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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

A schematic representation of the human RNF182 gene, transcript and protein structures. Solid lines refer to introns or non-transcribed genomic DNA. Hatched bars represent exons. The RNF182 gene (GenBank AL138718) contains four exons. The open reading frame (black bar) and 3' untranslated region of both transcripts are solely encoded by exon 4. Structural motifs of the encoded protein were predicted by ExPASy tools.
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Figure 1: A schematic representation of the human RNF182 gene, transcript and protein structures. Solid lines refer to introns or non-transcribed genomic DNA. Hatched bars represent exons. The RNF182 gene (GenBank AL138718) contains four exons. The open reading frame (black bar) and 3' untranslated region of both transcripts are solely encoded by exon 4. Structural motifs of the encoded protein were predicted by ExPASy tools.

Mentions: We have isolated a 300 bp cDNA fragment, 360nh, by subtractive hybridization using a pooled mRNA population from AD brains as a "tester" and the first strand cDNAs from a pooled age-matched control brains as a "driver" [10]. BLAST searches revealed that this fragment showed a significant sequence identity with human genomic clone RP11-127P7 on chromosome 6 (GenBank AL138718). No matching EST or mRNA for this 360nh sequence was found in GenBank. Genome BLAT analysis indicated that there exists a mRNA sequence (GenBank AK090576) that appeared to be transcribed from the same region of genomic DNA on chromosome 6. This mRNA contains an open reading frame (ORF) encoding a protein with a RING finger domain, dubbed RNF182. Although this mRNA had a 5' end sequence matching the genomic sequence upstream of where 360nh was derived, it did not contain the 360nh sequence. We speculated that the 360nh sequence might be on an alternatively spliced exon of the same gene. We, therefore, used a forward primer on the 5' end of 360nh and a reverse primer within the coding region of RNF182 to amplify any alternative transcript sequence from first strand cDNA synthesized from human brain mRNA. Sequence analysis of the resulting PCR fragment indicated that genomic fragment AL138718, indeed, contained a gene of four exons giving rise to two alternatively spliced transcripts by swapping exons 1 and 2 (Fig. 1). Both transcripts contain the same ORF, thus encoding the same protein of 247 amino acids, with calculated molecular mass of 27.4 kDa. The 360nh sequence was located in exon 2, which constitutes part of the 5' untranslated region of transcript II. Protein sequence comparison revealed that human RNF182 is highly homologous to those of rodents', with 98% and 97% sequence identity to mouse and rat, respectively. The predicted primary structure of this protein contained a typical C3HC4-type RING finger domain between amino acids C20 and C67. There are two putative transmembrane helices located at the C-terminus, spanning amino acids 178 to 200, 212 to 234, respectively. In addition, the primary sequence of RNF182 also suggested four leucine repeats between amino acids 197 and 225. These repeats are within the two transmembrane domains, but do not correspond to a leucine zipper (Fig. 1).


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)

A schematic representation of the human RNF182 gene, transcript and protein structures. Solid lines refer to introns or non-transcribed genomic DNA. Hatched bars represent exons. The RNF182 gene (GenBank AL138718) contains four exons. The open reading frame (black bar) and 3' untranslated region of both transcripts are solely encoded by exon 4. Structural motifs of the encoded protein were predicted by ExPASy tools.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A schematic representation of the human RNF182 gene, transcript and protein structures. Solid lines refer to introns or non-transcribed genomic DNA. Hatched bars represent exons. The RNF182 gene (GenBank AL138718) contains four exons. The open reading frame (black bar) and 3' untranslated region of both transcripts are solely encoded by exon 4. Structural motifs of the encoded protein were predicted by ExPASy tools.
Mentions: We have isolated a 300 bp cDNA fragment, 360nh, by subtractive hybridization using a pooled mRNA population from AD brains as a "tester" and the first strand cDNAs from a pooled age-matched control brains as a "driver" [10]. BLAST searches revealed that this fragment showed a significant sequence identity with human genomic clone RP11-127P7 on chromosome 6 (GenBank AL138718). No matching EST or mRNA for this 360nh sequence was found in GenBank. Genome BLAT analysis indicated that there exists a mRNA sequence (GenBank AK090576) that appeared to be transcribed from the same region of genomic DNA on chromosome 6. This mRNA contains an open reading frame (ORF) encoding a protein with a RING finger domain, dubbed RNF182. Although this mRNA had a 5' end sequence matching the genomic sequence upstream of where 360nh was derived, it did not contain the 360nh sequence. We speculated that the 360nh sequence might be on an alternatively spliced exon of the same gene. We, therefore, used a forward primer on the 5' end of 360nh and a reverse primer within the coding region of RNF182 to amplify any alternative transcript sequence from first strand cDNA synthesized from human brain mRNA. Sequence analysis of the resulting PCR fragment indicated that genomic fragment AL138718, indeed, contained a gene of four exons giving rise to two alternatively spliced transcripts by swapping exons 1 and 2 (Fig. 1). Both transcripts contain the same ORF, thus encoding the same protein of 247 amino acids, with calculated molecular mass of 27.4 kDa. The 360nh sequence was located in exon 2, which constitutes part of the 5' untranslated region of transcript II. Protein sequence comparison revealed that human RNF182 is highly homologous to those of rodents', with 98% and 97% sequence identity to mouse and rat, respectively. The predicted primary structure of this protein contained a typical C3HC4-type RING finger domain between amino acids C20 and C67. There are two putative transmembrane helices located at the C-terminus, spanning amino acids 178 to 200, 212 to 234, respectively. In addition, the primary sequence of RNF182 also suggested four leucine repeats between amino acids 197 and 225. These repeats are within the two transmembrane domains, but do not correspond to a leucine zipper (Fig. 1).

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