Proteomic profiling of mouse brain pyruvate kinase binding proteins: a hint for moonlighting functions of PKM1?

Affinity based proteomic profiling is widely used for identification of proteins involved in for-mation of various interactomes. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms, one of which, PKM2; pyruvate kinase muscle isoform M2 (expressed in actively dividing cells), exhibits many non-canonical (moonlighting) functions. Although the other muscle isoform, PKM1, predominantly expressed in adult differ-entiated tissues, lacks the moonlighting functions, certain evidence exists that it can also perform some non-canonical functions. In order to evaluate the proteomic profile of mouse brain pro-teins bound to PKM1, we have combined in this study affinity-based separation of mouse brain proteins followed by their mass spectrometry identification. Using the highly purified rabbit muscle pyruvate kinase, PKM1 and a 32-mer peptide (PK peptide), sharing high sequence ho-mology with the interface contact region of all PK isoforms, common proteins bound to both af-finity ligands have been recognized. Quantitative affinity binding to the affinity ligands of se-lected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bi-oinformatic 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 noncanonical (moonlighting) functions of PKM1