TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.     

The Bax Inhibitor-1 (BI-1) family in apoptosis and tumorigenesis

The signaling pathways that determine the fate of a cell regarding death or survival depend on a large number of regulatory proteins. The Bax Inhibitor-1 (BI-1) family is a highly preserved family of small transmembrane proteins located mostly in the endoplasmic reticulum (ER). Although most members of this family are still not characterized an antiapoptotic effect has been described for BI-1, Lifeguard (LFG), and the Golgi anti-apoptotic protein (GAAP). The cytoprotective activity has been associated to the control of ion homeostasis and ER stress but includes other cell death stimuli as well. Recent data describes multiple interactions between the proteins of the BI-1 family and the Bcl-2 family either stimulating the antiapoptotic function of Bcl-2 or inhibiting the proapoptotic effect of Bax. The potent cell death suppression makes this protein family an interesting target for the development of new drugs and gene therapeutic approaches for diseases caused by apoptotic dysregulation, such as cancer.     

Bax inhibitor-1 enhances survival and neuronal differentiation of embryonic stem cells via differential regulation of mitogen-activated protein kinases activities

Bax inhibitor-1 (BI-1), a member of the BI-1 family of integral membrane proteins, was originally identified as an inhibitor of stress-induced cell death in mammalian cells. Previous studies have shown that the withdrawal of leukemia inhibitory factor (LIF) results in differentiation of the majority of mouse embryonic stem (mES) cells into various cell lineages, while some ES cells die within 3days. Thus, to investigate the function of BI-1 in ES cell survival and neuronal differentiation, we generated mES cell lines that overexpress BI-1 or a carboxy-terminal BI-1ΔC mutant. Overexpression of BI-1 in mES cells significantly increased cell viability and resistance to apoptosis induced by LIF withdrawal, while the control vector or BI-1ΔC-overexpressing mES cells had no effect. Moreover, overexpression of BI-1 produced significant inhibition of the p38 mitogen-activated protein kinases (MAPK) pathway in response to LIF withdrawal, while activity of the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) MAPK pathway was increased. Interestingly, we found that BI-1-overexpressing cells showed higher expression levels of neuroectodermal markers (Otx1, Lmx1b, En1, Pax2, Wnt1, Sox1, and Nestin) and greater neuronal differentiation efficiency than control or BI-1ΔC-overexpressing mES cells did. Considering these findings, our results indicated that BI-1-modulated MAPK activity plays a key role in protecting mES cells from LIF-withdrawal-induced apoptosis and in promoting their differentiation toward neuronal lineages.     

Bifunctional apoptosis regulator (BAR), an endoplasmic reticulum (ER)-associated E3 ubiquitin ligase, modulates BI-1 protein stability and function in ER Stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.     

BI-1 regulates endoplasmic reticulum Ca2+ homeostasis downstream of Bcl-2 family proteins

BI-1 (Bax inhibitor-1) is an evolutionarily conserved multitransmembrane protein that resides in the endoplasmic reticulum (ER) and that has documented cytoprotective functions in both animals and plants. Recent studies indicate that BI-1 shares in common with Bcl-2/Bax family proteins the ability to regulate the amounts of Ca(2+) that can be released from the ER by agents, such as the ER-Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG). Using an ER-targeted, Ca(2+) indicator (cameleon), with characteristics optimized for measuring ER Ca(2+) ([Ca(2+)](er)), we studied the effects of BI-1 on [Ca(2+)](er) in resting and TG-treated cells. Similar to cells overexpressing antiapoptotic Bcl-2 or Bcl-X(L), overexpression of BI-1 resulted in lower resting [Ca(2+)](er), with concomitantly less Ca(2+) released into the cytosol upon stimulation by TG and with a higher rate of Ca(2+) leakage from the ER. Co-expression of SERCA restored levels of [Ca(2+)](er) to normal, showing opposing actions of the ER-Ca(2+)ATPase and BI-1 on ER Ca(2+) homeostasis. Conversely, cells with deficient BI-1 have increased [Ca(2+)](er), and release more Ca(2+) into the cytosol when challenged with TG. In BI-1-deficient cells, Bcl-X(L) fails to reduce [Ca(2+)](er), indicating that BI-1 functions downstream of Bcl-X(L). In bax(-/-)bak(-/-) double knock-out cells, both BI-1 and Bcl-X(L) retained their ability to reduce [Ca(2+)](er), suggesting that BI-1 and Bcl-X(L) operate downstream of or parallel to Bax/Bak. The findings reveal a hierarchy of functional interactions of BI-1 with Bcl-2/Bax family proteins in regulating ER Ca(2+) homeostasis.     

