The Biochemistry of Multimer Formation of the MEIG1 Protein

MEIG1 (Meiosis expressed gene) is a small, highly conserved, protein, rich in Lys, Ser/Thr and Tyr residues. It was first identified as being expressed in spermatocytes undergoing meiotic recombination, and as an important protein for tail formation during spermiogenic differentiation. Several lines of evidence suggest that MEIG1 is involved in the DDR of the cell, and Meig1-KO mice are predisposed to develop tumors, attributing tumor suppressive properties to MEIG1. Western-blot analysis of proteins from various biological systems (bacteria, cell cultures, tissue extracts) gave a ladder-like signal, where bands are separated by the putative size of MEIG1, as if it forms multimers. Indeed, preliminary MassSpec analysis revealed that all bands were composed of the MEIG1 protein. Given that none of these multimers could be reduced, we hypothesized that iso-peptide bonds (rather that S-S bonds) are involved in the formation of these multimers. To address this issue, we replaced three of the conserved Lys residues to Gly and expressed these mutant proteins in Meig1-KO cells. We found that all three mutant forms were able to form multimers in KO cells, suggesting that these Lys residues are not essential for iso-peptide bond formation. However, K57G mutant was less efficient in protecting plasmid DNA in a nuclease assay, compared to WT-MEIG1, suggesting that K57 is important for proper function of MEIG1. To further address the biochemistry of MEIG1 multimer formation, we plane to mutate all other conserved Lys residues, as well as to check a mutant form in which all Lys residues are mutated.