Nuclear Import Receptors Directly Bind to Arginine-Rich Dipeptide Repeat Proteins and Suppress Their Pathological Interactions - PubMed (original) (raw)

. 2020 Dec 22;33(12):108538.

doi: 10.1016/j.celrep.2020.108538.

Sinem Usluer 2, Benjamin Bourgeois 2, Francesca Simonetti 3, Hana M Odeh 4, Charlotte M Fare 5, Mareike Czuppa 6, Marian Hruska-Plochan 7, Mario Hofweber 8, Magdalini Polymenidou 7, James Shorter 5, Dieter Edbauer 9, Tobias Madl 10, Dorothee Dormann 11

Affiliations

Nuclear Import Receptors Directly Bind to Arginine-Rich Dipeptide Repeat Proteins and Suppress Their Pathological Interactions

Saskia Hutten et al. Cell Rep. 2020.

Abstract

Nuclear import receptors, also called importins, mediate nuclear import of proteins and chaperone aggregation-prone cargoes (e.g., neurodegeneration-linked RNA-binding proteins [RBPs]) in the cytoplasm. Importins were identified as modulators of cellular toxicity elicited by arginine-rich dipeptide repeat proteins (DPRs), an aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mechanistically, the link between importins and arginine-rich DPRs remains unclear. Here, we show that arginine-rich DPRs (poly-GR and poly-PR) bind directly to multiple importins and, in excess, promote their insolubility and condensation. In cells, poly-GR impairs Impα/β-mediated nuclear import, including import of TDP-43, an RBP that aggregates in C9orf72-ALS/FTD patients. Arginine-rich DPRs promote phase separation and insolubility of TDP-43 in vitro and in cells, and this pathological interaction is suppressed by elevating importin concentrations. Our findings suggest that importins can decrease toxicity of arginine-rich DPRs by suppressing their pathological interactions.

Keywords: ALS; FTD; TDP-43; chaperone; dipeptide repeat proteins; importin; nneurodegeneration; nuclear import receptor; nuclear transport receptor; phase separation.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of Interests J.S. is a consultant for Dewpoint Therapeutics.

Figures

Figure 1.

Figure 1.. Importins Bind Directly to Arginine-Rich Dipeptide Repeat Proteins (DPRs)

(A) Western blot showing binding of endogenous importins from HeLa cytosol to immobilized, biotinylated GR25 and PR25, but not GP25, in a pull-down (PD). (B–E) Direct binding of R-rich DPRs to the importins TNPO1 (B), Impβ (C), Impα3 (D), but not CRM1 (E) in pull-downs using biotinylated GR25, PR25, and GP25 (bait) and purified importins (prey) visualized by Sypro-Ruby staining. Input represents 5% of the importin used in the PD. Ovalbumin (ovalb.) was used to prevent unspecific binding to the beads. (F) Fluorescence polarization measurements of fluorescein isothiocyanate (FITC)-labeled GR25 (black) or PR25 (red) with increasing concentrations of TNPO1. Table shows calculated _k_D values assuming a 1:1 complex formation representing the mean of three independent experiments performed in technical triplicate ± SEM of the fit. See also Figure S1.

Figure 2.

Figure 2.. Poly-GR and Poly-PR Reduce the Solubility of Importins and Cause Their Condensation and Oligomerization

(A) Sedimentation assay to measure precipitation of recombinant His-Impα3/His-S-Impβ by 10-fold molar excess of TMR-GR25, TMR-PR25, TMR-GP25, or TMR-GR10 visualized by Sypro-Ruby staining. (B) Percentage of the respective importin being soluble shown as mean of three independent experiments ± SEM; *p < 0.0322 and ***p < 0.0002 by one-way ANOVA with Dunnett’s multiple-comparison test to GP25. (C) Sedimentation assay to quantify precipitation of recombinant His-Imα3, His-S-Imβ, His-TNPO1, or CRM1 in the presence of 10-fold molar excess of either TMR-GR25, TMR-PR25, or TMR-GP25 visualized by Sypro-Ruby staining. (D) Percentage of the respective NTR being soluble as the mean of three independent experiments ± SEM; *p < 0.0322, **p < 0.021, and ***p < 0.0002 by one-way ANOVA with Dunnett’s multiple-comparison test to GP25. (E) GFP-TNPO1 forms condensates in the presence of a 10-fold molar excess of TMR-labeled GR25 and PR25, but not with TMR-GP25 or a 50-fold excess of TMR-GR10. Scale bar, 10μmm. (F) Condensate formation of GFP-Impβ in the presence of a 10-fold molar excess of TMR-labeled GR25 or TMR-PR25, but not TMR-GP25 or a 50-fold excess of TMR-GR10, is enhanced by equimolar amounts of Impα3 (unlabeled). Scale bar, 10μm. (G) FRAP demonstrating reduced mobility of GFP-TNPO1 condensates induced by 10-fold molar excess of TMR-GR25. Scale bar, 5μm. (H) Anti-His western blot demonstrating oligomer formation of His-Impα3 and His-TNPO1, but not His-S-Impβ, by 10-fold molar excess of TMR-GR25 and TMR-PR25 shown by SDD-AGE. See also Figure S2.

Figure 3.

