Identifying the molecular function of MacroD2 and TARG1 [thesis]

Giuliana Katharina Möller, Andreas Ladurner
2020
Human cells need to react to environmental stimuli in a timely manner. One way to achieve this fast reaction time is the attachment of small chemical or biological units to proteins, so-called post-translational modifications. Post-translational modifications are added, read, and removed by different sets of enzymes dependent on the type of post-translational modification. One type of post-translational modification is ADP-ribosylation, a modification where either single or multiple units of
more » ... ultiple units of ADP-ribose are added to proteins by a family of enzymes called PARPs. ADP-ribosylation is involved in a plethora of cellular pathways and in a multitude of essential cellular functions such as DNA damage repair, transcription, and the cell cycle. Proteins modified with a single ADP-ribose moiety are called mono-ADP-ribosylated (MARylated). In MARylated proteins, where the ADP-ribose moiety is linked to the protein via acidic amino acids, the modification can be reversed by three enzymes - MacroD1, MacroD2, and TARG1. While MacroD1 is exclusively mitochondrial, both MacroD2 and TARG1 are present in the nucleus and cytoplasm. Not much is known about the function of MacroD2 and TARG1 so far. Both enzymes are connected to the response to DNA damage and to neurological defects in literature. Therefore, the aim of this thesis was to identify which functions both enzymes possess in human cells. To this end, I utilized a two-pronged approach. Firstly, I identified protein interaction partners of MacroD2 with the BioID approach. I used BioID since this system was generated to identify weak and transient interactions which is necessary since ADP-ribosylation is rapidly added and removed. With the interactors of MacroD2 identified with the BioID approach, I found that many proteins with gene ontology terms related to actin and focal adhesions were enriched. This led to the hypothesis that MacroD2 might be involved in the regulation of the actin cytoskeleton. As a second prong, I generated and validated CRISPR/Cas knockout cell lines lac [...]
doi:10.5282/edoc.25538 fatcat:huvqcpedcfglpbll2ucobxf5ti