Publications by authors named "Kazutoshi Mori"

96 Publications

Effects of cordierite particulate filters on diesel engine exhaust emissions in terms of pollution prevention approaches for better environmental management.

J Environ Manage 2021 May 31;293:112873. Epub 2021 May 31.

Department of Mechanical and Precision System Engineering, Faculty of Science and Engineering, Teikyo University, 320-8551, Utsunomiya, Tochigi, Japan.

In this study, a specific diesel fuel is experimentally tested in a 4-cylindered diesel engine with and without a cordierite-based diesel particulate filter (CPF) to show the prevention of emissions by using an after treatment system (ATS). In this context, engine exhaust emissions, total particle concentration (TPC) and soot concentration are investigated. The diesel engine is firstly evaluated with the data directly measured from the engine output (DEO) (without after treatment option), and then the changes in the exhaust emission are examined by using an ATS which is a cordierite-based diesel particulate filter to prevent pollution. In this regard, total particle concentration of DEO option is found to be 6134041.20 1/cm and total particle concentration by using CPF is obtained to be 707.84 1/cm. 99.99% reduction in TPC is achieved thanks to the use of CPF. The soot concentration of DEO option is calculated to be 2.158 mg/m. However, the soot concentration is found to be 0.014 mg/m by using the CPF. The particulate matters are burned at high temperatures after being filtered at the exhaust output thanks to the regeneration process within the CPF after treatment. CO emissions decreased from 0.7489 g/kWh to 0.7273 g/kWh with the CPF utilization, while HC emissions decreased from 0.0965 g/kWh to 0.0900 g/kWh via CPF. However, an increase in CO and NO emissions are observed due to oxidation in the CPF.
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http://dx.doi.org/10.1016/j.jenvman.2021.112873DOI Listing
May 2021

Endoplasmic reticulum-associated degradation controls virus protein homeostasis that is required for the flavivirus propagation.

J Virol 2021 May 12. Epub 2021 May 12.

Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Aomori, Japan

Many positive-stranded RNA viruses encode polyproteins and viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though their required amounts are different. In this study, we found that the extra membrane-anchored non-structural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 predominantly localized in the convoluted membrane (CM) of viral replication organelle, and viral NS proteins degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses.The results of this study reveal that the cellular ERAD system controls the amount of each viral protein in virus-infected cells; this "viral protein homeostasis" is critical for viral propagation. Furthermore, we clarified that the "convoluted membrane (CM)," which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/Sel1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.
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http://dx.doi.org/10.1128/JVI.02234-20DOI Listing
May 2021

Mechanisms of productive folding and endoplasmic reticulum-associated degradation of glycoproteins and non-glycoproteins.

Biochim Biophys Acta Gen Subj 2021 03 11;1865(3):129812. Epub 2020 Dec 11.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan. Electronic address:

Background: The quality of proteins destined for the secretory pathway is ensured by two distinct mechanisms in the endoplasmic reticulum (ER): productive folding of newly synthesized proteins, which is assisted by ER-localized molecular chaperones and in most cases also by disulfide bond formation and transfer of an oligosaccharide unit; and ER-associated degradation (ERAD), in which proteins unfolded or misfolded in the ER are recognized and processed for delivery to the ER membrane complex, retrotranslocated through the complex with simultaneous ubiquitination, extracted by AAA-ATPase to the cytosol, and finally degraded by the proteasome.

Scope Of Review: We describe the mechanisms of productive folding and ERAD, with particular attention to glycoproteins versus non-glycoproteins, and to yeast versus mammalian systems.

Major Conclusion: Molecular mechanisms of the productive folding of glycoproteins and non-glycoproteins mediated by molecular chaperones and protein disulfide isomerases are well conserved from yeast to mammals. Additionally, mammals have gained an oligosaccharide structure-dependent folding cycle for glycoproteins. The molecular mechanisms of ERAD are also well conserved from yeast to mammals, but redundant expression of yeast orthologues in mammals has been encountered, particularly for components involved in recognition and processing of glycoproteins and components of the ER membrane complex involved in retrotranslocation and simultaneous ubiquitination of glycoproteins and non-glycoproteins. This may reflect an evolutionary consequence of increasing quantity or quality needs toward mammals.

General Significance: The introduction of innovative genome editing technology into analysis of the mechanisms of mammalian ERAD, as exemplified here, will provide new insights into the pathogenesis of various diseases.
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http://dx.doi.org/10.1016/j.bbagen.2020.129812DOI Listing
March 2021

Antipsychotic olanzapine-induced misfolding of proinsulin in the endoplasmic reticulum accounts for atypical development of diabetes.

Elife 2020 11 17;9. Epub 2020 Nov 17.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.

