bims-indpro 2022-12-18 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–12–18
seventeen papers selected by
Sara Mingu, Johannes Gutenberg University

Condensates formed by prion-like low-complexity domains have small-world network structures and interfaces defined by expanded conformations.
Surface electrostatics dictate RNA-binding protein CAPRIN1 condensate concentration and hydrodynamic properties.
Assessment of models for calculating the hydrodynamic radius of intrinsically disordered proteins.
How viral proteins bind short linear motifs and intrinsically disordered domains.
Unravelling the microscopic characteristics of intrinsically disordered proteins upon liquid-liquid phase separation.
Shapeshifting proteins: the role of structural disorder and conformational plasticity in physiology and disease.
Unveiling the Metal-Dependent Aggregation Properties of the C-terminal Region of Amyloidogenic Intrinsically Disordered Protein Isoforms DPF3b and DPF3a.
In Silico Studies to Predict the Role of Solvent in Guiding the Conformations of Intrinsically Disordered Peptides and Their Aggregated Protofilaments.
RNA-Binding Proteins as Epigenetic Regulators of Brain Functions and Their Involvement in Neurodegeneration.
Homeodomain complex formation and biomolecular condensates in Hox gene regulation.
Modulation Effects of Fe3+, Zn2+, and Cu2+ Ions on the Amyloid Fibrillation of α-Synuclein: Insights from a FTIR Investigation.
Phosphorylation of the novel mTOR substrate Unkempt regulates cellular morphogenesis.
The biomedical potential of tardigrade proteins: A review.
HNF1B-driven three-dimensional chromatin structure for molecular classification in pancreatic cancers.
Different Intermolecular Interactions Drive Nonpathogenic Liquid-Liquid Phase Separation and Potentially Pathogenic Fibril Formation by TDP-43.
Chromatin sensing by the auxiliary domains of KDM5C regulates its demethylase activity and is disrupted by X-linked intellectual disability mutations.
Structure-based discovery of a novel small-molecule inhibitor of TEAD palmitoylation with anticancer activity.

Nat Commun. 2022 Dec 13. 13(1): 7722

Condensates formed by prion-like low-complexity domains have small-world network structures and interfaces defined by expanded conformations.

Mina Farag, Samuel R Cohen, Wade M Borcherds, Anne Bremer, Tanja Mittag, Rohit V Pappu.

Biomolecular condensates form via coupled associative and segregative phase transitions of multivalent associative macromolecules. Phase separation coupled to percolation is one example of such transitions. Here, we characterize molecular and mesoscale structural descriptions of condensates formed by intrinsically disordered prion-like low complexity domains (PLCDs). These systems conform to sticker-and-spacers architectures. Stickers are cohesive motifs that drive associative interactions through reversible crosslinking and spacers affect the cooperativity of crosslinking and overall macromolecular solubility. Our computations reproduce experimentally measured sequence-specific phase behaviors of PLCDs. Within simulated condensates, networks of reversible inter-sticker crosslinks organize PLCDs into small-world topologies. The overall dimensions of PLCDs vary with spatial location, being most expanded at and preferring to be oriented perpendicular to the interface. Our results demonstrate that even simple condensates with one type of macromolecule feature inhomogeneous spatial organizations of molecules and interfacial features that likely prime them for biochemical activity.

DOI: https://doi.org/10.1038/s41467-022-35370-7
J Biol Chem. 2022 Dec 07. pii: S0021-9258(22)01219-4. [Epub ahead of print] 102776

Surface electrostatics dictate RNA-binding protein CAPRIN1 condensate concentration and hydrodynamic properties.

Yuki Toyama, Atul Kaushik Rangadurai, Julie D Forman-Kay, Lewis E Kay.

