Bovine Papillomavirus Type 2 (BPV-2) E5 Oncoprotein Binds to the Subunit D of the V1-ATPase Proton Pump in Naturally Occurring Urothelial Tumors of the Urinary Bladder of Cattle

Background Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. Methods and Findings In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor β receptor. PDGFβR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as “V1-ATPase subunit D”, a component of the central stalk of the V1-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. Conclusion For the first time, a tri-component complex composed of E5/PDGFβR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V0-ATPase sector. We suggest that the E5/PDGFβR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

It has been suggested that BPV-2, a closely related serotype to BPV-1 [19], causes a latent infection of the urothelium, which can be activated by the chemical carcinogens of bracken fern ultimately resulting in bladder cancer [7].
The major transforming protein encoded by BPV-2 is the 44amino acid polypeptide E5. Bovine and human papillomavirus E5 proteins appear to be localized in the membranes of the endoplasmic reticulum, the Golgi apparatus and in the plasma membrane of the host cell [20]. It has been shown that E5 oncoprotein of bovine papillomavirus is responsible for cell transformation via several pathways [21,22] including the impairment of the V 0 -ATPase [23]. Furthermore, papillomavirus E5 protein is a powerful proteotoxic factor causing severe swelling and fragmentation of the Golgi apparatus and extensive vacuolization of the cytoplasm [24].
In vitro studies have revealed that BPV E5 oncoprotein can impair the vacuolar H+-ATPase proton pump as it is able to bind to its component, the cellular protein 16 k ductin/subunit c of the V 0 domain [25]. This pump is essential for the acidification of the intracellular organelle compartments and may have an important role in protein sorting and processing [26]. Dysfunction of the H+-ATPase proton pump can result in the perturbation of acidifica- tion of the endomembrane components and the cytosol. Furthermore, it has been suggested that the 16 k protein allows E5 to bind to the PDGFbR, the activation of which has a central role in bovine bladder carcinogenesis [15,18,21,26,27].
Herein we present in vivo data showing that E5 binds to the subunit D of the V 1 -ATPase proton pump in naturally occurring urothelial bladder tumors in cattle.

Ethics Statement
In this study we did not perform any animal experiments. We collected the samples directly from public slaughterhouses; the animals were slaughtered following a mandatory clinical antemortem examination as required by European Union legislation.

Tumor Samples
Fifteen bovine urothelial tumor samples and three normal (control) bladder samples were collected with the permission of the medical authorities in public and private slaughterhouses named ''Macello Comunale'' of Muro Lucano (PZ), ''Barbara Rocco sas'' of Simbario (VV), ''Real Beef srl'' of Flumeri (AV).
Bladder samples were routinely divided into several parts. Some parts were fixed in 10% buffered formalin for microscopic investigations. The remaining parts were immediately frozen in liquid nitrogen and stored at 280uC for subsequent biomolecular analysis.

Histopathology
The tissues fixed in 10% buffered formalin were routinely paraffin embedded. Histologic diagnosis was assessed on 5-mmthick hematoxylin-eosin (HE)-stained sections using morphologic criteria suggested in the recent report on the new histological classification of urothelial tumors of the urinary bladder of cattle [4].

