Search: onr:"swepub:oai:gup.ub.gu.se/84233" >
Identification of i...
Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry.
-
- Forsman, Alma, 1979 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin,Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
-
- Rüetschi, Ulla, 1962 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin,Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
-
- Ekholm, Josefine (author)
- Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin,Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
-
show more...
-
- Rymo, Lars, 1940 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin,Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
-
show less...
-
(creator_code:org_t)
- 2008-05-06
- 2008
- English.
-
In: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:6, s. 2309-19
- Related links:
-
https://gup.ub.gu.se...
-
show more...
-
https://doi.org/10.1...
-
show less...
Abstract
Subject headings
Close
- 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.
Subject headings
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)
Keyword
- Affinity Labels
- Cell Line
- Cell Line
- Tumor
- Chromatography
- Affinity
- methods
- Chromatography
- Gel
- Epstein-Barr Virus Nuclear Antigens
- genetics
- metabolism
- Genetic Vectors
- genetics
- HSC70 Heat-Shock Proteins
- analysis
- metabolism
- HSP70 Heat-Shock Proteins
- analysis
- metabolism
- Heterogeneous-Nuclear Ribonucleoprotein Group M
- analysis
- metabolism
- Humans
- Immunoprecipitation
- Peptides
- genetics
- Protein Binding
- Protein Interaction Mapping
- methods
- Protein Isoforms
- analysis
- metabolism
- Reproducibility of Results
- Staphylococcal Protein A
- genetics
- Tandem Mass Spectrometry
- methods
- Transfection
- Tumor Necrosis Factor Receptor-Associated Peptides and Proteins
- analysis
- metabolism
- Viral Proteins
- genetics
- metabolism
Publication and Content Type
- ref (subject category)
- art (subject category)
Find in a library
To the university's database