  Biomolecular condensates concentrate proteins, nucleic acids and small molecules, and play an essential role in many biological processes. Their formation is tuned by a balance between energetically favorable and unfavorable contacts, with charge-charge interactions playing a central role in some systems. The positively charged intrinsically disordered carboxy-terminal region of the RNA-binding protein CAPRIN1 is one such example, phase separating upon addition of negatively charged ATP or high concentrations of sodium chloride. Using solution NMR spectroscopy we measured residue-specific near-surface electrostatic potentials (ϕENS) of CAPRIN1 along its sodium chloride-induced phase separation trajectory to compare with those obtained using ATP. In both cases, electrostatic shielding decreases ϕENS values, yet surface potentials of CAPRIN1 in the two condensates can be different, depending on the amount of sodium chloride or ATP added. Our results establish that even small differences in ϕENS can significantly affect the level of protein enrichment and the mechanical properties of the condensed phase, leading, potentially, to the regulation of biological processes.

Keywords:  biomolecular condensates; electrostatics; intrinsically disordered protein; nuclear magnetic resonance (NMR); phase separation

DOI:  https://doi.org/10.1016/j.jbc.2022.102776
Biophys J. 2022 Dec 14. pii: S0006-3495(22)03923-6. [Epub ahead of print]

Assessment of models for calculating the hydrodynamic radius of intrinsically disordered proteins.

Francesco Pesce, Estella A Newcombe, Pernille Seiffert, Emil E Tranchant, Johan G Olsen, Christy R Grace, Birthe B Kragelund, Kresten Lindorff-Larsen.

Diffusion measurements by pulsed field gradient NMR and fluorescence correlation spectroscopy can be used to probe the hydrodynamic radius of proteins, which contains information about the overall dimension of a protein in solution. The comparison of this value with structural models of intrinsically disordered proteins is nonetheless impaired by the uncertainty of the accuracy of the methods for computing the hydrodynamic radius from atomic coordinates. To tackle this issue, we here build conformational ensembles of 11 intrinsically disordered proteins that we ensure are in agreement with measurements of compaction by small-angle X-ray scattering. We then use these ensembles to identify the forward model that more closely fits the radii derived from pulsed field gradient NMR diffusion experiments. Of the models we examined, we find that the Kirkwood-Riseman equation provides the best description of the hydrodynamic radius probed by pulsed field gradient NMR experiments. While some minor discrepancies remain, our results enable better use of measurements of the hydrodynamic radius in integrative modelling and for force field benchmarking and parameterization.

DOI: https://doi.org/10.1016/j.bpj.2022.12.013
Essays Biochem. 2022 Dec 12. pii: EBC20220047. [Epub ahead of print]

How viral proteins bind short linear motifs and intrinsically disordered domains.

Priyanka Madhu, Norman E Davey, Ylva Ivarsson.

  Viruses are the obligate intracellular parasites that exploit the host cellular machinery to replicate their genome. During the viral life cycle viruses manipulate the host cell through interactions with host proteins. Many of these protein-protein interactions are mediated through the recognition of host globular domains by short linear motifs (SLiMs), or longer intrinsically disordered domains (IDD), in the disordered regions of viral proteins. However, viruses also employ their own globular domains for binding to SLiMs and IDDs present in host proteins or virus proteins. In this review, we focus on the different strategies adopted by viruses to utilize proteins or protein domains for binding to the disordered regions of human or/and viral ligands. With a set of examples, we describe viral domains that bind human SLiMs. We also provide examples of viral proteins that bind to SLiMs, or IDDs, of viral proteins as a part of complex assembly and regulation of protein functions. The protein-protein interactions are often crucial for viral replication, and may thus offer possibilities for innovative inhibitor design.

Keywords:  Intrinsically disordered domains; Protein-Protein Interactions; Short Linear Motifs; Viruses; host-virus interactions; molecular interactions

DOI:  https://doi.org/10.1042/EBC20220047
Essays Biochem. 2022 Dec 16. 66(7): 891-900

Unravelling the microscopic characteristics of intrinsically disordered proteins upon liquid-liquid phase separation.

Si Wu, Jitao Wen, Sarah Perrett.

