| 1. |
- Boreström, Cecilia, 1974, et al.
(author)
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E2F1, ARID3A/Bright and Oct-2 factors bind to the Epstein-Barr virus C promoter, EBNA1 and oriP, participating in long-distance promoter-enhancer interactions.
- 2012
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In: The Journal of general virology. - : Microbiology Society. - 1465-2099 .- 0022-1317. ; 93, s. 1065-75
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Journal article (peer-reviewed)abstract
- The Epstein-Barr virus (EBV) C promoter (Cp) regulates several genes required for B-cell proliferation in latent EBV infection. The family of repeats (FR) region of the latent origin of plasmid replication (oriP) functions as an Epstein-Barr nuclear antigen 1 (EBNA1)-dependent distant enhancer of Cp activity, and the enhancer-promoter interaction is mediated by a higher-order multi-protein complex containing several copies of EBNA1. Using DNA-affinity purification with a 170 bp region of the Cp in combination with mass spectrometry, we identified the cell cycle-regulatory protein E2F1, the E2F-binding protein ARID3A, and the B-cell-specific transcription factor Oct-2 as components of this multi-protein complex. Binding of the three factors to the FR region of oriP was determined by DNA-affinity and immunoblot analysis. Co-immunoprecipitation and proximity ligation analysis revealed that the three factors, E2F1, ARID3A and Oct-2, interact with each other as well as with EBNA1 in the nuclei of EBV-positive cells. Using the chromatin immunoprecipitation assay, we showed that E2F1 and Oct-2 interacted with the FR part of oriP and the Cp, but the ARID3A interaction was, however, only detected at the Cp. Our findings support the hypothesis that EBNA1 initiates transcription at the Cp via interactions between multiple EBNA1 homodimers and cellular transcription factors in a large molecular machinery that forms a dynamic interaction between Cp and FR.
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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. |
- Forsman, Alma, 1979
(author)
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The Epstein-Barr nuclear antigens 1 & 5; Study of virus-host cellular protein interactions
- 2009
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Doctoral thesis (other academic/artistic)abstract
- The Epstein-Barr virus (EBV) is the causative agent or cofactor in the aetiology of several human malignancies such as Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma (NPC) and lymphoproliferative disorder in immunocompromised patients. EBV is a lymphotropic γ-herpes virus infecting more than 90 percent of the population worldwide. Following acute infection the virus establishes a life-long latency in resting memory B cells. The virus is remarkable for the efficiency with which it causes proliferation and immortalization of the infected B cells through expression of several latent gene products. All of the viral EBNA proteins have been proposed to play a role in the control of gene expression in the EBV infected lymphoblastoid cell. The present thesis is mainly focused on further elucidating the molecular mechanisms of the EBNA1 and EBNA5 proteins using proteomic technologies as approach. In paper I we used an improved tandem affinity purification procedure for identification and characterization of factors in the EBNA5 interaction proteome. The majority of the 37 validated interactors could be assigned to one of three groups according to function: protein folding and degradation, pre mRNA processing, or ribosomal proteins, implicating functional relationships with EBNA5 in these processes. We also showed that EBNA5 is part of high molecular protein complexes, supporting the notion that functional units in the cell are not single proteins but well-structured complexes composed of multiple proteins i.e. modules. The previously reported repressor activity of EBNA5 was further investigated in paper II. The study identified the novel interactor BAG2 as a major target for the function of EBNA5 via the chaperone-mediated folding and proteasome-degradation pathways. Taken together, the results are consistent with the hypothesis that EBNA5 tune the balance between protein rescue and destruction in a way that disfavour the path of degradation. The constituents of the large macromolecular complex that initiates transcription from the viral C promoter were investigated in paper III. Using a DNA affinity procedure we showed that the transcription factors E2F1, ARID3A/Bright and Oct-2 binds the Cp as well as EBNA1 and oriPI, possibly facilitating long-distance promoter-enhancer interactions. While the study of genes and proteins continues to be important, looking at isolated components is not enough to understand most biological processes. Modularity has been proposed as a general principle for the molecular architecture of living systems. These assemblies interact with other large protein complexes, thus the proteins are part of a protein-protein interaction network inside the cell. A common feature of these interaction networks is that it contains junctions of proteins that are highly interconnected, also called hubs. Hubs have a tendency of being essential and involved in cancer development. Two central pathways in cancer biology are the Rb- and p53-pathways, which are targets for both EBNA1 and EBNA5 action. This is consistent with the hypothesis that several viral proteins target the same hubs in the host, which ensures the takeover of the cellular machineries essential for the viral infection and persistence processes, and contribute to the robustness of the viral infectious system.
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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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