The oncogenic transcription factor FUS-CHOP can undergo nuclear liquid-liquid phase separation.

Myxoid liposarcoma is caused by a chromosomal translocation resulting in a fusion protein comprised of the N terminus of FUS (fused in sarcoma) and the full-length transcription factor CHOP (CCAAT/enhancer-binding protein homologous protein, also known as DDIT3). FUS functions in RNA metabolism, and CHOP is a stress-induced transcription factor. The FUS-CHOP fusion protein causes unique gene expression and oncogenic transformation. Although it is clear that the FUS segment is required for oncogenic transformation, the mechanism of FUS-CHOP-induced transcriptional activation is unknown. Recently, some transcription factors and super enhancers have been proposed to undergo liquid-liquid phase separation and form membraneless compartments that recruit transcription machinery to gene promoters. Since phase separation of FUS depends on its N terminus, transcriptional activation by FUS-CHOP could result from the N terminus driving nuclear phase transitions. Here, we characterized FUS-CHOP in cells and in vitro, and observed novel phase-separating properties relative to unmodified CHOP. Our data indicate that FUS-CHOP forms phase-separated condensates that colocalize with BRD4, a marker of super enhancer condensates. We provide evidence that the FUS-CHOP phase transition is a novel oncogenic mechanism and potential therapeutic target for myxoid liposarcoma. This article has an associated First Person interview with the first author of the paper.

DDX17 is involved in DNA damage repair and modifies FUS toxicity in an RGG-domain dependent manner.

Mutations in the RNA binding protein, Fused in Sarcoma (FUS), lead to amyotrophic lateral sclerosis (ALS), the most frequent form of motor neuron disease. Cytoplasmic aggregation and defective DNA repair machinery are etiologically linked to mutant FUS-associated ALS. Although FUS is involved in numerous aspects of RNA processing, little is understood about the pathophysiological mechanisms of mutant FUS. Here, we employed RNA-sequencing technology in Drosophila brains expressing FUS to identify significantly altered genes and pathways involved in FUS-mediated neurodegeneration. We observed the expression levels of DEAD-Box Helicase 17 (DDX17) to be significantly downregulated in response to mutant FUS in Drosophila and human cell lines. Mutant FUS recruits nuclear DDX17 into cytoplasmic stress granules and physically interacts with DDX17 through the RGG1 domain of FUS. Ectopic expression of DDX17 reduces cytoplasmic mislocalization and sequestration of mutant FUS into cytoplasmic stress granules. We identified DDX17 as a novel regulator of the DNA damage response pathway whose upregulation repairs defective DNA damage repair machinery caused by mutant neuronal FUS ALS. In addition, we show DDX17 is a novel modifier of FUS-mediated neurodegeneration in vivo. Our findings indicate DDX17 is downregulated in response to mutant FUS, and restoration of DDX17 levels suppresses FUS-mediated neuropathogenesis and toxicity in vivo.

N-terminal acetylation modestly enhances phase separation and reduces aggregation of the low-complexity domain of RNA-binding protein fused in sarcoma.

The RNA-binding protein fused in sarcoma (FUS) assembles via liquid-liquid phase separation (LLPS) into functional RNA granules and aggregates in amyotrophic lateral sclerosis associated neuronal inclusions. Several studies have demonstrated that posttranslational modification (PTM) can significantly alter FUS phase separation and aggregation, particularly charge-altering phosphorylation of the nearly uncharged N-terminal low complexity domain of FUS (FUS LC). However, the occurrence and impact of N-terminal acetylation on FUS phase separation remains unexplored, even though N-terminal acetylation is the most common PTM in mammals and changes the charge at the N-terminus. First, we find that FUS is predominantly acetylated in two human cell types and stress conditions. Next, we show that recombinant FUS LC can be acetylated when co-expressed with the NatA complex in Escherichia coli. Using NMR spectroscopy, we find that N-terminal acetylated FUS LC (FUS LC Nt-Ac) does not notably alter monomeric FUS LC structure or motions. Despite no difference in structure, Nt-Ac-FUS LC phase separates more avidly than unmodified FUS LC. More importantly, N-terminal acetylation of FUS LC reduces aggregation. Our findings highlight the importance of N-terminal acetylation of proteins that undergo physiological LLPS and pathological aggregation.

