REVIEW ARTICLE ER stress and diseases Hiderou Yoshida 1,2 1 Department of Biophysics, Graduate School of Science, Kyoto University, Japan 2 PRESTO-SORST, Japan Science and Technology Agency, Japan Keywords conformational disease; cytoplasmic splicing; ER stress response; ER-associated protein degradation (ERAD); Golgi stress response Correspondence H. Yoshida, Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan Fax: +81 75 753 3718 Tel: +81 75 753 4201 E-mail: hide@biophysics.mbox.media. kyoto-u.ac.jp (Received 11 September 2006, revised 14 November 2006, accepted 8 December 2006) doi:10.1111/j.1742-4658.2007.05639.x Proteins synthesized in the endoplasmic reticulum (ER) are properly folded with the assistance of ER chaperones. Malfolded proteins are disposed of by ER-associated protein degradation (ERAD). When the amount of unfolded protein exceeds the folding capacity of the ER, human cells acti- vate a defense mechanism called the ER stress response, which induces expression of ER chaperones and ERAD components and transiently attenuates protein synthesis to decrease the burden on the ER. It has been revealed that three independent response pathways separately regulate induction of the expression of chaperones, ERAD components, and trans- lational attenuation. A malfunction of the ER stress response caused by aging, genetic mutations, or environmental factors can result in various dis- eases such as diabetes, inflammation, and neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease, and bipolar disorder, which are collectively known as ‘conformational diseases’. In this review, I will summarize recent progress in this field. Molecules that regulate the ER stress response would be potential candidates for drug targets in various conformational diseases. Abbreviations AIGP, axotomy-induced glyco ⁄ Golgi protein; APP, amyloid precursor protein; ASK1, apoptosis signal-regulating kinase 1; ATF, activating transcription factor; BAK, Bcl-2 homologous antagonist ⁄ killer; BAP, BiP-associated protein; Bap31, B cell receptor-associated protein 31; Bax, Bcl2-associated X protein; Bcl2, B cell leukemia 2; BI-1, Bax inhibitor 1; Bim, Bcl2-interacting mediator of cell death; BiP, binding protein; bZIP, basic leucine zipper; c-Abl, Abelson murine leukemia viral oncogene homolog 1; C ⁄ EBP, CCAAT ⁄ enhancer-binding protein; CHOP, C ⁄ EBP- homologous protein; CREB, cAMP response element-binding protein; CREBH, cAMP response element-binding protein H; CReP, constitutive repressor of eIF2a phosphorylation; DAP, death-associated protein; Der1, degradation in the endoplasmic reticulum protein 1; Derlin-1, Der1- like protein 1; Doa10, degradation in the endoplasmic reticulum protein 10; DR5, death receptor 5; EDEM, ER degradation enhancing a)mannosidase-like protein; eIF2 a, a-subunit of eukaryotic translational initiation factor 2; ER, endoplasmic reticulum; ERAD, ER-associated degradation; ERdj, ER dnaJ; ERO1, ER oxidoreductin; ERp72, ER protein 72; ERSE, ER stress response element; FKBP13, FK506-binding protein 13; GADD, growth arrest and DNA damage; gp78, glycoprotein 78; GRP, glucose-regulated protein; HEDJ, human ER-associated dnaJ; HIAP2, human inhibitor of apoptosis 2; HRD1, HMG-CoA reductase degradation protein 1; HSP, heat shock protein; IAP, inhibitor of apoptosis; IDDM, insulin-dependent diabetes mellitus; IRE1, inositol requirement 1; JNK, Jun kinase; Keap1, Kelch-like Ech-associated protein 1; LZIP, basic leucine zipper protein; NIDDM, noninsulin-dependent diabetes mellitus; NOXA, neutrophil NADPH oxidase factor; Npl4, nuclear protein localization 4; NRF, nuclear respiratory factor; ORP150, oxygen-regulated protein 150; OS9, osteosarcoma 9; p58IPK, 58 kDa-inhibitor of protein kinase; pATF6(N), the nuclear form of ATF6 protein; PDI, protein disulfide isomerase; PERK, PRKR-like endoplasmic reticulum kinase; PKR, double stranded RNA-dependent protein kinase; PLP1, proteolipid protein 1; polyQ, polyglutamine; PrP, pion protein; PrP c , cellular PrP; PrP Sc , scrapie PrP; PS1, presenillin 1; PUMA, p53 up-regulated modulator of apoptosis; pXBP1(S), the spliced form of XBP1 protein; pXBP1(U), the unspliced form of XBP1 protein; RIP, regulated intramembrane proteolysis; RseA, regulator of s E ; S1P, site 1 protease; S2P, site 2 protease; SAPK, stress-activated protein kinase; SEL1, suppressor of lin12-like; SREBP, sterol response element-binding protein; TDAG51, T cell death-associated gene 51; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TRAF2, TNF receptor-associated factor 2; TRB3, Tribbles homolog 3; UBC6, ubiquitin conjugase 6; UBC7, ubiquitin conjugase 7; UBE1, ubiquitin-activating enzyme 1; UBE2G2, ubiquitin-activating enzyme 2G2; UBX2, UBX domain-containing protein 2; UCH-L1, ubiquitin C-terminal esterase L1; Ufd1, ubiquitin fusion degradation protein 1; UPRE, unfolded protein response element; VCP, valocin-containing protein; WFS1, Wolfram syndrome 1; XBP1, x-box binding protein 1; XIAP, inhibitor of apoptosis, x-linked; XTP3B, XTP3-transactivated gene B. 630 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS Introduction The endoplasmic reticulum (ER) is an organelle where secretory or membrane proteins are synthesized. Nas- cent