Second-generation antipsychotics are widely used to medicate patients with schizophrenia, but may cause metabolic side effects such as diabetes, which has been considered to result from obesity-associated insulin resistance. Olanzapine is particularly well known for this effect. However, clinical studies have suggested that olanzapine-induced hyperglycemia in certain patients cannot be explained by such a generalized mechanism. Here, we focused on the effects of olanzapine on insulin biosynthesis and secretion by mouse insulinoma MIN6 cells. Olanzapine reduced maturation of proinsulin, and thereby inhibited secretion of insulin; and specifically shifted the primary localization of proinsulin from insulin granules to the endoplasmic reticulum. This was due to olanzapine's impairment of proper disulfide bond formation in proinsulin, although direct targets of olanzapine remain undetermined. Olanzapine-induced proinsulin misfolding and subsequent decrease also occurred at the mouse level. This mechanism of olanzapine-induced β-cell dysfunction should be considered, together with weight gain, when patients are administered olanzapine.
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http://dx.doi.org/10.7554/eLife.60970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671685PMC
November 2020

EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD.

Elife 2020 02 17;9. Epub 2020 Feb 17.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.

Sequential mannose trimming of N-glycan (ManGlcNAc -> ManGlcNAc -> ManGlcNAc) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no α1,2-mannosidase activity toward ManGlcNAc in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted ManGlcNAc to ManGlcNAc(isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins.
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http://dx.doi.org/10.7554/eLife.53455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039678PMC
February 2020

Development of a Rapid in vivo Assay to Evaluate the Efficacy of IRE1-specific Inhibitors of the Unfolded Protein Response Using Medaka Fish.

Cell Struct Funct 2020 Feb 26;45(1):23-31. Epub 2019 Dec 26.

Department of Biophysics, Graduate School of Science, Kyoto University.

Three types of transmembrane protein, IRE1α/IRE1β, PERK, and ATF6α/ATF6β, are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1α inhibitors. IRE1α, IRE1β, ATF6α and ATF6β are ubiquitously expressed in medaka. We found that IRE1α/ATF6α-double knockout is lethal, similarly to IRE1α/IRE1β- and ATF6α/ATF6β-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6α-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1α in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1α inhibitors in vivo.Key words: endoplasmic reticulum, inhibitor screening, mRNA splicing, phenotypic assay, unfolded protein response.
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http://dx.doi.org/10.1247/csf.19032DOI Listing
February 2020

Reinvestigation of Disulfide-bonded Oligomeric Forms of the Unfolded Protein Response Transducer ATF6.

Cell Struct Funct 2020 Jan 19;45(1):9-21. Epub 2019 Dec 19.

Department of Biophysics, Graduate School of Science, Kyoto University.

ATF6α is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6α occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6α is reduced upon ER stress, such that only reduced monomer ATF6α is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6α and its relevant ATF6β, and found that ATF6α dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6α monomer physically associates with another ATF6α monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6α, namely monomer, C618-dimer and C467-dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6α activation.Key words: disulfide-bonded structure, endoplasmic reticulum, membrane-bound transcription factor, non-reducing SDS-PAGE, unfolded protein response.
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http://dx.doi.org/10.1247/csf.19030DOI Listing
January 2020

Cartilage endoplasmic reticulum stress may influence the onset but not the progression of experimental osteoarthritis.

Arthritis Res Ther 2019 09 11;21(1):206. Epub 2019 Sep 11.

Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health and Manchester Academic Health Science Centre, The University of Manchester, Manchester, M13 9PT, UK.

Background: Osteoarthritis has been associated with a plethora of pathological factors and one which has recently emerged is chondrocyte endoplasmic reticulum (ER) stress. ER stress is sensed by key ER-resident stress sensors, one of which is activating transcription factor 6 (ATF6). The purpose of this study is to determine whether increased ER stress plays a role in OA.

Methods: OA was induced in male wild-type (+/+), ColIITg (c/c) and Atf6α mice by destabilisation of the medial meniscus (DMM). c/c mice have increased ER stress in chondrocytes via the collagen II promoter-driven expression of ER stress-inducing Tg. Knee joints were scored histologically for OA severity. RNA-seq was performed on laser-micro-dissected RNA from cartilage of +/+ and c/c DMM-operated mice.

Results: In situ hybridisation demonstrated a correlation between the upregulation of ER stress marker, BiP, and early signs of proteoglycan loss and cartilage damage in DMM-operated +/+ mice. Histological analysis revealed a significant reduction in OA severity in c/c mice compared with +/+ at 2 weeks post-DMM. This chondroprotective effect in c/c mice was associated with a higher ambient level of BiP protein prior to DMM and a delay in chondrocyte apoptosis. RNA-seq analysis suggested Xbp1-regulated networks to be significantly enriched in c/c mice at 2 weeks post-DMM. Compromising the ER through genetically ablating Atf6α, a key ER stress sensor, had no effect on DMM-induced OA severity.

