Maintenance Of Endoplasmic Reticulum Homeostasis Research Articles

Gradual ER calcium depletion induces a progressive and reversible UPR signaling.

The unfolded protein response (UPR) is a widespread signal transduction pathway triggered by endoplasmic reticulum (ER) stress. Because calcium (Ca2+) is a key factor in the maintenance of ER homeostasis, massive Ca2+ depletion of the ER is a potent inducer of ER stress. Although moderate changes in ER Ca2+ drive the ubiquitous Ca2+ signaling pathways, a possible incremental relationship between UPR activation and Ca2+ changes has yet to be described. Here, we determine the sensitivity and time-dependency of activation of the three ER stress sensors, inositol-requiring protein 1 alpha (IRE1α), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 alpha (ATF6α) in response to controlled changes in the concentration of ER Ca2+ in human cultured cells. Combining Ca2+ imaging, fluorescence recovery after photobleaching experiments, biochemical analyses, and mathematical modeling, we uncover a nonlinear rate of activation of the IRE1α branch of UPR, as compared to the PERK and ATF6α branches that become activated gradually with time and are sensitive to more important ER Ca2+ depletions. However, the three arms are all activated within a 1 h timescale. The model predicted the deactivation of PERK and IRE1α upon refilling the ER with Ca2+. Accordingly, we showed that ER Ca2+ replenishment leads to the complete reversion of IRE1α and PERK phosphorylation in less than 15 min, thus revealing the highly plastic character of the activation of the upstream UPR sensors. In conclusion, our results reveal a dynamic and dose-sensitive Ca2+-dependent activation/deactivation cycle of UPR induction, which could tightly control cell fate upon acute and/or chronic stress.

8-OR: Novel Mechanism of Lipoprotein Lipase Folding and Maturation in Adipocytes

Lipoprotein lipase (LPL) plays a central role in systemic lipid clearance by catalyzing intravascular hydrolysis of lipoprotein triglycerides, hence representing an important target in the treatment of hyperlipidemia in diabetes. Nascent LPL protein undergoes folding and maturation in the endoplasmic reticulum (ER), with only the properly folded ones exiting the ER and reaching to the cell surface. However, the maturation process for nascent LPL in the ER remains largely unknown. Here we show that LPL maturation in the ER is controlled by compensatory actions of two ER protein quality control mechanisms, ER-associated degradation (ERAD) and ER-phagy, in adipocytes. Specifically, we show that LPL is misfolding prone and degraded by ERAD, and when ERAD is compromised, LPL forms aggregates which is cleared by ER-phagy. In adipocytes lacking both ERAD and ER-phagy, ER fragments containing LPL aggregates cluster and fuse to form a novel cellular architecture, termed as Coalescence of ER Fragments (CERFs), where LPL undergo soluble-to-insoluble phase transition catalyzed by disulfide bond-mediated interactions. Lastly, mice with adipocyte specific ERAD and ER-phagy deficiency exhibited postprandial hyperlipidemia. In summary, this study provides novel insights into not only LPL biogenesis, but also the crosstalk between protein quality control pathways in the maintenance of ER homeostasis, as well as the disease pathogenesis, more broadly, associated with protein folding defects in the ER. Disclosure S.Wu: None. L.Qi: None.

OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice

Starch and storage proteins are the main components of rice (Oryza sativa L.) grains. Despite their importance, the molecular regulatory mechanisms of storage protein and starch biosynthesis remain largely elusive. Here, we identified a rice opaque endosperm mutant, opaque3 (o3), that overaccumulates 57-kDa proglutelins and has significantly lower protein and starch contents than the wild type. The o3 mutant also has abnormal protein body structures and compound starch grains in its endosperm cells. OPAQUE3 (O3) encodes a transmembrane basic leucine zipper (bZIP) transcription factor (OsbZIP60) and is localized in the endoplasmic reticulum (ER) and the nucleus, but it is localized mostly in the nucleus under ER stress. We demonstrated that O3 could activate the expression of several starch synthesis-related genes (GBSSI, AGPL2, SBEI, and ISA2) and storage protein synthesis-related genes (OsGluA2, Prol14, and Glb1). O3 also plays an important role in protein processing and export in the ER by directly binding to the promoters and activating the expression of OsBIP1 and PDIL1-1, two major chaperones that assist with folding of immature secretory proteins in the ER of rice endosperm cells. High-temperature conditions aggravate ER stress and result in more abnormal grain development in o3 mutants. We also revealed that OsbZIP50 can assist O3 in response to ER stress, especially under high-temperature conditions. We thus demonstrate that O3 plays a central role in rice grain development by participating simultaneously in the regulation of storage protein and starch biosynthesis and the maintenance of ER homeostasis in endosperm cells.

