Degradation Of Unfolded Proteins Research Articles

Sel1l May Contributes to the Determinants of Neuronal Lineage and Neuronal Maturation Regardless of Hrd1 via Atf6-Sel1l Signaling.

The endoplasmic reticulum (ER) is the primary site of intracellular quality control involved in the recognition and degradation of unfolded proteins. A variety of stresses, including hypoxia and glucose starvation, can lead to accumulation of unfolded proteins triggering the ER-associated degradation (ERAD) pathway. Suppressor Enhancer Lin12/Notch1 Like (Sel1l) acts as a "gate keeper" in the quality control of de novo synthesized proteins and complexes with the ubiquitin ligase Hrd1 in the ER membrane. We previously demonstrated that ER stress-induced aberrant neural stem cell (NSC) differentiation and inhibited neurite outgrowth. Inhibition of neurite outgrowth was associated with increased Hrd1 expression; however, the contribution of Sel1l remained unclear. To investigate whether ER stress is induced during normal neuronal differentiation, we semi-quantitatively evaluated mRNA expression levels of unfolded protein response (UPR)-related genes in P19 embryonic carcinoma cells undergoing neuronal differentiation in vitro. Stimulation with all-trans retinoic acid (ATRA) for 4days induced the upregulation of Nestin and several UPR-related genes (Atf6, Xbp1, Chop, Hrd1, and Sel1l), whereas Atf4 and Grp78/Bip were unchanged. Small-interfering RNA (siRNA)-mediated knockdown of Sel1l uncovered that mRNA levels of the neural progenitor marker Math1 (also known as Atoh1) and the neuronal marker Math3 (also known as Atoh3 and NeuroD4) were significantly suppressed at 4days after ATRA stimulation. Consistent with this result, Sel1l silencing significantly reduced protein levels of immature neuronal marker βIII-tubulin (also known as Tuj-1) at 8days after induction of neuronal differentiation, whereas synaptogenic factors, such as cell adhesion molecule 1 (CADM1) and SH3 and multiple ankyrin repeat domain protein 3 (Shank3) were accumulated in Sel1l silenced cells. These results indicate that neuronal differentiation triggers ER stress and suggest that Sel1l may facilitate neuronal lineage through the regulation of Math1 and Math3 expression.

Abstract A20: 17-Aminogeldanamycin inhibits cytoprotective UPR pathways and cooperates with inhibitors of the MAPK signaling cascade in apoptosis induction

Abstract Geldanamycin is an inhibitor of Heat Shock Protein 90 (HSP90). Attenuation of HSP90 activity results in accumulation of unfolded proteins, induction of endoplasmic reticulum (ER) stress, and activation of unfolded protein response (UPR). HSP90 is a chaperone protein participating in appropriate folding of more than 100 proteins, including IRE1α and BRAFV600E. In melanoma cells, BRAFV600E was correlated with chronically induced ER stress and involved in UPR-induced resistance to apoptosis. As geldanamycin is highly hepatotoxic, geldanamycin derivatives exerting similar proapoptotic properties but higher selectivity towards cancer cells are needed. In the present study, 17-aminogeldanamycin exerted higher proapoptotic activity than geldanamycin in patient-derived melanoma cell populations as demonstrated by using fluorescence time-lapse imaging (IncuCyte ZOOM). We have also shown that 17-aminogeldanamycin inhibits ERK1/2 activity and cell proliferation,as well as induces apoptosis transiently, enhancing expression of HSP70 and GRP78. GRP78 regulates activity of the UPR pathway, and we have demonstrated that 17-aminogeldanamycin inhibits IRE1α-dependent pathway of UPR responsible for degradation of unfolded proteins and reduces levels of 36 kDa product of ATF6 cleavage in selected melanoma cell populations. Moreover, we have shown that combination of 17-aminogeldanamycin and trametinib induces apoptosis earlier and more efficiently. Our study suggests that cooperation of 17-aminogeldanamycin and trametinib might result from concurrent inhibition of the MAPK and protective UPR pathways but can be also associated with the basal UPR activity, which is diverse among drug-naïve melanoma cell populations. Further studies are necessary to fully explore antimelanoma capabilities of 17-aminogeldanamycin and its combination with targeted therapy. This work was financially supported by Grant 2014/15/B/NZ7/00947 from National Science Centre (Poland), and 502-03/1-156-01/502-14-318 from Medical University of Lodz. Citation Format: Aleksandra H. Mielczarek-Lewandowska, Mariusz L. Hartman, Malgorzata M. Sztiller-Sikorska, Marta Osrodek, Anna E. Gajos-Michniewicz, Malgorzata E. Czyz. 17-Aminogeldanamycin inhibits cytoprotective UPR pathways and cooperates with inhibitors of the MAPK signaling cascade in apoptosis induction [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A20.