proteins are folded with the assistance of molecu- lar chaperones and folding enzymes located in the ER (collectively called ER chaperones), and only correctly folded proteins are transported to the Golgi apparatus (Fig. 1). Unfolded or malfolded proteins are retained in the ER, retrotranslocated to the cytoplasm by the machinery of ER-associated degradation (ERAD), and degraded by the proteasome. ER chaperones and ERAD components are constitutively expressed in the ER to deal with nascent proteins. When cells synthes- ize secretory proteins in amounts that exceed the capa- city of the folding apparatus and ERAD machinery, unfolded proteins are accumulated in the ER. Unfol- ded proteins expose hydrophobic amino-acid residues that should be located inside the protein and tend to form protein aggregates. Protein aggregates are so toxic that they induce apoptotic cell death and cause ‘conformational diseases’ such as neurodegenerative disorders and diabetes mellitus. To alleviate such a stressful situation (ER stress), eukaryotic cells activate a series of self-defense mechanisms referred to collec- tively as the ER stress response or unfolded pro- tein response [1–4]. The mammalian ER stress response consists of four mechanisms. The first is attenuation of protein synthe- sis, which prevents any further accumulation of un- folded proteins. The second is the transcriptional induction of ER chaperone genes to increase folding capacity, and the third is the transcriptional induction of ERAD component genes to increase ERAD ability. The fourth is the induction of apoptosis to safely dis- pose of cells injured by ER stress to ensure the survival of the organism. In this article, I will describe the basics of the mam- malian ER stress response that are essential to under- standing conformational diseases. I will review hot topics such as ERAD, regulated intramembrane pro- teolysis (RIP) and cytoplasmic splicing, and briefly summarize the ER stress-related diseases. ER stress-inducing chemicals Chemicals such as tunicamycin, thapsigargin, and dithiothreitol are usually used to evoke ER stress in cultured cells or animals for experimental purposes. I will briefly summarize the ER stress-inducing chemicals below. The first group of ER stressors comprises glycosyla- tion inhibitors. Most of the proteins synthesized in the ER are N-glycosylated, and the N-glycosylation is cytoplasm ER ER chaperone degraded ribosome mRNA unfolded protein aggregation ER stresss nascent protein Golgi apparatus apoptosis folding disease ERAD translational attenuation Fig. 1. Mammalian ER stress response. An accumulation of unfolded proteins in the ER evokes ER stress, and cells induce the ER stress response to cope. The mammalian ER stress response consists of four mechanisms: (1) translational attenuation; (2) expression of ER chap- erones; (3) enhanced ERAD; (4) apoptosis. H. Yoshida ER stress and diseases FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 631 often essential for protein folding. Thus, chemicals that disturb N-glycosylation have the potential to induce ER stress. Tunicamycin is an antibiotic produced by Streptomyces lysosuperificus that inhibits N-glycosyla- tion by preventing UDP-GlcNAc–dolichol phosphate GlcNAc-phosphate transferase activity [5,6]. 2-Deoxy- d-glucose is also used to inhibit N-glycosylation [7], but is less efficient than tunicamycin. Another class of ER stressors is Ca 2+ metabolism disruptors. As the concentration of Ca 2+ ion in the ER is kept at a high level and ER chaperones such as BiP require Ca 2+ ions, chemicals that perturb Ca 2+ metabolism in the ER induce ER stress. Ca 2+ ionoph- ores such as A23187 and the Ca 2+ pump inhibitor, thapsigargin, are often used to evoke ER stress [5,8]. The third category of ER stressors is reducing agents. As the lumen of the ER is highly oxidative, proteins synthesized there can form intermolecular or intramolecular disulfide bonds between their cysteine residues. As the formation of disulfide bonds is important for the folding of secretory proteins, redu- cing agents that disrupt disulfide bonds evoke ER stress. Dithiothreitol and 2-mercaptoethanol are often used to this end [9,10]. Hypoxia is also known to induce ER stress, although the underlying mechanism is unknown. It is speculated that a decrease in glucose concentration induced by hypoxia (because hypoxia induces glyco- lytic enzymes to sustain ATP production and then cells consume glucose) inhibits N-glycosylation, leading to ER stress [11]. ER chaperones ER chaperones include molecular chaperones and fold- ing enzymes located in the ER, which are responsible for the folding of nascent proteins [4,12]. They are also involved in the unfolding of malfolded proteins in ERAD. In this section, I will review mammalian ER chaperones, focusing on recent discoveries. Binding protein (BiP) ⁄glucose-regulated protein (GRP)78 is a well-known ER chaperone that belongs to the heat shock protein (HSP)70 family. BiP binds to the hydro- phobic region of unfolded proteins via a substrate- binding domain and facilitates folding through conformational change evoked by the hydrolysis of ATP by the ATPase domain. Oxygen-regulated pro- tein (ORP)150 ⁄ GRP170 is an