bims-indpro Biomed News
on Intrinsically disordered proteins
Issue of 2022–10–09
fourteen papers selected by
Sara Mingu, Johannes Gutenberg University
  1. pH Induced Switch in the Conformational Ensemble of Intrinsically Disordered Protein Prothymosin-α and Its Implications for Amyloid Fibril Formation.
  2. A simple method to purify intrinsically disordered proteins by adjusting trichloroacetic acid concentration.
  3. Linear discriminant analysis reveals hidden patterns in NMR chemical shifts of intrinsically disordered proteins.
  4. Trehalose and tardigrade CAHS proteins work synergistically to promote desiccation tolerance.
  5. Surfactancy in a tadpole model of proteins.
  6. Reweighting methods for elucidation of conformation ensembles of proteins.
  7. Condensation of the fusion focus by the intrinsically disordered region of the formin Fus1 is essential for cell-cell fusion.
  8. Specialized germline P-bodies are required to specify germ cell fate in C. elegans embryos.
  9. Tryptophan mutations in G3BP1 tune the stability of a cellular signaling hub by weakening transient interactions with Caprin1 and USP10.
  10. Identification of chicken LOC420478 as Bucky ball equivalent and potential germ plasm organizer in birds.
  11. A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.
  12. An in silico study of how histone tail conformation affects the binding affinity of ING family proteins.
  13. Aging RNA granule dynamics in neurodegeneration.
  14. Keeping up with the condensates: The retention, gain, and loss of nuclear membrane-less organelles.
  1. J Phys Chem Lett. 2022 Oct 07. 9589-9598
    pH Induced Switch in the Conformational Ensemble of Intrinsically Disordered Protein Prothymosin-α and Its Implications for Amyloid Fibril Formation.
    Lipika Baidya, Govardhan Reddy.
      Aggregation of intrinsically disordered proteins (IDPs) can lead to neurodegenerative diseases. Although there is experimental evidence that acidic pH promotes IDP monomer compaction leading to aggregation, the general mechanism is unclear. We studied the pH effect on the conformational ensemble of prothymosin-α (proTα), which is involved in multiple essential functions, and probed its role in aggregation using computer simulations. We show that compaction in the proTα dimension at low pH is due to the protein's collapse in the intermediate region (E41-D80) rich in glutamic acid residues, enhancing its β-sheet content. We observed by performing dimer simulations that the conformations with high β-sheet content could act as aggregation-prone (N*) states and nucleate the aggregation process. The simulations initiated using N* states form dimers within a microsecond time scale, whereas the non-N* states do not form dimers within this time scale. This study contributes to understanding the general principles of pH-induced IDP aggregation.
    DOI:  https://doi.org/10.1021/acs.jpclett.2c01972
  2. Protein Expr Purif. 2022 Sep 28. pii: S1046-5928(22)00140-1. [Epub ahead of print] 106183
    A simple method to purify intrinsically disordered proteins by adjusting trichloroacetic acid concentration.
    Sofia P Romero-Perez, Alejandra A Covarrubias, Francisco Campos.
      Late embryogenic abundant proteins (LEA) are a group of proteins that accumulate during the desiccation phase of the seed and in response to water deficit in the plant. Most LEA proteins are highly hydrophilic and have physicochemical characteristics similar to those of intrinsically disordered proteins (IDPs). Although the function of LEA proteins is not fully understood, there is evidence indicating that these proteins have an important role in reducing the effects caused by water limitation. The analysis of the biochemical and physicochemical characteristics of LEA proteins is crucial to determine their function, for which it is necessary to obtain large amounts of pure protein. Within this current work, we have improved our previous TCA purification method used for basic recombinant LEA proteins to obtain acidic IDPs, the method reported here is fast and simple and is based on the enrichment of the protein of interest by boiling of the bacterial extract followed by a precipitation with different concentrations of TCA and salt. This protocol was applied to acidic and basic IDPs, represented by eight recombinant LEAs, resulting in milligram quantities of highly enriched proteins, which keep their in vitro functionality.
    Keywords:  Enzyme protection; Intrinsically disordered proteins (IDPs); Late embryogenesis abundant proteins (LEAPs); Protein purification; TCA precipitation
    DOI:  https://doi.org/10.1016/j.pep.2022.106183
  3. PLoS Comput Biol. 2022 Oct 06. 18(10): e1010258
    Linear discriminant analysis reveals hidden patterns in NMR chemical shifts of intrinsically disordered proteins.
