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

Jeff X Zhu,Hester A Doyle,Mark J Mamula,Dana W Aswad

doi:10.1074/jbc.m606958200

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 isoaspartate-related 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.

Highlights

Formation of isoaspartate3 is a major source of spontaneous protein damage under physiological conditions, arising in conjunction with deamidation of asparaginyl residues and isomerization of aspartyl residues [1,2,3,4,5,6]
Development of Methodology for Global Separation and Detection of Isoaspartyl Proteins in Brain Extracts of the Protein L-isoaspartyl methyltransferase (PIMT)-KO Mouse—To achieve a global analysis of proteins that serve as major substrates for PIMT in mammalian brain we considered two strategies, both of which are based on the fact that isoaspartyl proteins accumulate to high levels in the PIMT-KO mouse brain and that such proteins can be selectively labeled by PIMT-catalyzed methylation using [methyl3H]AdoMet as the methyl donor
Because PIMT has a low catalytic turnover number (ϳ0.4 minϪ1 at 30 °C) we reasoned that isoaspartyl proteins might form a sufficiently stable ternary complex to bind a number of isoaspartyl proteins to the column

Summary

Introduction

Formation of isoaspartate (isoAsp) is a major source of spontaneous protein damage under physiological conditions, arising in conjunction with deamidation of asparaginyl residues and isomerization of aspartyl residues [1,2,3,4,5,6]. For the following 10 years, it was generally assumed that methylation occurred on the side-chain carboxyls of normal aspartyl or glutamyl residues and that methylation was probably serving to regulate protein activity in a manner analogous to protein phosphorylation It was not until 1984 that this widely distributed methyltransferase was found to have a high degree of specificity for atypical L-isoaspartyl residues [11, 12]. Despite considerable progress made over the past 41 years in elucidating the function of “methanol-forming enzyme,” there has been relatively little information regarding the identity of its major endogenous substrates This situation is attributable to the fact that isoAsp levels are normally held to very low levels by PIMT action, and the fact that the protein methyl ester products of the PIMT reaction are so unstable that they do not survive most commonly used methods of high resolution protein separation. The discovery of all four of these proteins came not from systematic proteomics but rather from a combination of serendipity and guess work

Results

Discussion

Conclusion