A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation
© Liu et al; licensee BioMed Central Ltd. 2008
Received: 15 November 2007
Accepted: 25 February 2008
Published: 25 February 2008
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.
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.
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.
Alterations in multiple biological pathways contribute to the development of a sporadic Alzheimer's disease (AD). Amongst these are excessive oxidative stress and insufficient antioxidant defenses, disrupted calcium homeostasis, altered cholesterol synthesis, inappropriate hormonal and growth factor signaling, chronic inflammation, aberrant re-entry of neurons into the cell cycle and, especially, altered protein processing, folding and turnover. The later abnormalities lead to β-amyloid peptide production and senile plagues development, tau hyperphosphorylation and neurofibrillary tangles (NFTs) formation [1, 2]. Collectively these changes contribute the loss of synapse, neuronal death and ultimately brain atrophy and dementia characteristic of this disease. However, the scope and complexity of these changes are such that the etiology of sporadic AD still remains elusive.
Recent advances in molecular biology have introduced new, high-throughput tools for the analysis of differential gene expression in complex diseases such as AD. They allow simultaneous overviews of the changes in gene expressions or protein levels for multiple cellular pathways. The most commonly used technology for the assessment of gene expression changes in postmortem brains is the DNA microarray [3–7]. However, this method requires prior knowledge of gene sequences and cannot be applied as a discovery tool for novel transcripts. Furthermore, the expression levels of low abundance genes cannot readily be assessed by DNA microarray hybridization, as reliable results are usually obtained only for genes that are expressed in high or moderate levels. This is a significant limitation as many transcripts expressed preferentially in the brain (e.g., neurotransmitter receptors and their regulatory factors) are present at very low levels [8, 9]. Recently, we employed a subtractive hybridization and RNA amplification method to enrich and isolate rare and novel transcripts from AD brains . Using this approach, we have isolated more than 200 genes, which are deferentially expressed, amongst these was a novel brain-enriched sequence that not only was up regulated in AD brains, but also in neuronal cells subjected to injuries.
Here we have described the cloning and characterization of this gene, which encodes a RING finger domain containing protein, resembling an ubiquitin E3 ligase and designated RNF182. We have established that RNF182 can stimulate E2-dependent polyubiquitination in vitro and identified an interaction between RNF182 and ATP6V0C. This interaction facilitates the degradation of ATP6V0C via the ubiquitin-proteosome pathway. Overexpression of RNF182 in N2a cells accelerated cell death and it's downregulation reduced cells' response to injurious insults.
RNF182 is a novel RING finger-containing transmembrane protein
The expression patterns of RNF182
RNF182 is up regulated in AD brains and in NT2 neurons subjected to injuries
Description of brain samples used for qRT-PCR analysis of RNF182
Probable AD, according to CERAD
Senile dementia of AD type
Definite AD, possible multi-infarct dementia
Senile changes of AD type
Moderate senile changes of AD type, dementia
Overexpression of RNF182 triggers cell death and its downregulation reduces cell death caused by OGD in N2a cells
RNF182 exhibits ubiquitin E3 ligase activity
The C-terminal domain of RNF182 interacts with ATPV0C
RNF182 facilitates ATP6V0C degradation via the ubiquitin-proteosome pathway
We have isolated a novel brain-enriched protein, RNF182, which was up regulated in AD brain tissues. Further study of its activities in an NT2 cell model revealed that this gene was barely detectable in undifferentiated NT2 cells, but it clearly expressed in differentiated neurons and astrocytes. Nevertheless, it was still a gene expressed at low abundance as compared with other cellular constituents (such as structural proteins). Consistent with the results obtained from AD brains, treatments of NT2 neurons with OGD or OGD plus β-amyloid peptide caused apparent upregulation of RNF182. Furthermore, overexpression of RNF182 in N2a cells by itself triggered cell death and it's downregulation reduced cell death caused by OGD, suggesting that this gene might have a specific function in brain cells under stress conditions.