Conclusion: Our studies indicate that an increased capacity to effectively manage increases in ER stress in articular cartilage due either to pre-conditioning as a result of prior exposure to ER stress or to genetic pre-disposition may be beneficial in delaying the onset of OA, but once established, ER stress plays no significant role in disease progression.
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http://dx.doi.org/10.1186/s13075-019-1988-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737683PMC
September 2019

Environmental pollution cost analyses of biodiesel and diesel fuels for a diesel engine.

J Environ Manage 2019 Aug 13;243:218-226. Epub 2019 May 13.

Department of Mechanical and Precision System Engineering, Faculty of Science and Engineering, Teikyo University, 320-8551, Utsunomiya, Tochigi, Japan.

In this study, Japanese Industrial Standard diesel no 2 and waste cooking oil biodiesel fuels are compared in terms of environmental pollution cost analysis. The experiments of the diesel and biodiesel fueled diesel engine are done at 100 Nm, 200 Nm and full load (294 Nm), while engine speed is constant at 1800 rpm. The method used in this study consists of a combination of economic and environmental parameters. According to the analyses, the total environmental pollution cost of the biodiesel is higher than the diesel fuel. On the other hand, the total cost of the CO emissions of the diesel fuel is generally found to be higher than biodiesel fuel in terms of the life cycle based environmental pollution cost. The specific environmental pollution cost is found as minimum at full load to be 2.217 US cent/kWh for the diesel fuel and 2.449 US cent/kWh for the biodiesel fuel at full load. On the other hand, the life cycle based specific environmental pollution cost is determined as minimum at full load to be 5.050 US cent/kWh for the diesel fuel and 5.309 US cent/kWh for the biodiesel fuel. The biodiesel fuel has higher values than diesel fuel in terms of the specific environmental pollution cost rates. The maximum total carbon dioxide emission rate is found as 0.2405 × 10 kg/kJ for the biodiesel fuel at 100 Nm engine torque and the minimum one is obtained as 0.1884 × 10 kg/kJ for the diesel fuel at full load. When the payback periods of the fuels are examined, biodiesel has longer period than diesel. The environmental payback period and life cycle based environmental payback period are also compared for fuels. In this context, the biodiesel has longer environmental payback periods rates than diesel. Consequently, the biodiesel fueled engine has higher environmental pollution cost rates than the diesel fueled engine, while the total carbon dioxide parameter of the diesel fuel is close to the biodiesel fuel's rate. Also, all of the other environmental parameters of diesel fuel is generally better than biodiesel. Consequently, the diesel fuel is generally better option than the biodiesel considering environmental aspects. For better environmental management, the diesel fuel utilization in the diesel engine is slightly better option than biodiesel fuel in terms of environmental pollution cost analysis.
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http://dx.doi.org/10.1016/j.jenvman.2019.05.002DOI Listing
August 2019

ATF6α downregulation of PPARα promotes lipotoxicity-induced tubulointerstitial fibrosis.

Kidney Int 2019 03 11;95(3):577-589. Epub 2019 Jan 11.

Division of Chronic Kidney Disease Pathophysiology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan. Electronic address:

Tubulointerstitial fibrosis is a strong predictor of progression in patients with chronic kidney disease, and is often accompanied by lipid accumulation in renal tubules. However, the molecular mechanisms modulating the relationship between lipotoxicity and tubulointerstitial fibrosis remain obscure. ATF6α, a transcription factor of the unfolded protein response, is reported to be an upstream regulator of fatty acid metabolism. Owing to their high energy demand, proximal tubular cells (PTCs) use fatty acids as their main energy source. We therefore hypothesized that ATF6α regulates PTC fatty acid metabolism, contributing to lipotoxicity-induced tubulointerstitial fibrosis. Overexpression of activated ATF6α transcriptionally downregulated peroxisome proliferator-activated receptor-α (PPARα), the master regulator of lipid metabolism, leading to reduced activity of fatty acid β-oxidation and cytosolic accumulation of lipid droplets in a human PTC line (HK-2). ATF6α-induced lipid accumulation caused mitochondrial dysfunction, enhanced apoptosis, and increased expression of connective tissue growth factor (CTGF), as well as reduced cell viability. Atf6α-/- mice had sustained expression of PPARα and less tubular lipid accumulation following unilateral ischemia-reperfusion injury (uIRI), resulting in the amelioration of apoptosis; reduced expression of CTGF, α-smooth muscle actin, and collagen I; and less tubulointerstitial fibrosis. Administration of fenofibrate, a PPARα agonist, reduced lipid accumulation and tubulointerstitial fibrosis in the uIRI model. Taken together, these findings suggest that ATF6α deranges fatty acid metabolism in PTCs, which leads to lipotoxicity-mediated apoptosis and CTGF upregulation, both of which promote tubulointerstitial fibrosis.
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http://dx.doi.org/10.1016/j.kint.2018.09.023DOI Listing
March 2019

A Collection of Transgenic Medaka Strains for Efficient Site-Directed Transgenesis Mediated by phiC31 Integrase.

