In the present study, we demonstrate a requirement for the proteasome in ER stress-induced cell death. We determined that the pro-apoptotic activity of the proteasome lies in a pathway upstream of Bcl-2, Bcl-w, and Bcl-xL degradation, mitochondrial release of cytochrome c, caspase-3 maturation, and phosphatidylserine exposure. In each case, the proteasome inhibitors lactacystin and Pefabloc are effective at blocking these markers of a pathway that connects protracted ER stress to cellular destruction. As further indication that the proteasome is a player in this pathway, we utilized a mutant cell line that harbors a temperature-sensitive allele of the E1 ubiquitin-activating enzyme. ER stress-induced cell death proceeded normally at the permissive temperature, but was blocked at the restrictive temperature, indicating that one of the functions of the ubiquitin proteasome system is to initiate a pro-apoptotic signaling cascade in response to prolonged ER stress.
Previously, ER stress-induced apoptosis was shown to be mediated by a serine protease-like activity that was blocked by Pefabloc, a general inhibitor of serine proteases.11
We therefore sought to elucidate the identity of the Pefabloc-inhibitable activity required for ER stress-induced cell death. Toward this end, we developed two affinity labeling methods, one with biotinylated and the other with fluorogenic active site-directed serine protease inhibitors and subsequently analyzed the samples by mass spectrometry (Supplementary Figure 1
). Although we were unable to identify any serine proteases in either approach, we repeatedly obtained hits for proteasomal subunits among the proteins found both in the biotinylated inhibitor-enriched fraction as well as those that were labeled by the fluorogenic active site-directed serine protease inhibitor, albeit near the limit of detection in both cases. That the proteaseome, a threonine protease, could be identified in our mass spectrometry approaches with serine protease inhibitors serving as bait is not surprising because several serine protease inhibitors, among them Pefabloc, have been shown to inhibit the proteasome non-selectively.14,15
Whereas it was previously proposed that the inhibitory effect of Pefabloc on ER stress-induced cell death was due to inhibition of a serine protease,11
we now believe that at the concentrations used (300 μM), Pefabloc inhibits the activity of the proteasome, and that proteasomal inhibition prevents cells from undergoing apoptosis in response to ER stress. This hypothesis is supported by the fact that Pefabloc treatment results in an accumulation of an engineered proteasomal substrate, GFPu
. However, we cannot exclude the possibility that additional Pefabloc-inhibitable serine protease activities might also play a role in the novel ER stress-induced apoptotic cascade described herein.
To date, the mediators that link ER stress to the apoptotic machinery have not been fully elucidated. Caspase-12 was proposed to function as the apical caspase responsible for initiating an apoptotic cascade in response to ER stress. However, caspase-12−/−
mouse embryonic fibroblasts (MEFs) were shown to be only partially protected from ER stress-inducing agents.16
Furthermore, caspase-12 processing was shown to be downstream of the Bax/Bak gateway, as indicated by the fact that maturation of caspase-12 was blocked when Bax
double knockout MEFs were treated with BFA.9
These findings make it unlikely that caspase-12 could be a principal mediator of ER stress-induced cell death. CHOP/GADD153, the pro-apoptotic translational-dependent mediator of ER stress-induced cell death,17
is not a player in our model system because we co-treated cells with CHX so as to bypass the expression of protective UPR genes, as was done previously.8,11
Therefore, we have defined an ER stress-induced cell death pathway that is independent of CHOP/GADD153. We also excluded calpains from playing a role in this paradigm because none of the calpain inhibitors tested had any protective effect on ER stress-induced apoptosis (data not shown).
