Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry.

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Forsman, Alma,1979Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin,Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine (författare)

Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry.

Artikel/kapitelEngelska2008

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2008-05-06
American Chemical Society (ACS),2008

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LIBRIS-ID:oai:gup.ub.gu.se/84233
https://gup.ub.gu.se/publication/84233URI
https://doi.org/10.1021/pr700769eDOI

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Språk:engelska

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Ämneskategori:art swepub-publicationtype

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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.

Ämnesord och genrebeteckningar

MEDICIN OCH HÄLSOVETENSKAP Medicinsk bioteknologi Medicinsk bioteknologi hsv//swe
MEDICAL AND HEALTH SCIENCES Medical Biotechnology Medical Biotechnology hsv//eng
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

Biuppslag (personer, institutioner, konferenser, titlar ...)

Rüetschi, Ulla,1962Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin,Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine(Swepub:gu)xrueul (författare)
Ekholm, JosefineGothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin,Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine(Swepub:gu)xekbjo (författare)
Rymo, Lars,1940Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin,Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine(Swepub:gu)xrymla (författare)
Göteborgs universitetInstitutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin (creator_code:org_t)

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Ingår i:Journal of proteome research: American Chemical Society (ACS)7:6, s. 2309-191535-38931535-3907

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Av författaren/redakt...: Forsman, Alma, 1 ...; Rüetschi, Ulla, ...; Ekholm, Josefine; Rymo, Lars, 1940

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Av lärosätet: Göteborgs universitet

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