Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation - PubMed (original) (raw)

Comparative Study

. 2015 May 19;112(20):E2620-9.

doi: 10.1073/pnas.1504459112. Epub 2015 May 4.

Affiliations

Comparative Study

Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation

Liliana Malinovska et al. Proc Natl Acad Sci U S A. 2015.

Abstract

Many protein-misfolding diseases are caused by proteins carrying prion-like domains. These proteins show sequence similarity to yeast prion proteins, which can interconvert between an intrinsically disordered and an aggregated prion state. The natural presence of prions in yeast has provided important insight into disease mechanisms and cellular proteostasis. However, little is known about prions in other organisms, and it is not yet clear whether the findings in yeast can be generalized. Using bioinformatics tools, we show that Dictyostelium discoideum has the highest content of prion-like proteins of all organisms investigated to date, suggesting that its proteome has a high overall aggregation propensity. To study mechanisms regulating these proteins, we analyze the behavior of several well-characterized prion-like proteins, such as an expanded version of human huntingtin exon 1 (Q103) and the prion domain of the yeast prion protein Sup35 (NM), in D. discoideum. We find that these proteins remain soluble and are innocuous to D. discoideum, in contrast to other organisms, where they form cytotoxic cytosolic aggregates. However, when exposed to conditions that compromise molecular chaperones, these proteins aggregate and become cytotoxic. We show that the disaggregase Hsp101, a molecular chaperone of the Hsp100 family, dissolves heat-induced aggregates and promotes thermotolerance. Furthermore, prion-like proteins accumulate in the nucleus, where they are targeted by the ubiquitin-proteasome system. Our data suggest that D. discoideum has undergone specific adaptations that increase the proteostatic capacity of this organism and allow for an efficient regulation of its prion-like proteome.

Keywords: Dictyostelium discoideum; molecular chaperones; prion; protein aggregation; proteostasis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Bioinformatic analysis reveals a highly aggregation-prone proteome in D. discoideum. (A) Length distribution of N and Q runs in D. discoideum, S. cerevisiae, and H. sapiens. (B) Expression levels of polyQ-containing proteins in cell lysates of D. discoideum, S. cerevisiae, and H. sapiens. Equal amounts of total proteins, as judged by Ponceau staining, were separated by SDS/PAGE, and Q-rich proteins were detected by immunoblotting with a polyQ-specific antibody. (C) Prion-like proteins were predicted based on their compositional similarity with known yeast prion proteins (12). The pie chart shows the total number of prion-like proteins in the proteome of budding yeast or D. discoideum. (D) Prion-like proteins were analyzed for their association with SMART domains using DAVID (27). (E and F) Comparison of prion-like proteins from D. discoideum and D. purpureum. The Venn diagrams show protein orthologs and protein superfamily domains that are associated with prion-like domains. Protein ortholog information was obtained from DictyBase (90).

Fig. 2.

Fig. 2.

Aggregation-prone prion-like proteins do not aggregate in the cytosol of D. discoideum. (A) Fluorescence microscopy of D. discoideum cells expressing GFP-tagged aggregation-prone prion-like proteins (Q103, N47) or control peptides (Q25, N25). The numbers give the percentage of cells showing the observed distribution pattern. The majority of cells showed a diffuse cytosolic fluorescent signal. Small punctate structures in the cytosol are fixation artifacts and were not detectable in unfixed living cells. (B) Biochemical analysis of whole-cell extracts using semidenaturing detergent–agarose gel electrophoresis (SDD-AGE) (34). Cell lysates were denatured by boiling at 95 °C and applied to the gel to verify the position of the monomers. SDS-insoluble material is distinguishable by its retarded migration behavior. Numbers below the SDD-AGE immunoblot give the amount of SDS-insoluble material. (C) The nucleus in an N47-GFP–expressing cell was visualized by DAPI staining. (D) Fluorescent microscopy and biochemical analysis of D. discoideum cells expressing GFP-tagged NM and a highly aggregation-prone variant of NM (NM*). The position of the monomer (1-mer) was determined by boiling (not shown). (E) Same as D, except that the aggregation behavior of candidate prion domains from D. discoideum is shown. Fluorescent microscopy and biochemical analysis of D. discoideum (Left) and S. cerevisiae (Right) cells expressing GFP-tagged candidate PrDs from D. discoideum. Note that the tested PrDs are mainly soluble in D. discoideum cells, whereas they aggregate in the cytosol of S. cerevisiae cells. (F) Analysis of the PrD of the D. discoideum protein mekA. D. discoideum cells expressing GFP-tagged mekA-PrD show distinct fluorescent foci that reside in the nucleus. Because mekA-PrD could not be expressed in yeast, only the results for D. discoideum are shown. The nucleus was visualized by DAPI staining.

Fig. 3.

Fig. 3.