Ufl1 deficiency causes kidney atrophy associated with disruption of endoplasmic reticulum homeostasis

The UFMylation modification is a novel ubiquitin-like conjugation system, consisting of UBA5 (E1), UFC1 (E2), UFL1 (E3), and the conjugating molecule UFM1. Deficiency in this modification leads to embryonic lethality in mice and diseases in humans. However, the function of UFL1 is poorly characterized. Studies on Ufl1 conditional knockout mice have demonstrated that the deletion of Ufl1 in cardiomyocytes and in intestinal epithelial cells causes heart failure and increases susceptibility to experimentally induced colitis, respectively, suggesting an essential role of UFL1 in the maintenance of the homeostasis in these organs. Yet, its physiological function in other tissues and organs remains completely unknown. In this study, we generate the nephron tubules specific Ufl1 knockout mice and find that the absence of Ufl1 in renal tubular results in kidney atrophy and interstitial fibrosis. In addition, Ufl1 deficiency causes the activation of unfolded protein response and cell apoptosis, which may be responsible for the kidney atrophy and interstitial fibrosis. Collectively, our results have demonstrated the crucial role of UFL1 in regulating kidney function and maintenance of endoplasmic reticulum homeostasis, providing another layer of understanding kidney atrophy.

Zinc Attenuates the Cytotoxicity of Some Stimuli by Reducing Endoplasmic Reticulum Stress in Hepatocytes.

Zinc is an essential trace element and plays critical roles in cellular integrity and biological functions. Excess copper induced both oxidative stress and endoplasmic reticulum (ER) stress in liver-derived cultured cells. Excess copper also induced impairment of autophagic flux at the step of autophagosome–lysosome fusion, as well as Mallory–Denk body (MDB)-like inclusion body formation. Zinc ameliorated excess copper-induced impairment of autophagic flux and MDB-like inclusion body formation via the maintenance of ER homeostasis. Furthermore, zinc also ameliorated free fatty acid-induced impairment of autophagic flux. These results indicate that zinc may be able to protect hepatocytes from various ER stress-related conditions.

C. elegans MANF Homolog Is Necessary for the Protection of Dopaminergic Neurons and ER Unfolded Protein Response.

Neurotrophic factors (NTFs) are important for the development, function, and survival of neurons in the mammalian system. Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are two recently identified members of a novel family of NTFs in vertebrates that function to protect dopaminergic neurons. Although these genes are conserved across eukaryotes, their mechanism of neuroprotection is not fully understood. Sequence searches for MANF/CDNF homologs in invertebrates have identified a single ortholog that is most related to MANF. Here we report the in vivo characterization of the MANF gene, manf-1, in the nematode Caenorhabditis elegans. We found that manf-1 mutants have an accelerated, age-dependent decline in the survival of dopaminergic neurons. The animals also show increased endoplasmic reticulum (ER) stress, as revealed by reporter gene expression analysis of hsp-4, an ER chaperone BiP/GRP78 homolog, suggesting that a failure to regulate the ER unfolded protein response (ER-UPR) may be a contributing factor to dopaminergic neurodegeneration. Expression studies of manf-1 revealed that the gene is broadly expressed in a pattern that matches closely with hsp-4. Consistent with the requirements of manf-1 in the ER-UPR, we found that aggregates of α-Synuclein, a major constituent of Lewy bodies, were significantly increased in body wall muscles of manf-1 mutant animals. Overall, our work demonstrates the important role of manf-1 in dopaminergic neuronal survival and the maintenance of ER homeostasis in C. elegans.

