Medizinische Universität Graz - Research portal
Selected Publication:
Lenard, A.
Unraveling the complexity of intrinsically disordered protein regions: Investigating regulatory mechanisms governing structure, dynamics, and function.
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medizinische Universität Graz; 2024. pp.
- Authors Med Uni Graz:
- Advisor:
- Madl Tobias
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- Abstract:
- Aberrant protein liquid-liquid phase separation (LLPS) is a vital and widespread phenomenon underlying the assembly of membraneless organelles (MLOs) in cells. The presence of missense mutations and defective nucleocytoplsmic transport of arginine- glycine(-glycine)-rich (RG/RGG) protein regions, which are highly abundant in RNA- binding proteins (RBPs), have been associated with misregulated LLPS and MLO formation, implying their contribution to the onset and progression of neurodegeneration. LLPS and MLO recruitment of these regions are intricately regulated by post-translational modifications, such as arginine methylation and phosphorylation, as well as binding to nuclear import receptors, such as transportin-1. An emerging evidence suggests that numerous RBPs harbor citrullination sites in their disordered arginine-rich regions. Protein citrullination, catalyzed by calcium-dependent protein arginine deiminases (PADs), converts peptidyl-arginine residues to peptidyl-citrullines, resulting in the loss of positive charge. Elevated levels of protein citrullination are frequently observed in patients suffering from multiple sclerosis and various autoimmune, neurodegenerative and cancer diseases. The first part of this dissertation aimed at unravelling the effects of PAD4-mediated citrullination on model arginine-rich regions derived from FUS, G3BP1, and Nucleoprotein from SARS-CoV-2. Solution NMR data offers valuable insights into PAD4-mediated citrullination in vitro, facilitating residue-level assignment of modified sites, elucidation of citrullination kinetics and its modulation, and the derivation and comparison of structural and dynamic parameters for both an unmodified and citrullinated protein. Through a combination of solution NMR spectroscopy, ITC, DIC, and turbidity assays, it is demonstrated that citrullination of these regions reduces their RNA-induced LLPS, RNA- binding, and interaction with Transportin-1 in vitro. Furthermore, the obtained findings unveiled a reduced stress granules association of FUS upon its citrullination in semi- permeabilized cells. Additionally, the undertaken investigation revealed the lack of demethylation or citrullination of methylarginines by PAD4 in vitro. In conclusion, these results suggest that citrullination of disordered arginine-rich regions regulates their LLPS and transportin-1 mediated nuclear import. In the second part of this work, I introduce OptoFluidic Force Induction (OF2i) technology as a novel tool for real-time monitoring of particle concentration, and temporal evolution. This is motivated by our incomplete understanding of the molecular details underlying the transition of soluble single molecules into oligomeric complexes with liquid-like droplet properties, as well as the mechanisms regulating changes in the size distribution of nanoclusters formed by phase separating proteins. In this study, OF2i was utilized to monitor DTT-dependent aggregation of insulin, and investigate the role of RNA and salt concentration for RNA-mediated condensate formation of disordered regions from SARS- CoV-2 Nucleoprotein and FUS. In the summary, OF2i technology shows a promise as a tool to quantitatively characterize the size distribution and dynamics of condensates and protein aggregates.