Nuclear accumulations are dynamic and associated with the ubiquitin/proteasome system. (A) Light microscopy and correlative light and electron microscopy (CLEM) analysis of nuclear NM* accumulations. Two different nuclear patterns were observed. Numbers indicate the percentage of cells (with nuclear signal) showing the depicted pattern. Electron-dense structures within the nucleus are reminiscent of the nucleolus. (N, nucleus; Nu, nucleolus-like structure). (B) Colocalization of NM*-GFP and ubiquitin. Fixed D. discoideum cells expressing NM*-GFP (green) were stained with DAPI (blue) and an antibody against ubiquitin (red). NM*-GFP and ubiquitin signals show a strong overlap but are excluded from DAPI-stained areas. (C) Fluorescence recovery after photobleaching (FRAP) analysis of punctate NM*-GFP and corresponding time-lapse microscopy (Upper). A FRAP analysis of the nuclear NM*-GFP signal is shown for comparison (Lower). Punctate structures display a high mobile fraction (88%; Left), whereas the diffuse nuclear signal showed no recovery (Right). Time-lapse microscopy images are shown in false color. The legend on the Left indicates intensities (error bars represent SD). (D) Immunoblot analysis of NM*-GFP–expressing cells treated with MG132 (100 µM; Sigma), compared with control cells. NM*-GFP was detected using a GFP-specific antibody. (E) NM*-GFP stability in cells treated with MG132, compared with control cells. NM*-GFP–expressing cells were treated with cycloheximide (250 µg/mL; Applichem), and NM* levels were determined by immunoblotting over time. The levels were normalized to the loading control (streptavidin conjugates). Note that NM*-GFP levels decline more slowly in cells treated with MG132. (F) Effect of proteasomal inhibition (MG132) on the fraction of cells with nuclear NM*-GFP puncta. Cells were treated with proteasomal inhibitor for 14 h [error bars represent SD; n = 25 field of view (FOV)].

Fig. 4.

Fig. 4.

Cytosolic chaperones control protein aggregation in the cytoplasm. (A) Time-lapse microscopy of cells coexpressing NM*-GFP (green) and H2B-mRFPmars (red) during mitosis. The GFP signal relocates to the cytosol 10 min before cell division and 5 min before nuclear division. (B) Effect of inhibiting molecular chaperones in cells expressing NM*-GFP. Inhibition of Hsp90 [6 µM geldanamycin (Santa Cruz), 10 µM radicicol (Santa Cruz)] leads to formation of cytosolic foci and an increase in SDS-insoluble material. Inhibition of Hsp70 [10 µM VER-155088 (Sigma)] shows no measurable difference to control cells. This could be due to a poor intake of the inhibitor, low specificity of the inhibitor, or the fact that Hsp70 has no major role in regulating protein aggregation (error bars represent SD; n = 30 FOV). The position of the monomers as indicated on the SDD-AGE immunoblot was determined by boiling of the samples.

Fig. 5.

Fig. 5.

Effects of expressing prion-like proteins on health and development of D. discoideum. (A) Comparison of the cell division time of D. discoideum cells expressing aggregation-prone prion-like proteins (N47, Q103, NM*) and control proteins (N25, Q25). No significant differences were detected (error bars represent SD; n = 7). (B) Different developmental states of the strains shown in A. The expected developmental stage is schematically depicted on the Right. No measurable effect on developmental timing was detected. (C) Effects of heat stress (3 h, 30 °C) on the aggregation behavior of Q103-GFP and NM*-GFP. Fluorescence microscopy reveals cytosolic foci after heat stress. (D) Immunofluorescence of heat-stressed cells expressing NM*-GFP. Note that all heat-induced cytosolic GFP aggregates colocalize with ubiquitin. Additional ubiquitin-positive foci lacking a GFP signal indicate that other proteins are ubiquitinated and aggregate during heat stress. Nuclear localization is visualized by DAPI stain. (E) SDD-AGE analysis of whole-cell extracts from cells shown in C. Note the increase in SDS-insoluble material upon heat stress. The position of the monomers as indicated on the immunoblot was determined by boiling the sample. (F) Fluorescence recovery after photobleaching (FRAP) analysis of cytosolic NM*-GFP foci. The cytosolic NM* foci show a low mobile fraction (20%) (n = 4, error bars represent SD). (G) Comparison of cell death rates in cells expressing aggregation-prone prion-like marker proteins and control proteins. Cells are grown at normal temperature (25 °C) or elevated temperatures (34 °C). The amount of dead cells was determined as percentage of propidium iodide (PI)-positive cells (Upper). The increase in cell death was determined as ratio of dead cells at 23 °C and 34 °C (Lower) (error bars represent SD; n = 30,000).

Fig. 6.

Fig. 6.

Hsp101 is a major player in the control of heat-induced protein aggregation. (A) Fluorescence time-lapse microscopy analysis of D. discoideum cells expressing Q103-GFP (Upper) or NM*-GFP (Lower) during stress. Heat stress triggers formation of cytosolic foci; release of stress leads to dissolution of cytosolic aggregates. The time point is indicated below and the temperature is indicated above. Arrows mark cytosolic foci. (B) Quantification of time-lapse microscopy in A (error bars represent SD; n = 9 FOV). (C) Formation of cytosolic aggregates was followed by time-lapse fluorescence microscopy of cells expressing Q103 (black) or coexpressing Q103 and Hsp101 (red) during heat stress and recovery. Constitutive expression of Hsp101 prevents formation of cytosolic Q103 aggregation. The number of cells with aggregates was quantified (error bars represent SD; n = 9 FOV). (D) Time-lapse fluorescence microscopy of Q103-GFP cells coexpressing dominant-negative Hsp101 variants (black, gray) during recovery from heat stress (error bars represent SD; n = 9 FOV). Functional inhibition of Hsp101 results in slower aggregate dissolution rates. (E) Effects of Hsp101 overexpression and inactivation on thermotolerance in D. discoideum. Cells were grown at 25 °C or 34 °C. The amount of dead cells was determined as percentage of propidium iodide (PI)-positive cells (Upper). The increase in cell death was determined as ratio of dead cells at 23 °C and 34 °C (Lower). Overexpression of Hsp101 reduces cell death, whereas functional inhibition leads to elevated levels of cell death.

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