bims-indpro Biomed News
on Intrinsically disordered proteins
Issue of 2022‒07‒31
twenty-two papers selected by
Sara Mingu
Johannes Gutenberg University
  1. Single-droplet surface-enhanced Raman scattering decodes the molecular determinants of liquid-liquid phase separation.
  2. Compositional Bias of Intrinsically Disordered Proteins and Regions and Their Predictions.
  3. IDPsBind: a repository of binding sites for intrinsically disordered proteins complexes with known 3D structures.
  4. Editorial: Intrinsically Disordered Proteins and Regions: The Challenge to the Structure-Function Relationship.
  5. In-Silico Analysis of pH-Dependent Liquid-Liquid Phase Separation in Intrinsically Disordered Proteins.
  6. CARs-DB: A Database of Cryptic Amyloidogenic Regions in Intrinsically Disordered Proteins.
  7. NMR Reveals Specific Tracts within the Intrinsically Disordered Regions of the SARS-CoV-2 Nucleocapsid Protein Involved in RNA Encountering.
  8. A Mechanistic Model for Cell Cycle Control in Which CDKs Act as Switches of Disordered Protein Phase Separation.
  9. Lighting up Nobel Prize-winning studies with protein intrinsic disorder.
  10. JmjC Family of Histone Demethylases Form Nuclear Condensates.
  11. The stickers and spacers of Rubiscondensation: assembling the heartpiece of biophysical CO2 concentrating mechanisms.
  12. Charting the human disease condensate dysregulome.
  13. Assessment of Optimal Conditions for Marine Invertebrate Cell-Mediated Mineralization of Organic Matrices.
  14. Structure and assembly of cargo Rubisco in two native α-carboxysomes.
  15. Zinc controls PML nuclear body formation through regulation of a paralog specific auto-inhibition in SUMO1.
  16. PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora.
  17. Heparin promotes rapid fibrillation of the basic Parathyroid Hormone at physiological pH.
  18. The Molten Globule State of a Globular Protein in a Cell Is More or Less Frequent Case Rather than an Exception.
  19. Speculation on How RIC-3 and Other Chaperones Facilitate α7 Nicotinic Receptor Folding and Assembly.
  20. Essential Roles and Risks of G-Quadruplex Regulation: Recognition Targets of ALS-Linked TDP-43 and FUS.
  21. Phase Separation-Mediated Chromatin Organization and Dynamics: From Imaging-Based Quantitative Characterizations to Functional Implications.
  22. Probing Liquid-Liquid Phase Separation of RNA-Binding Proteins In Vitro and In Vivo.
  1. Nat Commun. 2022 Jul 28. 13(1): 4378
    Single-droplet surface-enhanced Raman scattering decodes the molecular determinants of liquid-liquid phase separation.
    Anamika Avni, Ashish Joshi, Anuja Walimbe, Swastik G Pattanashetty, Samrat Mukhopadhyay.
      Biomolecular condensates formed via liquid-liquid phase separation (LLPS) are involved in a myriad of critical cellular functions and debilitating neurodegenerative diseases. Elucidating the role of intrinsic disorder and conformational heterogeneity of intrinsically disordered proteins/regions (IDPs/IDRs) in these phase-separated membrane-less organelles is crucial to understanding the mechanism of formation and regulation of biomolecular condensates. Here we introduce a unique single-droplet surface-enhanced Raman scattering (SERS) methodology that utilizes surface-engineered, plasmonic, metal nanoparticles to unveil the inner workings of mesoscopic liquid droplets of Fused in Sarcoma (FUS) in the absence and presence of RNA. These highly sensitive measurements offer unprecedented sensitivity to capture the crucial interactions, conformational heterogeneity, and structural distributions within the condensed phase in a droplet-by-droplet manner. Such an ultra-sensitive single-droplet vibrational methodology can serve as a potent tool to decipher the key molecular drivers of biological phase transitions of a wide range of biomolecular condensates involved in physiology and disease.
    DOI:  https://doi.org/10.1038/s41467-022-32143-0
  2. Biomolecules. 2022 Jun 25. pii: 888. [Epub ahead of print]12(7):
    Compositional Bias of Intrinsically Disordered Proteins and Regions and Their Predictions.