Our finding that the proteasome is a mediator of ER stress-induced cell death is surprising because many reports document that proteasome inhibitors alone are toxic,18,19
and toxicity is exacerbated when cells also experience ER stress.20
What is more, proteasomal inhibitors can actually cause ER stress, as shown by the upregulation of Grp78/Bip and CHOP/GADD153, thus providing further evidence that proteasomal activity is intimately coupled to ERAD as the last leg of the UPR.21,22
Recently, a potent proteasome inhibitor, PS-341 (Bortezomib), was shown to be effective in the treatment of multiple myeloma, and is now approved by the US Food and Drug Administration. The toxicity of PS-341 in multiple myeloma cells is very likely to be the result of ER stress-induced apoptosis.22
Pro-apoptotic functions of the proteasome have been described in several instances including the constitutive death of neutrophils as well as NGF withdrawal, DNA damage, glucocorticoid treatment and reduced extracellular potassium.23–27
Additionally, the proteasome has been implicated in the early stages of wallerian degeneration after axotomy.28
In response to DNA damage, the large Bcl-2 homology domain-only protein Mule/ARF-BP1 was shown to ubiquitylate Mcl-1 – an anti-apoptotic Bcl-2 family member – thereby causing its degradation via the proteasome.29
Also, the Bcl-2 family members Bcl-2, Mcl-1, and Bfl-1 have been shown to undergo proteasomal degradation when critical serine or threonine residues become dephosphorylated in response to treatment with paclitaxel or TNF.30–35
In these examples, proteasomal activity tips the balance of pro-and anti-apoptotic Bcl-2 family members toward apoptosis, and thereby serves as a mediator of apoptosis.
The induction of UPR and ERAD in response to ER stress is protective. But at some point, after failing to achieve cellular homeostasis, cells induce programmed cell death. The mechanism of this switch is not fully understood, but likely involves the expression of CHOP/GADD153, which can cause downregulation of Bcl-2.36
However, even under translation-inhibitory conditions, where CHOP/GADD153 is not expressed, we find that proteasomal activity is required for ER stress-induced cell death. Despite the fact that the prosurvival function of ERAD requires proteasomal activity, it seems that the later pro-apoptotic arm of ER stress also requires the activity of the proteasome. Therefore, the proteasome is involved both in protecting cells from ER stress (as in the case of ERAD) and in the activation of programmed cell death. We have speculated that these two opposing activities of the proteasome could be the result of the alternative regulation of multiple E3 ubiquitin-ligase activities. In the protective phase of ER stress (UPR and ERAD), the E3 ubiquitin ligases SCFFBS1
recognize misfolded proteins that have been dislocated from the ER, thereby directing the proteasome to degrade these malfolded - and potentially toxic-proteins.5
Later, the cell could commit to cell death by upregulating an E3 ubiquitin-ligase activity that may target an anti-apoptotic molecule, such as the anti-apoptotic molecules Bcl-2, Bcl-w, and Bcl-xL. One candidate for such an activity is Mule/ARF-BP1, which has been shown to ubiquitylate Mcl-1, thus tagging Mcl-1 for proteasomal degradation and tipping the balance toward apoptosis.29
However, we did not find that full-length Mcl-1 accumulates in the presence of proteasomal inhibitors (data not shown). Therefore, the putative ER stress-activated E3 ubiquitin-ligase activity is not likely to be Mule/ARF-BP1 ().
Figure 5 Putative model of proteasome-mediated ER stress-induced cell death. ER stress activates a pro-apoptotic ER ubiquitin ligase which tags anti-apoptotic Bcl-2 family members with ubiquitin. Subsequently, the proteasome degrades these anti-apoptotic molecules, (more ...)
Manipulation of the cell's decision to induce cell death in response to protracted ER stress may be possible with the development of specific E3 ubiquitin ligase inhibitors: Those that would inhibit SCFFBS1
would be toxic, whereas those that would inhibit pro-apoptotic E3 ubiquitin ligases – like Mule/ARF-BP1 – would be protective. As evidence has emerged that ER stress plays a role in neurodegenerative diseases like Alzheimer's, Huntington's, Parkinson's and ALS,37
reagents that block the proteasomal degradation of anti-apoptotic molecules may prove to be neuroprotective.