ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid–liquid phase separation and liquid-to-solid transition

Ishiguro, Akira; Lü, Jun; Ozawa, Daisaku; Nagai, Yoshitaka; Ishihama, Akira

doi:10.1016/j.jbc.2021.101284

Cited by 32 publications

(66 citation statements)

References 83 publications

(134 reference statements)

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“…A wealth of evidence shows that increasing cytoplasmic concentration of FUS or mutations in the prion-like domain at the N-terminus promotes the protein’s conversion into an aggregated state ( Shelkovnikova et al, 2014 ; Patel et al, 2015 ). Conversely, the interaction of the NLS-PY domain with either TNPO-1 or with the G quadruplex sequences on mRNAs bound by the protein facilitate maintaining a liquid-liquid state ( Yoshizawa et al, 2018 ; Ishiguro et al, 2021 ). Mutations at the NLS-PY domain, e.g., the P525L, revert this condition by inducing a liquid-to-solid transition (i.e., an aggregated state).…”

Section: Discussionmentioning

confidence: 99%

A Deletion of the Nuclear Localization Signal Domain in the Fus Protein Induces Stable Post-stress Cytoplasmic Inclusions in SH-SY5Y Cells

2021

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Mutations in Fused-in-Sarcoma (FUS) gene involving the nuclear localization signal (NLS) domain lead to juvenile-onset Amyotrophic Lateral Sclerosis (ALS). The mutant protein mislocalizes to the cytoplasm, incorporating it into Stress Granules (SG). Whether SGs are the first step to the formation of stable FUS-containing aggregates is still unclear. In this work, we used acute and chronic stress paradigms to study the SG dynamics in a human SH-SY5Y neuroblastoma cell line carrying a deletion of the NLS domain of the FUS protein (homozygous: ΔNLS–/–; heterozygous: ΔNLS+/–). Wild-type (WT) cells served as controls. We evaluated the subcellular localization of the mutant protein through immunoblot and immunofluorescence, in basal conditions and after acute stress and chronic stress with sodium arsenite (NaAsO2). Cells were monitored for up to 24 h after rescue. FUS was expressed in both nucleus and cytoplasm in the ΔNLS+/– cells, whereas it was primarily cytoplasmic in the ΔNLS–/–. Acute NaAsO2 exposure induced SGs: at rescue,>90% of ΔNLS cells showed abundant FUS-containing if compared to less than 5% of the WT cells. The proportion of FUS-positive SGs remained 15–20% at 24 h in mutant cells. Cycloheximide did not abolish the long-lasting SGs in mutant cells. Chronic exposure to NaAsO2 did not induce significant SGs formation. A wealth of research has demonstrated that ALS-associated FUS mutations at the C-terminus facilitate the incorporation of the mutant protein into SGs. We have shown here that mutant FUS-containing SGs tend to fail to dissolve after stress, facilitating a liquid-to-solid phase transition. The FUS-containing inclusions seen in the dying motor neurons might therefore directly derive from SGs. This might represent an attractive target for future innovative therapies.

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Section: Discussionmentioning

confidence: 99%

A Deletion of the Nuclear Localization Signal Domain in the Fus Protein Induces Stable Post-stress Cytoplasmic Inclusions in SH-SY5Y Cells

2021

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“…Research on molecular mechanisms of G4s and G4 binding proteins is still in its infancy stage. In fact, some RNA binding proteins have recently been re-identified one after another as G4-RNA binding proteins ( Byrd et al, 2016 ; Ishiguro et al, 2016 ; Ramesh et al, 2020 ; Simko et al, 2020 ; Zheng et al, 2020 ; He et al, 2021 ; Ishiguro et al, 2021 ). Attempts to identify bound proteins with known G4s have been performed by mass spectrometry and proteome microarray ( Mori et al, 2013 ; Zhang et al, 2021 ).…”

Section: Dedicated Methods Of G4-rnamentioning

confidence: 99%

“…These gene products are involved in various cellular processes such as redox regulation, signal transduction, and axonal transport of RNAs. The most abundant gene species encode RNAs and RNA-binding proteins, such as intronic expansion of the human C9orf72 (chromosome 9 open reading frame 72) gene, TDP-43, FUS, hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), hnRNP A2/B1, hnRNPA3, EWSR1 (Ewing’s sarcoma RNA binding protein 1), and TIA1 (T cell-restricted intracellular antigen-1) ( Taylor et al, 2016 ; Ishiguro et al, 2021 ). Surprisingly, these RNA binding-proteins recognize and bind to mRNA containing G4 motifs ( Ishiguro et al, 2021 ).…”

