bims-indpro Biomed News
on Intrinsically disordered proteins
Issue of 2022–07–24
twelve papers selected by
Sara Mingu, Johannes Gutenberg University
  1. Conformational Characteristics and Phase Behavior of Intrinsically Disordered Proteins─Where Physical Chemistry Meets Biology.
  2. Phase Separation of Heterogeneous Nuclear Ribonucleoprotein A1 upon Specific RNA-Binding Observed by Magnetic Resonance.
  3. Structural characterisation of amyloidogenic intrinsically disordered zinc finger protein isoforms DPF3b and DPF3a.
  4. Intrinsically Disordered Protein Micelles as Vehicles for Convection-Enhanced Drug Delivery to Glioblastoma Multiforme.
  5. Membrane shape deformation induced by curvature-inducing proteins consisting of chiral crescent binding and intrinsically disordered domains.
  6. Nanoscale regulation of Ca2+ dependent phase transitions and real-time dynamics of SAP97/hDLG.
  7. Coacervation of poly-electrolytes in the presence of lipid bilayers: mutual alteration of structure and morphology.
  8. Counting Degrons: Lessons From Multivalent Substrates for Targeted Protein Degradation.
  9. Editorial: Protein Phase Separation and Aggregation in (Patho)Physiology of Neurons.
  10. Systematic discovery of biomolecular condensate-specific protein phosphorylation.
  11. Hyperosmotic-stress-induced liquid-liquid phase separation of ALS-related proteins in the nucleus.
  12. Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export.
  1. J Phys Chem B. 2022 Jul 21. 126(28): 5137-5139
    Conformational Characteristics and Phase Behavior of Intrinsically Disordered Proteins─Where Physical Chemistry Meets Biology.
    Lisha Arora, Samrat Mukhopadhyay.
      
    DOI:  https://doi.org/10.1021/acs.jpcb.2c04017
  2. Angew Chem Int Ed Engl. 2022 Jul 22.
    Phase Separation of Heterogeneous Nuclear Ribonucleoprotein A1 upon Specific RNA-Binding Observed by Magnetic Resonance.
    Irina Ritsch, Elisabeth Lehmann, Leonidas Emmanouilidis, Maxim Yulikov, Frédéric Allain, Gunnar Jeschke.
      Interaction of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with specific single-stranded RNA and its relation to liquid-liquid phase separation (LLPS) were studied in vitro by magnetic resonance based on site-directed spin labelling. An ensemble model of dispersed hnRNP A1 in the absence of RNA was derived from distance distributions between spin labelled sites and small angle X-ray scattering. This model revealed a compact state of the low-complexity domain and its interaction with the RNA recognition motifs. Paramagnetic relaxation enhancement NMR spectroscopy confirmed this interaction. Addition of RNA to dispersed hnRNP A1 induced liquid-droplet formation. Such LLPS depended on RNA concentration and sequence, with continuous wave EPR spectroscopy showing an influence of RNA point mutations on local protein dynamics. We propose that an interplay of sequence-specific RNA binding and LLPS contributes to regulation of specific RNA segregation during stress response.
    Keywords:  EPR spectroscopy; Intrinsically disordered domain; NMR spectroscopy; Protein models; liquid-liquid phase separation
    DOI:  https://doi.org/10.1002/anie.202204311
  3. Int J Biol Macromol. 2022 Jul 18. pii: S0141-8130(22)01534-3. [Epub ahead of print]
    Structural characterisation of amyloidogenic intrinsically disordered zinc finger protein isoforms DPF3b and DPF3a.
    Julien Mignon, Denis Mottet, Tanguy Leyder, Vladimir N Uversky, Eric A Perpète, Catherine Michaux.
      Double PHD fingers 3 (DPF3) is a zinc finger protein, found in the BAF chromatin remodeling complex, and is involved in the regulation of gene expression. Two DPF3 isoforms have been identified, respectively named DPF3b and DPF3a. Very limited structural information is available for these isoforms, and their specific functionality still remains poorly studied. In a previous work, we have demonstrated the first evidence of DPF3a being a disordered protein sensitive to amyloid fibrillation. Intrinsically disordered proteins (IDPs) lack a defined tertiary structure, existing as a dynamic conformational ensemble, allowing them to act as hubs in protein-protein interaction networks. In the present study, we have more thoroughly characterised DPF3a in vitro behaviour, as well as unravelled and compared the structural properties of the DPF3b isoform, using an array of predictors and biophysical techniques. Predictions, spectroscopy, and dynamic light scattering have revealed a high content in disorder: prevalence of random coil, aromatic residues partially to fully exposed to the solvent, and large hydrodynamic diameters. DPF3a appears to be more disordered than DPF3b, and exhibits more expanded conformations. Furthermore, we have shown that they both time-dependently aggregate into amyloid fibrils, as revealed by typical circular dichroism, deep-blue autofluorescence, and amyloid-dye binding assay fingerprints. Although spectroscopic and microscopic analyses have unveiled that they share a similar aggregation pathway, DPF3a fibrillates at a faster rate, likely through reordering of its C-terminal domain.
