Molecular Determinants and Genetic Modifiers of Aggregation and Toxicity for the ALS Disease Protein FUS/TLS

University of California San Francisco/Howard Hughes Medical Institute, United States of America
PLoS Biology (Impact Factor: 9.34). 04/2011; 9(4):e1000614. DOI: 10.1371/journal.pbio.1000614
Source: PubMed


TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.

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    • "Next, we confirmed that the full-length FUS protein forms a liquid phase-separated state as observed for FUS LC. Fulllength FUS (residues 1–526) is even more aggregation prone than the isolated LC domain (Sun et al., 2011) but is kept highly soluble by incorporation of an N-terminal fusion of maltose binding protein tag (MBP). Upon release of MBP by TEV cleavage leaving the native protein, samples as low as 1 mM full-length FUS assemble into an opalescent, phase-separated liquid (Figure 2B) resembling that formed by FUS LC. "
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    ABSTRACT: Phase-separated states of proteins underlie ribonucleoprotein (RNP) granules and nuclear RNA-binding protein assemblies that may nucleate protein inclusions associated with neurodegenerative diseases. We report that the N-terminal low-complexity domain of the RNA-binding protein Fused in Sarcoma (FUS LC) is structurally disordered and forms a liquid-like phase-separated state resembling RNP granules. This state directly binds the C-terminal domain of RNA polymerase II. Phase-separated FUS lacks static structures as probed by fluorescence microscopy, indicating they are distinct from both protein inclusions and hydrogels. We use solution nuclear magnetic resonance spectroscopy to directly probe the dynamic architecture within FUS liquid phase-separated assemblies. Importantly, we find that FUS LC retains disordered secondary structure even in the liquid phase-separated state. Therefore, we propose that disordered protein granules, even those made of aggregation-prone prion-like domains, are dynamic and disordered molecular assemblies with transiently formed protein-protein contacts.
    Molecular cell 10/2015; 60(2). DOI:10.1016/j.molcel.2015.09.006 · 14.02 Impact Factor
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    • "About FUS/TLS, closely related to TDP-43, the work done by Sun and collaborators is remarkable showing that FUS/TLS is readily aggregated in vitro in yeast model expressing human FUS/TLS when a yeast prion-like domain together with other molecular determinants is added. However, this aggregation capacity is not enhanced at least in vitro by the disease-linked mutations (Sun et al., 2011). The close relation between FUS and TDP-43 needs to be further assessed, as both are implicated in stress granules together with other prion-like domain showing proteins, and aggregates have been shown able to recruit native FUS and TDP-43 proteins possibly due to their yeast prion-like Q/N rich segment seeding (Polymenidou and Cleveland, 2011; Fuentealba et al., 2010). "
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    ABSTRACT: Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of fatal neurodegenerative disorders affecting several mammalian species being Creutzfeldt-Jacob Disease (CJD) the most representative in human beings, scrapie in ovine, Bovine Spongiform Encephalopathy (BSE) in bovine and Chronic Wasting Disease (CWD) in cervids. As stated by the "protein-only hypothesis", the causal agent of TSEs is a self-propagating aberrant form of the prion protein (PrP) that through a misfolding event acquires a β-sheet rich conformation known as PrP(Sc) (from scrapie). This isoform is neurotoxic, aggregation prone and induces misfolding of native cellular PrP. Compelling evidence indicates that disease-specific protein misfolding in amyloid deposits could be shared by other disorders showing aberrant protein aggregates such as Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic lateral sclerosis (ALS) and systemic Amyloid A amyloidosis (AA amyloidosis). Evidences of shared mechanisms of the proteins related to each disease with prions will be reviewed through the available in vivo models. Taking prion research as reference, typical prion-like features such as seeding and propagation ability, neurotoxic species causing disease, infectivity, transmission barrier and strain evidences will be analyzed for other protein-related diseases. Thus, prion-like features of amyloid β peptide and tau present in AD, α-synuclein in PD, SOD-1, TDP-43 and others in ALS and serum α-amyloid (SAA) in systemic AA amyloidosis will be reviewed through models available for each disease. Copyright © 2015. Published by Elsevier B.V.
    Virus Research 04/2015; 207. DOI:10.1016/j.virusres.2015.04.014 · 2.32 Impact Factor
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    • "This unexpected function is mediated by its unusual structural switch from a folded, ubiquitin-like domain upon binding to single stranded DNA and in low protein concentrations, to an unfolded and aggregation-prone structure when protein concentrations are increased (Qin et al., 2014). FUS is characterized by even higher aggregation propensity compared to SOD1 or TDP-43, as it was shown to spontaneously aggregate into fibrils in a cell-free system within minutes (Sun et al., 2011). Aggregates reconstituted in vitro from recombinant wild type or mutant SOD1 (Furukawa et al., 2013; Münch et al., 2011) and TDP-43 (Furukawa et al., 2011) triggered aggregation of endogenously expressed protein in cell culture (Fig. 2D). "
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    ABSTRACT: Propagation of pathological protein assemblies via a prion-like mechanism has been suggested to drive neurodegenerative diseases, such as Parkinson's and Alzheimer's. Recently, amyotrophic lateral sclerosis (ALS)-linked proteins, such as SOD1, TDP-43 and FUS were shown to follow self-perpetuating seeded aggregation, thereby adding ALS to the group of prion-like disorders. The cell-to-cell spread of these pathological protein assemblies and their pathogenic mechanism is poorly understood. However, as ALS is a non-cell autonomous disease and pathology in glial cells was shown to contribute to motor neuron damage, spreading mechanisms are likely to underlie disease progression via the interplay between affected neurons and their neighboring glial cells.
    Virus Research 02/2015; 8. DOI:10.1016/j.virusres.2014.12.032 · 2.32 Impact Factor
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