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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 physically interacts with ATP6V0C. A. Interaction between RNF182 and ATP6V0C in yeast two-hybrid system. Empty or RNF182 containing pGBKT7 bait vector and empty or ATP6V0C containing pACT2 library vector were co-transformed into yeast host cells AH109 and plated onto SD/-Trp -Leu -Ade -His +X-α-gal plate. In: a – a negative test of empty bait vector and ATP6V0C; b – a positive test showing the interaction between RNF182 and ATP6V0C; c – a negative test of RNF182 bait plus empty library vector. B. Total cellular proteins were extracted from HEK-293 cells co-transfected with flag-tagged ATP6V0C and GST-tagged RNF182 constructs and precipitated with glutathione-sepharose beads as described in the Materials and Methods. The precipitates were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane. The presence of RNF182 fragments in the complex was revealed by Western blotting with anti-GST antibody. Lanes 1, 3, 5, 7 represent total cellular proteins extracted from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. Lanes 2, 4, 6, 8 indicate GST fused protein fragments precipitated by glutathione-sepharose beads from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. C. The presence of ATP6V0C in the co-precipitated protein complexes shown in B (lanes 2, 4 6, 8) was revealed by Western blotting using anti-flag antibody. In: lane RINGΔ69–247 – GST-RNF182 RING finger domain, lane C-endΔ1–68 – GST-RNF182 C-end domain. D. Co-localization of RNF182 and ATP6V0C. N2a cells were co-transfected with flag tagged ATP6V0C and EGFP tagged RNF182 for 24 h. Cells were fixed and stained with anti-flag antibodies. 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 × 40 fluorescence microscope. a. DAPI stained nuclei. b. EGFP tagged RNF182. c. Flag tagged ATP6V0C. d. a, b and c overlay.
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Figure 7: RNF182 physically interacts with ATP6V0C. A. Interaction between RNF182 and ATP6V0C in yeast two-hybrid system. Empty or RNF182 containing pGBKT7 bait vector and empty or ATP6V0C containing pACT2 library vector were co-transformed into yeast host cells AH109 and plated onto SD/-Trp -Leu -Ade -His +X-α-gal plate. In: a – a negative test of empty bait vector and ATP6V0C; b – a positive test showing the interaction between RNF182 and ATP6V0C; c – a negative test of RNF182 bait plus empty library vector. B. Total cellular proteins were extracted from HEK-293 cells co-transfected with flag-tagged ATP6V0C and GST-tagged RNF182 constructs and precipitated with glutathione-sepharose beads as described in the Materials and Methods. The precipitates were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane. The presence of RNF182 fragments in the complex was revealed by Western blotting with anti-GST antibody. Lanes 1, 3, 5, 7 represent total cellular proteins extracted from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. Lanes 2, 4, 6, 8 indicate GST fused protein fragments precipitated by glutathione-sepharose beads from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. C. The presence of ATP6V0C in the co-precipitated protein complexes shown in B (lanes 2, 4 6, 8) was revealed by Western blotting using anti-flag antibody. In: lane RINGΔ69–247 – GST-RNF182 RING finger domain, lane C-endΔ1–68 – GST-RNF182 C-end domain. D. Co-localization of RNF182 and ATP6V0C. N2a cells were co-transfected with flag tagged ATP6V0C and EGFP tagged RNF182 for 24 h. Cells were fixed and stained with anti-flag antibodies. 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 × 40 fluorescence microscope. a. DAPI stained nuclei. b. EGFP tagged RNF182. c. Flag tagged ATP6V0C. d. a, b and c overlay.

