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Kazutoshi Mori - One of the best experts on this subject based on the ideXlab platform.
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reinvestigation of disulfide bonded oligomeric forms of the unfolded protein response transducer ATF6
Cell Structure and Function, 2020Co-Authors: Hibiki Koba, Tetsuya Okada, Tokiro Ishikawa, Satoshi Ninagawa, Shengyu Jin, Nanami Imada, Tetsushi Sakuma, Takashi Yamamoto, Kazutoshi MoriAbstract: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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development of a rapid in vivo assay to evaluate the efficacy of ire1 specific inhibitors of the unfolded protein response using medaka fish
Cell Structure and Function, 2019Co-Authors: Byungseok Jin, Tetsuya Okada, Tokiro Ishikawa, Mai Taniguchi, Satoshi Ninagawa, Shigehide Kagaya, Kazutoshi MoriAbstract: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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Luminal Domain of ATF6 Alone Is Sufficient for Sensing Endoplasmic Reticulum Stress and Subsequent Transport to the Golgi Apparatus
Cell structure and function, 2010Co-Authors: Yoshimi Sato, Tetsuya Okada, Satomi Nadanaka, Katsuya Okawa, Kazutoshi MoriAbstract:The transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). When unfolded proteins accumulate in the ER, ATF6 senses such ER stress via an as yet undetermined mechanism and relocates to the Golgi apparatus where it is cleaved by sequential action of Site-1 and Site-2 proteases, allowing liberated N-terminal fragments to translocate into the nucleus. This ATF6-mediated transcriptional induction of ER-localized molecular chaperones and folding enzymes together with components of ER-associated degradation leads to the maintenance of ER homeostasis in mammals. Here, we demonstrated that the luminal domain of ATF6 alone is sufficient for sensing ER stress and subsequent transportation to the Golgi apparatus. This domain of ATF6 was inserted between the N-terminal signal sequence and C-terminal tandem affinity purification tag. The resulting ATF6(C)-TAP translocated into the ER, where it was glycosylated and disulfide bonded. ATF6(C)-TAP occurred as monomer and dimer, and exhibited a relatively short half-life, similar to full-length ATF6. On application of dithiothreitol- or thapsigargin-induced ER stress, the ER chaperone BiP dissociated from ATF6(C)-TAP, and ATF6(C)-TAP was transported to the Golgi apparatus and then secreted into medium. Calnexin and protein disulfide isomerase were identified as cellular proteins capable of binding to ATF6(C)-TAP in addition to BiP, and subsequent analysis revealed that protein disulfide isomerase was bound to ATF6(C)-TAP with chaperone activity. These findings indicate that ATF6(C)-TAP can be used as a tool to isolate protein(s) that escort ATF6 from the ER to the Golgi apparatus in response to ER stress.
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pxbp1 u a negative regulator of the unfolded protein response activator pxbp1 s targets ATF6 but not atf4 in proteasome mediated degradation
Cell Structure and Function, 2009Co-Authors: Hiderou Yoshida, Aya Uemura, Kazutoshi MoriAbstract:Cells from yeast to humans activate unconventional mRNA splicing when unfolded proteins accumulate in the endoplasmic reticulum (ER) under ER stress conditions. The substrate of this splicing in mammalian cells is XBP1 mRNA, which encodes the unfolded protein response (UPR)-specific transcription factor XBP1. The C-terminal region of XBP1 is switched as a result of the splicing. Thus, unspliced and spliced mRNAs produce pXBP1(U) of 261 aa and pXBP1(S) of 376 aa, respectively, with the N-terminal region containing the DNA-binding domain shared. As the pXBP1(S)-specific C-terminal region functions as an activation domain, pXBP1(S) can activate transcription efficiently. We recently found that pXBP1(U) shuttles between the nucleus and cytoplasm, owing to the presence of a nuclear exclusion signal in the pXBP1(U)-specific C-terminal region, in marked contrast to the exclusively nuclear localization of pXBP1(S). pXBP1(U) can associate with pXBP1(S), and pXBP1(U)-pXBP1(S) complex is rapidly degraded by the proteasome. Two other transcription factors are activated in response to ER stress, namely ATF6 and ATF4. ATF6 is a UPR-specific transcription factor, whereas ATF4 is activated by not only ER stress but also various other stimuli. In this study, we show that pXBP1(U) targets the active form of ATF6 but not ATF4 for destruction by the proteasome via direct association. This enhanced degradation is mediated by the degradation domain located at the pXBP1(U)-specific C-terminal end. We conclude that pXBP1(U) functions as a negative regulator of the UPR-specific transcription factors ATF6 and pXBP1(S).
