The sarcoplasmic reticulum luminal thiol oxidase ERO1 regulates cardiomyocyte excitation-coupled calcium release and response to hemodynamic load.

Two related ER oxidation 1 (ERO1) proteins, ERO1α and ERO1ß, dynamically regulate the redox environment in the mammalian endoplasmic reticulum (ER). Redox changes in cysteine residues on intralumenal loops of calcium release and reuptake channels have been implicated in altered calcium release and reuptake. These findings led us to hypothesize that altered ERO1 activity may affect cardiac functions that are dependent on intracellular calcium flux. We established mouse lines with loss of function insertion mutations in Ero1l and Ero1lb encoding ERO1α and ERO1ß. The peak amplitude of calcium transients in homozygous Ero1α mutant adult cardiomyocytes was reduced to 42.0 ± 2.2% (n=10, P ≤ 0.01) of values recorded in wild-type cardiomyocytes. Decreased ERO1 activity blunted cardiomyocyte inotropic response to adrenergic stimulation and sensitized mice to adrenergic blockade. Whereas all 12 wild-type mice survived challenge with 4 mg/kg esmolol, 6 of 8 compound Ero1l and Ero1lb mutant mice succumbed to this level of ß adrenergic blockade (P ≤ 0.01). In addition, mice lacking ERO1α were partially protected against progressive heart failure in a transaortic constriction model [at 10 wk postprocedure, fractional shortening was 0.31 ± 0.02 in the mutant (n=20) vs. 0.23 ± 0.03 in the wild type (n=18); P ≤ 0.01]. These findings establish a role for ERO1 in calcium homeostasis and suggest that modifying the lumenal redox environment may affect the progression of heart failure.

Disulphide production by Ero1α-PDI relay is rapid and effectively regulated.

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cells are incompletely understood. Here, we show that after reductive challenge the ER steady-state disulphide content is restored on a time scale of seconds. Both the oxidase Ero1α and the oxidoreductase protein disulphide isomerase (PDI) strongly contribute to the rapid recovery kinetics, but experiments in ERO1-deficient cells indicate the existence of parallel pathways for disulphide generation. We find PDI to be the main substrate of Ero1α, and mixed-disulphide complexes of Ero1 primarily form with PDI, to a lesser extent with the PDI-family members ERp57 and ERp72, but are not detectable with another homologue TMX3. We also show for the first time that the oxidation level of PDIs and glutathione is precisely regulated. Apparently, this is achieved neither through ER import of thiols nor by transport of disulphides to the Golgi apparatus. Instead, our data suggest that a dynamic equilibrium between Ero1- and glutathione disulphide-mediated oxidation of PDIs constitutes an important element of ER redox homeostasis.

A small molecule inhibitor of endoplasmic reticulum oxidation 1 (ERO1) with selectively reversible thiol reactivity.

Endoplasmic reticulum oxidation 1 (ERO1) is a conserved eukaryotic flavin adenine nucleotide-containing enzyme that promotes disulfide bond formation by accepting electrons from reduced protein disulfide isomerase (PDI) and passing them on to molecular oxygen. Although disulfide bond formation is an essential process, recent experiments suggest a surprisingly broad tolerance to genetic manipulations that attenuate the rate of disulfide bond formation and that a hyperoxidizing ER may place stressed cells at a disadvantage. In this study, we report on the development of a high throughput in vitro assay for mammalian ERO1alpha activity and its application to identify small molecule inhibitors. The inhibitor EN460 (IC(50), 1.9 mum) interacts selectively with the reduced, active form of ERO1alpha and prevents its reoxidation. Despite rapid and promiscuous reactivity with thiolates, EN460 exhibits selectivity for ERO1. This selectivity is explained by the rapid reversibility of the reaction of EN460 with unstructured thiols, in contrast to the formation of a stable bond with ERO1alpha followed by displacement of bound flavin adenine dinucleotide from the active site of the enzyme. Modest concentrations of EN460 and a functionally related inhibitor, QM295, promote signaling in the unfolded protein response and precondition cells against severe ER stress. Together, these observations point to the feasibility of targeting the enzymatic activity of ERO1alpha with small molecule inhibitors.

