Dihydrolipoamide dehydrogenase moonlighting activity as a DNA chelating agent

Avraham Dayan, Adva Yeheskel, Raphael Lamed, Gideon Fleminger, Osnat Ashur-Fabian

Research output: Contribution to journalArticlepeer-review

Abstract

Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO2 as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin-mediated drug-delivery of RGD-modified DLDH (DLDHRGD), leading to apoptotic cell death. In these experiments, DLDHRGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA-binding capability of this enzyme. To explore this interaction under cell-free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel-shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA-binding. A Docking algorithm for prediction of protein-DNA complexes, Paradoc, identified a putative DNA binding site at the C-terminus of DLDH. Our finding that TiO2-bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.

Original languageEnglish
Pages (from-to)21-28
Number of pages8
JournalProteins: Structure, Function and Bioinformatics
Volume89
Issue number1
DOIs
StatePublished - Jan 2021

Keywords

  • DNA binding
  • dihydrolipoamide dehydrogenase
  • lambda DNA
  • moonlight activity

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