Protein-Folding Homeostasis in the Endoplasmic Reticulum and Nutritional Regulation

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David Ron and Heather P. Harding

Metabolic Research Laboratories, University of Cambridge, and NIHR Cambridge Biomedical Research Centre, Addenbrookes Hospital, Cambridge CB2 0QQ, United Kingdom Correspondence: [email protected]

The ﬂux of newly synthesized proteins entering the endoplasmic reticulum (ER) is under negative regulation by the ER-localized PKR-like ER kinase (PERK). PERK is activated by unfolded protein stress in the ER lumen and inhibits new protein synthesis by the phosphorylation of translation initiation factor eIF2a. This homeostatic mechanism, shared by all animal cells, has proven to be especially important to the well-being of professional secretory cells, notably the endocrine pancreas. PERK, its downstream effectors, and the allied branches of the unfolded protein response intersect broadly with signaling pathways that regulate nutrient assimilation, and ER stress and the response to it have been implicated in the development of the metabolic syndrome accompanying obesity in mammals. Here we review our current understanding of the cell biology underlying these relationships.

nsulin was among the first proteins to be se- through its amino-terminal signal sequence Iquenced, among the first to have its structure (Mandon et al. 2013). Oxidative folding and solved, and therefore among the first to provide signal sequence removal yield mature pro-insu- clues to the diversity of modifications that affect lin, whose tertiary structure is stabilized by three secreted proteins. The b cell of the pancreas, disulfide bonds (Bulleid 2012). Folded pro-in- which produces insulin, is one of the best-stud- sulin clears ER quality control (Braakman and ied secretory cells, and the role of the secretory Hebert 2013) and traffics distally (Lord et al. pathway in insulin biosynthesis has been recog- 2013). nized from the dawn of modern cell biology. The peptidase involved in post-ER steps of Years later, when the stress pathways that con- pro-insulin maturation has long been recog- tribute to protein-folding homeostasis in the nized as playing a key role in its secretion, but endoplasmic reticulum (the unfolded protein the sensitivity of insulin biosynthesis to integri- response, UPR) came under scrutiny (Gardner ty of ER steps was not recognized until later. An et al. 2013; Olzmann et al. 2013), it was revealed early clue came from study of a naturally occur- that their integrity is important to insulin ring mutation in mouse Ins2. The Akita muta- metabolism and to the function of b cells. tion results in a Cys-92!Tyr substitution, dis- The precursor of insulin, prepro-insulin, is rupting an essential disulfide bond and leading recruited to the ER membrane cotranslationally to misfolding of proinsulin 2 (Wanget al. 1999).

Editors: Susan Ferro-Novick, Tom A. Rapoport, and Randy Schekman Additional Perspectives on The Endoplasmic Reticulum available at www.cshperspectives.org Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a013177 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a013177

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D. Ron and H.P. Harding

Interestingly, a single copy of the mutation is ogy of the common, type II form of diabetes sufficient to compromise b cells, whereas ho- mellitus by limiting both the production of in- mozygosity for a null mutation in Ins2 is with- sulin and the body’s sensitivity to it. out an obvious phenotype in mice (because of redundancy between a rodent’s two insulin genes) (Duvillie et al. 1997). The biochemical CONTROLLING THE FLUX OF CLIENT PROTEINS INTO THE ANIMAL CELL ER (and phenotypic) dominance of the Akita mutation in mice (Colombo et al. 2008) fit well In unicellular eukaryotes and plants, the UPR is with retention of the mutant pro-insulin in predominantly atranscriptional program. IRE1, the ER, high levels of UPR signaling, and with an ER stress sensor that responds to an imbal- a progressive decline in b-cell mass and insulin ance between unfolded client protein load and stores as the mutant mice age. Thus, a pertur- chaperone reserve, activates a downstream tran- bation to ER protein-folding homeostasis in- scription factor by an unconventional splicing duced by the misfolding-prone mutant pro-in- event (Gardneret al. 2013). Metazoans evolved a sulin has a long-term negative effect on b-cell translational strand to their UPR, whereby ER function. stress attenuates new protein synthesis. Thus, Unbiased human genetics provided an addi- the UPR in animal cells is bipartite, with an tional clue to the importance of protein-folding acute program that attenuates the load on the homeostasis in the ER; the Wolcott–Rallison ER and a more latent transcriptional pro- syndrome is a rare recessive monogenic form gram—mediated jointly by IRE1, PERK, and of hypoinsulinaemic neonatal diabetes asso- ATF6—that builds ER capacity. ciated with bone dysplasia and episodic liver failure (Julier and Nicolino 2010). Positional a cloning revealed that the causative mutations eIF2 PHOSPHORYLATION—AN ANCIENT MECHANISM FOR REGULATING in EIF2AK3 severely disrupted the expression TRANSLATION or function of PERK (Delepine et al. 2000), an ER-localized stress-activated kinase that Translational regulation in the animal cell UPR tunes rates of new protein synthesis to the un- is achieved by coupling ER stress to the phos- folded protein load in the ER (Harding et al. phorylation of the a-subunit of translation 1999). Although known to be enriched in b initiation factor 2 (eIF2a). The key to this inno- cells, PERK expression is ubiquitous (Shi et al. vation is PERK, a type I ER-localized transmem- 1998). Therefore, the prominence of diabetes brane protein that fuses a stress-sensing luminal in the phenotype associated with loss-of-func- domain that is functionally interchangeable tion mutations in a ubiquitous component of with IRE1, with an eIF2a kinase cytosolic effec- the unfolded protein response (UPR) pointed tor domain that most certainly arose by dupli- to a special role for ER homeostasis in b-cell cation of an ancestral eIF2a kinase gene (likely health. GCN2) (Harding et al. 1999). More surprising has been the link between The signaling pathway downstream from chronic ER stress and the ability of insulin tar- PERKwas already in existence in the unicellular get tissues to respond to the hormone; it has ancestor of animals. Known as the general con- emerged that nutrient excess and obesity are trol response, this signaling pathway comprises associated with higher levels of UPR signaling eIF2, a translation initiation factor that escorts in the liver and fat and that steps that mitigate the charged initiator methionyl-tRNA to the ER stress in these tissues ameliorate the insulin small ribosomal subunit, and, as a ternary com- resistance that is part of the metabolic syndrome plex of eIF2–GTP–tRNAmet, contributes to the linked to nutrient excess. Thus, ER stress and 43S-ribosomes’ ability to recognize AUG start the response to it affect both the insulin-pro- codons and initiate translation (Hinnebusch ducing b cell and the insulin-responsive tissues 2000). AUG codon recognition and forma- and may therefore influence the pathophysiol- tion of the first peptide bond leads to GTP

