bims-indpro Biomed News
on Intrinsically disordered proteins
Issue of 2022‒02‒13
twenty-four papers selected by
Sara Mingu
Johannes Gutenberg University
  1. Determinants for intrinsically disordered protein recruitment into phase-separated protein condensates.
  2. Conformational ensembles of intrinsically disordered proteins and flexible multidomain proteins.
  3. Intrinsically disordered proteins/regions and insight into their biomolecular interactions.
  4. A covalent strategy to target intrinsically disordered proteins: Discovery of novel tau aggregation inhibitors.
  5. Single molecule probing of disordered RNA binding proteins.
  6. Structure-Activity Relationships of Novel Tau Ligands: Passive Fibril Binders and Active Aggregation Inhibitors.
  7. Multiscale Computational Study of the Conformation of the Full-Length Intrinsically Disordered Protein MeCP2.
  8. Transactivation domain of Adenovirus Early Region 1A (E1A): Investigating folding dynamics and aggregation.
  9. TAPASS: Tool for Annotation of Protein Amyloidogenicity in the context of other Structural States.
  10. Molecular Basis of Small-Molecule Binding to α-Synuclein.
  11. Phase separation of the mammalian prion protein: physiological and pathological perspectives.
  12. A unifying framework for amyloid-mediated membrane damage: The lipid-chaperone hypothesis.
  13. Telomere length regulation by Rif1 protein from Hansenula polymorpha.
  14. The aqueous humor proteome is intrinsically disordered.
  15. 14-3-3 Proteins are Potential Regulators of Liquid-Liquid Phase Separation.
  16. Prediction of structural alphabet protein blocks using data mining.
  17. The pathogenic R5L mutation disrupts formation of Tau complexes on the microtubule by altering local N-terminal structure.
  18. Regulation of the activity of bacterial histidine kinase PleC by the scaffolding protein PodJ.
  19. Bex1 is essential for ciliogenesis and harbours biomolecular condensate-forming capacity.
  20. Phase Separation by the SARS-CoV-2 Nucleocapsid Protein: Consensus and Open Questions.
  21. RNA-assisted sequestration of RNA-binding proteins by cytoplasmic inclusions of the C-terminal 35-kDa fragment of TDP-43.
  22. Liquid-liquid phase separation drives cellular function and dysfunction in cancer.
  23. Impaired nuclear transport induced by juvenile ALS causing P525L mutation in NLS domain of FUS: A molecular mechanistic study.
  24. Protein phase separation hotspots at the presynapse.
  1. Chem Sci. 2022 Jan 05. 13(2): 522-530
    Determinants for intrinsically disordered protein recruitment into phase-separated protein condensates.
    Yongsang Jo, Jinyoung Jang, Daesun Song, Hyoin Park, Yongwon Jung.
      Multivalent interactions between amino acid residues of intrinsically disordered proteins (IDPs) drive phase separation of these proteins into liquid condensates, forming various membrane-less organelles in cells. These interactions between often biased residues of IDPs are also likely involved in selective recruitment of many other IDPs into condensates. However, determining factors for this IDP recruitment into protein condensates are not understood yet. Here, we quantitatively examined recruitment tendencies of various IDPs with different sequence compositions into IDP-clustered condensates both in vitro as well as in cells. Condensate-forming IDP scaffolds, recruited IDP clients, and phase separation conditions were carefully varied to find key factors for selective IDP partitioning in protein condensates. Regardless of scaffold sequences, charged residues in client IDPs assured potent IDP recruitment, likely via strong electrostatic interactions, where positive residues could further enhance recruitment, possibly with cation-pi interactions. Notably, poly-ethylene glycol, a widely used crowding reagent for in vitro phase separation, abnormally increased IDP recruitment, indicating the need for careful use of crowding conditions. Tyrosines of IDP clients also strongly participated in recruitment both in vitro and in cells. Lastly, we measured recruitment degrees by more conventional interactions between folded proteins instead of disordered proteins. Surprisingly, recruitment forces by an even moderate protein interaction (K d ∼ 5 μM) were substantially stronger than those by natural IDP-IDP interactions. The present data offer valuable information on how cells might organize protein partitioning on various protein condensates.
    DOI:  https://doi.org/10.1039/d1sc05672g
  2. Biochem Soc Trans. 2022 Feb 07. pii: BST20210499. [Epub ahead of print]
    Conformational ensembles of intrinsically disordered proteins and flexible multidomain proteins.
    F Emil Thomasen, Kresten Lindorff-Larsen.