G3 (Bethesda) 2018 07 31;8(8):2585-2593. Epub 2018 Jul 31.

Department of Biophysics, Graduate School of Science.

Genetic analysis is facilitated by the efficient production of transgenic strains expressing a DNA of interest as a single copy at a designated chromosomal location. However, technical progress toward this goal in medaka fish (), a vertebrate model organism, has been slow. It is well known that phiC31 integrase enables efficient site-directed transgenesis by catalyzing the recombination of an attP DNA motif in a host genome with an attB motif in a targeting vector. This system was pioneered in medaka using the transposon system, and the attP site was established at three chromosomal locations. However, this number appeared insufficient with regard to genetic linkage between the attP-landing site and a genetically modified locus of interest. Here, to establish a collection of transgenic strains of medaka, we introduced an attP motif into the medaka genome using the maize transposon system and established 12 independent transgenic strains harboring a single copy of the attP motif in at least 11 of the 24 medaka chromosomes. We designed an attB-targeting vector that was integrated efficiently and precisely into the attP-landing site, and with which the DNA of interest was efficiently transmitted to germline cells. Extraneous sequences in the integrants derived from the bacterial backbone of the attB-targeting vector as well as a transgenic fluorescence marker present in the attP-landing site were removable through flippase-mediated recombination. Further, an advanced targeting vector with a heart-specific recombination marker served as a useful tool for easily screening phiC31 integrase-mediated recombinant G embryos, leading to the efficient establishment of transgenic strains. Thus, our resources advance genetic research in medaka.
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http://dx.doi.org/10.1534/g3.118.200130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071608PMC
July 2018

The exercise-inducible bile acid receptor Tgr5 improves skeletal muscle function in mice.

J Biol Chem 2018 06 17;293(26):10322-10332. Epub 2018 May 17.

From the Food Biochemistry Laboratory and

TGR5 (also known as G protein-coupled bile acid receptor 1, GPBAR1) is a G protein-coupled bile acid receptor that is expressed in many diverse tissues. TGR5 is involved in various metabolic processes, including glucose metabolism and energy expenditure; however, TGR5's function in skeletal muscle is not fully understood. Using both gain- and loss-of-function mouse models, we demonstrate here that Tgr5 activation promotes muscle cell differentiation and muscle hypertrophy. Both young and old transgenic mice with muscle-specific Tgr5 expression exhibited increased muscle strength. Moreover, we found that Tgr5 expression is increased by the unfolded protein response (UPR), which is an adaptive response required for maintenance of endoplasmic reticulum (ER) homeostasis. Both ER stress response element (ERSE)- and unfolded protein response element (UPRE)-like sites are present in the 5' upstream region of the gene promoter and are essential for expression by Atf6α (activating transcription factor 6α), a well known UPR-activated transcriptional regulator. We observed that in the skeletal muscle of mice, exercise-induced UPR increases expression, an effect that was abrogated in α KO mice, indicating that Atf6α is essential for this response. These findings indicate that the bile acid receptor Tgr5 contributes to improved muscle function and provide an additional explanation for the beneficial effects of exercise on skeletal muscle activity.
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http://dx.doi.org/10.1074/jbc.RA118.002733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028981PMC
June 2018

Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish.

Elife 2017 09 27;6. Epub 2017 Sep 27.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.

When activated by the accumulation of unfolded proteins in the endoplasmic reticulum, metazoan IRE1, the most evolutionarily conserved unfolded protein response (UPR) transducer, initiates unconventional splicing of XBP1 mRNA. Unspliced and spliced mRNA are translated to produce pXBP1(U) and pXBP1(S), respectively. pXBP1(S) functions as a potent transcription factor, whereas pXBP1(U) targets pXBP1(S) to degradation. In addition, activated IRE1 transmits two signaling outputs independent of XBP1, namely activation of the JNK pathway, which is initiated by binding of the adaptor TRAF2 to phosphorylated IRE1, and regulated IRE1-dependent decay (RIDD) of various mRNAs in a relatively nonspecific manner. Here, we conducted comprehensive and systematic genetic analyses of the IRE1-XBP1 branch of the UPR using medaka fish and found that the defects observed in XBP1-knockout or IRE1-knockout medaka were fully rescued by constitutive expression of pXBP1(S). Thus, the JNK and RIDD pathways are not required for the normal growth and development of medaka. The unfolded protein response sensor/transducer IRE1-mediated splicing of XBP1 mRNA encoding its active downstream transcription factor to maintain the homeostasis of the endoplasmic reticulum is sufficient for growth and development of medaka fish.
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http://dx.doi.org/10.7554/eLife.26845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636610PMC
September 2017

SEL1L-dependent Substrates Require Derlin2/3 and Herp1/2 for Endoplasmic Reticulum-associated Degradation.

