scholarly journals TDP-43 oligomers detected as initial intermediate species during aggregate formation

2018 ◽  
Author(s):  
Rachel L. French ◽  
Ashley N. Reeb ◽  
Himani Aligireddy ◽  
Niraja Kedia ◽  
Dhruva D. Dhavale ◽  
...  
Keyword(s):  
Self Assembly ◽  
Rna Binding ◽  
Initial Time ◽  
Intermediate Step ◽  
Liquid Droplets ◽  
Clinical Genetic ◽  
Lateral Sclerosis ◽  
Cytoplasmic Aggregate ◽  
Liquid Liquid Phase Separation

ABSTRACTAggregates of the RNA binding protein TDP-43 are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are neurodegenerative disorders with overlapping clinical, genetic and pathological features. Mutations in the TDP-43 gene are causative of ALS, supporting its central role in pathogenesis. The process of TDP-43 aggregation remains poorly understood and whether this includes formation of intermediate complexes is unknown. We characterized aggregates derived from purified TDP-43 as a function of time and analyzed them under semi-denaturing conditions. Our assays identified oligomeric complexes at the initial time points prior to the formation of large aggregates, suggesting that ordered oligomerization is an intermediate step of TDP-43 aggregation. In addition, we analyzed liquid-liquid phase separation of TDP-43 and detected similar oligomeric assembly upon the maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, ALS-linked mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that the aggregates generated from purified protein seed intracellular aggregation, which is detected by established markers of TDP-43 pathology. Remarkably, cytoplasmic aggregate propagation is detected earlier with A315T and M337V and is 50% more widespread than with wild-type aggregates. Our findings provide evidence for a controlled process of TDP-43 self-assembly into intermediate structures that provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.

scholarly journals Fused in Sarcoma: Properties, Self-Assembly and Correlation with Neurodegenerative Diseases

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1622 ◽  
Author(s):  
Chen Chen ◽  
Xiufang Ding ◽  
Nimrah Akram ◽  
Song Xue ◽  
Shi-Zhong Luo
Keyword(s):  
Neurodegenerative Diseases ◽  
Self Assembly ◽  
Frontotemporal Lobar Degeneration ◽  
Rna Binding ◽  
Mutual Transformation ◽  
Polyglutamine Diseases ◽  
Fused In Sarcoma ◽  
Self Assembling ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

Fused in sarcoma (FUS) is a DNA/RNA binding protein that is involved in RNA metabolism and DNA repair. Numerous reports have demonstrated by pathological and genetic analysis that FUS is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and polyglutamine diseases. Traditionally, the fibrillar aggregation of FUS was considered to be the cause of those diseases, especially via its prion-like domains (PrLDs), which are rich in glutamine and asparagine residues. Lately, a nonfibrillar self-assembling phenomenon, liquid–liquid phase separation (LLPS), was observed in FUS, and studies of its functions, mechanism, and mutual transformation with pathogenic amyloid have been emerging. This review summarizes recent studies on FUS self-assembling, including both aggregation and LLPS as well as their relationship with the pathology of ALS, FTLD, and other neurodegenerative diseases.

scholarly journals RNA Is a Double-Edged Sword in ALS Pathogenesis

2021 ◽  
Vol 15 ◽  
Author(s):  
Benjamin L. Zaepfel ◽  
Jeffrey D. Rothstein
Keyword(s):  
Motor Neurons ◽  
Cortical Neurons ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Metabolism ◽  
Small Subset ◽  
Toxic Rna ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that affects upper and lower motor neurons. Familial ALS accounts for a small subset of cases (<10–15%) and is caused by dominant mutations in one of more than 10 known genes. Multiple genes have been causally or pathologically linked to both ALS and frontotemporal dementia (FTD). Many of these genes encode RNA-binding proteins, so the role of dysregulated RNA metabolism in neurodegeneration is being actively investigated. In addition to defects in RNA metabolism, recent studies provide emerging evidence into how RNA itself can contribute to the degeneration of both motor and cortical neurons. In this review, we discuss the roles of altered RNA metabolism and RNA-mediated toxicity in the context of TARDBP, FUS, and C9ORF72 mutations. Specifically, we focus on recent studies that describe toxic RNA as the potential initiator of disease, disease-associated defects in specific RNA metabolism pathways, as well as how RNA-based approaches can be used as potential therapies. Altogether, we highlight the importance of RNA-based investigations into the molecular progression of ALS, as well as the need for RNA-dependent structural studies of disease-linked RNA-binding proteins to identify clear therapeutic targets.