Immunohistochemistry
All samples were stained and sections of normal bovine urinary bladder mucosa were tested in parallel as controls. Briefly, sections were deparaffinized, and blocked for endogenous peroxidase in 0.3% H 2 O 2 in methanol for 20 min. Antigen enhancement was performed by pretreating with microwave heating (twice for 5 min each at 750 W). The slides were washed three times with phosphate buffered saline (PBS, pH 7.4, 0.01 M). They were incubated for 1 h at room temperature with donkey serum (Santa Cruz Biotechnology Inc., CA, USA) diluted at 1 in 20 in PBS for the E5 detection and with protein block serum-free (DakoCytomation, Denmark) for V 1 -ATPase subunit D and pPDGFbR detection. The following primary antibodies were used: a purified polyclonal sheep anti-BPV-2 E5 (a kind gift by Dr. L. Nasir, Glasgow University), a monoclonal mouse anti-V 1 -ATPase subunit D (Santa Cruz Biotechnology Inc., CA, USA) and a polyclonal goat anti-pPDGFbR (phosphorylated at Tyr 770 ) (Santa Cruz Biotechnology Inc., CA, USA). They were diluted at 1 in 5000, at 1 in 50, at 1 in 200 in phosphate buffered saline (PBS;    Total protein extracts from tissue lysates were generated and used in Western blot analysis with an antibody specific for total PDGFbR and a phosphospecific PDGFbR antibody that recognized pPDGFbR phosphorylated at Tyr770. Lanes 1-3: urinary bladder from healthy animals. Lanes 4-6: representative neoplastic tissues from three cows with papillomavirusassociated tumors of the urinary bladder. Actin protein levels were detected to ensure equal protein loading. (y) Quantitative densitometric analysis of the filters was performed with Image Lab software (ChemiDoc; Bio-Rad Laboratories) and significance determined by the Student T-test (***, p, 0.001). doi:10.1371/journal.pone.0088860.g004 BPV-2 E5 Interacts with the V 1 -ATPase Proton Pump PLOS ONE | www.plosone.org Immunofluorescence All samples were stained and sections of normal bovine urinary bladder mucosa were tested in parallel as control. For two-color immunofluorescence, sections were deparaffinized, rehydrated and heated in a microwave oven in citrate buffer (twice for 5 min each at 750 W) to allow antigen unmasking. Briefly, the sections were rinsed in PBS, pre-incubated for 1 h with normal donkey serum (diluted at 1 in 20) and then overlaid with the purified polyclonal sheep anti-BPV-2 E5 primary antibody diluted at 1 in 500 in phosphate buffered saline (PBS; pH 7.4, 0.01 M) (a kind gift by Dr. L. Nasir, Glasgow University) and the polyclonal goat anti-pPDGFbR (phosphorylated at Tyr 770 ) primary antibodies (Santa Cruz Biotechnology Inc., CA, USA) diluted at 1 in 25 in phosphate buffered saline (PBS; pH 7.4, 0.01 M) were applied overnight at room temperature in a humified chamber.
Before the exposure to secondary antibodies, all the slides were washed for 20 min with PBS. A secondary antibody Alexa Fluor 488 donkey anti-sheep (Invitrogen, Molecular Probes) and a secondary antibody Alexa Fluor 546 donkey anti-goat (Invitrogen, Molecular Probes), diluted at 1 in 50 in PBS, were applied for 2 h at room temperature.
After washing 3 times with PBS, the slides were mounted under aqueous medium (Sigma-Aldrich, Milan, Italy).
An immunofluorescence staining was performed to detect V 1 -ATPase subunit D. The sections were treated as above, then the monoclonal mouse anti-V 1 -ATPase subunit D primary antibody (Santa Cruz Biotechnology Inc., CA, USA) diluted at 1 in 20 in phosphate buffered saline (PBS; pH 7.4, 0.01 M) was applied overnight at room temperature in a humid chamber. Before the exposure to secondary antibodies, all the slides were washed for 20 min with PBS. A secondary antibody Alexa Fluor 546 donkey anti-mouse (Invitrogen, Molecular Probes), diluted at 1 in 50 in PBS, was applied for 2 h at room temperature. After washing 3 times with PBS, the slides were mounted under aqueous medium (Sigma-Aldrich, Milan, Italy).
For two-color immunofluorescence staining of BPV-2 E5 and V 1 -ATPase subunit D, the sections were treated as above, then the polyclonal sheep anti-BPV-2 E5 (a kind gift by Dr. L. Nasir, Glasgow University) and the monoclonal mouse anti-V 1 -ATPase subunit D primary antibodies (Santa Cruz Biotechnology Inc., CA, USA) diluted respectively at 1 in 50 and 1 in 20 in phosphate buffered saline (PBS; pH 7.4, 0.01 M) were applied overnight at room temperature in a humid chamber. Before the exposure to secondary antibodies, all the slides were washed for 20 min with PBS. A secondary antibody Alexa Fluor 488 donkey anti-sheep  (Invitrogen, Molecular Probes) and a secondary antibody Alexa Fluor 546 donkey anti-mouse (Invitrogen, Molecular Probes), diluted at 1 in 50 in PBS, were applied for 2 h at room temperature.
After washing 3 times with PBS, the slides were mounted under aqueous medium (Sigma-Aldrich, Milan, Italy).
For two-color immunofluorescence staining of V 1 -ATPase subunit D and pPDGFbR (phosphorylated at Tyr 770 ), the sections were treated as above, then the monoclonal mouse anti-V 1 -ATPase subunit D primary antibody (Santa Cruz Biotechnology Inc., CA, USA) diluted at 1 in 20 in phosphate buffered saline (PBS; pH 7.4, 0.01 M) and the polyclonal goat anti-pPDGFbR (phosphorylated at Tyr 770 ) primary antibody (Santa Cruz Biotechnology Inc., CA, USA) diluted at 1 in 25 in phosphate buffered saline (PBS; pH 7.4, 0.01 M) were applied overnight at room temperature in a humid chamber. Before the exposure to secondary antibodies, all the slides were washed for 20 min with PBS. A secondary antibody Alexa Fluor 546 donkey anti-mouse (Invitrogen, Molecular Probes) and a secondary antibody Alexa Fluor 488 donkey anti-goat (Invitrogen, Molecular Probes), diluted at 1 in 50 in PBS, were applied for 2 h at room temperature.
After washing 3 times with PBS, the slides were mounted under aqueous medium (Sigma-Aldrich, Milan, Italy).
For all immunofluorescence observations and photography, a laser scanning confocal microscope LSM-510 (Zeiss, Göttingen, Germany) was used.