    Bi Zhao, Lukasz Kurgan.
      Intrinsically disordered regions (IDRs) carry out many cellular functions and vary in length and placement in protein sequences. This diversity leads to variations in the underlying compositional biases, which were demonstrated for the short vs. long IDRs. We analyze compositional biases across four classes of disorder: fully disordered proteins; short IDRs; long IDRs; and binding IDRs. We identify three distinct biases: for the fully disordered proteins, the short IDRs and the long and binding IDRs combined. We also investigate compositional bias for putative disorder produced by leading disorder predictors and find that it is similar to the bias of the native disorder. Interestingly, the accuracy of disorder predictions across different methods is correlated with the correctness of the compositional bias of their predictions highlighting the importance of the compositional bias. The predictive quality is relatively low for the disorder classes with compositional bias that is the most different from the "generic" disorder bias, while being much higher for the classes with the most similar bias. We discover that different predictors perform best across different classes of disorder. This suggests that no single predictor is universally best and motivates the development of new architectures that combine models that target specific disorder classes.
    Keywords:  amino acid bias; amino acids; disorder prediction; disorder propensity; disorder scale; intrinsic disorder; intrinsic disordered regions; intrinsically disordered proteins; predictive performance
    DOI:  https://doi.org/10.3390/biom12070888
  3. BMC Mol Cell Biol. 2022 Jul 26. 23(1): 33
    IDPsBind: a repository of binding sites for intrinsically disordered proteins complexes with known 3D structures.
    CanZhuang Sun, YongE Feng, GuoLiang Fan.
      BACKGROUND: Intrinsically disordered proteins (IDPs) lack a stable three-dimensional structure under physiological conditions but play crucial roles in many biological processes. Intrinsically disordered proteins perform various biological functions by interacting with other ligands.RESULTS: Here, we present a database, IDPsBind, which displays interacting sites between IDPs and interacting ligands by using the distance threshold method in known 3D structure IDPs complexes from the PDB database. IDPsBind contains 9626 IDPs complexes and 880 intrinsically disordered proteins verified by experiments. The current release of the IDPsBind database is defined as version 1.0. IDPsBind is freely accessible at http://www.s-bioinformatics.cn/idpsbind/home/ .
    CONCLUSIONS: IDPsBind provides more comprehensive interaction sites for IDPs complexes of known 3D structures. It can not only help the subsequent studies of the interaction mechanism of intrinsically disordered proteins but also provides a suitable background for developing the algorithms for predicting the interaction sites of intrinsically disordered proteins.
    Keywords:  Binding sites; Intrinsically Disordered Proteins; Intrinsically Disordered Proteins Complexes; PDB
    DOI:  https://doi.org/10.1186/s12860-022-00434-5
  4. Front Mol Biosci. 2022 ;9 962643
    Editorial: Intrinsically Disordered Proteins and Regions: The Challenge to the Structure-Function Relationship.
    Angelo Toto, Pietro Sormanni, Cristina Paissoni, Vladimir N Uversky.
      
    Keywords:  intrinsically disordered protein; intrinsically disordered region; multifunctional protein; protein structure; protein-protein interaction; structure-function continuum
    DOI:  https://doi.org/10.3389/fmolb.2022.962643
  5. Biomolecules. 2022 Jul 12. pii: 974. [Epub ahead of print]12(7):
    In-Silico Analysis of pH-Dependent Liquid-Liquid Phase Separation in Intrinsically Disordered Proteins.
    Carlos Pintado-Grima, Oriol Bárcenas, Salvador Ventura.
      Intrinsically disordered proteins (IDPs) are essential players in the assembly of biomolecular condensates during liquid-liquid phase separation (LLPS). Disordered regions (IDRs) are significantly exposed to the solvent and, therefore, highly influenced by fluctuations in the microenvironment. Extrinsic factors, such as pH, modify the solubility and disorder state of IDPs, which in turn may impact the formation of liquid condensates. However, little attention has been paid to how the solution pH influences LLPS, despite knowing that this process is context-dependent. Here, we have conducted a large-scale in-silico analysis of pH-dependent solubility and disorder in IDRs known to be involved in LLPS (LLPS-DRs). We found that LLPS-DRs present maximum solubility around physiological pH, where LLPS often occurs, and identified significant differences in solubility and disorder between proteins that can phase-separate by themselves or those that require a partner. We also analyzed the effect of mutations in the resulting solubility profiles of LLPS-DRs and discussed how, as a general trend, LLPS-DRs display physicochemical properties that permit their LLPS at physiologically relevant pHs.