      Intrinsically disordered proteins (IDPs) and multidomain proteins with flexible linkers show a high level of structural heterogeneity and are best described by ensembles consisting of multiple conformations with associated thermodynamic weights. Determining conformational ensembles usually involves the integration of biophysical experiments and computational models. In this review, we discuss current approaches to determine conformational ensembles of IDPs and multidomain proteins, including the choice of biophysical experiments, computational models used to sample protein conformations, models to calculate experimental observables from protein structure, and methods to refine ensembles against experimental data. We also provide examples of recent applications of integrative conformational ensemble determination to study IDPs and multidomain proteins and suggest future directions for research in the field.
    Keywords:  conformational ensemble; molecular dynamics; protein structure
    DOI:  https://doi.org/10.1042/BST20210499
  3. Biophys Chem. 2022 Feb 01. pii: S0301-4622(22)00011-4. [Epub ahead of print]283 106769
    Intrinsically disordered proteins/regions and insight into their biomolecular interactions.
    Pinak Chakrabarti, Devlina Chakravarty.
      Proteins may vary from being rigid to having flexible regions to being completely disordered, either as an intrinsically disordered protein (IDP) or having specific intrinsically disordered regions (IDRs). IDPs/IDRs can form complexes otherwise impossible, such as wrapping around the binding partner, hence providing the plasticity needed for achieving assemblies with specific functions. IDRs can exhibit promiscuity, using the same region in the sequence to bind multiple partners, and act as hubs in protein-protein interaction network (an essential part of the cell signalling network). Disorder-to-order transition on binding provides specificity with affinity, optimum for reversibility of the binding, thus offering suitability for regulation and signalling processes. IDRs interactions may be modulated by the environment or covalent modifications; mis-signalling or their unnatural or non-native folding may lead to diseases. This article aims to provide an overview of structural heterogeneity, as seen in IDPs/IDRs, and their role in biological recognition, binding and function.
    Keywords:  Allostery; Disorder-to-order transition; Flexibility; Intrinsically disordered proteins/regions; Molecular recognition; Promiscuity of binding
    DOI:  https://doi.org/10.1016/j.bpc.2022.106769
  4. Eur J Med Chem. 2022 Jan 29. pii: S0223-5234(22)00065-4. [Epub ahead of print]231 114163
    A covalent strategy to target intrinsically disordered proteins: Discovery of novel tau aggregation inhibitors.
    László Petri, Péter Ábrányi-Balogh, Darius Vagrys, Tímea Imre, Nikolett Varró, István Mándity, Anita Rácz, Lucia Wittner, Kinga Tóth, Estilla Zsófia Tóth, Tünde Juhász, Ben Davis, György Miklós Keserű.
      Intrinsically disordered proteins (IDPs) play important roles in disease pathologies; however, their lack of defined stable 3D structures make traditional drug design strategies typically less effective against these targets. Based on promising results of targeted covalent inhibitors (TCIs) on challenging targets, we have developed a covalent design strategy targeting IDPs. As a model system we chose tau, an endogenous IDP of the central nervous system that is associated with severe neurodegenerative diseases via its aggregation. First, we mapped the tractability of available cysteines in tau and prioritized suitable warheads. Next, we introduced the selected vinylsulfone warhead to the non-covalent scaffolds of potential tau aggregation inhibitors. The designed covalent tau binders were synthesized and tested in aggregation models, and inhibited tau aggregation effectively. Our results revealed the usefulness of the covalent design strategy against therapeutically relevant IDP targets and provided promising candidates for the treatment of tauopathies.
    Keywords:  Covalent inhibition; Electrophilic warhead; Tau aggregation; Tauopathy
    DOI:  https://doi.org/10.1016/j.ejmech.2022.114163
  5. STAR Protoc. 2022 Mar 18. 3(1): 101131
    Single molecule probing of disordered RNA binding proteins.
    Kevin Rhine, Sua Myong.
      Liquid-liquid phase separation of intrinsically disordered proteins is known to underlie diverse pathologies such as neurodegeneration, cancer, and aging. The nucleation step of condensate formation is of critical importance for understanding how healthy and disease-associated condensates differ. Here, we describe four orthogonal single-molecule techniques that enable molecular tracking of the RNA-protein interaction, RNA-induced oligomerization, and kinetics of nucleation. These approaches allow researchers to directly interrogate the initial steps of liquid-liquid phase separation. For complete details on the use and execution of this profile, please refer to Niaki et al. (2020), Rhine et al. (2020), and Rhine et al. (2022).
    Keywords:  Biophysics; Biotechnology and bioengineering; Molecular Biology; Single-molecule Assays
    DOI:  https://doi.org/10.1016/j.xpro.2022.101131
  6. ACS Chem Biol. 2022 Feb 11.
    Structure-Activity Relationships of Novel Tau Ligands: Passive Fibril Binders and Active Aggregation Inhibitors.
    David W Baggett, Abhinav Nath.