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ER Stress, PERK, and Insulin

hydrolysis. Replenishment of ternary complex selectivity of PERK action for pools of translat- pools requires exchange of GDP for GTP on ing ribosomes thus awaits the development of eIF2. This is a highly regulated step, with an more refined tools to measure its effects. exchange factor, eIF2B, and an anti-exchange factor, eIF5. eIF2(aP), phosphorylated on ser- eIF2(aP) DEPHOSPHORYLATION ine 51 of its a-subunit, inhibits the exchange factor eIF2B and thus attenuates the availability In animals, eIF2(aP) dephosphorylation is per- of ternary complexes for translation. Because formed by two phosphatase complexes. These protein synthesis has a major impact on energy consist of a common catalytic subunit, PP1, and nutrient stores, it is not surprising that sig- and a regulatory subunit, encoded either by naling by eIF2a phosphorylation has evolved to PPP1R15a (GADD34) or PPP1R15b (CReP). influence intermediary metabolism broadly. The regulatory subunit avidly binds the catalyt- Protein-folding homeostasis in the ER ben- ic PP1 subunit and endows the complex with efits from PERK-mediated eIF2a phosphory- specificity toward phosphorylated eIF2(aP). lation, because translational attenuation by ER In mammals, PPP1R15b (the ancestral metazo- stress contributes to the balance between un- an protein) is constitutively expressed (Jousse folded proteins entering the ER and chaperone et al. 2003), whereas expression of PPP1R15a, reserve of the compartment. The PERK-me- a mammalian invention, is tightly regulated by diated translational arm of the UPR gained stress, being induced by conditions that pro- importance with the rising complexity of the mote eIF2a phosphorylation. Thus, PPP1R15a secretory agenda of metazoans. Whereas worms contributes to a negative-feedback loop con- lacking PERK (pek-1) are impaired only after trolling levels of eIF2(aP) in mammals (Novoa ire-1 deletion (Shen et al. 2001), otherwise et al. 2001; Brush et al. 2003; Ma and Hendershot wild-type mammalian cells lacking PERK are 2003; Novoa et al. 2003). exquisitely sensitive to ER stress (Harding et al. The two regulatory subunits are apparently 2000b). dedicated to the dephosphorylation of eIF2 (aP), because the early embryonic lethality of their combined deficiency can be complete- LOCALIZATION OF PERK ACTION? ly suppressed by an S51A mutation in eIF2a Simple teleological considerations suggest that (Eif2s1tm1Rjk),whichprecludes phosphorylation PERK-mediated translation repression would by upstream kinases (Harding et al. 2009). Le- be restricted to the pool of mRNAs encoding thality of the combined deficiency of PPP1R15 secreted proteins, thereby focusing translation also confirms that levels of phosphorylated repression on the relevant organelle. PERK lo- eIF2a are tightly regulated. cation on the ER membrane suggests that this Negative feedback at the level of eIF2(aP) might be readily achieved by restricting the ki- dephosphorylation is mediated by transcrip- nase activity to a vicinal pool of eIF2. However, tional control of PPP1R15a, which is mediated there is no experimental evidence to suggest by eIF2(aP). Rare mRNAs are exempt from such selectivity in PERK action and, to date, the global repression of translation because of only global repression has been observed. This declining levels of ternary complexes in cells may reflect the bluntness of the pharmacologi- with increasing levels of eIF2(aP). These include cal tools used: a comparison of membrane-as- mRNAs encoding the transcription factors ATF4 sociated and cytosolic translation in wild-type and ATF5 that are endowed with a peculiar ar- and PERK knockout cells subjected to agents rangement of upstream open reading frames that promote high levels of ER stress. However, and are thereby subject to regulated transla- in yeast, eIF2 pools are not segregated, and ter- tion reinitiation. This results in repressed trans- nary complex formation presumably entails lation when ternary complexes are abundant recycling through a small number of eIF2B and enhanced translation when their levels de- clusters (Campbell et al. 2005). The issue of crease. This mechanism, first worked out inyeast