    Keywords:  bioinformatics; intrinsically disordered proteins; liquid-liquid phase separation; mutations; pH; protein disorder; protein solubility
    DOI:  https://doi.org/10.3390/biom12070974
  6. Front Mol Biosci. 2022 ;9 882160
    CARs-DB: A Database of Cryptic Amyloidogenic Regions in Intrinsically Disordered Proteins.
    Carlos Pintado-Grima, Oriol Bárcenas, Zoe Manglano-Artuñedo, Rita Vilaça, Sandra Macedo-Ribeiro, Irantzu Pallarès, Jaime Santos, Salvador Ventura.
      Proteome-wide analyses suggest that most globular proteins contain at least one amyloidogenic region, whereas these aggregation-prone segments are thought to be underrepresented in intrinsically disordered proteins (IDPs). In recent work, we reported that intrinsically disordered regions (IDRs) indeed sustain a significant amyloid load in the form of cryptic amyloidogenic regions (CARs). CARs are widespread in IDRs, but they are necessarily exposed to solvent, and thus they should be more polar and have a milder aggregation potential than conventional amyloid regions protected inside globular proteins. CARs are connected with IDPs function and, in particular, with the establishment of protein-protein interactions through their IDRs. However, their presence also appears associated with pathologies like cancer or Alzheimer's disease. Given the relevance of CARs for both IDPs function and malfunction, we developed CARs-DB, a database containing precomputed predictions for all CARs present in the IDPs deposited in the DisProt database. This web tool allows for the fast and comprehensive exploration of previously unnoticed amyloidogenic regions embedded within IDRs sequences and might turn helpful in identifying disordered interacting regions. It contains >8,900 unique CARs identified in a total of 1711 IDRs. CARs-DB is freely available for users and can be accessed at http://carsdb.ppmclab.com. To validate CARs-DB, we demonstrate that two previously undescribed CARs selected from the database display full amyloidogenic potential. Overall, CARs-DB allows easy access to a previously unexplored amyloid sequence space.
    Keywords:  amyloid; database; disease; intrinsically disordered proteins; protein-protein interactions
    DOI:  https://doi.org/10.3389/fmolb.2022.882160
  7. Biomolecules. 2022 Jul 02. pii: 929. [Epub ahead of print]12(7):
    NMR Reveals Specific Tracts within the Intrinsically Disordered Regions of the SARS-CoV-2 Nucleocapsid Protein Involved in RNA Encountering.
    Letizia Pontoriero, Marco Schiavina, Sophie M Korn, Andreas Schlundt, Roberta Pierattelli, Isabella C Felli.
      The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. 13C-detected NMR experiments, acquired simultaneously to 1H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1-248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction.
    Keywords:  COVID-19; IDP; NMR; RNA; SARS-CoV-2
    DOI:  https://doi.org/10.3390/biom12070929
  8. Cells. 2022 Jul 13. pii: 2189. [Epub ahead of print]11(14):
    A Mechanistic Model for Cell Cycle Control in Which CDKs Act as Switches of Disordered Protein Phase Separation.
    Liliana Krasinska, Daniel Fisher.
      Cyclin-dependent kinases (CDKs) are presumed to control the cell cycle by phosphorylating a large number of proteins involved in S-phase and mitosis, two mechanistically disparate biological processes. While the traditional qualitative model of CDK-mediated cell cycle control relies on differences in inherent substrate specificity between distinct CDK-cyclin complexes, they are largely dispensable according to the opposing quantitative model, which states that changes in the overall CDK activity level promote orderly progression through S-phase and mitosis. However, a mechanistic explanation for how such an activity can simultaneously regulate many distinct proteins is lacking. New evidence suggests that the CDK-dependent phosphorylation of ostensibly very diverse proteins might be achieved due to underlying similarity of phosphorylation sites and of the biochemical effects of their phosphorylation: they are preferentially located within intrinsically disordered regions of proteins that are components of membraneless organelles, and they regulate phase separation. Here, we review this evidence and suggest a mechanism for how a single enzyme's activity can generate the dynamics required to remodel the cell at mitosis.