Leveraging the Role of the Metastatic Associated Protein Anterior Gradient Homologue 2 in Unfolded Protein Degradation: A Novel Therapeutic Biomarker for Cancer.

Effective diagnostic, prognostic and therapeutic biomarkers can help in tracking disease progress, predict patients’ survival, and considerably affect the drive for successful clinical management. The present review aims to determine how the metastatic-linked protein anterior gradient homologue 2 (AGR2) operates to affect cancer progression, and to identify associated potential diagnostic, prognostic and therapeutic biomarkers, particularly in central nervous system (CNS) tumors. Studies that show a high expression level of AGR2, and associate the protein expression with the resilience to chemotherapeutic treatments or with poor cancer survival, are reported. The primary protein structures of the seven variants of AGR2, including their functional domains, are summarized. Based on experiments in various biological models, this review shows an orchestra of multiple molecules that regulate AGR2 expression, including a feedback loop with p53. The AGR2-associated molecular functions and pathways including genomic integrity, proliferation, apoptosis, angiogenesis, adhesion, migration, stemness, and inflammation, are detailed. In addition, the mechanisms that can enable the rampant oncogenic effects of AGR2 are clarified. The different strategies used to therapeutically target AGR2-positive cancer cells are evaluated in light of the current evidence. Moreover, novel associated pathways and clinically relevant deregulated genes in AGR2 high CNS tumors are identified using a meta-analysis approach.

Potential therapeutic application of PERK inhibitors

Recent studies aimed at understanding the molecular mechanisms of human disease indicate that in the pathogenesis of many metabolic disorders, including inflammatory processes, aging of the organism, as well as cancer and neurodegenerative disorders, endoplasmic reticulum stress plays a significant role that is associated with the accumulation of misfolded proteins in the lumen of endoplasmic reticulum. In response to endoplasmic reticulum stress, the unfolded protein response pathway, that has a dualistic role, is induced. The unfolded protein response can restore endoplasmic reticulum homeostasis by degradation of unfolded proteins, inhibition of translation, and mobilization of chaperons, but it can also promote apoptosis when endoplasmic reticulum stress is prolonged. The unfolded protein response signaling pathways may be activated via three transmembrane receptors such as: PERK, IRE1 and ATF6. The most promising for development of new therapies of many human diseases, in particular cancer and neurodegeneration is PERK pathway, that inhibition shows positive therapeutic effects both in in vitro and in vivo studies.

Ubiquitin ligase HMG-CoA reductase degradation 1 (HRD1) prevents cell death in a cellular model of Parkinson's disease

Endoplasmic reticulum (ER) stress may play a role in the etiology of Parkinson's disease (PD). We have previously reported that ubiquitin ligase 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase degradation 1 (HRD1) involved in ER stress degrades unfolded protein that accumulates in the ER due to loss of function of Parkin, which is a causative factor in familial PD. We have also demonstrated that cell death is suppressed by the degradation of unfolded proteins. These findings indicate that HRD1 may serve as a compensatory mechanism for the loss of function of Parkin in familial PD patients. However, the role of HRD1 in sporadic PD has not yet been identified. This study aimed to reveal the roles of HRD1 and associated molecules in a cellular model of PD. We demonstrated that expressions of HRD1 and Suppressor/Enhancer Lin12 1-like (SEL1L: a HRD1 stabilizer) increased in SH-SY5Y human neuroblastoma cells upon exposure to 6-hydroxydopamine (6-OHDA). The 6-OHDA-induced cell death was suppressed in cells overexpressing wt-HRD1, whereas cell death was enhanced in cells with knockdown of HRD1 expression. These results suggest that HRD1 is a key molecule involved in 6-OHDA-induced cell death. By contrast, suppression of SEL1L expression decreased the amount of HRD1 protein. As a result, 6-OHDA-induced cell death was enhanced in cells suppressing SEL1L expression, and this cell death was much more evident than that in cells with suppression of HRD1 expression. These findings strongly indicate that SEL1L is necessary for maintaining and stabilizing the amount of HRD1 protein, and stabilizing the amount of HRD1 protein through SEL1L may serve to protect against 6-OHDA-induced cell death. Furthermore, the expression of Parkin was reinforced when HRD1 mRNA had been suppressed in cells, but was not observed when SEL1L mRNA had been restrained. It is possible that Parkin expression is induced as a compensatory mechanism when HRD1 mRNA decreases. This intracellular transduction may suppress the enhancement of 6-OHDA-induced cell death caused by the loss of HRD1. Taken together with these results, it is suggested that HRD1 and its stabilizer (SEL1L) are key molecules for elucidating the pathogenesis and treatment of PD.