Role of BI-1 (TEGT)-mediated ERK1/2 activation in mitochondria-mediated apoptosis and splenomegaly in BI-1 transgenic mice

Bax Inhibitor-1 (BI-1) is an evolutionally conserved apoptotic suppressor and belongs to the BI-1 family of proteins, which contain BI-1-like transmembrane domains. As their cellular functions and regulatory mechanisms remain incompletely understood, we compared their anti-apoptotic properties. Forced expression of BI-1 resulted in the most effective suppression of stress-induced apoptosis, compared with other family members, together with significant extracellular signal-regulated kinase (ERK)1/2 activation. BI-1-mediated ERK1/2 activation led to the suppression of mitochondria-mediated reactive oxygen species (ROS) production. Involvement of the ERK signaling pathway in BI-1-induced anti-apoptotic effects was confirmed by knockdown studies with ERK- or BI-1-specific siRNA. Moreover, we produced transgenic (TG) mice overexpressing BI-1, and the relationship between ERK1/2 activation and the suppression of ROS production or apoptosis was confirmed in mouse embryonic fibroblast (MEF) cells derived from these mice. Interestingly, we found that BI-1 TG mice showed splenomegaly and abnormal megakaryopoiesis. Taken together, our results suggest that BI-1-induced ERK1/2 activation plays an important role in the modulation of intracellular ROS generation and apoptotic cell death and may also affect autoimmune response.     

The ancient cell death suppressor BAX inhibitor-1

Bax inhibitor-1 (BI-1) was initially identified for its ability to inhibit BAX-induced apoptosis in yeast cells and is the founding member of a family of highly hydrophobic proteins localized in diverse cellular membranes. It is evolutionarily conserved and orthologues from plants can substitute for mammalian BI-1 in regard to its anti-apoptotic function suggesting a high degree of functional conservation. BI-1 interacts with BCL-2 and BCL-XL and, similar to these two anti-apoptotic proteins, the effect of BI-1 on cell death involves changes in the amount of Ca²⁺ releasable from intracellular stores. However, BI-1 is also a negative regulator of the endoplasmic reticulum stress sensor IRE1 α, it interacts with G-actin and increases actin polymerization, enhances cancer metastasis by altering glucose metabolism and activating the sodium-hydrogen exchanger, and reduces the production of reactive oxygen species through direct interaction with NADPH-P450 reductase. In this contribution, we summarize what is known about the expression, intracellular localization and structure of BI-1 and specifically illuminate its effects on the intracellular Ca²⁺ homeostasis and how this might relate to its other functions. We also present a thorough phylogenetic analysis of BI-1 proteins from major phyla together with paralogues from all BI-1 family members.     

Six-transmembrane topology for Golgi anti-apoptotic protein (GAAP) and Bax inhibitor 1 (BI-1) provides model for the transmembrane Bax inhibitor-containing motif (TMBIM) family

The Golgi anti-apoptotic protein (GAAP) is a hydrophobic Golgi protein that regulates intracellular calcium fluxes and apoptosis. GAAP is highly conserved throughout eukaryotes and some strains of vaccinia virus (VACV) and camelpox virus. Based on sequence, phylogeny, and hydrophobicity, GAAPs were classified within the transmembrane Bax inhibitor-containing motif (TMBIM) family. TMBIM members are anti-apoptotic and were predicted to have seven-transmembrane domains (TMDs). However, topology prediction programs are inconsistent and predicted that GAAP and other TMBIM members have six or seven TMDs. To address this discrepancy, we mapped the transmembrane topology of viral (vGAAP) and human (hGAAP), as well as Bax inhibitor (BI-1). Data presented show a six-, not seven-, transmembrane topology for vGAAP with a putative reentrant loop at the C terminus and both termini located in the cytosol. We find that this topology is also conserved in hGAAP and BI-1. This places the charged C terminus in the cytosol, and mutation of these charged residues in hGAAP ablated its anti-apoptotic function. Given the highly conserved hydrophobicity profile within the TMBIM family and recent phylogenetic data indicating that a GAAP-like protein may have been the ancestral progenitor of a subset of the TMBIM family, we propose that this vGAAP topology may be used as a model for the remainder of the TMBIM family of proteins. The topology described provides valuable information on the structure and function of an important but poorly understood family of proteins.     

