ER-Associated Degradation in Health and Disease – from Substrate to Organism Asmita Bhattacharya1,2 and Ling Qi1,3,*

REVIEW SUBJECT COLLECTION: CELL BIOLOGY AND DISEASE ER-associated degradation in health and disease – from substrate to organism Asmita Bhattacharya1,2 and Ling Qi1,3,*

ABSTRACT note Sel1L is also known as Hrd3, and Hrd1 as SYVN1) – the focus – The recent literature has revolutionized our view on the vital of this Review is central to the most-conserved and best- importance of endoplasmic reticulum (ER)-associated degradation characterized branch of mammalian ERAD (Mueller et al., 2008, (ERAD) in health and disease. Suppressor/enhancer of Lin-12-like 2006; Ye et al., 2004). The ERAD process begins with the selection (Sel1L)–HMG-coA reductase degradation protein 1 (Hrd1)-mediated of the substrate protein; this is based on either glycosylation tags, ERAD has emerged as a crucial determinant of normal physiology mannose trimming status and/or conformational change, and is and as a sentinel against disease pathogenesis in the body, in a aided by chaperones, such as 78-kDa glucose-regulated protein α largely substrate- and cell type-specific manner. In this Review, we (Grp78; also known as HSPA5), ER degradation-enhancing - highlight three features of ERAD, constitutive versus inducible ERAD, mannosidase-like protein (Edem) family proteins and osteosarcoma quality versus quantity control of ERAD and ERAD-mediated amplified 9 (Os9) (Araki and Nagata, 2011; Bernasconi et al., 2010; regulation of nuclear gene transcription, through which ERAD Christianson et al., 2008; Hegde and Ploegh, 2010; Olzmann et al., exerts a profound impact on a number of physiological processes. 2013; Smith et al., 2011; van der Goot et al., 2018). In a second step, the substrate is retro-translocated into the cytosol through the KEY WORDS: Sel1L-Hrd1 ERAD, Health, Disease, Constitutive polytopic dislocon Hrd1; Hrd1 itself forms a ubiquitin-gated ERAD, Inducible ERAD, ERAD substrate, Quality control, Quantity channel activated by auto-ubiquitylation (Baldridge and Rapoport, control, Nuclear gene transcription 2016; Carvalho et al., 2010; Christianson et al., 2012). Other potential dislocon proteins, such as degradation in endoplasmic Introduction reticulum protein (Derlin) family members – Derlin-1, Derlin-2 or Secreted proteins, such as hormones and growth factors, as well as Derlin-3 – have been identified as possibly working together with transmembrane receptors, critically regulate nearly all aspects of Hrd1, but their role needs further characterization in mammalian life, including food intake, water balance, growth, metabolism and systems (Lilley and Ploegh, 2004; Ye et al., 2004). The adaptor immunity. The endoplasmic reticulum (ER) is a specialized cellular protein Sel1L is indispensable for the stability and function of Hrd1 compartment where the folding and maturation of most of these (Mueller et al., 2008, 2006; Sun et al., 2014; Vashistha et al., 2016). proteins take place (Hegde and Lingappa, 1997). Aberrations in Subsequent to the retro-translocation, substrates are ubiquitylated these complex thermodynamic folding processes and kinetic by Hrd1 (Kikkert et al., 2004) and targeted for proteasomal parameters can disrupt cellular homeostasis and lead to degradation by the ATPase valosin-containing protein Vcp (also debilitating diseases, such as liver and lung diseases, and diabetes known as p97) and other ubiquitin-modifying enzymes (Ernst et al., (Braakman and Bulleid, 2011; Wiseman et al., 2007). ER- 2009; Meyer et al., 2012) (Fig. 1A). In addition, other E3 ligases, associated degradation (ERAD) is a highly conserved, major such as glycoprotein 78 (Gp78, also known as AMFR), membrane- regulatory system that guards against such events, thereby associated ring-CH-type finger 6 (March6), ring finger protein 5 maintaining proteostasis within the ER (Aridor and Balch, 1999; (Rnf5, also known as Rma1) and tripartite motif containing 13 Guerriero and Brodsky, 2012). Despite being well-characterized at (Trim13), may work either in parallel or together with Hrd1; the biochemical level, which has been reviewed extensively however, these systems remain poorly characterized (Altier et al., elsewhere (Christianson and Ye, 2014; Merulla et al., 2013; 2011; Fang et al., 2001; Hassink et al., 2005; Younger et al., 2006; Olzmann et al., 2013), the significance of ERAD on a systemic Zhang et al., 2015) and will not be discussed here. Biochemical (patho)physiological scale has, until recently, remained unknown studies have identified some proteins as possible ERAD substrates (Hwang and Qi, 2018; Qi et al., 2017). This Review discusses the in mammalian cell lines, such as mutant α-antitrypsin, mutant emerging roles of ERAD in health and disease, without which, our transthyretin, cystic fibrosis transmembrane conductance regulator understanding of pathogenesis of a large number of diseases (CFTR) and unassembled Cd147 (also known as BSG) remains incomplete. (Christianson et al., 2012; Christianson et al., 2008; Liu et al., The suppressor/enhancer of lin-12-like (Sel1L)–HMG-coA 1997; Sifers et al., 1988; Tyler et al., 2012; Ward et al., 1995); reductase degradation protein 1 (Hrd1) protein complex (Fig. 1A; however, whether or not these proteins are bona fide endogenous ERAD substrates remains to be verified in vivo. Deletion of any key component of ERAD, such as Sel1L, Hrd1, 1Department of Molecular & Integrative Physiology, University of Michigan Medical Derlin or Vcp, results in embryonic lethality in mice (Dougan et al., School, Ann Arbor, MI 48105, USA. 2Graduate Program of Genetics, Genomics and 2011; Eura et al., 2012; Francisco et al., 2010; Müller et al., 2007), Development, Cornell University, Ithaca, NY 14853, USA. 3Division of Metabolism, Endocrinology & Diabetes, Department of Internal Medicine, University of Michigan underscoring the physiological significance of ERAD in Medical School, Ann Arbor, MI 48105, USA. development. Recently, cell type-specific knockout mice of specific ERAD genes have been reported, revealing that ERAD is *Author for correspondence ([email protected]) linked to a plethora of physiological conditions, often in a substrate-

A.B., 0000-0002-5669-8036; L.Q., 0000-0001-8229-0184 specific manner (Qi et al., 2017). These findings have allowed us to Journal of Cell Science

1 REVIEW Journal of Cell Science (2019) 132, jcs232850. doi:10.1242/jcs.232850

A B Fig. 1. Overview over physiological ERAD and emerging paradigms. (A) Sel1L–Hrd1- ER ER mediated ERAD. ERAD is a highly conserved cellular system that is responsible for the Chaperones Substrates Chemical agents retrotranslocation of substrate proteins from the Substrates proAvp Pre-BCR Tm Tg ER to the cytosol for proteasomal degradation. This process starts with substrate recruitment by chaperones (Grp78, Os9, Xtp3b, Edem1– UPR Edem3) and adaptor proteins (Sel1L, Derlin Pomc Sel1L proteins), followed by their retrotranslocation through the dislocon channel (Hrd1 or Derlin), and ubiquitylation by an E3 ligase (Hrd1) and ERAD ATPase (Vcp), before degradation at the proteasome. (B–D) Recent in vivo studies have Derlin highlighted three major conceptual advances in Crebh mammalian ERAD biology. (B) Constitutive versus inducible ERAD. ERAD may function in Hrd1 a constitutive capacity to maintain optimal Vcp Food B cell Nucleus levels of key proteins regulating critical maturation intake physiological process such as food intake, Proteasome water and energy balance, and B cell development, or in an inducible capacity, Fgf21 whereby ERAD gene expression is triggered by drug-induced UPR. (C) Quality versus quantity Water Systemic balance energy control by ERAD. Physiological ERAD has balance been reported to perform both quality control of misfolded substrates, thus ensuring fidelity of Constitutive Inducible production and protecting against their Cytosol Cytosol aggregation and loss-of-function effects, and quantity control of folding-competent C D substrates, thereby regulating their abundance and guarding against over-activation and gain- Cytosol α ER Ire1 of-function effects. (D) ERAD-regulated transcription. ERAD influences gene expression in the nucleus through the direct ERAD manipulation of ER-related transcription ERAD α– X factors; for example, the Ire1 Xbp1s axis of P0 XBP1u UPR, and the Crebh–Fgf21 axis in metabolism and growth. Direct degradation of nuclear proAvp Crebh transcription factors by ERAD is not shown proINS Xbp1s here. Tg, thapsigargin; Tm, tunicamycin; Pomc proINS, proinsulin; uXBP1, unspliced XBP1; S1P and S2P, Golgi site-specific proteases. S1P, S2P Quality control ER Quantity control ERAD, ER Golgi chaperones, Pre-BCR Xbp1s lipid Dgat2 metabolism Mogat2 Ire1α Gpat3 Fas

Crebh-N Fgf21, Apcs, ERAD Apoa4, Cidec

Crebh Cytosol Cytosol Nucleus map out the stipulations that are necessary for the identification of a fidelity and abundance of ER protein production, and communicate bona fide ERAD substrate in vivo (Box 1), and significantly external cues to gene expression in the nucleus. changed our view of the physiological and pathological importance of ERAD. In this Review, we highlight the hallmark features Constitutive versus inducible ERAD obtained from these recent animal studies: constitutive versus Accumulation of misfolded proteins triggers the ‘unfolded protein inducible ERAD, quality versus quantity control by ERAD, and response’ (UPR), which acts to reduce ER load and increase the ERAD-mediated regulation of gene transcription (Fig. 1). These expression of ER chaperones and ERAD components (Ron and characteristics enable ERAD to function in a basal capacity Walter, 2007; Walter and Ron, 2011). This ‘UPR-centric’ regulating key physiological processes in the body, ensure both mechanism of stress response and ERAD induction and/or Journal of Cell Science