  Biomolecular condensate formation via liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the spatiotemporal organization of biomolecules in the cell. These membraneless condensates form and disperse dynamically in response to environmental stimuli. Growing evidence indicates that the liquid-like condensates not only play functional physiological roles but are also implicated in a wide range of human diseases. As a major component of biomolecular condensates, intrinsically disordered proteins (IDPs) are intimately involved in the LLPS process. During the last decade, great efforts have been made on the macroscopic characterization of the physicochemical properties and biological functions of liquid condensates both in vitro and in the cellular context. However, characterization of the conformations and interactions at the molecular level within phase-separated condensates is still at an early stage. In the present review, we summarize recent biophysical studies investigating the intramolecular conformational changes of IDPs upon LLPS and the intermolecular clustering of proteins undergoing LLPS, with a particular focus on single-molecule fluorescence detection. We also discuss how these microscopic features are linked to the macroscopic phase transitions that are relevant to the physiological and pathological roles of the condensates.

Keywords:  fluorescence resonance energy transfer; phase separation; protein aggregation; protein conformation; protein dynamics; single molecule

DOI:  https://doi.org/10.1042/EBC20220148
Essays Biochem. 2022 Dec 16. 66(7): 817-819

Shapeshifting proteins: the role of structural disorder and conformational plasticity in physiology and disease.

Samrat Mukhopadhyay.

  Intrinsically disordered proteins (IDPs) defy the conventional structure-function paradigm and do not autonomously fold up into unique 3D structures for carrying out functions. They exist as rapidly interconverting conformational ensembles and are thought to expand the functional repertoire of proteins. Such shapeshifting proteins are associated with a multitude of biological functions and a wide range of human diseases. The thematic issue on 'Shapeshifting Proteins' in Essays in Biochemistry includes some exciting and emerging aspects of this class of proteins. Articles in this issue provide current trends and contemporary views on various intriguing features of these proteins involving their unique structural and dynamical characteristics, misfolding and aggregation behavior, and their phase transitions into biomolecular condensates. I hope that this thematic issue will be of considerable interest to the practitioners in protein biochemistry and biophysics as well as to the researchers in other allied areas involving cell and molecular biology, neuroscience, virology, pathophysiology, and so forth.

Keywords:  Intrinsically disordered proteins; aggregation; misfolding; phase transitions

DOI:  https://doi.org/10.1042/EBC20220197
Int J Mol Sci. 2022 Dec 04. pii: 15291. [Epub ahead of print]23(23):

Unveiling the Metal-Dependent Aggregation Properties of the C-terminal Region of Amyloidogenic Intrinsically Disordered Protein Isoforms DPF3b and DPF3a.

Tanguy Leyder, Julien Mignon, Denis Mottet, Catherine Michaux.

  Double-PHD fingers 3 (DPF3) is a BAF-associated human epigenetic regulator, which is increasingly recognised as a major contributor to various pathological contexts, such as cardiac defects, cancer, and neurodegenerative diseases. Recently, we unveiled that its two isoforms (DPF3b and DPF3a) are amyloidogenic intrinsically disordered proteins. DPF3 isoforms differ from their C-terminal region (C-TERb and C-TERa), containing zinc fingers and disordered domains. Herein, we investigated the disorder aggregation properties of C-TER isoforms. In agreement with the predictions, spectroscopy highlighted a lack of a highly ordered structure, especially for C-TERa. Over a few days, both C-TERs were shown to spontaneously assemble into similar antiparallel and parallel β-sheet-rich fibrils. Altered metal homeostasis being a neurodegeneration hallmark, we also assessed the influence of divalent metal cations, namely Cu2+, Mg2+, Ni2+, and Zn2+, on the C-TER aggregation pathway. Circular dichroism revealed that metal binding does not impair the formation of β-sheets, though metal-specific tertiary structure modifications were observed. Through intrinsic and extrinsic fluorescence, we found that metal cations differently affect C-TERb and C-TERa. Cu2+ and Ni2+ have a strong inhibitory effect on the aggregation of both isoforms, whereas Mg2+ impedes C-TERb fibrillation and, on the contrary, enhances that of C-TERa. Upon Zn2+ binding, C-TERb aggregation is also hindered, and the amyloid autofluorescence of C-TERa is remarkably red-shifted. Using electron microscopy, we confirmed that the metal-induced spectral changes are related to the morphological diversity of the aggregates. While metal-treated C-TERb formed breakable and fragmented filaments, C-TERa fibrils retained their flexibility and packing properties in the presence of Mg2+ and Zn2+ cations.

