1. Research Background:

A vital factor in comprehending ageing and age-related diseases involves deciphering the signaling pathways that dictate crucial fate decisions. Key among these pathways is regulation by ADP-ribosylation (ADPr), a biologically and clinically significant yet challenging post-translational modification known for its crucial role in maintaining genome stability. The overarching aim of our research group is to investigate the molecular mechanisms of DNA repair and ageing by elucidating the role of ADPr in these biologicalprocesses. Our group's seminal discoveries, including the identification of Serine ADPr by the HPF1/PARP1 writer complex as a novel type of histone mark (Nature Chem Biol 2016, Mol Cell 2017, eLife 2018, and CellReports 2018), have opened a broad and dynamic research area, with an increasing number of laboratories shifting their focus towards Ser-ADPr to understand DNA repair signaling, chromatin dynamics and develop new inhibitors. Our second major contribution is the transformation of these fundamental discoveries into afoundational technology, enabling the generation of essential and broadly applicable ADPr tools (Cell 2020, Mol Cell 2023, Trends Biochem Sci. 2023 and Nat Comms 2024). This places our laboratory at the forefront of ADPr research and has enabled us to uncover DNA damage-induced mono-ADPr as a second wave of PARP1 signaling that recruits, among others, the ubiquitin E3 ligase RNF114 (Mol Cell 2023).

2. Research questions addressed by the group:

Future breakthroughs in aging research, from basic biology to clinical applications, depend on understanding the precise molecular mechanisms that govern the many biological pathways involved in aging and age-related diseases. Yet, uncovering the biochemical basis of specific biological processes often presents significant challenges. Our ambition is to make transformative discoveries by shedding light on the complex molecular mechanisms of aging, using advanced proteomics and innovative chemical biology approaches.

Our first major direction is to elucidate the crosstalk between ADPr and ubiquitination that has emerged over the last few years. Although the fascinating discovery of phosphoribose-linked attachment of ubiquitin by Legionella, to which we contributed (Cell 2016), established a connection between these two PTMs, our research points to a more profound interplay. RNF114, which we identified as the reader of the mono- ADPr wave of PARP1 signaling (Mol Cell 2023), along with additional E3 ligases of the same family, recognizes both ubiquitin and ADPr. Therefore, we plan to dissect the pathways that write, erase and read these composite ubiquitin/ADPr signals.

A second significant avenue for future research involves investigating SIRT6, a mono-ADP-ribosyl transferase involved in multiple biological pathways relevant to aging. Despite being its first identified enzymatic activity, the mono-ADPr activity of SIRT6 has received less attention than its deacylation activity,largely due to a lack of reliable antibodies for detecting mono-ADPr. We anticipate that applying our novel tools (Cell 2020; Mol Cell 2023; Nat Comms 2024) will reveal the importance of SIRT6's mono-ADPr activity in aging.

3. Possible project(s):

1. ADP-ribosylation in ageing: molecular mechanisms of the mono-ADP-ribosyl transferase activity of SIRT6
   SIRT6 has been in the spotlight of research on aging because of its ability to influence multiple physiological and pathological processes  associated to organismal and cellular aging, most notably maintenance of genomic stability and regulation of the cellular epigenetic landscape. Interestingly, mice overexpressing Sirt6 display longer lifespan, while mice lacking Sirt6 show lifespan reduction. SIRT6 maintains genomic stability via improved efficiency of DNA repair and is particularly relevant under oxidative stress, which may be the source of persistent DNA damage in aged tissues. Upon its recruitment to chromatin under oxidative stress, SIRT6 mono-ADP-ribosylates PARP1, thereby stimulating its activity and enhancing DNA repair. Mechanistically the effect of SIRT6 on PARP1 has not been studied in the context of Ser-ADPr signaling and histone ADPr. Thus, our chemical biology approaches combined with advanced proteomics would make it possible to test the hypothesis that SIRT6 activation increases the levels of histone Ser-mono-ADPr and that this promotes chromatin remodeling and recruitment of repair proteins to chromatin possibly through crosstalk with other histone marks. These histone-focused studies could be complemented with global proteomic analyses of mono-ADPr sites (using our high-affinity mono-ADPr specific antibodies forpeptide immunoprecipitation) to determine the substrates of wild type (WT) SIRT6 as well its centenarian variant. Similarly to our transformative discovery of Ser-ADPr, I expect that the elucidation of the exact biochemical basis of SIRT6 would finally allow us to make sense of the debated dual deacetylase/mono-ADPr activities of SIRT6 and fully unveil the exact molecular mechanisms by which SIRT6 exerts its well-known aging-related functions.
2. Interplay between mono-ADPr and ubiquitination signaling
3. Other possible projects to be discussed (ADPr in stem cell biology, immune signaling…)

4. Applied Methods and model organisms:

• Methods: advanced proteomics, computational data analyses, chemical biology approaches, phage display for generating modular antibodies, biochemical enrichment (immunoprecipitation/pull downs), cell culture, western blotting, live-cell imaging, immunofluorescence, cell culture.
• Model organisms: mammalian cell culture and the model organism African killifish.

