Abstract
Isoaspartate (isoAsp) formation is a major source of protein damage that is kept in check by the repair function of protein L-isoaspartyl methyltransferase (PIMT). Mice deficient in PIMT accumulate isoAsp-containing proteins, resulting in cognitive deficits, abnormal neuronal physiology and cytoarchitecture, and fatal epileptic seizures 30–60 days after birth. Synapsins I and II, dynamin-1, collapsin response mediator protein 2 (CRMP2), and α/β-tubulin are major targets of PIMT in brain. To investigate links between isoAsp accumulation and the neurological phenotype of the KO mice, we used Western blotting to compare patterns of in vivo phosphorylation or acetylation of the major PIMT targets listed above. Phosphorylations of synapsins I and II at Ser-9 were increased in female KO vs. WT mice, and acetylation of tubulin at Lys-40 was decreased in male KO vs. WT mice. Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs. WT mice, but the statistical significance (P>0.1) was low. No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241. General levels of phosphorylation assessed with Pro-Q Diamond stain, or an anti-phosphotyrosine antibody, appeared similar in the WT and KO mice. We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage. We also uncovered unexpected differences in how male and female mice respond to isoAsp accumulation in the brain.
Highlights
Isoaspartate formation, through deamidation of asparaginyl residues or isomerization of aspartyl residues, constitutes a major source of spontaneous protein damage occurring in vivo and in vitro [1,2,3,4,5,6,7]
Generation of isoAsp sites is initiated by nucleophilic attack on the Asx side-chain carbonyl by the C-flanking amide bond nitrogen resulting in an intermediate succinimide, hydrolysis of which generates a mixture of α-linked L-aspartyl (~15-30%) and β-linked Lisoaspartyl (~70-85%) residues
We report here the use of modification-specific antibodies to monitor the effect of Protein L-isoaspartate O-methyltransferase (PIMT) deficiency on site-specific phosphorylation of synapsins I and II, dynamin-1, and collapsin response mediator protein 2 (CRMP2), as well as acetylation of α-tubulin
Summary
Isoaspartate (isoAsp) formation, through deamidation of asparaginyl residues or isomerization of aspartyl residues, constitutes a major source of spontaneous protein damage occurring in vivo and in vitro [1,2,3,4,5,6,7]. Continuing cycles of PIMT action have been shown to efficiently repair L-isoAsp sites in a number of peptides and proteins [13,14,15,16,17]. A repair function for PIMT in vivo is supported by observations that reduction of PIMT activity in cultured cells or knockout (KO) mice dramatically increase the level of isoaspartyl proteins [18,19,20,21]. A critical need for PIMT action in the brain is indicated by its high specific activity in this tissue [22,23] as well as the overt neurological phenotype of PIMT KO mice: increased brain size with abnormal neuro-anatomical and electrophysiological properties, impaired learning, and fatal epileptic seizures beginning at 4 weeks of age [19,20,24,25,26]
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