Abstract 2620: Aquaporin 11 as a new predictive biomarker of overall survival and platinum-based chemotherapy response in lung adenocarcinoma patients

Abstract Background: Biomarkers predictive of response to chemotherapy are critically needed for the precise selection of treatment protocols in lung adenocarcinoma (LUAD). Aquaporins (AQPs), transmembrane water channels, are emerging targets in cancer. Recently discovered, non-ubiquitous family member aquaporin 11 (AQP11), a tissue-specific endoplasmic reticulum (ER) resident, was identified as a cellular pro-survival factor implicated in the maintenance of ER homeostasis. AQP11 is mapped to 11q13-q14 amplicon harboring oncogenic drivers and associated with poor prognosis in cancer patients. We recently showed that high AQP11 expression is an in-vitro therapeutic biomarker of cisplatin therapy, which directly interferes with AQP11 functional structure. In addition, silencing of AQP11 expression in human lung cancer cell lines significantly increased response to cisplatin treatment. We hypothesized that LUAD tumors expressing high levels of AQP11 depend on AQP11-mediated cytoprotection and would be more responsive to platinum-based chemotherapy targeting AQP11. Method: We downloaded and curated matched mRNA expression, survival, and drug response data from The Cancer Genome Atlas (TCGA) LUAD dataset (N=369). Results: Analysis of PRECOG and TCGA databases showed that high AQP11 mRNA expression is negatively prognostic in patients with LUAD. TCGA LUAD cases were categorized by AQP11 mRNA levels into high and low expression (high = AQP11 mRNA expression mean + 1 standard deviation). Patients with high tumor AQP11 mRNA expression (10.3%) had significantly worse overall survival (OS) compared to patients with low AQP11 expression (p=0.0015). An analysis of patients treated with platinum-based chemotherapy (N=74) showed that patients with high AQP11 mRNA expression (7%) had significantly higher OS then patients with LUAD expressing low levels of AQP11 (p=0.0263). Conclusions: This study identifies AQP11 as a new biomarker of OS and chemotherapy in LUAD patients. It is conceivable that lung tumors expressing high levels of AQP11 are dependent on its function and elevated AQP11 expression renders tumor resistance to microenvironment and therapy-induced stress and associates with lower OS of patients. At the same time, as cisplatin efficiently targets AQP11 functional multimeric structure, these AQP11-depending tumors are more prone to platinum based chemotherapy. This study provides a rationale for combination anti-AQP11 and chemotherapy in LUAD tumors with high AQP11 expression. Citation Format: Michael Sharpnack, David P. Carbone, Mikhail M. Dikov, Elena E. Tchekneva. Aquaporin 11 as a new predictive biomarker of overall survival and platinum-based chemotherapy response in lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2620.

Amyotrophic lateral sclerosis (ALS) and Alzheimer\u2019s disease (AD) are characterised by differential activation of ER stress pathways: focus on UPR target genes

The endoplasmic reticulum (ER) plays an important role in maintenance of proteostasis through the unfolded protein response (UPR), which is strongly activated in most neurodegenerative disorders. UPR signalling pathways mediated by IRE1α and ATF6 play a crucial role in the maintenance of ER homeostasis through the transactivation of an array of transcription factors. When activated, these transcription factors induce the expression of genes involved in protein folding and degradation with pro-survival effects. However, the specific contribution of these transcription factors to different neurodegenerative diseases remains poorly defined. Here, we characterised 44 target genes strongly influenced by XBP1 and ATF6 and quantified the expression of a subset of genes in the human post-mortem spinal cord from amyotrophic lateral sclerosis (ALS) cases and in the frontal and temporal cortex from frontotemporal lobar degeneration (FTLD) and Alzheimer’s disease (AD) cases and controls. We found that IRE1α-XBP1 and ATF6 pathways were strongly activated both in ALS and AD. In ALS, XBP1 and ATF6 activation was confirmed by a substantial increase in the expression of both known and novel target genes involved particularly in co-chaperone activity and ER-associated degradation (ERAD) such as DNAJB9, SEL1L and OS9. In AD cases, a distinct pattern emerged, where targets involved in protein folding were more prominent, such as CANX, PDIA3 and PDIA6. These results reveal that both overlapping and disease-specific patterns of IRE1α-XBP1 and ATF6 target genes are activated in AD and ALS, which may be relevant to the development of new therapeutic strategies.Graphical abstractThe endoplasmic reticulum (ER) plays an important role in maintenance of proteostasis through the unfolded protein response (UPR). Two major UPR signalling pathways are mediated by IRE1α and ATF6. Here, we demonstrate that these pathways activate differential gene sets in human post-mortem tissues derived from amyotrophic lateral sclerosis (ALS) compared to Alzheimer’s disease (AD) cases. Our results identify IRE1α and ATF6 specific targets that can have major implications in the development of new therapeutic strategies and potential biomarkers.