Keywords:  aggregation; amyloid fibrillation; deep-blue autofluorescence; double-PHD fingers 3 (DPF3); electron microscopy; intrinsically disordered protein; metal cations; neurodegenerative diseases; spectroscopy

DOI:  https://doi.org/10.3390/ijms232315291
ACS Omega. 2022 Dec 06. 7(48): 43337-43345

In Silico Studies to Predict the Role of Solvent in Guiding the Conformations of Intrinsically Disordered Peptides and Their Aggregated Protofilaments.

Prabir Khatua, Souvik Mondal, Madhulika Gupta, Sanjoy Bandyopadhyay.

The formation of amyloids due to the self-assembly of intrinsically disordered proteins or peptides is a hallmark for different neurodegenerative diseases. For example, amyloids formed by the amyloid beta (Aβ) peptides are responsible for the most devastating neuropathological disease, namely, Alzheimer's disease, while aggregation of α-synuclein peptides causes the etiology of another neuropathological disease, Parkinson's disease. Characterization of the intermediates and the final amyloid formed during the aggregation process is, therefore, crucial for microscopic understanding of the origin behind such diseases, as well as for the development of proper therapeutics to combat those. However, most of the research activities reported in this area have been directed toward examining the early stages of the aggregation process, including probing the conformational characteristics of the responsible protein/peptide in the monomeric state or in small oligomeric forms. This is because the small soluble oligomers have been found to be more deleterious than the final insoluble amyloids. This review discusses some of the recent findings obtained from our simulation studies on Aβ and α-synuclein monomers and small preformed Aβ aggregates. A molecular-level insight of the aggregation process with a special emphasis on the role of water in inducing the aggregation process has been provided.

DOI: https://doi.org/10.1021/acsomega.2c06235
Int J Mol Sci. 2022 Nov 23. pii: 14622. [Epub ahead of print]23(23):

RNA-Binding Proteins as Epigenetic Regulators of Brain Functions and Their Involvement in Neurodegeneration.

Carlo Maria Di Liegro, Gabriella Schiera, Giuseppe Schirò, Italia Di Liegro.

  A central aspect of nervous system development and function is the post-transcriptional regulation of mRNA fate, which implies time- and site-dependent translation, in response to cues originating from cell-to-cell crosstalk. Such events are fundamental for the establishment of brain cell asymmetry, as well as of long-lasting modifications of synapses (long-term potentiation: LTP), responsible for learning, memory, and higher cognitive functions. Post-transcriptional regulation is in turn dependent on RNA-binding proteins that, by recognizing and binding brief RNA sequences, base modifications, or secondary/tertiary structures, are able to control maturation, localization, stability, and translation of the transcripts. Notably, most RBPs contain intrinsically disordered regions (IDRs) that are thought to be involved in the formation of membrane-less structures, probably due to liquid-liquid phase separation (LLPS). Such structures are evidenced as a variety of granules that contain proteins and different classes of RNAs. The other side of the peculiar properties of IDRs is, however, that, under altered cellular conditions, they are also prone to form aggregates, as observed in neurodegeneration. Interestingly, RBPs, as part of both normal and aggregated complexes, are also able to enter extracellular vesicles (EVs), and in doing so, they can also reach cells other than those that produced them.