    Keywords:  Amyloid fibril; Double PHD finger 3 (DPF3); Intrinsically disordered protein; Protein aggregation; Spectroscopy
    DOI:  https://doi.org/10.1016/j.ijbiomac.2022.07.102
  4. ACS Appl Bio Mater. 2022 Jul 20.
    Intrinsically Disordered Protein Micelles as Vehicles for Convection-Enhanced Drug Delivery to Glioblastoma Multiforme.
    Jamie M Gleason, Sarah H Klass, Paul Huang, Tomoko Ozawa, Raquel A Santos, Miko M Fogarty, David R Raleigh, Mitchel S Berger, Matthew B Francis.
      Lipid and micelle-based nanocarriers have been explored for anticancer drug delivery to improve accumulation and uptake in tumor tissue. As an experimental opportunity in this area, our lab has developed a protein-based micelle nanocarrier consisting of a hydrophilic intrinsically disordered protein (IDP) domain bound to a hydrophobic tail, termed IDP-2Yx2A. This construct can be used to encapsulate hydrophobic chemotherapeutics that would otherwise be too insoluble in water to be administered. In this study, we evaluate the in vivo efficacy of IDP-2Yx2A by delivering a highly potent but water-insoluble cancer drug, SN38, into glioblastoma multiforme (GBM) tumors via convection-enhanced delivery (CED). The protein carriers alone are shown to elicit minimal toxicity effects in mice; furthermore, they can encapsulate and deliver concentrations of SN38 that would otherwise be lethal without the carriers. CED administration of these drug-loaded micelles into mice bearing U251-MG GBM xenografts resulted in slowed tumor growth and significant increases in median survival times compared to nonencapsulated SN38 and PBS controls.
    Keywords:  SN38; biomaterials; convection-enhanced delivery; drug delivery; glioblastoma multiforme; intrinsically disordered protein; protein micelle
    DOI:  https://doi.org/10.1021/acsabm.2c00215
  5. J Chem Phys. 2022 Jul 21. 157(3): 034901
    Membrane shape deformation induced by curvature-inducing proteins consisting of chiral crescent binding and intrinsically disordered domains.
    Hiroshi Noguchi.
      Curvature-inducing proteins containing a bin/amphiphysin/Rvs domain often have intrinsically disordered domains. Recent experiments have shown that these disordered chains enhance curvature sensing and generation. Here, we report on the modification of protein-membrane interactions by disordered chains using meshless membrane simulations. The protein and bound membrane are modeled together as a chiral crescent protein rod with two excluded-volume chains. As the chain length increases, the repulsion between them reduces the cluster size of the proteins. It induces spindle-shaped vesicles and a transition between arc-shaped and circular protein assemblies in a disk-shaped vesicle. For flat membranes, an intermediate chain length induces many tubules owing to the repulsion between the protein assemblies, whereas longer chains promote perpendicular elongation of tubules. Moreover, protein rods with zero rod curvature and sufficiently long chains stabilize the spherical buds. For proteins with a negative rod curvature, an intermediate chain length induces a rugged membrane with branched protein assemblies, whereas longer chains induce the formation of tubules with periodic concave-ring structures.
    DOI:  https://doi.org/10.1063/5.0098249
  6. Nat Commun. 2022 Jul 22. 13(1): 4236
    Nanoscale regulation of Ca2+ dependent phase transitions and real-time dynamics of SAP97/hDLG.
    Premchand Rajeev, Nivedita Singh, Adel Kechkar, Cory Butler, Narendrakumar Ramanan, Jean-Baptiste Sibarita, Mini Jose, Deepak Nair.
      Synapse associated protein-97/Human Disk Large (SAP97/hDLG) is a conserved, alternatively spliced, modular, scaffolding protein critical in regulating the molecular organization of cell-cell junctions in vertebrates. We confirm that the molecular determinants of first order phase transition of SAP97/hDLG is controlled by morpho-functional changes in its nanoscale organization. Furthermore, the nanoscale molecular signatures of these signalling islands and phase transitions are altered in response to changes in cytosolic Ca2+. Additionally, exchange kinetics of alternatively spliced isoforms of the intrinsically disordered region in SAP97/hDLG C-terminus shows differential sensitivities to Ca2+ bound Calmodulin, affirming that the molecular signatures of local phase transitions of SAP97/hDLG depends on their nanoscale heterogeneity and compositionality of isoforms.
