Proteomic analysis of fatty liver induced by starvation of medaka fish larvae.

When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or β-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions.

Activation of XBP1 but not ATF6α rescues heart failure induced by persistent ER stress in medaka fish.

The unfolded protein response is triggered in vertebrates by ubiquitously expressed IRE1α/β (although IRE1β is gut-specific in mice), PERK, and ATF6α/β, transmembrane-type sensor proteins in the ER, to cope with ER stress, the accumulation of unfolded and misfolded proteins in the ER. Here, we burdened medaka fish, a vertebrate model organism, with ER stress persistently from fertilization by knocking out the gene encoding an ATP/ADP exchanger in the ER membrane, leading to decreased ATP concentration-mediated impairment of the activity of Hsp70- and Hsp90-type molecular chaperones in the ER lumen. ER stress and apoptosis were evoked from 4 and 6 dpf, respectively, leading to the death of all -KO medaka by 12 dpf because of heart failure (medaka hatch at 7 dpf).

Loss of ATF6α in a human carcinoma cell line is compensated not by its paralogue ATF6β but by sustained activation of the IRE1 and PERK arms for tumor growth in nude mice.

To survive poor nutritional conditions, tumor cells activate the unfolded protein response, which is composed of the IRE1, PERK, and ATF6 arms, to maintain the homeostasis of the endoplasmic reticulum, where secretory and transmembrane proteins destined for the secretory pathway gain their correct three-dimensional structure. The requirement of the IRE1 and PERK arms for tumor growth in nude mice is established. Here we investigated the requirement for the ATF6 arm, which consists of ubiquitously expressed ATF6α and ATF6β, by constructing ATF6α-knockout (KO), ATF6β-KO, and ATF6α/β-double KO (DKO) in HCT116 cells derived from human colorectal carcinoma.

A motor neuron disease-associated mutation produces non-glycosylated Seipin that induces ER stress and apoptosis by inactivating SERCA2b.

A causal relationship between endoplasmic reticulum (ER) stress and the development of neurodegenerative diseases remains controversial. Here, we focused on Seipinopathy, a dominant motor neuron disease, based on the finding that its causal gene product, Seipin, is a protein that spans the ER membrane twice. Gain-of-function mutations of Seipin produce non-glycosylated Seipin (ngSeipin), which was previously shown to induce ER stress and apoptosis at both cell and mouse levels albeit with no clarified mechanism.

Drop-Dry Deposition of Ni(OH) Precursors for Fabrication of NiO Thin Films.

Drop-dry deposition (DDD) is a method of depositing thin films by heating and drying the deposition solution dropped on a substrate. We prepared Ni(OH) precursor thin films by DDD and annealed them in air to prepare NiO thin films. The appropriate deposition conditions were found by changing the number of drop-dry cycles and the concentrations of chemicals in the solution, and the Ni(OH) precursor film with a thickness of 0.

Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation.

Many positive-stranded RNA viruses encode polyproteins from which viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though the required amounts for each protein are not the same. In this study, we found the extra membrane-anchored nonstructural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway.

Allosteric regulation accompanied by oligomeric state changes of Trypanosoma brucei GMP reductase through cystathionine-β-synthase domain.

Guanosine 5'-monophosphate reductase (GMPR) is involved in the purine salvage pathway and is conserved throughout evolution. Nonetheless, the GMPR of Trypanosoma brucei (TbGMPR) includes a unique structure known as the cystathionine-β-synthase (CBS) domain, though the role of this domain is not fully understood. Here, we show that guanine and adenine nucleotides exert positive and negative effects, respectively, on TbGMPR activity by binding allosterically to the CBS domain.

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

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.

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

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.

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

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.

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

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).

Correction: 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.

[This corrects the article DOI: 10.1371/journal.pone.

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.

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.

Novel Characteristics of Trypanosoma brucei Guanosine 5'-monophosphate Reductase Distinct from Host Animals.

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5'-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5'-monophosphate (GMP) to inosine 5'-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present.

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

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.

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

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.

Lipocalin-type prostaglandin D synthase scavenges biliverdin in the cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the second major protein in human cerebrospinal fluid (CSF) and belongs to the lipocalin superfamily composed of various secretory lipophilic ligand transporter proteins. However, the endogenous ligand of L-PGDS has not yet been elucidated. In this study, we purified L-PGDS from the CSF of aneurysmal subarachnoid hemorrhage (SAH) patients.

The unfolded protein response transducer ATF6 represents a novel transmembrane-type endoplasmic reticulum-associated degradation substrate requiring both mannose trimming and SEL1L protein.

Proteins misfolded in the endoplasmic reticulum (ER) are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed ER-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein that serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells.

The antipsychotic olanzapine induces apoptosis in insulin-secreting pancreatic β cells by blocking PERK-mediated translational attenuation.

Patients with schizophrenia receive medication to alleviate various symptoms, but some efficacious second generation antipsychotics, particularly olanzapine, can cause obesity, dyslipidemia, and diabetes mellitus. It has been generally considered that olanzapine contributes to the development of diabetes by inducing obesity and subsequent insulin resistance. In this study, we examined the effect of olanzapine and risperidone, another second generation antipsychotic, on a hamster pancreatic β cell line, and found that both evoked mild endoplasmic reticulum (ER) stress, as evidenced by mild activation of the ER stress sensor molecule PERK.

ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish.

ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice.

An IFN-γ-stimulated ATF6-C/EBP-β-signaling pathway critical for the expression of Death Associated Protein Kinase 1 and induction of autophagy.

The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The death-associated protein kinase 1 (DAPK1), an IFN-γ-induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis, and its expression is frequently down-regulated in a number of human tumors. Although the biochemical action of DAPK1 is well understood, mechanisms that regulate its expression are unclear.