ER chaperone belonging to the HSP110 family (a HSP70 subfamily), and facili- tates protein folding via a mechanism similar to that for BiP. It was originally identified as a pro- tein expressed in response to hypoxia. ER dnaJ (ERdj)1, ERdj3 ⁄ human ER-asociated dnaJ (HEDJ), ERdj4, ERdj5, SEC63, and p58IPK are ER chaper- ones belonging to the HSP40 family, and modulate the functions of BiP by regulating its ATPase activity as a cochaperone. BiP-associated protein (BAP), which is a member of the GrpE family, also modulates the func- tions of BiP by enhancing nucleotide exchange. GRP94 is an ER chaperone belonging to the HSP90 family, and facilitates folding through the hydrolysis of ATP. FKBP13 is a peptidyl-prolyl isomerase belonging to the FKBP family. These ER chaperones are involved in the general folding process of secretory proteins. Calnexin and calreticulin are ER chaperones specif- ically involved in the folding of glycoprotein. High- mannose type oligosaccharide is attached en bloc to most proteins synthesized in the ER, and then trimmed sequentially (Fig. 2). When two glucose residues are trimmed by glucosidase I or II and the protein con- tains only one glucose residue, calnexin and calreticulin bind and fold the client protein. When the last glucose residue is trimmed by glucosidase II, the client is released from calnexin and calreticulin, and binds to UDP-glucose–glycoprotein glucosyltransferase. If the protein is folded, it is released from the enzyme and transported to the Golgi apparatus. If it is not folded, UDP-glucose–glycoprotein glucosyltransferase attaches one glucose residue and returns it to calnexin and cal- reticulin. This folding process is called the calnexin cycle [13]. Calnexin and calreticulin share a similar molecular structure and function, although they are transmembrane and luminal proteins, respectively. Numerous folding enzymes are involved in the forma- tion of disulfide bonds in the ER, such as protein disul- fide isomerase (PDI), ERp72, ERp61, GRP58 ⁄ ERp57, ERp44, ERp29, and PDI-P5. These folding enzymes oxidize cysteine residues of nascent proteins and help proteins to form correct disulfide bonds. Reduced fold- ing enzymes are reoxidized by ER oxidoreductin (ERO1), which can use molecular oxygen as a terminal electron acceptor [14]. ERAD Unfolded or malfolded proteins are trapped by the ERAD machinery and transported to the cytoplasm [15–17]. Retrotranslocated proteins are ubiquitinated and degraded by the proteasome in the cytosol. Thus, the process of ERAD can be divided into four steps, recognition, retrotranslocation, ubiquitination, and degradation (Fig. 3). As ERAD is one of the hottest topics in the study of ER stress, I will sum- marize our current understanding of mammalian ERAD systems. ER stress and diseases H. Yoshida 632 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS Recognition During the calnexin cycle, the oligosaccharide of nascent polypeptides contains nine mannose residues. When one mannose residue is trimmed by a-mannosidase I, nas- cent polypeptides with eight mannose residues are released from calnexin or calreticulin and bind to ER degradation-enhancing a-mannosidase-like pro- tein (EDEM) (Fig. 2), which discriminates unfolded proteins from folded proteins [18–22]. There are three genes for EDEM, and both EDEM1 and EDEM2 are involved in ERAD. EDEM1 is an ER membrane protein, whereas EDEM2 and EDEM3 are luminal pro- teins [23–25]. All EDEMs contain the mannosidase-like Fig. 3. Mammalian ERAD machinery. Unfolded proteins released from the calnexin cycle are captured by a recognition complex containing EDEM and OS9, moved to the cytosol through retrotranslocation machinery, polyubiquitinated by the E1–E2–E3 system, and degraded by the proteasome. The precise function of each ERAD component is described in the text. Glucose Mannose GlucNAc Glc3Man9GlcNAc2-unfolded protein glucosidase I, II CNX / CRT glucosidase II UDP-GP Glc1Man9GlcNAc2-unfolded protein Man9GlcNAc2-unfolded protein Man9GlcNAc2-folded protein Man8GlcNAc2-folded protein Glc1Man8GlcNAc2-unfolded protein Man8GlcNAc2-unfolded protein ERAD Golgi apparatus ER EDEM mannosidase I mannosidase I Fig. 2. Folding and degradation of glycoprotein. Sugar chains of nascent glycoproteins synthesized in the ER are trimmed by glucosidase I or II, and polypeptides containing one glucose residue are folded by the calnexin cycle. One mannose residue of polypeptides that is unable to be folded by the calnexin cycle is removed by mannosidase I, and then the polypeptides are recognized by EDEM and degraded by ERAD. H. Yoshida ER stress and diseases FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 633 domain, which may be responsible for recognition of mannose residues. Osteosarcoma 9 (OS9) and XTP3-transactivated gene B (XTP3B) are other ERAD components respon- sible for the recognition of unfolded proteins [26–28]. OS9 specifically binds to unfolded glycoproteins con- taining eight (or five) mannose residues. OS9 also binds to unglycosylated unfolded proteins, suggesting that it plays a critical role in the recognition of both glycosylated and unglycosylated proteins. OS9 and XTP3B [29] contain the mannose-6-phosphate recep- tor-like