The prion-like domain of Fused in Sarcoma is phosphorylated by multiple kinases affecting liquid- and solid-phase transitions.

Fused in Sarcoma (FUS) is a ubiquitously expressed protein that can phase-separate from nucleoplasm and cytoplasm into distinct liquid-droplet structures. It is predominantly nuclear and most of its functions are related to RNA and DNA metabolism. Excessive persistence of FUS within cytoplasmic phase-separated assemblies is implicated in the diseases amyotrophic lateral sclerosis and frontotemporal dementia. Phosphorylation of FUS's prion-like domain (PrLD) by nuclear phosphatidylinositol 3-kinase-related kinase (PIKK)-family kinases following DNA damage was previously shown to alter FUS's liquid-phase and solid-phase transitions in cell models and in vitro. However, proteomic data suggest that FUS's PrLD is phosphorylated at numerous additional sites, and it is unknown if other non-PIKK and nonnuclear kinases might be influencing FUS's phase transitions. Here we evaluate disease mutations and stress conditions that increase FUS accumulation into cytoplasmic phase-separated structures. We observed that cytoplasmic liquid-phase structures contain FUS phosphorylated at novel sites, which occurred independent of PIKK-family kinases. We engineered phosphomimetic substitutions within FUS's PrLD and observed that mimicking a few phosphorylation sites strongly inhibited FUS solid-phase aggregation, while minimally altering liquid-phase condensation. These effects occurred independent of the exact location of the phosphomimetic substitutions, suggesting that modulation of PrLD phosphorylation may offer therapeutic strategies that are specific for solid-phase aggregation observed in disease.

The Role of Post-Translational Modifications in the Phase Transitions of Intrinsically Disordered Proteins.

Advances in genomics and proteomics have revealed eukaryotic proteomes to be highly abundant in intrinsically disordered proteins that are susceptible to diverse post-translational modifications. Intrinsically disordered regions are critical to the liquid-liquid phase separation that facilitates specialized cellular functions. Here, we discuss how post-translational modifications of intrinsically disordered protein segments can regulate the molecular condensation of macromolecules into functional phase-separated complexes.

The prionlike domain of FUS is multiphosphorylated following DNA damage without altering nuclear localization.

FUS (fused in sarcoma) is an abundant, predominantly nuclear protein involved in RNA processing. Under various conditions, FUS functionally associates with RNA and other macromolecules to form distinct, reversible phase-separated liquid structures. Persistence of the phase-separated state and increased cytoplasmic localization are both hypothesized to predispose FUS to irreversible aggregation, which is a pathological hallmark of subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. We previously showed that phosphorylation of FUS's prionlike domain suppressed phase separation and toxic aggregation, proportionally to the number of added phosphates. However, phosphorylation of FUS's prionlike domain was previously reported to promote its cytoplasmic localization, potentially favoring pathological behavior. Here we used mass spectrometry and human cell models to further identify phosphorylation sites within FUS's prionlike domain, specifically following DNA-damaging stress. In total, 28 putative sites have been identified, about half of which are DNA-dependent protein kinase (DNA-PK) consensus sites. Custom antibodies were developed to confirm the phosphorylation of two of these sites (Ser-26 and Ser-30). Both sites were usually phosphorylated in a subpopulation of cellular FUS following a variety of DNA-damaging stresses but not necessarily equally or simultaneously. Importantly, we found DNA-PK-dependent multiphosphorylation of FUS's prionlike domain does not cause cytoplasmic localization.

Study of Amyloids Using Yeast.

We detail some of the genetic, biochemical, and physical methods useful in studying amyloids in yeast, particularly the yeast prions. These methods include cytoduction (cytoplasmic mixing), infection of cells with prion amyloids, use of green fluorescent protein fusions with amyloid-forming proteins for cytology, protein purification and amyloid formation, and electron microscopy of filaments.

