Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on August 15, 2005
Human Molecular Genetics 2005 14(19):2787-2799; doi:10.1093/hmg/ddi312

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Endoplasmic reticulum stress compromises the ubiquitin–proteasome system

Victoria Menéndez-Benito¹, Lisette G.G.C. Verhoef¹, Maria G. Masucci² and Nico P. Dantuma^1,*

¹Department of Cell and Molecular Biology, The Medical Nobel Institute, Karolinska Institutet, PO Box 285, Von Eulers väg 3, S-171 77 Stockholm, Sweden and ²Microbiology and Tumor Biology Center, Karolinska Institutet, Nobels väg 16, S-171 77 Stockholm, Sweden

^* To whom correspondence should be addressed. Tel: +46 852487384; Fax: +46 8313529; Email: nico.dantuma{at}cmb.ki.se

Received May 2, 2005; Revised July 21, 2005; Accepted August 9, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
The presence of endoplasmic reticulum (ER) stress and impaired ubiquitin–proteasome system (UPS) activity has been independently implicated in the pathophysiology of conformational diseases. Here, we reveal a link between ER stress and the functionality of the UPS. Treatment of cells with different ER stressors delayed the degradation of an ER reporter substrate and caused a subtle but consistent accumulation of three independent nuclear/cytosolic UPS reporter substrates. A similar signature increase was observed upon induction of ER stress in transgenic mice expressing a reporter substrate. Cells undergoing ER stress failed to clear efficiently UBB⁺¹, an aberrant ubiquitin found in conformational diseases, which in turn caused general impairment of the UPS. We conclude that ER stress has a general inhibitory effect on the UPS. The compromised UPS during ER stress may explain the long-term gradual accumulation of misfolded proteins as well as the selective vulnerability of particular cell populations in conformational diseases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Conformational diseases are a heterogeneous group of disorders that includes polyglutamine diseases, Alzheimer's disease, Parkinson's disease, prion encephalopathy, retinitis pigmentosa and 1-antitrypsin deficiency (1). A common denominator in these diseases is the presence of proteins that undergo a conformational change from a soluble to a misfolded, aggregation-prone structure, which eventually leads to the formation of the characteristic nuclear or cytosolic protein inclusions (2). A major task of the ubiquitin–proteasome system (UPS) is to identify and degrade such aberrant proteins (3,4). Substrates of the UPS, which can be either misfolded proteins or normal short-lived proteins like p53, cyclins and IkB, are recognized by the presence of degradation signals. Degradation signals can be misfolded structures, small domains or motifs that recruit specific ubiquitin ligases resulting in ubiquitination and proteasomal degradation of the target protein (5). Functional tests show that cells are equipped with a largely excessive UPS activity (6,7), which enables the cells to promptly respond to acute proteotoxic stress, reflecting the crucial role of this system in protein quality control and cellular protein homeostasis (3,5).

Although ubiquitin/proteasome-dependent proteolysis is exclusively executed in the nucleus and cytosol, it has become increasingly clear that there is a tight connection between the protein quality control in the endoplasmic reticulum (ER) and the UPS (8). Given the substantial protein load in the ER, its delicate environment and the broad array of post-translational modifications taking place in this organelle, it is of uttermost importance for the cell to promptly eliminate abnormal and potentially hazardous ER proteins (9). In a process known as ER-associated degradation (ERAD), proteins that fail the ER quality control are transported back to the cytosol where they are rapidly destroyed by the UPS (8). Under conditions that negatively affect the environment in the ER, the pool of aberrant ER proteins rapidly increases resulting in the induction of the unfolded protein response (UPR). The UPR is a cellular program which is activated by a complex signaling cascade and involves at least three different mechanisms to re-establish homeostasis: attenuation of protein synthesis, optimization of chaperone-assisted protein folding and augmentation of protein degradation (10). Stimulation of translocation of aberrant ER proteins to the cytosol followed by UPS-dependent degradation is an integrated part of the UPR and is an important means to relieve the burden of abnormal proteins in the ER (9,10). Cells that fail to restore a proper ER homeostasis will eventually be eliminated by ER stress-induced, programmed cell death (11,12). Notably, cells that heavily depend on the ER, such as professional secretory cells, display a chronic UPR to facilitate production of large amounts of ER client proteins without overloading this organelle (13,14). Recent studies suggest that ER stress and induction of the UPR are common phenomena in conformational diseases. It has been reported that ER stress might participate in the pathogenesis of conformational diseases such as Alzheimer's disease (12), Parkinson's disease (15), Huntington's disease (16), amyotrophic lateral sclerosis (17), spongiform encephalopathies (18) and 1-antitrypsin deficiency (19).

An important unresolved question is why the UPS fails to clear misfolded, aggregation-prone proteins in conformational diseases and allows these proteins to form the characteristic cytoplasmic and nuclear inclusions (2). Several studies have identified disease-associated proteins, such as mutant huntingtin (6,20) and the aberrant ubiquitin UBB⁺¹ (21,22), that negatively affect the efficacy of the UPS supporting a role for UPS dysfunction in some conformational diseases. Impairment of the UPS as a consequence of these proteins is usually rapidly followed by cell death (6,22,23). It seems, therefore, unlikely that impairment of the UPS by these toxic proteins account for the long-term gradual accumulation of misfolded proteins observed in these diseases. Moreover, this model does not explain why particular cells perish, whereas others cope rather well with high levels of the mutant protein.

