Medizinische Universität Graz - Research portal
Selected Publication:
Usluer, S.
Regulation of Nuclear Import and Phase Separation
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medizinische Universität Graz; 2023. pp.
- Authors Med Uni Graz:
- Advisor:
- Höfler Gerald
- Madl Tobias
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- Abstract:
- Protein interactions are crucial in many cellular processes, including nuclear import and DNA repair. Intrinsically disordered regions (IDRs) enhance the complexity of these interactions due to their flexibility and lack of structure, enabling them to interact with multiple partners. This multivalence allows IDRs to form transient interactions, which allows for liquid-liquid phase separation (LLPS) in vitro and association with membrane-less organelles (MLOs) in cells. In my thesis, I aimed to show the importance of IDRs on protein interactions and how the sequence context of IDRs governs their biophysical features. Many RNA-binding proteins (RBPs), harboring disordered arginine-glycine/glycine-rich (RG/RGG) regions, are linked to various (patho)physiology processes. RG/RGG regions can regulate nuclear import and condensate formation by interacting with nuclear import receptors like transportin-1 (TNPO1). Considering the differences in RG/RGG protein behavior in terms of condensate formation, MLOs recruitment and nuclear import, I systematically investigated how the sequence context of RG/RGG regions alters condensate formation and TNPO1 binding, using model and natural peptides, and the RBP FUS. I identified that net charge, aromatic residues and charge density are important for condensate formation, stress granule recruitment and TNPO1-mediated chaperoning. Also, mutations in FUS RG/RGG regions interfered with its TNPO1-mediated nuclear import. Our findings help to understand how the intricate sequence ‘code’ of disordered RG/RGG proteins determines their diverse cellular functions. In the second part of my thesis, I focused on the interaction of p53 with arginine-rich IDRs. p53, a tumor suppressor gene, mediates several pathways after DNA damage. The double-strand break (DSB) protein MRE11, harboring the disordered glycine-arginine-rich (GAR) domain, plays an important role in DSB repair. There is preliminary evidence suggesting colocalization and interaction between p53, PML protein, and MRE11 at DSB sites. To reveal the molecular details of these interactions, I aimed to identify the domains mediating the p53-MRE11 binding and elucidate how post-translational modifications (PTMs) regulate it. I discovered that, in vitro, p53 binds to MRE11GAR mainly via p53TAD2. Phosphorylation of p53TAD2 enhances this interaction and methylation of MRE11GAR still allows binding to p53. I provide new insights into the molecular details of the p53-MRE11 complex formation and its regulation through PTMs, elucidating potential regulatory mechanisms that will promote our understanding of the DNA damage response. Moreover, the previous study showed that poly-PR dipeptide repeats encoded by chromosome 9 open reading frame 72 are linked to p53 stabilization in neuronal cells. In this work, I aimed to understand molecular mechanisms of poly-PR-mediated p53 stabilization. I showed that poly-PR/GR interact with p53 via its TAD2 domain and mediate its phase separation in vitro. These results may help uncover the role of p53 in neurodegeneration linked to poly-PR/GR. In general, my thesis highlights the significance of IDRs, their sequence context, and PTMs in protein interactions and biological functions. My findings suggest how the sequence code of RG/RGG regions and IDRs itself can regulate their cellular localization, potentially affecting their stability and functions in pathological contexts.