Sec61\u03b2 facilitates the maintenance of endoplasmic reticulum homeostasis by associating microtubules

Sec61β, a subunit of the Sec61 translocon complex, is not essential in yeast and commonly used as a marker of endoplasmic reticulum (ER). In higher eukaryotes, such as Drosophila, deletion of Sec61β causes lethality, but its physiological role is unclear. Here, we show that Sec61β interacts directly with microtubules. Overexpression of Sec61β containing small epitope tags, but not a RFP tag, induces dramatic bundling of the ER and microtubule. A basic region in the cytosolic domain of Sec61β is critical for microtubule association. Depletion of Sec61β induces ER stress in both mammalian cells and Caenorhabditis elegans, and subsequent restoration of ER homeostasis correlates with the microtubule binding ability of Sec61β. Loss of Sec61β causes increased mobility of translocon complexes and reduced level of membrane-bound ribosomes. These results suggest that Sec61β may stabilize protein translocation by linking translocon complex to microtubule and provide insight into the physiological function of ER-microtubule interaction.

Abstract 5468: Expression of aquaporin 11 as a potential predictor of cisplatin sensitivity

Abstract Platinum-based combination is a standard of care treatment for lung cancer patients. Accumulating evidence indicates that aquaporins are emerging targets in cancer. Aquaporin 11 (AQP11), a non-ubiquitous member of aquaporins family, is an endoplasmic reticulum (ER)-specific water channel mostly present in epithelial cells and is implicated in the maintenance of ER homeostasis. In our recent publications, we demonstrated that under the stress the reduction of AQP11 expression is a pro-apoptotic factor in normal epithelial cells. We revealed that under normal conditions AQP11 is heavily S-nitrosylated forming functionally important multimeric structures (> 1000 kDa), whereas cisplatin treatment interferes with AQP11 causing its structural and post-translational modification. We hypothesized that if tumor cells express AQP11 then cells with lower levels of AQP11 are not protected from cisplatin-associated ER stress and develop higher sensitivity to drug treatment. In normal lung AQP11 is expressed at low levels. We evaluated the expression of AQP11 in lung cancer cells to determine whether the AQP11 expression correlates with cisplatin sensitivity and whether lower AQP11 expression in lung tumor associates with positive overall survival in patients with lung adenocarcinoma. A strong link between the level of AQP11 protein expression and cell sensitivity to CP was detected using MTT assay. Screening lung cancer cell lines we found that all tested cancer cell lines expressed AQP11 protein at various levels. Western blot of stress markers showed that cisplatin-treated cells with higher AQP11 expression had lowered cellular and ER stress. Reduction of AQP11 expression using shRNA AQP11 silencing in cispaltin-resistant A549 and HCC827 cell lines, resulted in the significantly increased up to 3-fold sensitivity to cisplatin compared with control shRNA transfected or parental non-transfected cells (p<0.001). In sensitive H460 cells, knocking down AQP11 did not change sensitivity to cisplatin. TCGA database analysis of previously untreated lung adenocarcinoma, detected 13% tumors with elevated AQP11 mRNA expression. Lower AQP11 expression was significantly associated with higher OS and higher median survival rate (52.5 vs. 34.47 mo) than in patients with high AQP11 expression (p<0.05). AQP11 gene is located within oncogenic 11q13-q14 amplicon, which is a common aberration in solid tumors associated with poor prognosis. The AQP11 is a pro-survival factor protecting tumor cells from cisplatin-induced stress. Low AQP11 expression associates with higher OS in lung adenocarcinoma patients and with higher cispaltin sensitivity in lung cancer cell. The ER stress aggravation via interference with AQP11 could become a prospective therapeutic approach in anti-tumor treatment. Citation Format: Jason V. Evans, Anwari Akhter, David P. Carbone, Mikhail M. Dikov, Elena E. Tchekneva. Expression of aquaporin 11 as a potential predictor of cisplatin sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5468. doi:10.1158/1538-7445.AM2017-5468