Figure 3.. Poly-GR Interferes with Nuclear Import of TDP-43 and Other cNLS-Containing Cargoes

(A) Experimental design of the hormone-inducible nuclear import reporter assay. GCR2-GFP2 is fused to a protein of interest carrying a NLS. Upon cellular expression, the reporter remains cytoplasmic but is actively imported into the nucleus over time upon addition of the steroid hormone dexamethasone. (B) Delayed import of GCR2-GFP2-TDP-43 by GR25, but not PR25, by live cell imaging compared with untreated cells (control). Scale bar, 20 μm. Uptake of TMR-labeled DPR peptides (red) by GCR2-GFP2TDP-43-expressing cells (green) before dexamethasone addition was verified by confocal imaging (top row). (C) Nuclear/cytoplasmic (N/C) ratio of fluorescence intensities of GCR2-GFP2-TDP-43 import over time shown as mean of three independent experiments (20–51 cells each) ± SEM. Statistical significance was calculated by repeated-measures (RM) one-way ANOVA for the area under the curve with Tu-key’s multiple comparison test (*p < 0.0332). (D) Nuclear/cytoplasmic (N/C) ratio of fluorescence intensities of GCR2-GFP2-MBP-cNLS import over time in either untreated cells (untr) or cells pre-incubated TMR-GR25 (GR25) as mean of three independent experiments (19–69 cells each) ± SEM. Statistical significance was calculated by comparing the area under the curve by unpaired t test (**p < 0.0021). See also Figure S3.

Figure 4.

Figure 4.. Poly-GR and Poly-PR Promote Phase Separation of TDP-43

(A) Condensate formation upon Tev cleavage of TDP-43-_Tev_-MBP/MBP-_Tev_-TDP-43-GFP in the absence (-) or presence of equimolar (1x) or two-fold (2x) molar excess of TMR-labeled DPRs. Scale bar, 20 μm. (B) Anti-TDP-43 western blot showing that both TMR-GR25 and TMR-PR25 promote sedimentation of TDP-43 upon Tev cleavage of TDP-43-_Tev_-MBP more strongly than TMR-GP25. Note that Tev cleavage of the MBP tag is not complete in all reactions but that uncleaved TDP-43-MBP mirrors the sedimentation behavior of cleaved TDP-43 (likely because of multimerization). (C) Quantification of the amount of soluble, cleaved TDP-43 as mean of three independent experiments ± SEM; **p < 0.0021 by one-way ANOVA with Dunnett’s multiple-comparison test to untreated (-). (D) Experimental design to address RIPA-buffer solubility of endogenous TDP-43 upon incubation of intact cells with GR25/PR25 peptides shown in (E). (E) RIPA-soluble (S) and insoluble (P) fractions were analyzed using TDP-43 western blot (WB). Note that for visibility, the pellet fraction in the WB is overrepresented 4.5x. (F) Normalized S/P ratio for TDP-43 after correction for overrepresentation of the pellet fraction as mean of three independent experiments ± SEM; **p < 0.0021 by one-way ANOVA with Dunnett’s multiple-comparison test to untreated (-).

Figure 5.

Figure 5.. Importins Shield R-Rich DPRs and Suppress Condensate Formation with TDP-43 or RNA

(A) Equimolar concentrations of TNPO1, Impβ, or Impα3/β, but not CRM1 or Impα3 alone, prevent poly-GR-induced phase separation of TDP-43. Scale bar, 20 μm. (B) Anti-TDP-43 western blot showing sedimentation of TDP-43 upon Tev cleavage in the presence of GST-GR25 and the absence or presence of various NTRs. Note that Tev-mediated cleavage of the MBP tag is not complete in all reactions but that uncleaved TDP-43-MBP mirrors the sedimentation behavior of cleaved TDP-43 (likely because of multimerization). (C) Percentage of soluble, cleaved TDP-43 as mean of three independent experiments ± SEM; ***p < 0.0002 and *p < 0.0332 by one-way ANOVA with Dunnett’s multiple-comparison test to untreated (-) in the absence of GST-GR25. (D) Anti-GST western blot showing sedimentation of GST-GR25 in the absence or presence of TDP-43 and various NTRs. (E) Percentage of soluble GST-GR25 shown as mean of three independent experiments ± SEM; ***p < 0.0002 by one-way ANOVA with Dunnett’s multiple-comparison test to untreated (-) in the absence of TDP-43. (F) Condensation of TMR-GR25 by RNA is suppressed by equimolar concentrations of TNPO1 or Impβ, but not CRM1 or Impα3. Scale bar, 10 μm. See also Figure S4.

Figure 6.

Figure 6.. Importins Can Suppress Poly-GR-Induced TDP-43 Precipitation from Cytosol

(A) Western blot demonstrating that importins and TDP-43, but not GAPDH, are precipitated by poly-GR and poly-PR, but not poly-GP, in a concentration-dependent manner from HeLa cytosol; asterisk marks an unspecific band. (B) Percentage of soluble protein shown as mean of three or four independent experiments ± SEM; **p < 0.0021 and ***p < 0.0002 by one-way ANOVA with Dunnett’s multiple-comparison test to GP25. (C) Anti TDP-43 western blot demonstrating poly-GR-induced precipitation of TDP-43 from HeLa cytosol can be prevented by addition of TNPO1, Impα, or Impα/β, but not by CRM1. (D) Percentage of soluble TDP-43 in the presence of poly-GR and NTRs as mean of three independent experiments ± SEM; **p < 0.0021 by one-way ANOVA with Dunnett’s multiple comparison test to TDP-43/GR25.

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