribosomal proteins). GO analysis for the 49 proteins in the interactome is presented, including cellular components, molecular functions, and biological processes. (B) Results of the analysis of TSPYL5 interactome for disorder, potential propensity for LLPS, and the role of the protein in LLPS, shown as pie charts.
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