One of the structural characteristics of the RNF182 protein is the RING finger domain located at the N-terminus, resembling ubiquitin E3 ligases. Our in vitro ubiquitination assay showed that RNF182, indeed, exhibited substrate-independent, E2-dependent ubiquitin ligase activity, which placed this protein in the ubiquitin-proteosome pathway. The most common role of E3 ubiquitin ligase in neurodegenerative diseases is to facilitate the degradation of unwanted, toxic proteins, thus preventing neuronal cell death caused by protein aggregation. For example, synaphilin-1, one of the major components of Lewy Bodies in Parkinson's disease, is a substrate for three RING finger containing E3 ligases [15–17]. Parkin mutations disrupting its E3 activity have been directly linked to autosomal recessive juvenile Parkinsonism . Similarly, a tripartite motif protein, TRIM11, negatively regulates Humanin, a neuroprotective peptide, against AD-related insults, through ubiquitin-mediated protein-degradation pathways . A recent report demonstrates that the upregulation of E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) collaborates with Hsp70 to attenuate tau aggregation in AD brains . Quantitative analyses of CHIP in different regions of AD and transgenic mouse brains show that CHIP level is inversely proportional to sarkosyl-insoluble tau accumulation, suggesting that the upregulation of CHIP may protect against the formation of NFTs. Another cytosolic RING finger protein, Dactylidin, is also found up regulated in highly vulnerable regions of AD brains . Although E3 activity of Dactylidin has not yet been demonstrated, these authors speculate that its upregulation in those regions might be related to a putative E3 function. The evidence documented above all seems to indicate a protective function of these proteins due to proteolysis of toxic proteins. However, RNF182 was found to be up regulated during neuronal cell apoptosis and its overexpression alone killed cells, suggesting a role in promoting cell death. This is in agreement with the recent findings that overexpression of a RING finger protein, SIAH-1, triggers apoptotic cell death in various cell types . These authors also find that accumulation of SIAH-1 protein is promoted by its interaction with a scaffold protein POSH upon receiving of apoptotic stimuli. SIAH-1, in turn, activates the JNK pathway, thereby contributing to the death of neurons and other cell types. The E3 ligase activity is essential for SIAH-1-evoked cell death. Based on our RT-PCR and Western blotting results, endogenous RNF182 was low at both mRNA and protein levels and its upregulation during apoptosis was reflected at both the transcription and translation levels. It is not yet clear whether its contribution to cell death is accomplished through collaborations with other proteins. This is still under investigation in our laboratory. Thus far, we found additional brain proteins interacting with RNF182 and their further characterization might shed new light on the biological significance of these interactions.
The interaction of RNF182 with ATP6V0C is intriguing since ATP6V0C is a multi-functional protein that appears to function at the intersection of a number of biological processes. The vacuolar H+ -ATPase (V-ATPase) is a multi-subunit enzyme present in intracellular membrane compartments such as endosomes, lysosomes, clathrin-coated vesicles and the Golgi complex, where it plays a role in their acidification and maintenance of endocytic and exocytic pathways . Its 16 kDa subunit (ATP6V0C) is a membrane spanning protein that folds into four trans-membrane helices and assembles into a hexamer, forming the membrane proton channel of the enzyme . In addition to its role in V-ATPase, ATP6V0C functions independently to form gap junction complexes and neurotransmitter release channels, playing an important role in neurotransmitter release [25, 26]. Based on homology comparison with yeast V-ATPase, the 4th trans-membrane domain of ATP6V0C should be located on the exterior of the proton channel where it could easily interact with adjacent protein trans-membrane domains . Scanning the primary structure of RNF182 revealed two typical trans-membrane helices, which could make this E3 ligase unique and placed it in close proximity with ATP6V0C in the membrane, where other E3s might not have easy access. Our results demonstrated that the physical interaction of these two proteins led to the degradation of ATP6V0C through the ubiquitin-proteosome pathway, making the present findings a new illustration of a novel RING finger protein that targets an essential component of neurotransmitter release machinery. This is in agreement with the report of Chin et al. , who demonstrate that the RING finger protein, Staring, targets syntaxin 1 for proteosomal degradation, implying an important role of the ubiquitin-proteosome pathway in the degradation of membrane proteins at the nerve terminals.
We have isolated a novel Ring finger E3 ubiquitin ligase, RNF182, that is up regulated in AD brain and in neuronal cells subjected to cell death-inducing stresses. It's overexpression in N2a cells by itself triggered cell death. It is unlikely that this killing is directly related to its promotion of ATP6V0C degradation, but it might be related to the interactions of NRF182 with other key signaling proteins that are currently investigated in our laboratory. Since ATP6V0C is a key component of gap junctions and neurotransmitter release channels, and RNF182 is up regulated in AD brains, it would be tempting to speculate that RNF182-mediated ATP6V0C degradation contributes to the pathophysiology of this disease. Further study of the molecular mechanism controlling such degradation of synaptic proteins will undoubtedly enhance our understanding of neurodegeneration in AD.
