Structure-function relationship of the bovine papillomavirus E2 protein

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Proteomic and Biochemical Analysis of Extracellular Vesicles Isolated from Blood Serum of Patients with Melanoma

Background: Malignant melanoma is the most serious type of skin cancer with the highest mortality rate. Extracellular vesicles (EVs) have potential as new tumor markers that could be used as diagnostic and prognostic markers for early detection of melanoma. Methods: EVs were purified from the blood serum of melanoma patients using two methods—ultracentrifugation and PEG precipitation—and analyzed by mass spectrometry and immunoblot. Results: We identified a total of 585 unique proteins; 334 proteins were detected in PEG-precipitated samples and 515 in UC-purified EVs. EVs purified from patients varied in their size and concentration in different individuals. EVs obtained from stage II and III patients were, on average, smaller and more abundant than others. Detailed analysis of three potential biomarkers—SERPINA3, LGALS3BP, and gelsolin—revealed that the expression of SERPINA3 and LGALS3BP was higher in melanoma patients than healthy controls, while gelsolin exhibited higher expression in healthy controls. Conclusion: We suggest that all three proteins might have potential to be used as biomarkers, but a number of issues, such as purification of EVs, standardization, and validation of methods suitable for everyday clinical settings, still need to be addressed

Depletion of WBSCR22 reduces cell growth.

<p>(A) Protein expression of siWBSCR22 and siNeg. transfected cells was determined by western blot analysis using anti-WBSCR22 and anti-tubulin antibodies. Proteins from 10⁵ cells are loaded on each lane. (B) HeLa cells were transfected with siWBSCR22 or a control, siNeg, and the cell growth was monitored for five days. Average of three independent transfection experiments is shown.</p

Complementation of yeast <i>bud23Δ</i> strain with human WBSCR22.

<p>(A) Growth dilution assays of <i>bud23Δ</i> carrying plasmids <i>pRS315</i>, <i>pRS315-BUD23</i> and <i>pRS315</i>-WBSCR22. Cultures were grown overnight and diluted to final optical density at OD₆₀₀ 0.1, from which further 10-fold serial dilutions were spotted. Plates were incubated at 30°C for 3 days. (B) Polysome profiles of <i>bud23Δ</i> carrying plasmids <i>pRS315</i>, <i>pRS315-BUD23</i> and <i>pRS315</i>-WBSCR22. Cell lysates were centrifuged on 10-45% sucrose gradient. Absorbance at 254 nm was measured across the gradient, and the positions corresponding to the 40S, 60S and 80S ribosomal particles are indicated.</p

Characterization of the Functional Activities of the Bovine Papillomavirus Type 1 E2 Protein Single-Chain Heterodimers

Papillomaviruses are small DNA viruses which establish persistent infection in the epithelial tissue of various animal species. Three papillomavirus proteins encoded by the bovine papillomavirus type 1 E2 open reading frame have a common C-terminal DNA binding and dimerization domain and function as dimeric proteins in the regulation of viral gene expression, genome replication, and maintenance. The full-length E2 protein, expressed usually at the lowest level of the three, is an activator, while shorter forms of E2, lacking the transactivation domain, serve as repressors of replication and transcription. In virally infected cells, the full-length E2 protein forms heterodimers with repressor forms of the E2 protein and the biological activities of such heterodimers are poorly known. In order to study the functionality of E2 heterodimers, we joined the full-length E2 protein and E2 repressor by a flexible polypeptide hinge so that they formed a single-chain intramolecular dimer. The single-chain E2 heterodimers folded correctly to form genuine pseudodimers capable of binding to the specific E2 protein binding site with high affinity. Characterization of the activities of this protein in transcription showed that it functions as an effective transcriptional activator, which is comparable to what was found for the full-length E2 protein. The single-chain heterodimer is dependent to some extent on Brd4 protein and is able to support papillomavirus origin replication; however, it does not support the partitioning of the multimeric E2 binding site containing plasmids in dividing cells. Our results suggest that E2 heterodimers serve as activators of transcription and replication during the viral life cycle

The WBSCR22 protein in WBS patient lymhoblastoid cell lines.

