Lysine acetylation of TDP-43 drives phase separation and pathological aggregation

AbstractThe trans-activation response DNA-binding protein TDP-43 regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Confirming the results from site-directed mutagenesis, we succeeded to introduce authentic acetyl-lysine at the identified sites via amber suppression. [AcK84]TDP-43 showed cytoplasmic mislocalization and the aggregation propensity of [acK136]TDP-43 was confirmed. With newly developed antibodies, we found that the nuclear sirtuin SIRT1 can potently deacetylate [acK136]TDP-43. Moreover, SIRT1 reduced the aggregation propensity of [acK136]TDP-43. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting novel regulatory mechanisms for TDP-43 pathogenesis.

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Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation

10.1101/2021.04.30.442163 ◽

2021 ◽

Author(s):

Lara Gruijs da Silva ◽

Francesca Simonetti ◽

Saskia Hutten ◽

Henrick Riemenschneider ◽

Erin L. Sternburg ◽

...

Keyword(s):

Phase Separation ◽

Neurodegenerative Disorders ◽

Nuclear Import ◽

Cellular Response ◽

Rna Binding ◽

Low Complexity ◽

Post Translational Modifications ◽

Multi Scale ◽

Regulatory Functions ◽

Lateral Sclerosis

AbstractPost-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the RNA-binding protein TDP-43, is hyperphosphorylated in disease on several C-terminal serine residues, which is generally believed to promote TDP-43 aggregation. Here, we show that hyperphosphorylation by Casein kinase 1δ or C-terminal phosphomimetic mutations surprisingly reduce TDP-43 phase separation and aggregation and render TDP-43 condensates more liquid-like and dynamic. Multi-scale simulations reveal reduced homotypic interactions of TDP-43 low complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We propose that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.

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O-GlcNAcylation reduces phase separation and aggregation of the EWS N-terminal low complexity region

10.1101/2021.05.11.443654 ◽

2021 ◽

Author(s):

Michael L Nosella ◽

Maria Tereshchenko ◽

Iva Pritisanac ◽

Andrew Chong ◽

Jeffrey A Toretsky ◽

...

Keyword(s):

Phase Separation ◽

Rna Binding ◽

Low Complexity ◽

General Role ◽

Post Translational Modifications ◽

Phase Separation Behavior ◽

Separation Of Proteins ◽

Separation Behavior

Many membraneless organelles are thought to be biomolecular condensates formed by phase separation of proteins and other biopolymers. Post-translational modifications (PTMs) can impact protein phase separation behavior, although for many PTMs this aspect of their function is unknown. O-linked β-D-N-acetylglucosaminylation (O-GlcNAcylation) is an abundant form of intracellular glycosylation whose roles in regulating biomolecular condensate assembly and dynamics have not been delineated. Using an in vitro approach, we found that O-GlcNAcylation reduces the phase separation propensity of the EWS N-terminal low complexity region (LCRN) under different conditions, including in the presence of the arginine- and glycine-rich RNA-binding do- mains (RBD). O-GlcNAcylation enhances fluorescence recovery after photobleaching (FRAP) within EWS LCRN condensates and causes the droplets to exhibit more liquid-like relaxation following fusion. Following extended incubation times, EWS LCRN+RBD condensates exhibit diminished FRAP, indicating a loss of fluidity, while condensates containing the O-GlcNAcylated LCRN do not. In HeLa cells, EWS is less O-GlcNAcylated following OGT knockdown and more prone to aggregation based on a filter retardation assay. Relative to the human proteome, O-GlcNAcylated proteins are enriched with regions that are predicted to phase separate, suggesting a general role of O-GlcNAcylation in regulation of biomolecular condensates.

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Gestörter Kerntransport und Phasentrennung von RNA-Bindeproteinen

BIOspektrum ◽

10.1007/s12268-021-1599-z ◽

2021 ◽

Vol 27 (4) ◽

pp. 365-367

Author(s):

Saskia Hutten ◽

Dorothee Dormann

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Phase Separation ◽

Frontotemporal Dementia ◽

Nuclear Import ◽

Binding Proteins ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Rna ◽

Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disordes, whose underlying molecular mechanisms are only beginning to emerge. A common molecular hallmark of both diseases is the relocalization of nuclear RNA-binding proteins (RBP) into cytoplasmic aggregates. Defects in nuclear import and aberrant phase separation appear to underlie RBP mislocalization and aggregation and could potentially be targeted in future therapies.

