Protection from cytosolic prion protein toxicity by modulation of protein translocation - PubMed (original) (raw)

Protection from cytosolic prion protein toxicity by modulation of protein translocation

Neena S Rane et al. EMBO J. 2004.

Abstract

Failure to promptly dispose of undesirable proteins is associated with numerous diseases. In the case of cellular prion protein (PrP), inhibition of the proteasome pathway can generate a highly aggregation-prone, cytotoxic form of PrP implicated in neurodegeneration. However, the predominant mechanisms that result in delivery of PrP, ordinarily targeted to the secretory pathway, to cytosolic proteasomes have been unclear. By accurately measuring the in vivo fidelity of protein translocation into the endoplasmic reticulum (ER), we reveal a slight inefficiency in PrP signal sequence function that generates proteasomally degraded cytosolic PrP. Attenuating this source of cytosolic PrP completely eliminates the dependence on proteasomes for PrP degradation. This allows cells to tolerate both higher expression levels and decreased proteasomal capacity without succumbing to the adverse consequences of misfolded PrP. Thus, the generation of potentially toxic cytosolic PrP is controlled primarily during its initial translocation into the ER. These results suggest that a substantial proportion of the cell's constitutive proteasomal burden may consist of proteins that, like PrP, fail to cotranslationally enter the secretory pathway with high fidelity.

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Figures

Figure 1

Figure 1

Measurement and modulation of protein translocation in vivo. (A) Varying amounts of plasmid encoding TF or PrP-TF were transfected into MDCK cells and the amount of luciferase reporter activation measured (in relative light units (RLU)). (B) Duplicate samples from panel A were analyzed in parallel by immunoblotting with antibodies against NF-κB. The position of exogenously expressed TF on faint and dark exposures of the blot is indicated with arrows. Endogenous NF-κB (indicated by an asterisk) serves as a loading control. (C) Comparison of translocation efficiencies of the Opn, PrP, and Prl signal sequences fused to TF. Transfection of an unrelated plasmid (mock) resulted in no reporter activation. Varying amounts of TF lacking a signal sequence were analyzed in parallel (note 10-fold difference in _y_-axis scales). (D) Parallel immunoblots for TF expression of samples from panel C.

Figure 2

Figure 2

Reduced susceptibility to aggregate formation by modulation of PrP translocation. (A) Biosynthesis and maturation of PrP fused to different signal sequences was assessed by pulse-chase analysis (as described in Materials and methods) of transfected N2a cells in the absence (top panel) or presence (bottom panel) of proteasome inhibition. Samples after a 15 min pulse labeling (‘P' lanes) and following a 1 h chase in unlabeled media (‘C' lanes) are shown. The positions of different species of PrP are indicated: U, unglycosylated, *, singly glycosylated, **, doubly glycosylated, and M, mature. The percent of total PrP synthesized in the unglycosylated form is indicated below each set of lanes. (B) N2a cells expressing PrP with different signal sequences were assessed for aggregate formation by a solubility and sedimentation assay. The total proteins and immunoblots for GFP (a cotransfected control protein) and PrP in the soluble and insoluble fractions are shown (Sup. and Pel., respectively). The position of unglycosylated PrP (U) is indicated. Untreated and MG132-treated cells were analyzed in parallel. Cells not transfected with a PrP construct are indicated (nt).

Figure 3

Figure 3

Effect of prolonged proteasome inhibition on the metabolism of PrP and Opn-PrP. (A) N2a cells transfected with either wild-type PrP (upper panel) or Opn-PrP (lower panel) were treated for between 0 and 8 h with 5 μM MG132 before lysis and fractionation into detergent-soluble (Sup.) and insoluble (Pel.) fractions and analysis by immunoblotting. Note that, with wild-type PrP, an insoluble, unglycosylated species of PrP accumulates over 8 h (U); this is not observed with Opn-PrP, which only shows soluble, mature PrP (M). Results identical to those observed with Opn-PrP were also seen for Prl-PrP (unpublished results; see also Figure 2B). (B) Effect of signal sequence on ‘propagation' of cytoplasmic PrP aggregates. Two parallel plates each of N2a cells transfected with either wild-type PrP or Opn-PrP were treated for 2 h with 5 μM MG132. One plate (left panel) was harvested immediately and analyzed for PrP detergent solubility and aggregation. The second set of dishes was rinsed to remove the MG132, and incubated an additional 21 h in normal media (right panel) before harvesting for analysis of PrP detergent solubility and aggregation. After a 2 h ‘pulse' of MG132, note that the immunoblots of PrP and Opn-PrP are largely indistinguishable: both show nearly quantitative solubility and little unglycosylated, insoluble PrP. After a 21 h chase, wild-type PrP was nearly all unglycosylated and insoluble, as has been proposed to occur by a ‘self-propagation' mechanism (Ma and Lindquist, 2002). Even under these conditions, however, relatively little Opn-PrP was found in the unglycosylated, insoluble form.

Figure 4

Figure 4

Visualization and modulation of PrP metabolism in single live cells. (A) Expression and colocalization of PrP-mYFP (red) and Opn-PrP-mCFP (green) in N2a cells. Similar results were obtained with PrP-mCFP and Opn-PrP-mYFP (unpublished results). (B) Comparison of localization patterns in N2a cells expressing PrP-mYFP or Opn-PrP-mYFP after proteasome inhibition (with MG132) for 8 h. Two exposures (upper and lower panels) are shown for each representative field to facilitate visualization of cells expressing high (arrows) and low (asterisks) levels of PrP. The PrP-mYFP and Opn-PrP-mYFP cells were visualized using identical imaging conditions to allow direct comparisons between them. (C) Quantitative analysis of subcellular localization relative to expression level for PrP-mYFP (closed circles) and Opn-PrP-mYFP (open circles) after proteasome inhibition as in panel B. (D) Visualization of PrP-mYFP (red) and Opn-PrP-mCFP (green) following proteasome inhibition.