Section: G4-rnas and G4-binding Rna Proteins As The Genetic Causes Of...mentioning

confidence: 99%

“…It has been suggested that the G4 structure supports the formation of condensates ( Rhine et al, 2020 ; Roden and Gladfelter, 2021 ), which is the opposite to the inhibition of condensation by promiscuous RNA assembly ( Maharana et al, 2018 ). Eventually, in 2021, four laboratories experimentally demonstrated that G4 promotes the condensation of DNA/RNA-protein complexes through LLPS (DNA: Liu et al, 2021 ; Gao et al, 2021 ; DNA: Mimura et al, 2021 ; RNA: Ishiguro et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

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Essential Roles and Risks of G-Quadruplex Regulation: Recognition Targets of ALS-Linked TDP-43 and FUS

Ishiguro

Ishihama

2022

Front. Mol. Biosci.

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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.

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“…Finally, it was suggested that liquid-liquid phase separation (LLPS) that forms membraneless organelles by molecular reversible self-assembly, might be the missing link between protein misfolding, aggregation and pathogenesis associated to neurodegenerative disorders ( Nedelsky and Taylor, 2019 ; Babinchak and Surewicz, 2020 ; Alberti and Hyman, 2021 ). Notably, many amyloidogenic proteins are prone to phase transition that can initiate protein misfolding and aggregation, and modulate their biological function as shown for tau ( Ambadipudi et al, 2017 ; Zhang et al, 2017a ; Hernández-Vega et al, 2017 ; Wegmann et al, 2018 ; Boyko et al, 2019 ; Majumdar et al, 2019 ; Kanaan et al, 2020 ; Singh et al, 2020 ; Rai et al, 2021 ), TDP-43 ( Li et al, 2018c ; Wang et al, 2018 ; Babinchak et al, 2019 ; Conicella et al, 2020 ; Watanabe et al, 2020 ; Dang et al, 2021 ; Grese et al, 2021 ; Hallegger et al, 2021 ; Pakravan et al, 2021 ), α-synuclein ( Sawner et al, 2021 ), the amyloidogenic type II diabetes-associated IAPP ( Pytowski et al, 2020 ) and the fused in sarcoma (FUS) protein ( Patel et al, 2015 ; Monahan et al, 2017 ; Murthy et al, 2019 ; Ishiguro et al, 2021 ; Levone et al, 2021 ; Reber et al, 2021 ). Understanding the molecular mechanisms of aberrant phase separation should provide new strategies to control protein aggregation in neurodegeneration.…”

Section: Assembly Properties and Propagation Of Amyloid Fibrils In Ne...mentioning

confidence: 99%

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools

et al. 2022

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Protein aggregation into highly ordered, regularly repeated cross-β sheet structures called amyloid fibrils is closely associated to human disorders such as neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases, or systemic diseases like type II diabetes. Yet, in some cases, such as the HET-s prion, amyloids have biological functions. High-resolution structures of amyloids fibrils from cryo-electron microscopy have very recently highlighted their ultrastructural organization and polymorphisms. However, the molecular mechanisms and the role of co-factors (posttranslational modifications, non-proteinaceous components and other proteins) acting on the fibril formation are still poorly understood. Whether amyloid fibrils play a toxic or protective role in the pathogenesis of neurodegenerative diseases remains to be elucidated. Furthermore, such aberrant protein-protein interactions challenge the search of small-molecule drugs or immunotherapy approaches targeting amyloid formation. In this review, we describe how chemical biology tools contribute to new insights on the mode of action of amyloidogenic proteins and peptides, defining their structural signature and aggregation pathways by capturing their molecular details and conformational heterogeneity. Challenging the imagination of scientists, this constantly expanding field provides crucial tools to unravel mechanistic detail of amyloid formation such as semisynthetic proteins and small-molecule sensors of conformational changes and/or aggregation. Protein engineering methods and bioorthogonal chemistry for the introduction of protein chemical modifications are additional fruitful strategies to tackle the challenge of understanding amyloid formation.

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ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid–liquid phase separation and liquid-to-solid transition

Cited by 32 publications

References 83 publications

A Deletion of the Nuclear Localization Signal Domain in the Fus Protein Induces Stable Post-stress Cytoplasmic Inclusions in SH-SY5Y Cells

A Deletion of the Nuclear Localization Signal Domain in the Fus Protein Induces Stable Post-stress Cytoplasmic Inclusions in SH-SY5Y Cells

Essential Roles and Risks of G-Quadruplex Regulation: Recognition Targets of ALS-Linked TDP-43 and FUS

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools

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