N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H.

CREB-H is a liver-enriched bZIP transcription factor of the CREB3 subfamily. CREB-H is activated by intramembrane proteolysis that removes a C-terminal transmembrane domain. Aberrant expression of CREB-H is implicated in liver cancer. In this study we characterized N-linked glycosylation of CREB-H in the luminal domain at the C-terminus. We found that CREB-H is modified at three N-linked glycosylation sites in this region. Disruption of all three sites by site-directed mutagenesis completely abrogated N-linked glycosylation of CREB-H. The unglycosylated mutant of CREB-H was not unstable, unfolded or aggregated. Upon stimulation with an activator of intramembrane proteolysis such as brefeldin A and KDEL-tailed site 1 protease, unglycosylated or deglycosylated CREB-H was largely uncleaved, retained in an inactive form in the endoplasmic reticulum, and less capable of activating transcription driven by unfolded protein response element or C-reactive protein promoter. Taken together, our findings suggest that N-linked glycosylation is required for full activation of CREB-H through intramembrane proteolysis. Our work also reveals a novel mechanism for the regulation of CREB-H-dependent transcription.

ERO1-beta, a pancreas-specific disulfide oxidase, promotes insulin biogenesis and glucose homeostasis.

Mammals have two genes encoding homologues of the endoplasmic reticulum (ER) disulfide oxidase ERO1 (ER oxidoreductin 1). ERO1-beta is greatly enriched in the endocrine pancreas. We report in this study that homozygosity for a disrupting allele of Ero1lb selectively compromises oxidative folding of proinsulin and promotes glucose intolerance in mutant mice. Surprisingly, concomitant disruption of Ero1l, encoding the other ERO1 isoform, ERO1-alpha, does not exacerbate the ERO1-beta deficiency phenotype. Although immunoglobulin-producing cells normally express both isoforms of ERO1, disulfide bond formation and immunoglobulin secretion proceed at nearly normal pace in the double mutant. Moreover, although the more reducing environment of their ER protects cultured ERO1-beta knockdown Min6 cells from the toxicity of a misfolding-prone mutant Ins2(Akita), the diabetic phenotype and islet destruction promoted by Ins2(Akita) are enhanced in ERO1-beta compound mutant mice. These findings point to an unexpectedly selective function for ERO1-beta in oxidative protein folding in insulin-producing cells that is required for glucose homeostasis in vivo.

Role of ERO1-alpha-mediated stimulation of inositol 1,4,5-triphosphate receptor activity in endoplasmic reticulum stress-induced apoptosis.

Endoplasmic reticulum (ER) stress-induced apoptosis is involved in many diseases, but the mechanisms linking ER stress to apoptosis are incompletely understood. Based on roles for C/EPB homologous protein (CHOP) and ER calcium release in apoptosis, we hypothesized that apoptosis involves the activation of inositol 1,4,5-triphosphate (IP3) receptor (IP3R) via CHOP-induced ERO1-alpha (ER oxidase 1 alpha). In ER-stressed cells, ERO1-alpha is induced by CHOP, and small interfering RNA (siRNA) knockdown of ERO1-alpha suppresses apoptosis. IP3-induced calcium release (IICR) is increased during ER stress, and this response is blocked by siRNA-mediated silencing of ERO1-alpha or IP3R1 and by loss-of-function mutations in Ero1a or Chop. Reconstitution of ERO1-alpha in Chop(-/-) macrophages restores ER stress-induced IICR and apoptosis. In vivo, macrophages from wild-type mice but not Chop(-/-) mice have elevated IICR when the animals are challenged with the ER stressor tunicamycin. Macrophages from insulin-resistant ob/ob mice, another model of ER stress, also have elevated IICR. These data shed new light on how the CHOP pathway of apoptosis triggers calcium-dependent apoptosis through an ERO1-alpha-IP3R pathway.

Thioredoxin-related Protein 32 is an arsenite-regulated Thiol Reductase of the proteasome 19 S particle.