ER-localized protein-Herpud1 is a new mediator of IL-4-induced macrophage polarization and migration

ER-localized proteins have been reported function in endoplasmic reticulum, unfolded protein degradation and destruction of misfolded proteins by the ER-associated protein degradation (ERAD) system, but their function in the chemotaxis of macrophage cells remained un-addressed. Here, we showed that ER protein with ubiquitin like domain 1(Herpud1) was upregulated in IL-4-treated M2 macrophage cells and its expression pattern was similar with macrophage polarization markers, such as Arg1, Mrc1 and Fizz1. Inhibition of Herpud1 by using specific target shRNA decreased these marker's expression at mRNA and protein level in IL-4-treated or -untreated M2 macrophage cells. IL-4 treatment promoted M2 macrophage cell migration and polarization, but this promotion was weakened by Herpud1 depletion and we got similar results by inhibition of ER stress response with chemical molecule 4-phenylbutyric acid (4-PBA) in IL-4-treated or untreated-M2 macrophage cells with Herpud1 overexpression. These results indicated that depending on ER-associated protein degradation (ERAD) to help unfolded protein degradation or destruction is not the only function of Herpud1 and acting as a mediator of IL-4 induced macrophage activation and polarization maybe another unrevealed function, elucidating the role of Herpud1-associated M2 macrophage cell polarization and activation are helpful for exploration the function of macrophage cells in immune response.

Abstract B057: Plitidepsin inhibits autophagy, the main mechanism of acquired resistance to bortezomib

Abstract Background: Plitidepsin (APL), an antitumor agent isolated from the marine tunicate Aplidium albicans, has been tested with positive results in multiple myeloma (MM) patients in a phase III pivotal trial in combination with dexamethasone (clinicaltrials.gov identifier: NCT01102426) and in a phase I trial in combination with bortezomib and dexamethasone (clinicaltrials.gov identifier: NCT02100657). Plitidepsin targets eEF1A2, one of two isoforms of the alpha subunit of the eEF1 complex. In mammals, eEF1A2 has a selective pattern of expression in those tissues that do not express the A1 isoform, namely brain and muscle. Nonetheless, eEF1A2 is aberrantly expressed in many cancers, including solid tumors (1-3) and MM (4), and has been shown to hold oncogenic properties (5). We have previously demonstrated through several means the interaction of plitidepsin with its target, eEF1A2 and calculated a Kd of around 80 nM for this interaction (6). Indeed, we have found that plitidepsin exclusively binds to the GTP-bound form of eEF1A2 Aims: Here we investigated whether eEF1A2 levels had any effect on the sensitivity of MM cells to both plitidepsin and bortezomib, to better understand the synergistic effect these two drugs yield when combined (7). Methods: RPMI-8226, OPM1, U266-B1, MM1S and NCI-NH929 multiple myeloma cells were cultured in optimal conditions. eEF1A2 (GTX102326) and autophagy (L7543) were followed by Western blot. Dose-response experiments were performed in 96 well plates, treating cells with increasing doses of the compounds for 24 hours and measuring cell growth with MTT. Curves were then fitted with GraphPad Prism5 software. For combination experiments, the Chou-Talalay procedure was used. Results: We first analyzed the level of eEF1A2 protein in a panel of multiple myeloma cell lines. Then, we selected a cell line with low expression, MM-1S, and one with high expression, OPM-1, of the elongation factor and checked the sensitivity to both, bortezomib and plitidepsin through dose-response experiments. Interestingly, the overexpression of eEF1A2, the target of plitidepsin, seems to be related to an increased resistance to bortezomib, a phenomenon usually related to enhanced autophagy since this latter pathway compensates the inhibition of proteasome. eEF1A, in its GTP-bound form, has been shown to inhibit chaperone-mediated autophagy (CMA), essential for the degradation of unfolded proteins (8). Since plitidepsin binds to the GTP-bound eEF1A2, probably stabilizing this form of the elongation factor, it seems likely that, in such a way, it may inhibit CMA. We explored this possibility and, indeed, we observed that plitidepsin inhibited autophagy in MM cells. Summary/Conclusion: Plitidepsin treatment presumably counteracts the main mechanism of acquired resistance to bortezomib, namely autophagy. (1)Sun et al., 2014, Biochem Biophys Res Commun 450:1-6. (2)Xu et al., 2013, Clin Exp Metastasis 30:933-44. (3)Anand et al., 2002, Nat Genet 31:301-5. (4)Li et al., 2010, PLOS One 5, e10755. (5)Lee and Surh, 2009, Ann N Y Acad Sci 1171:87-93. (6)Losada et al., 2004, Br J Cancer 91:1405-13. (7)Mitsiades et al., 2008, Cancer Res 68:5216-25. (8)Bandyopadhyay et al., 2010, Mol Cel 39:535-47. Citation Format: Juan F. Martínez-Leal, Gaëlle Quiniou, Alejandro Losada, María José Muñoz, Rafael Sanchez-Mesa, Patricia Martinez-Rivas, Juan Manuel Dominguez, Carlos M. Galmarini. Plitidepsin inhibits autophagy, the main mechanism of acquired resistance to bortezomib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B057.