    Keywords:  cyclin-dependent kinase; intrinsic disorder; phase separation
    DOI:  https://doi.org/10.3390/cells11142189
  9. Cell Mol Life Sci. 2022 Jul 26. 79(8): 449
    Lighting up Nobel Prize-winning studies with protein intrinsic disorder.
    Lolita Piersimoni, Marina Abd El Malek, Twinkle Bhatia, Julian Bender, Christin Brankatschk, Jaime Calvo Sánchez, Guy W Dayhoff, Alessio Di Ianni, Jhonny Oscar Figueroa Parra, Dailen Garcia-Martinez, Julia Hesselbarth, Janett Köppen, Luca M Lauth, Laurin Lippik, Lisa Machner, Shubhra Sachan, Lisa Schmidt, Robin Selle, Ioannis Skalidis, Oleksandr Sorokin, Daniele Ubbiali, Bruno Voigt, Alice Wedler, Alan An Jung Wei, Peter Zorn, Alan Keith Dunker, Marcel Köhn, Andrea Sinz, Vladimir N Uversky.
      Intrinsically disordered proteins and regions (IDPs and IDRs) and their importance in biology are becoming increasingly recognized in biology, biochemistry, molecular biology and chemistry textbooks, as well as in current protein science and structural biology curricula. We argue that the sequence → dynamic conformational ensemble → function principle is of equal importance as the classical sequence → structure → function paradigm. To highlight this point, we describe the IDPs and/or IDRs behind the discoveries associated with 17 Nobel Prizes, 11 in Physiology or Medicine and 6 in Chemistry. The Nobel Laureates themselves did not always mention that the proteins underlying the phenomena investigated in their award-winning studies are in fact IDPs or contain IDRs. In several cases, IDP- or IDR-based molecular functions have been elucidated, while in other instances, it is recognized that the respective protein(s) contain IDRs, but the specific IDR-based molecular functions have yet to be determined. To highlight the importance of IDPs and IDRs as general principle in biology, we present here illustrative examples of IDPs/IDRs in Nobel Prize-winning mechanisms and processes.
    Keywords:  Computational methods; Disorder prediction; Intrinsically disordered proteins and regions; Nobel Prize
    DOI:  https://doi.org/10.1007/s00018-022-04468-y
  10. Int J Mol Sci. 2022 Jul 11. pii: 7664. [Epub ahead of print]23(14):
    JmjC Family of Histone Demethylases Form Nuclear Condensates.
    Marta Vicioso-Mantis, Samuel Aguirre, Marian A Martínez-Balbás.
      The Jumonji-C (JmjC) family of lysine demethylases (KDMs) (JMJC-KDMs) plays an essential role in controlling gene expression and chromatin structure. In most cases, their function has been attributed to the demethylase activity. However, accumulating evidence demonstrates that these proteins play roles distinct from histone demethylation. This raises the possibility that they might share domains that contribute to their functional outcome. Here, we show that the JMJC-KDMs contain low-complexity domains and intrinsically disordered regions (IDR), which in some cases reached 70% of the protein. Our data revealed that plant homeodomain finger protein (PHF2), KDM2A, and KDM4B cluster by phase separation. Moreover, our molecular analysis implies that PHF2 IDR contributes to transcription regulation. These data suggest that clustering via phase separation is a common feature that JMJC-KDMs utilize to facilitate their functional responses. Our study uncovers a novel potential function for the JMJC-KDM family that sheds light on the mechanisms to achieve the competent concentration of molecules in time and space within the cell nucleus.
    Keywords:  IDR; JmjC; epigenetic regulation; histone demethylases (HDM); phase separation
    DOI:  https://doi.org/10.3390/ijms23147664
  11. J Exp Bot. 2022 Jul 29. pii: erac321. [Epub ahead of print]
    The stickers and spacers of Rubiscondensation: assembling the heartpiece of biophysical CO2 concentrating mechanisms.