Keywords:  EVs; RNA-binding proteins (RBPs); intrinsically disordered regions (IDRs); learning; memory; neurodegeneration; post-transcriptional regulation of gene expression; synaptic plasticity

DOI:  https://doi.org/10.3390/ijms232314622
Semin Cell Dev Biol. 2022 Dec 12. pii: S1084-9521(22)00356-1. [Epub ahead of print]

Homeodomain complex formation and biomolecular condensates in Hox gene regulation.

Joseph Salomone, Edward Farrow, Brian Gebelein.

  Hox genes are a family of homeodomain transcription factors that regulate specialized morphological structures along the anterior-posterior axis of metazoans. Over the past few decades, researchers have focused on defining how Hox factors with similar in vitro DNA binding activities achieve sufficient target specificity to regulate distinct cell fates in vivo. In this review, we highlight how protein interactions with other transcription factors, many of which are also homeodomain proteins, result in the formation of transcription factor complexes with enhanced DNA binding specificity. These findings suggest that Hox-regulated enhancers utilize distinct combinations of homeodomain binding sites, many of which are low-affinity, to recruit specific Hox complexes. However, low-affinity sites can only yield reproducible responses with high transcription factor concentrations. To overcome this limitation, recent studies revealed how transcription factors, including Hox factors, use intrinsically disordered domains (IDRs) to form biomolecular condensates that increase protein concentrations. Moreover, Hox factors with altered IDRs have been associated with altered transcriptional activity and human disease states, demonstrating the importance of IDRs in mediating essential Hox output. Collectively, these studies highlight how Hox factors use their DNA binding domains, protein-protein interaction domains, and IDRs to form specific transcription factor complexes that yield accurate gene expression.

Keywords:  Biomolecular condensates; Hox; Intrinsically disordered regions; Low-affinity binding sites; Short linear interaction motifs; Transcription factor

DOI:  https://doi.org/10.1016/j.semcdb.2022.11.016
Molecules. 2022 Dec 01. pii: 8383. [Epub ahead of print]27(23):

Modulation Effects of Fe3+, Zn2+, and Cu2+ Ions on the Amyloid Fibrillation of α-Synuclein: Insights from a FTIR Investigation.

Yan Li, Yang Yu, Gang Ma.

  Amyloid fibrillation of α-synuclein is implicated in the pathogenesis of Parkinson's disease and heavy metal ions such as Fe3+, Zn2+, and Cu2+ are known to be involved in the process. In this work, we explored the use of FTIR spectroscopy to look into the modulation effects of Fe3+, Zn2+, and Cu2+ on the amyloid fibrillation of α-synuclein. We performed a curve-fitting analysis on the FTIR amide I bands of these α-synuclein fibril systems, namely, the pristine fibril and the fibrils prepared in the presence of Fe3+, Zn2+, and Cu2+. We found that the α-synuclein fibrils under the influences of metal ions all possessed a parallel β-sheet structure, turn structure, and disordered structure, similar to that of pristine α-synuclein fibril. We also observed metal-induced increases in the proportions of the β-sheet secondary structure within the α-synuclein fibrils, with Fe3+ being the most effective inducer. We performed second derivative analysis of the side chain carboxylic groups of α-synuclein fibrils and found that the side chain microenvironment of the α-synuclein fibrils was more influenced by Fe3+ than Zn2+, and Cu2+. In addition, our atomic force microscopic study revealed that the morphologies of α-synuclein fibrils under the influence of Fe3+ was quite different from that of the Zn2+ and Cu2+ systems. Our FTIR results suggested that the modulation effects of Fe3+, Zn2+, and Cu2+ on α-synuclein fibrillation occurred at both secondary and quaternary structural levels. At last, we proposed a mechanistic hypothesis to interpret how metal ions could affect the morphology of α-synuclein amyloid fibril based on the conformational plasticity properties of intrinsically disordered proteins.

Keywords:  FTIR; amyloid; fibril; fibrillation; metal ion; secondary structure; α-synuclein

DOI:  https://doi.org/10.3390/molecules27238383
J Biol Chem. 2022 Dec 09. pii: S0021-9258(22)01231-5. [Epub ahead of print] 102788

Phosphorylation of the novel mTOR substrate Unkempt regulates cellular morphogenesis.

