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Protein science : a publication of the Protein Society




dUTPase is an enzyme found in all organisms that have thymine as a constituent of DNA. Through evolution, humans have two major isoforms of dUTPase: a mitochondrial (mDut) and a nuclear (nDut) isoform. The nuclear isoform of dUTPase is a 164-amino-acids-long protein containing three cysteine residues. nDut's starting methionine is post-translationally cleaved, leaving four unique amino acids on its amino-terminus including one cysteine residue (C3). These are not present in the mitochondrial isoform (mDut). Using mass spectrometry analyses of recombinant dUTPase constructs, we have discovered an intermolecular disulfide bridge between cysteine-3 of each nDut monomer. We have found that these two residues stabilize a dimer configuration that is unique to the nDut isoform. We have also uncovered an intramolecular disulfide linkage between cysteine residues C78 and C134, stabilizing the monomeric state of the protein. Of note, both disulfide linkages are essential for nDut's enzymatic activity and dimeric formation can be augmented by the addition of the oxidizing agent, hydrogen peroxide to cells. Analyses of endogenous cellular dUTPase proteins confirm these differences between the two isoforms. We observed that mDut appears to be a mixture of monomer, dimer, and trimer conformations, as well as higher-order subunit interactions. In contrast, nDut appeared to exist only in monomeric and dimeric forms. Cysteine-based redox "switches" have recently emerged as a distinct class of post-translational modification. In light of this and our results, we propose that nDut possesses a redox switch whereby cysteine interactions regulate nDut's dUTP-hydrolyzing activity.


Copyright © 2018 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society. This is an open access article under the terms of the License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Creative Commons Attribution-NonCommercial 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License