      Intrinsically disordered proteins (IDPs) are core components of many biological processes and are central players in several pathologies. Despite being important drug targets, attempts to design small-molecule ligands that would help understand and attenuate their behavior are frustrated by the structural diversity exhibited by these flexible proteins. To accommodate the dynamic nature of IDPs, we developed a procedure that efficiently identifies active small-molecule ligands for disordered proteins. By exploring the chemical space around these ligands, we refined their effect on aggregation and identified molecular features critical for activity and affinity. Notably, the discovery of this new family of disordered protein ligands was achieved more quickly and with less expense than conventional high-throughput screening (HTS) or docking alone would have allowed. The resulting ligands include tau aggregation inhibitors as well as at least one compound that binds fibrils potently but does not appear to perturb the extent of kinetics of aggregation.
    DOI:  https://doi.org/10.1021/acschembio.2c00012
  7. J Chem Inf Model. 2022 Feb 07.
    Multiscale Computational Study of the Conformation of the Full-Length Intrinsically Disordered Protein MeCP2.
    Cecilia Chávez-García, Jérôme Hénin, Mikko Karttunen.
      The malfunction of the methyl-CpG binding protein 2 (MeCP2) is associated with the Rett syndrome, one of the most common causes of cognitive impairment in females. MeCP2 is an intrinsically disordered protein (IDP), making its experimental characterization a challenge. There is currently no structure available for the full-length MeCP2 in any of the databases, and only the structure of its MBD domain has been solved. We used this structure to build a full-length model of MeCP2 by completing the rest of the protein via ab initio modeling. Using a combination of all-atom and coarse-grained simulations, we characterized its structure and dynamics as well as the conformational space sampled by the ID and transcriptional repression domain (TRD) domains in the absence of the rest of the protein. The present work is the first computational study of the full-length protein. Two main conformations were sampled in the coarse-grained simulations: a globular structure similar to the one observed in the all-atom force field and a two-globule conformation. Our all-atom model is in good agreement with the available experimental data, predicting amino acid W104 to be buried, amino acids R111 and R133 to be solvent-accessible, and having a 4.1% α-helix content, compared to the 4% found experimentally. Finally, we compared the model predicted by AlphaFold to our Modeller model. The model was not stable in water and underwent further folding. Together, these simulations provide a detailed (if perhaps incomplete) conformational ensemble of the full-length MeCP2, which is compatible with experimental data and can be the basis of further studies, e.g., on mutants of the protein or its interactions with its biological partners.
    DOI:  https://doi.org/10.1021/acs.jcim.1c01354
  8. Curr Res Struct Biol. 2022 ;4 29-40
    Transactivation domain of Adenovirus Early Region 1A (E1A): Investigating folding dynamics and aggregation.
    Nitin Sharma, Kundlik Gadhave, Prateek Kumar, Rajanish Giri.
      Transactivation domain of Adenovirus Early region 1A (E1A) oncoprotein is an intrinsically disordered molecular hub protein. It is involved in binding to different domains of human cell transcriptional co-activators such as retinoblastoma (pRb), CREB-binding protein (CBP), and its paralogue p300. The conserved region 1 (TAD) of E1A is known to undergo structural transitions and folds upon interaction with transcriptional adaptor zinc finger 2 (TAZ2). Previous reports on Taz2-E1A studies have suggested the formation of helical conformations of E1A-TAD. However, the folding behavior of the TAD region in isolation has not been studied in detail. Here, we have elucidated the folding behavior of E1A peptide at varied temperatures and solution conditions. Further, we have studied the effects of macromolecular crowding on E1A-TAD peptide. Additionally, we have also predicted the molecular recognition features of E1A using MoRF predictors. The predicted MoRFs are consistent with its structural transitions observed during TAZ2 interactions for transcriptional regulation in literature. Also, as a general rule of MoRFs, E1A undergoes helical transitions in alcohol and osmolyte solution. Finally, we studied the aggregation behavior of E1A, where we observed that the E1A could form amyloid-like aggregates that are cytotoxic to mammalian cells.
    Keywords:  E1A; Intrinsically disordered proteins; Molecular recognition elements; Protein aggregation; Protein folding
    DOI:  https://doi.org/10.1016/j.crstbi.2022.01.001
  9. J Struct Biol. 2022 Feb 08. pii: S1047-8477(22)00010-7. [Epub ahead of print] 107840
    TAPASS: Tool for Annotation of Protein Amyloidogenicity in the context of other Structural States.
    Théo Falgarone, Étienne Villain, Abdelkader Guettaf, Jérémy Leclercq, Andrey V Kajava.