Figure 5

Figure 5

Analysis of PrP and Opn-PrP localization. N2a cells were transfected with Opn-PrP-mYFP (panels A and B) or PrP-mYFP (panels C and D) and either left untreated (panels A and C) or treated with 5 μM MG132 for 4 h. The cells were fixed and processed for double immunofluorescence analysis using antibodies against an endosome marker (EEA1, in red) and a Golgi marker (beta-COP, in blue). Green indicates the position of YFP-tagged PrPs. Note that, in panels A–C, mYFP is localized primarily to the cell surface and perinuclear structures that overlap partially with Golgi and endosome structures. By contrast, panel D shows many cells (indicated with arrows) that have additional accumulations of PrP in other regions of the cytoplasm. Higher magnification images with separated color channels of representative cells from panels B and D are shown in Supplementary Figures S8 and S9, respectively.

Figure 6

Figure 6

Effect of expression level on cytosolic PrP generation. (A) N2a cells were transfected with either wild-type PrP or a larger amount of Prl-PrP. In both cases, equal amounts of a plasmid expressing GFP were cotransfected. A fixed amount of cell lysate from PrP-expressing cells (1 ×; lane 2) was compared to different amounts of cell lysate from the Prl-PrP-expressing cells (from 0.1 × to 1 ×; lanes 3–6). Cells transfected with just GFP (nt; lane 1) are also shown as a control. Immunoblots were probed with antibodies against PrP, GFP, and an endogenous protein, the beta subunit of Sec61. Note that GFP and Sec61 levels are comparable when equal amounts of nt, PrP, and Prl-PrP cell lysates are compared (lanes 1–3). By contrast, Prl-PrP is expressed at ∼5–10-fold higher levels than PrP, as indicated by the observation that equal intensities are observed when 0.1–0.2 × of Prl-PrP (lanes 5 and 6) is compared to 1 × of PrP (lane 2). (B) Duplicate dishes of cells from lanes 1 and 2 of panel A were either left untreated or treated for 4 h with 5 μM MG132 prior to harvesting and fractionation of detergent-soluble PrP (Sup.) from insoluble PrP (Pel.). One-tenth the amount of Prl-PrP lysate was analyzed on the gel to allow direct comparisons between the samples. Note that, with PrP, unglycosylated, insoluble PrP (U) is generated upon MG132 treatment. However, despite the ∼10 × overexpression of Prl-PrP, this form is not generated. (C) Effect of proteasome inhibition on endogenously expressed PrP. Untransfected N2a cells were treated for between 0 and 8 h with 5 μM MG132 before analysis of total proteins by immunoblotting with the 7D9 anti-PrP antibody (which detects mouse PrP). Note that an unglycosylated species of PrP (U) accumulates over 8 h (left panel). Samples from the 8 h treatment were also analyzed for PrP detergent solubility and aggregation (right panel). Note that a substantial fraction of the unglycosylated PrP is in the detergent insoluble pellet (P), while all of the mature, glycosylated PrP (M) is in the detergent-soluble supernatant (S).

Figure 7

Figure 7

Relationship between CtmPrP and cytosolic PrP generation. (A) Wild-type PrP, PrP(G123P) (a mutant that cannot generate CtmPrP; Hegde et al, 1998) and PrP(AV3) (a mutant that generates increased levels of CtmPrP) were transfected into N2a cells and analyzed for CtmPrP levels as described in Materials and methods. Cell lysates were either left untreated or digested with proteinase K under ‘mild' or ‘harsh' conditions. Protease-digested samples were then deglycosylated with PNGase F prior to SDS–PAGE and immunoblotting with the 3F4 anti-PrP antibody. The position of a diagnostic CtmPrP-specific 18 kDa proteolytic fragment observed after ‘mild' but not ‘harsh' digestion (Hegde et al, 1998, 1999) is indicated to the right of the gel. The positions of molecular weight markers are indicated to the left. Overexposed images of the blot (Figure S10) confirmed the complete absence of a CtmPrP-specific band for the PrP(G123P) mutant. (B) Wild-type PrP, Opn-PrP, or the indicated PrP mutants were transfected into N2a cells and analyzed for the generation of unglycosylated aggregates of PrP. In each case, the cell lysates were separated into detergent-soluble (Sup.) and insoluble (Pel.) fractions before analysis. Samples of untreated cells (top panel) or cells treated with 5 μM MG132 for 8 h (bottom panel) were analyzed in parallel. The positions of mature (M) and unglycosylated PrP (U) are indicated. PrP(A117V), to a lesser extent than PrP(AV3), generates increased levels of CtmPrP relative to wild-type PrP (see Hegde et al, 1998). Similar results were observed with other proteasome inhibitors (Supplementary Figure S7).

Figure 8

Figure 8

Protection from PrP-mediated cytotoxicity by modulation of protein translocation. (A) N2a cells cotransfected with GFP (a transfection marker, indicated by green cells) and either PrP, Opn-PrP, or an unrelated control plasmid (coding for preprolactin) were analyzed for apoptosis using Annexin V staining (red) before and after treatment with MG132. (B) Quantitative analysis of Annexin V staining before (white bars) and after (black bars) proteasome inhibition (4 h) of N2a cells expressing PrP, Opn-PrP, or an unrelated protein. Equal transfection efficiency and PrP expression levels were confirmed among samples within each experiment (unpublished results; see Materials and methods for details). (C) Quantitative analysis of total cell death (judged by vital dye exclusion) before and after proteasome inhibition for 12 or 20 h of N2a cells expressing PrP (black bars), Opn-PrP (gray bars), or a control protein (white bars).

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