    DOI:  https://doi.org/10.1038/s41467-022-31912-1
  7. Chem Sci. 2022 Jul 06. 13(26): 7933-7946
    Coacervation of poly-electrolytes in the presence of lipid bilayers: mutual alteration of structure and morphology.
    Sayantan Mondal, Qiang Cui.
      Many intrinsically disordered peptides have been shown to undergo liquid-liquid phase separation and form complex coacervates, which play various regulatory roles in the cell. Recent experimental studies found that such phase separation processes may also occur at the lipid membrane surface and help organize biomolecules during signaling events; in some cases, phase separation of proteins at the membrane surface was also observed to lead to significant remodeling of the membrane morphology. The molecular mechanisms that govern the interactions between complex coacervates and lipid membranes and the impacts of such interactions on their structure and morphology, however, remain unclear. Here we study the coacervation of poly-glutamate (E30) and poly-lysine (K30) in the presence of lipid bilayers of different compositions. We carry out explicit-solvent coarse-grained molecular dynamics simulations by using the MARTINI (v3.0) force-field. We find that more than 20% anionic lipids are required for the coacervate to form stable contact with the bilayer. Upon wetting, the coacervate induces negative curvature to the bilayer and facilitates local lipid demixing, without any peptide insertion. The magnitude of negative curvature, extent of lipid demixing, and asphericity of the coacervate increase with the concentration of anionic lipids. Overall, we observe a decrease in the number of contacts among the polyelectrolytes as the droplet spreads over the bilayer. Therefore, unlike previous suggestions, interactions among polyelectrolytes do not constitute a driving force for the membrane bending upon wetting by the coacervate. Rather, analysis of interaction energy components suggests that bending of the membrane is favored by enhanced interactions between polyelectrolytes with lipids as well as with counterions. Kinetic studies reveal that, at the studied polyelectrolyte concentrations, the coacervate formation precedes bilayer wetting.
    DOI:  https://doi.org/10.1039/d2sc02013k
  8. Front Physiol. 2022 ;13 913063
    Counting Degrons: Lessons From Multivalent Substrates for Targeted Protein Degradation.
    Cynthia N Okoye, Pamela J E Rowling, Laura S Itzhaki, Catherine Lindon.
      E3s comprise a structurally diverse group of at least 800 members, most of which target multiple substrates through specific and regulated protein-protein interactions. These interactions typically rely on short linear motifs (SLiMs), called "degrons", in an intrinsically disordered region (IDR) of the substrate, with variable rules of engagement governing different E3-docking events. These rules of engagement are of importance to the field of targeted protein degradation (TPD), where substrate ubiquitination and destruction require tools to effectively harness ubiquitin ligases (E3s). Substrates are often found to contain multiple degrons, or multiple copies of a degron, contributing to the affinity and selectivity of the substrate for its E3. One important paradigm for E3-substrate docking is presented by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ligase that targets hundreds of proteins for destruction during mitotic exit. APC/C substrate targeting takes place in an ordered manner thought to depend on tightly regulated interactions of substrates, with docking sites provided by the substoichiometric APC/C substrate adaptors and coactivators, Cdc20 or Cdh1/FZR1. Both structural and functional studies of individual APC/C substrates indicate that productive ubiquitination usually requires more than one degron, and that degrons are of different types docking to distinct sites on the coactivators. However, the dynamic nature of APC/C substrate recruitment, and the influence of multiple degrons, remains poorly understood. Here we review the significance of multiple degrons in a number of E3-substrate interactions that have been studied in detail, illustrating distinct kinetic effects of multivalency and allovalency, before addressing the role of multiple degrons in APC/C substrates, key to understanding ordered substrate destruction by APC/C. Lastly, we consider how lessons learnt from these studies can be applied in the design of TPD tools.
    Keywords:  E3-substrate interaction; SLiM; degron; multivalency; targeted protein degradation; ubiquitin ligase
    DOI:  https://doi.org/10.3389/fphys.2022.913063
  9. Front Physiol. 2022 ;13 959570
    Editorial: Protein Phase Separation and Aggregation in (Patho)Physiology of Neurons.
    Dragomir Milovanovic, Silvio O Rizzoli.
      
    Keywords:  AMPA and NMDA-type receptors; TDP43; alpha-synuclein; neurodegenerative diseases; neuron; phase separation; synapse; synaptic vesicles
    DOI:  https://doi.org/10.3389/fphys.2022.959570
  10. Nat Chem Biol. 2022 Jul 21.
    Systematic discovery of biomolecular condensate-specific protein phosphorylation.