      Numerous studies have demonstrated that the propensity of a protein to form amyloids or amorphous aggregates is encoded by its amino acid sequence. This led to the emergence of several computational programs to predict amyloidogenicity from amino acid sequences. However, a growing number of studies indicate that an accurate prediction of the protein aggregation can only be achieved when also accounting for the overall structural context of the protein, and the likelihood of transition between the initial state and the aggregate. Here, we describe a computational pipeline called TAPASS, which was designed to do just that. The pipeline assigns each residue of a protein as belonging to a structured region or an intrinsically disordered region (IDR). For this purpose, TAPASS uses either several state-of-the-art programs for prediction of IDRs, of transmembrane regions and of structured domains or the artificial intelligence program AlphaFold. In the next step, this assignment is crossed with amyloidogenicity prediction. As a result, TAPASS allows the detection of Exposed Amyloidogenic Regions (EARs) located within intrinsically disordered regions (IDRs) and carrying high amyloidogenic potential. TAPASS can substantially improve the prediction of amyloids and be used in proteome-wide analysis to discover new amyloid-forming proteins. Its results, combined with clinical data, can create individual risk profiles for different amyloidoses, opening up new opportunities for personalised medicine. The architecture of the pipeline is designed so that it makes it easy to add new individual predictors as they become available. TAPASS can be used through the web interface (https://bioinfo.crbm.cnrs.fr/index.php?route=tools&tool=32).
    Keywords:  Aggregation; AlphaFold; Amyloid; Bioinformatics; Intrinsically Disordered Regions; Proteome-wide analysis
    DOI:  https://doi.org/10.1016/j.jsb.2022.107840
  10. J Am Chem Soc. 2022 Feb 07.
    Molecular Basis of Small-Molecule Binding to α-Synuclein.
    Paul Robustelli, Alain Ibanez-de-Opakua, Cecily Campbell-Bezat, Fabrizio Giordanetto, Stefan Becker, Markus Zweckstetter, Albert C Pan, David E Shaw.
      Intrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-time-scale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson's disease) binding the small-molecule drug fasudil in which the observed protein-ligand interactions were found to be in good agreement with previously reported NMR chemical shift data. In our simulations, fasudil, when bound, favored certain charge-charge and π-stacking interactions near the C terminus of α-synuclein but tended not to form these interactions simultaneously, rather breaking one of these interactions and forming another nearby (a mechanism we term dynamic shuttling). Further simulations with small molecules chosen to modify these interactions yielded binding affinities and key structural features of binding consistent with subsequent NMR experiments, suggesting the potential for MD-based strategies to facilitate the rational design of small molecules that bind with disordered proteins.
    DOI:  https://doi.org/10.1021/jacs.1c07591
  11. J Neurochem. 2022 Feb 11.
    Phase separation of the mammalian prion protein: physiological and pathological perspectives.
    Mariana J do Amaral, Maria Heloisa O Freire, Marcius S Almeida, Anderson S Pinheiro, Yraima Cordeiro.
      Abnormal phase transitions have been implicated in the occurrence of proteinopathies. Disordered proteins with nucleic acid binding ability drive the formation of reversible micron-sized condensates capable of controlling nucleic acid processing/transport. This mechanism, achieved via liquid-liquid phase separation (LLPS), underlies the formation of long-studied membraneless organelles (e.g., nucleolus) and various transient condensates formed by driver proteins. The prion protein (PrP) is not a classical nucleic acid-binding protein. However, it binds nucleic acids with high affinity, undergoes nucleocytoplasmic shuttling, contains a long intrinsically disordered region rich in glycines and evenly spaced aromatic residues, among other biochemical/biophysical properties of bona fide drivers of phase transitions. Because of this, our group and others have characterized LLPS of recombinant PrP. In vitro phase separation of PrP is modulated by nucleic acid aptamers, and, depending on the aptamer conformation, the liquid droplets evolve to solid-like species. Herein we discuss recent studies and previous evidence supporting PrP phase transitions. We focus on the central role of LLPS related to PrP physiology and pathology, with a special emphasis on the interaction of PrP with different ligands, such as proteins and nucleic acids, which can play a role in prion disease pathogenesis. Finally, we comment on therapeutic strategies directed at the nonfunctional phase separation that could potentially tackle prion diseases or other protein misfolding disorders.
    Keywords:  aptamer; liquid-liquid phase separation; nucleic acids; phase transitions; prion protein; protein misfolding diseases
    DOI:  https://doi.org/10.1111/jnc.15586
  12. Biochim Biophys Acta Proteins Proteom. 2022 Feb 07. pii: S1570-9639(22)00014-0. [Epub ahead of print] 140767
    A unifying framework for amyloid-mediated membrane damage: The lipid-chaperone hypothesis.
    Carmelo Tempra, Federica Scollo, Martina Pannuzzo, Fabio Lolicato, Carmelo La Rosa.