2 REVIEW Journal of Cell Science (2019) 132, jcs232850. doi:10.1242/jcs.232850

misfolded wild-type Avp prohormone (proAvp) proteins from the Box 1. How to identify a bona fide endogenous ERAD ER (Shi et al., 2017). Accordingly, deletion of Sel1L in Avp substrate neurons under basal conditions causes diabetes insipidus with Hallmarks that are required for a protein to be classed as an ERAD diluted urine and low urine osmolality. Constitutive ERAD activity substrate are that there is: (1) a stabilization of substrate protein towards misfolded nascent wild-type proAvp protein is a key step in (elongation of half-life) in the absence of ERAD; (2) a significant increase proAvp maturation to ensure its exit from the ER (Shi et al., 2017). in substrate protein levels in the absence of ERAD; (3) no significant ProAvp protein contains 16 cysteine residues with eight disulfide upregulation of substrate mRNA levels; (4) E3 ligase-dependent poly- ubiquitylation of the substrate is seen; (5) localization of substrate protein bonds and is thus likely prone to misfolding. Indeed, many in the ER during at least some part of its maturation; (6) interaction with mutations that cause the retention of nascent proAvp proteins in the core ERAD components (E3 ligase or adaptor protein). ER have been identified in humans with diabetes insipidus (Birk et al., 2009; Ito et al., 1997, 1999; Phillips, 2003; Rittig et al., 1996), although the link between ERAD and these pathogenic mutants function is thought to occur via inositol-requiring enzyme 1α remains unknown. (Ire1α)- or activating transcription factor 6 (Atf6)-mediated Similarly, in hypothalamic pro-opiomelanocortin (Pomc)- transcriptional control of ERAD gene expression (such as genes expressing neurons, constitutive ERAD functions to oversee the encoding Hrd1, Sel1L, Edem or Derlin), and is largely based on maturation of nascent Pomc in the ER (Kim et al., 2018). Pomc is a studies that used chemical agents, which cause massive ER stress metabolic prohormone with two disulfide bonds, which produces (Hampton, 2000; Zhang et al., 2011). However, whether or not such derivatives that regulate key physiological processes including scenarios apply in vivo under physiological conditions (where ER food intake and post-feeding satiety. Pomc neuron-specific stress is likely to be very mild, if present at all, in many cell types) deficiency of Sel1L in mice causes them to be hyperphagic and remains to be determined. become obese at three to four months of age on a normal chow diet. Several recent studies using animal models have suggested that Mechanistically, Sel1L deletion in Pomc neurons leads to the ERAD mediated by Sel1L–Hrd1 plays a critical constitutive role accumulation of misfolded wild-type Pomc protein, which, in a within the cell (Fig. 1B). Indeed, ERAD genes are constitutively and dominant-negative fashion, retains otherwise folded wild-type ubiquitously expressed, and are also active, independently of any Pomc protein in the ER (Kim et al., 2018). UPR activation. This constitutive ERAD degrades substrate These studies collectively demonstrate the importance of proteins, which may be misfolded or even folding or maturation constitutive ERAD in controlling the maturation and abundance competent, to not only ensure fidelity of production, but to also of specific ER proteins, thereby fine-tuning their activities regulate the abundance of the substrate. within the cell (Fig. 1B). This constitutive function of ERAD is Recent publications by our group and others have independently likely to be independent of the UPR and is sufficient to ensure shown that Sel1L–Hrd1-mediated ERAD is constitutively active in ER proteostasis in certain cell types under (patho-)physiological the murine liver under basal conditions (with very mild, if any, conditions. This process exerts a key homeostatic control of UPR) (Bhattacharya et al., 2018; Wei et al., 2018a). Sel1L–Hrd1 basic physiological processes in a substrate-specific manner, expression in hepatocytes increases with age or feeding. This including, but not limited to, food intake, water balance, constitutive ERAD function in mice fed with regular chow diet is systemic energy homeostasis and cellular development. instrumental in regulating the abundance of its substrate cAMP- To further elaborate this point, we have compared the phenotypes responsive element-binding protein, hepatocyte specific (Crebh; of animal models that are deficient in either ERAD or UPR also known as CREB3L3), an ER-resident transcription factor, published to date to assess the relative contribution of UPR and which in turn induces fibroblast growth factor 21 (Fgf21), a ERAD in various cell types (Table 1). As an example, unlike Pomc powerful metabolic hormone (Bhattacharya et al., 2018; Wei et al., neuron-specific Sel1L-deficient mice, which show early-onset 2018a). In the absence of this constitutive ERAD in the liver, mice obesity with a normal chow diet (Kim et al., 2018), Pomc neuron- exhibit growth retardation with significantly altered systemic energy specific Ire1α-deficient mice exhibit no significant phenotype on a homeostasis – largely owing to the over-activation of the Crebh– normal chow diet (Xiao et al., 2016; Yao et al., 2017). Only when Fgf21 axis (Bhattacharya et al., 2018; Wei et al., 2018a). placed on a high-fat diet for an extended period of time, were Ire1α- Other examples of constitutive ERAD are the Sel1L–Hrd1- deficient mice found in one study to become obese (Yao et al., mediated degradation of the UPR sensor protein Ire1α (Sun et al., 2017), but this observation was not recapitulated in another study 2015) and the pre-B cell receptor protein (pre-BCR) (Ji et al., 2016; (Xiao et al., 2016), possibly in part due to different Ire1α floxed Yang et al., 2018). The first study demonstrated that Ire1α is an mouse models used in these two studies. Therefore, we can endogenous substrate of Sel1L–Hrd1-mediated ERAD (Sun et al., conclude that UPR- and ERAD-deficient mouse models exhibit 2015). Here, the degradation of Ire1α by Sel1L–Hrd1 (with the help distinct phenotypes in most, if not all, cases (Table 1), suggesting of the ER chaperones Os9 and Grp78) occurs constitutively to that these two processes may exert disparate effects in vivo. restrain Ire1α activity under basal physiological conditions in many Additionally, in several instances, ERAD-deficient models cell types. In the gut epithelium, ERAD-mediated regulation of showcased more severe phenotypes than the corresponding UPR- Ire1α activity protects the intestines from inflammatory disease deficient models (Table 1), suggesting that constitutive ERAD may (Sun et al., 2016, 2015). Similarly, two independent studies have actually play a more pertinent role than UPR in these cell types in shown that in developing B cells, Sel1L–Hrd1-mediated ERAD the context of normal physiology. constitutively controls the abundance of surface pre-BCR, thereby restraining its signaling during the transition from large to small pre- Quality versus quantity control by ERAD B cell stage (Ji et al., 2016; Yang et al., 2018). Classically, ERAD has largely been associated with quality control, Moreover, in neurons expressing the antidiuretic hormone that is, the clearance of ‘misfolded’ proteins in the ER (Christianson arginine vasopressin (Avp), Sel1L–Hrd1-mediated ERAD and Ye, 2014; Ye et al., 2004). Recent work using Avp and Pomc constitutively acts to maintain systemic water balance by clearing neuron-specific Sel1L-knockout mice and cells has delineated that Journal of Cell Science

3 REVIEW Journal of Cell Science (2019) 132, jcs232850. doi:10.1242/jcs.232850

Table 1. Comparison between cell type-specific mouse models that are deficient in either ERAD or UPR Cell type In vivo loss of ERAD In vivo loss of UPR Pomc neuron Tg(Pomc-Cre); Sel1L-flox/flox: Early-onset obesity under normal Tg(Pomc-Cre); Ire1a-flox/flox: Obesity after 3-month high-fat diet chow diet, hyperphagia (Kim et al., 2018). (no phenotype under chow diet), hyperphagia (Yao et al., 2017); resistance to high-fat diet-induced obesity, adipose tissue browning (Xiao et al., 2016). Hepatocyte Tg(Alb-Cre); Sel1L/Hrd1-flox/flox: High serum Fgf21 levels, Tg(Alb-Cre); Ire1a-flox/flox: Lower serum Fgf21 levels, impaired decreased growth, activity and female fertility, increased insulin β-oxidation and ketogenesis, chemical- or diet-induced hepatic sensitivity and fat browning (Bhattacharya et al., 2018; Wei et al., steatosis (Jiang et al., 2014; Shao et al., 2014; Wang et al., 2018; 2018a). Zhang et al., 2011). Enterocyte Tg(Villin-Cre); Sel1L-flox/flox: Paneth cell defect, spontaneous Tg(Villin-Cre); Xbp1-flox/flox: Paneth cell defect, spontaneous enteritis, inflammation-associated dysbiosis, susceptibility to enteritis, hyper-proliferative stem cells, susceptibility to colitis experimental colitis (Sun et al., 2016, 2015). and intestinal tumors (Kaser et al., 2008; Niederreiter et al., 2013). Adipocyte Tg(Adipoq-Cre); Sel1L-flox/flox: Resistance to diet-induced obesity, rTA(Adipoq);Tre-Xbp1s, Tg(Adipoq-Cre); Xbp1-flox/flox: postprandial hypertriglyceridemia (Fujita et al., 2015; Sha et al., Regulation of UMP pathway, lipolysis, obesity (Deng et al., 2014a). 2018). Tg(Xbp1s): Improved glucose homeostasis, adiponectin maturation/assembly (Sha et al., 2014b). B cell Cd19-Cre; Sel1L/Hrd1-flox/flox: Blocked development of large pre-B Cd19-Cre; Xbp1/Ire1-flox/flox: Normal B cell development, cells, reduced mature plasma cells; increased activation-induced reduced secretory function of plasma cells (Benhamron et al., B-cell death (AICD) (Ji et al., 2016; Kong et al., 2016; Yang et al., 2014; Goldfinger et al., 2009). 2018). Dendritic cell Cd11c-Cre; Hrd1-flox/flox: Impaired CD4+ T cell priming, resistance to Cd11c-Cre; Ire1-flox/flox: Impaired CD8+T cell proliferation/ experimental autoimmune encephalomyelitis (EAE) by myelin priming to melanoma-associated antigen (Medel et al., 2018). oligodendrocyte glycoprotein (MOG) (Yang et al., 2014). Pancreatic β cell Tg(Ins2-Cre); Sel1L-flox/flox: Blunted glucose-stimulated insulin Tg(Ins2-Cre); Ire1-flox/flox or Xbp1-flox/flox: Blunted GSIS, secretion (GSIS), mild hyperglycemia, glucose intolerance impaired insulin production, chronic hyperglycemia, glucose (Hu et al., 2019). intolerance (Lee et al., 2011; Tsuchiya et al., 2018; Xu et al., 2014) Whole pancreas Tg(βActin-ERTCre); Sel1L-flox/flox: Profound pancreatic atrophy, Ela-Ert2-Cre/Ngn3-Cre/Pdx1-Cre; Perk-flox/flox (exocrine/ exocrine pancreatic insufficiency/atrophy, apoptosis (Sun et al., endocrine/pancreas): Hyperglycemia, exocrine pancreatic 2014). atrophy, apoptosis, diabetes mellitus (Iida et al., 2007; Zhang et al., 2002, 2006). Mist1-Ert2-Cre; Xbp1-flox/flox (acinar cells): Severe apoptosis followed by extensive expansion/recovery (Hess et al., 2011). Mouse models are denoted as ‘(promoter-Cre); floxed gene’; Tg, transgenic; Alb, albumin; Adipoq, adiponectin; Ins2, insulin2; Ela, elastase; Ngn3, noggin3; Pdx1, pancreatic and duodenal homeobox 1.