Roles of endoplasmic reticulum stress and apoptosis signaling pathways in gynecologic tumor cells: A systematic review

Abstract Efficient functioning of the endoplasmic reticulum (ER) is very important for most cellular activities, such as protein folding and modification. The ER closely interacts with other organelles, including the Golgi body, endosome, membrane, and mitochondria, providing lipids and proteins for the repair of these organelles. ER stress can be induced by various abnormal materials in the cell. ER stress is a compensatory intracellular environment disorder that occurs during areaction. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system, but excessive ER activation can cause cell death. The Pubmed and Web of Science databases were searched for full-text articles, and the terms “endoplasmic reticulum stress / unfolded protein response / gynecologic tumor cell apoptosis” were used as key words. Thirty-five studies of ER stress and unfolded protein response published from 2000 to 2016 were analyzed. Stress triggers apoptosis through a variety of signaling pathways. Increasing evidence has shown that the ER plays an important role in tumor cell diseases. The present review discusses the molecular mechanisms underlying unfolded protein response and its ability to promote survival and proliferation in gynecologic tumor cells. List of abbreviations: IRE1α: inositol-requiring enzyme 1α; UPR: unfolded protein response; XBP-1: X binding protein; PERK: protein kinase RNA-like ER kinase; GRP78: glucose-regulated protein 78; Tun: tunicamycin; CHOP: CCAAT-enhancer-binding protein homologous protein; ERSE: endoplasmic reticulum stress element; ASK1: apoptosis signal-regulating kinase 1; eIF-2alpha: eukaryotic translation initiation factor 2; IP3: inositol 1,4,5-trisphosphat; ERO1: endoplasmic reticulum oxidoreductin 1; TMEM214: transmembrane protein 214; GADD34: cofactor of eIF2.phosphatase; H1299: human hung carcinoma H1299 cells; SH-SY5Y: human neuroblastoma cells; JNK: c-Jun N-terminal kinase; VEGFA: vascular endothelial growth factor-A; SERCA: sarcoplasmic reticulum Ca2+-ATPase; NAFLD: nonalcoholic fatty liver disease; ATF4: activating transcription factor 4; Pim-1: proviral integration moloney virus; PUMA: p53-upregulated modulator of apoptosis