Pranetha Baskaran, Simeon R Mihaylov, Elin Vinsland, Kriti Shah, Lucy Granat, Sila K Ultanir, Andrew R Tee, Jernej Murn, Joseph M Bateman.

  Mechanistic target of rapamycin (mTOR) is a protein kinase that integrates multiple inputs to regulate anabolic cellular processes. For example , mTOR complex I (mTORC1) has key functions in growth control, autophagy and metabolism. However, much less is known about the signaling components that act downstream of mTORC1 to regulate cellular morphogenesis. Here we show that the RNA-binding protein Unkempt, a key regulator of cellular morphogenesis, is a novel substrate of mTORC1. We show that Unkempt phosphorylation is regulated by nutrient levels and growth factors via mTORC1. To analyze Unkempt phosphorylation, we immunoprecipitated Unkempt from cells in the presence or absence of the mTORC1 inhibitor rapamycin and used mass spectrometry to identify mTORC1-dependent phosphorylated residues. This analysis showed that mTORC1-dependent phosphorylation is concentrated in a serine-rich intrinsically disordered region in the C-terminal half of Unkempt. We also found that Unkempt physically interacts with and is directly phosphorylated by mTORC1 through binding to the regulatory-associated protein of mTOR, Raptor. Furthermore, analysis in the developing brain of mice lacking TSC1 expression showed that phosphorylation of Unkempt is mTORC1-dependent in vivo. Finally, mutation analysis of key serine/threonine residues in the serine-rich region indicates that phosphorylation inhibits the ability of Unkempt to induce a bipolar morphology. Phosphorylation within this serine-rich region thus profoundly affects the ability of Unkempt to regulate cellular morphogenesis. Taken together, our findings reveal a novel molecular link between mTORC1 signaling and cellular morphogenesis.

Keywords:  Raptor; Unkempt; cellular morphogenesis; intrinsically disordered region; mTOR; phosphorylation

DOI:  https://doi.org/10.1016/j.jbc.2022.102788
Biomed Pharmacother. 2022 Dec 07. pii: S0753-3322(22)01452-4. [Epub ahead of print]158 114063

The biomedical potential of tardigrade proteins: A review.

Nadiia Kasianchuk, Piotr Rzymski, Łukasz Kaczmarek.

  Tardigrades are ubiquitous microinvertebrates exhibiting extreme tolerance to various environmental stressors like low and high temperatures, lack of water, or high radiation. Although exact pathways behind the tardigrade extremotolerance are yet to be elucidated, some molecules involved have been identified. Their evidenced properties may lead to novel opportunities in biomedical and pharmacological development. This review aims to present the general characteristics of tardigrade intrinsically disordered proteins (TDPs: Dsup, CAHS, SAHS, MAHS) and late embryogenesis-abundant proteins (LEA) and provide an updated overview of their features and relevance for potential use in biomedicine and pharmacology. The Dsup reveals a promising action in attenuating oxidative stress, DNA damage, and pyrimidine dimerization, as well as increasing radiotolerance in transfected human cells. Whether Dsup can perform these functions when delivered externally is yet to be understood by in vivo preclinical testing. In turn, CAHS and SAHS demonstrate properties that could benefit the preservation of pharmaceuticals (e.g., vaccines) and biomaterials (e.g., cells). Selected CAHS proteins can also serve as inspiration for designing novel anti-apoptotic agents. The LEA proteins also reveal promising properties to preserve desiccated biomaterials and can act as anti-osmotic agents. In summary, tardigrade molecules reveal several potential biomedical applications advocating further research and development. The challenge of extracting larger amounts of these molecules can be solved with genetic engineering and synthetic biology tools. With new species identified each year and ongoing studies on their extremotolerance, progress in the medical use of tardigrade proteins is expected shortly.

