The regulation of the ADP-ribosyl-hydrolase MacroD2 upon DNA damage

To cope with the rapid changes in the environment, all the cellular processes must be carefully regulated and this is often achieved by means of the posttranslational modifications. These chemical modifications added to the side chains of amino acids in protein sequences have a variety of consequences, ranging from the stability to the change in protein-protein interactions. One of these post-translational modifications is ADP-ribosylation. ADPribosylation is mainly associated to the regulation of DNA damage repair, although it is important for the modulation of a large variety of cellular processes. Being a complex signaling pathway, ADP-ribosylation requires a likely complex metabolism and a number of transferases and hydrolases orchestrate its spatial and temporal dynamics. Among the latter, MacroD2 removes the most proximal ADP-ribosyl-moiety from the glutamate of a substrate protein. My host lab is interested in the understanding of the modulation of the DNA damage repair response by focusing on the ADP-ribosyl-hydrolases. This brought my direct supervisor, Dr. G. Timinszky, to discover a particular phenomenon associated to MacroD2 protein: when the protein is tagged with EGFP, upon DNA damage it recruits to the DNA lesion, while its nuclear signal decreases over time. The work of my PhD aimed to understand and characterize the strange DNA damage-dependent behavior of MacroD2. For my work, I use a combination of microscopy and biochemical technics. Thus, I show that the decrease in MacroD2 nuclear protein levels is due to its regulated nuclear export. This behavior of EGFP-tagged MacroD2 construct is also performed by the endogenous protein. I then characterize the stimulus that triggers MacroD2 nuclear export. Upon DNA damage, mostly upon formation of double-strand breaks, the kinase ATM, master regulator of genotoxic stress response, is activated. ATM induces the phosphorylation of MacroD2 on two specific serine residues located in the MacroD2 intrinsically disordered C-terminus region. This event triggers the nuclear export of MacroD2. I also show that MacroD2 nuclear export is able to affect its own recruitment dynamics at the DNA lesion, suggesting a potential role in the regulation of the DNA damage response. Although I defined the events inducing MacroD2 nuclear export, to understand the mechanism with which MacroD2 crosses the nuclear envelope, I performed a co-immunopurification experiment associated to peptide mass fingerprinting. By comparing genotoxic stress condition to the control, and a series of MacroD2 constructs (full-length protein, macrodomain or Cterminus fragments), it is possible to draw the interactome of the protein for the specific constructs in the specific conditions. The experiment does not indicate any candidate that could drive MacroD2 into the cytoplasm. On the other hand, the co-immunopurification experiment suggests few hypotheses about MacroD2 functional roles in the cells. In fact, although little is known about MacroD2 functions, by combining the analyses on the enriched proteins with published studies, I formulate testable hypotheses able to connect the enzymatic activity of MacroD2 to its genomic association with autism syndrome.