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human hrd1 promoter carries a functional unfolded protein response element to which xbp1 but not ATF6 directly binds
Journal of Biochemistry, 2008Co-Authors: Keisuke Yamamoto, Tetsuya Okada, Hiderou Yoshida, Randal J Kaufman, Natsumi Suzuki, Tadashi Wada, Kazutoshi MoriAbstract:Quality control of proteins in the endoplasmic reticulum (ER) is achieved by two mechanisms, the productive folding mechanism, which is assisted by a number of ER-localized molecular chaperones and folding enzymes (collectively termed ER chaperones), and the ER-associated degradation (ERAD) mechanism, by which misfolded proteins are degraded by the ubiquitin-dependent proteasome system in the cytosol. Accumulation of unfolded proteins in the ER activates the unfolded protein response (UPR), resulting in transcriptional induction of ER chaperones and ERAD components. In mammals, three signalling pathways operate for the UPR, namely the IRE1-XBP1, PERK-ATF4 and ATF6 pathways. Analysis of mouse embryonic fibroblasts deficient in UPR signalling molecule indicates that transcriptional induction of ERAD components depends on the IRE1-XBP1 pathway. However, the molecular basis of this finding remains unclear. Here, we analysed the promoter of human HRD1, which encodes an E3 ubiquitin ligase, an important component of ERAD. We found that induction of HRD1 is mediated by two cis-acting elements, a canonical ER stress response element and a novel element we designate as UPR element II. The presence of UPR element II to which XBP1 but not ATF6 directly binds explains at least in part the dependency of HRD1 induction on the IRE1-XBP1 pathway.
Amy S Lee - One of the best experts on this subject based on the ideXlab platform.
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ATF6 modulates srebp2 mediated lipogenesis
The EMBO Journal, 2004Co-Authors: Lingfang Zeng, Amy S Lee, Kazutoshi Mori, Shengzhan Luo, Yi Zhu, John Y.-j. ShyyAbstract:Activating transcription factor 6 (ATF6) and sterol regulatory element‐binding proteins (SREBPs) are activated by proteolytic cleavage. The ensuing nuclear translocation of their N‐termini (i.e., ATF6(N) and SREBP(N)) activates the respective target genes involved in unfolded protein response and lipogenesis. Here, we report that glucose deprivation activated ATF6 but suppressed the SREBP2‐regulated transcription. Overexpression of ATF6(N) had similar inhibitory effects on SREBP2‐targeted genes. The blockade of ATF6 cleavage by BiP/grp78 reversed this inhibitory effect. GST pull‐down and immunoprecipitation assays revealed that ATF6(N) bound to SREBP2(N). Deletion analysis of the various functional domains of ATF6 indicated that the interaction was through its leucine‐zipper domain. Chromatin immunoprecipitation assays revealed that ATF6(N) formed a complex with the SRE‐bound SREBP2(N). The attenuated transcriptional activity of SREBP2 was due, in part, to the recruitment of HDAC1 to the ATF6–SREBP2 complex. As a functional consequence, the lipogenic effect of SREBP2(N) in liver cells was suppressed by ATF6(N). Our results provide a novel mechanism by which ATF6 antagonizes SREBP2 to regulate the homeostasis of lipid and glucose.