In the present study, we have investigated the effect of ER stress on the functionality of the UPS. We found that ER stress has an overall effect on the functional status of the UPS as it causes accumulation of four different UPS substrates in the ER, cytosol and nucleus. Our data identify an unexpected link between ER stress and UPS functionality and suggest that the load of ER client proteins and the status of the ER environment may be important parameters for the gradual progressive accumulation of misfolded proteins in conformational diseases.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Generation and characterization of a panel of UPS reporter cell lines
It has been previously shown that functional analysis of the UPS can be accomplished by following the steady-state levels of reporter substrates (6,7). These reporter substrates are typically based on an intrinsic fluorescent protein provided with a constitutively active degradation signal that targets the proteins for ubiquitination and proteasomal degradation (24). Cells expressing reporter substrates respond to functional impairment of the UPS by accumulation of the readily detectable fluorescent reporter substrate. We generated a collection of four yellow fluorescent protein (YFP)-based reporters representing different classes of UPS substrates (Fig. 1A). These include the ERAD substrate CD3-YFP and three previously described cytosolic/nuclear reporters: the N-end rule substrate ubiquitin-arginine-YFP (Ub-R-YFP), the ubiquitin fusion degradation substrate Ub^G76V-YFP and the YFP-CL1 substrate (6,7). The T-cell receptor subunit CD3 is a well-established ERAD substrate (25). Notably, while Ub-R-YFP and Ub^G76V-YFP are properly folded proteins, YFP-CL1 is targeted for degradation through the presence of the C-terminal bulky hydrophobic motif CL1, which resembles a misfolded domain (26).

View larger version (47K): [in this window] [in a new window]   Figure 1. Generation and characterization of a panel of UPS reporter cell lines. (A) Schematic representation of the YFP-based reporter substrates. (B) Pulse-chase analysis of the turnover of the reporter substrates. MelJuSo reporter cell lines were labeled with [35S]methionine for 30 min and chased in complete medium. The reporter proteins were quantified with a phospho-imager. The intensity at time 0 was normalized to 100%. (C) Flow cytometric analysis of MelJuSo parental (blue filled) and MelJuSo reporter cell lines that were left untreated (pink line) or incubated with 200 nM of the proteasome inhibitor epoxomicin for 14 h (green line). One representative experiment out of five is shown. (D) Flow cytometric analysis of MelJuSo reporter cell lines after treatment with the indicated concentrations of epoxomicin. Triplicate measurements are shown from a representative experiment. Values are the mean of the fluorescent intensity±standard deviations. (E–H) Representative micrographs of MelJuSo reporter cell lines that were left untreated (left panels) or incubated for 14 h with 200 nM epoxomicin (right panels): (E) CD3-YFP, (F) Ub-R-YFP, (G) UbG76V-YFP and (H) YFP-CL1. YFP fluorescence (upper panels) and Hoechst staining (lower panels) are shown. Scale bar represents 10 µm.

 
Stable reporter cell lines were generated by transfection of the human melanoma cell line MelJuSo with plasmids encoding the reporter substrates. Pulse-chase analysis confirmed that the fusion proteins are short-lived with approximate half-lives of 10 min for Ub-R-YFP and Ub^G76V-YFP, 25 min for YFP-CL1 and 2 h for CD3-YFP (Fig. 1B, see also Fig. 3B and C). Treatment of the four reporter cell lines with the proteasome inhibitors epoxomicin (Fig. 1C and D), MG132 and MG262 (data not shown) resulted in a dose-dependent increase in the fluorescent intensities of the cells. Microscopic analysis of the untreated CD3-YFP cell line revealed a weak YFP fluorescence associated with the ER, which was clearly enhanced upon treatment with proteasome inhibitors (Fig. 1E). The YFP fluorescence of untreated Ub-R-YFP (Fig. 1F) and Ub^G76V-YFP (Fig. 1G) cell lines was close to the detection threshold but strongly increased in both cytosol and nucleus after inhibitor treatment. In inhibitor-treated YFP-CL1 cells, the accumulated reporter was sequestered in juxtanuclear structures (Fig. 1H) that co-stained with the centrosome marker -tubulin (Supplementary Material, Fig. S1). These characteristic structures, known as aggresomes (27), are often observed with hydrophobic aggregation-prone UPS substrates, which is in line with the misfolded nature of the CL1 degradation signal. Together, these data confirm that the reporter cell lines provide a versatile system to monitor independently three major pools of UPS substrates: ERAD substrates (CD3-YFP), soluble cytosolic/nuclear substrates (Ub-R-YFP and Ub^G76V-YFP) and misfolded cytosolic/nuclear substrates (YFP-CL1).

View larger version (30K): [in this window] [in a new window]   Figure 3. ER stress-induced accumulation of UPS reporters is caused by a delay in degradation. (A) [35S]methionine labeling of MelJuSo reporter cell lines showing newly synthesized YFP reporter in control and thapsigargin-treated cells. MelJuSo reporter cell lines were left untreated or treated for 17 h with 600 nM thapsigargin and were then labeled with [35S]methionine for 3 min. (B) Pulse-chase analysis of turnover of CD3-YFP reporter substrate. CD3-YFP MelJuSo reporter cell lines were left untreated or treated for 17 h with 600 nM thapsigargin and were then labeled with [35S]methionine for 30 min and chased in complete medium for the indicated times. (C) Quantification of the pulse-chase analysis as shown in (B). The intensity at time 0 was normalized to 100%. (D) Pulse-chase analysis of turnover of Ub-R-YFP reporter substrate in Ub-R-YFP MelJuSo cell line was performed as indicated in (B). (E) Quantification of the pulse-chase analysis as shown in (D). (F) Flow cytometric analysis showing the degradation of accumulated Ub-R-YFP. Ub-R-YFP MelJuSo cells were left untreated or treated with 600 nM thapsigargin for 17 h and were then incubated for 1 h with 1 µM MG132 in order to accumulate Ub-R-YFP. After four washes with medium, the cells were cultured in the absence of MG132 to allow diffusion of the proteasome inhibitor, and fluorescent intensity was determined by flow cytometry at the indicated times. Note that in the control cells, cells rapidly convert from the high GFP fluorescence (right peak) to the low GFP fluorescence (left peak), whereas in ER stress cells, there is a gradual reduction in fluorescence. The mean fluorescent intensity for untreated and thapsigargin-treated cells before addition of MG132 is marked by a dotted line.