    Javier A Romero, Paulina Putko, Mateusz Urbańczyk, Krzysztof Kazimierczuk, Anna Zawadzka-Kazimierczuk.
      NMR spectroscopy is key in the study of intrinsically disordered proteins (IDPs). Yet, even the first step in such an analysis-the assignment of observed resonances to particular nuclei-is often problematic due to low peak dispersion in the spectra of IDPs. We show that the assignment process can be aided by finding "hidden" chemical shift patterns specific to the amino acid residue types. We find such patterns in the training data from the Biological Magnetic Resonance Bank using linear discriminant analysis, and then use them to classify spin systems in an α-synuclein sample prepared by us. We describe two situations in which the procedure can greatly facilitate the analysis of NMR spectra. The first involves the mapping of spin systems chains onto the protein sequence, which is part of the assignment procedure-a prerequisite for any NMR-based protein analysis. In the second, the method supports assignment transfer between similar samples. We conducted experiments to demonstrate these cases, and both times the majority of spin systems could be unambiguously assigned to the correct residue types.
    DOI:  https://doi.org/10.1371/journal.pcbi.1010258
  4. Commun Biol. 2022 10 01. 5(1): 1046
    Trehalose and tardigrade CAHS proteins work synergistically to promote desiccation tolerance.
    Kenny Nguyen, Shraddha Kc, Tyler Gonzalez, Hugo Tapia, Thomas C Boothby.
      Tardigrades are microscopic animals renowned for their ability to survive extreme desiccation. Unlike many desiccation-tolerant organisms that accumulate high levels of the disaccharide trehalose to protect themselves during drying, tardigrades accumulate little or undetectable levels. Using comparative metabolomics, we find that despite being enriched at low levels, trehalose is a key biomarker distinguishing hydration states of tardigrades. In vitro, naturally occurring stoichiometries of trehalose and CAHS proteins, intrinsically disordered proteins with known protective capabilities, were found to produce synergistic protective effects during desiccation. In vivo, this synergistic interaction is required for robust CAHS-mediated protection. This demonstrates that trehalose acts not only as a protectant, but also as a synergistic cosolute. Beyond desiccation tolerance, our study provides insights into how the solution environment tunes intrinsically disordered proteins' functions, many of which are vital in biological contexts such as development and disease that are concomitant with large changes in intracellular chemistry.
    DOI:  https://doi.org/10.1038/s42003-022-04015-2
  5. J R Soc Interface. 2022 Oct;19(195): 20220172
    Surfactancy in a tadpole model of proteins.
    O T Dyer, R C Ball.
      We model the environment of eukaryotic nuclei by representing macromolecules by only their entropic properties, with globular molecules represented by spherical colloids and flexible molecules by polymers. We put particular focus on proteins with both globular and intrinsically disordered regions, which we represent with 'tadpole' constructed by grafting single polymers and colloids together. In Monte Carlo simulations, we find these tadpoles support phase separation via depletion flocculation, and demonstrate several surfactant behaviours, including being found preferentially at interfaces and forming micelles in single phase solution. Furthermore, the model parameters can be tuned to give a tadpole a preference for either bulk phase. However, we find entropy too weak to drive these behaviours by itself at likely biological concentrations.
    Keywords:  depletion flocculation; intrinsically disordered region; liquid–liquid phase separation; surfactant
    DOI:  https://doi.org/10.1098/rsif.2022.0172
  6. Curr Opin Struct Biol. 2022 Sep 29. pii: S0959-440X(22)00149-X. [Epub ahead of print]77 102470
    Reweighting methods for elucidation of conformation ensembles of proteins.
    Raquel Gama Lima Costa, David Fushman.
      Proteins are inherently dynamic macromolecules that exist in equilibrium among multiple conformational states, and motions of protein backbone and side chains are fundamental to biological function. The ability to characterize the conformational landscape is particularly important for intrinsically disordered proteins, multidomain proteins, and weakly bound complexes, where single-structure representations are inadequate. As the focus of structural biology shifts from relatively rigid macromolecules toward larger and more complex systems and molecular assemblies, there is a need for structural approaches that can paint a more realistic picture of such conformationally heterogeneous systems. Here, we review reweighting methods for elucidation of structural ensembles based on experimental data, with the focus on applications to multidomain proteins.