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ATF6 modulates SREBP2‐mediated lipogenesis
The EMBO journal, 2004Co-Authors: Lingfang Zeng, Amy S Lee, Kazutoshi Mori, Shengzhan Luo, Yi Zhu, John Y.-j. ShyyAbstract:Activating transcription factor 6 (ATF6) and sterol regulatory element‐binding proteins (SREBPs) are activated by proteolytic cleavage. The ensuing nuclear translocation of their N‐termini (i.e., ATF6(N) and SREBP(N)) activates the respective target genes involved in unfolded protein response and lipogenesis. Here, we report that glucose deprivation activated ATF6 but suppressed the SREBP2‐regulated transcription. Overexpression of ATF6(N) had similar inhibitory effects on SREBP2‐targeted genes. The blockade of ATF6 cleavage by BiP/grp78 reversed this inhibitory effect. GST pull‐down and immunoprecipitation assays revealed that ATF6(N) bound to SREBP2(N). Deletion analysis of the various functional domains of ATF6 indicated that the interaction was through its leucine‐zipper domain. Chromatin immunoprecipitation assays revealed that ATF6(N) formed a complex with the SRE‐bound SREBP2(N). The attenuated transcriptional activity of SREBP2 was due, in part, to the recruitment of HDAC1 to the ATF6–SREBP2 complex. As a functional consequence, the lipogenic effect of SREBP2(N) in liver cells was suppressed by ATF6(N). Our results provide a novel mechanism by which ATF6 antagonizes SREBP2 to regulate the homeostasis of lipid and glucose.
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induction of grp78 bip by translational block activation of the grp78 promoter by atf4 through an upstream atf cre site independent of the endoplasmic reticulum stress elements
Journal of Biological Chemistry, 2003Co-Authors: Shengzha Luo, Pete Aumeiste, Shujie Yang, Steve F Abcouwe, Amy S LeeAbstract:Abstract Mammalian cells respond to endoplasmic reticulum (ER) stress by attenuation of protein translation mediated through the PERK-eIF2α pathway and transcriptional activation of genes such as Grp78/BiP encoding ER chaperone proteins. The disruption of PERK function or the blocking of eIF2α Ser51 phosphorylation fails to attenuate translation after ER stress and also results in substantial impairment of Grp78/BiP induction by ER stress. While the activation of the Grp78 promoter by the ATF6 pathway through the endoplasmic reticulum stress elements (ERSEs) is well documented, the molecular mechanism linking PERK activation to Grp78 stress induction is unknown. We report here that ATF4, a transcription factor whose translation is up-regulated by the PERK-eIF2α pathway, can activate the Grp78 promoter independent of the ERSE. The ATF4-activating site is localized to an ATF/CRE sequence upstream of the ERSEs and is distinct from the C/EBP-ATF composite site previously identified as the ATF4 binding site in the ER stress-inducible chop promoter. In vitro translated ATF4 binding to the ATF/CRE site requires other nuclear co-factors from non-stressed cells, forming a complex that exhibits identical electrophoretic mobility as a thapsigargin-stress induced complex. Here we have identified the closely related ATF1 and CREB1 as nuclear co-factors that form in vivo complexes with endogenous ATF4. ER stress induces CREB1 phosphorylation and ATF1/CREB1 binding to the Grp78 promoter. Through the use of adenoviral vector expression systems, we provide evidence that when ATF4 function is suppressed and its binding partners are not able to compensate for its function, Grp78 induction by Tg and Tu is partially inhibited. Our studies resolve a mechanism responsible for inhibition of Grp78 mRNA induction by ER stress in cells that are functionally null for PERK or devoid of eIF2α phosphorylation.
