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InVivo_DDR_ADPR Report Summary

Project ID: 657501
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - InVivo_DDR_ADPR (Decoding the DNA damage signalling in C. elegans by proteomic analyses of ADP-ribosylation)

Reporting period: 2016-04-01 to 2018-03-31

Summary of the context and overall objectives of the project

Preservation of genome integrity and stability is critical for survival and propagation of individuals and species. Organisms have thus evolved rapid and efficient mechanisms –collectively termed the DNA damage response (DDR)– to combat threats posed by DNA damage. Among these, the post-translational modification (PTM) ADP-ribosylation (ADPr) plays a decisive role in effective DDR.

What is the issue being addressed?
Although much is known about the relevance of ADPr upon DNA injury, the underlying molecular mechanisms are still poorly understood. In contrast to the restricted amino acid specificities of most PTMs, nearly all chemically reactive amino acid side chains have been reported as targets of ADPr. Functional and mechanistic understanding of ADPr requires knowledge of its target amino acids and the exact conjugation sites within the substrate proteins.

What are the overall objectives?
The most challenging and innovative goal of this proposal is to elucidate the molecular mechanisms of ADPr in the DNA damage response. Initially, we aim to develop a mass spectrometric approach for unambiguous and unbiased ADPr site mapping in endogenous samples. Then, we will profile ADP-ribosylated peptides during DDR in cells. Finally, we aim to advance the understanding of the underlying molecular mechanisms as a prelude to improving prevention, diagnosis and treatment of many common health problems.

Why is it important?
Understanding the molecular mechanisms underlying such a complex biological process as ADPr will provide new insights for improved treatment of DNA damage-related diseases, including cancer. This is an ambitious, innovative, cross-disciplinary project at the forefront of two exciting fields, biology and proteomics.

With our unambiguous and unbiased ADPr site mapping, we uncovered serine ADPr (Ser-ADPr) as a novel protein modification and described the molecular mechanisms by characterizing the “writers” (proteins responsible for the attachment of the modification onto target proteins) and the “eraser” (protein responsible for the removal of the modification from ADP-ribosylated proteins). We have also shown that Ser-ADPr is a widespread modification and that is the major form of ADPr under DNA damage. Our findings have challenged 50 years of consensus understanding on ADPr biology and have opened a large and novel research area into how ADPr regulates the DNA damage response, chromatin dynamics and transcription.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

From the beginning of the project we have worked to advance proteomic technology into the largely uncharted territory of endogenous ADPr sites and to characterize the molecular mechanisms underlying ADPr in cells. These efforts have culminated in:

• Establishment of a sophisticated methodology for truly unbiased and unambiguous identification of ADPr sites on any reactive amino acid (Bonfiglio et al., Nucleic Acids Research 2017)

• Discovery of Serine ADP-ribosylation as a new DNA damage-dependent protein modification that had been missed for decades (Leidecker*, Bonfiglio* et al., Nature Chemical Biology 2016). * equal contribution

• Elucidation of the conjugation mechanisms (“writers”) for Serine ADP-ribosylation: identification of HPF1 as the Ser-ADPr-inducing factor (Bonfiglio et al, Molecular Cell 2017).

• Demonstration that Serine ADPr is a widespread molecular signal that targets hundreds of proteins during DNA repair (Bonfiglio et al, Molecular Cell 2017 and Palazzo*, Leidecker*, Prokhorova et al, eLife 2018). * equal contribution

• Elucidation of the molecular mechanisms underlying Ser-ADPr reversal (“erasers”): identification of ARH3 as the factor responsible of Ser-ADPr removal factor (Fontana*, Bonfiglio* et al, eLife 2017). * equal contribution

• Development of a method for the generation of novel and inventive tools that will be useful not only for our scientific projects but also for everyone, including the scientific community in general (Patent application EP17197550)

In addition to the scientific publications and presentations at key international conferences, our findings have been disseminated through press releases, highlights and comments:

• Highlight in the weekly magazine Chemical & Engineering News, October 2016

• Comment in the popular blog “News in Proteomics Research”, October 2016

• Highlight in the Max Planck Society Research News, February 2017

• Cover of Molecular Cell: It takes two to Serine ADP-ribosylate, February 2017

• Highlighted in a Molecular Cell Preview and as a “Featured Article” on the Molecular Cell website, March 2017

• Interview with Cell Press, March 2017

• Highlighted by an “Insight“ in eLife, August 2017

• eLife Digest, October 2017

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our discovery of Ser-ADPr revealed how important discoveries may be hidden in scientific “blind spots”. In fact, our experience highlighted the difficulty in studying this biologically and clinically important PTM at the molecular level due to a lack of suitable reagents and techniques. The need to progress with our research forced us to come up with our own approach for the generation of novel and inventive tools. This work led to a first patent application from the project (EP17197550). We are convinced that the generation of these tools will be useful not only for our scientific projects but also for everyone, including the scientific community in general, that will have access to these new reagents.
In addition, our work on the biology of ADPr opens up new possibilities to improve and increase the efficiency of the DNA repair machinery. Drugs that inhibit some of the enzymes that attach ADPr to proteins are already being used to treat some types of breast, ovarian and prostate cancers. A deeper understanding of the molecular mechanisms underlying these processes (writers and erasers) may aid the development of new therapies for these conditions.

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