      Over the past thirty years, researchers have highlighted the role played by a class of proteins or polypeptides that forms pathogenic amyloid aggregates in vivo, including i) the amyloid Aβ peptide, which is known to form senile plaques in Alzheimer's disease; ii) α-synuclein, responsible for Lewy body formation in Parkinson's disease and iii) IAPP, which is the protein component of type 2 diabetes-associated islet amyloids. These proteins, known as intrinsically disordered proteins (IDPs), are present as highly dynamic conformational ensembles. IDPs can partially (mis) fold into (dys) functional conformations and accumulate as amyloid aggregates upon interaction with other cytosolic partners such as proteins or lipid membranes. In addition, an increasing number of reports link the toxicity of amyloid proteins to their harmful effects on membrane integrity. Still, the molecular mechanism underlying the amyloidogenic proteins transfer from the aqueous environment to the hydrocarbon core of the membrane is poorly understood. This review starts with a historical overview of the toxicity models of amyloidogenic proteins to contextualize the more recent lipid-chaperone hypothesis. Then, we report the early molecular-level events in the aggregation and ion-channel pore formation of Aβ, IAPP, and α-synuclein interacting with model membranes, emphasizing the complexity of these processes due to their different spatial-temporal resolutions. Next, we underline the need for a combined experimental and computational approach, focusing on the strengths and weaknesses of the most commonly used techniques. Finally, the last two chapters highlight the crucial role of lipid-protein complexes as molecular switches among ion-channel-like formation, detergent-like, and fibril formation mechanisms and their implication in fighting amyloidogenic diseases.
    Keywords:  Aggregation; Amyloid; Ion channel-like; Lipid-chaperone; Model membrane; Toxic oligomer
    DOI:  https://doi.org/10.1016/j.bbapap.2022.140767
  13. Elife. 2022 02 07. pii: e75010. [Epub ahead of print]11
    Telomere length regulation by Rif1 protein from Hansenula polymorpha.
    Alexander N Malyavko, Olga A Petrova, Maria I Zvereva, Vladimir I Polshakov, Olga A Dontsova.
      Rif1 is a large multifaceted protein involved in various processes of DNA metabolism - from telomere length regulation and replication to double-strand break repair. The mechanistic details of its action, however, are often poorly understood. Here, we report functional characterization of the Rif1 homologue from methylotrophic thermotolerant budding yeast Hansenula polymorpha DL-1. We show that, similar to other yeast species, H. polymorpha Rif1 suppresses telomerase-dependent telomere elongation. We uncover two novel modes of Rif1 recruitment at H. polymorpha telomeres: via direct DNA binding and through the association with the Ku heterodimer. Both of these modes (at least partially) require the intrinsically disordered N-terminal extension - a region of the protein present exclusively in yeast species. We also demonstrate that Rif1 binds Stn1 and promotes its accumulation at telomeres in H. polymorpha.
    Keywords:  DNA replication; Hansenula polymorpha; Rif1; chromosomes; gene expression; genome stability; telomerase; telomere
    DOI:  https://doi.org/10.7554/eLife.75010
  14. Biochem Biophys Rep. 2022 Mar;29 101202
    The aqueous humor proteome is intrinsically disordered.
    Mak Djulbegovic, Vladimir N Uversky.
      •Our study demonstrated that intrinsic disorder is abundant in the aqueous humor.•The 749 aqueous proteins analyzed were enriched with disorder-promoting residues.•208 aqueous humor proteins were predicted to be highly intrinsically disordered.•Misregulation of IDPs may promote pathology in the aqueous humor.•IDPs in aqueous humor may serve as future targets for novel therapeutics.
    DOI:  https://doi.org/10.1016/j.bbrep.2022.101202
  15. Cell Biochem Biophys. 2022 Feb 10.
    14-3-3 Proteins are Potential Regulators of Liquid-Liquid Phase Separation.
    Xianlong Huang, Zhiwen Zheng, Yixin Wu, Meng Gao, Zhengding Su, Yongqi Huang.
      The 14-3-3 family proteins are vital scaffold proteins that ubiquitously expressed in various tissues. They interact with numerous protein targets and mediate many cellular signaling pathways. The 14-3-3 binding motifs are often embedded in intrinsically disordered regions which are closely associated with liquid-liquid phase separation (LLPS). In the past ten years, LLPS has been observed for a variety of proteins and biological processes, indicating that LLPS plays a fundamental role in the formation of membraneless organelles and cellular condensates. While extensive investigations have been performed on 14-3-3 proteins, its involvement in LLPS is overlooked. To date, 14-3-3 proteins have not been reported to undergo LLPS alone or regulate LLPS of their binding partners. To reveal the potential involvement of 14-3-3 proteins in LLPS, in this review, we summarized the LLPS propensity of 14-3-3 binding partners and found that about one half of them may undergo LLPS spontaneously. We further analyzed the phase separation behavior of representative 14-3-3 binders and discussed how 14-3-3 proteins may be involved. By modulating the conformation and valence of interactions and recruiting other molecules, we speculate that 14-3-3 proteins can efficiently regulate the functions of their targets in the context of LLPS. Considering the critical roles of 14-3-3 proteins, there is an urgent need for investigating the involvement of 14-3-3 proteins in the phase separation process of their targets and the underling mechanisms.
