| 1. |
- Ekholm, Josefine
(author)
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The interactome of the Epstein-Barr virus nuclear antigen 5 suggests novel roles in RNA and protein metabolism
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Epstein-Barr virus (EBV) is a human herpes virus that infects B and epithelial cells of the oropharynx. EBV is transmitted by saliva and establishes a lifelong latency in over 90% of the world's population. During latency, the virus exists predominantly as multicopy episomes in the nuclei of memory B cells. If these memory B cells are activated they can produce EBV virions and the person may shed the virus. Most people are exposed to the virus as children, when the disease produces no noticeable symptoms or only flu-like symptoms. Latent infection is associated with several malignancies, including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, and lymphoproliferative disorders in the immunosuppressed patients. A compromised immune system and an aberrant EBV latent gene expression are thought to play roles in the aetiology of EBV malignancies. EBV immortalizes human B cells through expression of at least 12 viral genes, which include the EBNA5 protein. Elucidation of EBNA5 functions has been guided by identification of interacting cellular and viral proteins. The functions of these cofactors implicate EBNA5 as a potential modulator of apoptosis, cell cycle processes, and transcriptional pathways. This thesis will add to the knowledge about how EBNA5 contributes to EBV biology. By using luciferase and CAT as model proteins to study EBNA5-regulated gene expression in lymphoid cells, we found that EBNA5, at high but still biologically relevant levels, acted as a promiscuous repressor of gene expression from transfected plasmids. However, EBNA5 was more selective in the regulation of chromosomal genes; only two out of 588 human genes, thereof the pro-apoptotic BNIP3 gene, were down-regulated. The decrease in expression from transfected plasmids was partly explained by the ability of EBNA5 to inhibit luciferase pre-mRNA 3´-end cleavage and polyadenylation. To gain further insight into molecular pathways in which EBNA5 may have a regulatory role, we searched for cellular targets for EBNA5. By using an improved tandem affinity purification procedure, we identified 147 novel interaction partners of which 37 were validated with independent methods. The validated proteins could be grouped into three main classes depending on their biological function: I) protein folding and degradation, II) pre-mRNA processing, and III) ribosome biogenesis. We further showed that EBNA5 is part of high molecular complexes. Of particular interest, we verified interaction between EBNA5, Hsp70, and BAG2, a co-chaperone with the ability to inhibit ubiquitinylation, and as a consequence, protein degradation of Hsp70 clients. Further studies on the effect of EBNA5 on luciferase activity demonstrated a correlation between luciferase activity and level of soluble luciferase protein. In addition, substantial amounts of insoluble luciferase had accumulated in the nuclei. Insolubilisation was accompanied by translocation of luciferase, EBNA5, Hsp70, and BAG2 to the nucleoli and to a small number of nuclear foci. An EBNA5 mutant (ΔCR3) was neither able to induce a similar translocation nor able to associate with BAG2. Therefore, we identified BAG2 as a major target of EBNA5. Moreover, over-expression of Hsp70 blocked EBNA5-induced insolubilisation of luciferase. Our results indicate that EBNA5 is likely to play a regulatory role in the decision between degradation and folding.
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| 2. |
- Forsman, Alma, 1979, et al.
(author)
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Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry.
- 2008
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In: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:6, s. 2309-19
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Journal article (peer-reviewed)abstract
- Epstein-Barr virus nuclear antigen 5 (EBNA5) is one of the first viral proteins detected after primary EBV infection and has been shown to be required for efficient transformation of B lymphocytes. EBNA5 is a protein that has many suggested functions but the underlying biology remains to be clarified. To gain further insight into the biological roles of the proposed multifunctional EBNA5, we isolated EBNA5 containing protein complexes using a modified tandem affinity purification (TAP) method and identified the protein components by LC-MS/MS analysis of tryptic digests on a LTQ-FT-ICR mass spectrometer. The modified TAP tag contained a Protein A domain and a StrepTagII sequence separated by two Tobacco Etch Virus protease cleavage sites and was fused to the C-terminus of EBNA5. Our results confirmed the wide applicability of this two-step affinity purification strategy for purification of protein complexes in mammalian cells. A total of 147 novel putative EBNA5 interaction partners were identified, 37 of which were validated with LC-MS/MS in split-tag experiments or in co-immuno precipitates from HEK293 cell extracts. This subgroup included the Bcl2-associated Athanogene 2 (BAG2) co-chaperone involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. These EBNA5 interactors were further verified by co-immunoprecipitations from cell extracts of three EBV-positive lymphoblastoid lines. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 interactors suggests that EBNA5 might have a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Our study also confirms previously identified interactors including HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, and alpha- and beta-tubulin.
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| 3. |
- Löfgren, Maria, et al.
(author)
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Conditioned serum in vitro treatment of chondrocyte pellets and osteoarthritic explants
- 2023
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In: Equine Veterinary Journal. - : Wiley. - 0425-1644 .- 2042-3306. ; 55:2, s. 325-335
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Journal article (peer-reviewed)abstract
- Background: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. Objectives: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1 beta and cartilage explants with mild osteoarthritis. Study design: In vitro experimental study. Methods: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h] and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1 beta and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1 beta prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6 and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage. Results: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS-treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated-compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups. Main limitations: Results from in vitro models cannot be directly translated to in vivo situations. Conclusions: In vitro treatment with conditioned serum did not alleviate IL-1 beta-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants.
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| 4. |
- Nguyen, Duong, 1986, et al.
(author)
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Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink
- 2017
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Journal article (peer-reviewed)abstract
- Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes.
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