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Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses
Malwina Michalak1,2, Viola Golde1, Dominik Helm3
1Department of Applied Tumor Biology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany.
Abstract:
Galectin-4 (Gal4) has been suggested to function as a tumor suppressor in colorectal cancer (CRC). In order to systematically explore its function in CRC, we established a CRC cell line where Gal4 expression can be regulated via the doxycycline (dox)-inducible expression of a single copy wildtype LGALS4 transgene generated by recombinase-mediated cassette exchange (RMCE). Using this model and applying in-depth proteomic and phosphoproteomic analyses, we systematically screened for intracellular changes induced by Gal4 expression. Overall, 3083 cellular proteins and 2071 phosphosites were identified and quantified, of which 1603 could be matched and normalized to their protein expression levels. A bioinformatic analysis revealed that most of the regulated proteins and phosphosites can be localized in the nucleus and are categorized as nucleic acid-binding proteins. The top candidates whose expression was modulated by Gal4 are PURB, MAPKAPK3, BTF3 and BCAR1, while the prime candidates with altered phosphorylation included ZBTB7A, FOXK1, PURB and CK2beta. In order to validate the (phospho)proteomic data, we confirmed these candidates by a radiometric metabolic-labelling and immunoprecipitation strategy. All candidates exert functions in the transcriptional or translational control, indicating that Gal4 might be involved in these processes by affecting the expression or activity of these proteins.
Insights
Galectin-4 (Gal4) may suppress colorectal cancer (CRC) by regulating nuclear proteins involved in gene expression. This study used proteomic analysis to identify key proteins affected by Gal4 in CRC cells.
Area of Science:
- Molecular Biology
- Cancer Research
- Proteomics
Background:
- Colorectal cancer (CRC) is a significant health concern.
- Galectin-4 (Gal4) is a potential tumor suppressor in CRC.
- Understanding Gal4's molecular mechanisms is crucial for CRC treatment.
Purpose of the Study:
- To systematically investigate the intracellular changes induced by Gal4 expression in CRC.
- To identify proteins and phosphosites regulated by Gal4.
- To elucidate Gal4's role in CRC pathogenesis.
Main Methods:
- Established a doxycycline-inducible CRC cell line for Gal4 expression using recombinase-mediated cassette exchange (RMCE).
- Performed in-depth proteomic and phosphoproteomic analyses to screen for intracellular changes.
- Utilized bioinformatic analysis to interpret proteomic data and identify key candidates.
- Validated proteomic findings using radiometric metabolic labeling and immunoprecipitation.
Main Results:
- Identified and quantified 3083 cellular proteins and 2071 phosphosites, with 1603 matched to protein levels.
- Most regulated proteins and phosphosites are nuclear and involved in nucleic acid binding.
- Key candidates modulated by Gal4 include PURB, MAPKAPK3, BTF3, BCAR1, ZBTB7A, FOXK1, and CK2beta.
- Validated candidates confirmed to be involved in transcriptional or translational control.
Conclusions:
- Gal4 expression significantly impacts the proteome and phosphoproteome in CRC cells.
- Gal4 likely influences CRC progression by modulating nuclear proteins involved in gene expression.
- These findings provide insights into Gal4's tumor-suppressive function and potential therapeutic applications in CRC.
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