 
ER stress causes accumulation of four different UPS substrates
To investigate the effect of ER stress on the UPS, we treated the reporter cell lines with the ER stressors thapsigargin, which depletes the ER-calcium storage by specific inhibition of the ER-calcium ATPase, and tunicamycin, which inhibits N-linked glycosylation. The presence of alternative spliced XBP1 transcripts (Fig. 2A) and induction of the transcription factor CHOP (Fig. 2B), two markers for induction of the UPR, confirmed the robustness of ER stress upon treatment with thapsigargin and tunicamycin. Both treatments induced an increase in the CD3-YFP levels that exceeded the effect obtained with proteasome inhibitors, suggesting that ER stress severely impairs the degradation of ERAD substrates (Fig. 2C). Interestingly, treatment with these ER stressors gave a subtle but highly significant increase in the levels of both the soluble and misfolded cytosolic/nuclear substrates. Notably, the increase was very similar for each of the reporters and corresponded with 5–15% of the effect obtained by full obstruction of the UPS with proteasome inhibitors (Fig. 2D). The accumulations corresponded with an approximate 7-fold increase for CD3-YFP, 3-fold increase for Ub-R-YFP and a 2-fold increase for Ub^G76V-YFP and YFP-CL1. A similar effect was observed in SH-SY5Y human neuroblastoma cells expressing Ub^G76V-green fluorescent protein (GFP) or Ub-R-GFP underscoring the general nature of this response (data not shown). A third ER stressor, the reducing agent dithiothreitol (DTT), was found to be too toxic for MelJuSo cells to perform any type of functional analysis on the UPS. However, a human cervix carcinoma cell line (HeLa) stably expressing the Ub^G76V-GFP could be treated with DTT and showed a reporter accumulation that was very similar to the levels obtained with thapsigargin and tunicamycin (Fig. 2E). Time course experiments revealed a gradual accumulation of Ub-R-YFP upon induction of ER stress that was detectable 6 h after thapsigargin administration (Fig. 2F). A maximal effect on Ub-R-YFP levels was already obtained at low levels of tunicamycin (data not shown) and thapsigargin (Fig. 2G) and did not further increase at higher concentrations of these ER stressors. Even these low doses of thapsigargin induced ER stress as evidenced by the presence of alternatively spliced XBP1 transcripts (Supplementary Material, Fig. S2).

View larger version (35K): [in this window] [in a new window]   Figure 2. ER stress causes accumulation of four different UPS substrates. (A) RT–PCR analysis using primers for XBP1 in MelJuSo cells that were left untreated and treated for 17 h with thapsigargin (600 nM) or tunicamycin (5 µg/ml). Upper band corresponds to the unspliced XBP1 variant (uXBP1) and lower band corresponds to the unconventional spliced variant (sXBP1) produced during activation of the UPR. RT–PCR reactions with reverse transcriptase and controls without reverse transcriptase are indicated. (B) Western blot analysis probed with an anti-CHOP antibody showed CHOP activation in tunicamycin and thapsigargin-treated MelJuSo cells. (C) Flow cytometric analysis of MelJuSo reporter cell lines that were left untreated (gray filled), treated with ER stressors for 17 h (continuous line) and incubated with 200 nM epoxomicin for 14 h (dotted line). The ER stressor in the left panel is thapsigargin (600 nM) and in the right panel is tunicamycin (5 µg/ml). One representative experiment out of five is shown. (D) Quantification of flow cytometric analysis as shown in (C). Values are the mean fluorescence±standard deviation of triplicate measurements from a representative experiment. ***Student's t-test, P<0.005. (E) Quantification of flow cytometric analysis of UbG76V-GFP HeLa cell lines that were left untreated, treated with the ER stressors thapsigargin (600 nM), tunicamycin (5 µg/ml) and DTT (5 mM) for 25 h and with epoxomicin (200 nM) for 14 h. Values are the mean fluorescence±standard deviation of triplicate measurements from a representative experiment. ***Student's t-test, P<0.005. (F) Quantification of flow cytometric analysis of Ub-R-YFP MelJuSo cell line treated with thapsigargin (600 nM) for the indicated time points. One representative experiment of four is shown. (G) Quantification of flow cytometric analysis of Ub-R-YFP MelJuSo cell line after treatment with the indicated concentrations of thapsigargin for 17 h. Values are the mean of the fluorescent intensity±standard deviations of triplicate measurements from a representative experiment. (H–L) Representative micrographs from MelJuSo reporter cell lines that were left untreated (left panel) and incubated with 600 nM thapsigargin for 17 h (right panel): (H) CD3-YFP, (I) Ub-R-YFP, (J) UbG76V-YFP and (K) YFP-CL1. YFP fluorescence (upper panels) and Hoechst staining (lower panels) are shown. (L) Micrograph from YFP-CL1 MelJuSo cells treated with 600 nM thapsigargin for 17 h. YFP fluorescence (upper panel) and Hoechst staining (lower panel) are shown. Aggregated YFP-CL1 reporter is indicated with an arrow. Scale bars represent 10 µm.