Keywords:  Biomaterial storage; Damage suppressor protein; Late embryogenesis-abundant proteins; Oxidative stress; Tardigrade Disordered Proteins

DOI:  https://doi.org/10.1016/j.biopha.2022.114063
Cancer Sci. 2022 Dec 13.

HNF1B-driven three-dimensional chromatin structure for molecular classification in pancreatic cancers.

Hiroyuki Kato, Keisuke Tateishi, Dosuke Iwadate, Keisuke Yamamoto, Hiroaki Fujiwara, Takuma Nakatsuka, Yotaro Kudo, Yoku Hayakawa, Hideaki Ijichi, Motoyuki Otsuka, Takahiro Kishikawa, Ryota Takahashi, Koji Miyabayashi, Yousuke Nakai, Yoshihiro Hirata, Atsushi Toyoda, Shinichi Morishita, Mitsuhiro Fujishiro.

  The molecular subtypes of pancreatic cancer (PC), either classical/progenitor-like or basal/squamous-like, are currently a major topic of research because of their direct association with clinical outcomes. Some transcription factors (TFs) have been reported to be associated with these subtypes. However, the mechanisms by which these molecular signatures of PCs are established remain unknown. Epigenetic regulatory processes, supported by dynamic changes in the chromatin structure, are essential for transcriptional profiles. Previously, we reported the importance of open chromatin profiles in the biological features and transcriptional status of PCs. Here, we aimed to analyze the relationships between three-dimensional (3D) genome structures and the molecular subtypes of human PCs using Hi-C analysis. We observed a correlation of the specific elements of 3D genome modules, including compartments, topologically associating domains, and enhancer-promoter loops, with the expression of related genes. We focused on HNF1B, a TF that is implicated in the progenitor subtype. Forced expression of HNF1B in squamous-type PC organoids induced the upregulation and downregulation of genes associated with progenitor and squamous subtypes, respectively. Long-range genomic interactions induced by HNF1B were accompanied by compartment modulation and H3K27ac redistribution. We also found that these HNF1B-induced changes in subtype-related gene expression required an intrinsically disordered region, suggesting a possible involvement of phase separation in compartment modulation. Thus, mapping of 3D structural changes induced by TFs, such as HNF1B, may become a useful resource for further understanding the molecular features of PCs.

Keywords:  HNF1B; Hi-C; intrinsically disordered region; pancreatic cancer; patient-derived organoid

DOI:  https://doi.org/10.1111/cas.15690
Int J Mol Sci. 2022 Dec 03. pii: 15227. [Epub ahead of print]23(23):

Different Intermolecular Interactions Drive Nonpathogenic Liquid-Liquid Phase Separation and Potentially Pathogenic Fibril Formation by TDP-43.

Yu-Teng Zeng, Lu-Lu Bi, Xiao-Feng Zhuo, Ling-Yun Yang, Bo Sun, Jun-Xia Lu.

  The liquid-liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, and is critical in the control of many biological processes by forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to de-mix in solution, forming the protein condensed droplets, and amyloid aggregates would form from the droplets after incubation. The molecular interactions regulating TDP-43 LCD LLPS were investigated at the protein fusion equilibrium stage, when the droplets stopped growing after incubation. We found the molecules in the droplet were still liquid-like, but with enhanced intermolecular helix-helix interactions. The protein would only start to aggregate after a lag time and aggregate slower than at the condition when the protein does not phase separately into the droplets, or the molecules have a reduced intermolecular helix-helix interaction. In the protein condensed droplets, a structural transition intermediate toward protein aggregation was discovered involving a decrease in the intermolecular helix-helix interaction and a reduction in the helicity. Our results therefore indicate that different intermolecular interactions drive LLPS and fibril formation. The discovery that TDP-43 LCD aggregation was faster through the pathway without the first protein phase separation supports that LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.

Keywords:  TDP-43; liquid–liquid phase separation; solution-state NMR

DOI:  https://doi.org/10.3390/ijms232315227
J Mol Biol. 2022 Dec 07. pii: S0022-2836(22)00539-3. [Epub ahead of print] 167913

Chromatin sensing by the auxiliary domains of KDM5C regulates its demethylase activity and is disrupted by X-linked intellectual disability mutations.