Sel1L–Hrd1-mediated ERAD is responsible for the quality control demonstrate the significance of substrate-specific ERAD in of the prohormone maturation process in neuroendocrine cells physiology. (Fig. 1C). As described above, in the absence of Sel1L, aberrantly Recent studies suggest that ERAD may also work in a capacity folded Pomc or proAvp molecules accumulate and abrogate the that controls the quantity of proteins by degrading folding- maturation of their otherwise properly folded counterparts by competent proteins (e.g. Crebh, Ire1α and pre-BCR), and thereby engaging them in aggregate formation in a dominant-negative optimizing their associated downstream processes (Fig. 1C). In the manner (Table 2). Interestingly, neither of these Pomc- or Avp- absence of ERAD, these substrate proteins accumulate, which specific Sel1L-knockout mouse models show any overt signs of ultimately leads to gain-of-function phenotypes (Table 2). Although neuronal death, inflammation or ER stress (Kim et al., 2018; Shi quantity control by ERAD has been demonstrated in the past in the et al., 2017), consistent with the notion that protein aggregates in the context of HMG-coA reductase (Hmgcr) degradation in yeast and ER are less toxic (than cytosolic aggregates) and can be well mammalian cells in vitro (Foresti et al., 2013; Hegde and Ploegh, tolerated by the cell (Vincenz-Donnelly et al., 2018). 2010; Johnson and DeBose-Boyd, 2018), in vivo evidence remains Similarly, Sel1L deficiency in adipocytes leads to their faulty limited. Indeed, accounts from animal models depicting Hmgcr maturation, and subsequent aggregation of lipoprotein lipase (Lpl) degradation through Gp78-mediated ERAD in the context of in the ER (Sha et al., 2014a). Accordingly, adipocyte-specific hepatic cholesterol production remain controversial, as one study Sel1L-knockout mice exhibit postprandial hypertriglyceridemia demonstrated Hmgcr to be a substrate of Gp78 (Liu et al., 2012), and are resistant to diet-induced obesity. In Schwann cells, deletion whereas another negated it (Tsai et al., 2012). of Derlin-2 results in ER retention of misfolded myelin protein zero By using proteomic and biochemical approaches in the liver, (P0, also known as MPZ), leading to defective myelin morphology we and others have shown that Sel1L-Hrd1 ERAD is able to recruit and function, and increased propensity to Charcot–Marie–Tooth 1B and ubiquitylate Crebh for proteasomal degradation (Bhattacharya (CMT1B) neuropathy (Volpi et al., 2019). Moreover, deletion of et al., 2018; Wei et al., 2018a). In Sel1L- and Hrd1-deficient Sel1L in pancreatic β-cells leads to impaired proinsulin maturation hepatocytes, Crebh accumulates in the hepatic ER and, following in the ER, glucose-stimulated insulin secretion and mild proteolysis at the Golgi, its N-terminal domain, which encodes an hyperglycemia in mice (Hu et al., 2019). However, fully active transcription factor, translocates to the nucleus where it understanding the relevance and importance of Sel1L–Hrd1 induces the expression of Fgf21. Consequently, these mice ERAD in β-cell biology and proinsulin maturation requires further phenocopy mouse models with gain-of-function of Fgf21 in studies. These examples of ERAD-mediated quality control exhibiting growth retardation, lower serum lipid levels, increased Journal of Cell Science

4 REVIEW Journal of Cell Science (2019) 132, jcs232850. doi:10.1242/jcs.232850

Table 2. Overview over endogenous ERAD substrates and their localization and pathology ERAD Substrate system Subcellular localization Cell type Pathology Pre-BCR Sel1L Hrd1 Plasma membrane, B cell Developmental block in transition from large to small pre-B cells (Ji et al., 2016; ER membrane Yang et al., 2018)

Fas Hrd1 Plasma membrane, B cell Activation-induced B-cell death (AICD), reduced mature B cells (Kong et al., 2016) ER membrane

Protein0 Derlin2 Plasma membrane, Schwann cell Impaired myelin homeostasis, Charcot-Marie-Tooth 1B neuropathy (Volpi et al., ER membrane 2019)

Crebh Sel1L Hrd1 ER membrane, Hepatocyte Highly elevated Fgf21 levels, altered growth and metabolism (Bhattacharya et al., Nucleus 2018; Wei et al., 2018a)

Hmgcr gp78 ER membrane Hepatocyte Altered lipid metabolism; substrate status disputed (Liu et al., 2012; Tsai et al., 2012) Ire1α Sel1L Hrd1 ER membrane Ubiquitous Spontaneous ileitis, susceptibility to experimental colitis, dysbiosis (Sun et al., 2016, 2015)

Mogat2 Aida Hrd1 ER lumen Enterocyte Increased fatty acid re-esterification and lipid absorption in intestine, postprandial Dgat2 Aida Hrd1 ER lumen Enterocyte hypertriglyceridemia, severe obesity in the absence of increased adipogenesis Gpat3 Aida Hrd1 ER lumen Enterocyte (Luo et al., 2018) ProAvp Sel1L Hrd1 ER lumen Avp neuron Central diabetes insipidus, polydipsia, polyuria (Shi et al., 2017)

Pomc Sel1L Hrd1 ER lumen Pomc neuron Hyperphagia, hyperleptinemia, age-associated obesity (Kim et al., 2018)

Nrf2 Hrd1 Nucleus Hepatocyte Suppression of anti-oxidation pathway, liver cirrhosis (Wu et al., 2014)

Pgc1β Hrd1 Nucleus White adipocyte Altered mitochondrial dynamics, weight gain and obesity (Fujita et al., 2015) p27kip1 Hrd1 Nucleus T cell Altered proliferation program (Xu et al., 2016)

Blimp1 Hrd1 Nucleus Dendritic cell Experimental autoimmune encephalomyelitis from myelin oligodendrocyte glycoprotein (Yang et al., 2014)

Hsd17b4 Hrd1 Peroxisome Hepatocyte Over activation of Ampk/Akt, resistance to diet-induced obesity, insulin sensitivity, Entpd5 Hrd1 ER lumen Hepatocyte fatty liver disease (Entpd5 aids in ATP hydrolysis, Cpt2, Rmnd1 in lipid/protein Rmnd1 Hrd1 Mitochondria Hepatocyte translocation, Hsd17b4 in fatty acid oxidation) (Wei et al., 2018b) Cpt2 Hrd1 Mitochondrial inner Hepatocyte membrane

insulin sensitivity (Inagaki et al., 2007; Inagaki et al., 2008; However, the relevance and importance of Aida for the function of Kharitonenkov et al., 2005; Owen et al., 2014), reduced Hrd1 remains to be established. female fertility (Owen et al., 2013), adipose tissue browning In the immune system, Sel1L–Hrd1-mediated ERAD is critical (BonDurant et al., 2017; Fisher et al., 2012; Hondares et al., 2010; for the proper maturation and function of developing B Owen et al., 2014), and resistance to diet-induced obesity lymphocytes. In immature B cells, Sel1L–Hrd1-mediated (Kharitonenkov et al., 2005). In addition, Hrd1 also degrades ubiquitylation and turnover of the pre-BCR complex is critical certain metabolic enzymes in hepatocytes, such as ectonucleoside for the developing B cell to transition from the large to small pre- triphosphate diphosphohydrolase 5 (Entpd5) in the ER, carnitine B stage. In the absence of ERAD, the pre-BCR complex palmitoyltransferase 2 (Cpt2) and required for meiotic nuclear continually accumulates and migrates to the B cell surface, division 1 homolog (Rmnd1) in mitochondria, and hydroxysteroid leading to excessive signaling and developmental blockade at the 17-β dehydrogenase 4 (Hsd17b4) in peroxisomes (Wei et al., large pre-B stage (Ji et al., 2016; Yang et al., 2018). Moreover, in 2018b); this may also contribute to the phenotype of hepatocyte- mature B cells, Hrd1 degrades the cell death receptor Fas (also specific ERAD-deficient mice. known as cluster of differentiation 95, Cd95) to protect B cells Similarly, in many cell types, including intestinal epithelium, from Fas-mediated apoptosis (Kong et al., 2016). adipocytes and pancreas, Sel1L–Hrd1-mediated ERAD regulates In summary, while ERAD function has previously mostly been the quantity of the UPR-sensor Ire1α, as deletion of either Sel1L or associated with the triage of misfolded proteins in the ER, thus Hrd1 leads to the accumulation and mild activation of Ire1α (Sun exerting a quality control function (e.g. of prohormones), several et al., 2015). Furthermore, a more-recent study reported the recent studies in physiological settings now demonstrate that ERAD identification of a Hrd1–axin interactor, dorsalization-associated also works as a quantity control mechanism to regulate the (Aida) ERAD complex, which ubiquitylates and degrades key abundance of folding-competent substrates, such as Crebh, Ire1α triglyceride synthesis enzymes, including glycerol-3-phosphate and the pre-BCR. Unlike its role in quality control, which acyltransferase 3 (Gpat3), monoacylglycerol o-acyltransferase 2 safeguards protein maturation processes in the ER, ERAD- (Mogat2) and diacylglycerol o-acyltransferase 2 (Dgat2), in mediated quantity control restricts or restrains the abundance enterocytes (Luo et al., 2018). The authors showed that these and function of its substrates and so fine-tunes associated degradation events protect mice from excessive lipid absorption, downstream processes to their optimal levels. It is important postprandial hypertriglyceridemia and obesity (Luo et al., 2018). to note here that it is difficult to state definitively whether or not Journal of Cell Science