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

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.

scholarly journals SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72

2021 ◽  
Vol 134 (4) ◽  
pp. jcs256602 ◽  
Author(s):  
Mirjana Malnar ◽  
Boris Rogelj
Keyword(s):  
Antisense Rna ◽  
Rna Binding ◽  
Dipeptide Repeat Proteins ◽  
Rna Transcripts ◽  
Repeat Proteins ◽  
Rna Foci ◽  
Hek Cells ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

ABSTRACTThe expanded GGGGCC repeat mutation in the C9orf72 gene is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion is transcribed to sense and antisense RNA, which form RNA foci and bind cellular proteins. This mechanism of action is considered cytotoxic. Translation of the expanded RNA transcripts also leads to the accumulation of toxic dipeptide repeat proteins (DPRs). The RNA-binding protein splicing factor proline and glutamine rich (SFPQ), which is being increasingly associated with ALS and FTD pathology, binds to sense RNA foci. Here, we show that SFPQ plays an important role in the C9orf72 mutation. Overexpression of SFPQ resulted in higher numbers of both sense and antisense RNA foci and DPRs in transfected human embryonic kidney (HEK) cells. Conversely, reduced SPFQ levels resulted in lower numbers of RNA foci and DPRs in both transfected HEK cells and C9orf72 mutation-positive patient-derived fibroblasts and lymphoblasts. Therefore, we have revealed a role of SFPQ in regulating the C9orf72 mutation that has implications for understanding and developing novel therapeutic targets for ALS and FTD.This article has an associated First Person interview with the first author of the paper.

scholarly journals Translational inhibition and phase separation primes the epigenetic silencing of transposons

2020 ◽  
Author(s):  
Eun Yu Kim ◽  
Ling Wang ◽  
Zhen Lei ◽  
Hui Li ◽  
Wenwen Fan ◽  
...  
Keyword(s):  
Phase Separation ◽  
Epigenetic Silencing ◽  
Regulatory Mechanisms ◽  
Genome Integrity ◽  
Liquid Droplets ◽  
Ribosome Stalling ◽  
Insight Into ◽  
Liquid Liquid Phase Separation

AbstractTransposons are mobile DNAs that can cause fatal mutations. To counteract these genome invaders, the host genomes deploy small interfering (si) RNAs to initiate and establish the epigenetic silencing. However, the regulatory mechanisms for the selective recognition of transposons by the host genomes remain still elusive. Here we show that plant transposon RNAs undergo frequent ribosome stalling caused by their inherently unfavourable codon sequence usage. The ribosome stalling then causes the RNA truncation and the localization to siRNA bodies, which are both critical prerequisites for the siRNA processing. In addition, SGS3, the key protein in the siRNA biogenesis pathway, forms liquid droplets in vitro through its prion-like domains implicating the role of liquid-liquid phase separation in the formation of the siRNA bodies. Our study provides a novel insight into the regulatory mechanisms for the recognition of invasive genetic elements which is essential for the maintenance of genome integrity.

scholarly journals Redox-mediated regulation of an evolutionarily conserved cross-β structure formed by the TDP43 low complexity domain

2020 ◽  
Vol 117 (46) ◽  
pp. 28727-28734
Author(s):  
Yi Lin ◽  
Xiaoming Zhou ◽  
Masato Kato ◽  
Daifei Liu ◽  
Sina Ghaemmaghami ◽  
...  
Keyword(s):  
Rna Binding ◽  
Methionine Sulfoxide ◽  
Low Complexity ◽  
Methionine Sulfoxide Reductase ◽  
Methionine Oxidation ◽  
Evolutionarily Conserved ◽  
Self Association ◽  
Insight Into ◽  
Lateral Sclerosis

A methionine-rich low complexity (LC) domain is found within a C-terminal region of the TDP43 RNA-binding protein. Self-association of this domain leads to the formation of labile cross-β polymers and liquid-like droplets. Treatment with H2O2caused phenomena of methionine oxidation and droplet melting that were reversed upon exposure of the oxidized protein to methionine sulfoxide reductase enzymes. Morphological features of the cross-β polymers were revealed by H2O2-mediated footprinting. Equivalent TDP43 LC domain footprints were observed in polymerized hydrogels, liquid-like droplets, and living cells. The ability of H2O2to impede cross-β polymerization was abrogated by the prominent M337V amyotrophic lateral sclerosis-causing mutation. These observations may offer insight into the biological role of TDP43 in facilitating synapse-localized translation as well as aberrant aggregation of the protein in neurodegenerative diseases.

scholarly journals Post-Translational Modifications Modulate Proteinopathies of TDP-43, FUS and hnRNP-A/B in Amyotrophic Lateral Sclerosis

