Journal: Nature Communications

Article Title: Metamorphism in TDP-43 prion-like domain determines chaperone recognition

doi: 10.1038/s41467-023-36023-z

Figure Lengend Snippet: A Cartoon representation of the domain architecture of TDP-43 (top). The disordered PLD (region 274–414) is represented by a purple line. PLD primary structure, with the Met residues highlighted in red (bottom). The double α-helices are limited by orange boxes. B Turbidity measurements showing concentration- and salt-dependent LLPS for the PLD. C Differential Interference Contrast (DIC) microscopy images showing liquid condensates formed by the PLD in the specified conditions. Scale bars correspond to 25 μM. D LLPS of MetO PLD in the corresponding samples (circles), compared to unmodified PLD (gray stars), as measured by the area under the turbidity curves after 24 h of incubation. Gray broken line corresponds to the averaged LLPS of unmodified PLD. E Detailed region of the overlay of the 15 N-HSQC spectra from de-mixed PLD (purple) and MetO PLD (green) showing the large shifts for the Met moieties upon methionine sulfoxidation. MetO cross peaks are highlighted in red. F Comparison of the NMR signal intensity of unmodified PLD (purple) and MetO PLD (green) upon de-mixing shows a reduced broadening in the region 305-345 for MetO PLD. The preceding region (280–305) is also partially broadened in both proteins. The plot at the bottom shows the hydrophobicity of the PLD. G Secondary chemical shifts (ΔCα-ΔCβ) analysis for 300 μM PLD (top), 25 μM PLD (middle), and 300 μM MetO PLD (bottom). In these plots, positive values indicate acquisition of α-helical conformations, while negative values correspond to β-strand structures. Each plot shows the overlay with the structural propensities at 300 μM (broken purple line) for comparison. The schematic cartoon at the top highlights the two α-helices (in cylinders) and β-strands (arrows) formed in the PLD. Met residues are located with asterisks. For clarity, Met residues were removed from the MetO PLD plot (bottom) due to the strong shifts upon oxidation (Supplementary Fig. ). Unless otherwise stated, turbidimetry and microscopy samples ( B–D ) contained 150 mM KCl. NaCl in B–D refers to 150 mM NaCl. NMR samples ( E–G ) contained 10 mM KCl.

Article Snippet: Thawed TDP-43 and PLD aliquots were clarified by centrifugation at 15,000 g for 15 min, followed by filtration through a 100 kDa filter (Microcon) at 10,000 g to remove preformed aggregates.

Techniques: Concentration Assay, Microscopy, Incubation, Comparison

Journal: Nature Communications

Article Title: Metamorphism in TDP-43 prion-like domain determines chaperone recognition

doi: 10.1038/s41467-023-36023-z

Figure Lengend Snippet: TDP-43 PLD phase separation is strictly controlled by HSP70 and JDPs, whose interaction is mediated by structured elements present in the PLD. A liquid-to-solid transition will promote aggregate and fibril formation. Under oxidative stress, methionine sulfoxidation of the PLD will promote structural changes that will abrogate chaperone control and impact PLD phase separation, leading to the formation of alternative mature amyloid fibrils. While CK1δ phosphorylation promotes the aggregation of the PLD and hampers its recognition by HSP70, phosphorylation of soluble PLD is prevented after methionine sulfoxidation. Overall, modifications in the PLD trigger metamorphism which determines chaperone recognition, with impact on TDP-43’s pathophysiology.

Article Snippet: Thawed TDP-43 and PLD aliquots were clarified by centrifugation at 15,000 g for 15 min, followed by filtration through a 100 kDa filter (Microcon) at 10,000 g to remove preformed aggregates.

Techniques: Control, Phospho-proteomics