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ATF6 as a transcription activator of the endoplasmic reticulum stress element thapsigargin stress induced changes and synergistic interactions with nf y and yy1
Molecular and Cellular Biology, 2000Co-Authors: Peter Baumeister, Shengzhan Luo, Binayak Roy, Trevor Phan, Dolly M Foti, Amy S LeeAbstract:ATF6, a member of the leucine zipper protein family, can constitutively induce the promoter of glucose-regulated protein (grp) genes through activation of the endoplasmic reticulum (ER) stress element (ERSE). To understand the mechanism of grp78 induction by ATF6 in cells subjected to ER calcium depletion stress mediated by thapsigargin (Tg) treatment, we discovered that ATF6 itself undergoes Tg stress-induced changes. In nonstressed cells, ATF6, which contains a putative short transmembrane domain, is primarily associated with the perinuclear region. Upon Tg stress, the ATF6 protein level dropped initially but quickly recovered with the additional appearance of a faster-migrating form. This new form of ATF6 was recovered as soluble nuclear protein by biochemical fractionation, correlating with enhanced nuclear localization of ATF6 as revealed by immunofluorescence. Optimal ATF6 stimulation requires at least two copies of the ERSE and the integrity of the tripartite structure of the ERSE. Of primary importance is a functional NF-Y complex and a high-affinity NF-Y binding site that confers selectivity among different ERSEs for ATF6 inducibility. In addition, we showed that YY1 interacts with ATF6 and in Tg-treated cells can enhance ATF6 activity. The ERSE stimulatory activity of ATF6 exhibits properties distinct from those of human Ire1p, an upstream regulator of the mammalian unfolded protein response. The requirement for a high-affinity NF-Y site for ATF6 but not human Ire1p activity suggests that they stimulate the ERSE through diverse pathways.
Hiderou Yoshida - One of the best experts on this subject based on the ideXlab platform.
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pxbp1 u a negative regulator of the unfolded protein response activator pxbp1 s targets ATF6 but not atf4 in proteasome mediated degradation
Cell Structure and Function, 2009Co-Authors: Hiderou Yoshida, Aya Uemura, Kazutoshi MoriAbstract:Cells from yeast to humans activate unconventional mRNA splicing when unfolded proteins accumulate in the endoplasmic reticulum (ER) under ER stress conditions. The substrate of this splicing in mammalian cells is XBP1 mRNA, which encodes the unfolded protein response (UPR)-specific transcription factor XBP1. The C-terminal region of XBP1 is switched as a result of the splicing. Thus, unspliced and spliced mRNAs produce pXBP1(U) of 261 aa and pXBP1(S) of 376 aa, respectively, with the N-terminal region containing the DNA-binding domain shared. As the pXBP1(S)-specific C-terminal region functions as an activation domain, pXBP1(S) can activate transcription efficiently. We recently found that pXBP1(U) shuttles between the nucleus and cytoplasm, owing to the presence of a nuclear exclusion signal in the pXBP1(U)-specific C-terminal region, in marked contrast to the exclusively nuclear localization of pXBP1(S). pXBP1(U) can associate with pXBP1(S), and pXBP1(U)-pXBP1(S) complex is rapidly degraded by the proteasome. Two other transcription factors are activated in response to ER stress, namely ATF6 and ATF4. ATF6 is a UPR-specific transcription factor, whereas ATF4 is activated by not only ER stress but also various other stimuli. In this study, we show that pXBP1(U) targets the active form of ATF6 but not ATF4 for destruction by the proteasome via direct association. This enhanced degradation is mediated by the degradation domain located at the pXBP1(U)-specific C-terminal end. We conclude that pXBP1(U) functions as a negative regulator of the UPR-specific transcription factors ATF6 and pXBP1(S).
