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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

RNF182 exhibits ubiquitin E3 activity. His-tagged recombinant RNF182 protein was incubated with or without E1, E2 or ubiquitin, and ubiquitination patterns were detected using an anti-ubiquitin antibody. The GST-SIAH-1 protein was used as a positive control.
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Figure 6: RNF182 exhibits ubiquitin E3 activity. His-tagged recombinant RNF182 protein was incubated with or without E1, E2 or ubiquitin, and ubiquitination patterns were detected using an anti-ubiquitin antibody. The GST-SIAH-1 protein was used as a positive control.

Mentions: Many proteins that contain RING finger domains exhibit ubiquitin E3 ligase activity. Some of these RING finger proteins catalyze substrate-independent, but E2-dependent assembly of multi-ubiquitin chains in reaction mixtures containing ubiquitin, E1, E2 and the RING finger protein itself [12-14]. E3, GST and bacterial proteins from the cell lysate can all serve as potential substrates. With this in mind, we performed an in vitro ubiquitination assay to assess whether RNF182 has an E2-dependent E3 ligase activity. An ubiquitination pattern consisting of a high molecular weight smear was obtained from the reactions containing his-tag RNF182 (Fig. 6), whereas none was detected in the absence of RNF182, indicating that RNF182 can function as an E3 ubiquitin ligase. A reaction mixture of E1, E2 and GST-SIAH-1 was used as a positive control. Other controls included RNF182 alone, reactions omitting E3 (RNF182 or GST-SiAH-1), E1, E2 or ubiquitin, all gave negative results. We did not observe apparent auto-ubiquitination of RNF182 by Western blotting of the same blot probed with anti-RNF182 antibody (data not shown), suggesting that the pattern observed in figure 6 was mostly from the ubiquitination of proteins from the bacterial cell lysate. Adding proteins extracted from bacterial clones expressing GST-ATP6V0C or GST alone gave a similar intensity of smears (data not shown). These results confirmed the hypothesis that RNF182 could function as a substrate-independent, E2-dependent E3 ubiquitin ligase.


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)

RNF182 exhibits ubiquitin E3 activity. His-tagged recombinant RNF182 protein was incubated with or without E1, E2 or ubiquitin, and ubiquitination patterns were detected using an anti-ubiquitin antibody. The GST-SIAH-1 protein was used as a positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: RNF182 exhibits ubiquitin E3 activity. His-tagged recombinant RNF182 protein was incubated with or without E1, E2 or ubiquitin, and ubiquitination patterns were detected using an anti-ubiquitin antibody. The GST-SIAH-1 protein was used as a positive control.
Mentions: Many proteins that contain RING finger domains exhibit ubiquitin E3 ligase activity. Some of these RING finger proteins catalyze substrate-independent, but E2-dependent assembly of multi-ubiquitin chains in reaction mixtures containing ubiquitin, E1, E2 and the RING finger protein itself [12-14]. E3, GST and bacterial proteins from the cell lysate can all serve as potential substrates. With this in mind, we performed an in vitro ubiquitination assay to assess whether RNF182 has an E2-dependent E3 ligase activity. An ubiquitination pattern consisting of a high molecular weight smear was obtained from the reactions containing his-tag RNF182 (Fig. 6), whereas none was detected in the absence of RNF182, indicating that RNF182 can function as an E3 ubiquitin ligase. A reaction mixture of E1, E2 and GST-SIAH-1 was used as a positive control. Other controls included RNF182 alone, reactions omitting E3 (RNF182 or GST-SiAH-1), E1, E2 or ubiquitin, all gave negative results. We did not observe apparent auto-ubiquitination of RNF182 by Western blotting of the same blot probed with anti-RNF182 antibody (data not shown), suggesting that the pattern observed in figure 6 was mostly from the ubiquitination of proteins from the bacterial cell lysate. Adding proteins extracted from bacterial clones expressing GST-ATP6V0C or GST alone gave a similar intensity of smears (data not shown). These results confirmed the hypothesis that RNF182 could function as a substrate-independent, E2-dependent E3 ubiquitin ligase.

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