Medizinische Universität Graz - Research portal
Selected Publication:
Grissmann, M.
Structural and functional studies of protein citrullination.
[ Diplomarbeit/Master Thesis (UNI) ] Universität Graz; 2023.
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- Authors Med Uni Graz:
- Advisor:
- Madl Tobias
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- Abstract:
- Citrullination is a post-translational modification. The modification is a catalytic process that converts peptidyl-arginine to peptidyl-citrulline in a calcium-dependent manner and is catalysed by the peptidylarginine deiminase (PAD) enzyme family. Misregulation of protein citrullination has been implicated in the onset and progression of several inflammatory and neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease, rheumatoid arthritis (RA), and cancer. The RG/RGG motif, which refers to protein regions that are enriched in arginine (R) and glycine (G), is a consensus site for citrullination. To investigate how citrullination regulates the structural and functional properties of targeted RG/RGG proteins, I applied biomolecular nuclear magnetic resonance spectroscopy (NMR) as well as other biophysical methods such as turbidity assays, differential interference contrast (DIC) microscopy and isothermal titration calorimetry (ITC). Furthermore, I utilized a novel NMR-based metabolomics method that allows us to study citrullination levels in different biological matrices. The results show that proteins containing the disordered RG/RGG motif are robustly citrullinated by PAD4 in vitro. In addition, the findings of this study indicate that arginines within intrinsically disordered regions (IDRs) are preferentially targeted for PAD4-mediated citrullination in vitro. Furthermore, the results demonstrate that citrullination of RG/RGG proteins impairs their liquid-liquid phase separation in vitro as well as reduces the binding to the nuclear import receptor Transportin-1. In addition, the performed metabolomics analysis demonstrated that basal levels of citrullination are very low in several mammalian cell lines without any stimulation. Interestingly, the investigation of the citrullination levels of HeLa cell lysates after adding recombinantly active PAD4 revealed time-dependent alternating dynamic changes in citrullination. This work also demonstrates that studying PTMs by NMR is an interesting and great way to gain more insight into these protein modifications.