    DOI:  https://doi.org/10.1016/j.sbi.2022.102470
  7. Curr Biol. 2022 Sep 29. pii: S0960-9822(22)01470-1. [Epub ahead of print]
    Condensation of the fusion focus by the intrinsically disordered region of the formin Fus1 is essential for cell-cell fusion.
    Ingrid Billault-Chaumartin, Olivia Muriel, Laetitia Michon, Sophie G Martin.
      Secretory vesicle clusters transported on actin filaments by myosin V motors for local secretion underlie various cellular processes, such as neurotransmitter release at neuronal synapses,1 hyphal steering in filamentous fungi,2,3 and local cell wall digestion preceding the fusion of yeast gametes.4 During fission yeast Schizosaccharomyces pombe gamete fusion, the actin fusion focus assembled by the formin Fus1 concentrates secretory vesicles carrying cell wall digestive enzymes.5-7 The position and coalescence of the vesicle focus are controlled by local signaling and actin-binding proteins to prevent inappropriate cell wall digestion that would cause lysis,6,8-10 but the mechanisms of focusing have been elusive. Here, we show that the regulatory N terminus of Fus1 contains an intrinsically disordered region (IDR) that mediates Fus1 condensation in vivo and forms dense assemblies that exclude ribosomes. Fus1 lacking its IDR fails to concentrate in a tight focus and causes cell lysis during attempted cell fusion. Remarkably, the replacement of Fus1 IDR with a heterologous low-complexity region that forms molecular condensates fully restores Fus1 focusing and function. By contrast, the replacement of Fus1 IDR with a domain that forms more stable oligomers restores focusing but poorly supports cell fusion, suggesting that condensation is tuned to yield a selectively permeable structure. We propose that condensation of actin structures by an IDR may be a general mechanism for actin network organization and the selective local concentration of secretory vesicles.
    Keywords:  FUS; IDR; actin cytoskeleton; cell-cell fusion; condensate; cryptochrome CRY2; formin; fused in sarcoma; intrinsically disordered region; myosin V Myo52; optogenetics; yeast Schizosaccharomyces pombe
    DOI:  https://doi.org/10.1016/j.cub.2022.09.026
  8. Development. 2022 Oct 05. pii: dev.200920. [Epub ahead of print]
    Specialized germline P-bodies are required to specify germ cell fate in C. elegans embryos.
    Madeline Cassani, Geraldine Seydoux.
      In animals with germ plasm, specification of the germline involves "germ granules", cytoplasmic condensates that enrich maternal transcripts in the germline founder cells. In C. elegans embryos, P granules enrich maternal transcripts, but surprisingly P granules are not essential for germ cell fate specification. Here we describe a second condensate in the C. elegans germ plasm. Like canonical P-bodies found in somatic cells, "germline P-bodies" contain regulators of mRNA decapping and deadenylation and, in addition, the intrinsically-disordered proteins MEG-1 and MEG-2 and the TIS11-family RNA-binding protein POS-1. Embryos lacking meg-1 and meg-2 do not stabilize P-body components, miss-regulate POS-1 targets, miss-specify the germline founder cell, and do not develop a germline. Our findings suggest that specification of the germ line involves at least two distinct condensates that independently enrich and regulate maternal mRNAs in the germline founder cells.
    Keywords:  C. elegans; Germ plasm; Germline; P bodies; Primordial germ cells; RNP granules
    DOI:  https://doi.org/10.1242/dev.200920
  9. J Biol Chem. 2022 Sep 29. pii: S0021-9258(22)00996-6. [Epub ahead of print] 102552
    Tryptophan mutations in G3BP1 tune the stability of a cellular signaling hub by weakening transient interactions with Caprin1 and USP10.
    Colin T Sheehan, Thomas H Hampton, Dean R Madden.
      Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of these G3BP1 affinities, and show tryptophan substitutions of specific G3BP1 residues reduce its affinity for both USP10 and Caprin1. We show that these same mutations reduce the stability of complexes between the full-length proteins, suggesting that co-purification can serve as a surrogate measure of interaction strength. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with USP10 and Caprin1. Furthermore, an amino-acid scan of USP10 and Caprin1 peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific substitutions with the potential to create higher affinity peptides. Taken together, these data show that small changes in component binding affinities can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes.
    Keywords:  bacterial pathogenesis; cancer; crystal structure; crystallography; deubiquitination; intrinsically disordered protein; mutagenesis; mutagenesis in vitro; neurodegeneration; peptide interaction; protein complex; protein structure; protein-protein interaction
    DOI:  https://doi.org/10.1016/j.jbc.2022.102552
  10. Sci Rep. 2022 Oct 07. 12(1): 16858
    Identification of chicken LOC420478 as Bucky ball equivalent and potential germ plasm organizer in birds.
    Sabine Klein, Roland Dosch, Stefanie Altgilbers, Wilfried A Kues.
      Bucky ball was identified as germ plasm organizer in zebrafish and has proven crucial for Balbiani body condensation. A synteny comparison identified an uncharacterized gene locus in the chicken genome as predicted avian counterpart. Here, we present experimental evidence that this gene locus indeed encodes a 'Bucky ball' equivalent in matured oocytes and early embryos of chicken. Heterologous expression of Bucky ball fusion proteins both from zebrafish and chicken with a fluorescent reporter revealed unique patterns indicative for liquid-liquid phase separation of intrinsically disordered proteins. Immuno-labeling detected Bucky ball from oocytes to blastoderms with diffuse distribution in matured oocytes, aggregation in first cleavage furrows, and co-localization to the chicken vasa homolog (CVH). Later, Bucky ball translocated to the cytoplasm of first established cells, and showed nuclear translocation during the major zygotic activation together with CVH. Remarkably, during the phase of area pellucida formation, Bucky ball translocated back into the cytoplasm at stage EGK VI, whereas CVH remained within the nuclei. The condensation of Bucky ball and co-localization with CVH in cleavage furrows and nuclei of the centrally located cells strongly suggests chicken Bucky ball as a germ plasm organizer in birds, and indicate a special importance of the major zygotic activation for germline specification.
    DOI:  https://doi.org/10.1038/s41598-022-21239-8
  11. Comput Struct Biotechnol J. 2022 ;20 5098-5114
    A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.
    M Laura Darriba, Celeste Pueblas Castro, Lorena M Coria, Laura Bruno, M Laura Cerutti, Lisandro H Otero, Lucía B Chemes, Rodolfo M Rasia, Sebastián Klinke, Juliana Cassataro, Karina A Pasquevich.
      U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.
    Keywords:  CD, Circular dichroism; GdmCl, guanidinium chloride; IDP, intrinsically disordered protein; IMAC, immobilized metal ion affinity chromatography; MBP, maltose binding protein; MW, molecular weight; Mucosal adjuvant; NMR, nuclear magnetic resonance; Protease inhibitor; Protein crystallization; Protein structure; SEC, size exclusion chromatography; SLS, Static light scattering; Structure-activity relationship; TEV, Tobacco Etch Virus; U-Omp19, unlipidated outer membrane protein 19
    DOI:  https://doi.org/10.1016/j.csbj.2022.08.054
  12. PeerJ. 2022 ;10 e14029
    An in silico study of how histone tail conformation affects the binding affinity of ING family proteins.
    Nadir Gül, Ahmet Yıldız.
       Background: Due to its intrinsically disordered nature, the histone tail is conformationally heterogenic. Therefore, it provides specific binding sites for different binding proteins or factors through reversible post-translational modifications (PTMs). For instance, experimental studies stated that the ING family binds with the histone tail that has methylation on the lysine in position 4. However, numerous complexes featuring a methylated fourth lysine residue of the histone tail can be found in the UniProt database. So the question arose if other factors like the conformation of the histone tail affect the binding affinity.
    Methods: The crystal structure of the PHD finger domain from the proteins ING1, ING2, ING4, and ING5 are docked to four histone H3 tails with two different conformations using Haddock 2.4 and ClusPro. The best four models for each combination are selected and a two-sample t-test is performed to compare the binding affinities of helical conformations vs. linear conformations using Prodigy. The protein-protein interactions are examined using LigPlot.