    Warren Shou Leong Ang, Jian Ann How, Jian Boon How, Oliver Mueller-Cajar.
      Aquatic autotrophs that fix carbon using ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) frequently expend metabolic energy to pump inorganic carbon towards the enzyme's active site. A central requirement of this strategy is the formation of highly concentrated Rubisco condensates known as carboxysomes and pyrenoids, which have convergently evolved multiple times in prokaryotes and eukaryotes respectively. Recent data indicates these condensates form by the mechanism of liquid- liquid phase separation (LLPS). LLPS requires networks of weak multivalent interactions typically mediated by intrinsically disordered scaffold proteins. Here we comparatively review recent rapid developments that detail the determinants and precise interactions that underlie diverse Rubisco condensates. The burgeoning field of biomolecular condensates has few examples where LLPS can be linked to clear phenotypic outcomes. When present, Rubisco condensates are essential for photosynthesis and growth, and they are thus emerging as powerful and tractable models to investigate the structure function relationship of phase separation in biology.
    Keywords:  CO2 fixation; Rubisco; biomolecular condensates; carboxysome; phase separation; pyrenoid
    DOI:  https://doi.org/10.1093/jxb/erac321
  12. Dev Cell. 2022 Jul 25. pii: S1534-5807(22)00490-7. [Epub ahead of print]57(14): 1677-1679
    Charting the human disease condensate dysregulome.
    Bappaditya Chandra, Richard Kriwacki.
      Although it is understood that myriad proteins function in cells within biomolecular condensates, it is unclear how protein condensation is altered in human disease. In this issue of Developmental Cell, Banani et al. show that mutations in disease-associated proteins may map to phase-separation-prone regions and thereby alter condensate formation.
    DOI:  https://doi.org/10.1016/j.devcel.2022.07.001
  13. Biomimetics (Basel). 2022 Jun 26. pii: 86. [Epub ahead of print]7(3):
    Assessment of Optimal Conditions for Marine Invertebrate Cell-Mediated Mineralization of Organic Matrices.
    Jeremy Elias, Thomas Angelini, Mark Q Martindale, Laurie Gower.
      Cellular strategies and regulation of their crystallization mechanisms are essential to the formation of biominerals, and harnessing these strategies will be important for the future creation of novel non-native biominerals that recapitulate the impressive properties biominerals possess. Harnessing these biosynthetic strategies requires an understanding of the interplay between insoluble organic matrices, mineral precursors, and soluble organic and inorganic additives. Our long-range goal is to use a sea anemone model system (Nematostella vectensis) to examine the role of intrinsically disordered proteins (IDPs) found in native biomineral systems. Here, we study how ambient temperatures (25-37 °C) and seawater solution compositions (varying NaCl and Mg ratios) will affect the infiltration of organic matrices with calcium carbonate mineral precursors generated through a polymer-induced liquid-precursor (PILP) process. Fibrillar collagen matrices were used to assess whether solution conditions were suitable for intrafibrillar mineralization, and SEM with EDS was used to analyze mineral infiltration. Conditions of temperatures 30 °C and above and with low Mg:Ca ratios were determined to be suitable conditions for calcium carbonate infiltration. The information obtained from these observations may be useful for the manipulation and study of cellular secreted IDPs in our quest to create novel biosynthetic materials.
    Keywords:  PILP; biomimetic processing; biomineralization; collagen mineralization
    DOI:  https://doi.org/10.3390/biomimetics7030086
  14. Nat Commun. 2022 Jul 25. 13(1): 4299
    Structure and assembly of cargo Rubisco in two native α-carboxysomes.
    Tao Ni, Yaqi Sun, Will Burn, Monsour M J Al-Hazeem, Yanan Zhu, Xiulian Yu, Lu-Ning Liu, Peijun Zhang.
      Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. They encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase catalyzing carbon fixation inside a proteinaceous shell. How Rubisco complexes pack within the carboxysomes is unknown. Using cryo-electron tomography, we determine the distinct 3D organization of Rubisco inside two distant α-carboxysomes from a marine α-cyanobacterium Cyanobium sp. PCC 7001 where Rubiscos are organized in three concentric layers, and from a chemoautotrophic bacterium Halothiobacillus neapolitanus where they form intertwining spirals. We further resolve the structures of native Rubisco as well as its higher-order assembly at near-atomic resolutions by subtomogram averaging. The structures surprisingly reveal that the authentic intrinsically disordered linker protein CsoS2 interacts with Rubiscos in native carboxysomes but functions distinctively in the two α-carboxysomes. In contrast to the uniform Rubisco-CsoS2 association in the Cyanobium α-carboxysome, CsoS2 binds only to the Rubiscos close to the shell in the Halo α-carboxysome. Our findings provide critical knowledge of the assembly principles of α-carboxysomes, which may aid in the rational design and repurposing of carboxysome structures for new functions.
    DOI:  https://doi.org/10.1038/s41467-022-32004-w
  15. Nucleic Acids Res. 2022 Jul 25. pii: gkac620. [Epub ahead of print]
    Zinc controls PML nuclear body formation through regulation of a paralog specific auto-inhibition in SUMO1.
    Mathieu Lussier-Price, Haytham M Wahba, Xavier H Mascle, Laurent Cappadocia, Veronique Bourdeau, Christina Gagnon, Sebastian Igelmann, Kazuyasu Sakaguchi, Gerardo Ferbeyre, James G Omichinski.
      SUMO proteins are important regulators of many key cellular functions in part through their ability to form interactions with other proteins containing SUMO interacting motifs (SIMs). One characteristic feature of all SUMO proteins is the presence of a highly divergent intrinsically disordered region at their N-terminus. In this study, we examine the role of this N-terminal region of SUMO proteins in SUMO-SIM interactions required for the formation of nuclear bodies by the promyelocytic leukemia (PML) protein (PML-NBs). We demonstrate that the N-terminal region of SUMO1 functions in a paralog specific manner as an auto-inhibition domain by blocking its binding to the phosphorylated SIMs of PML and Daxx. Interestingly, we find that this auto-inhibition in SUMO1 is relieved by zinc, and structurally show that zinc stabilizes the complex between SUMO1 and a phospho-mimetic form of the SIM of PML. In addition, we demonstrate that increasing cellular zinc levels enhances PML-NB formation in senescent cells. Taken together, these results provide important insights into a paralog specific function of SUMO1, and suggest that zinc levels could play a crucial role in regulating SUMO1-SIM interactions required for PML-NB formation and function.
    DOI:  https://doi.org/10.1093/nar/gkac620
  16. Proc Natl Acad Sci U S A. 2022 Aug 02. 119(31): e2203078119
    PRD-2 mediates clock-regulated perinuclear localization of clock gene RNAs within the circadian cycle of Neurospora.
    Bradley M Bartholomai, Amy S Gladfelter, Jennifer J Loros, Jay C Dunlap.
      The transcription-translation negative feedback loops underlying animal and fungal circadian clocks are remarkably similar in their molecular regulatory architecture and, although much is understood about their central mechanism, little is known about the spatiotemporal dynamics of the gene products involved. A common feature of these circadian oscillators is a significant temporal delay between rhythmic accumulation of clock messenger RNAs (mRNAs) encoding negative arm proteins, for example, frq in Neurospora and Per1-3 in mammals, and the appearance of the clock protein complexes assembled from the proteins they encode. Here, we report use of single-molecule RNA fluorescence in situ hybridization (smFISH) to show that the fraction of nuclei actively transcribing the clock gene frq changes in a circadian manner, and that these mRNAs cycle in abundance with fewer than five transcripts per nucleus at any time. Spatial point patterning statistics reveal that frq is spatially clustered near nuclei in a time of day-dependent manner and that clustering requires an RNA-binding protein, PRD-2 (PERIOD-2), recently shown also to bind to mRNA encoding another core clock component, casein kinase 1. An intrinsically disordered protein, PRD-2 displays behavior in vivo and in vitro consistent with participation in biomolecular condensates. These data are consistent with a role for phase-separating RNA-binding proteins in spatiotemporally organizing clock mRNAs to facilitate local translation and assembly of clock protein complexes.