5 REVIEW Journal of Cell Science (2019) 132, jcs232850. doi:10.1242/jcs.232850 the substrate proteins of ERAD are misfolded or folding position Sel1L–Hrd1 ERAD as a central node in linking competent. Theoretically, even a small unfolded segment may physiological cues to gene transcription. More such examples of suffice to form a degron in the protein, which can then be targeted ERAD-mediated gene regulation in vivo will likely emerge from to the ERAD pathway. However, an ERAD deficiency could future investigations. affect the folding environment of the ER, and/or post-ER trafficking, which may depend on the intrinsic folding and ERAD in disease biochemical properties of the substrate, as well as the levels of Disease-causing mutations often give rise to protein misfolding and various chaperones in the ER. thus, in theory, would create perfect ERAD substrates. However, the question then is how these pathogenic mutants cause disease if ERAD in regulating gene transcription ERAD is functional. Two recent papers on prohormones have A direct communication from the ER to gene transcription in the provided some insights into the pathological significance of ERAD nucleus is a characteristic of UPR, such as the axes involving Ire1α in dealing with pathogenic mutations (Kim et al., 2018; Shi et al., and the spliced form of X-box binding protein 1 (Xbp1s), or protein 2017). The mutants obtained from diabetes insipidus patients kinase R (PKR)-like ER kinase (PERK), eukaryotic translation (proAVP-G57S, Gly57 to Ser and ΔE47, deletion of Glu47) and initiation factor 2A (eIF2α) and activating transcription factor 4 obese patients (POMC-C28F, Cys28 to Phe) are prone to forming (Atf4) or Atf6 activation (Walter and Ron, 2011). But whether or intracellular aggregates (Birk et al., 2009; Creemers et al., 2008; Ito not ERAD exerts a similar control over nuclear transcription et al., 1999; Rittig et al., 1996). ERAD may become insufficient in remains underappreciated, especially in vivo. In yeast, early studies the face of an overwhelming amount of misfolded mutant proteins have shown that ubiquitin and/or proteasome-dependent processing or, alternatively, certain disease-causing mutant proteins may evade regulates the abundance of the ER-associated transcription factor ERAD (Kim et al., 2018; Shi et al., 2017). These pathogenic suppressor of Ty 23 (Spt23) and its paralog Mga2 to regulate fatty mutations then may form high-molecular-mass protein aggregates, acid metabolism (Hoppe et al., 2000; Rape et al., 2001). Recent which interfere with their wild-type counterparts in a dominant- studies in mouse models highlight the importance of ER protein negative manner. In the case of C28F POMC, the mutant forms turnover in the regulation of gene transcription in various higher molecular mass protein aggregates via the unpaired cysteine physiological settings (Fig. 1D). residue at position 50 (Kim et al., 2018). Indeed, the pathogenic In the liver, Sel1L–Hrd1 regulates the transcription of Fgf21 gene effect of the C28F mutation can be suppressed by an intragenic via degradation of the ER-resident transcription factor Crebh. As mutation at C50 to a serine or alanine residue (Kim et al., 2018). mentioned above, in the absence of Sel1L–Hrd1, Crebh These recent revelations, and the implication that ERAD may accumulates and translocates to the nucleus following its become insufficient in the face of disease mutations, also point to proteolysis at Golgi to trigger the expression of many target the potential therapeutic intervention of targeting ERAD-mediated genes, including Fgf21 (Bhattacharya et al., 2018; Wei et al., turnover of disease mutants. 2018a). As Sel1L–Hrd1 expression is controlled by nutrient fasting- In this context, small molecules targeting Sel1L–Hrd1-mediated feeding and growth, these studies suggest that hepatic ERAD ERAD may have significant therapeutic value. LS-101 and LS-102 activity directly links physiological cues to gene transcription and are two classes of Hrd1 inhibitors that have been demonstrated to systemic energy metabolism in the body. In addition, as described suppress the progression of rheumatoid arthritis – a disease in which above, Sel1L-Hrd1-mediated ERAD regulates the turnover of Ire1α, Hrd1 expression is found to be highly elevated (Yagishita et al., which is responsible for the production of the key UPR transcription 2012). Additionally, selective inhibition of Hrd1 by LS-102 has factor Xbp1s (Sun et al., 2015). This ERAD–UPR interaction is been shown to increase Pgc1β levels in white adipose tissues, required for the control of ER capacity and function, pointing to a leading to reduced fat accumulation and increased mitochondrial feedback loop between ERAD and UPR within the cell. numbers in mouse models (Fujita et al., 2015), further underscoring In addition, Hrd1 may directly mediate the degradation of nuclear the therapeutic importance of this regulatory axis in obesity transcription factors. Examples include B lymphocyte-induced treatment. Furthermore, CB5083, an inhibitor of the ERAD- maturation protein-1 (Blimp1; also known as PRDM1), a associated Vcp ATPase, has shown promise as a therapeutic agent transcriptional repressor of the MHC-II gene in dendritic cells that in combating both solid and hematological tumor models is crucial for the pathogenesis of myelin oligodendrocyte (Anderson et al., 2015; Le Moigne et al., 2017). Finally, as Sel1L glycoprotein (MOG)-induced experimental autoimmune is indispensable for Hrd1 stability and ERAD function, small encephalomyelitis (EAE) (Yang et al., 2014), nuclear factor molecules that target the interaction between Sel1L and Hrd1 may erythroid 2-related factor 2 (Nrf2, also known as NFE2L2), a also have significant therapeutic potential. regulator of anti-oxidant pathways in the liver, conferring protection from ROS-induced liver cirrhosis (Wu et al., 2014), p27kip1 Conclusions and further questions (CDKN1B), a cyclin-dependent kinase inhibitor in T cells, which Sel1L–Hrd1-mediated ERAD has emerged as a crucial determinant regulates their proliferation potential (Xu et al., 2016), and of normal physiology and as a sentinel against disease pathogenesis peroxisome proliferator-activated receptor γ coactivator 1β in the body. Recent studies of ERAD-deficient animal models (Pgc1β), which regulates mitochondrial biogenesis in the adipose highlight three features of ERAD (Fig. 1B–D) that are crucial for tissue, protecting from obesity development (Fujita et al., 2015). maintaining physiological homeostasis. First, constitutive ERAD However, the underlying molecular mechanism with regard to how can function to maintain optimal levels of substrate proteins within Hrd1 recognizes nuclear substrates and whether this depends on the cell. Second, ERAD activity can perform both quality control of Sel1L remains unclear. misfolded proteins (e.g. proAvp, Pomc and Lpl) and quantity Collectively, these studies reveal exciting regulatory cascades control of maturation-competent proteins (e.g. Crebh, preBCR and from protein degradation at the ER membrane to gene transcription Ire1α); this ensures the abundance of substrate proteins and in the nucleus (Fig. 1D), which likely are the means for how ERAD maintains the desired levels of downstream processes. Third, controls the responses to in vivo physiological cues. They also ERAD is also capable of integrating extracellular cues to regulate Journal of Cell Science

