Abstract
Isoaspartate (isoAsp) formation is a common type of spontaneous protein damage that is normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). PIMT-KO (knockout) mice exhibit a pronounced neuropathology highlighted by death from an epileptic seizure at 30 to 60 days after birth. The mechanisms by which isoaspartyl damage disrupts normal brain function are incompletely understood. Proteomic analysis of the PIMT-KO mouse brain has shown that a number of key neuronal proteins accumulate high levels of isoAsp, but the extent to which their cellular functions is altered has yet to be determined. One of the major neuronal targets of PIMT is creatine kinase B (CKB), a well-characterized enzyme whose activity is relatively easy to assay. We show here that (1) the specific activity of CKB is significantly reduced in the brains of PIMT-deficient mice, (2) that in vitro aging of recombinant CKB results in significant accumulation of isoAsp sites with concomitant loss of enzymatic activity, and (3) that incubation of in vitro aged CKB with PIMT and its methyl donor S-adenosyl-L-methionine substantially repairs the aged CKB with regard to both its isoAsp content and its enzymatic activity. These results, combined with similarity in phenotypes of PIMT-KO and CKB-KO mice, suggests that loss of normal CKB structure and function contributes to the mechanisms by which isoAsp accumulation leads to CNS dysfunction in the PIMT-KO mouse.
Highlights
Creatine kinases (EC 2.7.3.2) are a family of enzymes that catalyze reversible transfer of the phosphate group between ATP and creatine, playing a crucial role in intracellular energy metabolism of cells with high and fluctuating energy requirements [1,2,3]
Creatine kinase B is highly susceptible to isoAsp formation, as demonstrated by proteomic analysis of major methyl-accepting substrates for protein-L-isoaspartyl methyltransferase (PIMT) in PIMT-KO mouse brain extracts
Our goal in the present study was to determine if isoAsp damage to creatine kinase B (CKB) might contribute to the neuropathology resulting from PIMT deficiency
Summary
Creatine kinases (EC 2.7.3.2) are a family of enzymes that catalyze reversible transfer of the phosphate group between ATP and creatine, playing a crucial role in intracellular energy metabolism of cells with high and fluctuating energy requirements [1,2,3]. Cellular mechanisms for dealing with isoaspartyl protein damage include urinary excretion of the damaged proteins [15], degradation by isoAsp-selective proteases [16,17], and enzymatic repair Regarding the latter mechanism, isoAsp residues in peptides and proteins are recognized and repaired by the action of protein Lisoaspartyl O-methyltransferase (PIMT, EC 2.1.1.77) [18,19,20,21,22,23]. Continuing cycles of PIMT action efficiently repair L-isoAsp sites in vitro [23,24,25], while reduction of PIMT activity in cultured cells or knockout (KO) mice dramatically increases the level of isoAsp-containing proteins [26,27,28,29]. A proteomic study utilizing the PIMT-KO mouse revealed CKB as one of 22 major targets for PIMT in the brain [9]
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