Cell Struct Funct 2017 Jul 26;42(2):81-94. Epub 2017 May 26.

Department of Biophysics, Graduate School of Science, Kyoto University.

Accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ATF6 pathway is one of the three major pathways in vertebrates. Although ATF6, a transmembrane-type glycoprotein in the ER, functions as a UPR sensor/transducer, it is an unstable protein with a half-life of approximately 2 h and is constitutively subjected to the ER-associated degradation system with the location of the misfolded part in the ER lumen (ERAD-L). ERAD-L substrates are delivered to the cytosol through the retrotranslocon, which likely contains HRD1 (E3), gp78 (E3), SEL1L (a partner of HRD1), Derlin1/2/3 and Herp1/2. We previously showed that ATF6 represents a novel transmembrane-type ERAD-L substrate requiring both EDEM1/2/3-mediated mannose trimming and SEL1L. Here, by constructing and analyzing chicken DT40 cells deficient in various components of the retrotranslocon, we show that degradation of ATF6 requires Derlin2 or Derlin3 and that Derlin2 and Derlin3 are redundant for ERAD-L of ATF6. We further show that degradation of ATF6 requires Herp1 or Herp2 and that Herp1 and Herp2 are redundant for ERAD-L of ATF6. Furthermore, by investigating five more ERAD-L substrates, we show that SEL1L-dependent substrates require Derlin2/3 and Herp1/2 regardless of their soluble or transmembrane nature. Our results suggest that ERAD-L substrates take several routes to the cytosol. The HRD1-engaged route 1 requires SEL1L, Derlin2 or Derlin3, and Herp1 or Herp2, whereas the HRD1-engaged route 2 does not require them functionally. It remains to be determined whether the latter requires Derlin1 and whether these two routes are compositionally distinct.Key words: endoplasmic reticulum, proteasome, protein degradation, protein misfolding, ubiquitin.
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http://dx.doi.org/10.1247/csf.17007DOI Listing
July 2017

UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage-specific manner.

J Cell Biol 2017 06 12;216(6):1761-1774. Epub 2017 May 12.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan

The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (, , , and ) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.
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http://dx.doi.org/10.1083/jcb.201609100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461018PMC
June 2017

A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models.

PLoS One 2016 29;11(8):e0161486. Epub 2016 Aug 29.

National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, United States of America.

The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress, however, results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts, implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings, we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ~425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR, including a compound with a 2,9-diazaspiro[5.5]undecane core, which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines, including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161486PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003374PMC
August 2017

Atf6α deficiency suppresses microglial activation and ameliorates pathology of experimental autoimmune encephalomyelitis.

J Neurochem 2016 12 18;139(6):1124-1137. Epub 2016 Jul 18.

Department of Neuroanatomy, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan.

Accumulating evidence suggests a critical role for the unfolded protein response in multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we investigated the relevance of activating transcription factor 6α (ATF6α), an upstream regulator of part of the unfolded protein response, in EAE. The expressions of ATF6α-target molecular chaperones such as glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94) were enhanced in the acute inflammatory phase after induction of EAE. Deletion of Atf6α suppressed the accumulation of T cells and microglia/macrophages in the spinal cord, and ameliorated the clinical course and demyelination after EAE induction. In contrast to the phenotypes in the spinal cord, activation status of T cells in the peripheral tissues or in the culture system was not different between two genotypes. Bone marrow transfer experiments and adoptive transfer of autoimmune CD4 T cells to recipient mice (passive EAE) also revealed that CNS-resident cells are responsible for the phenotypes observed in Atf6α mice. Further experiments with cultured cells indicated that inflammatory response was reduced in Atf6α microglia, but not in Atf6α astrocytes, and was associated with proteasome-dependent degradation of NF-κB p65. Thus, our results demonstrate a novel role for ATF6α in microglia-mediated CNS inflammation. We investigated the relevance of ATF6α, an upstream regulator of part of the UPR, in EAE. Deletion of Atf6α suppressed inflammation, and ameliorated demyelination after EAE. Bone marrow transfer experiments and adoptive transfer of autoimmune CD4 T cells revealed that CNS-resident cells are responsible for the phenotypes in Atf6α mice. Furthermore, inflammatory response was reduced in Atf6α microglia, and was associated with degradation of NF-κB p65. Our results demonstrate a novel role for ATF6α in microglia-mediated inflammation. Cover image for this issue: doi: 10.1111/jnc.13346.
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http://dx.doi.org/10.1111/jnc.13714DOI Listing
December 2016

Deletion of Atf6α enhances kainate-induced neuronal death in mice.

Neurochem Int 2016 Jan 25;92:67-74. Epub 2015 Dec 25.