 
The increase in fluorescent intensities of each of the reporters could be readily detected by confocal laser scanning microscopy. The distribution pattern of the accumulated CD3-YFP (Fig. 2H), Ub-R-YFP (Fig. 2I), Ub^G76V-YFP (Fig. 2J) and YFP-CL1 (Fig. 2K) was comparable to those found after proteasome inhibitor treatment. Although most of YFP-CL1 cells displayed a diffuse distribution after treatment with ER stressors, distinct inclusions were detectable in a small percentage of YFP-CL1 cells (<1%) (Fig. 2L). Thus, despite triggering an efficient UPR, ER stress impairs UPS-dependent degradation of a constitutive ERAD substrate and, in addition, causes accumulation of soluble and misfolded substrates in the cytosol and nucleus.

ER stress-induced accumulation of UPS reporters is caused by a delay in degradation
UPS reporter substrates are very sensitive to small fluctuations in transcription or translation because of their short half-lives (28,29). Therefore, we analyzed whether the increase in reporter levels during ER stress was because of an increase in reporter synthesis or a delay in reporter degradation. Because there is a linear correlation between the rate of synthesis and the steady-state levels of proteins, the increase in synthesis should match the rise in reporter levels (28). To address this issue, we metabolically labeled reporter-expressing MelJuSo cells and compared the synthesis of the reporters in control cells and cells that had been treated for 17 h with thapsigargin. We found that thapsigargin treatment caused a slight decrease in reporter synthesis (Fig. 3A), probably as an effect of UPR-induced downregulation of translation (10). This suggests that changes in protein synthesis are not responsible for the elevated reporter levels.

Next, we analyzed in more detail the effect of ER stress on the turnover of the reporter substrate. Pulse-chase analysis showed that the half-life of the CD3-YFP was increased from 1.5–4 h, which is in line with the dramatic accumulation of this reporter in cells undergoing ER stress (Fig. 3B and C). In contrast, the half-life of the Ub-R-YFP was very similar in control and thapsigargin-treated cells showing that ER stress does not have a dramatic effect on the degradation of this reporter (Fig. 3D and E). However, it should be emphasized that a very small increase in the half-life of Ub-R-YFP will be sufficient to obtain the observed 3-fold increase over a 17 h period.

We have shown before that small changes in the efficiency of the UPS can be revealed by monitoring how rapidly cells can clear substrates that have been accumulated in the cells by temporally stalling proteasomal degradation (7). In this experimental setup, substrates of the UPS are first accumulated during a short incubation with the reversible proteasome inhibitor MG132, and the clearance of the accumulated substrates is then monitored after releasing the UPS by removal of the inhibitor. We found that the clearance of the Ub-R-YFP reporter was delayed in cells undergoing ER stress (Fig. 3F). Notably, in control cells, we found that cells rapidly converted from a high fluorescent to low fluorescent intensity, whereas reduction of the fluorescent intensity in thapsigargin-treated cells proceeded very gradually. Thus, degradation of ERAD and soluble substrates is delayed in cells undergoing ER stress.

Normal proteasome levels and proteasome activity during ER stress
We investigated whether reduction of proteasome activity could account for the delayed degradation of UPS substrates during ER stress. By western blot analysis, we compared the levels of two subunits of the proteasome core complex in control and thapsigargin-treated cells. No differences between the levels of the constitutive 3-subunit or the inducible ß2i-subunit were found (Fig. 4A). Even though the proteasome levels were not affected, reduced proteolytic activity of the proteasome could still be responsible for reduced protein degradation. Therefore, we measured the levels of the chymotrypsin-like and trypsin-like activities of the proteasome using fluorogenic peptides. This experiment did not reveal any significant differences in these proteasomal activities between untreated cells and cells treated with tunicamycin or thapsigargin (Fig. 4B). The lack of changes in these proteolytic activities of the proteasome during ER stress suggests that the proteasome core complex is unaffected and therefore unlikely to be responsible for the delayed degradation.

View larger version (30K): [in this window] [in a new window]   Figure 4. Normal proteasome levels and proteasome activity during ER stress. (A) Western blot analysis of lysates from MelJuSo cells that were left untreated and treated for 17 h with the ER stressors thapsigargin (600 nM) and tunicamycin (5 µg/ml) and for 14 h with the proteasome inihibitor epoxomicin (200 nM). The samples were probed with antibodies directed against the 3 and ß2i-proteasome subunits. ß-actin is shown as loading control. (B) Proteasomal activities in MelJuSo cells that were left untreated or treated for 17 h with the ER stressors thapsigargin (600 nM) and tunicamycin (5 µg/ml) or treated for 14 h with the proteasome inhibitor MG-262 (1 µM). The chymotrypsin-like and trypsin-like activities were determined by the hydrolysis of the fluorogenic substrates suc-LLVY-AMC and Boc-LRR-AMC, respectively. The activities of untreated cells were normalized as 100%. Values are the mean of the fluorescent intensity±standard deviations of triplicate measurements from a representative experiment. ***Student's t-test, P<0.001.

 
Reporter accumulation is not caused by induction of apoptosis
Sun et al. (30) have recently reported that induction of apoptosis by the DNA topoisomerase II inhibitor etoposide causes caspase-3-dependent cleavage of subunits in the 19S regulatory complex of the proteasome, which in turn inhibits the UPS. Because persistent ER stress eventually results in induction of apoptosis (12), we considered the possibility that the effect observed on protein degradation after treatment with ER stressors was due to ER stress-induced apoptosis. Caspase-3 is an effector caspase that is activated in different forms of programmed cell death including ER stress-induced cell death (31). In cells that had been treated for 17 h with thapsigargin, there was only a small increase in caspase-3 activity arguing against massive apoptosis at this time point (Fig. 5A). Etoposide treatment gave robust activation of caspase-3 but only moderately affected degradation of the reporter (Fig. 5B and C). Thus, the accumulation of reporter in the presence of minimal levels of active caspase-3 during ER stress and the marginal effect on reporter degradation in etoposide-treated cells suggest that apoptosis is not responsible for the UPS dysfunction observed during ER stress.

