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A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation.

Liu QY, Lei JX, Sikorska M, Liu R - Mol Neurodegener (2008)

Bottom Line: Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults.It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada. qing_yan.liu@nrc.gc.ca.

ABSTRACT

Background: Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD). These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells.

Results: RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated in vitro in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination in vitro. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both in vitro and in vivo, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.

Conclusion: Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

No MeSH data available.


Related in: MedlinePlus

Cellular levels of RNF182 modulate the rates of cell death. N2a cells were transiently transfected with RNF182/pcDNA3.1/myc-his plasmid or mouse RNF182 on-target plus smart pool siRNAs. Cells were collected for total RNA extractions 24–48 h after transfections. Trypan Blue exclusion assay was performed 24 h after transfection or 16 h after a 7 h OGD treatment of the siRNA transfected samples. A. Over expression of RNF182 mRNA was assessed by RT-PCR. In: lane 1 – negative PCR control, lane 2 – mock transfection, lane 3-transfection with RNF182/pcDNA3.1/myc-his plasmid. B. Percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). C. Percentage of cell death before and after transfection with transfection reagent removed 6 h after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). D. Percentage of cell death of the control and 24 h transfected samples subjected to a 7 h OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). E. Assessment of the siRNA silencing efficiency. RNA samples were collected 24 and 48 h after transfection with siRNAs. Down regulation of RNF182 mRNA was analyzed by RT-PCR. In: lanes 1 and 3 – 24 and 48 h after transfection with RNF182 siRNAs, lanes 2 and 4 – 24 and 48 h after transfection with non-targeting pool negative control siRNAs, respectively. F. Percentage of cell death 24 h after siRNA transfection, with or with out OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisks indicate a significant difference (ρ < 0.005; t-test). C24 – 24 h after transfection with non-targeting pool negative control siRNAs, siRNA24 – 24 h after transfection with mouse RNF182 on-target plus smart pool siRNAs, c24-OGD – 24 h after transfection with non-targeting pool negative control siRNAs plus OGD treatment, siRNA24-OGD – 24 h after transfection with on-target plus smart pool siRNAs plus OGD treatment.
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Figure 5: Cellular levels of RNF182 modulate the rates of cell death. N2a cells were transiently transfected with RNF182/pcDNA3.1/myc-his plasmid or mouse RNF182 on-target plus smart pool siRNAs. Cells were collected for total RNA extractions 24–48 h after transfections. Trypan Blue exclusion assay was performed 24 h after transfection or 16 h after a 7 h OGD treatment of the siRNA transfected samples. A. Over expression of RNF182 mRNA was assessed by RT-PCR. In: lane 1 – negative PCR control, lane 2 – mock transfection, lane 3-transfection with RNF182/pcDNA3.1/myc-his plasmid. B. Percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). C. Percentage of cell death before and after transfection with transfection reagent removed 6 h after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). D. Percentage of cell death of the control and 24 h transfected samples subjected to a 7 h OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). E. Assessment of the siRNA silencing efficiency. RNA samples were collected 24 and 48 h after transfection with siRNAs. Down regulation of RNF182 mRNA was analyzed by RT-PCR. In: lanes 1 and 3 – 24 and 48 h after transfection with RNF182 siRNAs, lanes 2 and 4 – 24 and 48 h after transfection with non-targeting pool negative control siRNAs, respectively. F. Percentage of cell death 24 h after siRNA transfection, with or with out OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisks indicate a significant difference (ρ < 0.005; t-test). C24 – 24 h after transfection with non-targeting pool negative control siRNAs, siRNA24 – 24 h after transfection with mouse RNF182 on-target plus smart pool siRNAs, c24-OGD – 24 h after transfection with non-targeting pool negative control siRNAs plus OGD treatment, siRNA24-OGD – 24 h after transfection with on-target plus smart pool siRNAs plus OGD treatment.

Mentions: To better understand the role of RNF182 in neurodegeneration we examined the effects of gene overexpression in N2a cell line. We cloned the coding region of RNF182 into a mammalian expression vector, pEGFP-N1, with a stop codon inserted between the end of the RNF182 and the beginning of EGFP. As a result, a faint green fluorescent signal was observed in the cells transfected with the plasmid construct, but the RNF182 protein was free to perform its routine function without the possible interference of the EGFP. A significant increase in RNF182 mRNA was observed 24 h after transfection (Fig. 5A). The overexpression of RNF182 by itself triggered cell death in N2a cells as compared with mock transfection of empty vector (Fig. 5B). The cell death was not caused by the transfection reagent as removing it did not alter the outcome (Fig. 5C). The subsequent challenge with OGD caused additional loss of cells in both control and the RNF182 transfected cultures (5D). Next, we down regulated the endogenous RNF182 in N2a cells using a mixture of four siRNAs targeting mouse RNF182 gene. The RNF182 transcript was knocked down 24 h after the siRNA transfection (Fig. 5E, lane 1) and still remained low 48 h later (Fig. 5E, lane 3). The transfected cells (after 24 h) were subjected to 7 h OGD treatment and 16 h re-oxygenation in a normal culture chamber and examined for cell viability (Fig. 5F). The results showed that the downregulation of the endogenous RNF182 significantly reduced the percentage of cell death caused by OGD treatment.


A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation.