    Keywords:  Condensate; Intrinsically disordered region; Protein–protein interaction; Regulation; Scaffold protein
    DOI:  https://doi.org/10.1007/s12013-022-01067-3
  16. Biochimie. 2022 Feb 07. pii: S0300-9084(22)00029-3. [Epub ahead of print]
    Prediction of structural alphabet protein blocks using data mining.
    Mirjana M Maljković, Nenad S Mitić, Alexandre G de Brevern.
      3D protein structures determine proteins' biological functions. The 3D structure of the protein backbone can be approximated using the prototypes of local protein conformations. Sets of these prototypes are called structural alphabets (SAs). Amongst several approaches to the prediction of 3D structures from amino acid sequences, one approach is based on the prediction of SA prototypes for a given amino acid sequence. Protein Blocks (PBs) is the most known SA, and it is composed of 16 prototypes of five consecutive amino acids which were identified as optimal prototypes considering the ability to correctly approximate the local structure and the prediction accuracy of prototypes from an amino acid sequence. We developed models for PBs prediction from sequence information using different data mining approaches and machine learning algorithms. Besides the amino acid sequences, the results of the following tools were used to train the models: the Spider3 predictor of protein structure properties, several predictors of the protein's intrinsically disordered regions, and a tool for finding repeats in amino acid sequences. The highest accuracy of the constructed models is 80%, which is a significant improvement compared to the previous best available prediction, whose accuracy was 61%. Analyzing the models constructed by applying different algorithms, it was noticed that the significance of input attributes differs among the models constructed by algorithms. Using the information about amino acids belonging to intrinsically disordered regions and repeats improves the precision of prediction for some PBs using the CART classification algorithm, while this is not the case with the C5.0 classification algorithm. Improved prediction approaches can have interesting applications in protein structural model approaches or computational protein design.
    Keywords:  Amino acid sequence; Disorder predictors; Machine learning; Protein blocks; Repeats; Spider3
    DOI:  https://doi.org/10.1016/j.biochi.2022.01.019
  17. Proc Natl Acad Sci U S A. 2022 Feb 15. pii: e2114215119. [Epub ahead of print]119(7):
    The pathogenic R5L mutation disrupts formation of Tau complexes on the microtubule by altering local N-terminal structure.
    Alisa Cario, Adriana Savastano, Neil B Wood, Zhu Liu, Michael J Previs, Adam G Hendricks, Markus Zweckstetter, Christopher L Berger.
      The microtubule-associated protein (MAP) Tau is an intrinsically disordered protein (IDP) primarily expressed in axons, where it functions to regulate microtubule dynamics, modulate motor protein motility, and participate in signaling cascades. Tau misregulation and point mutations are linked to neurodegenerative diseases, including progressive supranuclear palsy (PSP), Pick's disease, and Alzheimer's disease. Many disease-associated mutations in Tau occur in the C-terminal microtubule-binding domain of the protein. Effects of C-terminal mutations in Tau have led to the widely accepted disease-state theory that missense mutations in Tau reduce microtubule-binding affinity or increase Tau propensity to aggregate. Here, we investigate the effect of an N-terminal arginine to leucine mutation at position 5 in Tau (R5L), associated with PSP, on Tau-microtubule interactions using an in vitro reconstituted system. Contrary to the canonical disease-state theory, we determine that the R5L mutation does not reduce Tau affinity for the microtubule using total internal reflection fluorescence microscopy. Rather, the R5L mutation decreases the ability of Tau to form larger-order complexes, or Tau patches, at high concentrations of Tau. Using NMR, we show that the R5L mutation results in a local structural change that reduces interactions of the projection domain in the presence of microtubules. Altogether, these results challenge both the current paradigm of how mutations in Tau lead to disease and the role of the projection domain in modulating Tau behavior on the microtubule surface.
    Keywords:  NMR spectroscopy; TIRF microscopy; Tau; microtubule; neurodegenerative diseases
    DOI:  https://doi.org/10.1073/pnas.2114215119
  18. J Biol Chem. 2022 Feb 03. pii: S0021-9258(22)00123-5. [Epub ahead of print] 101683
    Regulation of the activity of bacterial histidine kinase PleC by the scaffolding protein PodJ.
    Chao Zhang, Wei Zhao, Samuel W Duvall, Kimberly A Kowallis, W Seth Childers.