View larger version (22K): [in this window] [in a new window]   Figure 5. Reporter accumulation is not caused by induction of apoptosis. (A) Caspase-3 activity in Ub-R-YFP MelJuSo cells that were left untreated and treated for 17 h with the DNA topoisomerase II inhibitor etoposide (25 nM), with the ER stressor thapsigargin (600 nM) and with the proteasome inhibitor epoxomicin (200 nM). The caspase-3 activity was determined by the hydrolysis of the fluorogenic caspase substrate Ac-DEVD-AFC. Values are the mean of the fluorescent intensity±standard deviations of triplicate measurements from a representative experiment. (B) Flow cytometric analysis of Ub-R-YFP levels in the same cells as in (A). (C) Quantification of flow cytometric analysis of Ub-R-YFP MelJuSo reporter cells as shown in (B). Values are the mean of the fluorescent intensity±standard deviations of triplicate measurements from a representative experiment. *Student's t-test, P<0.05; **Student's t-test, P<0.01; ***Student's t-test, P<0.001.

 
ER stress compromises clearance of the aberrant ubiquitin UBB⁺¹
We next asked how a problematic and disease-associated substrate is handled by the compromised UPS in cells undergoing ER stress. The aberrant ubiquitin UBB⁺¹ is encoded by an abnormal transcript of the ubiquitin B gene, which is generated at a low frequency by an erroneous transcriptional event known as molecular misreading (32). Accumulation of the UBB⁺¹ protein has been detected in a number of neurological and non-neurological conformational disorders (33–35). We have shown that UBB⁺¹ is a substrate of the UPS but unlike normal substrates, it can also cause general impairment of the UPS by an unknown mechanism (22). Ub^G76V-GFP HeLa cells were transiently transfected with myc-tagged UBB⁺¹ (^mycUBB⁺¹) and analyzed in the absence or presence of ER stress. Biochemical analysis showed that treatment with thapsigargin provoked a considerable accumulation of ^mycUBB⁺¹ including a smear of high-molecular weight adducts (Fig. 6A), which has previously been identified as polyubiquitinated UBB⁺¹ (21,23). Metabolic labeling revealed that synthesis of UBB⁺¹, similar as the reporters, is slightly reduced in ER stress cells (data not shown). This suggests that ER stress interferes with the clearance of the aberrant UBB⁺¹. In the absence of ER stress, a small fraction of the ^mycUBB⁺¹-expressing cells displayed profound impairment of the UPS as evidenced by the appearance of a subpopulation of cells with up to 100-fold increase in GFP fluorescence (Fig. 6B). Induction of ER stress in cells expressing ^mycUBB⁺¹ resulted in a 5-fold increase in the percentage of cells with a severely impaired UPS (Fig. 6B and C). We conclude that ER stress can cause accumulation of the toxic proteasome substrate UBB⁺¹, resulting in a general blockade of the UPS.

View larger version (24K): [in this window] [in a new window]   Figure 6. ER stress induces accumulation of UBB+1 and impairs the UPS. (A) Western blot analysis of lysates from mycUBB+1 transfected UbG76V-GFP HeLa cells incubated in the absence or presence of 600 nM thapsigargin for 17 h, and untransfected UbG76V-GFP HeLa cells incubated with 600 nM thapsigargin for 17 h. The samples were probed with an anti-c-Myc antibody. Molecular weight marker, the band corresponding to mycUBB+1, and high molecular weight mycUBB+1 are indicated. (B) Flow cytometric analysis of mycUBB+1 transfected UbG76V-GFP HeLa cells that were left untreated (control) or incubated with thapsigargin (600 nM) for 17 h. The percentages of mycUBB+1 expressing cells that are GFP-positive are indicated. (C) Quantification of three independent experiments as shown in (B). The percentage of mycUBB+1 positive cells that accumulate UbG76V-GFP was determined. GFPhigh cells are defined as those cells in the upper right panel as shown in (B). For the untransfected control, the percentage of cells accumulating UbG76V-GFP on the total cell population was determined. *Student's t-test, P<0.05.

 
ER stress causes accumulation of a UPS reporter substrate in vivo
The results obtained in cell lines prompted us to investigate the effect of ER stress on the UPS in vivo. We took advantage of a recently developed transgenic mouse model for monitoring the UPS, which is based on the ubiquitous expression of the Ub^G76V-GFP reporter (36).

Treatment of primary fibroblasts obtained from Ub^G76V-GFP/1 reporter mice with thapsigargin caused a clear increase of Ub^G76V-GFP levels very similar to that observed in the reporter MelJuSo cell line (Fig. 7A). ER stress can be induced in vivo by injecting mice intraperitoneally with sublethal doses of tunicamycin, which is known to induce ER stress transiently in the tubular epithelium of the kidney (37). Histological analysis of kidneys from Ub^G76V-GFP/1 mice treated with tunicamycin revealed accumulated levels of the reporter substrate throughout the kidney (Fig. 7B, upper panel). Although in vivo administration of proteasome inhibitors induces accumulation of Ub^G76V-GFP levels in the kidney at levels that are readily detectable by native GFP fluorescence (36), visualization of GFP accumulation in the kidneys of tunicamycin-treated mice required enhancement of the signal by immunostaining. This is consistent with the results obtained with the reporter cell lines that also showed a more subtle increase in reporter levels during ER stress when compared with proteasome inhibitor treatment. No staining was observed in the kidneys of vehicle-treated Ub^G76V-GFP/1 mice or tunicamycin-treated non-transgenic littermates (Fig. 7B, upper panel). Immunostaining for the ER stress marker CHOP confirmed induction of UPR in kidneys of tunicamycin-treated mice (Fig. 7B, lower panel).

