Described as a response to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), the eponymously named unfolded protein response (UPR) is characterized by the activation of three distinct signal transduction pathways mediated by inositol requiring (IRE) 1α, PKR-like ER kinase (PERK) and activating transcription factor (ATF) 6α (). Alterations in ER function can be induced by myriad stimuli, pharmacologically by exogenously applied chemicals or physiologically such as by increased secretory protein demand [29
]. Altogether these perturbations are referred to as ER stress and are recognized by the activation of the UPR transducers (Key Message Box 1
). The ER lumenal domains of all three stress sensors are bound by the ER chaperone BiP in the unstressed state. Upon ER stress, BiP binding to unfolded proteins causes dissociation from the lumenal domains of the sensors leading to the activation of IRE1α and PERK by transautophosphorylation, and ATF6α by proteolytic processing [10
]. Following activation, UPR signaling pathways act to induce expression of genes that encode functions to ameliorate the stressed state of the ER. These adaptive mechanisms include global attenuation of mRNA translation via phosphorylation of eIF2α. eIF2α phosphorylation dramatically decreases the functional load on the ER by reducing synthesis of new proteins that would require folding. Additionally, transcriptional activation of ER chaperones and ER expansion occur to facilitate folding of the accumulated misfolded proteins. ER-associated degradation (ERAD) components are upregulated transcriptionally as well, facilitating degradation of terminally misfolded proteins. Sustained ER stress leads to apoptosis. Though the exact mechanisms that mediate ER-stress induced apoptosis have not yet been elucidated, the transcription factor C/EBP homologous protein (CHOP), the mitogen activated protein kinase c-jun N-terminal kinase (JNK), Bcl-2 family proteins, calcium and redox homeostasis, and caspase activation have all been implicated.
The unfolded protein response sensors
Key Message Box 1.
In addition to the accumulation of misfolded proteins in the ER, many stimuli that perturb ER homeostasis can cause protein misfolding. The mechanisms by which some of these agents can activate the UPR are known. Tunicamycin inhibits N-linked protein glycosylation and castanospermine inhibits N-linked oligosaccharide trimming, thus both drugs alter protein folding and trafficking. Nutrient depletion presumably lowers ATP levels to disrupt chaperone-dependent protein folding reactions. Thapsigargin disrupts calcium storage which is required for protein chaperone function and protein folding. Dithiothreitol disrupts oxidative protein folding to cause protein misfolding. Brefeldin A activates the UPR by inhibiting ER to Golgi transport. In type 2 diabetes increased proinsulin biosynthesis leads to UPR due to excess demands on the folding machinery. In the neurodegenerative disorders, Alzheimer's disease and Parkinson's disease, abnormal protein aggregates are observed and are implicated in the pathogenesis of disease. Free fatty acids and reactive oxygen species can activate the UPR, though the exact mechanisms are not defined. Pharmacologic inhibition of proteasomal degradation causes misfolded proteins to accumulate.
Inositol requiring (IRE)-1α is a dual function type I transmembrane protein with Ser/Thr protein kinase and endoribonuclease activities (Key Message Box 2
). It is the most archaic and conserved branch of the UPR. Initially the Ire1
gene was isolated on screening of Saccharomyces cerevisiae
genetic mutants for inositol autotrophy. It was characterized as essential for expression of ER chaperones and adaptation to ER stress in yeast [23
]. Subsequently two mammalian genes, Ire1α
were identified, with significant homology with yeast at the C-terminus, and divergence at the N-terminus [131
is widely expressed, and Ire1β
expression is limited to intestinal epithelium, though it may be expressed in other cell types [9
]. IRE1α protein has an amino-terminal ER lumenal domain, a transmembrane region, and a carboxy-terminal domain that contains both kinase and endoribonuclease catalytic activities. Upon dissociation from BiP binding, IRE1α dimerizes, transautophosphorylates and activates the endoribonuclease (RNase). The RNase activity of IRE1α is essential for the execution of the UPR. IRE1 endoribonuclease activity splices the transcription factor Hac1
mRNA in yeast and Xbp1
mRNA in mammals. Activated IRE1α also recruits tumor necrosis factor receptor (TNFR)-associated factor-2)(TRAF2) and apoptosis signaling kinase 1 (ASK1) to mediate the activation of c-jun N-terminal kinase (JNK) and activation of nuclear factor kappa B ((NFκB) [53
]. The IRE1α RNase activity is also implicated in the degradation of several cellular mRNAs, a process named regulated IRE1-dependenet decay (RIDD) [51
], including proinsulin mRNA and IRE1α
mRNA itself [42
]. Microsomal triglyceride transfer protein (MTTP) mRNA is degraded by IRE1β [55