In-gel Digestion of IP Protein
Gel bands for mass spectrometric analysis were basically processed according to Shevchenko et al. [39]. Sliced gel pieces were washed with 100 mM NH 4 HCO 3 and acetonitrile (1:1, v/v) (buffer A). HPLC-grade acetonitrile was obtained from Sigma-Aldrich (St. Louis, MO). Proteins were in-gel reduced by 10 mM DTT, and subsequently alkylated with 20 mM iodoacetamide. After a washing step with buffer A, the gel pieces were dried in a vacuum centrifuge, and rehydrated at 4uC in digestion buffer  (50 mM NH 4 HCO 3 , 5 mM CaCl 2 ) containing 25 ng/ml trypsin. After overnight incubation, the peptides were extracted from the gel using three separate washings with a mixture of acetonitrile/ water/formic acid 70/25/5 (v/v/v). The extracts were combined and dried down in a vacuum centrifuge. The lyophilized digest was reconstituted in 30 ml of loading pump solvent (see nano LC-MS/MS Section). Ten ml of the solution were then injected for nano LC-MS/MS analysis.

Nano LC-MS/MS and Database Search
Chromatography was performed using an Ultimate nanoscale liquid chromatography (nano LC) system from Dionex (Sunnyvale, CA). The analytical nano LC column used was an in-house packed 75 mm i.d., 40 cm long Integra Frit TM column obtained from New Objective (Cambridge, MA), filled with 4 mm C 12 silica particles Jupiter Proteo from Phenomenex (Torrence, CA). Ten mL of the peptide mixture were loaded onto an in-house packed 150 mm i.d., 3 cm long Integra Frit TM (New Objective) trapping column (packing bed length 1 cm) at 12 mL/min of loading pump solvent, consisting of H 2 O/acetonitrile/trifluoroacetic acid (TFA) 97.95:2:0.05 (v/v/v). After 2 minutes washing, the trapping column was switched on-line to the analytical column, and gradient separation started at 200 nL/min.
MS detection was performed on a QSTAR XL hybrid mass spectrometer from Applied Biosystems (Foster City, CA) operating in positive ion mode, with nanoelectrospray (nESI) potential at 1800 V, curtain gas at 15 units, CAD gas at 3 units. nESI ionization was achieved via distal coated Pico Tips TM 20 mm ID,   Protein hits based on two successful peptide identifications were considered valid. Protein hits based on a single peptide identification with Mascot score higher than the significance level (.14) were retained after manual validation.