70 XBP1 DYSREGULATION BY CRISPR/Cas9-MEDIATED GENE EDITING DURING PORCINE EMBRYO EARLY DEVELOPMENT

Early developing embryos are very sensitive to their developmental milieu. For instance, variations in temperature, pH, or culture media composition can trigger endoplasmic reticulum (ER) stress. Endoplasmic reticulum stress has been shown to reduce early embryo development and embryo quality. In response to ER stress, embryos activate coping mechanisms, such as the unfolded protein response, to re-establish ER homeostasis. The X box binding protein (XBP1) is one of the main transducers of the unfolded protein response. Under ER stress, XBP1 mRNA is unconventionally spliced by IRE1α to yield its activated isoform (XBP1s), which allows expression of genes involved in protein folding, transport, and degradation. XBP1s has been detected in oocytes and early stage embryos of different species, including Drosophila, Xenopus, zebrafish, mice, and pigs, suggesting an important role during early embryo development. In this study, we used the CRISPR/Cas9 gene editing technology to investigate the effect of XBP1 dysregulation during development of porcine embryos in vitro. Pig zygotes were produced by intracytoplasmic sperm injection using in vitro-matured oocytes. Treatments consisted of (a) Cas9 mRNA (Cas9) + 1 single guide RNAs targeting XBP1 gene region 1 (sgRNA-1); (b) Cas9 + 1 single guide RNAs targeting XBP1 gene region 2 (sgRNA-2); (c) Cas9 + sgRNA-1 + sgRNA-2; (d) Cas9 alone; and (e) sgRNA-1 + sgRNA-2. After injection, embryos were cultured in vitro for 5 to 7 days to assess development and cell numbers. Experiments were repeated 5 or more times, and data were analysed by ANOVA and means compared using Student’s t-test or Tukey–Kramer Honestly Significant Difference test. Embryo cleavage was similar between the groups (a = 59.8 ± 4.9%, b = 58.8 ± 5.3%, c = 68.86 ± 2.2%, d = 66.4 ± 5.9%, and e = 70.10 ± 1.9%), but development to the blastocyst stage was substantially reduced (P < 0.05) in the groups injected with Cas9 + sgRNAs (a = 18 ± 4.5%, b = 16 ± 1.5%, and c = 5.3 ± 2.8%) compared with controls (d = 33.7 ± 6.2% and e = 31.4 ± 1.2%). Moreover, we observed that only 22.7% of the embryos treated with Cas9 + sgRNA-1 + sgRNA-2 were able to develop beyond 8-cell stage compared with 62.5% in the control group injected with Cas9 alone. These findings suggest that XBP1 activity is required for maintenance of ER homeostasis and development of porcine embryos beyond the main period of embryo genome activation.

The Unfolded Protein Response in a Pair of Sensory Neurons Promotes Entry of C. elegans into Dauer Diapause

In response to unfavorable environmental conditions such as starvation, crowding, and elevated temperature, Caenorhabditis elegans larvae enter an alternative developmental stage known as dauer, which is characterized by adaptive changes in stress resistance and metabolism. The genetic dissection of the molecular mechanisms of the C. elegans dauer developmental decision has defined evolutionarily conserved signaling pathways of organismal neuroendocrine physiology. Here, we have identified a mechanism by which a dominant mutation in a neuronal insulin gene, daf-28(sa191), causes constitutive entry into dauer diapause. We demonstrate that expression of the mutant DAF-28 insulin peptide results in endoplasmic reticulum (ER) stress in the ASI pair of chemosensory neurons. The neuronal ER stress does not compromise cellular survival but activates PEK-1, the C. elegans ortholog of the mammalian eIF2α kinase PERK, which in turn phosphorylates Ser49 of eIF2α, specifically in the ASI neuron pair, to promote entry into dauer diapause. Our data establish a novel role for ER stress and the unfolded protein response (UPR) in promoting entry into dauer diapause and suggest that, in addition to cell-autonomous activities in the maintenance of ER homeostasis, the UPR may act in a non-cell-autonomous manner to promote organismal adaptation to stress during larval development.

