Stresses that perturb the folding of nascent endoplasmic reticulum (ER) proteins activate the ER stress response. Upon ER stress, ER-associated ATF6 is cleaved; the resulting active cytosolic ...fragment of ATF6 translocates to the nucleus, binds to ER stress response elements (ERSEs), and induces genes, including the ER-targeted chaperone, GRP78. Recent studies showed that nutrient and oxygen starvation during tissue ischemia induce certain ER stress response genes, including GRP78; however, the role of ATF6 in mediating this induction has not been examined. In the current study, simulating ischemia (sI) in a primary cardiac myocyte model system caused a reduction in the level of ER-associated ATF6 with a coordinate increase of ATF6 in nuclear fractions. An ERSE in the GRP78 gene not previously shown to be required for induction by other ER stresses was found to bind ATF6 and to be critical for maximal ischemia-mediated GRP78 promoter induction. Activation of ATF6 and the GRP78 promoter, as well as grp78 mRNA accumulation during sI, were reversed upon simulated reperfusion (sI/R). Moreover, dominant-negative ATF6, or ATF6-targeted miRNA blocked sI-mediated grp78 induction, and the latter increased cardiac myocyte death upon simulated reperfusion, demonstrating critical roles for endogenous ATF6 in ischemia-mediated ER stress activation and cell survival. This is the first study to show that ATF6 is activated by ischemia but inactivated upon reperfusion, suggesting that it may play a role in the induction of ER stress response genes during ischemia that could have a preconditioning effect on cell survival during reperfusion.
Endoplasmic reticulum (ER) stresses that reduce ER protein folding activate the unfolded protein response (UPR). One effector of the UPR is the transcription factor X-box binding protein-1 (XBP1), ...which is expressed on ER stress-mediated splicing of the XBP1 mRNA. XBP1 induces certain ER-targeted proteins, eg, glucose-regulated protein 78 (GRP78), that help resolve the ER stress and foster cell survival. In this study, we determined whether hypoxia can activate the UPR in the cardiac context. Neonatal rat ventricular myocyte cultures subjected to hypoxia (16 hours) exhibited increased XBP1 mRNA splicing, XBP1 protein expression, GRP78 promoter activation, and GRP78 protein levels; however, the levels of these UPR markers declined during reoxygenation, suggesting that the UPR is activated during hypoxia but not during reoxygenation. When cells were infected with a recombinant adenovirus (AdV) encoding dominant-negative XBP1 (AdV-XBP1dn), UPR markers were reduced; however, hypoxia/reoxygenation-induced apoptosis increased. Confocal immunocytofluorescence demonstrated that hypoxia induced GRP78 in neonatal rat and isolated adult mouse ventricular myocytes. Moreover, mouse hearts subjected to in vivo myocardial infarction exhibited increased GRP78 expression in cardiac myocytes near the infarct, but not in healthy cells distal to the infarct. These results indicate that hypoxia activates the UPR in cardiac myocytes and that XBP1-inducible proteins may contribute to protecting the myocardium during hypoxic stress.
In this study, we created a mouse model of methamphetamine cardiomyopathy that reproduces the chronic, progressive dosing commonly encountered in addicted subjects. We gradually increased the ...quantity of methamphetamine given to C57Bl/6 mice from 5 to 40 mg/kg over 2 or 5 months during two study periods. At the fifth month, heart weight was increased, echocardiograms showed a dilated cardiomyopathy and survival was lower in males, with less effect in females. Interestingly, these findings correspond to previous observations in human patients, suggesting greater male susceptibility to the effects of methamphetamine on the heart. Transcriptional analysis showed changes in genes dysregulated in previous methamphetamine neurological studies as well as many that likely play a role in cardiac response to this toxic stress. We expect that a deeper understanding of the molecular biology of methamphetamine exposure in the heart will provide insights into the mechanism of cardiomyopathy in addicts and potential routes to more effective treatment.
We developed a model of chronic methamphetamine abuse in mice. Our principal finding was that male mice had greater susceptibility to cardiomyopathy, much higher mortality than female mice, and greater dysregulation of cardiac mRNA transcripts that have been identified as responsive to meth treatment in the brain, as well as a surprising number of transcripts that encode proteins important in inflammation and immune response.
The C-type lectin dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) mediates the innate immune recognition of microbial carbohydrates. We investigated the ...function of this molecule in the host response to pathogens in vivo, by generating mouse lines lacking the DC-SIGN homologues SIGNR1, SIGNR3, and SIGNR5. Resistance to Mycobacterium tuberculosis was impaired only in SIGNR3-deficient animals. SIGNR3 was expressed in lung phagocytes during infection, and interacted with M. tuberculosis bacilli and mycobacterial surface glycoconjugates to induce secretion of critical host defense inflammatory cytokines, including tumor necrosis factor (TNF). SIGNR3 signaling was dependent on an intracellular tyrosine-based motif and the tyrosine kinase Syk. Thus, the mouse DC-SIGN homologue SIGNR3 makes a unique contribution to protection of the host against a pulmonary bacterial pathogen.
