Identifying the molecular function of MacroD2 and TARG1

Möller, Giuliana Katharina (2020): Identifying the molecular function of MacroD2 and TARG1. Dissertation, LMU München: Medizinische Fakultät

Abstract

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 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 lacking either MacroD2, TARG1 or both enzymes. With those cell lines I systematically screened for phenotypes related to the identified MacroD2 interactors. I screened all cell lines for defects in intensity or localization of the actin cytoskeleton and focal adhesions with immunofluorescence experiments. I could not identify any defects. Subsequently, I addressed if the knockout cell lines had defects in actin regulated processes such as cell migration and attachment. I realized that only cells lacking both MacroD2 and TARG1 had tremendous defects in cell migration and attachment. In order to identify how cell migration and attachment were deregulated in cells lacking MacroD2 and TARG1, I tested epidermal growth factor receptor (EGFR) signaling as a possible deregulated pathway. I found that cells lacking both enzymes did not increase cell migration in response to EGF treatment and that EGFR was accumulated in perinuclear foci after EGF treatment. In summary, I could show that cells lacking MacroD2 and TARG1 had defects in cell migration and attachment, as well as deregulated EGFR signaling. The fact that MacroD2 and TARG1 can compensate for each other in cell migration, attachment, and EGFR signaling suggests that they perform at least partially redundant functions in unstressed cells.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: MacroD2, TARG1, ADP-ribosylation
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 10. Januar 2020
• 1. Bericht­erstatter:in: Ladurner, Andreas