Mentions: Since the primary structure of RNF182 contained a RING finger domain and leucine repeats, which often participate in protein-protein interactions, we used yeast two-hybrid screening to identify a potential RNF182 interacting proteins in human brain. Yeast strain AH109 harboring the two-hybrid construct (pGBKT7-RNF182) expressing full length human RNF182 was used to screen a human brain cDNA library. Among 18 clones that displayed Ade/His prototype and β-galactosidase activity, 7 were found to represent a single unique gene encoding human ATPase, H+ transporting, lysosomal 16 kDa, V0 subunit C (ATP6V0C). The interaction between these two proteins was reproducibly reconstructed in the yeast two-hybrid system and it passed all required tests (Fig. 7A). Because the endogenous level of RNF182 was very low, in order to establish the interaction of these two proteins in vivo, we fused the RING finger domain, C-terminal domain and full length coding region of RNF182 with GST, which had previously been cloned into the mammalian expression vector pcDNA3.1. The coding region of ATP6V0C was sub-cloned into a pCMV-Tag1 vector carrying a flag tag. To perform the in vivo binding assay, HEK293 cells were transiently co-transfected with pCMV-Tag1-ATP6V0C and pcDNA3-GST-RNF182 or deletion constructs. The transfected cells were harvested and the protein extracts were incubated with Glutathione-Sepharose beads. The beads were precipitated by centrifugation, and the samples were boiled and separated by SDS/PAGE. The blot was first probed with anti-GST antibody to ensure that RNF182 protein and the deletion fragments were successfully precipitated by the procedure. We indeed observed fusion proteins of expected sizes after the Western analyses, including GST alone (Fig. 7B). The same protein samples were subsequently probed with anti-flag antibody for ATP6V0C. As shown in Fig. 7C, ATP6V0C bound to full length RNF182 and the C-end domain, but not to the RING finger domain or GST alone. These results confirmed the interaction detected through the yeast two-hybrid assay and also suggested that the RING finger domain did not play a role in this interaction. Next, we examined the sub-cellular localization of RNF182 and ATP6V0C. A EGFP fused RNF182 and flag tagged ATP6V0C were co-transfected into N2a cells. In transfected cells, co-localization of these proteins was detected in a punctuated pattern in the cytoplasmic and perinuclear regions. This is in agreement with the physical interaction detected in the yeast two-hybrid and co-precipitation assays.


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 physically interacts with ATP6V0C. A. Interaction between RNF182 and ATP6V0C in yeast two-hybrid system. Empty or RNF182 containing pGBKT7 bait vector and empty or ATP6V0C containing pACT2 library vector were co-transformed into yeast host cells AH109 and plated onto SD/-Trp -Leu -Ade -His +X-α-gal plate. In: a – a negative test of empty bait vector and ATP6V0C; b – a positive test showing the interaction between RNF182 and ATP6V0C; c – a negative test of RNF182 bait plus empty library vector. B. Total cellular proteins were extracted from HEK-293 cells co-transfected with flag-tagged ATP6V0C and GST-tagged RNF182 constructs and precipitated with glutathione-sepharose beads as described in the Materials and Methods. The precipitates were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane. The presence of RNF182 fragments in the complex was revealed by Western blotting with anti-GST antibody. Lanes 1, 3, 5, 7 represent total cellular proteins extracted from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. Lanes 2, 4, 6, 8 indicate GST fused protein fragments precipitated by glutathione-sepharose beads from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. C. The presence of ATP6V0C in the co-precipitated protein complexes shown in B (lanes 2, 4 6, 8) was revealed by Western blotting using anti-flag antibody. In: lane RINGΔ69–247 – GST-RNF182 RING finger domain, lane C-endΔ1–68 – GST-RNF182 C-end domain. D. Co-localization of RNF182 and ATP6V0C. N2a cells were co-transfected with flag tagged ATP6V0C and EGFP tagged RNF182 for 24 h. Cells were fixed and stained with anti-flag antibodies. 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 × 40 fluorescence microscope. a. DAPI stained nuclei. b. EGFP tagged RNF182. c. Flag tagged ATP6V0C. d. a, b and c overlay.