Stressed to death – mechanisms of ER stress-induced cell death

The endoplasmic reticulum (ER) is a highly dynamic organelle of fundamental importance present in all eukaryotic cells. The majority of synthesized structural and secreted proteins undergo post-translational modification, folding and oligomerization in the ER lumen, enabling proteins to carry out their physiological functions. Therefore, maintenance of ER homeostasis and function is imperative for proper cellular function. Physiological and pathological conditions can disturb ER homeostasis and thus negatively impact upon protein folding, resulting in an accumulation of unfolded proteins. Examples include hypoxia, hypo- and hyperglycemia, acidosis, and fluxes in calcium levels. Increased levels of unfolded/misfolded proteins within the ER lumen triggers a condition commonly referred to as 'ER stress'. To combat ER stress, cells have evolved a highly conserved adaptive stress response referred to as the unfolded protein response (UPR). UPR signaling affords the cell a 'window of opportunity' for stress resolution however, if prolonged or excessive the UPR is insufficient and ER stress-induced cell death ensues. This review discusses the role of ER stress sensors IRE1, PERK and ATF6, describing their role in ER stress-induced death signaling with specific emphasis placed upon the importance of the intrinsic cell death pathway and Bcl-2 family regulation.

Essential Roles of the Kar2/BiP Molecular Chaperone Downstream of the UPR Pathway in Cryptococcus neoformans

The endoplasmic reticulum (ER) is a central hub where secreted or membrane-bound proteins are maturated and folded properly in eukaryotes. Maintenance of ER homeostasis is particularly important for human fungal pathogens, such as Cryptococcus neoformans, which encounter a plethora of host-mediated stresses during infection. Our previous study demonstrated that the unfolded protein response (UPR) pathway, composed of the evolutionarily conserved Ire1 kinase and the unique Hxl1 transcription factor, has pleiotropic roles in ER stress response, thermotolerance, antifungal drug resistance, and virulence in C. neoformans. Here, we functionally characterized an ER-resident molecular chaperone, Kar2/BiP, in C. neoformans. Conditional expression of KAR2 by the copper-regulated promoter revealed that Kar2 is essential for the viability of C. neoformans. Constitutive expression of KAR2 by the strong histone H3 promoter partially restores resistance to ER stress, cell wall stress, thermotolerance, and genotoxic stress in ire1Δ and hxl1Δ mutants, suggesting that Kar2 mainly functions downstream of the UPR pathway. Furthermore, Kar2 appears to control azole resistance in C. neoformans downstream of the UPR pathway without regulation of ERG11 or ERG3. Interestingly, we discovered that azole treatment is sensed as ER-stress and subsequently activates the Ire1-dependent Hxl1 splicing event and induction of KAR2 by the UPR pathway. In contrast, the constitutive expression of Kar2 is not sufficient to restore the Ire1-mediated regulation of capsule production in C. neoformans UPR mutants. In conclusion, this study demonstrates that Kar2 is not only essential for vegetative growth but also required for response and adaptation to the environmental stresses and antifungal drugs downstream of the UPR pathway in C. neoformans.

Endoplasmic Reticulum Stress and Type 2 Diabetes

Given the functional importance of the endoplasmic reticulum (ER), an organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, the maintenance of ER homeostasis in insulin-secreting β-cells is very important. When ER homeostasis is disrupted, the ER generates adaptive signaling pathways, called the unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis fails to be restored, the ER initiates death signaling pathways. New observations suggest that both chronic hyperglycemia and hyperlipidemia, known as important causative factors of type 2 diabetes (T2D), disrupt ER homeostasis to induce unresolvable UPR activation and β-cell death. This review examines how the UPR pathways, induced by high glucose and free fatty acids (FFAs), interact to disrupt ER function and cause β-cell dysfunction and death.

Role of Pro-oncogenic Protein Disulfide Isomerase (PDI) Family Member Anterior Gradient 2 (AGR2) in the Control of Endoplasmic Reticulum Homeostasis

The protein-disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) is reportedly overexpressed in numerous cancers and plays a role in cancer development. However, to date the molecular functions of AGR2 remain to be characterized. Herein we have identified AGR2 as bound to newly synthesized cargo proteins using a proteomics analysis of endoplasmic reticulum (ER) membrane-bound ribosomes. Nascent protein chains that translocate into the ER associate with specific ER luminal proteins, which in turn ensures proper folding and posttranslational modifications. Using both imaging and biochemical approaches, we confirmed that AGR2 localizes to the lumen of the ER and indirectly associates with ER membrane-bound ribosomes through nascent protein chains. We showed that AGR2 expression is controlled by the unfolded protein response and is in turn is involved in the maintenance of ER homeostasis. Remarkably, we have demonstrated that siRNA-mediated knockdown of AGR2 significantly alters the expression of components of the ER-associated degradation machinery and reduces the ability of cells to cope with acute ER stress, properties that might be relevant to the role of AGR2 in cancer development.