domain, which may be critical to the recogni- tion of mannose residues. Retrotranslocation Nascent glycoproteins recognized by EDEM and OS9 as malfolded are destined for the retrotranslocation machinery [30,31]. Before their retrotranslocation, nas- cent proteins associate with PDI and BiP to cleave disulfide bonds and to unfold the partially folded struc- ture, respectively [32–34]. Although unfolded ER pro- teins were previously speculated to be retrotranslocated through the translocon containing Sec61, the molecular structure of the retrotranslocation machinery remains elusive. Derlin-1 is a mammalian homolog of yeast Der1, and thought to be a critical component of the machinery. Derlin-1 may form a retrotranslocation channel in the ER membrane and associates with p97 through an adaptor protein, valocin-containing protein (VCP)-interacting membrane protein 1 (VIMP1) [35]. Derlin-2 and Derlin-3, other Der1 homo- logs, are also involved in ERAD [35–37], although the exact underlying mechanism is still unclear. p97 ⁄ cdc48 ⁄ VCP is a cytosolic AAA-ATPase and recruits unfolded ER proteins to the cytosol [38,39]. Ubiquitin fusion degradation protein 1 (Ufd1) and nuclear protein localization 4 (Npl4) bind to p97 as a cofactor and help p97 to extract unfolded proteins. The polypeptide portion of unfolded proteins interacts with p97, whereas the polyubiquitin chains attached to them are recognized by both p97 and Ufd1 and may activate the ATPase activity of p97 [40–42]. Ubiquitination Retrotranslocated (or retrotranslocating) proteins are ubiquitinated by the E1–E2–E3 ubiquitin system. Ubiquitin is first conjugated to enzyme E2 by enzyme E1, and then transferred to ERAD substrates by enzyme E3. HMG-CoA reductase degradation pro- tein 1 (HRD1), gp78, and TEB4 ⁄ Doa10 are mem- brane-anchored E3 ligases involved in ERAD [43–46], whereas ubiquitin conjugase (UBC)6 and UBE2- G2 ⁄ UBC7 are E2 conjugase involved in ERAD. UBE1 is an E1 ubiquitin-activating enzyme that is ubiqui- tously involved in protein degradation by the protea- some. HRD1 shows a preference for substrates that contain misfolded luminal domains, whereas Doa10 prefers transmembrane proteins containing misfolded cytosolic domains (Doa10 also ubiquitinates cytosolic proteins). These two distinctive ERAD systems are called ERAD-L (luminal ERAD) and ERAD-C (cyto- solic ERAD) [47,48]. EDEM and OS9 are thought to specifically recognize ERAD-L substrates. Actually, they form distinct ubiquitin–ligase complexes: the HRD1 complex contains HRD1, OS9, HRD3, Derlin- 1, USA1, UBX2 and p97, whereas the Doa10 complex consists of Doa10, UBX2 and p97 [49–51]. Substrates containing misfolded transmembrane domains skip the interaction to OS9 and HRD3, and directly associate with the HRD1 complex, which is called the ERAD-M pathway [49]. However, there are a lot of other E3 ligases involved in the ERAD, and they preferentially recognize distinct ERAD substrates. FBX2 (F-box only protein 2) is another E3 ligase that specifically recognizes N-glycos- ylated proteins located in the cytosol [52,53]. Parkin is an E3 involved in Parkinson’s disease (see below). In the case of cystic fibrosis transmembrane conductance regulator, its folding status is sequentially monitored by the two E3 ligase complexes, such as the RMA1 complex and the CHIP (C-terminus of Hsc70-interact- ing protein) complex [54]. Molecules other than E1–E2–E3 enzymes are also involved in ubiquitination. UBX2 binds to both p97 and E3 ligases such as HRD1 and Doa10 to recruit E3 to p97 [55], whereas gp78 directly associates with p97 [56]. The ubiquitin-domain protein, Herp (homo- cysteine-induced endoplasmic reticulum protein), associates with a complex containing HRD1, p97, Derlin-1, and VCP-interacting membrane pro- tein [57,58]. Degradation Retrotranslocated and ubiquitinated proteins are deglycosylated by peptide–N-glycanase before their degradation by the proteasome, because bulky glycan chains may hamper the entrance of substrates into the proteasome pore. As peptide–N-glycanase is asso- ciated with Derlin-1, it is possible that deglycosylation occurs coretrotranslocationally [59]. Deglycosylated substrates are then delivered to the proteasome. Dsk2 and Rad23 facilitate this delivery of ERAD substrates [60]. ER stress and diseases H. Yoshida 634 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS Response pathways for ER stress The mammalian ER stress response has four mecha- nisms: (1) translational attenuation; the enhanced expression of (2) ER chaperones and (3) ERAD components; (4) induction of apoptosis. These four responses are regulated by the regulatory pathways as described below (Fig. 4). PERK pathway PERK is a type I transmembrane protein located in the ER, which senses the accumulation of unfolded pro- teins in the ER lumen [61–63]. The luminal portion of PERK is involved in sensing unfolded proteins, whereas the cytoplasmic portion contains a kinase domain. In the absence of ER stress, BiP binds to the luminal domain of PERK and keeps it from being acti- vated (Figs 4 and 5A). In response to ER stress, BiP is released from PERK, and PERK is activated through oligomerization and trans-phosphorylation [64]. Activa- ted PERK