View larger version (57K): [in this window] [in a new window]   Figure 7. ER stress induces reporter accumulation in vivo. (A) Flow cytometric analysis from UbG76V-GFP/1 primary mouse fibroblasts left untreated (blue filled), treated with the ER stressor thapsigargin (600 nM) for 24 h (pink line) or with the proteasome inhibitor epoxomicin (200 nM) for 17 h (green line). One representative experiment out of three is shown. (B) Micrographs of kidney cryosections from a UbG76V-GFP/1 mouse and a non-transgenic littermate, treated with vehicle only (control) or with tunicamycin (1 µg/g body weight). GFP (upper panel) and CHOP (lower panel) immunostainings are shown. (C and D) GFP (C) and CHOP (D) immunostainings of kidney cryosections from a tunicamycin-treated UbG76V-GFP/1 mouse. GFP and CHOP immunostainings (upper panel), Hoechst staining (middle panel) and merged (lower panel) micrographs are shown. Tubular epithelium (T) and glomeruli (G) are indicated with arrows. (E) Western blot analysis of kidney homogenates from UbG76V-GFP/1 mice injected with vehicle or with tunicamycin and probed with an anti-GFP antibody. ß-actin is shown as loading control. (F) RT–PCR for XBP1 from RNA isolated from UbG76V-GFP/1 mice injected with vehicle or with tunicamycin. Upper band corresponds to the unspliced XBP1 variant (uXBP1) and lower band corresponds to the unconventional spliced variant (sXBP1) produced during activation of the UPR. RT–PCR reactions with reverse transcriptase and controls without reverse transcriptase are indicated. (G) Fluorescence micrographs from liver cryosections from UbG76V-GFP/1 mice injected with vehicle or with tunicamycin. The cryosections were immunostained with an anti-GFP antibody. Scale bars in all panels represent 20 µm.

 
Induction of UPR by in vivo administration of tunicamycin is restricted to the tubular epithelium of the kidney (12,37). Detailed analysis of the kidneys of tunicamycin-treated reporter mice showed that accumulation of Ub^G76V-GFP reporter levels was also confined to the tubular epithelium, whereas the adjacent glomeruli were negative for the reporter substrate (Fig. 7C) and the UPR marker CHOP (Fig. 7D). Biochemical analysis of kidney samples confirmed the increase in Ub^G76V-GFP levels (Fig. 7E) as well as the induction of the UPR (Fig. 7F). In addition, accumulation of the reporter was also found in sporadic cells in the livers of tunicamycin-treated mice (Fig. 7G). No increase in reporter levels was found in the small intestine and pancreas. Thus, induction of ER stress in vivo is accompanied by an increase in the levels of a soluble nuclear/cytoplasmic reporter similar to what has been found in the reporter cell lines.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
An important unresolved question in the pathophysiology of conformational diseases is why the UPS fails to destroy the aggregation-prone proteins and allows the accumulation of these toxic proteins over time (3). Several reports have identified factors that can cause a general dysfunction of the UPS (6,20,38,39) and it appears that some of the disease-associated proteins themselves are problematic proteasome substrates that resist the UPS (22,40,41). Although a general impairment of the UPS could explain the uncontrolled accumulation of misfolded proteins, this is difficult to reconcile with the typical slow progression of these diseases as a general impairment of the UPS is usually promptly followed by the induction of apoptosis (7). Therefore, even though global UPS impairment may play a role in the final stage of the cellular pathology, it is unlikely to contribute to the early stages of the disease. In the first in vivo study addressing this issue, no substantial dysfunction of the UPS was detected during disease progression in a mouse model for spinocerebellar ataxia-7 (28).

In the present study, we show that ER stress has a subtle effect on the functionality of the UPS, which results in a modest accumulation of cytosolic/nuclear substrates. This accumulation is only 10% of the effect accomplished by a full obstruction of the pathway. Importantly, the UPS remains rather functional in these cells as the levels of the short-lived reporter proteins are kept within acceptable limits during the time frame of these experiments. This does not mean that the subtle changes in the UPS are without consequences. We found that for a small fraction of the cells expressing the YFP-CL1, reporter treatment with ER stressors was sufficient to provoke the formation of inclusions suggesting that the amounts of misfolded proteins in the cytosol exceed the capacity of the cell's refolding and degradation systems. Moreover, cells exposed to ER stressors failed to efficiently clear the aberrant ubiquitin UBB⁺¹ found in conformational diseases.

Several conformational diseases have been linked to the occurrence of ER stress (12,16,17,19). Although a lot of attention has been paid to the role of ER stress in diseases, chronic ER stress is not restricted to pathologic conditions. For example, plasma cells, after an initial UPR-independent adaptation process (42), use ER stress-induced UPR to facilitate increased production of immunoglobulins (14). UPR-dependent adaptation of the capacity of the ER in order to accommodate an increased load of ER client proteins seems to be a general mechanism in professional secretory cells (13). Interestingly, this appears to be the case also for some neurons because transport of glutamate receptors in nematodes heavily depend on constitutive induction of UPR (43). Our data suggest that the pressure to keep the ER operative while facing large amounts of aberrant ER proteins may at the same time corrupt the UPS and cause a chronic imbalance between generation and clearance of misfolded proteins in the nucleus and cytosol. Thus, the vulnerability of cells in conformational disorders may relate to the operative status of their ER and the levels of ER client proteins.