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D. Ron and H.P. Harding

(Hinnebusch 2000), is conserved in its many de- amino acid import, metabolism, and tRNA tails in mammalian cells (Lu et al. 2004a; Vattem charging. Thus, PERK leads to both short-term and Wek 2004; Zhou et al. 2008; Ingolia et al. repression of new protein synthesis—presum- 2011), and accounts for a substantial fraction ably to protect the ER from excessive unfolded of activated gene expression in the unfolded pro- protein load—but subsequently promotes the tein response of cultured mouse embryonic ﬁ- secretory agenda of the cell (Fig. 1). Although broblasts (Harding et al. 2003; Lu et al. 2004b). these conﬂicting activities are separated tempo- This eIF2(aP)-dependent gene expression rally,theycontaintheseedsofafailureofhomeo- program is not unique to ER stress. Mammals stasis that is considered below. have three additional kinases that phosphorylate eIF2a in response to other signals. Because it integrates signaling from diverse forms of cellu- ER STRESS AND b CELLS lar stress, the pathway has been named the “in- Translational Control in b Cells and PERK’s tegrated stress response” (Harding et al. 2003). Role in Defense of Their Well-Being Its target genes include PPP1R15a, which promotes recovery of protein synthesis following its Regulating synthesis rates is an important pro- repression by eIF2(aP), and genes involved in tective mechanism in coping with ER stress

ER Stress

IRE1 PERK

PPP1R15B eIF2(αP)

ATF4, ATF5, XBP1 ATF6 and others

CHOP

Amino-acid import Chaperones, ER enzymes PPP1R15A and assimilation

Figure 1. A schematic overview of the eIF2a-phosphorylation-dependent gene-expression program in the context of the UPR. In addition to attenuating global protein synthesis, eIF2(aP) also up-regulates the translation of rare mRNA, exempliﬁed by those encoding the transcription factors ATF4 and ATF5. This couples increased eIF2 phosphorylation to a gene expression program whose targets include the transcription factor CHOP,which activates a regulatory subunit of an eIF2(aP)-directed phosphatase, PPP1R15A/GADD34, and to enhanced expression of genes involved in amino acid import and assimilation and other more conventional target genes of the UPR that are coregulated by the two other branches of the UPR (IRE1, XBP1, and ATF6).The net effect of this program is to dephosphorylate eIF2a (a task that is aided by the constitutively expressed PPP1R15B/CReP) and thereby reverse translational repression and to enhance the capacity of the cell to synthesize and secrete proteins by its effects on amino acid metabolism and the ER. The near-immediate repression of protein synthesis upon PERK activation defends the cell against ER stress; however, the gene expression program has conﬂicting effects on levels of ER stress and appears to have evolved primarily to restore synthesis and secretion of proteins.

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ER Stress, PERK, and Insulin