BPV-2 E5 and PDGFbR Immunoprecipitation
Tissues were lysed in ice-cold buffer containing 50 mM Tris-HCl (pH 7.5), 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM PMSF, 1.7 mg/ml Aprotinin, 50 mM NaF, and 1 mM sodium orthovanadate. The protein concentration was measured using the Bradford assay (Bio-Rad Laboratories, Milan, Italy). Proteins (1000 mg) were immunoprecipitated using 2 mg of anti-E5 antibody (a kind gift of Dr L. Nasir, Glasgow University) or anti-PDGFbR antibody (Santa Cruz Biotechnology, CA, USA) and 30 ml of Protein A/G-Plus Agarose (Santa Cruz Biotechnology, CA, USA). Immunoprecipitates were washed four times in complete lysis buffer (above), finally heated in 1X Laemmli sample buffer at 100uC for 10 minutes. Immunoprecipitates were separated on polyacrylamide gels and transferred to nitrocellulose filter membranes (Ge Healthcare Life Sciences, Chalfont St Giles, UK) for 16 h at 30 mA in 192 mM glycine/25 mM Tris-HCl (pH 7.5)/10% methanol. Membranes were blocked for 1 h at room temperature in 5% nonfat dry milk, incubated with anti-E5 antibody, anti-PDGFbR, anti-pPDGFbR (phosphorylated at Tyr 770 ), anti-V 1 -ATPase subunit D (Santa Cruz Biotechnology, CA, USA), and anti V 0 -ATPase c subunit (Cosmo Bio CO, Japan) overnight at 4uC. After three washes in Tris-buffered saline, membranes were incubated with rabbit anti-sheep IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology, CA, USA) or with goat anti-rabbit or anti-mouse IgG (Bio-Rad Laboratories, Milan, Italy) for 60 min at room temperature. Proteins were visualized by enhanced chemiluminescence system (Western Blotting Luminol Reagent, Santa Cruz Biotechnology, CA, USA) and ChemiDoc XRS Plus (Bio-Rad Laboratories, Milan, Italy). Images were acquired with Image Lab Software version 2.0.1 (Bio-Rad Laboratories, Milan, Italy).

