Proteomic Profiling of Mouse Brain Pyruvate Kinase Binding Proteins: A Hint for Moonlighting Functions of PKM1?

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Since protein-protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321.", "claims": [{"id": "claim-method", "statement": "The method was used to calculate the FDR (False Discovery Rate) parameter; the FDR parameter of 1% was taken as a cutoff for protein registration.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:37108803", "locator": "sections[13] \"4.6. The Mass Spectrometric Analysis (LC-MS/MS)\" paragraph 3", "quote": "The method was used to calculate the FDR (False Discovery Rate) parameter; the FDR parameter of 1% was taken as a cutoff for protein registration."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD041321.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:37108803", "locator": "sections[25] \"notes\" paragraph 1", "quote": "The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD041321."}], "state": "complete"}, {"id": "claim-target-result", "statement": "Proteomic profiling using immobilized PKM1 identified 44 individual mouse brain proteins.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:37108803", "locator": "sections[3] \"2.1. 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