acutely, whereas induced gene expression is protein load and have suggested that b-cell fail- important for longer-term adaptation. This is ure in the mutant mice proceeds through the attested to by the hypersensitivity of cultured corruption of gene expression programs (Gupta PERK knockout cells to acute perturbation of et al. 2010). the protein-folding environment in the ER In regard to the aforementioned uncertain- (Harding et al. 2000b) and stands in contrast ty, it is worth noting that mutations in known to the relative minor contribution of IRE1 to mediators of the eIF2(aP)-dependent gene the resistance of cultured mammalian cells to expression program, the transcription factors acute ER stress (Cross et al. 2012). ATF4 and CHOP, do not share the b-cell defect Theoretical considerations predict that trans- of the PERK knockout; in some circumstances, lational control gains importance in circum- CHOP deletion is protective (see below)! How- stances associated with wide fluctuations in cli- ever, this argument against the primacy of tran- ent protein load (Trusina et al. 2008). Such scription is weakened by the observation that fluctuations are substantially restricted to the ATF4 accounts for less than half of the PERK- ER, because cellular growth and developmental dependent changes in mRNA abundance ob- programs that unfold gradually dictate protein served in ER-stressed fibroblasts (Harding et al. synthesis in other compartments. In contrast, 2003). The consequences of PERK deficiency the ER of a b cell may be called upon to accom- can be ameliorated by inhibition of protein syn- modate fluctuations of up to 10-fold in client thesis in cultured cells (Harding et al. 2000b), protein load over a period of ,1 h (Zucker and but this merely indicates that ER stress is a client Logothetopoulos 1975). protein-driven process. IRE1 signaling is en- The exuberant increase in pro-insulin trans- hanced in the tissues of PERK knockout mice lation in response to glycemic excursions occurs (Harding et al. 2001b), but it is impossible to despite PERK’s moderating influence. The latter tell if this reflects the loss of a gene expression accounts for 30% less pro-insulin translation program that sensitizes cellsto physiological lev- observed in glucose-stimulated islets isolat- els of client protein load or whether it reflects ed from wild-type mice compared with PERK inappropriately high levels of client protein knockouts (Fig. 2) (Harding et al. 2001b). This translation owing to lack of PERK. moderate difference in client protein synthesis The recent availability of potent and selec- between wild-type and the PERK mutant is tive PERK kinase inhibitors has allowed us to nonetheless associated with a progressive de- examine, for the first time, the consequences of cline in b-cell function and mass, leading to PERK inactivation in otherwise normal cells the rapid development of diabetes in mice and subject to physiological levels of ER stress. In humans lacking PERK (Delepine et al. 2000; fibroblasts, an increase in basal translation is Harding et al. 2001b; Zhang et al. 2002). How- noted within minutes of PERK inactivation. ever, given that eIF2a phosphorylation regulates In b cells, this is associated with rapid accu- both bulk synthesis of ER client proteins and an mulation of high-molecular-weight complexes important gene expression program (Harding containing misfolded wild-type pro-insulin. et al. 2003), it has been impossible to date to Together, these unpublished observations argue deconvolute their relative contribution to the in favor of the importance of translational con- phenotype of PERK deficiency. That mice ho- trol in PERK action. mozygous for an S51A mutation in eIF2a share the b-cell defect with the Perk knockout Pathophysiological Mechanisms in (Scheuner et al. 2001) is unhelpful in regard to ER-Stressed b Cells this issue, because all of the effects of PERK (transcriptional and translational) are mediated b cells of the pancreas are sensitive to ER stress through eIF2(aP) (Lu et al. 2004b). Indeed, and suffer serious consequences from impaired some investigators have argued against the pri- ability to regulate its levels. But how important macy of PERK’s role in attenuating ER client is ER stress to b cells that do not have an overtly

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D. Ron and H.P. Harding

A Insulin High iv secretion glucose

iii ER ER stress client-protein i load

PERK Proinsulin

eIF2(αP) ii

B 11 10 9 Δ 8 7 6 5 4 3 (fold induction) (fold 2 Insulin/total protein 1

Glucose (mM): 2.8 16.7 2.8 16.7 Perk +/+ Perk –/– C d12 d21 d42 +/+ Insulin PERK –/– PERK Glucagon

Figure 2. Translational control in b cells. (A) Schema of the interplay between glycemic excursions and translational control in b cells. High serum glucose (i) stimulates pro-insulin translation in the b cell, increasing ER client protein load and promoting a physiological ER stress. The latter is modulated by PERK-mediated eIF2a phosphorylation (ii), which protects the b cell from ER stress while capping insulin synthesis (iii). Insulin resistance, which impairs glycemic control (iv), burdens the b cell by increased signaling in i. (B) A comparison of the effects of glucose excursions on pro-insulin translation in islets of Langerhans isolated from wild-type and PERK knockout mice. The arrows point to the 30% difference (D) in pro-insulin translation affected by PERK signaling (point ii in A). (C) Photomicrographs of pancreas from wild-type and PERK knockout mice of the indicated age stained with antiserum to insulin and glucagon. Note the profound loss of insulin-positive b cells in the PERK mutant. (Panel from Harding et al. 2001a; reprinted, with permission, from the authors.)

compromised UPR or a mutation leading to demand for hormone. Elevated levels of UPR pro-insulin misfolding? The common adult target genes have been noted in the islets of form of diabetes mellitus (type II) arises in the Langerhans of human patients with type II di- context of peripheral resistance to insulin action abetes (Laybutt et al. 2007), suggesting that the and failure of the b cells to keep up with the overworked b cells of individuals with insulin