2021 ◽  
Vol 8 ◽  
Author(s):  
Stefania Farina ◽  
Francesca Esposito ◽  
Martina Battistoni ◽  
Giuseppe Biamonti ◽  
Sofia Francia
Keyword(s):  
Phase Transition ◽  
Amyotrophic Lateral Sclerosis ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cajal Bodies ◽  
Post Translational Modifications ◽  
P Granules ◽  
Sequence Complexity ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

It has been shown that protein low-sequence complexity domains (LCDs) induce liquid-liquid phase separation (LLPS), which is responsible for the formation of membrane-less organelles including P-granules, stress granules and Cajal bodies. Proteins harbouring LCDs are widely represented among RNA binding proteins often mutated in ALS. Indeed, LCDs predispose proteins to a prion-like behaviour due to their tendency to form amyloid-like structures typical of proteinopathies. Protein post-translational modifications (PTMs) can influence phase transition through two main events: i) destabilizing or augmenting multivalent interactions between phase-separating macromolecules; ii) recruiting or excluding other proteins and/or nucleic acids into/from the condensate. In this manuscript we summarize the existing evidence describing how PTM can modulate LLPS thus favouring or counteracting proteinopathies at the base of neurodegeneration in ALS.

scholarly journals The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation

2019 ◽  
Author(s):  
Stefan Reber ◽  
Helen Lindsay ◽  
Anny Devoy ◽  
Daniel Jutzi ◽  
Jonas Mechtersheimer ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Mitochondrial Gene ◽  
Liquid Phase Separation ◽  
Fused In Sarcoma ◽  
Damage Repair ◽  
Interaction Partners ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. One of the best studied proteins undergoing LLPS is Fused in Sarcoma (FUS), a predominantly nuclear RNA-binding protein. Mutations in FUS have been causally linked to Amyotrophic Lateral Sclerosis (ALS), an adult-onset motor neuron disease, and LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. In spite of this, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. In order to study the consequences of LLPS on FUS and its interaction partners, we developed a method that allows for the purification of phase separated FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome of FUS, depending on its biophysical state. While non-phase separated FUS interacts mainly with its well-known interaction partners involved in pre-mRNA processing, phase-separated FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, factors with function in mitochondria are strongly enriched with phase-separated FUS, providing a potential explanation for early changes in mitochondrial gene expression observed in mouse models of ALS-FUS. In summary, we present a methodology that allows to investigate the interactome of phase-separating proteins and provide evidence that LLPS strongly shapes the FUS interactome with important implications for function and disease.

scholarly journals Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Abner Gonzalez ◽  
Taro Mannen ◽  
Tolga Çağatay ◽  
Ayano Fujiwara ◽  
Hiroyoshi Matsumura ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Biochemical Analysis ◽  
Rna Binding ◽  
Arginine Methylation ◽  
Mutation Site ◽  
Amyotropic Lateral Sclerosis ◽  
Localization Signal ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractMutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid–liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-β2 (Kapβ2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapβ2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapβ2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapβ2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapβ2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapβ2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

The role of TDP-43 in the pathogenesis of ALS and FTLD

2013 ◽  
Vol 41 (6) ◽  
pp. 1536-1540 ◽  
Author(s):  
Marco Baralle ◽  
Emanuele Buratti ◽  
Francisco E. Baralle
Keyword(s):  
Rna Binding ◽  
Self Regulation ◽  
Binding Characteristics ◽  
Cellular Processes ◽  
Aggregation Processes ◽  
Main Component ◽  
Nuclear Ribonucleoprotein ◽  
Different Levels ◽  
Lateral Sclerosis

TDP-43 (TAR DNA-binding protein 43) is an hnRNP (heterogeneous nuclear ribonucleoprotein) protein whose role in cellular processes has come to the forefront of neurodegeneration research after the observation that it is the main component of brain inclusions in ALS (amyotrophic lateral sclerosis) and FTLD (frontotemporal lobar degeneration) patients. Functionally, this aberrant aggregation and mislocalization implies that, in the affected neurons, transcripts regulated by TDP-43 may be altered. Since then, a considerable amount of data has been gathered on TDP-43 interactions and on the genes that are influenced by its absence or overexpression. At present, however, most of these data come from high-throughput searches, making it problematic to separate the direct effects of TDP-43 from secondary misregulations occurring at different levels of the gene expression process. Furthermore, our knowledge of the biochemistry of TDP-43, its RNA-binding characteristics, its nuclear and cytoplasmic targets, and the details of its interactions with other proteins is still incomplete. The understanding of these features could hold the key for uncovering TDP-43′s role in ALS and FTLD pathogenesis. We describe in the present paper our work on TDP-43 RNA binding, self-regulation and aggregation processes, and attempt to relate them to the neurodegenerative pathologies.

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