Abstract 2770: DERL3 hypermethylation alters rhabdomyosarcoma cell metabolism

Abstract Physiological cellular activity requires a precise control of the proteome. However, cancer cells have a distorted protein profile. It is likely that intrinsic defects in protein homeostasis participate in human tumorigenesis, such as alterations in de novo protein synthesis, or defects in protein degradation. In this last case, the ubiquitin (Ub) proteasome system (UPS) targets a variety of proteins, including functional proteins that are no longer needed. Without appropriate protein homeostasis maintained by the UPS, healthy cells can undergo malignant transformation, and this observation has been therapeutically exploited by the development of proteasome inhibitors as anticancer agents. Many of the cytoplasm UPS-degraded proteins are retrotranslocated from the endoplasmic reticulum (ER). In this regard, ER possesses a quality control mechanism, termed the ER-associated degradation mechanism (ERAD), that is induced in response to ER stress by a transcriptional program, known as the unfolded protein response (UPR), which leads to the accelerated degradation of unfolded proteins. Within the ERAD pathway, the Derlin proteins play a critical role. It has been proposed that Derlins form an export channel in the membrane of the ER through which the ERAD substrates pass to reach the proteasome. A previous study in our lab has shown that DERL3 silencing by DNA methylation deregulate SLC2A1 (GLUT1) degradation, promoting the Warburg effect. SLC2A1 is a key glucose receptor up-regulated in most solid tumors, allowing cancer cells to get the glucose they need for tumor progression. In this study we use an embryonic rhabdomyosarcoma cell line as a model to find out the role of DERL3 in these tumors, as the percentage of promoter hypermethylation is these tumors is approximately 60%. Rhabdomyosarcoma (RMS) is one of the leading causes of cancer related deaths among children, being the histological variant of embryonic rhabdomyosarcoma the most common soft tissue sarcoma of childhood and adolescence, with 350 cases per year in USA. The restoration of DERL3 expression in vitro promotes GLUT1 degradation and GLUT4 up-regulation, becoming cells sensitive to insulin. Moreover, it has been shown that DERL3 confers sensitivity against an ERAD related drug, Eeyarestatin I, opening a new approach for RMS treatment. All together, these initial findings highlight the role of DERL3 in RMS metabolism. Citation Format: Pere Llinas, Paula Lopez, Manel Esteller. DERL3 hypermethylation alters rhabdomyosarcoma cell metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2770.

The Transcription Factor p8 Regulates Autophagy in Response to Palmitic Acid Stress via a Mammalian Target of Rapamycin (mTOR)-independent Signaling Pathway

Autophagy is an evolutionarily conserved degradative process that allows cells to maintain homoeostasis in numerous physiological situations. This process also functions as an essential protective response to endoplasmic reticulum (ER) stress, which promotes the removal and degradation of unfolded proteins. However, little is known regarding the mechanism by which autophagy is initiated and regulated in response to ER stress. In this study, different types of autophagy were identified in human gastric cancer MKN45 cells in response to the stress induced by nutrient starvation or lipotoxicity in which the regulation of these pathways is mammalian target of rapamycin (mTOR)-dependent or -independent, respectively. Interestingly, we found that p8, a stress-inducible transcription factor, was enhanced in MKN45 cells treated with palmitic acid to induce lipotoxicity. Furthermore, an increase in autophagy was observed in MKN45 cells stably overexpressing p8 using a lentivirus system, and autophagy induced by palmitic acid was blocked by p8 RNAi compared with the control. Western blotting analyses showed that autophagy was regulated by p8 or mTOR in response to the protein kinase-like endoplasmic reticulum kinase/activating transcription factor 6-mediated ER stress of lipotoxicity or the parkin-mediated mitochondrial stress of nutrient starvation, respectively. Furthermore, our results indicated that autophagy induced by palmitic acid is mTOR-independent, but this autophagy pathway was regulated by p8 via p53- and PKCα-mediated signaling in MKN45 cells. Our findings provide insights into the role of p8 in regulating autophagy induced by the lipotoxic effects of excess fat accumulation in cells.

Modification of ubiquitin C-terminal hydrolase L1 by reactive lipid species: role in neural regeneration and diseases of aging.

Modification of ubiquitin C-terminal hydrolase L1 by reactive lipid species: role in neural regeneration and diseases of aging.

Regulation of endoplasmic reticulum functions.