6 REVIEW Journal of Cell Science (2019) 132, jcs232850. doi:10.1242/jcs.232850 nuclear gene transcription by controlling the turnover of ER- abrogation of function of the properly folded ‘bystander’ resident transcriptional modulators. Future explorations into both proteins along the way, while only triggering very mild, if any, the physiological and pathological aspects of ERAD as part of a UPR owing to mechanisms including, but not limited to, an macroscale signaling network are thus indispensable. adaptation in the ER and/or compensation from other cellular Nevertheless, a number of key questions remain. For instance, we protein clearance systems. Further research emphasis on these still lack the means to accurately measure and quantify ERAD aspects is needed to delineate the emerging interplay between capacity and function in a (patho)physiological context. To better these mutant proteins and ERAD function in the context of elucidate ERAD biology, a reliable tool or readout is required that disease pathogenesis. allows to directly assess ERAD activity, especially in a Another challenging area that warrants further investigation is the physiological setting, akin to the tools available for measuring inevitable crosstalk among the three key quality control systems UPR activity (e.g. Ire1α and Perk phosphorylation, Xbp1 splicing) within the cell – ERAD, UPR and autophagy (or ER-phagy) – in (see Box 2), or autophagic flux (such as LC3 lipidation or p62 terms of how and when these processes complement each other, or degradation). act in a redundant or, possibly, competitive manner, especially in the Another important question is what defines ERAD substrate context of substrate choice, allocation of cellular resources and specificity. With the identification of many endogenous substrates disease development. Animal models that bear tissue-specific (Table 2), we are now at a better position to address whether deficiencies in ERAD, UPR and autophagy, either singly or in substrate recognition by ERAD is relatively stochastic, largely combination with each other, will be necessary to tease apart these driven by local stoichiometric concentrations, or whether specific challenging but critical questions. Probing into these outstanding chaperones (e.g. Os9 or Grp78) intervene to make it a deterministic questions will allow us to visualize, in the whole organism, the or ‘intelligent’ choice. Furthermore, while several recent studies intricate network formed by these cellular quality control systems in using mouse models have demonstrated that changes in the enabling cellular function in diverse contexts of health and disease. physiological states of the body (e.g. nutrient fasting-feeding, water deprivation and growth) induce the expression of ERAD Acknowledgements components in specific cell types (Bhattacharya et al., 2018; Kim We apologize to colleagues whose works were not cited due to the space limitations. et al., 2018; Shi et al., 2017; Wei et al., 2018a), an open area of Competing interests research is to identify the underlying signaling pathways. Achieving The authors declare no competing or financial interests. this might also make it possible to develop therapeutic approaches aimed at fine-tuning ERAD function within the cell. Funding One key issue in ERAD research is the popular, but misguided, The work in the Qi laboratory is supported by the National Institutes of Health (NIH; belief that ERAD deficiency is always associated with massive ER R01GM113188, R35DM130292, R01DK105393, R01DK111174, R01DK120047, R01DK120330, R01DK117639), the University of Michigan Protein Folding stress and cell death. However, that does not appear to hold true in Diseases Initiative, Juvenile Diabetes Research Foundation United States of certain cell types under physiological settings (Bhattacharya et al., America (JDRF) and American Diabetes Association (ADA). A.B. was a recipient of 2018; Kim et al., 2018; Shi et al., 2017; Wei et al., 2018a). Instead, an American Heart Association Predoctoral Fellowship (16PRE29750001). L.Q. is specific ERAD substrates, either via loss- or gain-of-function, the recipient of the Junior Faculty and Career Development Awards from the ADA. Deposited in PMC for release after 12 months. appear to contribute collectively to the phenotypes of ERAD- deficient mice. While current popular belief is that disease-causing References proteins are misfolded and exert their pathogenicity by triggering Altier, C., Garcia-Caballero, A., Simms, B., You, H., Chen, L., Walcher, J., ER stress and activating UPR, another possibility, as discussed Tedford, H. W., Hermosilla, T. and Zamponi, G. W. (2011). The cavbeta subunit above, is that at least some of these disease mutants may prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels. Nat. Neurosci. 14, 173-180. doi:10.1038/nn.2712 bypass ERAD-mediated quality control. This often leads to the Anderson, D. J., Le Moigne, R., Djakovic, S., Kumar, B., Rice, J., Wong, S., Wang, J., Yao, B., Valle, E., Kiss von Soly, S. et al. (2015). Targeting the AAA ATPase p97 as an approach to treat cancer through disruption of protein homeostasis. Cancer Cell 28, 653-665. doi:10.1016/j.ccell.2015.10.002 Box 2. How to accurately quantify stress levels in the ER Araki, K. and Nagata, K. (2011). Protein folding and quality control in the ER. Cold In order to accurately quantify stress levels in the ER, it is necessary to: (1) Spring Harb. Perspect. Biol. 3, a007526. doi:10.1101/cshperspect.a007526 measure the level of phosphorylation of the UPR sensors Ire1α and Perk to Aridor, M. and Balch, W. E. (1999). Integration of endoplasmic reticulum signaling accurately quantify the ‘level’ of ER stress (Yang et al., 2010); (2) measure in health and disease. Nat. Med. 5, 745-751. doi:10.1038/10466 Baldridge, R. D. and Rapoport, T. A. (2016). Autoubiquitination of the Hrd1 ligase activation of downstream effectors of Ire1α and Perk, namely, Xbp1 mRNA triggers protein retrotranslocation in ERAD. Cell 166, 394-407. doi:10.1016/j.cell. splicing assessment (via RT-PCR) and the ratio of phosphorylation of α α 2016.05.048 eIF2 to total eIF2 protein level (via western blotting) (Sha et al., 2009); Benhamron, S., Hadar, R., Iwawaky, T., So, J.-S., Lee, A.-H. and Tirosh, B. and (3) measure the expression of downstream target genes, although this (2014). Regulated IRE1-dependent decay participates in curtailing cannot be used alone, to assess ER stress level under pathophysiological immunoglobulin secretion from plasma cells. Eur. J. Immunol. 44, 867-876. conditions. doi:10.1002/eji.201343953 We and others have verified the following antibodies as dependable Bernasconi, R., Galli, C., Calanca, V., Nakajima, T. and Molinari, M. (2010). tools for UPR assessment (DeNicola et al., 2015; He et al., 2012; Sun Stringent requirement for HRD1, SEL1L, and OS-9/XTP3-B for disposal of ERAD- et al., 2019; Yang et al., 2013); other antibodies (e.g. against Atf6) require LS substrates. J. Cell Biol. 188, 223-235. doi:10.1083/jcb.200910042 further quality assessment and improvement in specificity: Bhattacharya, A., Sun, S., Wang, H., Liu, M., Long, Q., Yin, L., Kersten, S., • Ire1α, Cell Signaling #3294 (use together with phos-tag western Zhang, K. and Qi, L. (2018). Hepatic Sel1L-Hrd1 ER-associated degradation blotting) (ERAD) manages FGF21 levels and systemic metabolism via CREBH. EMBO J. • Xbp1s, Cell Signaling #83418 37, e99277. doi:10.15252/embj.201899277 Birk, J., Friberg, M. A., Prescianotto-Baschong, C., Spiess, M. and • Perk, Cell Signaling #3192 Rutishauser, J. (2009). Dominant pro-vasopressin mutants that cause • Phospho-eIF2α, Cell Signaling #3597 diabetes insipidus form disulfide-linked fibrillar aggregates in the endoplasmic • eIF2α-total, Cell Signaling #9722 reticulum. J. Cell Sci. 122, 3994-4002. doi:10.1242/jcs.051136 • Atf4, Cell Signaling #11815 BonDurant, L. D., Ameka, M., Naber, M. C., Markan, K. R., Idiga, S. O., Acevedo,

