Post-translational Modifications: Reversing ADP-ribosylation

Interactions between serines and molecules of ADP-ribose play an important role in signaling that the DNA in a cell has been damaged and needs to be repaired.
  1. Giuliana Katharina Moeller
  2. Gyula Timinszky  Is a corresponding author
  1. Ludwig-Maximilians-University of Munich, Germany
  2. Biological Research Center of the Hungarian Academy of Sciences, Hungary

Cells rapidly react to stimuli in their environment by making modifications to proteins that change the way those proteins interact with other molecules (Mann and Jensen, 2003). Once a stimulus has stopped, these 'post-translational modifications' are usually reversed and the cell’s life goes back to normal. For example, when a cell suffers damage to its DNA, the addition of a molecule called ADP-ribose – a process that is known as ADP-ribosylation – to certain proteins sends a signal that leads to the damage being repaired; drugs that inhibit the addition of ADP-ribose are also used in cancer therapy (see Li and Yu, 2015 for a review).

It was discovered in the 1960s that specialized enzymes called PARPs can add one or more units of ADP-ribose (ADPr) to specific amino acids within proteins. Over the decades, it became clear that these enzymes are involved in a wide range of cellular processes, including DNA repair, transcription, chromatin regulation and cell death. The first target sites for ADP-ribosylation to be identified were mostly glutamates, aspartates and lysines, and the enzymes responsible for the removal of the ADPr units were also established (Figure 1)(Barkauskaite et al., 2013).

Mono- and poly(ADP-ribosyl)ation and their reversal.

When a protein (top) undergoes mono(ADP-ribosyl)ation the ADP-ribose (red circle) can be added to a glutamate (Glu) or aspartate (Asp; left) or a serine (Ser; right). It is also possible for multiple units of ADP-ribose to be added to a protein at a given target site in a process known as poly(ADP-ribosyl)ation (bottom). The enzymes PARP1 and PARP2 are involved in ADP-ribosylation of both Glu/Asp and Ser, with a protein called HPF1 acting as a cofactor in the mono(ADP-ribosyl)ation of Ser. The enzymes involved in the reversal of both mono- and poly(ADP-ribosyl)ation are shown. Fontana et al. have shown that ARH3 is exclusively responsible for reversing the mono(ADP-ribosyl)ation of Ser, and that it is also involved (with PARG) in reversing the poly(ADP-ribosyl)ation of Ser.

More recently, it was shown that serines can be target sites for ADP-ribosylation, and that many of the proteins that contain such target sites have important roles in DNA damage repair (Bilan et al., 2017; Bonfiglio et al., 2017; Leidecker et al., 2016; Gibbs-Seymour et al., 2016). However, nothing was known about the enzymes or mechanisms responsible for the removal of the ADPr units from the serines. Now, in eLife, Ivan Ahel of the University of Oxford, Ivan Matic of the Max Planck Institute for Biology of Ageing in Cologne and co-workers – including Pietro Fontana, Juan José Bonfiglio and Luca Palazzo as joint first authors, along with Edward Bartlett – provide new insight into these matters (Fontana et al., 2017).

Using biochemical approaches and a technique called mass spectrometry, Fontana et al. screened a number of proteins that are known to bind to ADPr to find out if they could remove ADPr units that had been added to serines. They discovered that an enzyme called ARH3 could remove ADPr from serine on histone proteins (Figure 1). Previous research has shown that ARH3 and PARG work in similar ways. Both enzymes are able to break the ribose bonds that hold chains of ADPr units together, but ARH3 hydrolyses the chains less efficiently than PARG and also has a different structure (Mueller-Dieckmann et al., 2006; Oka et al., 2006). Fontana et al. discovered that unlike PARG, ARH3 was able to cleave both single ADPr units and chains of ADPr on histones and other proteins.

Since mass spectrometry is a rather expensive and laborious technique, Fontana et al. also used ARH3 in combination with western blotting – a basic technique to detect specific proteins or protein modifications – to track ADP-ribosylation on serines. These experiments confirmed the findings obtained with mass spectrometry, and proved that histone proteins are primarily – if not exclusively – modified on serine. Future studies could build on these findings and use ARH3 as a tool to detect the ADP-ribosylation of serines in proteins.

Despite these new insights, many outstanding questions remain. For example, how does adding ADPr to serine affect the role of a protein? And what happens when two neighboring amino acids experience post-translational modifications? A widely studied post-translational modification that regulates gene expression involves the methylation or acetylation of two lysines (K9 and K27) in histone three (Saksouk et al., 2015). However, these lysines are followed by a serine, which could undergo its own post-translation modification (which could be phosphorylation or ADP-ribosylation). Would these modifications influence each other? Probably, yes. This complex interplay may have far reaching consequences in the regulation of gene expression, and may play an important role in many diseases that depend on ADP-ribosylation pathways.

References

Article and author information

Author details

  1. Giuliana Katharina Moeller

    Giuliana Katharina Moeller is in the Department of Physiological Chemistry, Biomedical Center Munich, Ludwig-Maximilians-Universität München, Munich, Germany

    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2006-2335
  2. Gyula Timinszky

    Gyula Timinszky is in the Department of Physiological Chemistry, Biomedical Centre Munich, Ludwig-Maximilians-Universität München, Munich, Germany and the Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary

    For correspondence
    gyula.timinszky@med.uni-muenchen.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6342-8985

Publication history

  1. Version of Record published: August 10, 2017 (version 1)

Copyright

© 2017, Moeller et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,259
    views
  • 140
    downloads
  • 1
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Giuliana Katharina Moeller
  2. Gyula Timinszky
(2017)
Post-translational Modifications: Reversing ADP-ribosylation
eLife 6:e29942.
https://doi.org/10.7554/eLife.29942
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Thomas RM Germe, Natassja G Bush ... Anthony Maxwell
    Research Article

    DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (–1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface ‘swapping’ (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed ‘swivelling’ mechanism for DNA gyrase (Gubaev et al., 2016).

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Marian Brenner, Christoph Zink ... Antje Gohla
    Research Article

    Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.