phosphorylates and inactivates the a-subunit of eukaryotic translational initiation factor 2 (eIF2a), leading to translational attenuation. The phosphoryla- tion of PERK is transient as the protein is dephosphor- ylated by specific phosphatases such as CReP (constitutive repressor of eIF2a phosphorylation), pro- tein phosphatase 2C-GADD34, and p58IPK. CReP is constitutively expressed, whereas the expression of GADD34 and p58IPK is induced on ER stress by PERK and activating transcription factor (ATF)6 pathways, respectively. Interestingly, translation of the transcription factor ATF4 is up-regulated by eIF2a-mediated translational attenuation. There are several small ORFs in the 5¢-UTR of ATF4 mRNA (Fig. 5B). The ribosome first binds to a 5¢-cap structure, slides on the ATF4 mRNA, and then starts translation at the small ORFs with unphosphorylated (active) eIF2a. As the ribosome is released from the ATF4 mRNA upon the termin- ation of translation at the stop codon of small ORFs, the ATF4 ORF cannot be translated in the absence of ER stress. In contrast, as phosphorylated (inactive) eIF2a cannot start translation, the probability that the ribosome reaches the ATF4 ORF is increased in the presence of ER stress. Thus, the translation of ATF4 ER ER stresss nucleus AARE ERSE UPRE 4 SS 66 ER chaperone ERAD component CHOP anti-oxidative stress translation 6 GA 6 6 6 S2P S1P ATF6 PERK pATF6(N) P eIF2α GADD34 p58IPK CReP translational attenuation ? 4 ATF4 IRE1α S U DBD AD XBP1 pre-mRNA DBD-AD mature mRNA NF-Y pXBP1(S) pXBP1(U) Fig. 4. Mammalian response pathways for ER stress. Three response pathways (PERK, ATF6, and IRE1 pathways) regulate the mammalian ER stress response. PERK, a transmembrane kinase, phosphorylates eIF2a to attenuate translation, and to up-regulate expression of ATF4, leading to enhanced transcription of target genes such as CHOP. ATF6, a transmembrane transcription factor, is translocated to the Golgi apparatus and cleaved by proteases such as S1P and S2P, leading to enhanced transcription of ER chaperone genes. IRE1, a transmem- brane RNase, splices XBP1 pre-mRNA, and pXBP1(S) translated from mature XBP1 mRNA activates transcription of ERAD component genes. H. Yoshida ER stress and diseases FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 635 is remarkably enhanced in response to ER stress. The targets of ATF4 include CHOP (C ⁄ EBP homology protein), a transcription factor involved in the induc- tion of apoptosis, and proteins involved in amino-acid metabolism such as asparagine synthetase or those involved in resistance to oxidative stress [65]. eIF2a is also phosphorylated by other kinases, such as dsRNA-dependent protein kinase (PKR), GCN2 (general control of amino-acid synthesis 2) and heme- regulated translational inhibitor. These kinases are activated by viral infections, amino-acid starvation, and heme deficiency, respectively, indicating that trans- lational attenuation and ATF4 induction is induced by not only ER stress but also these physiological situa- tions. Thus, the cellular response mediated by the phosphorylation of eIF2a is called the integrated stress response and is essential for cell survival [66]. ATF6 pathway There is another sensor molecule, ATF6, on the ER membrane [67–70]. ATF6 is a type II transmembrane protein, the luminal domain of which is responsible for the sensing of unfolded proteins. The cytoplasmic portion of ATF6 has a DNA-binding domain con- taining the basic-leucine zipper motif (bZIP) and a PERK IRE1 BiP BiP BiP BiP phosphorylation oligomerization A ER ATF6 GLS BiP GLS BiP GLS translocation to Golgi B ATF4 mRNA ATF4 coding region small ORFs CAP - ER stress ATF4 mRNA CAP eIF2α small peptide + ER stress ATF4 mRNA CAP phosphorylated eIF2α ATF4 protein ribosome eIF2α Fig. 5. Activation of the PERK pathway. (A) Activation of PERK, IRE1, and ATF6. In the absence of ER stress, BiP prevents PERK, IRE1, and ATF6 from being activated by binding to these sensors. BiP prevents the activation of IRE1 and PERK by keeping them from being oligomerized, whereas BiP inhibits the translocation of ATF6 by mask- ing the Golgi-localization signal (GLS). When BiP is sequestered from sensors by unfol- ded proteins, these sensor molecules are activated. (B) Regulation of ATF4 expres- sion. In the absence of ER stress, most of the eIF2a is active (not phosphorylated), and translation starts at the small ORFs, leading to the release of ribosomes before they reach the ATF4 ORF. Upon ER stress, most of the eIF2a becomes inactive (phosphoryl- ated), and translation rarely starts at the small ORFs, thus ribosomes can reach the ATF4 ORF and induce translation of ATF4 protein. ER stress and diseases H. Yoshida 636 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS transcriptional activation domain. In the absence of ER stress, BiP binds to the luminal domain of ATF6 and hinders the Golgi-localization signal, leading to inhibition of ATF6 translocation (Fig. 5A) [71–75]. In response to the accumulation of unfolded proteins, BiP dissociates from ATF6, and ATF6 is moved to the Golgi apparatus by vesicular transport (Fig. 4). In the Golgi apparatus, ATF6 is sequentially cleaved by a pair of processing proteases called site 1 