      Scaffolding proteins can customize the response of signaling networks to support cell development and behaviors. PleC is a bifunctional histidine kinase whose signaling activity coordinates asymmetric cell division to yield a motile swarmer cell and a stalked cell in the gram-negative bacterium Caulobacter crescentus. Past studies have shown that PleC's switch in activity from kinase to phosphatase correlates with a change in PleC's subcellular localization pattern from diffuse to localized at the new cell pole. Here we investigated how the bacterial scaffolding protein PodJ regulates the subcellular positioning and activity of PleC. We reconstituted the PleC-PodJ signaling complex through both heterologous expressions in Escherichia coli and in vitro studies. In vitro, PodJ phase separates as a biomolecular condensate that recruits PleC and inhibits its kinase activity. We also constructed an in vivo PleC-CcaS chimeric histidine kinase reporter assay and demonstrated using this method that PodJ leverages its intrinsically disordered region (IDR) to bind to PleC's PAS sensory domain and regulate PleC-CcaS signaling. Regulation of the PleC-CcaS was most robust when PodJ was concentrated at the cell poles and was dependent on the allosteric coupling between PleC-CcaS's PAS sensory domain and its downstream histidine kinase domain. In conclusion, our in vitro biochemical studies suggest that PodJ phase separation may be coupled to changes in PleC enzymatic function. We propose that this coupling of phase separation and allosteric regulation may be a generalizable phenomenon amongst enzymes associated with biomolecular condensates.
    Keywords:  Caulobacter crescentus; PAS domain; biomolecular condensate; histidine kinase; intrinsically disordered region (IDR); phase separation; scaffold
    DOI:  https://doi.org/10.1016/j.jbc.2022.101683
  19. BMC Biol. 2022 Feb 10. 20(1): 42
    Bex1 is essential for ciliogenesis and harbours biomolecular condensate-forming capacity.
    Emi Hibino, Yusuke Ichiyama, Atsushi Tsukamura, Yosuke Senju, Takao Morimune, Masahito Ohji, Yoshihiro Maruo, Masaki Nishimura, Masaki Mori.
      BACKGROUND: Primary cilia are sensory organelles crucial for organ development. The pivotal structure of the primary cilia is a microtubule that is generated via tubulin polymerization reaction that occurs in the basal body. It remains to be elucidated how molecules with distinct physicochemical properties contribute to the formation of the primary cilia.RESULTS: Here we show that brain expressed X-linked 1 (Bex1) plays an essential role in tubulin polymerization and primary cilia formation. The Bex1 protein shows the physicochemical property of being an intrinsically disordered protein (IDP). Bex1 shows cell density-dependent accumulation as a condensate either in nucleoli at a low cell density or at the apical cell surface at a high cell density. The apical Bex1 localizes to the basal body. Bex1 knockout mice present ciliopathy phenotypes and exhibit ciliary defects in the retina and striatum. Bex1 recombinant protein shows binding capacity to guanosine triphosphate (GTP) and forms the condensate that facilitates tubulin polymerization in the reconstituted system.
    CONCLUSIONS: Our data reveals that Bex1 plays an essential role for the primary cilia formation through providing the reaction field for the tubulin polymerization.
    Keywords:  Bex1; Intrinsically disordered protein (IDP); Juvenility-associated genes (JAGs); Primary cilia; Tubulin polymerization
    DOI:  https://doi.org/10.1186/s12915-022-01246-x
  20. J Biol Chem. 2022 Feb 04. pii: S0021-9258(22)00117-X. [Epub ahead of print] 101677
    Phase Separation by the SARS-CoV-2 Nucleocapsid Protein: Consensus and Open Questions.
    Sean M Cascarina, Eric D Ross.
      In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have re-allocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein - an essential viral protein required for viral replication - and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs.
    Keywords:  Intrinsically disordered region; Phase separation; SARS-CoV-2; Stress granule; biomolecular condensate; innate immunity; membraneless organelle; nucleocapsid protein
    DOI:  https://doi.org/10.1016/j.jbc.2022.101677
  21. J Cell Sci. 2022 Feb 10. pii: jcs.259380. [Epub ahead of print]
    RNA-assisted sequestration of RNA-binding proteins by cytoplasmic inclusions of the C-terminal 35-kDa fragment of TDP-43.
    Lei-Lei Jiang, Wen-Liang Guan, Jian-Yang Wang, Shu-Xian Zhang, Hong-Yu Hu.
      TDP-43 is a nuclear splicing factor functioning in pre-mRNA processing. Its C-terminal 35-kDa fragment (TDP-35) forms inclusions or aggregates in cytoplasm, and sequesters full-length TDP-43 into the inclusions through binding with RNA. We extended the research to investigate whether TDP-35 inclusions sequester other RNA-binding proteins (RBPs) and how RNA-binding specificity exerts the function in this sequestration process. We have characterized TIA1 (T-cell restricted intracellular antigen-1) and other RBPs that can be sequestered into the TDP-35 inclusions through specific RNA binding, and found that this sequestration leads to dysfunction of TIA1 in maturation of target pre-mRNA. Moreover, we directly visualized the dynamic sequestration of TDP-43 by the cytoplasmic TDP-35 inclusions by live-cell imaging. Our results demonstrate that TDP-35 sequesters some specific RBPs and this sequestration is assisted by binding with sequence-specific RNA. This study provides further evidence in supporting the hijacking hypothesis for RNA-assisted sequestration and will be beneficial to further understanding of the TDP-43 proteinopathies.