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ER Stress, PERK, and Insulin

resistance may be experiencing more ER stress reduced b-cell mass. The theme of unfolded than those with normal insulin sensitivity. protein load is common to all of these perturba- An excess client protein-driven mechanism tions, but how is it linked to dysfunction of the for enhanced ER stress is also consistent with endocrine pancreas? A large bodyof research has experiments in which glucose, a potent driver been devoted to cell death in ER stress. Common of insulin biosynthesis, led to higher levels of downstream cell death effectors are implicated UPR markers in explanted rodent islets and cul- (e.g., see Wei et al. 2001; Cunha et al. 2012), but tured b cells (Wang et al. 2005; Lipson et al. proximal links remain obscure. It is also difficult 2006; Elouil et al. 2007). The b-cell ER’s ability to deconvolute the role of cell death from other to handle client proteins may be further im- mechanisms that lead to decline in b-cell mass. paired by lipid dysregulation, which prevails In this regard, it is worth noting that in bone and in individuals with insulin resistance who often cartilage, chronic ER stress is associated with experience hyperlipidemia and have enhanced transcriptional reprogramming to a less differ- non-adipose tissue lipid stores. Exposure of cul- entiated, less secretory phenotype (Tsang et al. tured b cells and islets to excess saturated fatty 2007; Murakami et al. 2009). Knockdown of acids is a potent inducer of UPR signaling (Kar- PERK in INS1 cells (a clonal b-cell line) leads askov et al. 2006). Altered lipid composition of to lower levels of pro-insulin mRNA (Gupta the ER membrane has been shown to lead to et al. 2010). A similar process in the islets of dysfunction of the ER calcium pump and pro- Langerhans could lead to lower insulin biosyn- mote depletion of lumenal calcium stores. This thesis with little or no excess cell death. It is our phenomenon, first observed in macrophages (Li impressionthatmany plausiblepathophysiolog- et al. 2004), has since been confirmed in b cells ical mechanisms exist for linking ER stress in b (Cunha et al. 2008) and liver (Fu et al. 2011). cells to islet dysfunction, but their contribution Giventheimportanceofcalciumtothefunc- to the pathophysiology of common forms of di- tion of diverse ER enzymes and chaperones, it is abetes mellitus remains to be determined. assumed that lumenal calcium depletion com- promises the capacity of the organelle to handle Failure of Homeostasis in the client proteins and activates the UPR via the lu- ER-Stressed Cell menal stress-sensing domains of IRE1, PERK, and ATF6.However, recent observations in yeast The CHOP/Ddit3 gene is strongly induced via suggest a more complex relationship of lipids to an eIF2(aP)-dependent signaling pathway, UPR signaling: UPR activation by manipula- whichinER-stressedcells islargelyPERKdepen- tions that perturb protein-folding homeostasis dent (Harding et al. 2000a). One of the more in the yeast ER lumen requires the lumenal do- enduring observations in the field is the survival main of Ire1p, as expected. However, a mutant benefit of CHOP deletion in ER-stressed mam- Ire1p lacking the lumenal domain can yet recog- malian cells and tissues (Zinszner et al. 1998). nize perturbation of membrane lipid composi- This benefit extends not only to Ins2AKITA mice tion (Promleket al. 2011). If a similar unconven- confronted with ER stress-mediated b-cell dys- tional mechanism for UPR activation by altered function (Oyadomari et al. 2002), but also to lipid composition exists in mammals, it opens Leprdb/db mice in which diabetes is induced by the door to the possibility that lipids tune the a mutation in the leptin receptor gene (Song et UPR independently of their effect on protein- al. 2008). Glucose intolerance arises in Leprdb/db folding homeostasisintheERlumen.Thisseems mice as a consequence of obesity and insulin an important area for future research. resistance. Therefore, the events that unfold in Misfolded mutant pro-insulin (Wang et al. their endocrine pancreas and prevent the or- 1999), loss of translational control (Harding gan from matching the demand for insulin are et al. 2001a), and lack of ER chaperones (Ladiges believed to be shared with obese, insulin-resis- et al. 2005) or enzymes (Zito et al. 2010) are all tant humans (the commonplace antecedents of associated with inadequate insulin secretion and diabetes mellitus in our species). Interestingly,

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D. Ron and H.P. Harding

CHOPknockoutLeprdb/db micehavelargerislets to resume secreted protein synthesis, by pro- and more insulin than the Leprdb/db mice with moting recovery in ternary complexes, by pro- intact CHOP.Therefore, the improved glycemic viding cysteine to fuel protein synthesis, and control and enhanced b-cell mass of the double- by enhancing disulfide-bond formation. In the mutant mice suggest that ER-stress-mediated context of severe ER stress, these otherwise ho- CHOP induction in b cells may contribute to meostatic effects of CHOP are counterproduc- the pathogenesis of common forms of type II tive. This is reflected in the benefits afforded to diabetes mellitus. severely stressed cells by deletion or inhibition This is an interesting possibility, but an im- of CHOPor its target GADD34 (Marciniak et al. portant limitation applies: The CHOP knock- 2004; Boyce et al. 2005; Tsaytler et al. 2011) and out was global; therefore, it is impossible to ex- may, in theory, represent a therapeutic oppor- clude that CHOPaction in an insulin-responsive tunity for intervention (Fig. 3). tissue may have contributed to the decline in b- cell mass and deterioration in glycemic control of the wild-type Leprdb/db mice. This is more than a pedantic concern. CHOP antagonizes A PPP1R15A AA import IRE1 RIDD normal adipogenesis by interfering with the ac- tRNA charging PERK eIF2(αP) tivity of members of the C/EBP family of tran-