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human hrd1 promoter carries a functional unfolded protein response element to which xbp1 but not ATF6 directly binds
Journal of Biochemistry, 2008Co-Authors: Keisuke Yamamoto, Tetsuya Okada, Hiderou Yoshida, Randal J Kaufman, Natsumi Suzuki, Tadashi Wada, Kazutoshi MoriAbstract:Quality control of proteins in the endoplasmic reticulum (ER) is achieved by two mechanisms, the productive folding mechanism, which is assisted by a number of ER-localized molecular chaperones and folding enzymes (collectively termed ER chaperones), and the ER-associated degradation (ERAD) mechanism, by which misfolded proteins are degraded by the ubiquitin-dependent proteasome system in the cytosol. Accumulation of unfolded proteins in the ER activates the unfolded protein response (UPR), resulting in transcriptional induction of ER chaperones and ERAD components. In mammals, three signalling pathways operate for the UPR, namely the IRE1-XBP1, PERK-ATF4 and ATF6 pathways. Analysis of mouse embryonic fibroblasts deficient in UPR signalling molecule indicates that transcriptional induction of ERAD components depends on the IRE1-XBP1 pathway. However, the molecular basis of this finding remains unclear. Here, we analysed the promoter of human HRD1, which encodes an E3 ubiquitin ligase, an important component of ERAD. We found that induction of HRD1 is mediated by two cis-acting elements, a canonical ER stress response element and a novel element we designate as UPR element II. The presence of UPR element II to which XBP1 but not ATF6 directly binds explains at least in part the dependency of HRD1 induction on the IRE1-XBP1 pathway.
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Reduction of disulfide bridges in the lumenal domain of ATF6 in response to glucose starvation.
Cell structure and function, 2006Co-Authors: Satomi Nadanaka, Hiderou Yoshida, Kazutoshi MoriAbstract:Mammalian transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). Upon ER stress ATF6 is transported to the Golgi apparatus where it is cleaved to release its cytoplasmic domain. This is then translocated into the nucleus where it activates transcription of ER-localized molecular chaperones and folding enzymes to maintain the homeostasis of the ER. We recently found that, owing to the presence of intra- and intermolecular disulfide bridges, ATF6 occurs in unstressed ER in monomer, dimer and oligomer forms. Disulfide-bonded ATF6 is reduced on treatment of cells with various chemical ER stress inducers, and only the reduced monomer ATF6 reaches the Golgi apparatus. In this study, we evoked ER stress under more physiological conditions, namely, glucose starvation, and analyzed its consequence for ATF6 activation. Glucose starvation activated ATF6 and induced the ER chaperone BiP, albeit weakly. ATF6 was thus dissociated from BiP, transported to the Golgi apparatus, and cleaved. Glucose starvation enhanced the synthesis of ATF6 approximately two-fold, probably via transcriptional induction. Importantly, reduction of disulfide bridges and transport of reduced monomer occurred in response to glucose starvation. We conclude that ER stress-induced reduction of ATF6 represents a general feature of the ATF6 activation process.
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a serine protease inhibitor prevents endoplasmic reticulum stress induced cleavage but not transport of the membrane bound transcription factor ATF6
Journal of Biological Chemistry, 2003Co-Authors: Tetsuya Okada, Ryuichiro Sato, Kyosuke Haze, Satomi Nadanaka, Hiderou Yoshida, Nabil G Seidah, Yuko Hirano, Manabu Negishi, Kazutoshi MoriAbstract:Abstract Mammalian cells express several transcription factors embedded in the endoplasmic reticulum (ER) as transmembrane proteins that are activated by proteolysis, and two types of these proteins have been extensively investigated. One type comprises the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). The other type comprises the activating transcription factors 6 (ATF6α and ATF6β), which are activated in response to ER stress. It was shown previously that both SREBP and ATF6 are cleaved sequentially first by the Site-1 protease (serine protease) and then by the Site-2 protease (metalloprotease) (Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355–1364). In this study, we examined various protease inhibitors and found that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, prevented ER stress-induced cleavage of ATF6α and ATF6β, resulting in inhibition of transcriptional induction of ATF6-target genes. AEBSF also inhibited production of the mature form of SREBP-2 that was induced in response to sterol depletion, and appeared to directly prevent cleavage of ATF6α and ATF6β by inhibiting Site-1 protease. As the Site-1 protease is localized in the Golgi apparatus, both SREBP and ATF6 must relocate to the Golgi apparatus to be cleaved. We showed here that AEBSF treatment had little effect on ER stress-induced translocation of ATF6 from the ER to the Golgi apparatus, but blocked nuclear localization of ATF6. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF.