The endoplasmic reticulum (ER) is a multifunctional organelle that is specialized for lipid synthesis, Ca2+ homeostasis, and protein synthesis. Protein synthesis is a multistep process involving folding and manipulation of proteins as well as the exportation of proteins to an appropriate location. Several stress responses, such as metabolic and anaerobic stress, induce ER dysfunction and protein unfolding, which accelerates the disruption of ER homeostasis. To prevent the production of unfolded proteins, cells induce the unfolded protein response (UPR), which is a highly regulated protein quality surveillance system. UPR maintains and restores ER homeostasis to increase the secretory function of the ER via the induction of the ER chaperone protein. This stimulates re-folding or degradation of unfolded proteins. However, irreparable ER stress induces a cell death response to eliminate these damaged cells [1]. Three types of ER transmembrane proteins, a protein-kinase and site-specific endoribonuclease, inositol-requiring enzyme 1 α (IRE1α), protein kinase R-like ER kinase/pancreatic eukaryotic initiation factor 2 kinase, and activating transcription factor 6 are essential for the mammalian UPR system. In particular, IRE1α is an essential signal transducer involved in the maintenance of ER function. The IRE1α pathway induces X-box binding protein 1(S) [XBP1(S)], which is the active form of XBP1 generated by IRE1α-dependent splicing of the XBP1 mRNA. XBP1(S) is a transcription factor that increases the expression of the ER chaperone, thus enhancing the secretory function of the ER and suppressing ER stress-mediated cell death [1]. Cancer cells alternate between glycolysis and protein synthesis pathways to survive and proliferate during stress associated with tumor growth and prolonged metabolic and anaerobic stress [2]. Cancer cells produce a lot of new proteins to satisfy their rapid growth using an energy source, such as the cancer-specific aerobic glycolysis pathway. Thus, the augmented function of the ER and resistance of ER stress-mediated cell death is essential for tumor proliferation and survival. Many reports have shown the enhancement of ER function in a variety of human cancers, but the mechanism for the cancer cell-specific regulation of ER function remains unresolved. Our recent study reveals that the tumor suppression gene, p53, is a key regulator of ER function [3]. To the best of our knowledge, this is the first report to connect the cancer-related gene to ER function. p53 is mutated in at least half of human cancers, and defects in the p53 response lead to tumor development. The functions of p53 influence many types of cell signaling transduction pathways, but the role of p53 in the ER is largely unknown [4]. We found that the loss of p53 function increased IRE1α, XBP1(S), and ER chaperone expression in non-stressed and ER-stressed conditions and also increased ER function. Interestingly, IRE1α was abundantly expressed in mutant p53 cell lines compared with wild-type p53 cell lines. The upregulation of IRE1α expression triggered the activation of the IRE1α/XBP1 pathway. Further, our experiments identified the mechanism for increased IRE1 expression with p53 deficiency, and our results revealed that suppressed p53-dependent IRE1α-synoviolin-1 (SYVN1) association induced IRE1α proteasomal degradation. Therefore, wild-type p53 suppresses ER function and adapts to ER stress by keeping IRE1α expression levels low, which highlights the anti-cancer effects of p53. However, loss of p53 function activates the IRE1α/XBP1 pathway and stimulates resistance to ER stress-mediated cell death. Thus, up-regulation of IRE1α expression via the loss of p53 function is one of the mechanisms by which a cancer-specific increase in ER function and activation of the IRE1α/XBP1 pathway is related to some parts of the aggressive cancer phenotype found in p53-mutant cancers. Another important finding of our current study is that an inhibitor of IRE1 selectively prevents the growth of p53-null tumors in vivo compared with that of wild type p53 tumors [3]. Several reports suggested that the inhibition of the IRE1α/XBP1 pathway is a new anti-cancer target. Cubillos-Ruiz et al. reported that the inhibition of activated XBP1 stimulates tumor-infiltrating dendritic cell-induced anti-tumor immunity [5]. The IRE1α inhibitor has an anti-cancer effect on human multiple myeloma and pancreatic cancers [6, 7]. Furthermore, DNA-damaging agents are widely used as treatments for various forms of cancer in the clinic, and its anti-cancer effects depend on the appropriate function of wild-type p53. Thus, mutant p53 cancer cells have been reported to show chemoresistance to many anti-cancer agents, and there is a need for novel therapies against mutant p53 cancers. Therefore, preventing the activation of the IRE1α/XBP1 pathway is a promising target for anti-tumor agents of mutant p53 cancers. In summary, cancer cells need to increase ER function to survive stressful environments. Loss of p53 function augments ER function via the activation of the IRE1α/XBP1 pathway by the disruption of the p53-SYVN1-IRE1α complex. Thus, targeting the IRE1α/XBP1 pathway has potential as a novel therapeutic strategy for malignancy and chemo-resistance of mutant p53 cancers.

Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production

Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes.

Stress-induced self-cannibalism: on the regulation of autophagy by endoplasmic reticulum stress.

Macroautophagy (autophagy) is a cellular catabolic process which can be described as a self-cannibalism. It serves as an essential protective response during conditions of endoplasmic reticulum (ER) stress through the bulk removal and degradation of unfolded proteins and damaged organelles; in particular, mitochondria (mitophagy) and ER (reticulophagy). Autophagy is genetically regulated and the autophagic machinery facilitates removal of damaged cell components and proteins; however, if the cell stress is acute or irreversible, cell death ensues. Despite these advances in the field, very little is known about how autophagy is initiated and how the autophagy machinery is transcriptionally regulated in response to ER stress. Some three dozen autophagy genes have been shown to be required for the correct assembly and function of the autophagic machinery; however; very little is known about how these genes are regulated by cellular stress. Here, we will review current knowledge regarding how ER stress and the unfolded protein response (UPR) induce autophagy, including description of the different autophagy-related genes which are regulated by the UPR.

791 RITONAVIR INTERACTS WITH BORTEZOMIB SYNERGISTICALLY TO ENHANCE ENDOPLASMIC RETICULUM STRESS IN PROSTATE CANCER CELLS

INTRODUCTION AND OBJECTIVES: Inducing endoplasmic reticulum (ER) stress is a novel approach to cancer therapy. The HIV protease inhibitor ritonavir induces ER stress by activating the unfolded protein response and we hypothesized that combining the proteasome inhibitor bortezomib with ritonavir would enhance this stress by inhibiting the degradation of unfolded proteins. METHODS: The viability and clonogenicity of the prostate cancer cells (LNCaP, PC3, and DU145) treated with ritonavir (25–50 M) and bortezomib (5–10 nM) were assessed by MTS assay and colony formation assay. The in vivo efficacy of the combination was evaluated using a murine subcutaneous tumor model using PC3 cells. Annexin-V assay was used to detect apoptosis. Induction of ER stress, histone acetylation, and the expression of p21 and apoptosis-associated proteins were evaluated by western blot analysis. RESULTS: The combination of ritonavir and bortezomib induced apoptosis and inhibited the prostate cancer growth synergistically. It also inhibited colony formation. In subcutaneous tumor models, 10-day treatment with the combination was well tolerated and inhibited tumor growth. The combination induced ER stress synergistically, as evidenced by the increased expression of glucose-regulated protein 78, heat shock protein 70, and cyclic AMP response element binding protein 2 (CREB2). It also induced histone acetylation and thereby increased p21 expression. This histone acetylation is attributable to the increased CREB2 expression because CREB2 is also a histone acetyltransferase. In addition, decreased expression of histone deacetylases by the combination was thought to enhance this histone acetylation. CONCLUSIONS: The combination of ritonavir and bortezomib induces apoptosis and inhibits the proliferation of prostate cancer cells by inducing ER stress. Our results provide a rationale for investigating this combination in patients with prostate cancer.

Restoring endoplasmic reticulum function by chemical chaperones: an emerging therapeutic approach for metabolic diseases

The endoplasmic reticulum (ER) is a eukaryotic organelle that plays important roles in protein synthesis, folding and trafficking, calcium homoeostasis and lipid and steroid synthesis. It is the major protein synthesis compartment for secreted, plasma membrane and organelle proteins. Perturbations of ER homeostasis such as the accumulation of unfolded or misfolded proteins cause ER stress. To alleviate this stress, ER triggers an evolutionarily conserved signalling cascade called the unfolded protein response (UPR). As an initial response, the UPR aims at adapting and restoring ER function by translational attenuation, upregulation of ER chaperones and degradation of unfolded proteins. However, if the ER function is severely impaired because of excessive or prolonged exposure to stress, then the inflicted cells may undergo programmed cell death. During ER stress, unstable or partially folded mutant proteins are prevented from trafficking to their proper subcellular localizations and usually rapidly degraded. The small molecules named chemical chaperones help to stabilize these mutant proteins and facilitate their folding and proper trafficking from the ER to their final destinations. Because increasing number of studies suggest that ER stress is involved in a number of disease pathogenesis including neurodegenerative diseases, cancer, obesity, diabetes and atherosclerosis, promoting ER folding capacity through chemical chaperones emerges as a novel therapeutic approach. In this review, we provide insight into the many important functions of chemical chaperones during ER stress, their impact on the ER-stress-related pathologies and their potential as a new drug targets, especially in the context of metabolic disorders.