    Keywords:  Neurospora; cell biology; circadian rhythms; liquid–liquid phase separation; smFISH
    DOI:  https://doi.org/10.1073/pnas.2203078119
  17. FEBS Lett. 2022 Jul 28.
    Heparin promotes rapid fibrillation of the basic Parathyroid Hormone at physiological pH.
    Luca M Lauth, Bruno Voigt, Twinkle Bhatia, Lisa Machner, Jochen Balbach, Maria Ott.
      In acidic secretory granules of mammalian cells, peptide hormones including the parathyroid hormone (PTH) are presumably stored in the form of functional amyloid fibrils. Mature PTH, however, is considerably positively charged in acidic environments, a condition known to impede unassisted self-aggregation into fibrils. Here, we studied the role of the polyanion heparin on promoting fibril formation of PTH. Employing ITC, CD spectroscopy, NMR, SAXS and fluorescence-based assays we could demonstrate that heparin binds PTH with submicromolar affinity and facilitates its conversion into fibrillar seeds, enabling rapid formation of amyloid fibrils under acidic conditions. In absence of heparin, PTH remained in a soluble monomeric state. We suspect that heparin-like surfaces are required in vivo to convert PTH efficiently into fibrillar deposits.
    Keywords:  acidic pH; amyloid fibrillation; functional amyloids; heparin interaction; intrinsically disordered proteins; parathyroid hormone
    DOI:  https://doi.org/10.1002/1873-3468.14455
  18. Molecules. 2022 Jul 07. pii: 4361. [Epub ahead of print]27(14):
    The Molten Globule State of a Globular Protein in a Cell Is More or Less Frequent Case Rather than an Exception.
    Valentina E Bychkova, Dmitry A Dolgikh, Vitalii A Balobanov, Alexei V Finkelstein.
      Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn's hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners.
    Keywords:  chaperone; coil; functional state; globular protein; intrinsically disordered; membrane; molten globule; post-translational modifications; rigid native state; unfolded state
    DOI:  https://doi.org/10.3390/molecules27144361
  19. Molecules. 2022 Jul 15. pii: 4527. [Epub ahead of print]27(14):
    Speculation on How RIC-3 and Other Chaperones Facilitate α7 Nicotinic Receptor Folding and Assembly.
    Ralph H Loring.
      The process of how multimeric transmembrane proteins fold and assemble in the endoplasmic reticulum is not well understood. The alpha7 nicotinic receptor (α7 nAChR) is a good model for multimeric protein assembly since it has at least two independent and specialized chaperones: Resistance to Inhibitors of Cholinesterase 3 (RIC-3) and Nicotinic Acetylcholine Receptor Regulator (NACHO). Recent cryo-EM and NMR data revealed structural features of α7 nAChRs. A ser-ala-pro (SAP) motif precedes a structurally important but unique "latch" helix in α7 nAChRs. A sampling of α7 sequences suggests the SAP motif is conserved from C. elegans to humans, but the latch sequence is only conserved in vertebrates. How RIC-3 and NACHO facilitate receptor subunits folding into their final pentameric configuration is not known. The artificial intelligence program AlphaFold2 recently predicted structures for NACHO and RIC-3. NACHO is highly conserved in sequence and structure across species, but RIC-3 is not. This review ponders how different intrinsically disordered RIC-3 isoforms from C. elegans to humans interact with α7 nAChR subunits despite having little sequence homology across RIC-3 species. Two models from the literature about how RIC-3 assists α7 nAChR assembly are evaluated considering recent structural information about the receptor and its chaperones.
    Keywords:  cys-loop receptors; intracellular domain; molecular modeling; multimeric protein assembly
    DOI:  https://doi.org/10.3390/molecules27144527
  20. Front Mol Biosci. 2022 ;9 957502
    Essential Roles and Risks of G-Quadruplex Regulation: Recognition Targets of ALS-Linked TDP-43 and FUS.
    Akira Ishiguro, Akira Ishihama.
