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

Abstract

Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. 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.

Keywords: PKM; PKM binding proteins; interactome; moonlighting functions; pyruvate kinase; pyruvate kinase isoforms.

Figure 1

Biosensor validation of PKM1 interaction with selected identified PKM binding proteins. In the case of A, B, and C, purified rabbit muscle PK1 was used as an analyte, and purified rabbit muscle GAPDH (A), LDH (B), and aldolase A (C) were immobilized onto a chip surface; in (D) PKM1 was immobilized on the chip surface and CK was used as the analyte.

Figure 2

The interactome map reflecting the interaction between mouse brain proteins bound to the full-length PKM1 and the PK peptide. Explanations are given in the text.