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identification of the g13 camp response element binding protein related protein gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response
Biochemical Journal, 2001Co-Authors: Kyosuke Haze, Tetsuya Okada, Hiderou Yoshida, Manabu Negishi, Hideki Yanagi, Takashi Yura, Kazutoshi MoriAbstract:Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis -acting ER stress response element consisting of 19nt (CCAATN 9 CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus; their basic regions seem to function as a nuclear localization signal. Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis -acting ER stress response element in vitro that hardly respond to ER stress in vivo . We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6α and the G13 gene product ATF6β.
Tetsuya Okada - One of the best experts on this subject based on the ideXlab platform.
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reinvestigation of disulfide bonded oligomeric forms of the unfolded protein response transducer ATF6
Cell Structure and Function, 2020Co-Authors: Hibiki Koba, Tetsuya Okada, Tokiro Ishikawa, Satoshi Ninagawa, Shengyu Jin, Nanami Imada, Tetsushi Sakuma, Takashi Yamamoto, Kazutoshi MoriAbstract: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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development of a rapid in vivo assay to evaluate the efficacy of ire1 specific inhibitors of the unfolded protein response using medaka fish
Cell Structure and Function, 2019Co-Authors: Byungseok Jin, Tetsuya Okada, Tokiro Ishikawa, Mai Taniguchi, Satoshi Ninagawa, Shigehide Kagaya, Kazutoshi MoriAbstract: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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Luminal Domain of ATF6 Alone Is Sufficient for Sensing Endoplasmic Reticulum Stress and Subsequent Transport to the Golgi Apparatus
Cell structure and function, 2010Co-Authors: Yoshimi Sato, Tetsuya Okada, Satomi Nadanaka, Katsuya Okawa, Kazutoshi MoriAbstract:The transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). When unfolded proteins accumulate in the ER, ATF6 senses such ER stress via an as yet undetermined mechanism and relocates to the Golgi apparatus where it is cleaved by sequential action of Site-1 and Site-2 proteases, allowing liberated N-terminal fragments to translocate into the nucleus. This ATF6-mediated transcriptional induction of ER-localized molecular chaperones and folding enzymes together with components of ER-associated degradation leads to the maintenance of ER homeostasis in mammals. Here, we demonstrated that the luminal domain of ATF6 alone is sufficient for sensing ER stress and subsequent transportation to the Golgi apparatus. This domain of ATF6 was inserted between the N-terminal signal sequence and C-terminal tandem affinity purification tag. The resulting ATF6(C)-TAP translocated into the ER, where it was glycosylated and disulfide bonded. ATF6(C)-TAP occurred as monomer and dimer, and exhibited a relatively short half-life, similar to full-length ATF6. On application of dithiothreitol- or thapsigargin-induced ER stress, the ER chaperone BiP dissociated from ATF6(C)-TAP, and ATF6(C)-TAP was transported to the Golgi apparatus and then secreted into medium. Calnexin and protein disulfide isomerase were identified as cellular proteins capable of binding to ATF6(C)-TAP in addition to BiP, and subsequent analysis revealed that protein disulfide isomerase was bound to ATF6(C)-TAP with chaperone activity. These findings indicate that ATF6(C)-TAP can be used as a tool to isolate protein(s) that escort ATF6 from the ER to the Golgi apparatus in response to ER stress.
