Limits...
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

Expression pattern of RNF182 gene. A. Changes in mRNA levels of RNF182 transcript during RA-induced differentiation of NT2 cells were determined by quantitative RT-PCR. The samples were measured against the cDNA of undifferentiated NT2 cells as a control, set at 100%. Percentage of each sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between test sample and the control sample. undiff – undifferentiated NT2 cells (control), NT2N – NT2 neurons, NT2A – NT2 astrocytes. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). B. Changes in RNF182 protein levels were determined by Western blotting with anti-RNF182 antibody using 100 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. C. Ethidium bromide stained agarose gel of RT-PCR products amplified from the coding region of RNF182 (top panel) and β-actin (bottom panel) from various mouse tissues: lane M – molecular size marker, lane 1 – kidney, lane 2 – skeletal muscle, lane 3 – liver, lane 4 – heart, lane 5 – cortex, lane 6 – hippocampus, lane 7 – cerebellum, lane 8 – spinal cord, lane 9 – negative PCR control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2279130&req=5

Figure 2: Expression pattern of RNF182 gene. A. Changes in mRNA levels of RNF182 transcript during RA-induced differentiation of NT2 cells were determined by quantitative RT-PCR. The samples were measured against the cDNA of undifferentiated NT2 cells as a control, set at 100%. Percentage of each sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between test sample and the control sample. undiff – undifferentiated NT2 cells (control), NT2N – NT2 neurons, NT2A – NT2 astrocytes. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). B. Changes in RNF182 protein levels were determined by Western blotting with anti-RNF182 antibody using 100 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. C. Ethidium bromide stained agarose gel of RT-PCR products amplified from the coding region of RNF182 (top panel) and β-actin (bottom panel) from various mouse tissues: lane M – molecular size marker, lane 1 – kidney, lane 2 – skeletal muscle, lane 3 – liver, lane 4 – heart, lane 5 – cortex, lane 6 – hippocampus, lane 7 – cerebellum, lane 8 – spinal cord, lane 9 – negative PCR control.

Mentions: RNF182 is a weakly expressed gene, not detectable by Northern blotting. Quantitative RT-PCR analysis indicated that the gene was up regulated during retinoic acid (RA) – induced differentiation of human NT2 cells. The increased level of RNF182 transcripts II was detected in both NT2 neurons and NT2 astrocytes (Fig. 2A). This was further confirmed by Western analysis using anti-RNF182 antibody (Fig. 2B). Next, we analyzed the tissue distribution of RNF182 by semi-quantitative RT-PCR using a primer pair from the coding region of the gene (Fig. 2C). A single band of 395 bp product was detected in the mouse cortex, hippocampus, cerebellum and spinal cord, but not in heart, liver, kidney or skeletal muscle, indicating that RNF182 was a brain-enriched gene.


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)

Expression pattern of RNF182 gene. A. Changes in mRNA levels of RNF182 transcript during RA-induced differentiation of NT2 cells were determined by quantitative RT-PCR. The samples were measured against the cDNA of undifferentiated NT2 cells as a control, set at 100%. Percentage of each sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between test sample and the control sample. undiff – undifferentiated NT2 cells (control), NT2N – NT2 neurons, NT2A – NT2 astrocytes. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). B. Changes in RNF182 protein levels were determined by Western blotting with anti-RNF182 antibody using 100 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. C. Ethidium bromide stained agarose gel of RT-PCR products amplified from the coding region of RNF182 (top panel) and β-actin (bottom panel) from various mouse tissues: lane M – molecular size marker, lane 1 – kidney, lane 2 – skeletal muscle, lane 3 – liver, lane 4 – heart, lane 5 – cortex, lane 6 – hippocampus, lane 7 – cerebellum, lane 8 – spinal cord, lane 9 – negative PCR control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2279130&req=5

Figure 2: Expression pattern of RNF182 gene. A. Changes in mRNA levels of RNF182 transcript during RA-induced differentiation of NT2 cells were determined by quantitative RT-PCR. The samples were measured against the cDNA of undifferentiated NT2 cells as a control, set at 100%. Percentage of each sample was calculated by 100x 2-ΔCt, where ΔCt is the cycle number difference between test sample and the control sample. undiff – undifferentiated NT2 cells (control), NT2N – NT2 neurons, NT2A – NT2 astrocytes. The experiments were performed in triplicate. Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). B. Changes in RNF182 protein levels were determined by Western blotting with anti-RNF182 antibody using 100 μg/lane of total cellular protein. The Western blotting of β-actin was shown as loading control. C. Ethidium bromide stained agarose gel of RT-PCR products amplified from the coding region of RNF182 (top panel) and β-actin (bottom panel) from various mouse tissues: lane M – molecular size marker, lane 1 – kidney, lane 2 – skeletal muscle, lane 3 – liver, lane 4 – heart, lane 5 – cortex, lane 6 – hippocampus, lane 7 – cerebellum, lane 8 – spinal cord, lane 9 – negative PCR control.
Mentions: RNF182 is a weakly expressed gene, not detectable by Northern blotting. Quantitative RT-PCR analysis indicated that the gene was up regulated during retinoic acid (RA) – induced differentiation of human NT2 cells. The increased level of RNF182 transcripts II was detected in both NT2 neurons and NT2 astrocytes (Fig. 2A). This was further confirmed by Western analysis using anti-RNF182 antibody (Fig. 2B). Next, we analyzed the tissue distribution of RNF182 by semi-quantitative RT-PCR using a primer pair from the coding region of the gene (Fig. 2C). A single band of 395 bp product was detected in the mouse cortex, hippocampus, cerebellum and spinal cord, but not in heart, liver, kidney or skeletal muscle, indicating that RNF182 was a brain-enriched gene.

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