We propose that the functional status of the UPS may progress through three distinct phases in conformational diseases (Fig. 8). Initially, the UPS is fully functional and can adequately cope with all cellular substrates. During the second phase, ER stress or other stress conditions may compromise the UPS causing a slow progressive buildup of a particular subset of demanding proteins, which are stored in nuclear inclusions. This phase may explain the slow progression of the diseases as it may start long before the onset of the disease and proceed over decades. The compromised UPS, however, does not necessarily have to reflect a pathologic condition but may be a part of the normal cell physiology. During the third and final phase, the accumulated substrates may cause a general blockade of the UPS rapidly followed by cell death. Whether cells will perish during the second phase or eventually face full UPS impairment may depend, among many other factors, on the toxicity of the proteins that prevail in the presence of the compromised UPS and the cell's capacity to handle and neutralize these proteins.

View larger version (16K): [in this window] [in a new window]   Figure 8. Model for UPS dysfunction in conformational diseases. Schematic representation of the possible roles of the UPS in conformational diseases. According to this model, the status of the UPS can be divided into three phases. Phase 1: The UPS functions properly and efficiently degrades all UPS substrates. Phase 2: Conditions such as ER stress compromises the UPS. Even though the UPS is functional in this phase, a particular subset of demanding substrates may accumulate gradually over time. During this phase, cellular dysfunction and cell death may be caused by moderate accumulation of toxic proteins. Phase 3: If cells survive phase 2, the gradual accumulation of substrates may cause an out-of-control positive feedback loop, where accumulated substrates further impair the UPS, resulting in a general impairment of the UPS, which will be rapidly followed by cell death.

 

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Constructs
For generation of the Ub-R-YFP and Ub^G76V-YFP reporters, the GFP open-reading frames in the previously described Ub-R-GFP and Ub^G76V-GFP plasmids (7) were replaced with YFP open-reading frames using the PinAI and NotI restriction sites. The YFP-CL1 reporter was generated by inserting an oligodimer encoding the 16 amino acid CL1 degradation signal (ACKNWFSSLSHFVIHL) (6,44) in enhanced GFP-C1 (BD Biosciences Clontech) using the EcoRI and BamH1 restriction sites, then the enhanced GFP open-reading frame was replaced with YFP using the Nhe1 and SspB1 restriction sites. CD3 was PCR amplified from a plasmid (gift from Dr Kikkert, Leiden University Medical Center, Leiden, The Netherlands) (45), introducing flanking BglII and EcoRI sites, and cloned in Ub-R-YFP digested with BamHI and EcoRI. The ^mycUBB⁺¹ open-reading frame was expressed from a cytomegalovirus (CMV) promoter in the mammalian expression vector pCMV-myc (BD Biosciences Clontech).

Transfections and tissue culture
The human melanoma cell line MelJuSo and the cervical epithelial carcinoma cell line HeLa were cultured in Iscove's modified Dulbecco's medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (Sigma-Aldrich), 10 U/ml penicillin and 10 µg/ml streptomycin (Sigma-Aldrich). Primary fibroblasts from Ub^G76V-GFP/1 mice were isolated and cultivated, as described previously (36). MelJuSo and HeLa cells were transfected with Lipofectamine (Invitrogen) according to the recommendation of the manufacturer. Stably transfected cell lines were selected in the presence of 0.5 mg/ml G418 (Gibco) and screened for YFP fluorescence upon administration of the proteasome inhibitor MG132 (Affiniti). Where indicated, the cells were treated with the proteasome inhibitors epoxomicin, MG132 and MG262 (Affiniti), the ER stressors tunicamycin, thapsigargin and DTT (Sigma-Aldrich) and the DNA topoisomerase II inhibitor etoposide (Sigma-Aldrich).

Metabolic labeling
MelJuSo cells were incubated for 1 h in methionine/cysteine free medium and then metabolically labeled with 40 µCi [³⁵S]methionine (Redivue Pro-Mix ³⁵S, Amersham Biosciences) at 37°C. To analyze newly synthesized proteins, the metabolic labeling was performed for 3 min and then the cells were harvested in lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris–HCl (pH 8), 20 mM N-ethylmaleimide (Sigma-Aldrich) and complete protease inhibitor cocktail (Roche). To determine degradation rates, the metabolic labeling was performed for 30 min, and after the labeling period, the cells were washed and chased with complete medium supplemented with 10 mM methionine and 1 mM cysteine. Cells were harvested at the indicated time points in lysis buffer. Lysates were centrifuged at 14 000g for 10 min at 4°C and the supernatant was used for immunoprecipitation. YFP-based reporters were immunoprecipitated for 1 h at 4°C with 0.5 µl rabbit polyclonal anti-GFP antibody (Molecular Probes), followed by incubation with protein-A–Sepharose beads (Amersham Biosciences) for 2 h at 4°C. Analysis and quantification of the pulse-chase experiments were performed with a Phospho-imager and Imagequant software (Molecular Dynamics).

Fluorescence microscopy and flow cytometry
MelJuSo cell lines were grown on cover slips for fluorescence microscopy. After fixation with 4% paraformaldehyde (Sigma) and staining with Hoechst 33258 (Molecular Probes), the cells were examined with a Zeiss LSM 510 laser scanning confocal microscope and the images were processed with Adobe Photoshop software. Flow cytometry was performed with a FACSort flow cytometer (Beckton and Dickinson) and analyzed with Cellquest software. For analysis of ^mycUBB⁺¹, cells were harvested 48 h after transfection, fixed with cytofix/cytoperm (BD Biosciences), washed with perm/wash (BD Biosciences) and stained with a mouse monoclonal anti-c-Myc antibody (9E10, Santa Cruz Biotechnology). After subsequent washing steps, the cells were incubated with APC labeled goat anti-mouse immunoglobulins (BD Biosciences Pharmingen).