Cell culture and oxygen-glucose deprivation (OGD) treatment
Human embryonal teratocarcinoma Atera2/D1 (NT2) cells (Stratagene, La Jolla, CA), mouse Neuro-2a (N2a) neuroblastoma cells (ATCC CCL-131) and human HEK 293 cells  were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Bethesda, MD) supplemented with 10% fetal calf serum (GCS, Wisent, Inc. St. Bruno, PQ), and 40 μg/ml gentamicin sulfate (Sigma Cell Culture, St. Louis, MO). NT2 cells were differentiated into neurons and astrocytes with all trans-retinoic acid (RA, Sigma, Oakville, ON) according to the method of Pleasure and Lee  as described previously . N2a cells were differentiated into neurons by replacing culture medium with DMEM containing 0.5% FBS and 20 μM RA for 3 days.
For OGD treatment, NT2 neurons in T75 flasks were washed once with glucose-free DMEM, and incubated in glucose-free DMEM with 10% FBS for 2 h in a Gas Pak 100 chamber (VWR, Montreal, OC, Canada) as described previously . At the end of the OGD treatment, cells were removed from the chamber and returned to the incubator for 16 h. In a parallel experiment, 20 μM β-amyloid peptide (25–35 Aβ amide, Bachem California, Inc) was added to the culture medium during the OGD treatment and re-oxygenation period. The same OGD treatment was performed with N2a cells except the incubation in OGD conditions was 7 h. Cell viability for both cell lines was assessed by the Trypan Blue (Sigma, Oakville, ON) exclusion assay. Labelled cells were counted using a hemocytometer.
RNA extraction, RT-RCR and real time quantitative RT-PCR (qRT-PCR)
RNA extraction, first strand cDNA synthesis, and qRT-PCR analysis were performed as described previously . RNA pools extracted from frontal cortex of postmortem human brain samples described previously  were used for subtractive hybridization and qRT-PCR. Additional brain samples (Table 1) were obtained from the Human Brain and Spinal Fluid Resource Center (VAMC, Los Angeles, CA), which is sponsored by NINDS/NIMN, National Multiple Sclerosis Society, VA Greater Los Angeles Healthcare System, and Veterans Health Services and Research Administration, Department of Veteran Affairs. To detect the expression level of the RNF182 transcript in brain tissue and NT2 neurons, equal amounts of cDNA (2 ng each) were used with the primers: 360nhF 5' TGCCCGTGTGAGCTAGCA 3' and 360nhR 5' AGAACGGAGATATCCATGGTGAA 3' located in exon 2 of the gene. For semi-quantitative RT-PCR, a 395 bp cDNA fragment within the coding region of RNF182, and the entire coding region of ATP6V0C (468 bp) were amplified from first-stranded cDNA using the primers: 395F 5' TTGTGCCAAATGCCTCTACA 3' and 395R 5' ACGTGCAGTTCCACACAGTC 3', vATPcF 5' ATGTCCGAGTCCAAGAGCGGC 3' and vATPcR 5' CTACTTTGTGGAGAGGATGAG 3', respectively. PCR was performed as follows: 1 cycle at 94°C for 5 min, 30 cycles of 94°C for 45 sec, 60°C for 45 sec and 72°C for 45 sec. In the last cycle, the incubation was extended for 5 min at 72°C. The samples were separated on a 1% agarose gel containing 0.5 μg/ml ethidium bromide and photographed.
Cloning of the FNR182 transcript
A 300 bp cDNA fragment, 360nh, was isolated by subtractive hybridization using the mRNA population from AD brains as a "tester" and the first strand cDNA from control brains as a "driver" . To amplify the cDNA fragment overlapping with both 360nh and the existing mRNA (acc # AK090576), we used a forward RT-PCR primer 5'TGTTGTGGCCCTTAATCTGAGTGCTG 3' and a reverse primer 5' GATGTTGTTGTCATCGGGCAGGCTAC 3'. The PCR conditions were as described above. The resulting PCR product was cloned into pCR-Blunt II-TOPO vector (Invitrogen, Burlington, ON), subsequently analyzed by DNA sequencing and Genbank searches.
Plasmids and transient transfections
Human cDNA encoding the full length RNF182 protein was cloned into the pBAD/HisA vector (Invitrogen, Burlington, ON) for his-tag RNF182 protein production. To clone the GST-RNF182 or GST-RNF82 RING finger domain, or the GST-RNF182 C terminal domain into a mammalian vector, we first inserted GST sequences into the HindIII/XhoI site of pcDNA3.1 to form a pcDNA-GST tag vector. The entire coding region of RNF182, the N terminal 68 aa containing the RING finger domain and the C terminal 179 aa were then cloned into the EcoRI and XhoI site of the pcDNA3-GST tag vector, in frame with the GST sequence. The coding region of the ATP6V0C cDNA was cloned into the pCMV-Tag1 vector. These plasmids were transfected into HEK 293 cells for the co-precipitation assay.
