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Copenhagen Team's Proteome-Wide Protein PARylation Study Could Aid PARP Inhibitor Development


A team led by researchers at the University of Copenhagen has completed a proteome-wide analysis of protein-polyADP-ribosylation, or PARylation, a post-translational modification thought to play a key role in DNA damage repair.

Detailed in a paper published last week in Molecular Cell, the study significantly expands the number of proteins known to be PARylated and could offer new insights into DNA damage repair pathways and the therapies targeting them, Michael Nielsen, a University of Copenhagen researcher and author on the paper, told ProteoMonitor.

Protein PARylation typically occurs in response to genotoxic stress and has been linked to processes including DNA damage repair, chromatin regulation, and transcription. The modification is generated by activation of PAR polymerases, or PARPs, which have emerged as potential drug targets, particularly in cancer.

PARP inhibitors have not yet made it to the market, but a number of such agents are currently in clinical trials, with companies including AstraZeneca, Abbott, and Clovis Oncology exploring the therapeutics.

There is particular interest in potentially using PARP inhibitors in breast and ovarian cancer patients featuring BRCA1 and BRCA2 mutations, which are thought to inhibit DNA damage repair.

Despite the research and drug development activity surrounding PARP inhibitors, though, scientists have to date identified relatively few PARylated proteins, Nielsen noted.

"Overall, in the literature, only around 20 proteins have been confirmed to be modified [by PARylation]," he said. In the Molecular Cell study, Nielsen and his colleagues upped this number substantially, identifying a total of 235 proteins that they characterized as "significantly PARylated" in response to genotoxic stress.

Key to their effort was utilization of a PAR-binding macrodomain of the protein Af1521 as an affinity agent for enriching PARylated proteins. While past work had suggested Af1521 as a suitable reagent for this purpose, this was the first time, Nielsen said, that it had been incorporated as part of a quantitative workflow looking at protein PARylation.

There are other PARylation-enrichment methods that rely on boronic acid beads, but, he said, that method is applicable only to modifications on glutamic acid and aspartic acid residues, whereas the AF1521 approach offers a more unbiased enrichment.

In combination with the AF1521-based enrichment, the researchers used SILAC labeling and mass spec analysis on a Thermo Fisher Scientific Q Exactive instrument to quantify protein PARylation levels in response to genotoxic stresses including treatment with hydrogen peroxide, methyl methane sulfonate, UV radiation, and ionizing radiation.

Use of SILAC labeling allowed the researchers to generate high-quality quantitative data, which was key to distinguishing between actual protein PARylation and background interference. While the team ultimately identified 235 proteins they deemed significantly PARylated, this was a small subset of the several thousand total proteins they identified as showing some level of PARylation.

"A lot of those were background contaminants, though," Nielsen said. "So that was the advantage of doing this quantitatively – we could apply a filter to identify those most strongly modified."

In their analysis of the identified proteins, the researchers found that they were enriched not only for DNA damage repair but for RNA metabolism, as well. This enrichment was particularly evident, Nielsen noted, in response to oxidative and alkylative stress.

The finding, Nielsen said, fits into emerging observations of the role of RNA metabolism in DNA damage repair and the potential role of protein PARylation as a link between the two.

The researchers then selected two specific RNA-related PARylation candidates identified via their analysis, the proteins TAF15 and THRAP3, and investigated their behavior in response to PARylation.

As the authors noted, TAF15 and THRAP3 are involved, respectively, in RNA transcritption and splicing. Upon exposure to genotoxic stress, TAF15 proteins form nuclear substructures known as nucleolar caps, while THRAP3 accumulates in other structures called nuclear speckles.

Using high-content imaging, Nielsen and his colleagues tracked the behavior of these proteins following exposure to genotoxic stress, finding that PARylation appeared to inhibit the formation of the TAF15 nucleolar caps while inducing the formation of the THRAP3 nuclear speckles.

This observation is essentially the first of a specific link between genotoxic stress, PARylation, and such RNA-linked nuclear substructures, Nielsen said.

"People have been describing how, not the PARylated [proteins] but the presence of these [PAR] chains are associated to a degree with some of these nuclear substructures," he said. "But this would be the first time that it has been shown to be playing a significant role in [response to] genotoxic stress."

In addition to offering insights into the emerging links between RNA metabolism and DNA damage repair, the study also provides data and methods that could prove useful for future PARP inhibitor work, Nielsen noted.

"Hopefully this can help people who are working with PARP inhibitors figure out what targets are affected by PARP inhibition," he said. "Our method provides the ability to investigate this question in more detail."

For instance, he added, "we can use this SILAC set-up to interrogate what the effect is of DNA damage with and without a PARP inhibitor and what specific targets are actually affected by a [given] PARP inhibitor."

Nielsen said he and his team are currently investigating these questions.