The mitochondrial mono-ADP-ribosylation landscape

Project Information

MITOMAR

Grant agreement ID: 627676

Project closed

Start date 1 April 2014

End date 23 June 2016

Funded under

• Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)

Total cost € 161 968,80

EU contribution € 161 968,80

Coordinated by

LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN
Germany

Final Report Summary - MITOMAR (The mitochondrial mono-ADP-ribosylation landscape)

The MitoMAR project (The mitochondrial mono-ADP-ribosylation landscape) aimed to uncover mitochondrial proteins and pathways that are dynamically influenced by mono-ADP-ribosylation.

The detailed research results (that are being published in the peer-reviewed literature) are summarized below:
By overexpressing a mitochondrially-localized, catalytically-dead ADP-ribose binding mutant macrodomain protein, we found that the turnover of endogenous mono-ADP-ribosylation is important for human mitochondrial morphology, indicating that reversible ADP-ribosylation reactions are required for mitochondrial homeostasis and function. In addition to this cell-biological evidence, we have been able to identify the mitochondrial ADP-ribosyl-proteome in human cells and have started to dissect its biological roles. Specifically, in addition to an already-known ADP-ribosylation-regulated protein, glutamate dehydrogenase, we found several novel ADP-ribosylated proteins, including glutamate pyruvate transaminase and malic enzyme 2, indicating a broad role of ribosylation in glutamate metabolism. In addition, we have purified other metabolic enzymes, broadening the known mitochondrial pathways regulated by mono-ADP-ribosylation. The new ADP-ribosylated targets are currently being analyzed in molecular and mechanistic detail in order to establish and validate the biological role that the ADP-ribosylation of these targets has. In sum, MitoMAR allowed us to define the human mitochondrial ADP-ribosylome, revealing interesting proteins whose ADP-ribosylation-dependent regulation can affect mitochondrial functionality and cellular homeostasis. A manuscript reporting these findings is expected to be prepared toward the end of 2016/beginning of 2017.

In addition, a collaborative effort with the laboratory of Dr. Dmtri Filippov at the University of Leiden in the Netherlands has been very successful and productive, allowing us to publish a paper in the prestigious chemistry journal Angewandte Chemie (Kistemaker et al., 2016). Our report shows that distinct macrodomain modules display unexpected selectivity toward novel, custom-synthesized mono-ADP-ribosylated-peptides, revealing that the local peptide sequence adjoining the ADP ribosylation site matters for the recognition of ADP-ribosylation signals by macrodomain effector proteins. However, because of technical limitations, the generation of the mono-ADP-ribosylated peptide array has not yet been achieved. As stated in the periodic report, we decided to move towards the synthesis of a ‘clickable handle’ ADP ribose, as alternative of the generation of the mono-ADP-ribosylated array. Although this compound is now “ready to use”, its functionality still needs to be tested. Work is on-going to establish this novel procedure.

TRAINING PROGRESS AND ACHIEVEMENTS

In addition to what already described in the periodic report, MitoMAR has been disseminated prior to publication to a specialized research community, through the following actions:
- Invited research talk: EMBL Group Leader Retreat on Current Topics in Cell Biology (04-08 November, 2015). Lake Como, Italy.
- Invited research talk: FASEB Meeting on NAD Metabolism and Signaling (09-14 August 2015). Timmendorfer Strand, Germany.
With regards to research training, I have attended a 2-day “Grant Proposal Writing workshop” (01-02 June 2016) in Munich, Germany.

Further, I have gained valuable teaching experience by partaking in our departmental teaching of biochemistry to 1st year medical students. I took an active role in supervising practical lab courses.

My communication and presentation skills have also sharpened by (i) the weekly meeting with Dr. Ladurner and his team, (ii) the 4-monthly departmental seminars where I have presented on my project and (iii) the 4-monthly departmental journal clubs. Together with a Mini-Review submitted to Molecular Cell, these latter two events have simultaneously aided my writing skills, since each meeting requires the preparation of a timely written summary.

Last but not least, the MitoMAR project has allowed me to build up and maintain strong and direct connections to other leading groups, including those of Matthias Mann (Max Planck Institute for Biochemistry, Munich) and Michael Nielsen (Novo Nordisk Center for Protein Research, Copenhagen), Dmitri Filippov (University of Leiden, Netherlands) and Gianni Cesareni (Rome).