158. THE UNFOLDED PROTEIN RESPONSE MAY CONTRIBUTE TO GLUCOCORTICOID-INDUCED PLACENTAL GROWTH RESTRICTION IN THE RAT VIA INCREASED PLACENTAL EXPRESSION OF HEAT SHOCK PROTEIN 70

Perturbations of normal endoplasmic reticulum (ER) physiology occur in a number of pathological conditions, including diabetes and preeclampsia. These pathologies are associated with elevated levels of inappropriately folded proteins and induction of ER stress. Accumulation of misfolded proteins induces the unfolded protein response which increases ER protein folding capacity and promotes ER-associated degradation of unfolded proteins. Glucocorticoids are essential for maturation of fetal organs, however excess exposure during pregnancy retards fetal and placental growth. Glucocorticoids also induce ER stress within macrophages, which reside within the placenta, and activate immune responses which can lead to oxidative stress and subsequent placental dysfunction. We hypothesised that excess glucocorticoid exposure would induce ER stress within the placenta and contribute to restriction of fetal and placental growth. This study compared placentas (n = 6/group) for control (Con) and dexamethasone-exposed pregnancies (Dex; 0.75 μg/mL drinking water from day 13 of gestation) at days 16 and 22 of gestation in the rat (term = 23 days). Placentas were dissected into junctional (JZ) and labyrinth (LZ) zones for separate analysis. Quantitative PCR was used to determine expression of mRNA for markers of ER stress, including heat shock factors (HSF-1 and HSF-2), heat shock proteins (HSP-70 and HSP-90) and C/EBP homologous protein (CHOP10). HSF-1 expression increased 2- to 4-fold from day 16 to 22 in both placental zones, but was not increased by glucocorticoids. Dex-exposure increased HSP-70 expression 2- to 3-fold in the LZ at both days of gestation, indicative of an ER stress response. Similar patterns for JZ expression of HSP-70 were observed. JZ expression of HSP-90 was also upregulated by Dex at day 22 but not day 16. CHOP10 was not induced by Dex-administration in either zone at either gestational time, which suggests that rather than activation of the ATF6/PERK pathway, the activation of ER stress is likely to be via XBP1 induction.

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.

Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana

Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). In this case, so-called unfolded protein response (UPR) genes are induced. We determined the transcriptional expression of Arabidopsis thaliana UPR genes by fluid microarray analysis of tunicamycin-treated plantlets. Two hundred and fifteen up-regulated genes and 17 down-regulated ones were identified. These genes were reanalyzed with functional DNA microarrays, using DNA fragments cloned through fluid microarray analysis. Finally, 36 up-regulated and two down-regulated genes were recognized as UPR genes. Among them, the up-regulation of genes related to protein degradation (HRD1, SEL-1L/HRD3 and DER1), regulation of translation (P58(IPK)), and apoptosis (BAX inhibitor-1) was reconfirmed by real-time reverse transcriptase-PCR. The induction of SEL-1L protein in an Arabidopsis membrane fraction on tunicamycin-treatment was demonstrated. Phosphorylation of initiation factor-2alpha, which was inhibited by P58(IPK), was decreased in tunicamycin-treated plantlets. However, regulatory changes in translation caused by ER stress were not detected in Arabidopsis. Plant cells appeared to have a strategy for overcoming ER stress through enhancement of protein folding activity, degradation of unfolded proteins, and regulation of apoptosis, but not regulation of translation.

Roles of CHOP/GADD153 in endoplasmic reticulum stress

Endoplasmic reticulum (ER) is the site of synthesis and folding of secretory proteins. Perturbations of ER homeostasis affect protein folding and cause ER stress. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system. However, when the ER function is severely impaired, the organelle elicits apoptotic signals. ER stress has been implicated in a variety of common diseases such as diabetes, ischemia and neurodegenerative disorders. One of the components of the ER stress-mediated apoptosis pathway is C/EBP homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153 (GADD153). Here, we summarize the current understanding of the roles of CHOP/GADD153 in ER stress-mediated apoptosis and in diseases including diabetes, brain ischemia and neurodegenerative disease.