Endoplasmic reticulum stress:
cell life and death decisions (original) (raw)
The adaptive responses to misfolded proteins in the ER provide protection from cell death, inasmuch as gene transfer–mediated overexpression of Grp78 or protein-disulfide isomerase (PDI) reduces cell death induced by oxidative stress, Ca2+ disturbances, and hypoxia (19, 20). However, when protein misfolding is persistent or excessive, ER stress triggers cell death, typically apoptosis. Several mechanisms, described below, have been proposed for linking the distressed ER to cell death (Figure 2), including direct activation of proteases, kinases, transcription factors, and Bcl-2–family proteins and their modulators.
Cell death mechanisms induced by ER stress. Several of the proposed pathways linking ER stress to cell death are depicted. Dashed lines indicate protein translocation events (c-Abl, Jafrac2). The mitochondrial permeability transition pore complex, which is Ca2+-sensitive, is not shown in the diagram. See the text for additional details.
Proteases. Caspases are required for apoptosis, and certain members of this family of cysteine proteases associate with the ER (reviewed in ref. 21). In rodents, caspase-12 associates with activated Ire1, resulting in proteolytic processing of caspase-12. Mice lacking caspase-12 genes display partial resistance to pharmacological inducers of ER stress, such as tunicamycin (inhibitor of N-linked protein glycosylation) and thapsigargin (inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases [SERCAs], which pump Ca2+ into the ER) (22). Because proteolytic activity has been difficult to demonstrate for caspase-12 (23), it is unknown whether the proteolytic processing of caspase-12 that occurs during ER stress results in its activation. Also, the mechanisms responsible for proteolysis of caspase-12 may be indirect, involving calpains activated by Ca2+ released in the vicinity of the ER (24), instead of an induced proximity mechanism where oligomers of Ire1 provide a scaffold for clustering caspase-12 zymogens. Caspase-7 also may activate caspase-12 by translocating from cytosol to ER (25). However, the relevance of caspase-12 to ER-induced apoptosis has been questioned because of an absence of caspase-12 in most humans. In this regard, the ancestral human CASPASE-12 gene is disrupted by a termination codon and thus is inactive (26). For persons with hereditary polymorphisms that leave the open reading frame intact (estimated at ∼1% of African populations), caspase-12 operates as a _trans_-dominant inhibitor of proinflammatory caspases, lacking conserved residues required for catalytic activity (23).
Human caspase-4, one of the closest paralogs of rodent caspase-12, may associate with ER (27), raising the possibility that this protease can perform the functions normally ascribed to rodent caspase-12 in the context of ER stress. But caspase-4 belongs to the group of proinflammatory caspases responsible for proteolytic activation of cytokines, rather than the apoptotic caspases. Nevertheless, small interfering RNA–mediated knock down of caspase-4 in human neuroblastoma cells partially reduces cell death caused by the ER stress inducers thapsigargin and amyloid β-peptide (AβP), but not inducers of mitochondria-dependent cell death (e.g., UV irradiation, DNA-damaging drugs). However, caspase-4 knock down in HeLa cells had little effect on apoptosis induced by ER stress, implying that the relevance of this protease to ER stress is tissue-specific.
The ER resident protein Bap31 contains 3 predicted transmembrane domains, followed by a leucine zipper and a death effector domain–like (DED-L) region that associates with certain isoforms of procaspase-8 in the cytosol (28). Bap31 can display either pro- or antiapoptotic phenotypes, depending on whether its cytosolic tail is removed by cleavage by caspases. Overexpression of full-length Bap31 blocks apoptosis induced by anti-Fas antibody and cycloheximide, while expression of the truncated 20-kDa protein induces apoptosis (29, 30). A mutant of Bap31 in which the caspase-8 cleavage site was mutated suppressed Fas-induced apoptosis (29). Since proximal steps in Fas signaling were not blocked by mutant Bap31, this suggests that the ER participates as an intermediary in death receptor–induced apoptosis in some cells.
The DED-L domain of Bap31 also binds a homologous DED-L domain in BAR, another ER-associated apoptosis regulator (31). The 52-kDa BAR protein contains a RING domain that binds ubiquitin-conjugating enzymes, followed by an α-helical region that binds Bcl-2 and Bcl-xL, the DED-L domain, and a C-terminal membrane-anchoring domain (32). Like the DED-L domain of Bap31, the DED-L domain of BAR binds caspase-8, sequestering it and thwarting apoptosis induction initiated by TNF/Fas-family death receptors (32, 33). BAR also binds the apoptosis regulators Hip and Hippi, which contain DED-L domains homologous to those found in BAR and Bap31. Hip associates with Huntingtin (Htt), the protein implicated in Huntington disease that causes degeneration of neurons containing Htt polyglutamine (polyQ) expansions (34). Hippi is a DED-L domain–containing Hip-interacting protein that binds procaspase-8. Htt with polyQ expansion has reduced affinity for Hip compared with the normal Htt protein, a circumstance under which it has been proposed that Hip is free to bind Hippi and trigger caspase-8 activation (35). Interactions of BAR, Bap31, Hip, and Hippi deserve further investigation on a number of fronts, including whether these proteins represent substrates for the E3 ligase activity of BAR, elucidation of their agonistic and antagonistic relations among each other, and evaluation of effects of these protein interactions on nonapoptotic functions of Htt and its interacting proteins.
