Phosphomimetic substitutions in TDP-43’s transiently α-helical region suppress phase separation

Haider, Raza; Penumutchu, Srinivasa; Boyko, Solomiia; Surewicz, Witold K.

doi:10.1016/j.bpj.2024.01.001

Cited by 8 publications

(3 citation statements)

References 61 publications

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“…Previous studies have examined the LLPS of TDP-43 at concentrations varying from 15-600µM 12,28,33,35 ; Garg and Bhat 28 determined the saturation concentration to be 6.6µM at pH 7.4, in the presence of NaCl 28 . Our study examined the behaviour of the protein at a 10-fold lower concentration, well within the sub-saturated regime, where nanocondensation is thought to occur.…”

Section: Discussionmentioning

confidence: 99%

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Quantification of nanocondensates formation at the single molecule level

Houx,

Copie,

Gambin

et al. 2024

Preprint

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Understanding the molecular mechanisms of biomolecular condensate formation through liquid-liquid phase separation is crucial for deciphering cellular cues in normal and pathological contexts. Recent studies have highlighted the existence of sub-micron assemblies, known as nanocondensates or mesoscopic clusters, in the organization of a significant portion of the proteome. However, as smaller condensates are invisible to classical microscopy, new tools must be developed to quantify their numbers and properties. Here, we establish a simple analysis framework using single molecule fluorescence spectroscopy to quantify the formation of nanocondensates diffusing in solution. We used the low-complexity domain of TAR DNA-binding protein 43 (TDP-43) as a model system to show that we can recapitulate the phase separation diagram of the protein in various conditions. Single molecule spectroscopy reveals rapid formation of TDP-43 nanoclusters at ten-fold lower concentrations than described previously by microscopy. We demonstrate how straightforward fingerprinting of individual nanocondensates provides an exquisite quantification of their formation, size, density, and their temporal evolution. Overall, this study highlights the potential of single molecule spectroscopy to investigate the formation of biomolecular condensates and liquid-liquid phase separation mechanisms in protein systems.

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Section: Discussionmentioning

confidence: 99%

“…Current research often uses long incubation times ranging from hours to multiple days 12,28 . This allows the droplets to grow enough to be observed using imaging techniques [29][30][31][32][33] . In contrast, our single molecule approach focusses on the early stages of phase transition.…”

Section: Kinetics Of Tdp-43 Lcd Llps Inductionmentioning

confidence: 99%

Quantification of nanocondensates formation at the single molecule level

Houx,

Copie,

Gambin

et al. 2024

Preprint

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“…Phosphorylation in SR-rich proteins has been observed to increase solubility or disrupt alpha helical regions inhibiting their ability to self-assemble in fibrils ( 60 ). In TDP-43, phosphorylation of few key residues disrupts the helical structure and regulates condensate formation ( 61 ).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation in the Ser/Arg-rich region of the nucleocapsid of SARS-CoV-2 regulates phase separation by inhibiting self-association of a distant helix

Stuwe,

Reardon,

et al. 2024

Journal of Biological Chemistry

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Pathological C‐terminal phosphomimetic substitutions alter the mechanism of liquid–liquid phase separation of TDP‐43 low complexity domain

Haider,

Shipley,

Surewicz

et al. 2024

Protein Science

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C‐terminally phosphorylated TAR DNA‐binding protein of 43 kDa (TDP‐43) marks the proteinaceous inclusions that characterize a number of age‐related neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal lobar degeneration and Alzheimer's disease. TDP‐43 phosphorylation at S403/S404 and (especially) at S409/S410 is, in fact, accepted as a biomarker of proteinopathy. These residues are located within the low complexity domain (LCD), which also drives the protein's liquid–liquid phase separation (LLPS). The impact of phosphorylation at these LCD sites on phase separation of the protein is a topic of great interest, as these post‐translational modifications and LLPS are both implicated in proteinopathies. Here, we employed a combination of experimental and simulation‐based approaches to explore this question on a phosphomimetic model of the TDP‐43 LCD. Our turbidity and fluorescence microscopy data show that phosphomimetic Ser‐to‐Asp substitutions at residues S403, S404, S409 and S410 alter the LLPS behavior of TDP‐43 LCD. In particular, unlike the LLPS of unmodified protein, LLPS of the phosphomimetic variants displays a biphasic dependence on salt concentration. Through coarse‐grained modeling, we find that this biphasic salt dependence is derived from an altered mechanism of phase separation, in which LLPS‐driving short‐range intermolecular hydrophobic interactions are modulated by long‐range attractive electrostatic interactions. Overall, this in vitro and in silico study provides a physiochemical foundation for understanding the impact of pathologically relevant C‐terminal phosphorylation on the LLPS of TDP‐43 in a more complex cellular environment.

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Phosphomimetic substitutions in TDP-43’s transiently α-helical region suppress phase separation

Cited by 8 publications

References 61 publications

Quantification of nanocondensates formation at the single molecule level

Quantification of nanocondensates formation at the single molecule level

Phosphorylation in the Ser/Arg-rich region of the nucleocapsid of SARS-CoV-2 regulates phase separation by inhibiting self-association of a distant helix

Pathological C‐terminal phosphomimetic substitutions alter the mechanism of liquid–liquid phase separation of TDP‐43 low complexity domain

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