Uncovering Protein Prenylation in Th1 Cells: Novel Prenylation Sites and Insights into Statin and Farnesyltransferase Inhibition

Title: Uncovering protein prenylation in Th1 cells: novel prenylation sites and insights into statin and farnesyltransferase inhibition

Journal: BMC biology

Published: 2025

Background

T helper 1 (Th1) cell activation is an essential process for immune responses and is tightly regulated, including the prenylation of proteins critical for T cell function. Prenylation facilitates membrane association and protein function and, according to current consensus, is confined to C-terminal prenylation motifs. However, the full extent of the prenylated proteome, a broader understanding of prenylation sites, and the effects of inhibiting prenylation or blocking isoprenoid synthesis using statins remain incompletely understood. To address these gaps, we aimed to comprehensively identify and characterise protein prenylation in Th1 cells.

Materials & Methods

Labelling of prenylated proteins in Th1 cells

For the comparison of activated and non-activated Th1 cells, Th1 cells were stimulated as described above on day 6 to activate them, while non-activated cells were kept resting. At the same time, to label prenylated proteins, cells were treated with F-Az dissolved in ethanol (25 nM) (Cayman Chemical, Ann Arbor, USA) or GG-Az dissolved in DMSO (25 nM) (Thermo Fisher Scientific Inc., Waltham, USA), or the respective solvent as a control, for 24 h before harvesting. Cells were washed in PBS, and pellets were frozen in liquid nitrogen and stored at − 80℃ until further use.

FTI treatment of Th1 cells

To identify proteins that are sensitive to farnesyl transferase inhibition, Th1 cells were cultured, stimulated on day 6 as described above, and treated with FTI-277 (20 µM) (Selleck Chemicals LLC, Houston, USA) for 1 h before adding F-Az or ethanol (control) for 23 h, followed by harvesting. Of note, due to a power failure, the culturing conditions during the last few hours of the experiment were not controlled. Cell proliferation and viability were assessed using an H3-thymidine incorporation assay and PI staining.

Preparation of total extracts

For the analysis of the total proteome of Th1 cells, 1 million cells per sample were lysed in urea extraction buffer and prepared for mass spectrometry measurements, essentially as previously described.

Enrichment of prenylated proteins

For the enrichment of prenylated proteins, the Click-iT™ Protein Enrichment Kit (Thermo Fisher Scientific Inc., Waltham, USA) was used. Per sample, 50 million cells were processed. In brief, azide-labelled proteins were covalently linked to resin-bound alkyne via a click reaction. The bound peptides were washed several times before on-resin digestion with trypsin. The resulting tryptic peptides were then purified and desalted using Pierce C18 Spin Columns (Thermo Fisher Scientific Inc., Waltham, USA).

Results

Figure 1. Distribution of the canonical prenylation motifs, the new C-terminal prenylation motifs, and prenylation without a C-terminal cysteine in known prenylated proteins (green), identified prenylated proteins (blue), and newly identified prenylated proteins (cyan) in samples treated with A GG-Az and B F-Az. C Log₂-transformed centred intensities ± 95% confidence intervals for the identified prenylated proteins, as indicated, in activated (+) and non-activated (−) Th1 cells treated with GG-Az, F-Az, or mock-treated as controls.

Prenylation Figure 2. For a selection of significantly prenylated proteins in enrichment samples of Th1 cells treated with A F-Az or B GG-Az, the log₂-transformed centered intensities ± 95% confidence intervals are shown, as indicated, in activated (+) and non-activated (−) Th1 cells treated with GG-Az, F-Az, or mock-treated as controls.

Conclusions

Our findings confirm that prenylation plays a key role in Th1 cell function, with more proteins undergoing prenylation than previously known, some of which exhibit activation-dependent changes. The identification of non-canonical prenylation events challenges current views on prenylation, expanding the repertoire of modification sites. Together, our molecular insights into protein prenylation in Th1 cells and the effects of prenyltransferase inhibition and statin treatment have important implications for therapeutic strategies targeting immune regulation.

Reference

Koch, J., Ruggia, A., Beha, C., Wipf, I., Zhakparov, D., Westermann, P., Schmelzer, S., Heider, A., Fröhlich, K., & Baerenfaller, K. (2025). Uncovering protein prenylation in Th1 cells: novel prenylation sites and insights into statin and farnesyltransferase inhibition. BMC biology, 23(1), 233. https://doi.org/10.1186/s12915-025-02345-1

* For Research Use Only. Not for use in diagnostic procedures.

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