BPV-2 DNA Detection and Sequencing
DNA was extracted from urinary bladder samples from frozen pathological and normal (control) bladder samples using the DNeasy Tissue kit (Qiagen, Milan, Italy) according to the manufacturer's protocol. All the samples were lysed using proteinase K. Lysates were loaded onto DNeasy spin columns. After two washing steps pure DNA was eluted with low salt buffer. To amplify the entire BPV-2 genome, the purified DNA was subjected to multiply primed rolling-circle amplification using a reaction mixture containing 20 ng sample DNA, 12.5 mM of each primer, 4 mM dNTPs and 10 U phi 29 DNA polymerase (Fermentas, Milan, Italy). The resulting linear dsDNA product was purified using MinElute PCR Purification kit (Qiagen, Milan, Italy). For the detection of BPV-2 DNA, specific primers for a 503 bp DNA amplicon encompassing the BPV-2 E5-L2 ORF sequence (nt 3723-4225) were designed by Primer BLAST  The reaction was carried out in a thermocycler (Veriti, Applied Biosystems, Monza, Italy) with an initial denaturation step of 3 min. Then, 35 cycles of amplification were carried out with a denaturation step at 94uC for 40 sec, an annealing step at 60uC, 30 sec, for b-actin or at 50uC,40 sec, for BPV-2, and an extension step at 72uC for 1 min. A final extension step at 72uC for 7 min was performed in each PCR assay. Detection of the amplified products was carried out by electrophoresis on ethidium bromide-stained agarose gel. In each experiment, a blank sample consisting of reaction mixture without DNA and a positive sample consisting of cloned BPV-2 (a kind gift by Dr. A. Venuti) were included. The amplified DNA was subjected to direct sequencing in an automated apparatus (ABI Prism 3100 Genetic Analyzer; Applied Biosystems, Monza, Italy).
Lysates were clarified at 5006g for 20 min. The protein concentration was measured using the Bradford assay (Bio-Rad Laboratories, Milan, Italy). For Western blotting, 50 mg of lysate proteins were heated at 100uC in 4X premixed Laemmli sample buffer (Bio-Rad Laboratories, Milan, Italy). Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. After electrophoresis, proteins were transferred onto nitrocellulose filter membranes (GE Healthcare Life Sciences, Chalfont St Giles, UK) for 1 h at 10 V in 192 mM glycine/25 mM Tris-HCl (pH 7.5)/10% methanol using a Trans-Blot SD Semi Dry cell (Bio-Rad Laboratories, Milan, Italy) according to the manufacturer's instructions. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS, pH 7.5) for 1 h at room temperature, washed with TBS-0.1% Tween. Then, filters were probed both with anti-PDGFbR, anti-pPDGFbR (phosphorylated at Tyr 770 ), and anti-V 1 -ATPase subunit D antibody (Santa Cruz   Biotechnology, CA, USA) for an overnight incubation at 4uC. After three washes in Tris-buffered saline, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Bio-Rad Laboratories, Milan, Italy) and antigoat IgG (Santa Cruz Biotechnology, CA, USA), for 1 h at room temperature. After appropriate washing steps, protein detection and image acquisition were performed as above reported.

RNA Extraction
Total RNA was extracted from urinary bladders of cows using the RNeasy Mini Kit (Qiagen, Milan, Italy), according to the manufacturer's instructions. The RNA quality was determined by agarose gel electrophoresis and ultraviolet spectrophotometer analysis. The RNA was treated with RNase-free DNase I Fermentas Life Sciences (Dasit, Milan, Italy) to remove potential DNA contamination.
cDNA Synthesis and Real Time-PCR Analysis (RT-PCR) for V 1 -ATPase Subunit D For Real Time-PCR analysis, 500 ng RNA were reversetranscribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Milan, Italy) and the reaction was incubated at 25uC for 5 min, 42uC for 30 min, 85uC for 5 min, and then kept at 4uC for 5 min. Real Time reactions were performed using SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Milan, Italy). For the detection of V 1 -ATPase subunit D specific primers (forward primer, 59-AAGACTCAGTGGCTGGGTTG -39; reverse primer, 59-AGGTTTCGACCTGTCTGTGC-39) were used. All reactions were performed in triplicate and b-actin was used as the internal standard (forward primer 59-TAGCA-CAGGCCTCTCGCCTTCG-39; reverse primer 59-GCA-CATGCCGGAGCCGTTGT-39).

Microscopical Pattern of the Tumors
Histological patterns of urothelial tumors of the urinary bladder of cattle were consistent with the diagnosis of papillary urothelial neoplasm of low malignant potential (PUNLMP) (two cases), papillary and invasive urothelial cancers (nine and four cases, respectively).

PCR Analysis and BPV-2 Sequencing
PCR yielded BPV-2 DNA fragments of anticipated size (503 bp) for all neoplastic lesions. No BPV-2 DNA was detected in normal (control) bladder samples (Figure 1). The presence of BPV-2 DNA was also confirmed by sequencing (Figure 1) according to BPV-2 sequence M20219.1.

