The Krebs cycle enzyme fumarase is a dual-targeted protein that is located in the mitochondria and cytoplasm of eukaryotic cells. Besides being involved in the TCA cycle and primary metabolism, fumarase is a tumour suppressor that aids DNA repair in human cells. Using mass spectrometry, we identified modifications in peptides of cytosolic yeast fumarase, some of which were absent when the cells were exposed to DNA damage (using the homing endonuclease system or hydroxyurea). We show that DNA damage increased the enzymatic activity of fumarase, which we hypothesized to be affected by post-translational modifications. Succinylation and ubiquitination of fumarase at lysines 78 and 79, phosphorylation at threonine 122, serine 124 and threonine 126 as well as deamidation at arginine 239 were found to be functionally relevant. Upon homology analysis, these residues were also found to be evolutionally conserved. Serine 128, on the other hand, is not evolutionary conserved and the Fum1S128D phosphorylation mimic was able to aid DNA repair. Our molecular model is that the above modifications inhibit the enzymatic activity of cytosolic fumarase under conditions of no DNA damage induction and when there is less need for the enzyme. Upon genotoxic stress, some fumarase modifications are removed and some enzymes are degraded while unmodified proteins are synthesized. This report is the first to demonstrate how post-translational modifications influence the catalytic and DNA repair functions of fumarase in the cell.
Bibliographical noteFunding Information:
We thank Ms Yardena Silas, Ms Rachel Teo, Ms Si Min Lang and Ms Chia Lynn Choo for critical comments on the manuscript and students from Clementi Town Secondary School for practical support. This work was supported by grants from the National Research Foundation (NRF) to OP and NL.
We thank Ms Yardena Silas, Ms Rachel Teo, Ms Si Min Lang and Ms Chia Lynn Choo for critical comments on the manuscript and students from Clementi Town Secondary School for practical support. This work was supported by grants from the National Research Foundation (NRF) to OP and NL. SW produced Figures S2?S6; DR repeated the experiments, produced Figure S7 and wrote part of the manuscript; JT produced Figures 5 and S1; JL repeated the experiments; JY and AC supported SW; ZY supported the MS experiments; TKL performed the MS experiments; QL supervised TKL; OP co-supervised SW, DR, LJ and ZY; NL supervised SW, DR, JT, JL, JY, AC, ZY and wrote the manuscript. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
© 2020 The Author(s)
- DNA repair
- TCA cycle
- enzymatic activity
- post-translational modification