Phase separation of toxic dipeptide repeat proteins related to C9orf72 ALS/FTD

Jafarinia, Hamidreza; Giessen, E. van der; Onck, Patrick

doi:10.1101/2020.01.31.926428

Cited by 4 publications

(4 citation statements)

References 69 publications

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“…We thus believe that RNA molecules—and not proteins—are the primary target of arginine‐rich CPPs, which in addition explains why the peptides accumulate at nucleoli as this is where the highest concentration of RNA exists in cells. The very high affinity of arginine‐rich peptides for RNA should also contribute to the reported property of these CPPs to undergo phase separation (Boeynaems et al, 2017; Jafarinia et al, 2020), as these reactions are seeded by RNA. In addition to their own phase separation, C9ORF72‐associated argine‐rich DPRs perturb the integrity of membrane‐less organelles such as the nucleolus or stress granules, which we reason can be driven by displacing the RNA‐binding proteins that mediate the phase separation of these structures (Lee et al, 2016; Boeynaems et al, 2017; White et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Widespread displacement of DNA‐ and RNA‐binding factors underlies toxicity of arginine‐rich cell‐penetrating peptides

et al. 2021

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Due to their capability to transport chemicals or proteins into target cells, cell‐penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine‐rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine‐rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA‐ and DNA‐binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine‐rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA‐ and DNA‐binding factors from chromatin and mRNA accounts for the toxicity of arginine‐rich CPPs, including those that have been recently associated with the onset of ALS.

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Section: Discussionmentioning

confidence: 99%

Widespread displacement of DNA‐ and RNA‐binding factors underlies toxicity of arginine‐rich cell‐penetrating peptides

et al. 2021

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“…The 1-BPA model accounts for the physicochemical properties (charge, hydrophobicity) of all 20 amino acids, and incorporates backbone potentials that distinguish between three groups of amino acids (i.e., Glycine, Proline or other residues) depending on their backbone stiffness. The interaction potentials between cationic residues (R,K) and aromatic residues (F,Y,W) have been recalibrated to accommodate for the effect of cation-pi interactions 64 . We refer the reader to Refs.…”

Section: Coarse-grained Model For Unfolded Proteinsmentioning

confidence: 99%

“…The secondary and tertiary structure of Kap95 were preserved using an elastic network: for all amino acid beads separated less than 1.2 nm, a harmonic potential with a binding constant of 8000 kJ/nm 2 was applied, with the original separation between amino acids beads in the crystal structure forming the reference distance for the harmonic potentials. Within the 1-BPA-CP ('cation-pi') forcefield 64 , interactions between Kap95 and unfolded proteins fall under one of four categories (see sections 4 and 5 of the SI, Tables S3-4): coulombic interactions, volume exclusion, cation-pi interactions and specific interactions between binding site regions and FG-residues that use the 1-BPA hydrophobic potential (Equation S1). We assigned Kap95 residues to binding site regions based on the following procedure: First, we considered all binding site residues identified in a computational study 39 that investigated the binding of FG-Nup segments and isolated FG/GLFGrepeats to evolutionarily conserved regions on the surface of a mouse homolog of Kap95 (PDB:…”

Section: Parametrization Of a Coarse-grained Model For Yeast Kap95mentioning

confidence: 99%

Transport receptor occupancy in Nuclear Pore Complex mimics

Fragasso

Vries

Andersson

et al. 2021

Preprint

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Nuclear Pore Complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess 'Kap-centric' NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.

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“…In addition, it is considered that a minimum sequence length of protein is required and below which LLPS cannot occur. For example, the LLPS of the poly-PR/poly-GA requires a minimum peptide length of 50/100 (23,24). In contrast, numerous experiments demonstrate that even very short peptides can self-assemble into ordered solid-like nanoarchitectures.…”

Section: Introductionmentioning

confidence: 99%

Dipeptides are minimalistic but sufficient for liquid-liquid phase separation

Tang¹,

Bera

Yao³

et al. 2021

Preprint

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Liquid-liquid phase separation (LLPS) of proteins mediates the assembly of biomolecular condensates involved in physiological and pathological processes. Identifying the minimalistic building blocks and the sequence determinant of protein phase separation is of urgent importance but remains challenging due to the enormous sequence space and difficulties of existing methodologies in characterizing the phase behavior of ultrashort peptides. Here we demonstrate computational tools to efficiently quantify the microscopic fluidity and density of liquid-condensates/solid-aggregates and the temperature-dependent phase diagram of peptides. Utilizing our approaches, we comprehensively predict the LLPS abilities of all 400 dipeptide combinations of coded amino acids based on 492 micro-second molecular dynamics simulations, and observe the occurrences of spontaneous LLPS. We identify 54 dipeptides that form solid-like aggregates and three categories of dipeptides with high LLPS propensity. Our predictions are validated by turbidity assays and differential interference contrast (DIC) microscopy on four representative dipeptides (WW, QW, GF, and VI). Phase coexistence diagrams are constructed to explore the temperature dependence of LLPS. Our results reveal that aromatic moieties are crucial for a dipeptide to undergo LLPS, and hydrophobic and polar components are indispensable. We demonstrate for the first time that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, suggesting that LLPS is a general property of peptides/proteins, independent of their sequence length. This study provides a computational and experimental approach to the prediction and characterization of the phase behavior of minimalistic peptides, and will be helpful for understanding the sequence-dependence and molecular mechanism of protein phase separation.

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Phase separation of toxic dipeptide repeat proteins related to C9orf72 ALS/FTD

Cited by 4 publications

References 69 publications

Widespread displacement of DNA‐ and RNA‐binding factors underlies toxicity of arginine‐rich cell‐penetrating peptides

Widespread displacement of DNA‐ and RNA‐binding factors underlies toxicity of arginine‐rich cell‐penetrating peptides

Transport receptor occupancy in Nuclear Pore Complex mimics

Dipeptides are minimalistic but sufficient for liquid-liquid phase separation

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