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Mimitin - a novel cytokine-regulated mitochondrial protein.

Wegrzyn P, Yarwood SJ, Fiegler N, Bzowska M, Koj A, Mizgalska D, Malicki S, Pajak M, Kasza A, Kachamakova-Trojanowska N, Bereta J, Jura J, Jura J - BMC Cell Biol. (2009)

Bottom Line: This was accompanied by a slight decrease in proliferation of HepG2 cells.Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin.We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. p.wegrzyn@uj.edu.pl

ABSTRACT

Background: The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin - a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners.

Results: By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18-24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway.

Conclusion: Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of apoptotic caspases 3/7, suggesting that this protein may indirectly participate in apoptosis.

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Effect of mimitin gene silencing on caspase 3/7 activities. HepG2 cells were transfected with mimitin-siRNA, GAPDH-siRNA, negative control siRNA, or transfection reagent (siPORT NeoFX) (blank). Apoptosis was induced 72 h post transfection by treatment of cells with TNF/CHX for 5 h. Caspase 3/7 activity was measured using Caspase-GloTM3/7 assay kit. A. Changes in caspase activity in cells with TNF/CHX-induced apoptosis were compared to cells treated with TNF/CHX and transfected with a negative control siRNA (assumed as equal 1). In each case the value of caspase 3/7 activity after TNF/CHX treatment was divided by the value of basal activity of non-stimulated cells. Values represent the mean of three independent experiments with error bars showing standard error (*p < 0.05). B. Representative Western blot analysis with 20 μg protein from caspase activity assay using anti-GAPDH and anti-Mimitin antibodies. Samples marked (+) were stimulated with TNF/CHX while samples marked (-) were not stimulated.
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Figure 6: Effect of mimitin gene silencing on caspase 3/7 activities. HepG2 cells were transfected with mimitin-siRNA, GAPDH-siRNA, negative control siRNA, or transfection reagent (siPORT NeoFX) (blank). Apoptosis was induced 72 h post transfection by treatment of cells with TNF/CHX for 5 h. Caspase 3/7 activity was measured using Caspase-GloTM3/7 assay kit. A. Changes in caspase activity in cells with TNF/CHX-induced apoptosis were compared to cells treated with TNF/CHX and transfected with a negative control siRNA (assumed as equal 1). In each case the value of caspase 3/7 activity after TNF/CHX treatment was divided by the value of basal activity of non-stimulated cells. Values represent the mean of three independent experiments with error bars showing standard error (*p < 0.05). B. Representative Western blot analysis with 20 μg protein from caspase activity assay using anti-GAPDH and anti-Mimitin antibodies. Samples marked (+) were stimulated with TNF/CHX while samples marked (-) were not stimulated.

Mentions: The mimitin gene was silenced in HepG2 cells using the established parameters and then caspase 3 and 7 activities were assayed, since they are known to be involved in programmed cell death [10-12]. The knockdown of mimitin gene had no effect on caspase 3 and 7 activities. This indicated that the level of mimitin has no direct influence on the basic activities of these caspases; however, it could still modulate them once they had been induced by a pro-apoptotic signal. Indeed, when we first induced apoptosis by treating the cells with TNF/CHX and then determined these caspases we found a striking effect of mimitin. HepG2 cells treated with siRNA specific for mimitin (mimitin-A) showed approximately 15 fold increase in the activity of caspases in comparison to negative control (Fig. 6A). Mimitin silencing was confirmed by Western blot analysis (Fig. 6B). However, treatment of cells with a GAPDH-specific siRNA had an even stronger effect on caspase activities (38-fold increase), although the protein level of GAPDH was only slightly reduced (Fig. 6B). The effects of both transfections show statistically significant differences in comparison to the negative control, with p < 0.02 for the mimitin-specific siRNA and p < 0.05 for GAPDH specific siRNA. The result obtained in HepG2 cells with the GAPDH-specific siRNA is not surprising because some reports indicate that silencing of the gene coding for GAPDH in tumour cells decreases the rate of glycolysis, inhibits cell proliferation and eventually leads to apoptosis [13,14].