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Liquid–Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation

Biomolecules ◽

10.3390/biom11040548 ◽

2021 ◽

Vol 11 (4) ◽

pp. 548

Author(s):

Donya Pakravan ◽

Emiel Michiels ◽

Anna Bratek-Skicki ◽

Mathias De Decker ◽

Joris Van Lindt ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Hydrophobic Interactions ◽

Low Complexity ◽

Liquid Phase Separation ◽

End Stage ◽

Positive Effect ◽

Self Aggregation ◽

Lateral Sclerosis ◽

Liquid Liquid Phase Separation

Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid–liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a “reentrant” fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics.

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Inherited and Sporadic Amyotrophic Lateral Sclerosis and Fronto-Temporal Lobar Degenerations arising from Pathological Condensates of Phase Separating Proteins

Human Molecular Genetics ◽

10.1093/hmg/ddz162 ◽

2019 ◽

Vol 28 (R2) ◽

pp. R187-R196 ◽

Cited By ~ 2

Author(s):

Michael Fernandopulle ◽

GuoZhen Wang ◽

Jonathon Nixon-Abell ◽

Seema Qamar ◽

Varun Balaji ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Recent Work ◽

Neurodegenerative Disorders ◽

Low Complexity ◽

Present Review ◽

Sporadic Amyotrophic Lateral Sclerosis ◽

Missense Mutations ◽

Post Translational Modifications ◽

Intrinsically Disordered ◽

Lateral Sclerosis

Abstract Recent work on the biophysics of proteins with low complexity, intrinsically disordered domains that have the capacity to form biological condensates has profoundly altered the concepts about the pathogenesis of inherited and sporadic neurodegenerative disorders associated with pathological accumulation of these proteins. In the present review, we use the FUS, TDP-43 and A11 proteins as examples to illustrate how missense mutations and aberrant post-translational modifications of these proteins cause amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD).

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The Impact of ALS-Associated Genes hnRNPA1, MATR3, VCP and UBQLN2 on the Severity of TDP-43 Aggregation

Cells ◽

10.3390/cells9081791 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1791

Author(s):

Ana Bajc Česnik ◽

Helena Motaln ◽

Boris Rogelj

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Rna Binding ◽

Neurodegenerative Disorder ◽

Low Complexity ◽

Wild Type ◽

Disease Heterogeneity ◽

Cytoplasmic Inclusions ◽

The Impact ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder, characterized by cytoplasmic inclusions of RNA-binding protein TDP-43. Despite decades of research and identification of more than 50 genes associated with amyotrophic lateral sclerosis (ALS), the cause of TDP-43 translocation from the nucleus and its aggregation in the cytoplasm still remains unknown. Our study addressed the impact of selected ALS-associated genes on TDP-43 aggregation behavior in wild-type and aggregation prone TDP-43 in vitro cell models. These were developed by deleting TDP-43 nuclear localization signal and stepwise shortening its low-complexity region. The SH-SY5Y cells were co-transfected with the constructs of aggregation-prone TDP-43 and wild-type or mutant ALS-associated genes hnRNPA1, MATR3, VCP or UBQLN2. The investigated genes displayed a unique impact on TDP-43 aggregation, generating distinct types of cytoplasmic inclusions, similar to those already described as resembling prion strains, which could represent the basis for neurodegenerative disease heterogeneity.

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RNA-binding proteins with prion-like domains in health and disease

Biochemical Journal ◽

10.1042/bcj20160499 ◽

2017 ◽

Vol 474 (8) ◽

pp. 1417-1438 ◽

Cited By ~ 152

Author(s):

Alice Ford Harrison ◽

James Shorter

Keyword(s):

Phase Transitions ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Low Complexity ◽