Perturbation of the cytoplasmic protein folding environment by exposure to oxidative stress-inducing As(III)-containing compounds challenges the ubiquitin-proteasome system. Here we report on mass spectrometric analysis of As(III)-induced changes in the proteasome's composition in samples prepared by stable isotope labeling with amino acids in cell culture, using mammalian cells in which TRP32 (thioredoxin-related protein of 32 kDa; also referred to as TXNL1) was identified as a novel subunit of the 26 S proteasome. Quantitative genetic interaction mapping, using the epistatic miniarray profiling approach, identified a functional connection between TRP32 and the proteasome. Deletion of txl1, the Schizosaccharomyces pombe homolog of TRP32, results in a slow growth phenotype when combined with deletion of cut8, a gene required for normal proteasome localization. Deletion analysis in vivo, chemical cross-linking, and manipulation of the ATP concentration in vitro during proteasome immunopurification revealed that the C-terminal domain of mammalian TRP32 binds the 19 S regulatory particle in proximity to the proteasome substrate binding site. Thiol modification with polyethylene glycol-maleimide showed disulfide bond formation at the active site of TRP32 in cells exposed to As(III). Pulse-chase labeling showed that TRP32 is a stable protein whose half-life of >6 h is surprisingly reduced to 1 h upon exposure of cells to As(III). These findings reveal a previously undescribed thiol reductase at the proteasome's regulatory particle.

Human T-cell leukemia virus oncoprotein tax represses nuclear receptor-dependent transcription by targeting coactivator TAX1BP1.

Human T-cell leukemia virus type 1 oncoprotein Tax is a transcriptional regulator that interacts with a large number of host cell factors. Here, we report the novel characterization of the interaction of Tax with a human cell protein named Tax1-binding protein 1 (TAX1BP1). We show that TAX1BP1 is a nuclear receptor coactivator that forms a complex with the glucocorticoid receptor. TAX1BP1 and Tax colocalize into intranuclear speckles that partially overlap with but are not identical to the PML oncogenic domains. Tax binds TAX1BP1 directly, induces the dissociation of TAX1BP1 from the glucocorticoid receptor-containing protein complex, and represses the coactivator function of TAX1BP1. Genetic knockout of Tax1bp1 in mice abrogates the influence of Tax on the activation of nuclear receptors. We propose that Tax-TAX1BP1 interaction mechanistically explains the previously reported repression of nuclear receptor activity by Tax.

Differential subcellular localization and activity of kelch repeat proteins KLHDC1 and KLHDC2.

We have previously identified and characterized human KLHDC2/HCLP-1, a kelch repeat protein that interacts with and inhibits transcription factor LZIP. In this study, we identified and characterized a paralog of KLHDC2 called KLHDC1. KLHDC1 and KLHDC2 share about 50% identity at the level of amino acid sequence and both gene loci localize to human chromosome 14q21.3. This cluster of KLHDC1 and KLHDC2 genes is highly conserved in vertebrates ranging from pufferfish to human. Both genes are expressed highly in skeletal muscle, but weakly in various other tissues. While KLHDC2 was predominantly found in the nucleus, KLHDC1 is a cytoplasmic protein. Neither KLHDC1 nor KLHDC2 binds to actin. In addition, KLHDC1 was unable to inhibit LZIP/CREB3-mediated transcriptional activation. Thus, KLHDC1 and KLHDC2 have differential localization and activity in cultured mammalian cells.

Modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein.

Perturbation of the function of endoplasmic reticulum (ER) causes stress leading to the activation of cell signaling pathways known as the unfolded protein response (UPR). Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) uses ER as a site for synthesis and processing of viral proteins. In this report, we demonstrate that infection with SARS-CoV induces the UPR in cultured cells. A comparison with M, E, and NSP6 proteins indicates that SARS-CoV spike (S) protein sufficiently induces transcriptional activation of several UPR effectors, including glucose-regulated protein 78 (GRP78), GRP94, and C/EBP homologous protein. A substantial amount of S protein accumulates in the ER. The expression of S protein exerts different effects on the three major signaling pathways of the UPR. Particularly, it induces GRP78/94 through PKR-like ER kinase but has no influence on activating transcription factor 6 or X box-binding protein 1. Taken together, our findings suggest that SARS-CoV S protein specifically modulates the UPR to facilitate viral replication.