]. X-box-binding protein 1 (Xbp1
) mRNA is the only characterized target of IRE1α RNase activity that is subject to ligation; in yeast the ligation is performed by the tRNA ligase Rlg1p and the mammalian ligase is yet to be identified [126
mRNA undergoes unconventional (cytoplasmic, spliceosome-independent) splicing to generate a potent basic leucine zipper domain (bZIP)-containing transcription factor [134
]. Removal of 26 nucleotides from the mammalian Xbp1
mRNA results in a translational frame switch, encoding a protein containing 376 amino acids, as compared with 261 amino acids encoded by the unspliced mRNA. Both forms of XBP1 can bind the ER stress element (ERSE); however, spliced XBP1 (sXBP1) activates the UPR far more potently than its unspliced form [156
]. Furthermore, upon ER stress, Xbp1
mRNA expression is enhanced by ATF6α, providing additional substrate for IRE1α to splice into the more transcriptionally active form [73
]. The unspliced XBP1 protein is unstable in the cell and can heterodimerize with ATF6 and sXBP1 to promote their proteasomal degradation [157
]. Spliced XBP1 can bind to three cis-acting elements, ERSE, unfolded protein response element (UPRE) and ERSE-II [151
]. Though ATF6α can bind some of these elements as well, sXBP1 homodimers and sXBP1-ATF6 heterodimers bind to ERSE activating the transcription of several XBP1 target genes, including Herp [151
], EDEM (ER degradation-enhancing alpha-mannosidase-like protein) [155
], and MDG1/ERdj4 [62
]. Transcriptional targets of sXBP1 include genes that encode functions in ER protein folding and quality control, ER-associated degradation (ERAD) and ER biogenesis [128
Key Message Box 2.
Inositol requiring 1α (IRE1α) is conserved from yeast to higher organisms. It is activated upon dissociation from BiP by dimerization and transautophosphorylation to elicit its endoribonuclease activity. IRE1α initiates cleavage of 26 nucleotides from X-box binding protein-1 (XBP1) mRNA in the cytosol. Spliced XBP 1 mRNA is ligated by an unidentified ligase and encodes for a potent transcription factor (sXBP1) that activates a subset of UPR genes involved with ER biogenesis and ER-associated degradation (ERAD). IRE1α also recruits the adaptor protein TRAF2 (tumor necrosis factor receptor (TNFR)-associated factor-2), leading to activation of c-jun N-terminal kinase (JNK). IRE1α-dependent JNK activation has been linked to insulin resistance and apoptosis. Caspase 12 may be recruited by the IRE1α-TRAF2 complex in ER stress induced apoptosis in mice.
The genetic absence of either IRE1α or XBP1 in the mouse results in embryonic lethality after gestational day ~11.5 that is associated with fetal liver hypoplasia in these embryos [113
]. In addition, hepatocyte-specific expression of a transgene encoding spliced XBP1 was able to rescue embryonic lethality associated with Xbp1
deletion, suggesting the embryonic lethality is due to a requirement for spliced XBP1 in hepatocytes [70
]. However, it was recently reported that Ire1α
deletion also causes placental defects that were proposed to be responsible for the embryonic lethality of conditional Ire1α
-null mice [56
deficiency is non-lethal, though the mice are more susceptible to dextran sulfate-induced colitis and demonstrate enhanced enterocyte chylomicron secretion secondary to increased expression of MTTP [9
ATF6α is a type II ER transmembrane protein, with a cytoplasmic N-terminus that contains a basic leucine zipper motif that functions as a transcription factor following regulated intramembrane proteolysis (RIP) in ER-stressed cells [46
] (Key Message Box 3
). The ER resident form is 90 kDa and has two Golgi localization sequences (GLS) that are masked by BiP binding [123
]. Upon dissociation from BiP, ATF6α translocates to the Golgi and its C-terminal half is cleaved by site-1 protease [152
]. The membrane anchored N-terminus is cleaved by site-2 protease and a 50 kDa protein is released into the cytosol. The 50 kDa N-terminus protein translocates to the nucleus to activate transcription by binding to the ATF (activating transcription factor)/cAMP response element (CRE) and ER stress response element (ERSE). Transcriptional induction of ER chaperone genes such as BiP and GRP94 (glucose regulated protein 94) is mostly mediated by ATF6α binding to the cis-acting ERSE consensus sequence CCAAT-N9-CCACG. ATF6α also transcriptionally activates ERAD components by heterodimerization with sXBP1. Two Atf6
genes, alpha(α) and beta(β) are expressed ubiquitously, with no obvious phenotype in mice lacking either Atf6α
individual isoform [150
]. However, challenge of Atf6α
-null mice, but not Atf6β
-null mice, with ER stress in the liver leads to hepatosteatosis and death [119
]. Although ATF6α mediates UPR gene induction in response to ER stress, the gene targets for ATF6β have not been identified. Interestingly, the combined deletion of Atf6α
causes a very early embryonic lethality, suggesting these genes provide an essential complementary function(s) in early mammalian development.