    Sindhuja Sridharan, Alberto Hernandez-Armendariz, Nils Kurzawa, Clement M Potel, Danish Memon, Pedro Beltrao, Marcus Bantscheff, Wolfgang Huber, Sara Cuylen-Haering, Mikhail M Savitski.
      Reversible protein phosphorylation is an important mechanism for regulating (dis)assembly of biomolecular condensates. However, condensate-specific phosphosites remain largely unknown, thereby limiting our understanding of the underlying mechanisms. Here, we combine solubility proteome profiling with phosphoproteomics to quantitatively map several hundred phosphosites enriched in either soluble or condensate-bound protein subpopulations, including a subset of phosphosites modulating protein-RNA interactions. We show that multi-phosphorylation of the C-terminal disordered segment of heteronuclear ribonucleoprotein A1 (HNRNPA1), a key RNA-splicing factor, reduces its ability to locate to nuclear clusters. For nucleophosmin 1 (NPM1), an essential nucleolar protein, we show that phosphorylation of S254 and S260 is crucial for lowering its partitioning to the nucleolus and additional phosphorylation of distal sites enhances its retention in the nucleoplasm. These phosphorylation events decrease RNA and protein interactions of NPM1 to regulate its condensation. Our dataset is a rich resource for systematically uncovering the phosphoregulation of biomolecular condensates.
    DOI:  https://doi.org/10.1038/s41589-022-01062-y
  11. Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00888-9. [Epub ahead of print]40(3): 111086
    Hyperosmotic-stress-induced liquid-liquid phase separation of ALS-related proteins in the nucleus.
    Chao Gao, Jinge Gu, Hong Zhang, Kai Jiang, Linlin Tang, Ren Liu, Li Zhang, Pengfei Zhang, Cong Liu, Bin Dai, Jie Song.
      Hyperosmotic stress as physiologic dysfunction can reduce the cell volume and then redistribute both protein concentration and ionic strength, but its effect on liquid-liquid phase separation (LLPS) is not well understood. Here, we map the hyperosmotic-stress-induced nuclear LLPS of amyotrophic lateral sclerosis (ALS)-related proteins (fused in sarcoma [FUS], TAR DNA-binding protein 43 [TDP-43]). The dynamic and reversibility of FUS granules are continuable with the increase of hypertonic stimulation time, but those of TDP-43 granules decrease significantly. Strikingly, FUS granules, but not TDP-43 granules, contain essential chaperone Hsp40, which can protect amyloid protein from solid aggregation. Moreover, FUS nuclear granules can co-localize with paraspeckles, but not promyelocytic leukemia (PML) bodies or nuclear speckles, while TDP-43 nuclear granules cannot co-localize with the above nuclear bodies. Together, these results may broaden our understanding of the LLPS of ALS-related proteins in response to cellular stress.
    Keywords:  ALS-related proteins; CP: Molecular biology; CP: Neuroscience; chaperones; hyperosmotic stress; liquid-liquid phase separation; nuclear granules; paraspeckles
    DOI:  https://doi.org/10.1016/j.celrep.2022.111086
  12. Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00908-1. [Epub ahead of print]40(3): 111106
    Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export.
    Lauren Duan, Benjamin L Zaepfel, Vasilisa Aksenova, Mary Dasso, Jeffrey D Rothstein, Petr Kalab, Lindsey R Hayes.
      Nuclear clearance of the RNA-binding protein TDP-43 is a hallmark of neurodegeneration and an important therapeutic target. Our current understanding of TDP-43 nucleocytoplasmic transport does not fully explain its predominantly nuclear localization or mislocalization in disease. Here, we show that TDP-43 exits nuclei by passive diffusion, independent of facilitated mRNA export. RNA polymerase II blockade and RNase treatment induce TDP-43 nuclear efflux, suggesting that nuclear RNAs sequester TDP-43 in nuclei and limit its availability for passive export. Induction of TDP-43 nuclear efflux by short, GU-rich oligomers (presumably by outcompeting TDP-43 binding to endogenous nuclear RNAs), and nuclear retention conferred by splicing inhibition, demonstrate that nuclear TDP-43 localization depends on binding to GU-rich nuclear RNAs. Indeed, RNA-binding domain mutations markedly reduce TDP-43 nuclear localization and abolish transcription blockade-induced nuclear efflux. Thus, the nuclear abundance of GU-RNAs, dictated by the balance of transcription, pre-mRNA processing, and RNA export, regulates TDP-43 nuclear localization.
    Keywords:  CP: Molecular biology; RNA; TDP-43; amyotrophic lateral sclerosis; frontotemporal dementia; neurodegeneration; nuclear transport; splicing; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2022.111106
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