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.
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.
Abstract 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.
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.
Synopsis
The N‐terminal globular domain (NTD) of TDP‐43 forms linear polymers whose disruption by phosphomimetic substitution reduces TDP‐43 liquid‐liquid phase separation and RNA splicing function.
TDP‐43 NTD polymerization is disrupted by the phosphomimetic substitution at S48.
NTD forms a linear polymer through low‐affinity head‐to‐tail intermolecular contacts.
TDP‐43 NTD polymerization‐disrupting mutations destabilize phase separation of full‐length TDP‐43 in vitro and in cells.
Disrupting NTD polymerization impedes TDP‐43's role in RNA splicing in cells.
Structural and functional analyses of ALS‐related protein TDP43 illustrates its higher order organisation and links splicing activity with the ability to undergo liquid–liquid phase separation.
A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an ...altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The PSI⁺ prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the structure of ...amyloid fibrils formed in vitro from purified recombinant$Sup35^{1-253}$, consisting of the glutamine- and asparagine-rich Nterminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole-dipole couplings among$^{13}C$nuclei in a series of Sup35NM fibril samples,$^{13}C-labeled$at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular$^{13}C-^{13}C$distances of ≈0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of ≈0.5 nm. These results indicate that an in-register parallel β-sheet structure underlies the PSI⁺ prion phenomenon.
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.
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.
Sex, prions, and plasmids in yeast Kelly, Amy C; Shewmaker, Frank P; Kryndushkin, Dmitry ...
Proceedings of the National Academy of Sciences - PNAS,
10/2012, Letnik:
109, Številka:
40
Journal Article
Recenzirano
Odprti dostop
Even deadly prions may be widespread in nature if they spread by infection faster than they kill off their hosts. The yeast prions PSI+ and URE3 (amyloids of Sup35p and Ure2p) were not found in 70 ...wild strains, while PIN+ (amyloid of Rnq1p) was found in ∼16% of the same population. Yeast prion infection occurs only by mating, balancing the detrimental effects of carrying the prion. We estimated the frequency of outcross mating as about 1% of mitotic doublings from the known detriment of carrying the 2-μm DNA plasmid (∼1%) and its frequency in wild populations (38/70). We also estimated the fraction of total matings that are outcross matings (∼23–46%) from the fraction of heterozygosity at the highly polymorphic RNQ1 locus (∼46%). These results show that the detriment of carrying even the mildest forms of PSI+, URE3, or PIN+ is greater than 1%. We find that Rnq1p polymorphisms in wild strains include several premature stop codon alleles that cannot propagate PIN+ from the reference allele and others with several small deletions and point mutations which show a small transmission barrier. Wild strains carrying PIN+ are far more likely to be heterozygous at RNQ1 and other loci than are pin− strains, probably reflecting its being a sexually transmitted disease. Because sequence differences are known to block prion propagation or ameliorate its pathogenic effects, we hypothesize that polymorphism of RNQ1 was selected to protect cells from detrimental effects of the PIN+ prion.
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