Mimitin - a novel cytokine-regulated mitochondrial protein.

Wegrzyn P, Yarwood SJ, Fiegler N, Bzowska M, Koj A, Mizgalska D, Malicki S, Pajak M, Kasza A, Kachamakova-Trojanowska N, Bereta J, Jura J, Jura J - BMC Cell Biol. (2009)

Effect of mimitin gene silencing on caspase 3/7 activities. HepG2 cells were transfected with mimitin-siRNA, GAPDH-siRNA, negative control siRNA, or transfection reagent (siPORT NeoFX) (blank). Apoptosis was induced 72 h post transfection by treatment of cells with TNF/CHX for 5 h. Caspase 3/7 activity was measured using Caspase-GloTM3/7 assay kit. A. Changes in caspase activity in cells with TNF/CHX-induced apoptosis were compared to cells treated with TNF/CHX and transfected with a negative control siRNA (assumed as equal 1). In each case the value of caspase 3/7 activity after TNF/CHX treatment was divided by the value of basal activity of non-stimulated cells. Values represent the mean of three independent experiments with error bars showing standard error (*p < 0.05). B. Representative Western blot analysis with 20 μg protein from caspase activity assay using anti-GAPDH and anti-Mimitin antibodies. Samples marked (+) were stimulated with TNF/CHX while samples marked (-) were not stimulated.
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Figure 6: Effect of mimitin gene silencing on caspase 3/7 activities. HepG2 cells were transfected with mimitin-siRNA, GAPDH-siRNA, negative control siRNA, or transfection reagent (siPORT NeoFX) (blank). Apoptosis was induced 72 h post transfection by treatment of cells with TNF/CHX for 5 h. Caspase 3/7 activity was measured using Caspase-GloTM3/7 assay kit. A. Changes in caspase activity in cells with TNF/CHX-induced apoptosis were compared to cells treated with TNF/CHX and transfected with a negative control siRNA (assumed as equal 1). In each case the value of caspase 3/7 activity after TNF/CHX treatment was divided by the value of basal activity of non-stimulated cells. Values represent the mean of three independent experiments with error bars showing standard error (*p < 0.05). B. Representative Western blot analysis with 20 μg protein from caspase activity assay using anti-GAPDH and anti-Mimitin antibodies. Samples marked (+) were stimulated with TNF/CHX while samples marked (-) were not stimulated.
Mentions: The mimitin gene was silenced in HepG2 cells using the established parameters and then caspase 3 and 7 activities were assayed, since they are known to be involved in programmed cell death [10-12]. The knockdown of mimitin gene had no effect on caspase 3 and 7 activities. This indicated that the level of mimitin has no direct influence on the basic activities of these caspases; however, it could still modulate them once they had been induced by a pro-apoptotic signal. Indeed, when we first induced apoptosis by treating the cells with TNF/CHX and then determined these caspases we found a striking effect of mimitin. HepG2 cells treated with siRNA specific for mimitin (mimitin-A) showed approximately 15 fold increase in the activity of caspases in comparison to negative control (Fig. 6A). Mimitin silencing was confirmed by Western blot analysis (Fig. 6B). However, treatment of cells with a GAPDH-specific siRNA had an even stronger effect on caspase activities (38-fold increase), although the protein level of GAPDH was only slightly reduced (Fig. 6B). The effects of both transfections show statistically significant differences in comparison to the negative control, with p < 0.02 for the mimitin-specific siRNA and p < 0.05 for GAPDH specific siRNA. The result obtained in HepG2 cells with the GAPDH-specific siRNA is not surprising because some reports indicate that silencing of the gene coding for GAPDH in tumour cells decreases the rate of glycolysis, inhibits cell proliferation and eventually leads to apoptosis [13,14].

Bottom Line: This was accompanied by a slight decrease in proliferation of HepG2 cells.Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin.We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. p.wegrzyn@uj.edu.pl

ABSTRACT

Background: The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin - a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners.

Results: By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18-24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway.

Conclusion: Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of apoptotic caspases 3/7, suggesting that this protein may indirectly participate in apoptosis.

Show MeSH
Related in: MedlinePlus