Department of Neuroanatomy, Graduate School of Medical Science, Kanazawa University, Japan; CREST (Core Research for Evolutionary Science and Technology), JST (Japan Science and Technology Agency), Japan. Electronic address:

Excessive amount of L-glutamate in the brain causes neuronal damage in various pathological conditions including epilepsy and stroke. We previously reported that the 150-kDa oxygen-regulated protein (ORP150), a molecular chaperone in the endoplasmic reticulum (ER), inhibited the L-glutamate-induced neuronal death, at least partly, by improving Ca(2+) homeostasis in the ER. In the present study, we analyzed the role of activating transcription factor 6α (ATF6α), an upstream transcriptional factor critical for the operation of the ER, using mouse intrahippocampal kainate (KA) injection model. Expression of Hspa5, which encodes the molecular chaperone 78 kDa glucose-regulated protein (GRP78), increased after KA injection in the wild type (WT) mice. Comparative analysis using WT and Atf6α(-/-) mice revealed that KA induced pronounced neuronal death in the CA3 region of Atf6α(-/-) mice. The enhanced neuronal death in Atf6α(-/-) mice was associated with reduced expression of molecular chaperones in the ER and significant induction of c-fos in the hippocampal neurons. Furthermore, an injection of dantrolene, an inhibitor of ryanodine receptor, partially rescued these effects in Atf6α(-/-) mice after KA injection. Our results suggest that ATF6α plays an important role in neuronal survival after KA-induced excitotoxicity through the regulation of Ca(2+) response and neuronal activity.
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http://dx.doi.org/10.1016/j.neuint.2015.12.009DOI Listing
January 2016

Unfolded protein response in hypothalamic cultures of wild-type and ATF6α-knockout mice.

Neurosci Lett 2016 Jan 18;612:199-203. Epub 2015 Dec 18.

Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. Electronic address:

Recent studies suggest that endoplasmic reticulum (ER) stress in the hypothalamus could affect systemic homeostatic regulation in areas such as energy and water balance. Activating transcription factor 6α (ATF6α) is an ER stress transducer which increases the expression of ER chaperones and ER-associated degradation (ERAD) components under ER stress. In the present study, we examined the regulation of the unfolding protein response (UPR) in mouse hypothalamic cultures of wild-type (WT) and ATF6α(-/-) mice. Thapsigargin (TG), an ER stressor, significantly increased the mRNA expression of immunoglobulin heavy chain binding protein (BiP), spliced X-box binding protein 1 (XBP1), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and ERAD components, in hypothalamic cultures of WT mice with the same threshold (0.1μM) and similar time courses. On the other hand, TG-induced upregulation of BiP and CHOP as well as most ERAD-related genes, but not spliced XBP1 or ATF4, was attenuated in ATF6α(-/-) mice compared with WT mice. Our data suggest that all the UPR arms are activated similarly in the mouse hypothalamus under ER stress conditions, where ATF6α regulates the expression of ER chaperones, CHOP, and ERAD components.
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http://dx.doi.org/10.1016/j.neulet.2015.12.031DOI Listing
January 2016

Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway.

J Cell Biol 2015 Nov 16;211(4):775-84. Epub 2015 Nov 16.

Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ-ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ-ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.
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http://dx.doi.org/10.1083/jcb.201504109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657166PMC
November 2015

The unfolded protein response: the dawn of a new field.

Authors:
Kazutoshi Mori

Proc Jpn Acad Ser B Phys Biol Sci 2015 ;91(9):469-80

Department of Biophysics, Graduate School of Science, Kyoto University.

Originating from cancer research in mammalian cultured cells, the entirely new field of the unfolded protein response (UPR) was born in 1988. The UPR is a transcriptional induction program coupled with intracellular signaling from the endoplasmic reticulum (ER) to the nucleus to maintain the homeostasis of the ER, an organelle which controls the quality of proteins destined for the secretory pathway. Extremely competitive analyses using the budding yeast Saccharomyces cerevisiae revealed that although signaling from both the ER and cell surface is initiated by activation of a transmembrane protein kinase, the mechanism downstream of ER-resident Ire1p, a sensor molecule of the UPR, is unique. Thus, unconventional spliceosome-independent mRNA splicing is utilized to produce the highly active transcription factor Hac1p. This is the autobiographical story of how a young and not yet independent scientist competed with a very famous full professor in the early days of UPR research, which ultimately lead to their sharing Lasker Basic Medical Research Award in 2014.
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http://dx.doi.org/10.2183/pjab.91.469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754504PMC
August 2016

Endoplasmic Reticulum (ER) Stress Induces Sirtuin 1 (SIRT1) Expression via the PI3K-Akt-GSK3β Signaling Pathway and Promotes Hepatocellular Injury.

J Biol Chem 2015 Dec 23;290(51):30366-74. Epub 2015 Oct 23.

From the Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan,

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3β signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage.
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http://dx.doi.org/10.1074/jbc.M115.664169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683260PMC
December 2015

XBP 1-Deficiency Abrogates Neointimal Lesion of Injured Vessels Via Cross Talk With the PDGF Signaling.