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Figure 7: RNF182 physically interacts with ATP6V0C. A. Interaction between RNF182 and ATP6V0C in yeast two-hybrid system. Empty or RNF182 containing pGBKT7 bait vector and empty or ATP6V0C containing pACT2 library vector were co-transformed into yeast host cells AH109 and plated onto SD/-Trp -Leu -Ade -His +X-α-gal plate. In: a – a negative test of empty bait vector and ATP6V0C; b – a positive test showing the interaction between RNF182 and ATP6V0C; c – a negative test of RNF182 bait plus empty library vector. B. Total cellular proteins were extracted from HEK-293 cells co-transfected with flag-tagged ATP6V0C and GST-tagged RNF182 constructs and precipitated with glutathione-sepharose beads as described in the Materials and Methods. The precipitates were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane. The presence of RNF182 fragments in the complex was revealed by Western blotting with anti-GST antibody. Lanes 1, 3, 5, 7 represent total cellular proteins extracted from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. Lanes 2, 4, 6, 8 indicate GST fused protein fragments precipitated by glutathione-sepharose beads from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. C. The presence of ATP6V0C in the co-precipitated protein complexes shown in B (lanes 2, 4 6, 8) was revealed by Western blotting using anti-flag antibody. In: lane RINGΔ69–247 – GST-RNF182 RING finger domain, lane C-endΔ1–68 – GST-RNF182 C-end domain. D. Co-localization of RNF182 and ATP6V0C. N2a cells were co-transfected with flag tagged ATP6V0C and EGFP tagged RNF182 for 24 h. Cells were fixed and stained with anti-flag antibodies. 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 × 40 fluorescence microscope. a. DAPI stained nuclei. b. EGFP tagged RNF182. c. Flag tagged ATP6V0C. d. a, b and c overlay.
Mentions: Since the primary structure of RNF182 contained a RING finger domain and leucine repeats, which often participate in protein-protein interactions, we used yeast two-hybrid screening to identify a potential RNF182 interacting proteins in human brain. Yeast strain AH109 harboring the two-hybrid construct (pGBKT7-RNF182) expressing full length human RNF182 was used to screen a human brain cDNA library. Among 18 clones that displayed Ade/His prototype and β-galactosidase activity, 7 were found to represent a single unique gene encoding human ATPase, H+ transporting, lysosomal 16 kDa, V0 subunit C (ATP6V0C). The interaction between these two proteins was reproducibly reconstructed in the yeast two-hybrid system and it passed all required tests (Fig. 7A). Because the endogenous level of RNF182 was very low, in order to establish the interaction of these two proteins in vivo, we fused the RING finger domain, C-terminal domain and full length coding region of RNF182 with GST, which had previously been cloned into the mammalian expression vector pcDNA3.1. The coding region of ATP6V0C was sub-cloned into a pCMV-Tag1 vector carrying a flag tag. To perform the in vivo binding assay, HEK293 cells were transiently co-transfected with pCMV-Tag1-ATP6V0C and pcDNA3-GST-RNF182 or deletion constructs. The transfected cells were harvested and the protein extracts were incubated with Glutathione-Sepharose beads. The beads were precipitated by centrifugation, and the samples were boiled and separated by SDS/PAGE. The blot was first probed with anti-GST antibody to ensure that RNF182 protein and the deletion fragments were successfully precipitated by the procedure. We indeed observed fusion proteins of expected sizes after the Western analyses, including GST alone (Fig. 7B). The same protein samples were subsequently probed with anti-flag antibody for ATP6V0C. As shown in Fig. 7C, ATP6V0C bound to full length RNF182 and the C-end domain, but not to the RING finger domain or GST alone. These results confirmed the interaction detected through the yeast two-hybrid assay and also suggested that the RING finger domain did not play a role in this interaction. Next, we examined the sub-cellular localization of RNF182 and ATP6V0C. A EGFP fused RNF182 and flag tagged ATP6V0C were co-transfected into N2a cells. In transfected cells, co-localization of these proteins was detected in a punctuated pattern in the cytoplasmic and perinuclear regions. This is in agreement with the physical interaction detected in the yeast two-hybrid and co-precipitation assays.

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