scription factors (Ron and Habener 1992; Cro- OK zat et al. 1993; Batchvarova et al. 1995). Insulin resistance is believed to develop when the ability Synthesis Chaperone to assimilate nutrients in adipose tissue stores is secretion reserve exceeded and lipids begin to accumulate else- Conventional load where (Virtue and Vidal-Puig 2010). By remov- ing a negative regulator of adipogenesis, CHOP B PPP1R15A AA import IRE1 RIDD deletion could improve insulin sensitivity and tRNA charging disrupt peripheral feed-forward mechanisms PERK eIF2(αP) that contribute to islet dysfunction. Indeed, a recent paper reports that CHOP deletion en- OK hances adiposity while improving insulin sensi- Synthesis Chaperone tivity (Maris et al. 2012). Therefore, it would be secretion reserve informative to compare the effects of nutrient Heavy load overload in mice with and without a b-cell-specific knockout of CHOP. Figure 3. Failure of homeostasis in the UPR. Faced These physiological caveats not withstand- with conventional levels of unfolded protein load ing, the evidence that CHOP contributes cell- (A), homeostasis in the ER lumen is maintained by autonomously to the dysfunction and death of a balance between factors that favor chaperone re- ER-stressed cells is compelling; less so is our serve by restraining protein synthesis, such as IRE1- mediated RIDD and PERK-mediated eIF2a phos- understanding of the mechanisms. CHOP over- phorylation and factors that favor protein synthesis expression leads to declining glutathione levels [such as amino transporters, tRNA synthetases, or and enhanced accumulation of reactive oxygen eIF2(aP)-phosphatases, PPP1R15A]. This balance species (McCullough et al. 2001). CHOP also was honed by years of natural selection. However, contributes to the activation of PPP1R15a/ under conditions of usually heavy unfolded protein load, for example, caused by a mutation in an abun- GADD34, the inducible regulatory subunit of AKITA the eIF2(aP)-directed phosphatase and to the dant secreted protein such as the Ins2 , homeostasis is favored by a different balance, by one with less expression of Ero1L encoding the ER disulfide PPP1R15A and more eIF2(aP) (B). This propensity oxidase ERO1a (Marciniak et al. 2004). These for failure of homeostasis at the UPR’s normal set activities suggest that CHOPassists its upstream points explains the beneficial effects of PPP1R15A activator ATF4in preparing the ER-stressed cells inactivation in certain unusual stressful situations.

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ER Stress, PERK, and Insulin