Arterioscler Thromb Vasc Biol 2015 Oct 27;35(10):2134-44. Epub 2015 Aug 27.

From the Cardiovascular Division, King's College London BHF Centre, London, United Kingdom (L.Z., Y.L., J.Y., A.Z., X.Y., M.M., Y.H., Q.X.); Institute of Bioengineering (J.Y., W.W.) and Centre for Clinical Pharmacology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry (Q.X.), Queen Mary University of London, London, United Kingdom; Department of Emergency Medicine, The Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China (G.W.); Centre for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom (A.M.); and Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan (K.M.).

Objective: Smooth muscle cell (SMC) migration and proliferation play an essential role in neointimal formation after vascular injury. In this study, we intended to investigate whether the X-box-binding protein 1 (XBP1) was involved in these processes.

Approach And Results: In vivo studies on femoral artery injury models revealed that vascular injury triggered an immediate upregulation of XBP1 expression and splicing in vascular SMCs and that XBP1 deficiency in SMCs significantly abrogated neointimal formation in the injured vessels. In vitro studies indicated that platelet-derived growth factor-BB triggered XBP1 splicing in SMCs via the interaction between platelet-derived growth factor receptor β and the inositol-requiring enzyme 1α. The spliced XBP1 (XBP1s) increased SMC migration via PI3K/Akt activation and proliferation via downregulating calponin h1 (CNN1). XBP1s directed the transcription of mir-1274B that targeted CNN1 mRNA degradation. Proteomic analysis of culture media revealed that XBP1s decreased transforming growth factor (TGF)-β family proteins secretion via transcriptional suppression. TGF-β3 but not TGF-β1 or TGF-β2 attenuated XBP1s-induced CNN1 decrease and SMC proliferation.

Conclusions: This study demonstrates for the first time that XBP1 is crucial for SMC proliferation via modulating the platelet-derived growth factor/TGF-β pathways, leading to neointimal formation.
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http://dx.doi.org/10.1161/ATVBAHA.115.305420DOI Listing
October 2015

Activation of the Unfolded Protein Response in Sporadic Inclusion-Body Myositis but Not in Hereditary GNE Inclusion-Body Myopathy.

J Neuropathol Exp Neurol 2015 Jun;74(6):538-46

From the USC Neuromuscular Center, Department of Neurology, University of Southern California Keck School of Medicine, Good Samaritan Hospital, Los Angeles, California (AN, CDA, WKE, MC, VA); and Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan (SA, KM).

Muscle fibers in patients with sporadic inclusion-body myositis (s-IBM),the most common age-associated myopathy, are characterized by autophagic vacuoles and accumulation of ubiquitinated and congophilic multiprotein aggregates that contain amyloid-β and phosphorylated tau. Muscle fibers of autosomal-recessive hereditary inclusion-body myopathy caused by the GNE mutation (GNE-h-IBM) display similar pathologic features, except with less pronounced congophilia. Accumulation of unfolded/misfolded proteins inside the endoplasmic reticulum (ER) lumen leads to ER stress, which elicits the unfolded protein response (UPR) as a protective mechanism. Here we demonstrate for the first time that UPR is activated in s-IBM muscle biopsies, since there was 1) increased activating transcription factor 4 (ATF4) protein and increased mRNA of its target C/EBP homologous protein; 2) cleavage of the ATF6 and increased mRNA of its target glucose-regulated protein 78; and 3) an increase of the spliced form of X-box binding protein 1 and increased mRNA of ER degradation-enhancing α-mannosidase-like protein, target of heterodimer of cleaved ATF6 and spliced X-box binding protein 1. In contrast, we did not find similar evidence of the UPR induction in GNE-h-IBM patient muscle, suggesting that different intracellular mechanisms might lead to similar pathologic phenotypes. Interestingly, cultured GNE-h-IBM muscle fibers had a robust UPR response to experimental ER stress stimuli, suggesting that the GNE mutation per se is not responsible for the lack of UPR in GNE-h-IBM biopsied muscle.
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http://dx.doi.org/10.1097/NEN.0000000000000196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506930PMC
June 2015

Deletion of Atf6α impairs astroglial activation and enhances neuronal death following brain ischemia in mice.

J Neurochem 2015 Feb 6;132(3):342-53. Epub 2015 Jan 6.

Department of Neurosurgery, Graduate School of Medical Sciences, Kanazawa University, Kanazawa City, Ishikawa, Japan.