Exposing cells to conditions that modulate growth can impair endoplasmic reticulum (ER) protein folding, leading to ER stress and activation of the transcription factor, ATF6. ATF6 binds to ER stress ...response elements in target genes, inducing expression of proteins that enhance the ER protein folding capacity, which helps overcome the stress and foster survival. To examine the mechanism of ATF6-mediated survival in vivo, we developed a transgenic mouse model that expresses a novel conditionally activated form of ATF6. We previously showed that activating ATF6 protected the hearts of ATF6 transgenic mice from ER stresses. In the present study, transcript profiling identified modulatory calcineurin interacting protein-1 (MCIP1), also known as regulator of calcineurin 1 (RCAN1), as a novel ATF6-inducible gene that encodes a known regulator of calcineurin/nuclear factor of activated T cells (NFAT)-mediated growth and development in many tissues. The ability of ATF6 to induce RCAN1 in vivo was replicated in cultured cardiac myocytes, where adenoviral (AdV)-mediated overexpression of activated ATF6 induced the RCAN1 promoter, up-regulated RCAN1 mRNA, inhibited calcineurin phosphatase activity, and exerted a striking growth modulating effect that was inhibited by RCAN1-targeted small interfering RNA. These results demonstrate that RCAN1 is a novel ATF6 target gene that may coordinate growth and ER stress signaling pathways. By modulating growth, RCAN1 may reduce the need for ER protein folding, thus helping to overcome the stress and enhance survival. Moreover, these results suggest that RCAN1 may also be a novel integrator of growth and ER stress signaling in many other tissues that depend on calcineurin/NFAT signaling for optimal growth and development.
The endoplasmic reticulum (ER)-transmembrane proteins, ATF6α and ATF6β, are cleaved during the ER stress response (ERSR). The resulting N-terminal fragments (N-ATF6α and N-ATF6β) have conserved ...DNA-binding domains and divergent transcriptional activation domains. N-ATF6α and N-ATF6β translocate to the nucleus, bind to specific regulatory elements, and influence expression of ERSR genes, such as glucose-regulated protein 78 (GRP78), that contribute to resolving the ERSR, thus, enhancing cell viability. We previously showed that N-ATF6α is a rapidly degraded, strong transcriptional activator, whereas β is a slowly degraded, weak activator. In this study we explored the molecular basis and functional impact of these isoform-specific characteristics in HeLa cells. Mutants in the transcriptional activation domain or DNA-binding domain of N-ATF6α exhibited loss of function and increased expression, the latter of which suggested decreased rates of degradation. Fusing N-ATF6α to the mutant estrogen receptor generated N-ATF6α-MER, which, without tamoxifen exhibited loss-of-function and high expression, but in the presence of tamoxifen N-ATF6α-MER exhibited gain-of-function and low expression. N-ATF6β conferred loss-of-function and high expression to N-ATF6α, suggesting that ATF6β is an endogenous inhibitor of ATF6α. In vitro DNA binding experiments showed that recombinant N-ATF6β inhibited the binding of recombinant N-ATF6α to an ERSR element from the GRP78 promoter. Moreover, siRNA-mediated knock-down of endogenous ATF6β increased GRP78 promoter activity and GRP78 gene expression, as well as augmenting cell viability. Thus, the relative levels of ATF6α and -β, may contribute to regulating the strength and duration of ATF6-dependent ERSR gene induction and cell viability.
In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by ...irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.
The endoplasmic reticulum (ER)-transmembrane proteins, ATF6α and ATF6β, are cleaved during the ER stress response (ERSR).
The resulting N-terminal fragments (N-ATF6α and N-ATF6β) have conserved ...DNA-binding domains and divergent transcriptional
activation domains. N-ATF6α and N-ATF6β translocate to the nucleus, bind to specific regulatory elements, and influence expression
of ERSR genes, such as glucose-regulated protein 78 (GRP78), that contribute to resolving the ERSR, thus, enhancing cell viability.