Immunoprecipitation and Immunohistochemistry for BPV-2 E5 Protein
The expression of E5 was detected by immunoprecipitation and immunohistochemistry in tumor samples. E5 immunoreactivity was evident in cells located in basal and suprabasal urothelial layers (Figure 2 and 3).

Coimmunoprecipitation and Colocalization of E5 Oncoprotein with PDGFbR
Previous in vivo studies have shown that E5 binds to the activated (phosphorylated) form of PDGFbR in bladder tumors [18,21,27]. Indeed, PDGFbR appeared to be constitutively expressed and its phosphorylation was increased in the tumor samples compared to the healthy ones as detected in total lysates ( Figure 4). The activation of the phosphorylated PDGFbR was also documented by immunohistochemical investigations ( Figure 5).
A coimmunoprecipitation experiment using an anti-E5 antibody was carried out and PDGFbR and pPDGFbR were detected by Western blot in the immunoprecipitates ( Figure 6). Morphologically, the E5/pPDGFbR complex was shown by confocal microscopy: E5 and the activated form of the PDGFbR appeared to co-localize as judged by the yellow fluorescence of the merged images ( Figure 7). Normal urothelium yielded no E5 signal.

PDGFbR Binds to the Component D of V 1 -ATPase: Proteomic Analysis
In vitro, PDGFbR can bind to the proteolipid ring of V 0 sector of the proton pump in absence of E5, it has been suggested that E5 binds to the PDGFbR via the subunit c of the ring. However, these data have been obtained for medium-adapted cultured cells and hence may not reflect the authentic in vivo situation, for which no information is available thus far [26].
Accordingly, we investigated whether a similar complex could take place in vivo and performed immunoprecipitation of PDGFbR on bladder carcinoma tissue and on control normal bladder tissue from healthy cattle.
Proteins contained in the two PDGFbR pull-downs from bladder tumor samples and from normal bladder tissue, were separated with SDS-PAGE and detected with Coomassie staining. Differential analysis between the protein bands contained in the two immunoprecipitates revealed a single band present in the PDGFbR pull-down from bladder tumor samples only. The differential band was excised and in-gel digested with trypsin. The peptides were injected for nano LC-MS/MS analysis. Database search of MS/MS spectra allowed the identification of the protein ''V1-ATPase subunit D'' (Table 1). Because the protein has been identified by a single peptide hit having a low Mascot score, this result needed to be validated by further investigations. For this purpose, we performed Western blot analysis using an anti-subunit D antibody on the PDGFbR immunoprecipitates. This allowed us to detect the presence of the subunit D ( Figure 8). We also performed Western blot analysis using an anti-subunit c antibody on these immunoprecipitates as the PDGFbR/subunit c complex was documented in in vitro studies [25]. We did not detect the subunit c of the V 0 -ATPase domain (Figure 8). The complex pPDGFbR/subunit D of the V 1 -ATPase was shown by immunofluorescence studies as the two proteins appeared to colocalize by confocal microscopy (Figure 9).

Western Blot Analysis of the Subunit D
We performed immunoblotting to reveal the total level of the subunit D of the V 1 -domain. Overexpression of subunit D could be shown by immunoblotting ( Figure 10) and morphologically documented by immunofluorescence ( Figure 11). Furthermore, a statistically significant increase of the transcripts of this subunit was also shown by RT-PCR ( Figure 12). Normal levels of the constitutively expressed subunit c were detected by Western blot both in healthy and tumor samples ( Figure 13).

Coimmunoprecipitation and Colocalization of E5
Oncoprotein with the Subunit D of the V1-ATPase Using Western blot, we detected the subunit D of the V 1domain of the proton pump in E5 immunoprecipitates ( Figure 14). Morphologically, this complex was demonstrated by confocal microscopy as E5 and subunit D appeared to colocalize ( Figure 15). Normal urothelium yielded no E5 signal.
Ultimately, both pPDGFbR and subunit D of the V 1 -ATPase were found by Western blot in E5 immunoprecipitates ( Figure 16).