5. Desirable skills and qualifications:

Ability to join an interdisciplinary, collaborative, and highly productive research environment, demonstrating flexibility to thrive in a rapidly advancing research field. The selected candidate will receive extensive training under the direct supervision of the group leader, made possible by the small size of the research group, gaining proficiency in a variety of highly sought-after advanced technologies. Such experience will significantly enhance their career prospects within academia and beyond.

6. References and key publications:

1. Longarini, E. J. Matic, I. Preserving ester-linked modifications reveals glutamate and aspartate mono-ADP-ribosylation by PARP1 and its reversal by PARG. (2024) Nature Communications, May 18, 2024
2. Longarini, E. J. Dauben, H. Locatelli, C. Wondisford, AR. Smith, R. Muench, R. Kolvenbach, Pope, A. Bonfiglio, J. J. Pinto Jurado, E. Fajka- Boja, R. Colby, T. Schuller, M. Ahel, I. Timinszhy, G. O’Sullivan, RJ., Huet, S. Matic, I. Modular antibodies reveal DNA damage-induced mono-ADP- ribosylation as a second wave of PARP1 signaling. Molecular Cell, 2023 May 18;83(10):1743-1760.e11
   ‘Tools of the trade’ highlight in Nature Reviews Molecular Cell Biology 25, page 3 (2024) ‘Deciphering ADP-ribosylation signalling’
   ‘Technology of the Month’ in Trends in Biochemical Sciences (2023) ‘A chemical biology/modular antibody platform for ADP-ribosylation signaling’ – Front cover featuring an artistic illustration of our technology
   Spotlight in Cell Reports Methods ‘Molecular tools unveil distinct waves of ADP-ribosylation during DNA repair’
   Selected for Molecular Cell’s ‘Best of 2023’ collection
3. Bonfiglio, J. J. Leidecker, O. Dauben, H. Longarini, E. J. Colby, T. San Segundo-Acosta, P. Perez, K. A. Matic, I*. (2020) An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation. Cell, 2020 Nov,183, 4, 1086-1102 e23
   Recommended by Polo S: Faculty Opinions (formerly F1000) of [Bonfiglio JJ et al., Cell 2020 183(4):1086-1102.e23]. In Faculty Opinions, 03 Dec 2020; 10.3410/f.739034237.793580334
   Granted EPO Patent EP3853241B1 (2039-09-17 anticipated expiration) “Site-specific serine adp-ribosylated proteins and peptides and method for producing the same” by Juan José Bonfiglio and Ivan Matic
4. Bonfiglio JJ, Fontana P, Zhang Q, Colby T, Gibbs-Seymour I, Atanassov I, Bartlett E, Zaja R, Ahel I, Matic I* (2017) Serine ADP-Ribosylation Depends on HPF1. Molecular Cell 65: 932-940 e6
   This study was selected by Molecular Cell as the best paper (“Featured Article”) of the March 2, 2017 issue
   Molecular Cell’s interview: “Meet the author Juan José Bonfiglio”
   Front Cover in Molecular Cell. It takes two to tango: PARP1 and HPF1
   Preview in Molecular Cell: 2 March 2017, Pages 777-778 “SERious Surprises for ADP-Ribosylation Specificity: HPF1 Switches PARP1 Specificity to Ser Residues”
   Preview in Cell Chemical Biology 2017 Apr 20;24(4):431-432 “ADP- Ribosylation Goes Normal: Serine as the Major Site of the Modification”
5. Leidecker O, Bonfiglio JJ, Colby T, Zhang Q, Atanassov I, Zaja R, Palazzo L, Stockum A, Ahel I, Matic I* (2016) Serine is a new target residue for endogenous ADP-ribosylation on histones. Nature Chemical Biology 12: 998-+
   Highlighted by the magazine Chemical & Engineering News (C&EN), the world's most comprehensive and authoritative source of news about chemistry and related fields: “Tracking a tricky chemical protein modification” (October 17, 2016 Volume 94, Issue 41)
   Interview by the New York-based GenomeWeb, the largest online newsroom focused on advanced molecular research tools: “Max Planck PTM Studies Suggest Benefits of Manual Inspection of Mass Spec Data” (Dec 16, 2016)