We previously showed that N-ATF6α is a rapidly degraded, strong transcriptional activator, whereas β is a slowly degraded,
weak activator. In this study we explored the molecular basis and functional impact of these isoform-specific characteristics
in HeLa cells. Mutants in the transcriptional activation domain or DNA-binding domain of N-ATF6α exhibited loss of function
and increased expression, the latter of which suggested decreased rates of degradation. Fusing N-ATF6α to the mutant estrogen
receptor generated N-ATF6α-MER, which, without tamoxifen exhibited loss-of-function and high expression, but in the presence
of tamoxifen N-ATF6α-MER exhibited gain-of-function and low expression. N-ATF6β conferred loss-of-function and high expression
to N-ATF6α, suggesting that ATF6β is an endogenous inhibitor of ATF6α. In vitro DNA binding experiments showed that recombinant N-ATF6β inhibited the binding of recombinant N-ATF6α to an ERSR element from
the GRP78 promoter. Moreover, siRNA-mediated knock-down of endogenous ATF6β increased GRP78 promoter activity and GRP78 gene
expression, as well as augmenting cell viability. Thus, the relative levels of ATF6α and -β, may contribute to regulating
the strength and duration of ATF6-dependent ERSR gene induction and cell viability.
Exposing cells to conditions that modulate growth can impair endoplasmic reticulum (ER) protein folding, leading to ER stress and activation of the transcription factor, ATF6. ATF6 binds to ER stress ...response elements in target genes, inducing expression of proteins that enhance the ER protein folding capacity, which helps overcome the stress and foster survival. To examine the mechanism of ATF6-mediated survival
in vivo
, we developed a transgenic mouse model that expresses a novel conditionally activated form of ATF6. We previously showed that activating ATF6 protected the hearts of ATF6 transgenic mice from ER stresses. In the present study, transcript profiling identified
modulatory calcineurin interacting protein-1
(
MCIP1
), also known as
regulator of calcineurin 1
(
RCAN1
), as a novel ATF6-inducible gene that encodes a known regulator of calcineurin/nuclear factor of activated T cells (NFAT)-mediated growth and development in many tissues. The ability of ATF6 to induce RCAN1
in vivo
was replicated in cultured cardiac myocytes, where adenoviral (AdV)-mediated overexpression of activated ATF6 induced the RCAN1 promoter, up-regulated RCAN1 mRNA, inhibited calcineurin phosphatase activity, and exerted a striking growth modulating effect that was inhibited by RCAN1-targeted small interfering RNA. These results demonstrate that
RCAN1
is a novel ATF6 target gene that may coordinate growth and ER stress signaling pathways. By modulating growth, RCAN1 may reduce the need for ER protein folding, thus helping to overcome the stress and enhance survival. Moreover, these results suggest that RCAN1 may also be a novel integrator of growth and ER stress signaling in many other tissues that depend on calcineurin/NFAT signaling for optimal growth and development.
Human α1,3/4-Fucosyltransferases Vo, Loc; Lee, Stephanie; Marcinko, Marie C. ...
The Journal of biological chemistry,
09/1998, Letnik:
273, Številka:
39
Journal Article
Recenzirano
Odprti dostop
An analysis of the acceptor substrate specificity of domain swap mutants of human α1,3/4-fucosyltransferases (FucTs) III and V has been carried out. The results demonstrate that changing Asp336 of ...FucT III to Ala (as in FucT V) produced a protein (III/V1) with a reduced activity with a variety of acceptors. An analysis of the kinetic properties of FucT III and the III/V1 mutant demonstrated that III/V1 had a 40-fold reduction in its affinity for the H-type 1 acceptor substrate (Fucα1,2Galβ1,3GlcNAc) and 4-fold reduction in its affinity for GDP-fucose when compared with FucT III. Further, the overall catalytic efficiency of III/V1 was ∼100-fold lower than that of FucT III with an H-type 1 acceptor substrate. The complementary domain swap resulting from the change of Ala349 of FucT V to Asp (V/III1) produced a FucT that had higher enzyme activity with a range of acceptor substrates and had a higher affinity for an H-type 2 acceptor substrate (Fucα1,2Galβ1,4GlcNAc) with an 8-fold higher overall catalytic efficiency than that of FucT V. No significant change occurred in the Km for GDP-fucose for this protein when compared with FucT V. Kinetic parameters of two other FucT domain swaps (III8/V and V8/III), resulting in proteins that differed from FucT III and V at the NH2terminus of their catalytic domain, were not significantly different from those of the parental enzymes when H-type 1 and H-type 2 acceptor substrates were utilized. Thus, substitution of an acidic amino acid for a nonpolar amino acid (i.e. Asp versus Ala) at the COOH terminus of FucTs produces an enzyme with enhanced enzyme activities. These results, together with the results presented in the accompanying papers (Nguyen, A. T., Holmes, E. H., Whitaker, J. M., Ho, S., Shetterly, S., and Macher, B. A. (1998)J. Biol. Chem. 273, 25244–25249; Sherwood, A. L., Nguyen, A. T., Whitaker, J. M., Macher, B. A., and Holmes, E. H. (1998) J. Biol. Chem. 273, 25256–25260), provide new insights into the structure/function relationships of human α1,3/4-FucT enzymes.
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