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human hrd1 promoter carries a functional unfolded protein response element to which xbp1 but not ATF6 directly binds
Journal of Biochemistry, 2008Co-Authors: Keisuke Yamamoto, Tetsuya Okada, Hiderou Yoshida, Randal J Kaufman, Natsumi Suzuki, Tadashi Wada, Kazutoshi MoriAbstract:Quality control of proteins in the endoplasmic reticulum (ER) is achieved by two mechanisms, the productive folding mechanism, which is assisted by a number of ER-localized molecular chaperones and folding enzymes (collectively termed ER chaperones), and the ER-associated degradation (ERAD) mechanism, by which misfolded proteins are degraded by the ubiquitin-dependent proteasome system in the cytosol. Accumulation of unfolded proteins in the ER activates the unfolded protein response (UPR), resulting in transcriptional induction of ER chaperones and ERAD components. In mammals, three signalling pathways operate for the UPR, namely the IRE1-XBP1, PERK-ATF4 and ATF6 pathways. Analysis of mouse embryonic fibroblasts deficient in UPR signalling molecule indicates that transcriptional induction of ERAD components depends on the IRE1-XBP1 pathway. However, the molecular basis of this finding remains unclear. Here, we analysed the promoter of human HRD1, which encodes an E3 ubiquitin ligase, an important component of ERAD. We found that induction of HRD1 is mediated by two cis-acting elements, a canonical ER stress response element and a novel element we designate as UPR element II. The presence of UPR element II to which XBP1 but not ATF6 directly binds explains at least in part the dependency of HRD1 induction on the IRE1-XBP1 pathway.
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a serine protease inhibitor prevents endoplasmic reticulum stress induced cleavage but not transport of the membrane bound transcription factor ATF6
Journal of Biological Chemistry, 2003Co-Authors: Tetsuya Okada, Ryuichiro Sato, Kyosuke Haze, Satomi Nadanaka, Hiderou Yoshida, Nabil G Seidah, Yuko Hirano, Manabu Negishi, Kazutoshi MoriAbstract:Abstract Mammalian cells express several transcription factors embedded in the endoplasmic reticulum (ER) as transmembrane proteins that are activated by proteolysis, and two types of these proteins have been extensively investigated. One type comprises the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). The other type comprises the activating transcription factors 6 (ATF6α and ATF6β), which are activated in response to ER stress. It was shown previously that both SREBP and ATF6 are cleaved sequentially first by the Site-1 protease (serine protease) and then by the Site-2 protease (metalloprotease) (Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355–1364). In this study, we examined various protease inhibitors and found that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, prevented ER stress-induced cleavage of ATF6α and ATF6β, resulting in inhibition of transcriptional induction of ATF6-target genes. AEBSF also inhibited production of the mature form of SREBP-2 that was induced in response to sterol depletion, and appeared to directly prevent cleavage of ATF6α and ATF6β by inhibiting Site-1 protease. As the Site-1 protease is localized in the Golgi apparatus, both SREBP and ATF6 must relocate to the Golgi apparatus to be cleaved. We showed here that AEBSF treatment had little effect on ER stress-induced translocation of ATF6 from the ER to the Golgi apparatus, but blocked nuclear localization of ATF6. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF.
Michael S Kilberg - One of the best experts on this subject based on the ideXlab platform.
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human chac1 protein degrades glutathione and mrna induction is regulated by the transcription factors atf4 and atf3 and a bipartite atf cre regulatory element
Journal of Biological Chemistry, 2015Co-Authors: Rebecca R Crawford, Jixiu Shan, Michael S Kilberg, Eugenia T Prescott, Charity F Sylvester, Ashlee N Higdon, Imran N MungrueAbstract:Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the -267 ATF/cAMP response element (CRE) site and a novel -248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein β binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease.
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Auto-activation of c-JUN Gene by Amino Acid Deprivation of Hepatocellular Carcinoma Cells Reveals a Novel c-JUN-mediated Signaling Pathway
Journal of Biological Chemistry, 2011Co-Authors: Lingchen Fu, Mukundh N. Balasubramanian, Elizabeth E. Dudenhausen, Jixiu Shan, Michael S KilbergAbstract:Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.