    Keywords:  Cytoplasmic Inclusions; Live-Cell Imaging; RNA-Assisted Sequestration; RNA-Binding Protein; TDP-43; TIA1
    DOI:  https://doi.org/10.1242/jcs.259380
  22. Nat Rev Cancer. 2022 Feb 11.
    Liquid-liquid phase separation drives cellular function and dysfunction in cancer.
    Sohum Mehta, Jin Zhang.
      Cancer is a disease of uncontrollably reproducing cells. It is governed by biochemical pathways that have escaped the regulatory bounds of normal homeostatic balance. This balance is maintained through precise spatiotemporal regulation of these pathways. The formation of biomolecular condensates via liquid-liquid phase separation (LLPS) has recently emerged as a widespread mechanism underlying the spatiotemporal coordination of biological activities in cells. Biomolecular condensates are widely observed to directly regulate key cellular processes involved in cancer cell pathology, and the dysregulation of LLPS is increasingly implicated as a previously hidden driver of oncogenic activity. In this Perspective, we discuss how LLPS shapes the biochemical landscape of cancer cells.
    DOI:  https://doi.org/10.1038/s41568-022-00444-7
  23. Biochim Biophys Acta Proteins Proteom. 2022 Feb 05. pii: S1570-9639(22)00013-9. [Epub ahead of print] 140766
    Impaired nuclear transport induced by juvenile ALS causing P525L mutation in NLS domain of FUS: A molecular mechanistic study.
    Sushmita Basu, K C Rajendra, Suresh Alagar, Ranjit Prasad Bahadur.
      Amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD) are progressive neurological disorders affecting motor neurons. Cellular aggregates of fused in sarcoma (FUS) protein are found in cytoplasm of ALS and FTLD patients. Nuclear localisation signal (NLS) domain of FUS binds to Karyopherin β2 (Kapβ2), which drives nuclear transport of FUS from cytoplasm. Several pathogenic mutations are reported in FUS NLS which are associated with its impaired nuclear transport and cytoplasmic mis-localisation. P525L mutation in NLS is most commonly found in cases of juvenile ALS (jALS), which affects individuals below 25 years of age. jALS progresses aggressively causing death within a year of its onset. This study elucidates the molecular mechanism behind jALS-causing P525L mutation hindering nuclear transport of FUS. We perform multiple molecular dynamics simulations in aqueous and hydrophobic solvent to understand the effect of the mutation at molecular level. Dynamics of Kapβ2-FUS complex is better captured in hydrophobic solvent compared to aqueous solvent. P525 and Y526 (PY-motif) of NLS exhibit fine-tuned stereochemical arrangement, which is essential for optimum Kapβ2 binding. P525L causes loss of several native contacts at interface leading to weaker binding, which promotes self-aggregation of FUS in cytoplasm. Native complex samples closed conformation, while mutant complex exhibits open conformation exposing hydrophilic residues of Kapβ2 to hydrophobic solvent. Mutant complex also fails to exhibit spring-like motion essential for its transport through nuclear pore complex. This study provides a mechanistic insight of binding affinity between NLS and Kapβ2 that inhibits self-aggregation of FUS preventing the disease condition.
    Keywords:  ALS and FTLD; Classical PY-NLS; Fused in sarcoma; Karyopherin β2; Molecular spring; Nuclear transport
    DOI:  https://doi.org/10.1016/j.bbapap.2022.140766
  24. Open Biol. 2022 02;12(2): 210334
    Protein phase separation hotspots at the presynapse.
    Janin Lautenschläger.
      Fundamental discoveries have shaped our molecular understanding of presynaptic processes, such as neurotransmitter release, active zone organization and mechanisms of synaptic vesicle (SV) recycling. However, certain regulatory steps still remain incompletely understood. Protein liquid-liquid phase separation (LLPS) and its role in SV clustering and active zone regulation now introduce a new perception of how the presynapse and its different compartments are organized. This article highlights the newly emerging concept of LLPS at the synapse, providing a systematic overview on LLPS tendencies of over 500 presynaptic proteins, spotlighting individual proteins and discussing recent progress in the field. Newly discovered LLPS systems like ELKS/liprin-alpha and Eps15/FCho are put into context, and further LLPS candidate proteins, including epsin1, dynamin, synaptojanin, complexin and rabphilin-3A, are highlighted.
    Keywords:  active zone; clathrin-mediated endocytosis; exocytosis; phase separation; synapse; synapsin
    DOI:  https://doi.org/10.1098/rsob.210334
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