Bax inhibitor-1 regulates endoplasmic reticulum stress-associated reactive oxygen species and heme oxygenase-1 expression

The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes and protects cells from ER stress-induced apoptosis. The ER is associated with generation of reactive oxygen species (ROS) through oxidative protein folding. This study examined the role of BI-1 in the regulation of ER stress-induced accumulation of ROS and expression of unfolded protein response-associated proteins. BI-1 reduced the expression levels of glucose response protein 78, C/EBP homologous protein, phospho-eukaryotic initiation factor 2alpha, IRE1alpha, XBP-1, and phospho-JNK and inhibited the cleavage of ATF-6alpha p-90, leading to the inhibition of ROS. Although ROS scavengers offer some protection against ER stress-induced apoptosis, the expression of pro-apoptotic ER stress proteins was not affected. This study shows that the response of unfolded proteins is followed by ROS accumulation under ER stress, which is regulated in BI-1 cells. The mechanism for these BI-1-associated functions involves the expression of heme oxygenase-1 (HO-1) through nuclear factor erythroid 2-related factor 2. In BI-1 cells, the transfection of HO-1 small interfering RNA completely abolished the BI-1-induced protection. The endogenous expression of HO-1 through ER stress-initiated ROS is believed to be as a protection signal. In conclusion, these observations suggest that BI-1 can inhibit the ER stress proteins as well as the accumulation of ROS, thereby protecting the cells. Moreover, HO-1 plays an important role in the BI-1-associated protection against ER stress.     

Molecular characterization of two plant BI-1 homologues which suppress Bax-induced apoptosis in human 293 cells

To date, few homologues of animal programmed cell death (PCD) regulators have been identified in plants. Among these is the plant Bax Inhibitor-1 (BI-1) protein, which possesses, like its human counterpart, the ability to suppress Bax-induced lethality in yeast cells. As the role of BI-1 in the regulation of plant PCD remains to be elucidated, we cloned BnBI-1 and NtBI-1 from cDNA libraries of oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.). The analysis of the deduced amino acid sequences of BnBI-1 and NtBI-1 indicated that these proteins share a relatively high level of identity with other plant BI-1 proteins (73-95%) as well as with animal BI-1 proteins (26-42%). Comparative analysis with other available plant BI-1 proteins allowed the establishment of a structural model presenting seven transmembrane domains. Moreover, transient co-transfection of Bax with BnBI-1 or NtBI-1 in human embryonic kidney 293 cells revealed that both proteins can substantially inhibit apoptosis induced by Bax overexpression. Localization studies were also conducted using stable transformation of tobacco BY-2 cells and Saccharomyces cerevisiae, or transient expression in tobacco leaves, with the fusion protein BnBI-1GFP under control of the cauliflower mosaic virus 35S promoter. All transformants showed a fluorescence pattern of distribution typical of an endoplasmic reticulum (ER) protein. Results from differential permeabilization experiments in BY-2 cells expressing BnBI-1GFP also showed that the C-terminus is located on the cytosolic side of the ER. Taken altogether, our results suggest that BI-1 is evolutionarily conserved and could act as a key regulator of a death pathway common to plants and animals.     

Proteomic profiling of differentially expressed proteins from Bax inhibitor-1 knockout and wild type mice

Bax inhibitor-1 (BI-1) is an anti-apoptotic protein located in the endoplasmic reticulum (ER). The role of BI-1 has been studied in different physiopathological models including ischemia, diabetes, liver regeneration and cancer. However, fundamental knowledge about the effects of BI-1 deletion on the proteome is lacking. To further explore this protein, we compared the levels of different proteins in bi-1 (-/-) and bi-1 (+/+) mouse tissues by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). In several bi-1 (-/-) mice, glucose-regulated protein 75 (GRP75/mortalin/ PBP74/mthsp70), peroxiredoxin6 (Prx6) and fumarylacetoacetate hydrolase (FAH) showed a pI shift that could be attributed to post-translational modifications. Selenium-binding protein 2 (SBP2) and ferritin light chain 1 levels were significantly increased. Phosphatidylethanolamine-binding protein-1 (PEBP-1) was dramatically decreased in bi-1 (-/-) mice, which was confirmed by Western blotting. The phosphorylation of GRP75, Prx6 and FAH were compared between bi-1 (+/+) and bi-1 (-/-) mice using liver tissue lysates. Of these three proteins, only one exhibited modified phosphorylation; Tyr phosphorylation of Prx6 was increased in bi-1 (-/-) mice. Our protein profiling results provide fundamental knowledge about the physiopathological function of BI-1.     

Ghitm is an ortholog of the Bombyx mori prothoracic gland-derived receptor (Pgdr) that is ubiquitously expressed in mammalian cells and requires an N-terminal signal sequence for expression

In a previous paper, we reported the cloning of a cDNA encoding a putative receptor, Pgdr, from the prothoracic gland of the silkworm, Bombyx mori. Few studies concerning the orthologous cDNA of Pgdr in mammals, a growth hormone-inducible transmembrane protein (Ghitm) that encodes a putative receptor, have been performed. Analysis of the distribution of Ghitm expression revealed ubiquitous expression in mouse embryo and adult tissues, as well as mammalian cell lines. The pattern of Ghitm expression suggested that once Ghitm mRNA was expressed in the putative brain region of mouse embryo, Ghitm-expressing cells spread ubiquitously throughout all tissues during embryonic development. In addition, Western blot analyses demonstrated that cleavage of the N-terminal portion in GHITM appears to regulate the expression level, suggesting that cleavage is essential for the proper expression of GHITM.     