protease (S1P) and site 2 protease (S2P), and the resultant cytoplas- mic portion of ATF6 [pATF6(N)] translocates into the nucleus. In the nucleus, pATF6(N) binds to a cis-act- ing element, the ER stress response element (ERSE), and activates the transcription of ER chaperone genes such as BiP, GRP94 and calreticulin [68]. The consen- sus sequence of the ER stress response element is CCAAT-(N9)-CCACG, and ATF6 binds to the CCACG portion, whereas a general transcription fac- tor, NF-Y (nuclear factor Y), binds to the CCAAT portion. The cleavage of ATF6 is unique, especially as the second cleavage by S2P occurs in the transmembrane region [75]. This process is called regulated intramem- brane proteolysis (RIP), which is well conserved from bacteria to mammals (Fig. 6). The most characterized substrate of RIP is sterol response element-binding protein (SREBP) [75]. SREBP is a transcription factor that is located in the ER membrane like ATF6. Upon a deficiency of sterol, SREBP is transported to the Golgi apparatus, cleaved by S1P and S2P, and activates the transcription of genes involved in the biosynthesis of sterol. Thus, the activation of ATF6 and SREBP is mainly regulated at the level of vesicular transport. The regulation of the transport of SREBP has been well characterized, and regulatory components such as the sensor-escort protein SCAP (SREBP cleavage-activa- ting protein) and the anchor protein INSIG (insulin- induced gene 1) have been identified [76]. There are two genes for ATF6, called ATF6a and ATF6b, which have a similar function and are ubiqui- tously expressed [68,77]. Recently, several bZIP tran- scription factors located in the ER and regulated by RIP have been reported. cAMP response element-bind- ing protein H (CREBH) is specifically expressed in liver, and processed by S1P and S2P in response to ER stress [78]. CREBH activates the transcription of acute-phase response genes involved in acute inflam- matory responses. OASIS (old astrocyte specifically induced substance) is also cleaved by S1P and S2P in response to ER stress in astrocytes and activates the transcription of BiP [79]. A spermatid-specific tran- scription factor, Tisp40 (transcript induced in spermio- genesis 40), is also severed by S1p and S2P and activates the transcription of EDEM [80]. These tissue- specific ATF6-like molecules may contribute to the ER stress response. Fig. 6. Molecules regulated by RIP. RIP is conserved from bacteria to mammals, and is involved in various biological processes. SREBP sen- ses a sterol deficiency and activates the transcription of genes involved in sterol synthesis. Cleavage of APP by RIP results in the production of antibody, which is responsible for the onset of Alzheimer’s disease. Notch is a cell surface protein that is cleaved by RIP upon binding Delta, leading to the activation of target genes involved in differentiation. Bacterial RseA protein anchors a transcription factor, r E , to keep it inactive. In response to accumulation of unfolded proteins in the periplasm, RseA is cleaved by RIP, leading to transcriptional activation of periplasmic chaperones. H. Yoshida ER stress and diseases FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 637 Luman ⁄ LZIP ⁄ CREB3 can be cut by S1P and S2P and activates the transcription of EDEM through a cis-acting element, unfolded protein response element (UPRE), although ER stress cannot induce Luman RIP [80–82]. CREB4 is transported to the Golgi apparatus in response to ER stress, is cleaved by S1P and S2P, and activates the transcription of BiP, although cleavage is not observed upon ER stress [83]. These ATF6-like molecules, which are insensitive to ER stress, might be activated in situations other than ER stress and activate transcription of ER chaperones. IRE1 pathway The third sensor molecule in the ER membrane is IRE1 (inositol requirement 1) [84–86]. The luminal domain of IRE1 is similar to that of PERK and involved in the sensing of unfolded proteins, whereas the cytoplasmic domain contains a kinase domain and an RNase domain. There are two genes for IRE1, IRE1a and IRE1b. Upon ER stress, BiP suppression of IRE1 activation is released, and IRE1 is activated through dimerization and transphosphorylation (Figs 4 and 5A) [64]. Activated IRE1a converts XBP1 (x-box binding protein 1) pre-mRNA into mature mRNA by an unconventional splicing mechanism [69,87]. As the DNA-binding domain and the activation domain are encoded in ORFs in XBP1 pre-mRNA, a pro- tein translated from pre-mRNA [pXBP1(U)] cannot activate transcription. In contrast, a protein translated from mature mRNA [pXBP1(S)] activates the tran- scription of ERAD component genes such as EDEM, HRD1, Derlin-2, and Derlin-3 through a cis-acting ele- ment, unfolded protein response element, as these two ORFs are joined in mature mRNA [37,88,89]. pXBP1(S) also induces the expression of proteins involved in lipid synthesis and ER biogenesis, as well as the expression of ER chaperones such as BiP, p58IPK, ERdj4, PDI-P5 and HEDJ [90,91]. Thus, XBP1 is essential to the function of cells that produce large amounts of secretory proteins such as pancreatic b-cells, hepatocytes, and antibody-producing plasma cells [92–95]. The splicing of