Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Zhou, Q.
Investigating the multitude of interactome for intrinsically disordered regions associated with phase separation
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medizinische Universität Graz; 2024. pp.
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Höfler Gerald
- Madl Tobias
- Altmetrics:
- Abstract:
- Intrinsically disordered regions (IDRs) are protein regions without a folded structure. They constitute a substantial part of the human proteome. Over the past decades, strategies based on diverse biophysical and biochemical approaches have been developed to investigate the structural and functional features of IDRs. However, despite the continuously increasing database of IDR-interactomes and sophisticated characterization of binding complex structures, most IDRs are still poorly understood. They form interactions with proteins, small molecules, and nucleic acids and play critical roles in essential cellular processes including nuclear transport, transcription regulation, and formation of membraneless organelles via liquid-liquid phase separation (LLPS) under the regulation by post-translational modifications (PTMs). Interactions between IDRs and nuclear transport receptors (NTRs) determine the nuclear localization of proteins, this includes transportin 3 (TNPO3) which recognizes IDRs consist of serine-arginine (SR)-repeats as its classical nuclear localization signal (NLS). Previous studies suggested the presence of its non-classical NLSs and here, our data described three different TNPO3-NLSs in which tyrosine-rich motif and positive charges are the driving force of the interactions. This includes a crystal structure showing the binding complex between TNPO3 and the NLS of cold-inducible RNA-binding protein (CIRBP), which is negatively regulated by serine and tyrosine phosphorylation. CIRBP is involved in the biological granule formation via LLPS, which harbours an RG/RGG region being an IDR rich in arginine-glycine/arginine-glycine-glycine motifs. We have identified that nucleotides and dinucleotides such as ATP and NADPH are the only binding partners of the RG/RGG region of CIRBP among human metabolites and can regulate its RNA-induced LLPS as a hydrotrope. Meanwhile, we used molecular dynamics (MD) simulations to highlight the transient low-affinity interactions mediated by phenylalanine aromatic cycles within artificially designed RG/RGG-mimicking peptide (FRGG)7. To interfere with IDR interactions, we further performed structure-based protein design using RosettaRemodel to design short peptides targeting IDRs that form transient α helical structures. The targets are the second transactivation domain (TAD2) of p53 and the IDR adjacent to the region of Nucleoporin 98kD (Nup98) rich in phenylalanine-glycine (FG) motif. The former protein, p53, is a transcription factor that regulates cell proliferation and senescence, whereas the latter protein, Nup98, is located in the nuclear pore complex (NPC) that maintains a permeability barrier via LLPS to regulate protein nuclear translocation. We succeeded in designing binders targeting both and demonstrated the feasibility of our design conception. Overall, our study used diverse approaches to investigate the multitude of IDR interaction mechanisms.