The Role of Post-Translational Modifications on Prion-Like Aggregation and Liquid-Phase Separation of FUS.

Subcellular mislocalization and aggregation of the human FUS protein occurs in neurons of patients with subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. FUS is one of several RNA-binding proteins that can functionally self-associate into distinct liquid-phase droplet structures. It is postulated that aberrant interactions within the dense phase-separated state can potentiate FUS's transition into solid prion-like aggregates that cause disease. FUS is post-translationally modified at numerous positions, which affect both its localization and aggregation propensity. These modifications may influence FUS-linked pathology and serve as therapeutic targets.

A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing.

TDP-43 is an RNA-binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C-terminal domain which mediates ALS inclusions, TDP-43 has a globular N-terminal domain (NTD). Here, we show that TDP-43 NTD assembles into head-to-tail linear chains and that phosphomimetic substitution at S48 disrupts TDP-43 polymeric assembly, discourages liquid-liquid phase separation (LLPS) in vitro, fluidizes liquid-liquid phase separated nuclear TDP-43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head-to-tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher-order polymerization. These data provide structural detail for the established mechanistic role of the well-folded TDP-43 NTD in splicing and link this function to LLPS. In addition, the fusion-tag solubilized, recombinant form of TDP-43 full-length protein developed here will enable future phase separation and in vitro biochemical assays on TDP-43 function and interactions that have been hampered in the past by TDP-43 aggregation.

Yeast Models of Prion-Like Proteins That Cause Amyotrophic Lateral Sclerosis Reveal Pathogenic Mechanisms.

Many proteins involved in the pathogenic mechanisms of amyotrophic lateral sclerosis (ALS) are remarkably similar to proteins that form prions in the yeast Saccharomyces cerevisiae. These ALS-associated proteins are not orthologs of yeast prion proteins, but are similar in having long, intrinsically disordered domains that are rich in hydrophilic amino acids. These so-called prion-like domains are particularly aggregation-prone and are hypothesized to participate in the mislocalization and misfolding processes that occur in the motor neurons of ALS patients. Methods developed for characterizing yeast prions have been adapted to studying ALS-linked proteins containing prion-like domains. These yeast models have yielded major discoveries, including identification of new ALS genetic risk factors, new ALS-causing gene mutations and insights into how disease mutations enhance protein aggregation.

Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity.

Neuronal inclusions of aggregated RNA-binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation-prone, yeast prion-like, low sequence-complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in-cell phosphorylation sites across FUS LC We show that both phosphorylation and phosphomimetic variants reduce its aggregation-prone/prion-like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self-interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS-associated cytotoxicity. Hence, post-translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.

Genetic Methods for Studying Yeast Prions.

The recognition that certain long-known nonchromosomal genetic elements were actually prions was based not on the specific phenotypic manifestations of those elements, but rather on their unusual genetic properties. Here, we outline methods of prion assay, methods for showing the nonchromosomal inheritance, and methods for determining whether a nonchromosomal trait has the unusual characteristics diagnostic of a prion. Finally, we discuss genetic methods often useful in the study of yeast prions.

Prion Transfection of Yeast.

Transfection of yeast with amyloid filaments, made from recombinant protein or prepared from extracts of cells infected with a prion, has become an important method in characterizing yeast prions. Here, we describe a method for transmission of [URE3] with Ure2p amyloid that is based on a previously published protocol for transfection with Sup35p filaments to make cells [PSI+]. This method may be used for other prions by changing just the amyloid source, host strain, and plating medium.

Isolation and Analysis of Prion and Amyloid Aggregates from Yeast Cells.