Further evidence for failure of homeostasis and fat were initially elevated and then declined in the UPR is provided by the observation that with weight loss following bariatric surgery both diminished and excessive eIF2(aP) con- (Gregor et al. 2009). Interestingly, partial com- tributes to cell dysfunction and death in con- promise of the XBP1 gene, encoding IRE1’s ef- ditions of severe ER stress (Cnop et al. 2007; fector, markedly enhanced insulin resistance in Lin et al. 2009). The IRE1 branch of the UPR obese mice (Ozcan et al. 2004), whereas chem- may also contribute to a failure of homeostasis. ical chaperones that are believed to lower the Animal cell IRE1 not only is endowed with se- level of ER stress restored insulin sensitivity to quence-specific RNase activity (directed to the obese animals (Ozcan et al. 2006). These earlier mRNA of its effector XBP1), but also possesses studies cited enhanced activation of the Jun ami- the ability to cleave and promote degradation no-terminal kinase (JNK) by hyperactive IRE1a of membrane-bound mRNAs promiscuously (Urano et al. 2000) as playing a role in insulin (Hollien and Weissman 2006; Hollien et al. resistance—JNK phosphorylation of the insulin 2009). This regulated IRE1-dependent degrada- receptor substrate-1 inhibits signaling down- tion (RIDD) may serve a homeostatic role by stream from its receptor (Ozcan et al. 2004). contributing to reprogramming the repertoire The picture has grown more complicated of mRNAs in stressed cells (Gardner et al. since, and the primacy of JNK activation has 2013) but when engaged too vigorously may been challenged. XBP1 has been reported to in- also contribute to decline in b-cell function activate the FOXO1 transcription factor and (Han et al. 2009; Lee et al. 2011). thereby attenuate the expression of genes in- We believe that failure of homeostasis de- volved in hepatic glucose production (Zhou serves careful attention in the apparent switch et al. 2011). The eIF2(aP) branch of the UPR between the adaptive and maladaptive UPR ob- also affects intermediary metabolism. Mice ho- served experimentally and that the benefit to mozygousforthederegulatingS51Amutationin organisms from eliminating cells that have ex- Eif2a succumb to neonatal hypoglycemia due perienced ER stress may be minimal. But cells to poor accretion of glycogen in the liver at the do die and malfunction during ER stress, and end of gestation (Scheuner et al. 2001). Adult regardless of whether this is contributed to by mice with compromised levels of liver eIF2(aP) agents that evolved specifically for that purpose due to a constitutively expressed PPP1R15a or by an imperfect system, understanding the transgene also have abnormally low liver glyco- underlying mechanisms may uncover therapeu- gen stores and low levels of blood sugar (Oyado- tic opportunities. mari et al. 2008). Thus, in the liver, the IRE1 and PERK arms of the UPR are pulling in different directions. The outcome of this tugging is influ- ER STRESS IN THE PERIPHERY: INSULIN enced further by the non-cell-autonomous ef- AND LEPTIN RESISTANCE fects of these effectors (e.g., see Birkenfeld et al. Despite the uncertainties and the gaps in our 2011). knowledge, it is easy to countenance a role for Leptin is a hormone produced by adipo- ER stress in the insulin-producing b cells in the cytes that contributes to a negative-feedback development of type II diabetes mellitus. But the loop that suppresses food intake when nutrient past8yearshavealsoseenaburgeoning literature stores are replete. Rodent models of obesity re- linking ER stress and the UPR in peripheral tis- capitulate the physiological resistance to circu- sues to their ability to respond to insulin and lating leptin that is observed in most obese hu- metabolize glucose and other nutrients. mans. Interestingly, in obese leptin-resistant Nutrient excess in the form of a high-fat diet mice, enhanced UPR activity has been mea- is associated with activation of the UPR in liver sured in the hypothalamic nuclei involved in and adipose tissue of mice (Ozcan et al. 2004) the neural circuitry by which leptin normally and man (Gregor et al. 2009). In a longitudinal represses appetite (Zhang et al. 2008; Ozcan study of obese humans, UPR markers in liver et al. 2009). The proximate cause of UPR

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D. Ron and H.P. Harding

hyperactivity in leptin’s target cells is unclear as REFERENCES are the mechanisms by which it might link to Reference is also in this collection. leptin resistance. Nonetheless, it will be interesting to learn if the two events are linked causally. Batchvarova N, Wang X-Z, Ron D. 1995. Inhibition of adipogenesis by the stress-induced protein CHOP Several of the UPR signal transducers (GADD153). EMBO J 14: 4654–4661. evolved from ancestors that were dedicated to Birkenfeld AL, Lee HY, Majumdar S, Jurczak MJ, metabolic regulation. PERK and its downstream Camporez JP, Jornayvaz FR, Frederick DW, Guigni B, effector ATF4are likely descendants from GCN2 Kahn M, Zhang D, et al. 2011. Influence of the hepatic eukaryotic initiation factor 2a (eIF2a) endoplasmic re- and GCN4, which regulate amino acid sufficien- ticulum (ER) stress response pathway on insulin-mediat- cy in unicellular eukaryotes. ATF6 is a distant ed ER stress and hepatic and peripheral glucose metabo- cousin of the sterol regulated transcription fac- lism. J Biol Chem 286: 36163–36170. tor SREBP (Ye et al. 2000). Therefore, it is no Boyce M, Bryant KF, Jousse C, Long K, Harding HP, Scheuner D, Kaufman RJ, Ma D, Coen D, Ron D, et al. surprise that UPR activity affects intermediary 2005. A selective inhibitor of eIF2a dephosphorylation metabolism. The difficulty lies in determining protects cells from ER stress. Science 307: 935–939. the importance of such links. Chemical chaper- Braakman I, Hebert DN. 2013. Protein folding in the endoplasmic reticulum. Cold Spring Harb Perspect Biol doi: ones that suppress UPR signaling and restore 10.1101/cshperpsect.a013201. insulin and leptin sensitivity have been cited as Brush MH, Weiser DC, Shenolikar S. 2003. Growth arrest evidence in support of the primacy of the link; and DNA damage-inducible protein GADD34 targets however, the mechanism of action of these com- protein phosphatase 1a to the endoplasmic reticulum and promotes dephosphorylation of the a subunit of pounds is far from clear, and there is a concern eukaryotic translation initiation factor 2. Mol Cell Biol that they are a somewhat blunt tool to dissect a 23: 1292–1303. complex problem. Bulleid NJ. 2012. Disulfide bond formation in the mammalian endoplasmic reticulum. Cold Spring Harb Perspect Biol 4: a013219. CONCLUDING REMARKS Campbell SG, Hoyle NP,Ashe MP.2005. Dynamic cycling of eIF2 through a large eIF2B-containing cytoplasmic body: Protein-folding homeostasis in the ER is precar- Implications for translation control. J Cell Biol 170: ious. Secretion is ubiquitous, and the flux of 925–934. protein into the ER is highly dynamic. Thus, Cnop M, Ladriere L, Hekerman P, Ortis F, Cardozo AK, Dogusan Z, Flamez D, Boyce M, Yuan J, Eizirik DL. ER stress is a normal feature of life, and the 2007. Selective inhibition of eukaryotic translation initi- response to it can affect nutritional regulation ation factor 2a dephosphorylation potentiates fatty acid- at multiple levels. The most coherent link is at induced endoplasmic reticulum stress and causes pancreatic b-cell dysfunction and apoptosis. J Biol Chem 282: the level of the pancreatic b cell, which produces 3989–3997. insulin, but enhanced ER stress in the insulin- Colombo C, Porzio O, Liu M, Massa O, Vasta M, Salardi S, responsive tissues also has the potential to affect Beccaria L, Monciotti C, Toni S, Pedersen O, et al. 2008. metabolism. The complex regulation of nutri- Seven mutations in the human insulin gene linked to permanent neonatal/infancy-onset diabetes mellitus. ent acquisition and assimilation has made it J Clin Invest 118: 2148–2156. difficult to assess the quantitative significance Cross BC, Bond PJ, Sadowski PG, Jha BK, Zak J, Goodman of ER stress and the response to it in metabolic JM, Silverman RH, Neubert TA, Baxendale IR, Ron D, et dysregulation. This research effort will benefit al. 2012. The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding from establishing in further detail the compo- small molecule. Proc Natl Acad Sci 109: E869–E878. nents of the UPR and the fundamentals of their Crozat AY, A˚ man P, Mandahl N, Ron D. 1993. Fusion of activity and then applying that knowledge to CHOP to a novel RNA-binding protein in human myx- oid liposarcoma with t(12;16)(q13;p11). Nature 363: complex physiological circumstances. 640–644. Cunha DA, Hekerman P, Ladriere L, Bazarra-Castro A, Ortis F, Wakeham MC, Moore F, Rasschaert J, ACKNOWLEDGMENTS Cardozo AK, Bellomo E, et al. 2008. Initiation and exe- cution of lipotoxic ER stress in pancreatic b-cells. J Cell This work is supported by a Wellcome Trust Prin- Sci 121: 2308–2318. cipal Research Fellowship to D.R. and by EU FP7 Cunha DA, Igoillo-Esteve M, Gurzov EN, Germano CM, BetaBatgrant277713 andbyNIHgrant DK47119. Naamane N, Marhfour I, Fukaya M, Vanderwinden