<p>(A) Extracts from wild-type (E001 and E004) and three WBS patient-derived lymphoblastoid cell lines (GM13472, GM13473, GM13482) were immunoblotted to visualise the indicated proteins. (B) Quantification of western blot analysis. Average of three independent experiments with standard deviations is shown. The P value is in all cases below 0.005 using Student’s t-test.</p

Analysis of pre-rRNA processing in WBSCR22-depleted cells.

<p>(A) Pre-rRNA processing in HeLa cells according to Carron et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075686#B19" target="_blank">19</a>]. (B) Northern blot analysis of HeLa (lanes 1-6) and HEK293 (lanes 7, 8) cells transfected with siWBSCR22 or siNeg. with the 5’-ITS1 probe. RNA was extracted 72 h after transfection and 3 µg of total (T), 6 µg of cytoplasmic (C) and 3 µg of nuclear (N) RNA were loaded on each lane. Lanes 7 and 8 show nuclear RNA. The positions of precursor rRNAs are indicated. (C) The amounts of pre-rRNAs in the nuclear fractions of HeLa and HEK293 cells were quantified by PhosphorImager and are represented relative to siNeg of appropriate cell line which was set as 1. Results shown are representative of three independent transfection experiments with standard deviations. The P value is 0.2 for 21S and 0.02 for 18S-E using Student’s t-test. (D) Northern blot analysis of rRNA from T (total), C (cytoplasmic) and N (nuclear) fractions with the 5’-ITS1 probe from HeLa cells transfected with siWBSCR22 and plasmids expressing E2Tag-WBSCR22 or epitope tag alone. Band corresponding to 18S-E pre-rRNA is shown. (E) Protein expression of siWBSCR22-depleted cells transfected with expression plasmid for E2Tag-WBSCR22 was determined by western blotting using anti-WBSCR22 and anti-tubulin antibodies.</p

Localization of the WBSCR22 protein within the cell.

<p>(A) Subcellular localization of WBSCR22 by immunofluorescence analysis. The localization of endogenous protein in HeLa cells was analyzed by anti-WBSCR22 antibody. DAPI was used to stain the nucleus of the cell. (B) Total extracts of HeLa cells were centrifuged on 10-50% sucrose gradient at 27 000 rpm for 4 hours and fractionated. The 18S and 28S rRNAs were detected by ethidium bromide and the WBSCR22 protein was analyzed by western blotting using anti-WBSCR22 antibodies. (C) HeLa cell extracts were centrifuged on 10-50% sucrose gradient at 27 000 rpm for 13 hours and analyzed by western blotting.</p

The Stability of Ribosome Biogenesis Factor WBSCR22 Is Regulated by Interaction with TRMT112 via Ubiquitin-Proteasome Pathway.

The human WBSCR22 protein is a 18S rRNA methyltransferase involved in pre-rRNA processing and ribosome 40S subunit biogenesis. Recent studies have shown that the protein function in ribosome synthesis is independent of its enzymatic activity. In this work, we have studied the WBSCR22 protein interaction partners by SILAC-coupled co-immunoprecipitation assay and identified TRMT112 as the interaction partner of WBSCR22. Knock-down of TRMT112 expression decreased the WBSCR22 protein level in mammalian cells, suggesting that the stability of WBSCR22 is regulated through the interaction with TRMT112. The localization of the TRMT112 protein is determined by WBSCR22, and the WBSCR22-TRMT112 complex is localized in the cell nucleus. We provide evidence that the interaction between WBSCR22/Bud23 and TRMT112/Trm112 is conserved between mammals and yeast, suggesting that the function of TRMT112 as a co-activator of methyltransferases is evolutionarily conserved. Finally, we show that the transiently expressed WBSCR22 protein is ubiquitinated and degraded through the proteasome pathway, revealing the tight control of the WBSCR22 protein level in the cells