Human cDNA encoding full length RNF182 protein was cloned in the pEGFP-N1 vector (Clontech, Palo Alto, CA, USA) with or without a stop codon added between the C-terminus of RNF182 and the EGFP sequence and the pcDNA3.1/myc-his vector (Invitrogen, Burlington, ON) to produce pRNF182*EGFP, pRNF182-EGFP and pcDNARNF182-myc-his constructs, respectively. For RNF182-EGFP localization analysis, N2a cells were plated on poly-lysine-coated cover slips in 6-well plates, at a density of 0.5 × 106 cells/well, 24 h before transfection. Cells were transfected with 5 μg/well of pRNF182-EGFP plasmid DNA and 15 μl LipofectAmine 2000 reagent (Invitrogen, Burlington, ON) according to the manufacturer's instructions. After 24 h, the cells were stained with anti-RNF182 antibody (dilution1:500 v/v) followed by Cy3-conjugated anti-rabbit IgG. The nuclei were counterstained with DAPI in PBS for 5 min and then mounted in Vectashield mounting medium (Vector laboratories, Burlingame CA, USA). The cells were viewed with a Zeiss Axiovert 200 M fluorescence microscope equipped with a Zeiss AxioCam camera (Zeiss, Midland, ON). The images were captured and analyzed using Zeiss Axiovision 3.1 software. For overexpression analysis, N2a cells were plated in 6-well plates at a density of 0.5 × 106 cells/well, 24 h before transfection. Cells were transfected with 5 μg pcDNARNF182-myc-his plasmid or pRNF182*EGFP plasmid and 15 μl lipofectAmine 2000 reagent, or co-transfected with 2.5 μg each of the pcDNARNF182-myc-his or pRNF182*EGFP and pCMV-Tag1-ATP6V0C plasmids plus 15 μl lipofectAmine 2000 reagent. Cells were collected for Trypan Blue exclusion assay as well as total RNA and protein extraction 24 h after transfection or treated with 30 μM MG132 (Sigma, Oakville, ON) for 8 h prior to total RNA and protein extraction. For siRNA silencing, the on-target plus smart pool siRNAs were purchased from Dharmacon (Dharmacon, Thermo Fisher Scientific, Inc). N2a cells were plated in 12-well plates at a density of 0.25 × 106cells/well, 24 h before transfection. Cell were transfected with 100 μM mouse RNF182 on-target plus smart pool siRNAs using Dharmafect1 transfection reagent according the manufacturer's instructions. Cells were subjected to 7 h OGD treatment 24–48 h after transfection and collected for Trypan Blue exclusion assay 16 h after re-oxygenation. For co-precipitation analysis, HEK 293 cells were plated in 10 cm plates at a density of 2 × 106 cells/plate, 24 h before transfection. Cells were co-transfected with 7.5 μg of pcDNA3 plasmid DNA harboring a GST-fused full-length, RING finger or C-end RNF182 cDNA fragment and 7.5 μg of pCMV-Tag1-ATP6V0C plasmid DNA mixed with 45 μl LipofectAmine 2000 reagent. Cells were collected for total protein extraction 48 h after transfection.
Antibody production and purification
Custom polyclonal antibody (GenScprit, Piscataway, NJ) was produced using synthetic peptide N'-ELLLTPKRLASLVSPSH (identical sequence between human and rodent). The immune serum was purified by immunoaffinity purification using recombinant his-tag RNF182 protein. Briefly, purified his-tag RNF182 protein was separated by SDS-PAGE and electro-blotted onto a nitrocellulose membrane. The Ponceau stained membrane portion containing the RNF182 antigen was excised and subjected to a Western blotting procedure using 2 mL original crude serum. The bound antigen-specific antibody was eluted with 0.1 M Glycine-HCl buffer, pH 2.7. The eluted antibody was neutralized by adding 1/10 volume of 1 M Tris, pH 8.5, concentrated using Amicon Ultra-15 Centrifugal Filter Device (Millipore, Fisher Scientific, Ottawa, ON).