The ability of BAR and Bap31 to bind procaspase-8 prompts speculation that perhaps these ER proteins could promote rather than inhibit caspase-8 activation, if induced to aggregate in ER membranes, thereby constituting a novel ER-associated “apoptosome.” If so, then the parallel ability of BAR and Bap31 to bind Bcl-2 and Bcl-xL through domains separate from the DED-L domain might supply a mechanism for preventing caspase activation, providing a long-sought analogy to the paradigm for caspase regulation seen in Caenorhabditis elegans, where the Bcl-2 ortholog Ced9 binds caspase activator Ced4, preventing activation of Ced3 protease (36).
Kinases. The kinase Ask1 has been implicated in apoptosis induction in the context of signaling by TNF-family receptors (reviewed in ref. 37). During ER stress, Ask1 is recruited to oligomerized Ire1 complexes containing TRAF2, activating this kinase and causing downstream activation of JNK and p38 MAPK. Consistent with a key role for Ask1 in apoptosis induced by ER stress, studies of ask1–/– neurons subjected to inducers of ER stress indicate a requirement for this kinase for JNK activation and cell death (38). The downstream death effectors of Ask1 are not clear. The kinase pathway initiated by Ask1 leads to JNK activation, and JNK-mediated phosphorylation activates the proapoptotic protein Bim (39–41), while inhibiting the antiapoptotic protein Bcl-2 (42).
Thus, Ire1 plays roles in all 3 of the ER responses to unfolded proteins (adaptation, alarm, and apoptosis), through its actions upon XBP-1 (adaptation), TRAF2 (alarm [NF-κB]), and apoptosis effectors caspase-12 and Ask1. How these 3 functions of Ire1 are integrated remains unclear.
The protein tyrosine kinase c-Abl can translocate from the ER surface to mitochondria in response to ER stress (43). Moreover, a functional role for c-Abl has been suggested by studies of c-Abl–/– fibroblasts, which display resistance to cell death induced by Ca2+ ionophores, brefeldin A, and tunicamycin (43). How c-Abl promotes apoptosis is unknown at present.
Transcription factors. CHOP (GADD153) is a member of the C/EBP family of bZIP transcription factors, and its expression is induced to high levels by ER stress (reviewed in ref. 44). The chop gene promoter contains binding sites for all of the major inducers of the UPR, including ATF4, ATF6, and XBP-1, and these transcription factors play causative roles in inducing chop gene transcription. Cause-and-effect roles in chop gene induction have been demonstrated for signaling molecules involved in ER stress by genetic manipulation of mice, showing that perk–/– and _atf4_–/– cells and eIF2α(S51A) knock-in cells fail to induce chop during ER stress (11, 12, 45). Cross-talk between the PERK/eIF2α pathway and the Ire1/TRAF2/Ask1 pathway may also enhance CHOP activity at a posttranscriptional level, given that Ask1 activates both JNK and p38 MAPKs, and phosphorylation of the CHOP protein on serine 78 and serine 81 by p38 MAPKs increases its transcriptional and apoptotic activity (46). In addition to the aforementioned regulators, upstream activators of chop also include ATF2, which is induced by hypoxia and which is required for chop induction during amino acid starvation (47).
Overexpression of CHOP protein induces apoptosis, through a Bcl-2–inhibitable mechanism (48, 49). Moreover, chop–/– mice are resistant to kidney damage induced by tunicamycin and to brain injury resulting from cerebral artery occlusion, demonstrating a role for CHOP in cell destruction when ER stress is involved (48, 50). How CHOP induces apoptosis is unclear. CHOP forms heterodimers with other C/EBP-family transcription factors via bZIP-domain interactions, which suppresses their binding to C/EBP sites in DNA, while promoting binding to alternative DNA sequences for target gene activation (51). Consequently, CHOP inhibits expression of genes responsive to C/EBP-family transcription factors, while enhancing expression of other genes containing the consensus motif 5′-(A/G)(A/G)(A/G)TGCAAT(A/C)CCC-3′. One relevant target may be bcl-2, whose expression is suppressed by CHOP, at least in some cellular contexts (49). CHOP may also have nontranscriptional actions, still poorly defined (44). While capable of inducing apoptosis and contributing to cell death in several scenarios involving ER stress, CHOP is not essential for cell death induced by ER stress, as demonstrated by the observation that perk–/– and eIF2α(S51A) knock-in cells are hypersensitive to ER stress–induced apoptosis but fail to induce chop gene expression (12, 45).
