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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

Subcellular localization of RNF182. N2a cells were transiently transfected with pEGFP-RNF182 plasmid DNA. Cells were fixed and stained with anti-RNF182 antibody. Cy3-conjugated anti-rabbit IgG was used to detect the specific immunostaining. The nuclei were stained with DAPI and viewed with a Zeiss Axiovert 200 M × 63 fluorescence microscope. Arrows indicate non-transfected cells. Asterisk represents a transfected cell.
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Figure 3: Subcellular localization of RNF182. N2a cells were transiently transfected with pEGFP-RNF182 plasmid DNA. Cells were fixed and stained with anti-RNF182 antibody. Cy3-conjugated anti-rabbit IgG was used to detect the specific immunostaining. The nuclei were stained with DAPI and viewed with a Zeiss Axiovert 200 M × 63 fluorescence microscope. Arrows indicate non-transfected cells. Asterisk represents a transfected cell.

Mentions: To examine the cellular localization of RNF182, we sub-cloned the coding region of RNF182 into the pEGFP-N1 vector where EGFP was fused to the carboxy terminus of RNF182. The plasmid construct was transiently transfected into N2a cells to allow fluorescence detection. The same cell culture was also stained with anti-RNF182 antibody. The non-transfected cell population demonstrated a speckled distribution of endogenous RNF182 protein in the cytoplasm (Fig. 3 panel RNF182, arrows). Fluorescent microscopy of transiently transfected N2a cells revealed a strong signal of the recombinant RNF182 protein throughout the cytoplasm in both differentiated and undifferentiated cells (panel EGFP). No fluorescent signal was observed in the nuclei. Transfected cells showed much stronger staining of RNF182 (Fig. 3 panel RNF182, asterisk), representing a combined signal of both endogenous and exogenous RNF182 protein.


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)

Subcellular localization of RNF182. N2a cells were transiently transfected with pEGFP-RNF182 plasmid DNA. Cells were fixed and stained with anti-RNF182 antibody. Cy3-conjugated anti-rabbit IgG was used to detect the specific immunostaining. The nuclei were stained with DAPI and viewed with a Zeiss Axiovert 200 M × 63 fluorescence microscope. Arrows indicate non-transfected cells. Asterisk represents a transfected cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Subcellular localization of RNF182. N2a cells were transiently transfected with pEGFP-RNF182 plasmid DNA. Cells were fixed and stained with anti-RNF182 antibody. Cy3-conjugated anti-rabbit IgG was used to detect the specific immunostaining. The nuclei were stained with DAPI and viewed with a Zeiss Axiovert 200 M × 63 fluorescence microscope. Arrows indicate non-transfected cells. Asterisk represents a transfected cell.
Mentions: To examine the cellular localization of RNF182, we sub-cloned the coding region of RNF182 into the pEGFP-N1 vector where EGFP was fused to the carboxy terminus of RNF182. The plasmid construct was transiently transfected into N2a cells to allow fluorescence detection. The same cell culture was also stained with anti-RNF182 antibody. The non-transfected cell population demonstrated a speckled distribution of endogenous RNF182 protein in the cytoplasm (Fig. 3 panel RNF182, arrows). Fluorescent microscopy of transiently transfected N2a cells revealed a strong signal of the recombinant RNF182 protein throughout the cytoplasm in both differentiated and undifferentiated cells (panel EGFP). No fluorescent signal was observed in the nuclei. Transfected cells showed much stronger staining of RNF182 (Fig. 3 panel RNF182, asterisk), representing a combined signal of both endogenous and exogenous RNF182 protein.

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