M. R., Walsh, S. A., Ornitz, D. M. and Potthoff, M. J. (2017). FGF21 regulates Journal of Cell Science

7 REVIEW Journal of Cell Science (2019) 132, jcs232850. doi:10.1242/jcs.232850

metabolism through adipose-dependent and -independent mechanisms. Cell Hegde, R. S. and Lingappa, V. R. (1997). Membrane protein biogenesis: regulated Metab. 25, 935-944.e4. doi:10.1016/j.cmet.2017.03.005 complexity at the endoplasmic reticulum. Cell 91, 575-582. doi:10.1016/S0092- Braakman, I. and Bulleid, N. J. (2011). Protein folding and modification in the 8674(00)80445-6 mammalian endoplasmic reticulum. Annu. Rev. Biochem. 80, 71-99. doi:10.1146/ Hegde, R. S. and Ploegh, H. L. (2010). Quality and quantity control at the annurev-biochem-062209-093836 endoplasmic reticulum. Curr. Opin. Cell Biol. 22, 437-446. doi:10.1016/j.ceb. Carvalho, P., Stanley, A. M. and Rapoport, T. A. (2010). Retrotranslocation of a 2010.05.005 misfolded luminal ER protein by the ubiquitin-ligase Hrd1p. Cell 143, 579-591. Hess, D. A., Humphrey, S. E., Ishibashi, J., Damsz, B., Lee, A. H., Glimcher, L. H. doi:10.1016/j.cell.2010.10.028 and Konieczny, S. F. (2011). Extensive pancreas regeneration following acinar- Christianson, J. C. and Ye, Y. (2014). Cleaning up in the endoplasmic reticulum: specific disruption of Xbp1 in mice. Gastroenterology 141, 1463-1472. doi:10. ubiquitin in charge. Nat. Struct. Mol. Biol. 21, 325-335. doi: 10.1038/nsmb.2793 1053/j.gastro.2011.06.045 **Christianson, J. C., Shaler, T. A., Tyler, R. E. and Kopito, R. R. (2008). OS-9 Hondares, E., Rosell, M., Gonzalez, F. J., Giralt, M., Iglesias, R. and Villarroya, F. and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase (2010). Hepatic FGF21 expression is induced at birth via PPARalpha in response complex for ERAD. Nat. Cell Biol. 10, 272-282. doi:10.1038/nsmb.2793 to milk intake and contributes to thermogenic activation of neonatal brown fat. Cell Christianson, J. C., Olzmann, J. A., Shaler, T. A., Sowa, M. E., Bennett, E. J., Metab. 11, 206-212. doi:10.1016/j.cmet.2010.02.001 Richter, C. M., Tyler, R. E., Greenblatt, E. J., Harper, J. W. and Kopito, R. R. Hoppe, T., Matuschewski, K., Rape, M., Schlenker, S., Ulrich, H. D. and Jentsch, (2012). Defining human ERAD networks through an integrative mapping strategy. S. (2000). Activation of a membrane-bound transcription factor by regulated Nat. Cell Biol. 14, 93-105. doi:10.1038/ncb2383 ubiquitin/proteasome-dependent processing. Cell 102, 577-586. doi:10.1016/ Creemers, J. W. M., Lee, Y. S., Oliver, R. L., Bahceci, M., Tuzcu, A., Gokalp, D., S0092-8674(00)00080-5 Keogh, J., Herber, S., White, A., O’Rahilly, S. et al. (2008). Mutations in the Hu, Y., Gao, Y., Zhang, M., Deng, K.-Y., Singh, R., Tian, Q., Gong, Y., Pan, Z., Liu, amino-terminal region of proopiomelanocortin (POMC) in patients with early- Q., Boisclair, Y. R. et al. (2019). Endoplasmic Reticulum-Associated onset obesity impair POMC sorting to the regulated secretory pathway. J. Clin. Degradation (ERAD) has a critical role in supporting glucose-stimulated insulin Endocrinol. Metab. 93, 4494-4499. doi:10.1210/jc.2008-0954 secretion in pancreatic beta-cells. Diabetes 68, 733-746. doi:10.2337/db18-0624 Deng, Y., Wang, Z. V., Gordillo, R., Zhu, Y., Ali, A., Zhang, C., Wang, X., Shao, M., Hwang, J. and Qi, L. (2018). Quality control in the endoplasmic reticulum: crosstalk Zhang, Z., Iyengar, P. et al. (2018). Adipocyte Xbp1s overexpression drives between ERAD and UPR pathways. Trends Biochem. Sci. 43, 593-605. doi:10. uridine production and reduces obesity. Mol. Metab. 11, 1-17. doi:10.1016/j. 1016/j.tibs.2018.06.005 molmet.2018.02.013 Iida, K., Li, Y., McGrath, B. C., Frank, A. and Cavener, D. R. (2007). PERK eIF2 DeNicola, G. M., Chen, P.-H., Mullarky, E., Sudderth, J. A., Hu, Z., Wu, D., Tang, alpha kinase is required to regulate the viability of the exocrine pancreas in mice. H., Xie, Y., Asara, J. M., Huffman, K. E. et al. (2015). NRF2 regulates serine BMC Cell Biol. 8, 38. doi:10.1186/1471-2121-8-38 biosynthesis in non-small cell lung cancer. Nat. Genet. 47, 1475-1481. doi:10. Inagaki, T., Dutchak, P., Zhao, G., Ding, X., Gautron, L., Parameswara, V., Li, Y., 1038/ng.3421 Goetz, R., Mohammadi, M., Esser, V. et al. (2007). Endocrine regulation of the Dougan, S. K., Hu, C.-C. A., Paquet, M.-E., Greenblatt, M. B., Kim, J., Lilley, fasting response by PPARalpha-mediated induction of fibroblast growth factor 21. B. N., Watson, N. and Ploegh, H. L. (2011). Derlin-2-deficient mice reveal an Cell Metab. 5, 415-425. doi:10.1016/j.cmet.2007.05.003 essential role for protein dislocation in chondrocytes. Mol. Cell. Biol. 31, Inagaki, T., Lin, V. Y., Goetz, R., Mohammadi, M., Mangelsdorf, D. J. and Kliewer, S. A. (2008). Inhibition of growth hormone signaling by the fasting- 1145-1159. doi:10.1128/MCB.00967-10 induced hormone FGF21. Cell Metab. 8, 77-83. doi:10.1016/j.cmet.2008.05.006 Ernst, R., Mueller, B., Ploegh, H. L. and Schlieker, C. (2009). The otubain YOD1 is Ito, M., Jameson, J. L. and Ito, M. (1997). Molecular basis of autosomal dominant a deubiquitinating enzyme that associates with p97 to facilitate protein dislocation neurohypophyseal diabetes insipidus. Cellular toxicity caused by the from the ER. Mol. Cell 36, 28-38. doi:10.1016/j.molcel.2009.09.016 accumulation of mutant vasopressin precursors within the endoplasmic Eura, Y., Yanamoto, H., Arai, Y., Okuda, T., Miyata, T. and Kokame, K. (2012). reticulum. J. Clin. Invest. 99, 1897-1905. doi:10.1172/JCI119357 Derlin-1 deficiency is embryonic lethal, Derlin-3 deficiency appears normal, and Ito, M., Yu, R. N., Jameson, J. L. and Ito, M. (1999). Mutant vasopressin precursors Herp deficiency is intolerant to glucose load and ischemia in mice. PLoS ONE 7, that cause autosomal dominant neurohypophyseal diabetes insipidus retain e34298. doi:10.1371/journal.pone.0034298 dimerization and impair the secretion of wild-type proteins. J. Biol. Chem. 274, Fang, S., Ferrone, M., Yang, C., Jensen, J. P., Tiwari, S. and Weissman, A. M. 9029-9037. doi:10.1074/jbc.274.13.9029 (2001). The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein Ji, Y., Kim, H., Yang, L., Sha, H., Roman, C. A., Long, Q. and Qi, L. (2016). The ligase implicated in degradation from the endoplasmic reticulum. Proc. Natl. Acad. Sel1L-Hrd1 endoplasmic reticulum-associated degradation complex manages a Sci. USA 98, 14422-14427. doi:10.1073/pnas.251401598 key checkpoint in b cell development. Cell Rep. 16, 2630-2640. doi:10.1016/j. Fisher, F. M., Kleiner, S., Douris, N., Fox, E. C., Mepani, R. J., Verdeguer, F., Wu, celrep.2016.08.003 J., Kharitonenkov, A., Flier, J. S., Maratos-Flier, E. et al. (2012). FGF21 Jiang, S., Yan, C., Fang, Q.-C., Shao, M.-L., Zhang, Y.-L., Liu, Y., Deng, Y.-P., regulates PGC-1alpha and browning of white adipose tissues in adaptive Shan, B., Liu, J.-Q., Li, H.-T. et al. (2014). Fibroblast growth factor 21 is regulated thermogenesis. Genes Dev. 26, 271-281. doi:10.1101/gad.177857.111 by the IRE1alpha-XBP1 branch of the unfolded protein response and counteracts Foresti, O., Ruggiano, A., Hannibal-Bach, H. K., Ejsing, C. S. and Carvalho, P. endoplasmic reticulum stress-induced hepatic steatosis. J. Biol. Chem. 289, (2013). Sterol homeostasis requires regulated degradation of squalene 29751-29765. doi:10.1074/jbc.M114.565960 monooxygenase by the ubiquitin ligase Doa10/Teb4. eLife 2, e00953. doi:10. Johnson, B. M. and DeBose-Boyd, R. A. (2018). Underlying mechanisms 7554/eLife.00953 for sterol-induced ubiquitination and ER-associated degradation of HMG Francisco, A. B., Singh, R., Li, S., Vani, A. K., Yang, L., Munroe, R. J., Diaferia, CoA reductase. Semin. Cell Dev. Biol. 81, 121-128. doi:10.1016/j.semcdb. G., Cardano, M., Biunno, I., Qi, L. et al. (2010). Deficiency of suppressor 2017.10.019 enhancer lin12 1 like (SEL1L) in mice leads to systemic endoplasmic reticulum Kaser, A., Lee, A.-H., Franke, A., Glickman, J. N., Zeissig, S., Tilg, H., stress and embryonic lethality. J. Biol. Chem. 285, 13694-13703. doi:10.1074/jbc. Nieuwenhuis, E. E. S., Higgins, D. E., Schreiber, S., Glimcher, L. H. et al. M109.085340 (2008). XBP1 links ER stress to intestinal inflammation and confers genetic risk for Fujita, H., Yagishita, N., Aratani, S., Saito-Fujita, T., Morota, S., Yamano, Y., human inflammatory bowel disease. Cell 134, 743-756. doi:10.1016/j.cell.2008. Hansson, M. J., Inazu, M., Kokuba, H., Sudo, K. et al. (2015). The E3 ligase 07.021 synoviolin controls body weight and mitochondrial biogenesis through negative Kharitonenkov, A., Shiyanova, T. L., Koester, A., Ford, A. M., Micanovic, R., regulation of PGC-1beta. EMBO J. 34, 1042-1055. doi:10.15252/embj. Galbreath, E. J., Sandusky, G. E., Hammond, L. J., Moyers, J. S., Owens, R. A. 201489897 et al. (2005). FGF-21 as a novel metabolic regulator. J. Clin. Invest. 115, Goldfinger, M., Laviad, E. L., Hadar, R., Shmuel, M., Dagan, A., Park, H., Merrill, 1627-1635. doi:10.1172/JCI23606 A. H., Jr., Ringel, I., Futerman, A. H. and Tirosh, B. (2009). De novo ceramide Kikkert, M., Doolman, R., Dai, M., Avner, R., Hassink, G., van Voorden, S., synthesis is required for N-linked glycosylation in plasma cells. J. Immunol. 182, Thanedar, S., Roitelman, J., Chau, V. and Wiertz, E. (2004). Human HRD1 is an 7038-7047. doi:10.4049/jimmunol.0802990 E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic Guerriero, C. J. and Brodsky, J. L. (2012). The delicate balance between secreted reticulum. J. Biol. Chem. 279, 3525-3534. doi:10.1074/jbc.M307453200 protein folding and endoplasmic reticulum-associated degradation in human Kim, G. H., Shi, G., Somlo, D. R. M., Haataja, L., Song, S., Long, Q., Nillni, E. A., physiology. Physiol. Rev. 92, 537-576. doi:10.1152/physrev.00027.2011 Low, M. J., Arvan, P., Myers, M. G. et al. (2018). Hypothalamic ER-associated Hampton, R. Y. (2000). ER stress response: getting the UPR hand on misfolded degradation regulates POMC maturation, feeding and age-associated obesity. proteins. Curr. Biol. 10, R518-R521. doi:10.1016/S0960-9822(00)00583-2 J. Clin. Invest. 128, 1125-1140. doi:10.1172/JCI96420 Hassink, G., Kikkert, M., van Voorden, S., Lee, S.-J., Spaapen, R., van Laar, T., Kong, S., Yang, Y., Xu, Y., Wang, Y., Zhang, Y., Melo-Cardenas, J., Xu, X., Gao, Coleman, C. S., Bartee, E., Früh, K., Chau, V. et al. (2005). TEB4 is a C4HC3 B., Thorp, E. B., Zhang, D. D. et al. (2016). Endoplasmic reticulum-resident E3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum. Biochem. J. ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death 388, 647-655. doi:10.1042/BJ20041241 receptor CD95/Fas. Proc. Natl. Acad. Sci. USA 113, 10394-10399. doi:10.1073/ He, Y., Beatty, A., Han, X., Ji, Y., Ma, X., Adelstein, R. S., Yates, J. R., Kemphues, pnas.1606742113 K. and Qi, L. (2012). Non-muscle myosin IIB links cytoskeleton to IRE1α signaling Le Moigne, R., Aftab, B. T., Djakovic, S., Dhimolea, E., Valle, E., Murnane, M.,

during ER stress. Dev. Cell 23, 1141-1152. doi:10.1016/j.devcel.2012.11.006 King, E. M., Soriano, F., Menon, M. K., Wu, Z. Y. et al. (2017). The p97 Inhibitor Journal of Cell Science