Discussion
Our results indicate, for the first time, that a ternary complex composed of BPV-2 E5 oncoprotein/PDGFbR/subunit D of V 1 -ATPase is present in urothelial cells of naturally occurring tumors of the bovine urinary bladder.
It has been shown that bovine papillomavirus E5 interacts with the subunit c of the V 0 domain in cultured cells [25]. As PDGFbR can bind to the subunit c in the absence of E5, it has been suggested that E5 binds to PDGFbR via its association with subunit of V 0 -ATPase proton pump [26].
Our in vivo findings appear to be different from previous in vitro results and show that the complex composed of E5 and the activated form of PDGFbR is associated with the overexpressed subunit D of the V 1 domain, which catalyzes ATP hydrolysis, but not with the subunit c of the V 0 domain responsible for H + translocation. Furthermore, the subunit c appeared to be constitutively expressed, as normal levels of expression were shown to occur both in normal and neoplastic tissues.
It has been suggested that cell perturbation resulting from binding of the subunit c of the proton pump and E5 oncoprotein is responsible for Golgi alkalinization which, in turn, leads to the activation of Golgi-associated Src molecules. Indeed, Golgi alkalinization and c-Src are involved in a common mechanism leading to E5-dependent NIH3T3 cell transformation [28].
The subunit D of the V 1 complex belongs to a central rotor stalk, which is composed also of F subunit. These proteins are bound directly to the subunit d of the V 0 sector that links to the c ring. Therefore, the central stalk (DFd complex) connects the V 1 and V 0 domains. It serves as a rotor that couples the energy that is released from the hydrolysis of ATP to the rotation of the proteolipid c ring of the V 0 -ATPase pump and causes active transport of protons thus regulating the pH (acidification) of intracellular organelles and cytosol [29][30][31]. The controlled pH of the intracellular compartments is crucial for many biological processes, including membrane trafficking and protein degradation. It is conceivable that in naturally occurring bovine bladder cancers, the complex E5/PDGFbR/subunit D could have an important role in perturbing proteostasis network as well as organelle and cytosol homeostasis. It is worthwhile noting that in bovine urothelial tumors we detected an overexpression of some of the most important markers of proteostasis stress such as heat shock proteins (HSPs) [32]. The latter are known to act as molecular chaperones to restore protein homeostasis [33,34]. Furthermore, we found an overexpression of the co-chaperone BAG3 (Bcl-2 associated athanogene 3), which depends on an altered degradation of the protein rather than the upregulation of gene transcription [35]. It is worth remembering that BAG3 protein degradation occurs via proteasome system only.
Cytosolic pH has been identified as a novel regulator that mediates the formation of proteasome storage granule (PSGs) and other protein aggregates. The regulation of proper partitioning of the proteasome into PSGs is essential for maintaining the correct level of the proteasome in the cytosol. It has been shown that the impaired ability of V-ATPase to regulate intracellular pH affects the kinetics of the PSG formation [30].
In our cases, the impairment of the vacuolar pump induced by E5 oncoprotein can be responsible for a proteasomal dysfunction resulting in a reduced clearance of specific protein such as proteasome-degraded BAG3 protein, known to be involved in a plethora of biological processes including the key role in mitigating the proteotoxicity via selective autophagy [36]. It has been shown that autophagy is activated when the proteasome function is reduced, thus constituting a strong functional link between autophagy and proteasome systems [37]. Our findings are consistent with several in vitro studies showing that the impairment of V-ATPase can induce autophagic responses and increase the formation of autophagosomes thus the autophagy represents a mechanism to overcome alteration of pH homeostasis mediated by proton pump perturbation [38].
Finally, the selective autophagy occurring in bovine urothelial tumor cells transformed by E5 oncoprotein that we have been studying (Roperto, unpublished data) seems to strengthen this suggestion and the emerging concept that the molecular chaperones, the ubiquitin-proteasome system (UPS) and the autophagy machinery are central elements of the proteostasis network in which the V-ATPase proton pump is also involved.