Scotin is another ER-targeted apoptosis inducer (52). The gene encoding Scotin is a direct target of p53, suggesting a way to link DNA damage to ER-mediated cell death mechanisms.
Bcl-2–family proteins and their modulators. Association of certain Bcl-2/Bax–family proteins with ER membranes dates back to the initial discovery of Bcl-2 (53). Though better known for their actions upon mitochondria, Bcl-2/Bax–family proteins also integrate into ER membranes, where they modulate ER Ca2+ homeostasis and control cell death induced by ER stress agents, including tunicamycin, brefeldin A (an inhibitor of ER-Golgi transport), thapsigargin, and oxidants (reviewed in ref. 54). Experiments in which the normal C-terminal transmembrane domain of Bcl-2 was swapped with membrane-targeting domains from ER resident proteins suggested that Bcl-2 targeted exclusively to the ER (as opposed to both ER and mitochondria) is more restricted in its antiapoptotic actions, suppressing cell death induced by ER stress agents and by c-Myc. Recent findings that apoptosis induced by c-Myc may be attributable to its induction of Bim suggest that ER-targeted Bcl-2 may sequester this BH3-only protein, preventing it from interacting with other members of the Bcl-2/Bax family (55).
Spike is a BH3-only protein anchored in the ER (56). The BH3-like domain of Spike is required for apoptosis induction, but dimerization partners among Bcl-2/Bax–family proteins have yet to be found. Several other Bcl-2/Bax–family proteins reside at least in part in association with or integrated into ER membranes, with some, such as the antiapoptotic protein Mcl-1 and proapoptotic Bik, found predominantly in the ER (57, 58). Given the preferences of certain BH3 domains for interactions with particular members of the Bcl-2/Bax family (59), it seems likely that a network of interactions among a subset of this family of apoptosis regulators takes place on ER membranes, the functional consequences of which are not yet fully understood. Recently, expression of at least 1 of the Bcl-2/Bax–family genes was linked to ER stress. The BH3-only protein Puma is induced by tunicamycin and thapsigargin in a p53-independent manner, with Puma–/– cells showing resistance to apoptosis induced by ER stress (60).
The BI-1 protein contains 6 transmembrane domains, resides in the ER (61), interacts functionally or physically with Bcl-2–family members, and is induced by hypoxia (62). This protein blocks cell death induced by oxidative stress in yeast, plants, and animals (63). Mice lacking BI-1 display increased sensitivity to tunicamycin-induced kidney damage and to stroke injury, implying a role for BI-1 in protection from insults known to trigger ER stress. In cultured cells, overexpression of BI-1 selectively reduces, while BI-1 deficiency selectively increases, sensitivity to cell death induced by agents that trigger ER stress, while having far less effect on apoptosis induced by agents that trigger cell death pathways linked to mitochondria (intrinsic pathway) or TNF/Fas-family death receptors (extrinsic pathway) (64). BI-1 associates with the antiapoptotic proteins Bcl-2 and Bcl-xL, but not proapoptotic Bax and Bak (61). Nevertheless, BI-1 inhibits cell death induced by Bax overexpression, in animal cells, plants, and yeast.
The ER protein Bap31 lacks homology with Bcl-2/Bax–family proteins and contains no recognizable BH3 dimerization domain, but it binds Bcl-2 and Bcl-xL and regulates apoptosis. BAR also binds Bcl-2 and Bcl-xL, and the responsible domain is required for BAR-mediated suppression of cell death (32). Interestingly, BAR is capable of suppressing Bax-induced death of yeast, implying caspase-independent functions for this protein, given that yeast lack bona fide caspases. This suggests that, mechanistically, BAR may share something in common with Bcl-2 and BI-1, which also suppress Bax-induced killing of yeast.
Other apoptosis regulators. Given that mitochondria release apoptogenic proteins into the cytosol, the ER might use similar mechanisms for linking ER stress to cell death. In insect cells, at least one example has been uncovered of a protein, called Jafrac2, that is normally sequestered in the ER but is released into the cytosol during apoptosis induced by certain stimuli (65). Like most proteins imported into the ER, the N-terminal leader peptide of Jafrac2 is removed by proteolysis. This proteolytic processing exposes an IAP-binding motif in Jafrac2, poising it to attack antiapoptotic IAP-family proteins upon accessing the cytosol, thereby freeing caspases. It remains to be determined whether examples of apoptogenic protein release from the ER of mammalian cells will be discovered.