Protein Repair in the Brain, Proteomic Analysis of Endogenous Substrates for Protein L-Isoaspartyl Methyltransferase in Mouse Brain

Abstract

Protein L-isoaspartyl methyltransferase (PIMT) catalyzes repair of L-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-3 H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30 -35 3 H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartaterelated damage to a number of the neuron-rich proteins detected in this study. Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases. Formation of isoaspartate (isoAsp)3 is a major source of spontaneous protein damage under physiological conditions, arising in conjunction with deamidation of asparaginyl residues and isomerization of aspartyl residues (1-6). This non-enzymatic process occurs via the formation of a succinimide intermediate (cyclic imide) following nucleophilic attack of the Asx side-chain carbonyl group by the amide nitrogen at the C-flanking amino acid (see Fig. 1). Hydrolysis of the succinimide leads to formation of isoaspartyl (isoAsp) and aspartyl products in a typical ratio of 3 to 1. The ␤-linkage characteristic of the predominant isoaspartyl form introduces a kink into the polypeptide backbone that can disrupt normal protein folding and activity. Isoaspartyl formation is strongly influenced by the amino acid that immediately follows (is C-flanking to) an Asn or Asp residue, by the degree of local polypeptide flexibility, and by environmental stressors such as high pH, heat shock, radiation, and oxidation.The enzyme activity we now call protein L-isoaspartyl methyltransferase (PIMT, EC 2.1.1.77) was first encountered in 1965 by Axelrod and Daly (7) as an S-adenosyl-L-methionine (AdoMet)-dependent...

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