TORC1 and TORC2 coactivators are required for tax activation of the human T-cell leukemia virus type 1 long terminal repeats.

Human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription from the long terminal repeats (LTR). Mechanisms through which Tax activates LTR have been established, but coactivators of this process remain to be identified and characterized. Here we show that all three members of the TORC family of transcriptional regulators are coactivators of Tax for LTR-driven expression. TORC coactivation requires CREB, but not ATF4 or other bZIP factors. Tax physically interacts with TORC1, TORC2, and TORC3 (TORC1/2/3), and the depletion of TORC1/2/3 inhibited Tax activity. TORC coactivation can be further enhanced by transcriptional coactivator p300. In addition, coactivators in the p300 family are required for full activity of Tax independently of TORC1/2/3. Thus, both TORC and p300 families of coactivators are essential for optimal activation of HTLV-1 transcription by Tax.

The liver-enriched transcription factor CREB-H is a growth suppressor protein underexpressed in hepatocellular carcinoma.

We have previously characterized transcription factor LZIP to be a growth suppressor targeted by hepatitis C virus oncoprotein. In search of proteins closely related to LZIP, we have identified a liver-enriched transcription factor CREB-H. LZIP and CREB-H represent a new subfamily of bZIP factors. CREB-H activates transcription by binding to cAMP responsive element, box B, and ATF6-binding element. Interestingly, CREB-H has a putative transmembrane (TM) domain and it localizes ambiently to the endoplasmic reticulum. Proteolytic cleavage that removes the TM domain leads to nuclear translocation and activation of CREB-H. CREB-H activates the promoter of hepatic gluconeogenic enzyme phosphoenolpyruvate carboxykinase. This activation can be further stimulated by cAMP and protein kinase A. CREB-H transcript is exclusively abundant in adult liver. In contrast, the expression of CREB-H mRNA is aberrantly reduced in hepatoma tissues and cells. The enforced expression of CREB-H suppresses the proliferation of cultured hepatoma cells. Taken together, our findings suggest that the liver-enriched bZIP transcription factor CREB-H is a growth suppressor that plays a role in hepatic physiology and pathology.

Specific TATAA and bZIP requirements suggest that HTLV-I Tax has transcriptional activity subsequent to the assembly of an initiation complex.

BACKGROUND: Human T-cell leukemia virus type I (HTLV-I) Tax protein is a transcriptional regulator of viral and cellular genes. In this study we have examined in detail the determinants for Tax-mediated transcriptional activation. RESULTS: Whereas previously the LTR enhancer elements were thought to be the sole Tax-targets, herein, we find that the core HTLV-I TATAA motif also provides specific responsiveness not seen with either the SV40 or the E1b TATAA boxes. When enhancer elements which can mediate Tax-responsiveness were compared, the authentic HTLV-I 21-bp repeats were found to be the most effective. Related bZIP factors such as CREB, ATF4, c-Jun and LZIP are often thought to recognize the 21-bp repeats equivalently. However, amongst bZIP factors, we found that CREB, by far, is preferred by Tax for activation. When LTR transcription was reconstituted by substituting either kappaB or serum response elements in place of the 21-bp repeats, Tax activated these surrogate motifs using surfaces which are different from that utilized for CREB interaction. Finally, we employed artificial recruitment of TATA-binding protein to the HTLV-I promoter in "bypass" experiments to show for the first time that Tax has transcriptional activity subsequent to the assembly of an initiation complex at the promoter. CONCLUSIONS: Optimal activation of the HTLV-I LTR by Tax specifically requires the core HTLV-I TATAA promoter, CREB and the 21-bp repeats. In addition, we also provide the first evidence for transcriptional activity of Tax after the recruitment of TATA-binding protein to the promoter.