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J. Biol. Chem., Vol. 281, Issue 44, 33802-33813, November 3, 2006

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

Jeff X. Zhu¹, Hester A. Doyle, Mark J. Mamula, and Dana W. Aswad²

From the Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697 and the Section of Rheumatology, Yale University School of Medicine, New Haven, Connecticut 06510

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-³H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 ³H-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.

Received for publication, July 21, 2006 , and in revised form, August 30, 2006.

^* This work was supported by National Institutes of Health Grant (NIH) NS17269 (to D. W. A.), NIH Grants AI36529 and AI48120 (to M. J. M.), and Arthritis Foundation (the Ethel F. Donaghue Foundation grant) and NIH postdoctoral fellowship F32-AR47759 (to H. A. D.). Pfizer La Jolla provided financial support and work release time (to J. X. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Biochemical Pharmacology, Pfizer La Jolla, 10628 Science Center Dr., San Diego, CA 92121.

² To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, 3205 McGaugh Hall, University of California, Irvine, CA 92697-3900. Tel.: 949-824-6866; Fax: 949-824-8551; E-mail: dwaswad{at}uci.edu.

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