    Results: The linear histone conformations in predicted INGs-histone H3 complexes exhibit statistically significant higher binding affinity than their helical counterparts (confidence level of 99%). The outputs of predicted models generated by the molecular docking programs Haddock 2.4 and ClusPro are comparable, and the obtained protein-protein interaction patterns are consistent with experimentally confirmed binding patterns.
    Conclusion: The results show that the conformation of the histone tail is significantly affecting the binding affinity of the docking protein. Herewith, this in silico study demonstrated in detail the binding preference of the ING protein family to histone H3 tail. Further research on the effect of certain PTMs on the final tail conformation and the interaction between those factors seem to be promising for a better understanding of epigenetics.
    Keywords:  Binding affinity; Epigenetics; Histone Tail; ING Protein; In silico; Intrinsically Disordered Proteins; Molecular docking
    DOI:  https://doi.org/10.7717/peerj.14029
  13. Front Mol Biosci. 2022 ;9 991641
    Aging RNA granule dynamics in neurodegeneration.
    Kevin Rhine, Norah Al-Azzam, Tao Yu, Gene W Yeo.
      Disordered RNA-binding proteins and repetitive RNA sequences are the main genetic causes of several neurodegenerative diseases, including amyotrophic lateral sclerosis and Huntington's disease. Importantly, these components also seed the formation of cytoplasmic liquid-like granules, like stress granules and P bodies. Emerging evidence demonstrates that healthy granules formed via liquid-liquid phase separation can mature into solid- or gel-like inclusions that persist within the cell. These solidified inclusions are a precursor to the aggregates identified in patients, demonstrating that dysregulation of RNA granule biology is an important component of neurodegeneration. Here, we review recent literature highlighting how RNA molecules seed proteinaceous granules, the mechanisms of healthy turnover of RNA granules in cells, which biophysical properties underly a transition to solid- or gel-like material states, and why persistent granules disrupt the cellular homeostasis of neurons. We also identify various methods that will illuminate the contributions of disordered proteins and RNAs to neurodegeneration in ongoing research efforts.
    Keywords:  RNA; RNA granules; liquid-liquid phase separation; neurodegeneration; stress granules
    DOI:  https://doi.org/10.3389/fmolb.2022.991641
  14. Front Mol Biosci. 2022 ;9 998363
    Keeping up with the condensates: The retention, gain, and loss of nuclear membrane-less organelles.
    Emma Lacroix, Timothy E Audas.
      In recent decades, a growing number of biomolecular condensates have been identified in eukaryotic cells. These structures form through phase separation and have been linked to a diverse array of cellular processes. While a checklist of established membrane-bound organelles is present across the eukaryotic domain, less is known about the conservation of membrane-less subcellular structures. Many of these structures can be seen throughout eukaryotes, while others are only thought to be present in metazoans or a limited subset of species. In particular, the nucleus is a hub of biomolecular condensates. Some of these subnuclear domains have been found in a broad range of organisms, which is a characteristic often attributed to essential functionality. However, this does not always appear to be the case. For example, the nucleolus is critical for ribosomal biogenesis and is present throughout the eukaryotic domain, while the Cajal bodies are believed to be similarly conserved, yet these structures are dispensable for organismal survival. Likewise, depletion of the Drosophila melanogaster omega speckles reduces viability, despite the apparent absence of this domain in higher eukaryotes. By reviewing primary research that has analyzed the presence of specific condensates (nucleoli, Cajal bodies, amyloid bodies, nucleolar aggresomes, nuclear speckles, nuclear paraspeckles, nuclear stress bodies, PML bodies, omega speckles, NUN bodies, mei2 dots) in a cross-section of organisms (e.g., human, mouse, D. melanogaster, Caenorhabditis elegans, yeast), we adopt a human-centric view to explore the emergence, retention, and absence of a subset of nuclear biomolecular condensates. This overview is particularly important as numerous biomolecular condensates have been linked to human disease, and their presence in additional species could unlock new and well characterized model systems for health research.
    Keywords:  compartmentalization; eukaryota; functional amyloid aggregation; liquid-liquid phase separation (LLPS); membraneless organelles (MLOs); nuclear condensate
    DOI:  https://doi.org/10.3389/fmolb.2022.998363
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