Liu QY, Lei JX, Sikorska M, Liu R - Mol Neurodegener (2008)

Cellular levels of RNF182 modulate the rates of cell death. N2a cells were transiently transfected with RNF182/pcDNA3.1/myc-his plasmid or mouse RNF182 on-target plus smart pool siRNAs. Cells were collected for total RNA extractions 24–48 h after transfections. Trypan Blue exclusion assay was performed 24 h after transfection or 16 h after a 7 h OGD treatment of the siRNA transfected samples. A. Over expression of RNF182 mRNA was assessed by RT-PCR. In: lane 1 – negative PCR control, lane 2 – mock transfection, lane 3-transfection with RNF182/pcDNA3.1/myc-his plasmid. B. Percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). C. Percentage of cell death before and after transfection with transfection reagent removed 6 h after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). D. Percentage of cell death of the control and 24 h transfected samples subjected to a 7 h OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). E. Assessment of the siRNA silencing efficiency. RNA samples were collected 24 and 48 h after transfection with siRNAs. Down regulation of RNF182 mRNA was analyzed by RT-PCR. In: lanes 1 and 3 – 24 and 48 h after transfection with RNF182 siRNAs, lanes 2 and 4 – 24 and 48 h after transfection with non-targeting pool negative control siRNAs, respectively. F. Percentage of cell death 24 h after siRNA transfection, with or with out OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisks indicate a significant difference (ρ < 0.005; t-test). C24 – 24 h after transfection with non-targeting pool negative control siRNAs, siRNA24 – 24 h after transfection with mouse RNF182 on-target plus smart pool siRNAs, c24-OGD – 24 h after transfection with non-targeting pool negative control siRNAs plus OGD treatment, siRNA24-OGD – 24 h after transfection with on-target plus smart pool siRNAs plus OGD treatment.
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Figure 5: Cellular levels of RNF182 modulate the rates of cell death. N2a cells were transiently transfected with RNF182/pcDNA3.1/myc-his plasmid or mouse RNF182 on-target plus smart pool siRNAs. Cells were collected for total RNA extractions 24–48 h after transfections. Trypan Blue exclusion assay was performed 24 h after transfection or 16 h after a 7 h OGD treatment of the siRNA transfected samples. A. Over expression of RNF182 mRNA was assessed by RT-PCR. In: lane 1 – negative PCR control, lane 2 – mock transfection, lane 3-transfection with RNF182/pcDNA3.1/myc-his plasmid. B. Percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). C. Percentage of cell death before and after transfection with transfection reagent removed 6 h after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). D. Percentage of cell death of the control and 24 h transfected samples subjected to a 7 h OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisk indicates a significant difference (ρ < 0.005; t-test). E. Assessment of the siRNA silencing efficiency. RNA samples were collected 24 and 48 h after transfection with siRNAs. Down regulation of RNF182 mRNA was analyzed by RT-PCR. In: lanes 1 and 3 – 24 and 48 h after transfection with RNF182 siRNAs, lanes 2 and 4 – 24 and 48 h after transfection with non-targeting pool negative control siRNAs, respectively. F. Percentage of cell death 24 h after siRNA transfection, with or with out OGD treatment. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisks indicate a significant difference (ρ < 0.005; t-test). C24 – 24 h after transfection with non-targeting pool negative control siRNAs, siRNA24 – 24 h after transfection with mouse RNF182 on-target plus smart pool siRNAs, c24-OGD – 24 h after transfection with non-targeting pool negative control siRNAs plus OGD treatment, siRNA24-OGD – 24 h after transfection with on-target plus smart pool siRNAs plus OGD treatment.
Mentions: To better understand the role of RNF182 in neurodegeneration we examined the effects of gene overexpression in N2a cell line. We cloned the coding region of RNF182 into a mammalian expression vector, pEGFP-N1, with a stop codon inserted between the end of the RNF182 and the beginning of EGFP. As a result, a faint green fluorescent signal was observed in the cells transfected with the plasmid construct, but the RNF182 protein was free to perform its routine function without the possible interference of the EGFP. A significant increase in RNF182 mRNA was observed 24 h after transfection (Fig. 5A). The overexpression of RNF182 by itself triggered cell death in N2a cells as compared with mock transfection of empty vector (Fig. 5B). The cell death was not caused by the transfection reagent as removing it did not alter the outcome (Fig. 5C). The subsequent challenge with OGD caused additional loss of cells in both control and the RNF182 transfected cultures (5D). Next, we down regulated the endogenous RNF182 in N2a cells using a mixture of four siRNAs targeting mouse RNF182 gene. The RNF182 transcript was knocked down 24 h after the siRNA transfection (Fig. 5E, lane 1) and still remained low 48 h later (Fig. 5E, lane 3). The transfected cells (after 24 h) were subjected to 7 h OGD treatment and 16 h re-oxygenation in a normal culture chamber and examined for cell viability (Fig. 5F). The results showed that the downregulation of the endogenous RNF182 significantly reduced the percentage of cell death caused by OGD treatment.

Bottom Line: Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults.It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada. qing_yan.liu@nrc.gc.ca.

ABSTRACT

Background: Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD). These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells.

Results: RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated in vitro in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination in vitro. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both in vitro and in vivo, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.

Conclusion: Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

No MeSH data available.


Related in: MedlinePlus