8 REVIEW Journal of Cell Science (2019) 132, jcs232850. doi:10.1242/jcs.232850

CB-5083 is a unique disrupter of protein homeostasis in models of multiple Sha, H., Sun, S., Francisco, A. B., Ehrhardt, N., Xue, Z., Liu, L., Lawrence, P., myeloma. Mol. Cancer Ther. 16, 2375-2386. doi:10.1158/1535-7163.MCT-17- Mattijssen, F., Guber, R. D., Panhwar, M. S. et al. (2014a). The ER-associated 0233 degradation adaptor protein Sel1L regulates LPL secretion and lipid metabolism. Lee, A.-H., Heidtman, K., Hotamisligil, G. S. and Glimcher, L. H. (2011). Dual and Cell Metab. 20, 458-470. doi:10.1016/j.cmet.2014.06.015 opposing roles of the unfolded protein response regulated by IRE1alpha and Sha, H., Yang, L., Liu, M., Xia, S., Liu, Y., Liu, F., Kersten, S. and Qi, L. (2014b). XBP1 in proinsulin processing and insulin secretion. Proc. Natl. Acad. Sci. USA Adipocyte spliced form of x-box-binding protein 1 promotes adiponectin 108, 8885-8890. doi:10.1073/pnas.1105564108 multimerization and systemic glucose homeostasis. Diabetes 63, 867-879. Lilley, B. N. and Ploegh, H. L. (2004). A membrane protein required for dislocation doi:10.2337/db13-1067 of misfolded proteins from the ER. Nature 429, 834-840. doi:10.1038/nature02592 Shao, M., Shan, B., Liu, Y., Deng, Y., Yan, C., Wu, Y., Mao, T., Qiu, Y., Zhou, Y., Liu, Y., Choudhury, P., Cabral, C. M. and Sifers, R. N. (1997). Intracellular Jiang, S. et al. (2014). Hepatic IRE1alpha regulates fasting-induced metabolic disposal of incompletely folded human alpha1-antitrypsin involves release from adaptive programs through the XBP1s-PPARalpha axis signalling. Nat. Commun. calnexin and post-translational trimming of asparagine-linked oligosaccharides. 5, 3528. doi:10.1038/ncomms4528 J. Biol. Chem. 272, 7946-7951. doi:10.1074/jbc.272.12.7946 Shi, G., Somlo, D., Kim, G. H., Prescianotto-Baschong, C., Sun, S., Beuret, N., Liu, T.-F., Tang, J.-J., Li, P.-S., Shen, Y., Li, J.-G., Miao, H.-H., Li, B.-L. and Song, Long, Q., Rutishauser, J., Arvan, P., Spiess, M. et al. (2017). ER-associated B.-L. (2012). Ablation of gp78 in liver improves hyperlipidemia and insulin degradation is required for vasopressin prohormone processing and systemic resistance by inhibiting SREBP to decrease lipid biosynthesis. Cell Metab. 16, water homeostasis. J. Clin. Invest. 127, 3897-3912. doi:10.1172/JCI94771 213-225. doi:10.1016/j.cmet.2012.06.014 Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H. and Woo, S. L. Luo, H., Jiang, M., Lian, G., Liu, Q., Shi, M., Li, T. Y., Song, L., Ye, J., He, Y., Yao, (1988). A frameshift mutation results in a truncated alpha 1-antitrypsin that is L. et al. (2018). AIDA selectively mediates downregulation of fat synthesis retained within the rough endoplasmic reticulum. J. Biol. Chem. 263, 7330-7335. enzymes by ERAD to retard intestinal fat absorption and prevent obesity. Cell Smith, M. H., Ploegh, H. L. and Weissman, J. S. (2011). Road to ruin: targeting Metab. 27, 843-853.e6. doi:10.1016/j.cmet.2018.02.021 proteins for degradation in the endoplasmic reticulum. Science 334, 1086-1090. Medel, B., Costoya, C., Fernandez, D., Pereda, C., Lladser, A., Sauma, D., doi:10.1126/science.1209235 Pacheco, R., Iwawaki, T., Salazar-Onfray, F. and Osorio, F. (2018). IRE1alpha Sun, S., Shi, G., Han, X., Francisco, A. B., Ji, Y., Mendonça, N., Liu, X., Locasale, activation in bone marrow-derived dendritic cells modulates innate recognition of J. W., Simpson, K. W., Duhamel, G. E. et al. (2014). Sel1L is indispensable for melanoma cells and favors CD8+ T cell priming. Front. Immunol. 9, 3050. doi:10. mammalian endoplasmic reticulum-associated degradation, endoplasmic 3389/fimmu.2018.03050 reticulum homeostasis, and survival. Proc. Natl. Acad. Sci. USA 111, Merulla, J., Fasana, E., Solda,̀ T. and Molinari, M. (2013). Specificity and E582-E591. doi:10.1073/pnas.1318114111 regulation of the endoplasmic reticulum-associated degradation machinery. Sun, S., Shi, G., Sha, H., Ji, Y., Han, X., Shu, X., Ma, H., Inoue, T., Gao, B., Kim, H. Traffic 14, 767-777. doi:10.1111/tra.12068 et al. (2015). IRE1a is an endogenous substrate of endoplasmic-reticulum- Meyer, H., Bug, M. and Bremer, S. (2012). Emerging functions of the VCP/p97 associated degradation. Nat. Cell Biol. 17, 1546-1555. doi:10.1038/ncb3266 AAA-ATPase in the ubiquitin system. Nat. Cell Biol. 14, 117-123. doi:10.1038/ Sun, S., Louri, R., Cohen, S. B., Ji, Y., Goodrich, J. K., Poole, A. C., Ley, R. E., ncb2407 Denkers, E. Y., Mcguckin, M. A., Long, Q. et al. (2016). Epithelial Sel1L is Mueller, B., Lilley, B. N. and Ploegh, H. L. (2006). SEL1L, the homologue of yeast required for the maintenance of intestinal homeostasis. Mol. Biol. Cell 27, 483-490. doi:10.1091/mbc.e15-10-0724 Hrd3p, is involved in protein dislocation from the mammalian ER. J. Cell Biol. 175, Sun, S., Kelekar, S., Kliewer, S. A. and Mangelsdorf, D. J. (2019). The orphan 261-270. doi:10.1083/jcb.200605196 nuclear receptor SHP regulates ER stress response by inhibiting XBP1s Mueller, B., Klemm, E. J., Spooner, E., Claessen, J. H. and Ploegh, H. L. (2008). degradation. Genes Dev. 33, 1083-1094. doi:10.1101/gad.326868.119 SEL1L nucleates a protein complex required for dislocation of misfolded Tsai, Y. C., Leichner, G. S., Pearce, M. M. P., Wilson, G. L., Wojcikiewicz, R. J. H., glycoproteins. Proc. Natl. Acad. Sci. USA 105, 12325-12330. doi:10.1073/pnas. Roitelman, J. and Weissman, A. M. (2012). Differential regulation of HMG-CoA 0805371105 reductase and Insig-1 by enzymes of the ubiquitin-proteasome system. Mol. Biol. Müller, J. M. M., Deinhardt, K., Rosewell, I., Warren, G. and Shima, D. T. (2007). Cell 23, 4484-4494. doi:10.1091/mbc.e12-08-0631 Targeted deletion of p97 (VCP/CDC48) in mouse results in early embryonic Tsuchiya, Y., Saito, M., Kadokura, H., Miyazaki, J.-I., Tashiro, F., Imagawa, Y., lethality. Biochem. Biophys. Res. Commun. 354, 459-465. doi:10.1016/j.bbrc. Iwawaki, T. and Kohno, K. (2018). IRE1-XBP1 pathway regulates oxidative 2006.12.206 proinsulin folding in pancreatic beta cells. J. Cell Biol. 217, 1287-1301. doi:10. Niederreiter, L., Fritz, T. M. J., Adolph, T. E., Krismer, A.-M., Offner, F. A., 1083/jcb.201707143 Tschurtschenthaler, M., Flak, M. B., Hosomi, S., Tomczak, M. F., Kaneider, Tyler, R. E., Pearce, M. M. P., Shaler, T. A., Olzmann, J. A., Greenblatt, E. J. and N. C. et al. (2013). ER stress transcription factor Xbp1 suppresses intestinal Kopito, R. R. (2012). Unassembled CD147 is an endogenous endoplasmic tumorigenesis and directs intestinal stem cells. J. Exp. Med. 210, 2041-2056. reticulum-associated degradation substrate. Mol. Biol. Cell 23, 4668-4678. doi:10. doi:10.1084/jem.20122341 1091/mbc.e12-06-0428 Olzmann, J. A., Kopito, R. R. and Christianson, J. C. (2013). The mammalian van der Goot, A. T., Pearce, M. M. P., Leto, D. E., Shaler, T. A. and Kopito, R. R. endoplasmic reticulum-associated degradation system. Cold Spring Harb. (2018). Redundant and antagonistic roles of XTP3B and OS9 in decoding glycan Perspect. Biol. 5, a013185. doi:10.1101/cshperspect.a013185 and non-glycan degrons in ER-associated degradation. Mol. Cell 70, 516-530.e6. Owen, B. M., Bookout, A. L., Ding, X., Lin, V. Y., Atkin, S. D., Gautron, L., Vashistha, N., Neal, S. E., Singh, A., Carroll, S. M. and Hampton, R. Y. (2016). Kliewer, S. A. and Mangelsdorf, D. J. (2013). FGF21 contributes to Direct and essential function for Hrd3 in ER-associated degradation. Proc. Natl. neuroendocrine control of female reproduction. Nat. Med. 19, 1153-1156. Acad. Sci. USA 113, 5934-5939. doi:10.1073/pnas.1603079113 doi:10.1038/nm.3250 Vincenz-Donnelly, L., Holthusen, H., Körner, R., Hansen, E. C., Presto, J., Owen, B. M., Ding, X., Morgan, D. A., Coate, K. C., Bookout, A. L., Rahmouni, K., Johansson, J., Sawarkar, R., Hartl, F. U. and Hipp, M. S. (2018). High capacity Kliewer, S. A. and Mangelsdorf, D. J. (2014). FGF21 acts centrally to induce of the endoplasmic reticulum to prevent secretion and aggregation of sympathetic nerve activity, energy expenditure, and weight loss. Cell Metab. 20, amyloidogenic proteins. EMBO J. 37, 337-350. doi:10.15252/embj.201695841 670-677. doi:10.1016/j.cmet.2014.07.012 Volpi, V. G., Ferri, C., Fregno, I., Del Carro, U., Bianchi, F., Scapin, C., Pettinato, Phillips, J. A.3rd. (2003). Dominant-negative diabetes insipidus and other E., Solda, T., Feltri, M. L., Molinari, M. et al. (2019). Schwann cells ER- endocrinopathies. J. Clin. Invest. 112, 1641-1643. doi:10.1172/JCI20441 associated degradation contributes to myelin maintenance in adult nerves and Qi, L., Tsai, B. and Arvan, P. (2017). New insights into the physiological role of limits demyelination in CMT1B mice. PLoS Genet. 15, e1008069. doi:10.1371/ endoplasmic reticulum-associated degradation. Trends Cell Biol. 27, 430-440. journal.pgen.1008069 doi:10.1016/j.tcb.2016.12.002 Walter, P. and Ron, D. (2011). The unfolded protein response: from stress pathway Rape, M., Hoppe, T., Gorr, I., Kalocay, M., Richly, H. and Jentsch, S. (2001). to homeostatic regulation. Science 334, 1081-1086. doi:10.1126/science. Mobilization of processed, membrane-tethered SPT23 transcription factor by 1209038 CDC48(UFD1/NPL4), a ubiquitin-selective chaperone. Cell 107, 667-677. doi:10. Wang, J. M., Qiu, Y., Yang, Z., Kim, H., Qian, Q., Sun, Q., Zhang, C., Yin, L., Fang, 1016/S0092-8674(01)00595-5 D., Back, S. H. et al. (2018). IRE1alpha prevents hepatic steatosis by processing Rittig, S., Robertson, G. L., Siggaard, C., Kovacs, L., Gregersen, N., Nyborg, J. and promoting the degradation of select microRNAs. Sci. Signal. 11, eaa04617. and Pedersen, E. B. (1996). Identification of 13 new mutations in the doi:10.1126/scisignal.aao4617 vasopressin-neurophysin II gene in 17 kindreds with familial autosomal Ward, C. L., Omura, S. and Kopito, R. R. (1995). Degradation of CFTR by the dominant neurohypophyseal diabetes insipidus. Am. J. Hum. Genet. 58, 107-117. ubiquitin-proteasome pathway. Cell 83, 121-127. doi:10.1016/0092- Ron, D. and Walter, P. (2007). Signal integration in the endoplasmic reticulum 8674(95)90240-6 unfolded protein response. Nat. Rev. Mol. Cell Biol. 8, 519-529. doi:10.1038/ Wei, J., Chen, L., Li, F., Yuan, Y., Wang, Y., Xia, W., Zhang, Y., Xu, Y., Yang, Z., nrm2199 Gao, B. et al. (2018a). HRD1-ERAD controls production of the hepatokine FGF21 Sha, H., He, Y., Chen, H., Wang, C., Zenno, A., Shi, H., Yang, X., Zhang, X. and through CREBH polyubiquitination. EMBO J. 37, e98942. doi:10.15252/embj. Qi, L. (2009). The IRE1alpha-XBP1 pathway of the unfolded protein response is 201898942 required for adipogenesis. Cell Metab. 9, 556-564. doi:10.1016/j.cmet.2009. Wei, J., Yuan, Y., Chen, L., Xu, Y., Zhang, Y., Wang, Y., Yang, Y., Peek, C. B.,