      A non-canonical DNA/RNA structure, G-quadruplex (G4), is a unique structure formed by two or more guanine quartets, which associate through Hoogsteen hydrogen bonding leading to form a square planar arrangement. A set of RNA-binding proteins specifically recognize G4 structures and play certain unique physiological roles. These G4-binding proteins form ribonucleoprotein (RNP) through a physicochemical phenomenon called liquid-liquid phase separation (LLPS). G4-containing RNP granules are identified in both prokaryotes and eukaryotes, but extensive studies have been performed in eukaryotes. We have been involved in analyses of the roles of G4-containing RNAs recognized by two G4-RNA-binding proteins, TDP-43 and FUS, which both are the amyotrophic lateral sclerosis (ALS) causative gene products. These RNA-binding proteins play the essential roles in both G4 recognition and LLPS, but they also carry the risk of agglutination. The biological significance of G4-binding proteins is controlled through unique 3D structure of G4, of which the risk of conformational stability is influenced by environmental conditions such as monovalent metals and guanine oxidation.
    Keywords:  FUS (fused in sarcoma); G-quadruplex (G4); RNP granule; TDP-43 (43 kDa TAR DNA-binding protein); amyotrophic lateral sclerosis (ALS); liquid-liquid phase separation (LLPS)
    DOI:  https://doi.org/10.3389/fmolb.2022.957502
  21. Int J Mol Sci. 2022 Jul 21. pii: 8039. [Epub ahead of print]23(14):
    Phase Separation-Mediated Chromatin Organization and Dynamics: From Imaging-Based Quantitative Characterizations to Functional Implications.
    Woei Shyuan Ng, Hendrik Sielaff, Ziqing Winston Zhao.
      As an effective and versatile strategy to compartmentalize cellular components without the need for lipid membranes, phase separation has been found to underpin a wide range of intranuclear processes, particularly those involving chromatin. Many of the unique physico-chemical properties of chromatin-based phase condensates are harnessed by the cell to accomplish complex regulatory functions in a spatially and temporally controlled manner. Here, we survey key recent findings on the mechanistic roles of phase separation in regulating the organization and dynamics of chromatin-based molecular processes across length scales, packing states and intranuclear functions, with a particular emphasis on quantitative characterizations of these condensates enabled by advanced imaging-based approaches. By illuminating the complex interplay between chromatin and various chromatin-interacting molecular species mediated by phase separation, this review sheds light on an emerging multi-scale, multi-modal and multi-faceted landscape that hierarchically regulates the genome within the highly crowded and dynamic nuclear space. Moreover, deficiencies in existing studies also highlight the need for mechanism-specific criteria and multi-parametric approaches for the characterization of chromatin-based phase separation using complementary techniques and call for greater efforts to correlate the quantitative features of these condensates with their functional consequences in close-to-native cellular contexts.
    Keywords:  DNA damage repair; chromatin organization; intrinsically disordered region; nuclear condensate; phase separation; quantitative imaging; super-enhancer; transcription
    DOI:  https://doi.org/10.3390/ijms23148039
  22. Methods Mol Biol. 2022 ;2537 307-333
    Probing Liquid-Liquid Phase Separation of RNA-Binding Proteins In Vitro and In Vivo.
    Stephanie Heinrich, Maria Hondele.
      Biomolecular condensates and the concept of liquid-liquid phase separation (LLPS) have transformed cell biology in recent years. Condensates organize cellular content and compartmentalize biochemical reactions, in particular many processes involving RNA. This protocol is aimed at readers new to the LLPS field who want to test their protein or cellular structure of interest. We describe the basic principles of liquid-liquid phase separation, and outline initial approaches-both in vitro and in yeast cells-for the characterization of a candidate cellular condensate. First, we focus on strategies to purify phase-separating proteins and to reconstitute condensates from recombinant proteins in vitro for observation by light microscopy. Second, we describe in vivo experiments (including fluorescence recovery after photobleaching (FRAP) microscopy and 1,6-Hexanediol treatment) to test whether a subcellular structure displays liquid-like behavior in cells.
    Keywords:  1,6-Hexanediol; Biomolecular condensates; FRAP; Light microscopy; Liquid–liquid phase separation; Protein purification; RNA; RNA binding proteins
    DOI:  https://doi.org/10.1007/978-1-0716-2521-7_18
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