XBP1 pre-mRNA by IRE1a is quite different from conventional mRNA splicing (Fig. 7A) [69]. Conventional splicing is catalyzed by the spliceo- some, and the consensus sequence at the exon–intron border is GU-AG or AU-AC (Chambon’s rule). The splicing reaction is sequential: the 5¢ site is cleaved first, then the 3¢ site after a lariat structure is formed. In con- trast, unconventional splicing of XBP1 pre-mRNA is catalyzed by IRE1a and RNA ligase, and there is a pair of stem–loop structures at the exon–intron border instead of GU-AG or AU-AC. Moreover, the splicing reaction is not sequential but random. The most important difference between conventional and unconventional splicing is where the reaction occurs (Fig. 7B). Conventional splicing (nuclear spli- cing) takes place in the nucleus, whereas unconven- tional splicing (cytoplasmic splicing) occurs in the cytoplasm. The biological significance of cytoplasmic splicing is that pre-mRNA used for translation in the cytoplasm can be spliced when it is necessary to change the nature of the protein translated from the mRNA, in response to extracellular or intracellular signaling. In contrast, as nuclear splicing cannot splice mRNA exported to the cytoplasm, it is necessary for pre-mRNA to be transcribed de novo and spliced. Thus, cytoplasmic splicing would be a very rapid, ver- satile, and energy-efficient mechanism with minimal waste as compared with conventional mRNA splicing. Recently, it was found that pXBP1(U) encoded in XBP1 pre-mRNA is a negative feedback regulator of pXBP1(S). Thus, in the case of XBP1, pre-mRNA and mature mRNA encode negative and positive regula- tors, respectively, and their expression is switched by cytoplasmic splicing in response to the situation in the ER [96]. IRE1b is specifically expressed in epithelial cells of the gastrointestinal tract, and thought to cleave rRNA to attenuate translation in response to ER stress [84]. When IRE1 b– ⁄ – mice were exposed to an inducer of inflammatory bowel disease, they actually developed colitis, possibly because of the enhanced ER stress [97]. Recently, the crystal structure of the luminal domain of IRE1a was solved [98]. The luminal domain is sim- ilar in structure to the peptide-binding domain of major histocompatibility complexes, suggesting the interesting possibility that it directly senses ER stress by directly binding unfolded proteins. Apoptosis-inducing pathways The accumulation of unfolded proteins in the ER is toxic to cells. Thus, if the PERK, ATF6, and IRE1 pathways cannot suppress ER stress, an apoptotic pathway is triggered to ensure survival of the organism as a last line of defense. A number of pathways have been reported to be involved in ER stress-induced apoptosis, and the full induction of apoptosis seems to require the concomitant activation of several death pathways, although there remain many arguments over ER stress-induced apoptosis [99–105]. In this section, I will briefly summarize the known death pathways, focusing on recent progress (Fig. 8). ER stress and diseases H. Yoshida 638 FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS The most characterized pathway is the CHOP path- way. CHOP ⁄ GADD153 (growth arrest and DNA damage 153) is a transcription factor, the expression of which is induced by the ATF6 and PERK pathways upon ER stress [70,106,107]. CHOP– ⁄ – cells exhibit less programmed cell death when faced with ER stress [108], suggesting that the CHOP pathway is a major regulator of ER stress-induced apoptosis. As for the target genes of CHOP, CHOP activates the transcrip- tion of GADD34, ERO1, DR5 (death receptor 5), and carbonic anhydrase VI, which seem to be responsible for apoptosis. GADD34 associated with protein phos- phatase 2C enhances dephosphorylation of eIF2a and promotes ER client protein biosynthesis [109], whereas ERO1, which encodes an ER oxidase, makes the ER a more hyper-oxidizing environment [110]. DR5, which encodes a cell surface death receptor, may activate caspase cascades [111]. Carbonic anhydrase VI may change the cellular pH, affecting various cellular pro- cesses [112,113]. However, the exact signaling mechan- ism from CHOP to apoptosis is still unclear. The second apoptotic pathway is the IRE1–TRAF2– ASK1 pathway. The cytoplasmic part of IRE1 binds to an adaptor protein, TRAF2 (tumor necrosis factor receptor-associated factor 2), which couples plasma membrane death receptor to Jun kinase (JNK) and stress-activated protein kinase (SAPK) [114]. IRE1 and TRAF2 form a complex with a mitogen-activated protein kinase kinase kinase, ASK1 (apoptosis signal- regulating kinase 1), and this IRE1–TRAF2–ASK1 complex is responsible for the phosphorylation and activation of JNK [115]. Actually, IRE1– ⁄ – cells as well as ASK1– ⁄ – cells are impaired in the activation of JNK and apoptosis by ER stress. In contrast, A B Fig. 7. Cytoplasmic splicing. (A) Comparison between nuclear and cytoplasmic splicing. Conventional splicing is catalyzed by the spliceosome in the nucleus, and there is a consensus sequence at the exon–intron boundary such as GU-AG or AU-AC. The splicing reaction is sequential: the 5¢ site is cleaved first, the lariat structure is formed, and then the 3¢ site is