Protein extraction, Western blotting and co-precipitation
Recombinant His-tag RNF182 protein was purified from Top10 cells using a HiTrap nickel column (Pharmacia Biotech, Baie d'Urfe', QC). The recombinant GST fusion SIAH-1 protein was purified from Rosetta cells harboring a pGEX4T-1/SIAH plasmid (a kind gift of Dr. M. Weissman, NIH, USA) . For total protein extraction from cultured cells, cells were trypsinized and collected by centrifugation. They were washed twice with PBS and lysed with RIPA buffer containing 1X protease Inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The lysate was vortexed and incubated on ice for 15 min, followed by sonication for 30 sec. In some cases the total cellular protein was freeze-dried and reconstituted in PBS in order to achieve a higher concentration. Western blotting analyses were performed as previously described . The blots were probed with the following primary antibodies: Rabbit polyclonal, affinity-purified anti-RNF182 (1:1000), rabbit polyclonal anti-flag (1:1000, Rockland, Gilbertsville, PA), goat polyclonal anti-GST (1:1000, Amersham Phamacia Biotech, Baie d'Urfe, QC), mouse monoclonal anti-ubiquitin (1:1000), and mouse monoclonal anti-β-actin (1:5000 v/v, both Sigma, Oakville, ON). The antigens were detected using horseradish peroxidase-conjugated secondary antibodies: anti-mouse IgG (1:5000 v/v), anti-rabbit IgG (1:5000 v/v, both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or anti-goat IgG (1:5000, Sigma, Oakville, ON). The antigen-antibody complexes were visualized by enhanced chemiluminescence using an ECL Plus detection kit (Amersham Phamacia Biotech, Baie d'Urfe, QC).
For the co-precipitation assay, flag-tagged ATP6V0C and GST-tagged RNF182 constructs were transiently co-transfected into HEK-293 cells and total cellular proteins were extracted as described above. The extracts were incubated with 200 μl of glutathione-sepharose beads for overnight at 4°C. Beads were precipitated by centrifugation at 10,000 g for 1 min and washed four times with PBST (1% Triton ×-100 in PBS), and samples were boiled in protein loading buffer and separated by 12% SDS-PAGE. The presence of RNF182 fragments and ATP6V0C in the complex was revealed by Western blotting as described above.
Yeast two-hybrid screening
Human cDNA encoding the full length RNF182 protein was cloned into the pGBKT7 vector (Clontch, Palo Alto, CA, USA) to generate a chimaeric open reading frame encoding the Gal4 DNA binding domain and RNF182 protein. This construct was introduced into Saccharomyces cerevisiae strain AH109. A single colony containing cells harboring the pGBKT7-RNF182 plasmid was then used to provide host cells for screening a human brain cDNA expression library constructed using the pACT2 vector (Clontech, Palo Alto, CA, USA). The protein-protein interaction was first screened by plating the transformants onto SD/-Trp-Leu-His-Ade selection plates. Positive clones were then re-screened for the presence of β-galactosidase activity to eliminate false interactions. Library plasmids harboring RNF182 interacting proteins were rescued and re-introduced into the RNF182/pGBKT7-containing host cells to further eliminate false interactions. The identity of the cDNA encoding RNF182-interacting protein was revealed by DNA sequencing and database searches.
In vitroubiquitination assay
Ubiquitination experiments were carried out according to a previously published report  with modifications. Thirty microliter, in vitro reactions were performed in ubiquitination buffer (50 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, 0.5 mM DTT, 2 mM ATP, 1 mM creatine phosphate) containing 0.5 units of creatine phosphokinase, 750 ng his-tag RNF182 or 1.3 μg GST-SIAH-1 (in the case of positive control reactions), 55 ng E1 (Boston Biochem, Cambridge, MA), 85 ng E2/Ubc5a (Boston Biochem, Cambridge, MA), 10 μg ubiquitin (Sigma-Aldrich, Oakville, ON), and 2 μL of bacterial lysate from Rosetta cells transformed with pGEX-3X. The mixture was incubated at 30°C for 90 min and the reaction was stopped by adding 5X SDS-PAGE loading buffer. The reaction mixture was resolved on 8% SDS-PAGE gel and analyzed by Western blotting using mouse anti-ubiquitin monoclonal antibody (Sigma-Aldrich, Oakville, ON).
The authors would like to thank Ms Stephanie Crosbie and Ms Julie LeBlanc for their technical assistance, and co-op students Amy Aylsworth, Sasha High, Katie Morse and Jill Taylor for their contribution to this project. We thank Dr. Hui Shen, Ms Caroline Sodja and Ms Maria Ribecco for providing total RNA samples of NT2 cells, Dr. Mahmud Bani for his assistance on fluorescence microscopy, and Dr. John van der Meer for editing this manuscript.
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