Key Message Box 3.
Activating transcription factor 6 α (ATF6α) is a basic leucine zipper domain (bZIP) family transcription factor that upon release from BiP transits to the Golgi compartment where it is processed by regulated intramembrane proteolysis. Cleaved ATF6α activates a subset of UPR genes, including XBP1, ER protein chaperones and CHOP.
PERK is a type I ER resident protein kinase that, upon ER stress is activated to phosphorylate the alpha subunit of eukaryotic translation-initiation factor 2 (eIF2α) on serine residue 51 [45
] (Key Message Box 4
). This leads to a rapid reduction in the initiation of mRNA translation thus reducing the load of new client proteins that require folding in the ER. Attenuation of translation promotes adaptation as PERK-null cells, as well as cells with Ser51Ala mutation at the PERK phosphorylation site in eIF2α, are unable to decrease protein synthesis upon ER stress and exhibit enhanced cell death [121
]. PERK dependent eIF2α phosphorylation decreases synthesis of cyclin D1 to mediate cell cycle arrest in stressed cells [13
]. PERK is expressed ubiquitously with highest levels in the pancreas [124
]. Loss of function mutations in PERK are the cause of Wolcot-Rallison syndrome in humans which stems from a loss of insulin production and beta cell failure [25
]. PERK deletion in mice also causes pancreatic insufficiency most prominent in the beta cell at 4 weeks of age and in acinar cells at 6-8 weeks of age [44
]. Further analysis of the role of eIF2α phosphorylation, by characterization of mice with Ser51Ala homozygous mutation in eIF2α demonstrated that eIF2α phosphorylation prevents neonatal lethality due to hypoglycemia and preserves pancreatic beta cell mass [121
]. In the absence of eIF2α phosphorylation, beta cells exhibit a higher rate of protein synthesis thus increasing the demand for protein folding. This includes increased proinsulin folding and misfolding; the later leads to accumulation of misfolded protein in the ER, and thereby enhanced oxidative stress [3
]. These observations lead to the notion that inhibition of translation by PERK-mediated eIF2α phosphorylation in response to ER stress is required for cell survival by limiting the protein-folding load thus preventing accumulation of misfolded proteins, and thereby the subsequent additional stress of oxidative protein folding. Phosphorylation of eIF2α can also be effected by three other kinases that respond to cellular and environmental stress; these kinases are protein kinase RNA-activated (PKR), heme-regulated inhibitor (HRI), and GCN2 kinase.
Key Message Box 4.
Protein kinase RNA (PKR)-like ER kinase (PERK) is activated by dimerization following dissociation from BiP in the stressed ER. It is activated by transautophosphorylation, leading to subsequent phosphorylation of its only known physiologically important substrate eukaryotic translation initiation factor α (eIF2α) on serine residue 51. This results in translational halt and reduces the client load of new proteins in the ER. Selective translation of activating transcription factor 4 (ATF4), and its downstream target C/EBP homologous protein (CHOP) occurs. ATF4 induces the expression of amino acid biosynthesis transporters and antioxidant stress response genes. CHOP activates the expression of GADD34 (growth arrest and DNA damage 34), which along with protein phosphatase 1 dephosphorylates eIF2α leading to resumption of mRNA translation.
Transcriptional profiling of homozygous Ser51Ala eIF2α mutant and wildtype cells demonstrated that the expression of several genes is dependent on PERK-mediated eIF2α phosphorylation [121
]. Key among these is activating transcription factor 4(ATF4) that is up regulated at the mRNA translational level upon eIF2α phosphorylation [43
translation is repressed by the presence of two upstream open reading frames (uORFs). Upon eIF2α phosphorylation ribosomes scan through the upstream uORFs to initiate Atf4
]. ATF4 transcriptionally activates numerous ER-stress response genes that promote adaptation, as inhibition of transcription in ER-stressed cells impairs viability. ATF4 induces genes responsible for the antioxidant response, amino acid metabolism, and apoptosis, including the C/EBP homologous protein (CHOP). CHOP, also known as growth arrest and DNA damage-inducible gene (GADD)153, was identified as a stress-induced negative regulator of other C/EBP-family proteins [6
]. Subsequently, CHOP expression was found to be potently induced by ER-stress inducing agents. Mice deleted in Chop
develop normally; however cells isolated from these mice are resistant to ER-stress induced cell death, implicating a requirement for CHOP in the apoptotic response to ER stress [163
]. CHOP upregulates the expression of GADD34, which complexes with protein phosphatase 1 (PP1c) to target dephosphorylation of eIF2α, thus forming a negative feedback loop [96
]. Phosphorylation of eIF2α with subsequent translation attenuation reduces synthesis of IκB with consequent activation of the transcription factor NFκB as part of the response to stress [60