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Mitochondrial Uncoupler Carbonyl Cyanide m-Chlorophenylhydrazone Induces the Multimer Assembly and Activity of Repair Enzyme Protein l-Isoaspartyl Methyltransferase

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Abstract

The protein l-isoaspartyl methyltransferase (PIMT) repairs damaged aspartyl residues in proteins. It is commonly described as a cytosolic protein highly expressed in brain tissues. Here, we report that PIMT is an active monomeric as well as a multimeric protein in mitochondria isolated from neuroblastoma cells. Upon treatments with mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), PIMT monomers level decreased by half while that of PIMT multimers was higher. Gel electrophoresis under reducing conditions of CCCP-induced PIMT multimers led to PIMT monomers accumulation, indicating that multimers resulted from disulfide-linked PIMT monomers. The antioxidant ascorbic acid significantly lowered CCCP-induced formation of PIMT multimers, suggesting that reactive oxygen species contributed to PIMT multimerization. In addition, the elevation of PIMT multimers catalytic activity upon treatments with CCCP was severely inhibited by the reducing agent dithiothreitol. This indicated that PIMT monomers have lower enzymatic activity following CCCP treatments and that activation of PIMT multimers is essentially dependent on the formation of disulfide-linked monomers of PIMT. Furthermore, the perturbation of mitochondrial function by CCCP promoted the accumulation of damaged aspartyl residues in proteins with high molecular weights. Thus, this study demonstrates the formation of active PIMT multimers associated with mitochondria that could play a key role in repairing damaged proteins accumulating during mitochondrial dysfunction.

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Abbreviations

AdoHcy:

S-Adenosylhomocysteine

AdoMet:

S-Adenosylmethionine

BCA:

Bicinchoninic acid

BSA:

Bovine serum albumin

BSO:

l-Buthionine sulfoximine

CCCP:

Carbonyl cyanide m-chlorophenylhydrazone

DCHF-DA:

2′,7′-Dichlorofluorescein diacetate

DSIPisoD:

(β-Asp5)-delta-sleep inducing peptide

DTT:

Dithiothreitol

ETC:

Electron transport chain

IEF:

Isoelectric focusing

PBS:

Phosphate-buffered saline

PIMT:

Protein l-isoaspartyl (d-aspartyl) methyltransferase

Rh123:

Rhodamine

ROS:

Reactive oxygen species

∆Ψ m :

Mitochondrial membrane potential

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Acknowledgments

This work was supported by a grant from the National Sciences and Engineering Research Council of Canada to Dr. Richard R. Desrosiers (grant number 121740-06). We thank Michel Marion for his technical assistance in two-dimensional gel analysis.

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Authors and Affiliations

  1. The Montreal General Hospital, McGill University Health Centre, 1650 Cedar Avenue, Montreal, QC, H3G 1A4, Canada

    Irvens Fanélus

  2. Université du Québec à Montréal, C.P. 8888, Succursale Centre-Ville, Montréal, QC, H3C 3P8, Canada

    Richard R. Desrosiers

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  1. Irvens Fanélus
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Correspondence to Richard R. Desrosiers.

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Fanélus, I., Desrosiers, R.R. Mitochondrial Uncoupler Carbonyl Cyanide m-Chlorophenylhydrazone Induces the Multimer Assembly and Activity of Repair Enzyme Protein l-Isoaspartyl Methyltransferase. J Mol Neurosci 50, 411–423 (2013). https://doi.org/10.1007/s12031-012-9946-7

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