Multiple oxidative stress-response members of the Adapt78 family

Center for Immunology and Microbial Disease, The Albany Medical College, Albany, NY 12208, USA.
Free Radical Biology and Medicine (Impact Factor: 5.74). 09/2004; 37(4):454-62. DOI: 10.1016/j.freeradbiomed.2004.05.014
Source: PubMed


Adapt78 is an oxidative and calcium stress-response gene. Its protein product is a potent natural inhibitor of the intracellular calcium signaling protein calcineurin. Much of what is known about Adapt78 protein is based on cell-transfection studies. Toward understanding natural endogenous Adapt78, we used an antibody raised against cellular Adapt78 and recently determined that endogenous Adapt78 protein, like its mRNA, is oxidative and calcium stress responsive. Here we report the identification of a second endogenous form of this protein family of 41 kDa. Subcellular fractionation of human HeLa cells revealed that in contrast to results of previous transfection studies, most endogenous Adapt78, characterized as 29 and 41 kDa electrophoretic doublets, resides in the cellular cytosol. The 41 kDa form of Adapt78 was abundant and found to exhibit many characteristics in common with the previously reported oxidative stress-responsive 29 kDa form, including hypo- and hyperphosphorylation variants, rapid loss of the hypophosphorylated form following oxidative stress, response to various kinase and phosphatase inhibitors, and localization. However, it also exhibited some unique characteristics, most notably the lack of calcium inducibility. Finally, the 29 kDa form exhibited a much shorter half-life and strong stabilization following oxidant exposure compared with the 41 kDa Adapt78 form. These data reveal the presence of a novel oxidative stress-responsive 41 kDa Adapt78 species, lend further insight into the Adapt78 family of proteins and their distribution, and challenge previous conclusions obtained using transfection protocols.

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    • "It was shown that Adapt78 up-regulation is mediated by c-Jun, a transcription factor downstream of the JNK signaling pathway, during response to thapsigargin-induced endoplasmic reticulum stress and contributes to protection against cell death (Zhao et al., 2008). Adapt78 is an inhibitor of calcineurin, an important intracellular phosphatase that mediates many cellular responses to calcium, and was identified as an oxidant-inducible gene (Michtalik et al., 2004). Ubiquitous overexpression of its Drosophila homologue, gene nla, leads to lifespan prolongation (Seong et al., 2001b). "
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    • "It encodes two major transcripts that are translated into the protein products isoform 1 (RCAN-1) and isoform 4 (RCAN1-4). Isoform 1 is 36–41 kDa and usually expressed at constant levels, whereas isoform 4 is 25–29 kDa and highly inducible by intracellular calcium (Crawford et al., 1997; Michtalik et al., 2004). Both forms inhibit calcineurin, an intracellular phosphatase that mediates many cellular responses to calcium (Gorlach et al., 2000; Kingsbury & Cunningham, 2000; Rothermel et al., 2000; Rusnak & Mertz, 2000). "
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    ABSTRACT: Studies on the role of regulator of calcineurin 1 (RCAN1) in immunity are limited, but have demonstrated an involvement in T-lymphocyte function. Here, we expand these studies to macrophages and in vivo infection. The treatment of RAW and primary mouse macrophages with lipopolysaccharide from Escherichia coli strongly induced RCAN1 isoform 4 (RCAN1-4), but not isoform 1. RCAN1-4 induction involved calcium, calcineurin, and reactive oxygen species. Subsequent analysis with whole bacteria including gram-negative E. coli and gram-positive Staphylococcus aureus revealed strong RCAN1-4 inductions by both, and where tested, dependence on calcium. Staphylococcus aureus cell wall components peptidoglycan and lipoteichoic acid also strongly induced RCAN1-4. In vivo, a significant induction in the proinflammatory cytokines monocyte chemotactic protein-1, interleukin-6, interferon-γ, and tumor necrosis factor-α was observed in knockout (KO) lung vs. wild-type (WT) mice 7 days after nasal infection with Fransicella tularensis. This induction was not accompanied by a significant increase in F. tularensis burden in the KO lung. Additionally, a modest increase in respiratory burst activity in KO vs. WT macrophages was observed. Combined, these studies indicate that RCAN1 is involved in macrophage and the overall in vivo immune response, and provide additional evidence that RCAN1 plays an important role in cell immunity and infectious disease.
    Preview · Article · Oct 2010 · FEMS Immunology & Medical Microbiology
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    • "In our previous study, we demonstrated that H 2 O 2 triggers proteolytic cleavage and irreversible inactivation of calcineurin (Lee et al., 2007). Since DSCR1 exhibits greater stability with H 2 O 2 (Michtalik et al., 2004), we hypothesized that DSCR1 may be involved in the proteolysis of calcineurin in conditions of oxidative stress. To test this hypothesis, we overexpressed or down-regulated DSCR1 and examined the calcineurin protein content in the presence of H 2 O 2 . "
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    ABSTRACT: Down syndrome critical region 1 (DSCR1), an oxidative stress-response gene, interacts with calcineurin and represses its phosphatase activity. Recently it was shown that hydrogen peroxide inactivates calcineurin by proteolytic cleavage. Based on these facts, we investigated whether oxidative stress affects DSCR1- mediated inactivation of calcineurin. We determined that overexpression of DSCR1 leads to increased proteolytic cleavage of calcineurin. Convertsely, knockdown of DSCR1 abolished calcineurin cleavage upon treatment with hydrogen peroxide. The PXIIXT motif in the COOH-terminus of DSCR1 is responsible for both binding and cleavage of calcineurin. The knockdown of overexpressed DSCR1 in DS fibroblast cells also abrogated calcineurin proteolysis by hydrogen peroxide. These results suggest that DSCR1 has the ability to inactivate calcineurin by inducing proteolytic cleavage of calcineurin upon oxidative stress.
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