To dissect the role of endoplasmic reticulum (ER) stress and unfolded protein response in brain ischemia, we investigated the relevance of activating transcription factor 6α (ATF6α), a master transcriptional factor in the unfolded protein response, after permanent middle cerebral artery occlusion (MCAO) in mice. Enhanced expression of glucose-regulated protein78, a downstream molecular chaperone of ATF6α, was observed in both neurons and glia in the peri-infarct region of wild-type mice after MCAO. Analysis using wild-type and Atf6α(-/-) mice revealed a larger infarct volume and increased cell death in the peri-ischemic region of Atf6α(-/-) mice 5 days after MCAO. These phenotypes in Atf6α(-/-) mice were associated with reduced levels of astroglial activation/glial scar formation, and a spread of tissue damage into the non-infarct area. Further analysis in mice and cultured astrocytes revealed that signal transducer and activator of transcription 3 (STAT3)-glial fibrillary acidic protein signaling were diminished in Atf6α(-/-) astrocytes. A chemical chaperone, 4-phenylbutyrate, restored STAT3-glial fibrillary acidic protein signaling, while ER stressors, such as tunicamycin and thapsigargin, almost completely abolished signaling in cultured astrocytes. Furthermore, ER stress-induced deactivation of STAT3 was mediated, at least in part, by the ER stress-responsive tyrosine phosphatase, TC-PTP/PTPN2. These results suggest that ER stress plays critical roles in determining the level of astroglial activation and neuronal survival after brain ischemia.
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http://dx.doi.org/10.1111/jnc.12981DOI Listing
February 2015

Lasker Award winner Kazutoshi Mori.

Authors:
Kazutoshi Mori

Nat Med 2014 Oct;20(10):1115-7

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http://dx.doi.org/10.1038/nm.3682DOI Listing
October 2014

Activating transcription factor 6α is required for the vasopressin neuron system to maintain water balance under dehydration in male mice.

Endocrinology 2014 Dec 9;155(12):4905-14. Epub 2014 Sep 9.

Departments of Endocrinology and Diabetes (Y.A., D.H., W.L., Y.M., H.S., M.G., R.B., Y.S., Y.O., H.A.) and Pathology (N.A., M.T.), Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Institute of Immunology Co., Ltd (A.S.), 1198-4 Iwazo, Utsunomiya 321-0973, Japan; Institute for Genome Research (S.O.), University of Tokushima, Tokushima 770-8503, Japan; and Department of Biophysics (K.M.), Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

Activating transcription factor 6α (ATF6α) is a sensor of endoplasmic reticulum (ER) stress and increases the expression of ER chaperones and molecules related to the ER-associated degradation of unfolded/misfolded proteins. In this study, we used ATF6α knockout (ATF6α(-/-)) mice to clarify the role of ATF6α in the arginine vasopressin (AVP) neuron system. Although urine volumes were not different between ATF6α(-/-) and wild-type (ATF6α(+/+)) mice with access to water ad libitum, they were increased in ATF6α(-/-) mice compared with those in ATF6α(+/+) mice under intermittent water deprivation (WD) and accompanied by less urine AVP in ATF6α(-/-) mice. The mRNA expression of immunoglobulin heavy chain binding protein, an ER chaperone, was significantly increased in the supraoptic nucleus in ATF6α(+/+) but not ATF6α(-/-) mice after WD. Electron microscopic analyses demonstrated that the ER lumen of AVP neurons was more dilated in ATF6α(-/-) mice than in ATF6α(+/+) mice after WD. ATF6α(-/-) mice that were mated with mice possessing a mutation causing familial neurohypophysial diabetes insipidus (FNDI), which is characterized by progressive polyuria and AVP neuronal loss due to the accumulation of mutant AVP precursor in the ER, manifested increased urine volume under intermittent WD. The aggregate formation in the ER of AVP neurons was further impaired in FNDI/ATF6α(-/-) mice compared with that in FNDI mice, and AVP neuronal loss was accelerated in FNDI/ATF6α(-/-) mice under WD. These data suggest that ATF6α is required for the AVP neuron system to maintain water balance under dehydration.
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http://dx.doi.org/10.1210/en.2014-1522DOI Listing
December 2014

EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step.

J Cell Biol 2014 Aug;206(3):347-56

Department of Biophysics, Graduate School of Science, and Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8502, Japan

Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) in which Htm1-mediated mannose trimming from the oligosaccharide Man8GlcNAc2 to Man7GlcNAc2 is the rate-limiting step in yeast. In contrast, the roles of the three Htm1 homologues (EDEM1/2/3) in mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or as lectins. We therefore conducted transcription activator-like effector nuclease-mediated gene knockout analysis in human cell line and found that all endogenous EDEMs possess mannosidase activity. Mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 is performed mainly by EDEM3 and to a lesser extent by EDEM1. Most surprisingly, the upstream mannose trimming from Man9GlcNAc2 to Man8GlcNAc2 is conducted mainly by EDEM2, which was previously considered to lack enzymatic activity. Based on the presence of two rate-limiting steps in mammalian gpERAD, we propose that mammalian cells double check gpERAD substrates before destruction by evolving EDEM2, a novel-type Htm1 homologue that catalyzes the first mannose trimming step from Man9GlcNAc2.
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http://dx.doi.org/10.1083/jcb.201404075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121980PMC
August 2014