Western blot analysis
Cell lysates were resolved on 10% SDS–PAGE and transferred to Protan BA 85 nitrocellulose filters (Schleicher and Schuell). The filters were blocked at room temperature for 1 h in phosphate-buffered saline (PBS) supplemented with 5% skim milk and 0.1% Tween-20 and incubated overnight at 4°C with primary antibody. We used the following primary antibodies: a rabbit polyclonal anti-GFP antibody (dilution 1:1000), a rabbit polyclonal anti-CHOP/GADD153 antibody (F-168, dilution 1:200; Santa Cruz Biotechnology), a mouse monoclonal anti-c-Myc antibody (dilution 1:1000), a mouse monoclonal anti-3 antibody (dilution 1:1000; Affinity) and a rabbit polyclonal anti-ß2i antibody (dilution 1:1000; Affinity). After subsequent washing steps and incubation with peroxidase-conjugated goat anti-rabbit or anti-mouse serum, the blots were developed by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).

Fluorometric assay for protease activity
Aliquots of 2x10⁶ cells were lysed with glass beads (Sigma) at 4°C in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 5 mM MgCl₂, 1 mM DTT and 250 mM sucrose, and the protein concentration of cleared supernatants was measured by BCA protein assay reagent (Pierce). The chymotrypsin-like and trypsin-like activity of the proteasome was determined by hydrolysis of fluorogenic substrates suc-LLVY-AMC (Affiniti) and Boc-LRR-AMC (Affiniti), as described previously (6). Caspase-3 activity was measured with the fluorogenic substrate Ac-DEVD-AFC (Enzyme System Products, Livermore, CA, USA). Ten microgram of protein from the cleared lysates was incubated with 100 µM Ac-DEVD-AFC in 200 µl buffer containing 100 mM HEPES buffer (pH 7.4), 20% glycerol, 0.5 mM EDTA and 5 mM DTT for 1 h at 37°C. Substrate hydrolysis was measured using a luminescence spectrometer (LS50B, Perkin–Elmer, Wellesley, MA, USA) at 400 nm excitation and 505 nm emission.

RT–PCR
RNA was isolated from 1x10⁶ MelJuSo cells or from 1 mg of mouse tissue, using RNase minikit (Qiagen). cDNA was generated by reverse transcription of 1 µg RNA, using random primers (Invitrogen) and M-MLV reverse transcriptase (Invitrogen). The XBP1 transcripts were PCR amplified from the cDNA using the sense primer 5'-CCA TGG GAA GAT GTT CTG GG-3' and the anti-sense primer 5'-ACA CGC TTG GGA ATG GAC AC-3' for mouse cDNA and the sense primer 5'-GGG GCT TGG TAT ATA TGT GG-3' and the anti-sense primer 5'-CCT TGT AGT TGA GAA CCA GG-3' for human cDNA.

In vivo analysis of the UPS in reporter mice
All animal experiments were approved by the Ethical Committee in Stockholm (Ethical permission number N-46/04). Adult Ub^G76VGFP/1 (36) and C57Bl/6 mice, matched for sex and age, were given a single intraperitoneal injection of 1 µg tunicamycin/g body weight, where tunicamycin was dissolved in 150 µl of PBS containing 150 mM dextrose. One day post-injection mice were anesthetized with isoflurane (4.4% isoflurane in oxygen) and transcardially perfused with 50 ml of PBS. The tissues were excised and fixed by immersion in 4% paraformaldehyde for 24 h at 4°C, cryoprotected by immersion in a graded series of sucrose solutions as follows: 3 h in 7% sucrose, 3 h in 15% sucrose and 17 h in 30% sucrose, and embedded in Tissue-Tek OCT compound (Sakura Finetek USA). Immunohistochemistry was performed in 10 µm cryosections fixed with 4% paraformaldehyde. The sections were incubated overnight at 4°C with rabbit polyclonal anti-GFP antibody (1 : 1000 dilution) or rabbit polyclonal anti-CHOP/GADD153 antibody (1 : 50 dilution). Staining was revealed with a secondary Alexa Fluor 488-conjugated anti-rabbit antibody (Molecular Probes) or with a secondary Alexa Fluor 543-conjugated anti-rabbit antibody (Molecular Probes) for 1 h at room temperature. Nuclei were counterstained with Hoechst 33258 (Molecular Probes).

	SUPPLEMENTARY MATERIAL

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Supplementary Material is available at HMG Online.

	ACKNOWLEDGEMENTS

 
The authors would like to thank Derek Dimcheff, John Portis, Jose Lucas, Aaron Bowman, Huda Zogbhi, Marjolein Kikkert and Roberto Sitia for critical reading of this manuscript, helpful suggestions and reagents; Florian Salomons and Christa Maynard for excellent technical advice and helpful suggestions; Margareta Hagelin, Maj-Britt Alter and Torunn Söderberg for technical assistance with the transgenic mice; and Juan Carlos Higuita-Vasquez for fruitful discussions and reagents. This work was supported by the Swedish Research Council, Swedish Cancer Society, the Wallenberg foundation, the HighQ foundation, the Nordic Center of Excellence Neurodegeneration and the Karolinska Institute. NPD is supported by the Swedish Research Council.

Conflict of Interest statement. The authors declare that there is no conflict of interest.

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