The key role of solvent in condensation: Mapping water in liquid-liquid phase-separated FUS

Ahlers, Jonas; Adams, Ellen M.; Bader, Verian; Pezzotti, Simone; Winklhofer, Konstanze F.; Tatzelt, Jörg; Havenith, Martina

doi:10.1016/j.bpj.2021.01.019

Cited by 77 publications

(98 citation statements)

References 81 publications

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“…As described previously ( 57 ), fluorescent imaging laser scanning microscopy on a microscope (ELYRA PS.1; Carl Zeiss) with an imaging detector (LSM 880; Carl Zeiss) was performed. For z-stack scanning, a 63× numerical aperture 1.4 oil-immersion objective was used to record a stack of 67.5 × 67.5 × 10 μm and 0.330 μm for each optical section.…”

Section: Methodsmentioning

confidence: 99%

The N-terminal domain of the prion protein is required and sufficient for liquid–liquid phase separation: A crucial role of the Aβ-binding domain

Kamps

Lin

Oliva

et al. 2021

Journal of Biological Chemistry

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Formation of biomolecular condensates through liquid–liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrP C ). PrP C is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrP C is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aβ oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aβ-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation–π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aβ-binding domain in this process.

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Section: Methodsmentioning

confidence: 99%

The N-terminal domain of the prion protein is required and sufficient for liquid–liquid phase separation: A crucial role of the Aβ-binding domain

Kamps

Lin

Oliva

et al. 2021

Journal of Biological Chemistry

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“…For this reason, the influence of inorganic salt ions (NaCl), metal ions (Ca 2+ and Zn 2+ ) and RNA in the FUS LLPS mechanism was tested by turbidity assays, following the OD at 595 nm. It is important to mention that despite turbidity assays of FL FUS have been previously reported, in most cases the assays are done with MBP-FUS in the presence of TEV protease (Burke et al , 2015; Ahlers et al , 2021; Kaur et al , 2019). Since it is known that FUS phase separation is highly sensitive to crowding and prone to unspecific interactions with proteins, it is possible that the presence of cleaved MBP and TEV protease in the solution could affect the turbidity results and analysis (Kaur et al , 2019; Kang et al , 2019; Lin et al , 2015).…”

Section: Resultsmentioning

confidence: 99%

Fused in sarcoma undergoes cold denaturation: Implications on phase separation

Felix

Laurents

Oroz

et al. 2022

Preprint

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The mediation of fused in sarcoma (FUS) protein liquid-liquid phase separation (LLPS) is generally attributed to the low-complexity and disordered domains, while the role of its folded domains remains unknown. In this work we questioned the role of the folded domains on the full-length (FL) FUS LLPS and studied the influence of several metabolites, ions and overall conditions on the LLPS process using turbidity assays, differential interference contrast microscopy and nuclear magnetic resonance spectroscopy. We demonstrate that FL FUS LLPS is highly responsive to the surrounding conditions, and that overall intrinsic disorder is crucial for LLPS. To promote such disorder, we reveal that the FUS RNA-recognition domain (RRM) and the zinc-finger motif (ZnF) undergo cold denaturation above 0°C, at a temperature that is determined by the conformational stability of the ZnF domain. We hypothesize that, in cold shock conditions, cold denaturation might provide a pathway that exposes additional residues to promote FUS self-assembly. Such findings mark the first evidence that FUS globular domains may have an active role in stress granule formation in cold stress.

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“…LLPS of IDRs in proteins enables the rapid formation of membraneless organelles without mechanical barriers but are distinctly segregated by chemical boundaries [ 210 , 211 ]. However, phase separation at its core is an entropically unfavorable thermodynamic process requiring a reduction or a negative change in global free energy enabled by energetically favorable multivalent protein–protein interactions that can offset energetic costs [ 183 , 212 , 213 ].…”

Section: Liquid–liquid Phase Separation May Regulate Prion Conversion...mentioning

confidence: 99%

Melatonin: Regulation of Prion Protein Phase Separation in Cancer Multidrug Resistance

Loh¹,

Reíter

2022

Molecules

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The unique ability to adapt and thrive in inhospitable, stressful tumor microenvironments (TME) also renders cancer cells resistant to traditional chemotherapeutic treatments and/or novel pharmaceuticals. Cancer cells exhibit extensive metabolic alterations involving hypoxia, accelerated glycolysis, oxidative stress, and increased extracellular ATP that may activate ancient, conserved prion adaptive response strategies that exacerbate multidrug resistance (MDR) by exploiting cellular stress to increase cancer metastatic potential and stemness, balance proliferation and differentiation, and amplify resistance to apoptosis. The regulation of prions in MDR is further complicated by important, putative physiological functions of ligand-binding and signal transduction. Melatonin is capable of both enhancing physiological functions and inhibiting oncogenic properties of prion proteins. Through regulation of phase separation of the prion N-terminal domain which targets and interacts with lipid rafts, melatonin may prevent conformational changes that can result in aggregation and/or conversion to pathological, infectious isoforms. As a cancer therapy adjuvant, melatonin could modulate TME oxidative stress levels and hypoxia, reverse pH gradient changes, reduce lipid peroxidation, and protect lipid raft compositions to suppress prion-mediated, non-Mendelian, heritable, but often reversible epigenetic adaptations that facilitate cancer heterogeneity, stemness, metastasis, and drug resistance. This review examines some of the mechanisms that may balance physiological and pathological effects of prions and prion-like proteins achieved through the synergistic use of melatonin to ameliorate MDR, which remains a challenge in cancer treatment.

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The key role of solvent in condensation: Mapping water in liquid-liquid phase-separated FUS

Cited by 77 publications

References 81 publications

The N-terminal domain of the prion protein is required and sufficient for liquid–liquid phase separation: A crucial role of the Aβ-binding domain

The N-terminal domain of the prion protein is required and sufficient for liquid–liquid phase separation: A crucial role of the Aβ-binding domain

Fused in sarcoma undergoes cold denaturation: Implications on phase separation

Melatonin: Regulation of Prion Protein Phase Separation in Cancer Multidrug Resistance

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