Effects of phenylalanine on the survival and neurite outgrowth of rat cortical neurons in primary cultures: possible involvement of brain-derived neurotrophic factor

Bax inhibitor-1 (BI-1), a newly identified apoptosis inhibitor, has recently been found to be overexpressed in several human carcinomas and its specific down-regulation by RNA interference (RNAi) could lead to cell death. The purpose of this study is to investigate the role of BI-1 in apoptosis-resistance and the underlying mechanisms in human nasopharyngeal carcinoma (NPC) cells. Our results showed that BI-1 was expressed in two different human NPC cell lines, CNE-2Z and CNE-1, and specific inhibition of BI-1 expression by siRNA caused a significant increase in spontaneous apoptosis in both cell lines. Mechanistically, we demonstrated that down-regulation of BI-1 protein expression decreased the ratio of Bcl-X_L/Bcl-2 with Bax protein as determined by Western blot and increased the activity of caspase-3 by colorimetric analysis, thus leading to the activation of the associated cell death pathways. Taken together, these results have provided evidence that BI-1 could serve as an important molecular target gene for the development of new therapeutic strategy against human NPCs.     

Enhanced lysosomal activity is involved in Bax inhibitor-1-induced regulation of the endoplasmic reticulum (ER) stress response and cell death against ER stress: involvement of vacuolar H+-ATPase (V-ATPase)

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H(+)-ATPase(V-ATPase), a proton pump, was activated, suggesting high H(+) uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.     

The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore

Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca(2+) homeostasis by acting as a Ca(2+)-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca(2+) pore responsible for its Ca(2+)-leak properties. We utilized a set of C-terminal peptides to screen for Ca(2+) leak activity in unidirectional (45)Ca(2+)-flux experiments and identified an α-helical 20-amino acid peptide causing Ca(2+) leak from the ER. The Ca(2+) leak was independent of endogenous ER Ca(2+)-release channels or other Ca(2+)-leak mechanisms, namely translocons and presenilins. The Ca(2+)-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca(2+)-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca(2+)-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca(2+)-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca(2+) leak. Thus, we elucidated residues critically important for BI-1-mediated Ca(2+) leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca(2+)-leak properties of BI-1 are an added function during evolution.     

BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response

Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.     

Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell death suppressor

In spite of fundamental differences between plant and animal cells, it is remarkable that some cell death regulators that were identified to control cell death in metazoans can also function in plants. The fact that most of these proteins do not have structural homologs in plant genomes suggests that they may be targeting a highly conserved 'core' mechanism with conserved functions that is present in all eukaryotes. The ubiquitous Bax inhibitor-1 (BI-1) is a common cell death suppressor in eukaryotes that has provided a potential portal to this cell death core. In this review, we will update the current status of our understanding on the function and activities of this intriguing protein. Genetic, molecular and biochemical studies have so far suggested a consistent view that BI-1 is an endoplasmic reticulum (ER)-resident transmembrane protein that can interact with multiple partners to alter intracellular Ca(2+) flux control and lipid dynamics. Functionally, the level of BI-1 protein has been hypothesized to have the role of a rheostat to regulate the threshold of ER-stress inducible cell death. Further, delineation of the cell death suppression mechanism by BI-1 should shed light on an ancient cell death core-control pathway in eukaryotes, as well as novel ways to improve stress tolerance.     

Yeast Bax inhibitor, Bxi1p, is an ER-localized protein that links the unfolded protein response and programmed cell death in Saccharomyces cerevisiae

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. Studies in both mammalian and plant cells suggest that the BI-1 protein resides in the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. It is thought to act via a mechanism involving altered calcium dynamics. In this paper, we provide evidence that the Saccharomyces cerevisiae protein encoded by the open reading frame, YNL305C, is a bona fide homolog for BI-1. First, we confirm that yeast cells from two different strain backgrounds lacking YNL305C, which we have renamed BXI1, are more sensitive to heat-shock induced cell death than wildtype controls even though they have indistinguishable growth rates at 30°C. They are also more susceptible both to ethanol-induced and to glucose-induced programmed cell death. Significantly, we show that Bxi1p-GFP colocalizes with the ER localized protein Sec63p-RFP. We have also discovered that Δbxi1 cells are not only more sensitive to drugs that induce ER stress, but also have a decreased unfolded protein response as measured with a UPRE-lacZ reporter. Finally, we have discovered that deleting BXI1 diminishes the calcium signaling response in response to the accumulation of unfolded proteins in the ER as measured by a calcineurin-dependent CDRE-lacZ reporter. In toto, our data suggests that the Bxi1p, like its metazoan homologs, is an ER-localized protein that links the unfolded protein response and programmed cell death.