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Protein-Folding Homeostasis in the Endoplasmic Reticulum and Nutritional Regulation

David Ron and Heather P. Harding

Cold Spring Harb Perspect Biol 2012; doi: 10.1101/cshperspect.a013177

Subject Collection The Endoplasmic Reticulum

Cell Biology of the Endoplasmic Reticulum and How Viruses Use the Endoplasmic Reticulum for the Golgi Apparatus through Proteomics Entry, Replication, and Assembly Jeffrey Smirle, Catherine E. Au, Michael Jain, et al. Takamasa Inoue and Billy Tsai The Mammalian Endoplasmic Expanding Proteostasis by Membrane Trafficking Reticulum-Associated Degradation System Networks James A. Olzmann, Ron R. Kopito and John C. Darren M. Hutt and William E. Balch Christianson Functional Insights from Studies on the Structure N-Linked Protein Glycosylation in the of the Nuclear Pore and Coat Protein Complexes Endoplasmic Reticulum Thomas Schwartz Jörg Breitling and Markus Aebi Endoplasmic Reticulum Targeting and Insertion of Retrograde Traffic from the Golgi to the Tail-Anchored Membrane Proteins by the GET Endoplasmic Reticulum Pathway Anne Spang Vladimir Denic, Volker Dötsch and Irmgard Sinning Lipid Transport between the Endoplasmic The Role of the Endoplasmic Reticulum in Reticulum and Mitochondria Peroxisome Biogenesis Vid V. Flis and Günther Daum Lazar Dimitrov, Sheung Kwan Lam and Randy Schekman Protein Folding in the Endoplasmic Reticulum Sphingolipid Homeostasis in the Endoplasmic Ineke Braakman and Daniel N. Hebert Reticulum and Beyond David K. Breslow Endoplasmic Reticulum Structure and The Contribution of Systematic Approaches to Interconnections with Other Organelles Characterizing the Proteins and Functions of the Amber R. English and Gia K. Voeltz Endoplasmic Reticulum Maya Schuldiner and Jonathan S. Weissman

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Endoplasmic Reticulum Stress Sensing in the Protein Translocation across the Rough Unfolded Protein Response Endoplasmic Reticulum Brooke M. Gardner, David Pincus, Katja Gotthardt, Elisabet C. Mandon, Steven F. Trueman and Reid et al. Gilmore

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