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transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and ire1 xbp1 arms of the pathway
Biochemical Journal, 2009Co-Authors: Altin Gjymishka, Michael S KilbergAbstract:The UPR (unfolded protein response) pathway comprises three signalling cascades mediated by the ER (endoplasmic reticulum) stress-sensor proteins PERK [PKR (double-stranded RNA-activated protein kinase)-like ER kinase], IRE1 (inositol-requiring kinase 1) and ATF6 (activating transcription factor 6). The present study shows that ASNS (asparagine synthetase) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. ChIP (chromatin immunoprecipitation) analysis demonstrated that during ER stress, ATF4, ATF3 and C/EBPbeta (CCAAT/enhancer-binding protein beta) bind to the ASNS proximal promoter region that includes the genomic sequences NSRE (nutrient-sensing response element)-1 and NSRE-2, previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signalling resulted in enhanced recruitment of general transcription factors, including RNA Pol II (polymerase II), to the ASNS promoter. The ASNS gene is also activated by the AAR (amino acid response) pathway following amino acid deprivation of tissue or cells. Immunoblot analysis of HepG2 cells demonstrated that simultaneous activation of the AAR and UPR pathways did not further increase the ASNS or ATF4 protein abundance when compared with triggering either pathway alone. In addition, siRNA (small interfering RNA)-mediated knockdown of XBP1 (X-box-binding protein 1), ATF6alpha or ATF6beta expression did not affect ASNS transcription, whereas siRNA against ATF4 suppressed ASNS transcription during UPR activation. Collectively, these results indicate that the PERK/p-eIF2alpha (phosphorylated eukaryotic initiation factor 2alpha)/ATF4 signalling cascade is the only arm of the UPR that is responsible for ASNS transcriptional induction during ER stress. Consequently, ASNS NSRE-1 and NSRE-2, in addition to ERSE (ER stress response element)-I, ERSE-II and the mUPRE (mammalian UPR element), function as mammalian ER-stress-responsive sequences.
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activation of the atf3 gene through a co ordinated amino acid sensing response programme that controls transcriptional regulation of responsive genes following amino acid limitation
Biochemical Journal, 2007Co-Authors: Yuan Xiang Pan, Hong Chen, Michelle M Thiaville, Michael S KilbergAbstract:Expression of ATF3 (activating transcription factor 3) is induced by a variety of environmental stress conditions, including nutrient limitation. In the present study, we demonstrate that the increase in ATF3 mRNA content following amino acid limitation of human HepG2 hepatoma cells is dependent on transcriptional activation of the ATF3 gene, through a highly co-ordinated amino acid-responsive programme of transcription factor synthesis and action. Studies using transient over-expression and knockout fibroblasts showed that several ATF and C/EBP (CCAAT/enhancer-binding protein) family members contribute to ATF3 regulation. Promoter analysis showed that a C/EBP-ATF composite site at -23 to -15 bp relative to the transcription start site of the ATF3 gene functions as an AARE (amino acid response element). Chromatin immunoprecipitation demonstrated that amino acid limitation increased ATF4, ATF3, and C/EBPbeta binding to the ATF3 promoter, but the kinetics of each was markedly different. Immediately following histidine removal, there was a rapid increase in histone H3 acetylation prior to an enhancement in ATF4 binding and in histone H4 acetylation. These latter changes closely paralleled the initial increase in RNA pol II (RNA polymerase II) binding to the promoter and in the transcription rate from the ATF3 gene. The increase in ATF3 and C/EBPbeta binding was considerably slower and more closely correlated with a decline in transcription rate. A comparison of the recruitment patterns between ATF and C/EBP transcription factors and RNA polymerase II at the AARE of several amino acid-responsive genes revealed that a highly co-ordinated response programme controls the transcriptional activation of these genes following amino acid limitation.