Physiological IRE-1-XBP-1 and PEK-1 Signaling in Caenorhabditis elegans Larval Development and Immunity

Endoplasmic reticulum (ER) stress activates the Unfolded Protein Response, a compensatory signaling response that is mediated by the IRE-1, PERK/PEK-1, and ATF-6 pathways in metazoans. Genetic studies have implicated roles for UPR signaling in animal development and disease, but the function of the UPR under physiological conditions, in the absence of chemical agents administered to induce ER stress, is not well understood. Here, we show that in Caenorhabditis elegans XBP-1 deficiency results in constitutive ER stress, reflected by increased basal levels of IRE-1 and PEK-1 activity under physiological conditions. We define a dynamic, temperature-dependent requirement for XBP-1 and PEK-1 activities that increases with immune activation and at elevated physiological temperatures in C. elegans. Our data suggest that the negative feedback loops involving the activation of IRE-1-XBP-1 and PEK-1 pathways serve essential roles, not only at the extremes of ER stress, but also in the maintenance of ER homeostasis under physiological conditions.

Mechanism and components of endoplasmic reticulum-associated degradation

The folding of secretory and membrane proteins takes place in the endoplasmic reticulum (ER). The quality of the proteins folded in the ER is carefully monitored by an ER quality control mechanism that allows only correctly folded proteins to be transported to their final destination, and misfolded or unassembled proteins to be retained in the ER and subsequently degraded in a process termed 'ER-associated degradation' (ERAD). The ERAD pathway is conserved from yeast to mammals, and plays an essential role in the maintenance of ER homeostasis, as well as in the prevention of various diseases that arise from the accumulation of aberrant proteins in the ER. In the ERAD pathway, molecular chaperones and lectin-like proteins are involved in the identification of misfolded proteins, ER-resident reductases cleave disulfide bonds in these proteins to facilitate retrograde transport to the cytosol and AAA(+) adenosine triphosphatase withdraws them from the retrotranslocation channel to the cytosol where they are degraded by the ubiquitin/proteasome system. The possible mechanisms that underlie ERAD and the various factors involved in this process are discussed in this article.

CHOP Mediates Endoplasmic Reticulum Stress-Induced Apoptosis in Gimap5-Deficient T Cells

Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from Gimap5−/− BBDP rats. We then discovered that ER stress-induced apoptotic signaling through C/EBP-homologous protein (CHOP) occurs in Gimap5−/− T cells. Knockdown of CHOP by siRNA protected Gimap5−/− T cells from ER stress-induced apoptosis, thereby identifying a role for this cellular pathway in the T cell lymphopenia of the BBDP rat. These findings indicate a direct relationship between Gimap5 and the maintenance of ER homeostasis in the survival of T cells.

Bcl-2 family on guard at the ER

The endoplasmic reticulum (ER) is the main site for protein folding, lipid biosynthesis, and calcium storage in the cell. Disturbances of these critical cellular functions lead to ER stress. The ER responds to disturbances in its homeostasis by launching an adaptive signal transduction pathway, known as the unfolded protein response (UPR). The UPR strives to maintain ER function during stress; however, if the stress is not resolved, apoptotic responses are activated that involve cross talk between the ER and mitochondria. In addition, ER stress is also known to induce autophagy to counteract XBP-1-mediated ER expansion and assist in the degradation of unfolded proteins. One family of proteins involved in the regulation of apoptosis is that of B-cell lymphoma protein 2 (Bcl-2). Complex interactions among the three subgroups within the Bcl-2 family [the antiapoptotic, the multidomain proapoptotic, and the Bcl-2 homology domain 3 (BH3)-only members] control the signaling events of apoptosis upstream of mitochondrial outer membrane permeabilization. These proteins were found to have diverse subcellular locations to aid in the response to varied intrinsic and extrinsic stimuli. Of recent interest is the presence of the Bcl-2 family at the ER. Here, we review the involvement of proteins from each of the three Bcl-2 family subgroups in the maintenance of ER homeostasis and their participation in ER stress signal transduction pathways.