Fused In Sarcoma ◽

Heterogeneous Nuclear Ribonucleoproteins ◽

Health And Disease ◽

Lateral Sclerosis

Approximately 70 human RNA-binding proteins (RBPs) contain a prion-like domain (PrLD). PrLDs are low-complexity domains that possess a similar amino acid composition to prion domains in yeast, which enable several proteins, including Sup35 and Rnq1, to form infectious conformers, termed prions. In humans, PrLDs contribute to RBP function and enable RBPs to undergo liquid–liquid phase transitions that underlie the biogenesis of various membraneless organelles. However, this activity appears to render RBPs prone to misfolding and aggregation connected to neurodegenerative disease. Indeed, numerous RBPs with PrLDs, including TDP-43 (transactivation response element DNA-binding protein 43), FUS (fused in sarcoma), TAF15 (TATA-binding protein-associated factor 15), EWSR1 (Ewing sarcoma breakpoint region 1), and heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNPA1 and hnRNPA2), have now been connected via pathology and genetics to the etiology of several neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Here, we review the physiological and pathological roles of the most prominent RBPs with PrLDs. We also highlight the potential of protein disaggregases, including Hsp104, as a therapeutic strategy to combat the aberrant phase transitions of RBPs with PrLDs that likely underpin neurodegeneration.

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Nonclassical nuclear localization signals mediate nuclear import of CIRBP

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1918944117 ◽

2020 ◽

Vol 117 (15) ◽

pp. 8503-8514 ◽

Cited By ~ 5

Author(s):

Benjamin Bourgeois ◽

Saskia Hutten ◽

Benjamin Gottschalk ◽

Mario Hofweber ◽

Gesa Richter ◽

...

Keyword(s):

Phase Separation ◽

Nuclear Localization ◽

Nuclear Import ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Interaction ◽

Stress Granule ◽

Rich Region ◽

Nuclear Localization Signals ◽

Cargo Proteins

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)–rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

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FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

Science Advances ◽

10.1126/sciadv.abf8660 ◽

2021 ◽

Vol 7 (30) ◽

pp. eabf8660

Author(s):

Nicol Birsa ◽

Agnieszka M. Ule ◽

Maria Giovanna Garone ◽

Brian Tsang ◽

Francesca Mattedi ◽

...

Keyword(s):

Phase Separation ◽

Motor Neurons ◽

Rna Binding ◽

Fragile X ◽

Protein Translation ◽

Fused In Sarcoma ◽

Mental Retardation Protein ◽

Lateral Sclerosis

FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation.

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Molecular structure and interactions within amyloid-like fibrils formed by a low-complexity protein sequence from FUS

Nature Communications ◽

10.1038/s41467-020-19512-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Myungwoon Lee ◽

Ujjayini Ghosh ◽

Kent R. Thurber ◽

Masato Kato ◽

Robert Tycko

Keyword(s):

Self Assembly ◽

Structural Model ◽

Rna Binding ◽

Low Complexity ◽

Growth Direction ◽

Structural Basis ◽

Amyloid Formation ◽

Hydrophobic Residues ◽

Dynamics Simulations ◽

Lateral Sclerosis

AbstractProtein domains without the usual distribution of amino acids, called low complexity (LC) domains, can be prone to self-assembly into amyloid-like fibrils. Self-assembly of LC domains that are nearly devoid of hydrophobic residues, such as the 214-residue LC domain of the RNA-binding protein FUS, is particularly intriguing from the biophysical perspective and is biomedically relevant due to its occurrence within neurons in amyotrophic lateral sclerosis, frontotemporal dementia, and other neurodegenerative diseases. We report a high-resolution molecular structural model for fibrils formed by the C-terminal half of the FUS LC domain (FUS-LC-C, residues 111-214), based on a density map with 2.62 Å resolution from cryo-electron microscopy (cryo-EM). In the FUS-LC-C fibril core, residues 112-150 adopt U-shaped conformations and form two subunits with in-register, parallel cross-β structures, arranged with quasi-21 symmetry. All-atom molecular dynamics simulations indicate that the FUS-LC-C fibril core is stabilized by a plethora of hydrogen bonds involving sidechains of Gln, Asn, Ser, and Tyr residues, both along and transverse to the fibril growth direction, including diverse sidechain-to-backbone, sidechain-to-sidechain, and sidechain-to-water interactions. Nuclear magnetic resonance measurements additionally show that portions of disordered residues 151-214 remain highly dynamic in FUS-LC-C fibrils and that fibrils formed by the N-terminal half of the FUS LC domain (FUS-LC-N, residues 2-108) have the same core structure as fibrils formed by the full-length LC domain. These results contribute to our understanding of the molecular structural basis for amyloid formation by FUS and by LC domains in general.

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