04.009 Diebold, L., Yang, Y. et al. (2018b). ER-associated ubiquitin ligase HRD1 Journal of Cell Science

9 REVIEW Journal of Cell Science (2019) 132, jcs232850. doi:10.1242/jcs.232850

programs liver metabolism by targeting multiple metabolic enzymes. Nat. priming during inflammation. J. Exp. Med. 211, 2467-2479. doi:10.1084/jem. Commun. 9, 3659. doi:10.1038/s41467-018-06091-7 20140283 Wiseman, R. L., Powers, E. T., Buxbaum, J. N., Kelly, J. W. and Balch, W. E. Yang, Y., Kong, S., Zhang, Y., Melo-Cardenas, J., Gao, B., Zhang, Y., Zhang, (2007). An adaptable standard for protein export from the endoplasmic reticulum. D. D., Zhang, B., Song, J., Thorp, E. et al. (2018). The endoplasmic reticulum- Cell 131, 809-821. doi:10.1016/j.cell.2007.10.025 resident E3 ubiquitin ligase Hrd1 controls a critical checkpoint in B cell Wu, T., Zhao, F., Gao, B., Tan, C., Yagishita, N., Nakajima, T., Wong, P. K., development in mice. J. Biol. Chem. 293, 12934-12944. doi:10.1074/jbc. Chapman, E., Fang, D. and Zhang, D. D. (2014). Hrd1 suppresses Nrf2- RA117.001267 mediated cellular protection during liver cirrhosis. Genes Dev. 28, 708-722. Yao, T., Deng, Z., Gao, Y., Sun, J., Kong, X., Huang, Y., He, Z., Xu, Y., Chang, Y., doi:10.1101/gad.238246.114 Yu, K.-J. et al. (2017). Ire1alpha in pomc neurons is required for thermogenesis Xiao, Y., Xia, T., Yu, J., Deng, Y., Liu, H., Liu, B., Chen, S., Liu, Y. and Guo, F. and glycemia. Diabetes 66, 663-673. doi:10.2337/db16-0533 (2016). Knockout of inositol-requiring enzyme 1alpha in pro-opiomelanocortin Ye, Y., Shibata, Y., Yun, C., Ron, D. and Rapoport, T. A. (2004). A membrane neurons decreases fat mass via increasing energy expenditure. Open Biol. 6, protein complex mediates retro-translocation from the ER lumen into the cytosol. 160131. doi:10.1098/rsob.160131 Nature 429, 841-847. doi:10.1038/nature02656 Xu, T., Yang, L., Yan, C., Wang, X., Huang, P., Zhao, F., Zhao, L., Zhang, M., Jia, Younger, J. M., Chen, L., Ren, H.-Y., Rosser, M. F. N., Turnbull, E. L., Fan, C.-Y., W., Wang, X. et al. (2014). The IRE1alpha-XBP1 pathway regulates metabolic Patterson, C. and Cyr, D. M. (2006). Sequential quality-control checkpoints stress-induced compensatory proliferation of pancreatic beta-cells. Cell Res. 24, triage misfolded cystic fibrosis transmembrane conductance regulator. Cell 126, 1137-1140. doi:10.1038/cr.2014.55 571-582. doi:10.1016/j.cell.2006.06.041 Xu, Y., Zhao, F., Qiu, Q., Chen, K., Wei, J., Kong, Q., Gao, B., Melo-Cardenas, J., Zhang, P., McGrath, B., Li, S., Frank, A., Zambito, F., Reinert, J., Gannon, M., Zhang, B., Zhang, J. et al. (2016). The ER membrane-anchored ubiquitin ligase Ma, K., McNaughton, K. and Cavener, D. R. (2002). The PERK eukaryotic Hrd1 is a positive regulator of T-cell immunity. Nat. Commun. 7, 12073. doi:10. initiation factor 2 alpha kinase is required for the development of the skeletal 1038/ncomms12073 system, postnatal growth, and the function and viability of the pancreas. Mol. Cell. Yagishita, N., Aratani, S., Leach, C., Amano, T., Yamano, Y., Nakatani, K., Biol. 22, 3864-3874. doi:10.1128/MCB.22.11.3864-3874.2002 Nishioka, K. and Nakajima, T. (2012). RING-finger type E3 ubiquitin ligase Zhang, W., Feng, D., Li, Y., Iida, K., McGrath, B. and Cavener, D. R. (2006). PERK inhibitors as novel candidates for the treatment of rheumatoid arthritis. Int. J. Mol. EIF2AK3 control of pancreatic beta cell differentiation and proliferation is required Med. 30, 1281-1286. doi:10.3892/ijmm.2012.1129 for postnatal glucose homeostasis. Cell Metab. 4, 491-497. doi:10.1016/j.cmet. Yang, L., Xue, Z., He, Y., Sun, S., Chen, H. and Qi, L. (2010). A Phos-tag-based 2006.11.002 approach reveals the extent of physiological endoplasmic reticulum stress. PLoS Zhang, K., Wang, S., Malhotra, J., Hassler, J. R., Back, S. H., Wang, G., Chang, ONE 5, e11621. doi:10.1371/journal.pone.0011621 L., Xu, W., Miao, H., Leonardi, R. et al. (2011). The unfolded protein response Yang, L., Sha, H., Davisson, R. L. and Qi, L. (2013). Phenformin activates the transducer IRE1alpha prevents ER stress-induced hepatic steatosis. EMBO J. 30, unfolded protein response in an AMP-activated protein kinase (AMPK)-dependent 1357-1375. doi:10.1038/emboj.2011.52 manner. J. Biol. Chem. 288, 13631-13638. doi:10.1074/jbc.M113.462762 Zhang, T., Xu, Y., Liu, Y. and Ye, Y. (2015). gp78 functions downstream of Hrd1 to Yang, H., Qiu, Q., Gao, B., Kong, S., Lin, Z. and Fang, D. (2014). Hrd1-mediated promote degradation of misfolded proteins of the endoplasmic reticulum. Mol. BLIMP-1 ubiquitination promotes dendritic cell MHCII expression for CD4 T cell Biol. Cell 26, 4438-4450. doi:10.1091/mbc.E15-06-0354 Journal of Cell Science