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. Keywords: FUS; ALS; FTLD; prion; amyloid; LLPS The Link between FUS and Neurodegenerative DiseaseFUS (fused in sarcoma) gets its name from forming oncogenic fusion proteins with specific transcription factors following chromosomal rearrangements [1]. Such rearrangement is common in liposarcomas, thus FUS also goes by the name TLS (translocated in liposarcoma). Since 2009, FUS has received more attention for its connection to neurodegenerative disease after missense mutations were discovered to cause a small percentage of cases of amyotrophic lateral sclerosis (ALS-FUS) [2,3]. In the motor neurons of these patients, the normally nuclear FUS protein was found in cytoplasmic proteinaceous inclusions. Since then, non-mutant FUS has been identified in cytoplasmic inclusions in cortical neurons of a subset of patients with frontotemporal dementia (FTD; the neuropathological diagnosis is termed frontotemporal lobar degeneration (FTLD-FUS)) [4]. Both ALS and FTD are incurable and their clinical and pathological overlap suggests that they are part of a disease continuum.Mutations in FUS are autosomal dominant causes of familial ALS. Most mutations alter the C-terminal nuclear localization signal, resulting in excess cytoplasmic FUS that can form inclusions with gain-of-function toxicity [5]. Whether ALS-FUS or FTLD-FUS, it is the accumulation of FUS into cytoplasmic aggregates that appears to cause neuronal loss. The clinical presentation likely depends on the specific type of neurons that are affected. Biophysical and histological analysis suggest that FUS cytoplasmic aggregation may spread across anatomical networks through a prion-like mechanism [6]. Therefore, future drugs may target FUS's ability to cytoplasmically localize and/or form proteinaceous aggregates. Extensive post-translational methylation and phosphorylation of FUS have been shown to influence localization and aggregation, respectively. Here we review the post-translational modifications (PTMs) of FUS in the context of how they affect function, self-association and pathology.

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.

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). 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. Though it is clear 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 were 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 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.

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.

Fused in Sarcoma (FUS) is a ubiquitously expressed protein that can phase-separate from nucleoplasm and cytoplasm into distinct liquid-droplet structures. It is predominately 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 (ALS) and frontotemporal dementia (FTD). Phosphorylation of FUS's prion-like domain (PrLD), by nuclear 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 FUS's PrLD is phosphorylated at numerous additional sites and it is unknown if other non-PIKK and non-nuclear kinases might be influencing FUS's phase transitions. Here we evaluated 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 occured independently 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 occured 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.