cleaved. In contrast, unconventional splicing is catalyzed by IRE1 and RNA ligase in the cytoplasm, there is a characteristic stem–loop structure at the boundary, and the splicing reaction is ran- dom without forming a lariat structure. (B) Biological significance of cytoplasmic spli- cing. As nuclear splicing cannot splice pre- mRNA exported to the cytoplasm, de novo transcription is required to change the char- acter of the protein encoded in the pre- mRNA. In contrast, as cytoplasmic splicing can splice pre-mRNA that is translated in the cytoplasm, it can rapidly change the character of a protein in response to exter- nal or internal stimuli, without de novo transcription. H. Yoshida ER stress and diseases FEBS Journal 274 (2007) 630–658 ª 2007 The Author Journal compilation ª 2007 FEBS 639 [...]... response to ER stress [143], whereas other pro-apoptotic factors, Bax (Bcl2-associated X protein) and Bak (Bcl-2 homologous antagonist ⁄ killer), are present in the ER membrane as well as the mitochondrial membrane [144,145] During ER stress, Bax and Bak oligomerize and activate caspase-12 Interestingly, Bax and Bak associate with IRE1a and modulate IRE1a function during ER stress [146] Bax and Bak are... the ERAD machinery, enhances degradation of polyQ proteins and suppresses polyQ protein-induced neurodegeneration [208] Judging from these findings, it is probable that ER stress is involved in the onset of polyQ diseases Pelizaeus-Merzbacher disease is a progressive neurodegenerative disorder characterized by a loss of coordination, motor abilities, and intellectual function [209] ER stress and diseases. .. [131] Transcription of IAP-2 and ER stress and diseases XIAP (inhibitor of apoptosis, X-linked), two other IAPs, is up-regulated during ER stress, and cells in which these IAPs have been knocked down are sensitive to ER stress- induced apoptosis [154] Cells overexpressing XIAP or HIAP1 are resistant to ER stress [122,155] These results suggest involvement of IAP proteins in ER stress- induced apoptosis c-Abl... ER quality control machinery and increased ER stress [265] Moreover, up-regulation of ER chaperones protected cardiomyocytes from ER stress- induced apoptosis [266] These findings strongly suggest that the ER stress response is essential for homeostasis of cardiomyocytes Liver diseases Hepatocytes have a well-developed ER structure that is essential for the vigorous synthesis of secretory proteins, and. .. response to ER stress, whereas an anti-apoptotic factor, Bcl-xL (Bcl-2-like 1), binds to Bim and inhibits its translocation [150] Bim-knockdown cells are resistant to ER stress The ER- localized anti-apoptotic factor BI-1 (Bax inhibitor-1) inhibits the activation of Bax during ER stress, and BI-1– ⁄ – mice are sensitive to ER stress, whereas mice overexpressing BI-1 are resistant [151] BIK (Bcl2-interacting... induced by ER stress, and knockdown or knockout of WFS1 causes ER stress in pancreatic b-cells [238,239] These findings strongly suggest that ER stress is deeply involved in the onset of IDDM There are several reports suggesting that ER stress is also involved in NIDDM First, obesity, one of the causes of NIDDM, evokes ER stress, and XBP1– ⁄ – mice develop insulin resistance [240], although the underlying... of inflammation is complicated, ER stress is involved in some types of inflammation In inflammation of the central nervous system, interferon-c induces ER stress and apoptosis of oligodendrocytes [250] Interestingly, PERK+ mice show enhanced central nervous system hypomyelination and oligodendrocyte loss, suggesting that the PERK pathway has a protective role against interferon-c-induced apoptosis In the... induces ER stress in neurons and activates the ATF6, IRE1 and PERK pathways [261], leading to the CHOP-mediated apoptosis of neurons [262] Ischemia also induces ER stress and the expression of ER chaperones in the heart, leading to degeneration of cardiomyocytes [263], suggesting that ER stress is involved in the development of ischemic heart disease (see below) Heart diseases It has been reported that ER. .. findings on how ER stress is involved in conformational diseases Neurodegenerative diseases Neurons are thought to be sensitive to protein aggregates, and there are many reports that ER stress is involved in neurodegenerative diseases [182–184] In fact, disruption of SIL1 ⁄ BAP, a cochaperone of BiP, results in the accumulation of protein aggregates and neurodegeneration [184] Most of these diseases are... FEBS 645 ER stress and diseases H Yoshida ER stress is also involved in hepatocarcinogenesis [272–274] In human hepatocellular carcinoma, the ATF6 and IRE1 pathways are activated, and expression of BiP is markedly increased, suggesting that the transformation of hepatocytes induces ER stress, and cells cope with the stress by activating the ER stress response pathways cells of patients with hereditary . endoplasmic reticulum; ERAD, ER- associated degradation; ERdj, ER dnaJ; ERO1, ER oxidoreductin; ERp72, ER protein 72; ERSE, ER stress response element; FKBP13,. hypoxia. ER dnaJ (ERdj)1, ERdj3 ⁄ human ER- asociated dnaJ (HEDJ), ERdj4, ERdj5, SEC63, and p58IPK are ER chaper- ones belonging to the HSP40 family, and modulate