Amyloid fibers are large and extremely stable structures that can resist denaturation by strong anionic detergents, such as sodium dodecyl sulfate or sarkosyl. Here, we present two complementary analytical methods that exploit these properties, enabling the isolation and characterization of amyloid/prion aggregates. The first technique, known as semidenaturating detergent agarose gel electrophoresis, is an immunoblotting technique, conceptually similar to conventional western blotting. It enables the targeted identification of large detergent-resistant protein aggregates using antibodies specific to the protein of interest. The second method, called the technique for amyloid purification and identification, is a nontargeted approach that can isolate amyloid aggregates for analysis by tandem mass spectrometry. The latter approach requires no special genetic tools or antibodies, and can identify amyloid-forming proteins, such as prions, as well as proteins tightly associated with amyloid, from a variety of cell sources.

Prions.

Infectious proteins (prions) are usually self-templating filamentous protein polymers (amyloids). Yeast prions are genes composed of protein and, like the multiple alleles of DNA-based genes, can have an array of "variants," each a distinct self-propagating amyloid conformation. Like the lethal mammalian prions and amyloid diseases, yeast prions may be lethal, or only mildly detrimental, and show an array of phenotypes depending on the protein involved and the prion variant. Yeast prions are models for both rare mammalian prion diseases and for several very common amyloidoses such as Alzheimer's disease, type 2 diabetes, and Parkinson's disease. Here, we describe their detection and characterization using genetic, cell biological, biochemical, and physical methods.

Stress granules at the intersection of autophagy and ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease caused by loss of upper and lower motor neurons. The majority of ALS cases are classified as sporadic (80-90%), with the remaining considered familial based on patient history. The last decade has seen a surge in the identification of ALS-causing genes - including TARDBP (TDP-43), FUS, MATR3 (Matrin-3), C9ORF72 and several others - providing important insights into the molecular pathways involved in pathogenesis. Most of the protein products of ALS-linked genes fall into two functional categories: RNA-binding/homeostasis and protein-quality control (i.e. autophagy and proteasome). The RNA-binding proteins tend to be aggregation-prone with low-complexity domains similar to the prion-forming domains of yeast. Many also incorporate into stress granules (SGs), which are cytoplasmic ribonucleoprotein complexes that form in response to cellular stress. Mutant forms of TDP-43 and FUS perturb SG dynamics, lengthening their cytoplasmic persistence. Recent evidence suggests that SGs are regulated by the autophagy pathway, suggesting a unifying connection between many of the ALS-linked genes. Persistent SGs may give rise to intractable aggregates that disrupt neuronal homeostasis, thus failure to clear SGs by autophagic processes may promote ALS pathogenesis. This article is part of a Special Issue entitled SI:Autophagy.

Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity.

Amyotrophic lateral sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other RNA-binding proteins, have been linked to ALS pathology. Recently, Pur-alpha, a DNA/RNA-binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA-mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics.

Hepatic expression of serum amyloid A1 is induced by traumatic brain injury and modulated by telmisartan.

Traumatic brain injury affects the whole body in addition to the direct impact on the brain. The systemic response to trauma is associated with the hepatic acute-phase response. To further characterize this response, we performed controlled cortical impact injury on male mice and determined the expression of serum amyloid A1 (SAA1), an apolipoprotein, induced at the early stages of the acute-phase response in liver and plasma. After cortical impact injury, induction of SAA1 was detectable in plasma at 6 hours post-injury and in liver at 1 day post-injury, followed by gradual diminution over time. In the liver, cortical impact injury increased neutrophil and macrophage infiltration, apoptosis, and expression of mRNA encoding the chemokines CXCL1 and CXCL10. An increase in angiotensin II AT1 receptor mRNA at 3 days post-injury was also observed. Administration of the AT1 receptor antagonist telmisartan 1 hour post-injury significantly decreased liver SAA1 levels and CXCL10 mRNA expression, but did not affect CXCL1 expression or the number of apoptotic cells or infiltrating leukocytes. To our knowledge, this is the first study to demonstrate that SAA1 is induced in the liver after traumatic brain injury and that telmisartan prevents this response. Elucidating the molecular pathogenesis of the liver after brain injury will assist in understanding the efficacy of therapeutic approaches to brain injury.

Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates.

Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥ 100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.