Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose)

Altmeyer, Matthias; Neelsen, Kai J.; Teloni, Federico; Pozdnyakova, Irina; Pellegrino, Stefania; Grøfte, Merete; Rask, Maj‐Britt; Streicher, Werner; Jungmichel, Stephanie; Nielsen, Michael L.; Lukáš, Jiří

doi:10.1038/ncomms9088

Cited by 503 publications

(587 citation statements)

References 51 publications

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“…Importantly, all of these compartments are membrane‐less organelles, which may have liquid properties. The liquid behavior of DNA damage foci was recently experimentally supported (Altmeyer et al , 2015; Patel et al , 2015). In conclusion, SPOP localizes to a variety of different nuclear membrane‐less organelles, but has not been found diffusely localized.…”

Section: Resultsmentioning

confidence: 92%

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

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Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

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Section: Resultsmentioning

confidence: 92%

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

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“…Complementing previous findings that DNA‐PK treatment disrupts FUS LC recruitment to hydrogel models of granules (Han et al , 2012), here we demonstrate that phosphorylation and phosphomimetic substitution of FUS LC disrupts domain self‐interaction and LLPS. The LC is required for nuclear self‐association of FUS into chromatin‐binding dynamic puncta (Yang et al , 2014) and cytoplasmic granules (Shelkovnikova et al , 2014), as well as robust recruitment at sites of DNA damage (Mastrocola et al , 2013; Altmeyer et al , 2015). Therefore, cells might flip a chemical “off switch” by using phosphorylation to regulate FUS function via disrupting FUS self‐assembly.…”

Section: Discussionmentioning

confidence: 99%

“…However, the effect of FUS phosphorylation on LLPS has not yet been determined. Modification of FUS with poly(ADP‐ribose; PAR) at sites of DNA damage induces phase separation of FUS (Altmeyer et al , 2015), while arginine methylation of the nuage protein DDX4 reduces LLPS (Nott et al , 2015). More generally, many of the low‐complexity prion‐like domains associated with aggregation in disease have been shown by proteomic screens to harbor many sites of post‐translational modification.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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“…In this sense, gH2AX is a common feature of several distinct DSB repair machineries because it builds the underlying platform for recruitment of the repair-associated proteins and their assembly at the lesion site (4). Hence, individual DSB repair units might be significantly larger than the gH2AX subfoci alone (55).…”

Section: Focus Structure and Implicationsmentioning

confidence: 99%

Superresolution light microscopy shows nanostructure of carbon ion radiation‐induced DNA double‐strand break repair foci

et al. 2016

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Carbon ion radiation is a promising new form of radiotherapy for cancer, but the central question about the biologic effects of charged particle radiation is yet incompletely understood. Key to this question is the understanding of the interaction of ions with DNA in the cell's nucleus. Induction and repair of DNA lesions including double-strand breaks (DSBs) are decisive for the cell. Several DSB repair markers have been used to investigate these processes microscopically, but the limited resolution of conventional microscopy is insufficient to provide structural insights. We have applied superresolution microscopy to overcome these limitations and analyze the fine structure of DSB repair foci. We found that the conventionally detected foci of the widely used DSB marker gH2AX (Ø 700-1000 nm) were composed of elongated subfoci with a size of ∼100 nm consisting of even smaller subfocus elements (Ø 40-60 nm). The structural organization of the subfoci suggests that they could represent the local chromatin structure of elementary DSB repair units at the DSB damage sites. Subfocus clusters may indicate induction of densely spaced DSBs, which are thought to be associated with the high biologic effectiveness of carbon ions. Superresolution microscopy might emerge as a powerful tool to improve our knowledge of interactions of ionizing radiation with

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Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose)

Cited by 503 publications

References 51 publications

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

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

Superresolution light microscopy shows nanostructure of carbon ion radiation‐induced DNA double‐strand break repair foci

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