Fatima S Ugur, Mark J S Kelly, Danica Galonić Fujimori.

  The H3K4me3 chromatin modification, a hallmark of promoters of actively transcribed genes, is dynamically removed by the KDM5 family of histone demethylases. The KDM5 demethylases have a number of accessory domains, two of which, ARID and PHD1, lie between the segments of the catalytic domain. KDM5C, which has a unique role in neural development, harbors a number of mutations adjacent to its accessory domains that cause X-linked intellectual disability (XLID). The roles of these accessory domains remain unknown, limiting an understanding of how XLID mutations affect KDM5C activity. Through in vitro binding and kinetic studies using nucleosomes, we find that while the ARID domain is required for efficient nucleosome demethylation, the PHD1 domain alone has an inhibitory role in KDM5C catalysis. In addition, the unstructured linker region between the ARID and PHD1 domains interacts with PHD1 and is necessary for nucleosome binding. Our data suggests a model in which the PHD1 domain inhibits DNA recognition by KDM5C. This inhibitory effect is relieved by the H3 tail, enabling recognition of flanking DNA on the nucleosome. Importantly, we find that XLID mutations adjacent to the ARID and PHD1 domains break this regulation by enhancing DNA binding, resulting in the loss of specificity of substrate chromatin recognition and rendering demethylase activity lower in the presence of flanking DNA. Our findings suggest a model by which specific XLID mutations could alter chromatin recognition and enable euchromatin-specific dysregulation of demethylation by KDM5C.

Keywords:  NMR; histone demethylase; intrinsically disordered region; nucleosome; reader domain

DOI:  https://doi.org/10.1016/j.jmb.2022.167913
Front Oncol. 2022 ;12 1021823

Structure-based discovery of a novel small-molecule inhibitor of TEAD palmitoylation with anticancer activity.

Artem Gridnev, Subhajit Maity, Jyoti R Misra.

  The paralogous oncogenic transcriptional coactivators YAP and TAZ are the distal effectors of the Hippo signaling pathway, which plays a critical role in cell proliferation, survival and cell fate specification. They are frequently deregulated in most human cancers, where they contribute to multiple aspects of tumorigenesis including growth, metabolism, metastasis and chemo/immunotherapy resistance. Thus, they provide a critical point for therapeutic intervention. However, due to their intrinsically disordered structure, they are challenging to target directly. Since YAP/TAZ exerts oncogenic activity by associating with the TEAD1-4 transcription factors, to regulate target gene expression, YAP activity can be controlled indirectly by regulating TEAD1-4. Interestingly, TEADs undergo autopalmitoylation, which is essential for their stability and function, and small-molecule inhibitors that prevent this posttranslational modification can render them unstable. In this article we report discovery of a novel small molecule inhibitor of YAP activity. We combined structure-based virtual ligand screening with biochemical and cell biological studies and identified JM7, which inhibits YAP transcriptional reporter activity with an IC50 of 972 nMoles/Ltr. Further, it inhibits YAP target gene expression, without affecting YAP/TEAD localization. Mechanistically, JM7 inhibits TEAD palmitoylation and renders them unstable. Cellular thermal shift assay revealed that JM7 directly binds to TEAD1-4 in cells. Consistent with the inhibitory effect of JM7 on YAP activity, it significantly impairs proliferation, colony-formation and migration of mesothelioma (NCI-H226), breast (MDA-MB-231) and ovarian (OVCAR-8) cancer cells that exhibit increased YAP activity. Collectively, these results establish JM7 as a novel lead compound for development of more potent inhibitors of TEAD palmitoylation for treating cancer.

Keywords:  TEAD; YAP; cancer; hippo signaling; small-molecule inhibitor; virtual ligand screening

DOI:  https://doi.org/10.3389/fonc.2022.1021823