Forsman Alma, Rüetschi Ulla, Ekholm Josefine, Rymo Lars
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden.
J Proteome Res. 2008 Jun;7(6):2309-19. doi: 10.1021/pr700769e. Epub 2008 May 6.
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.
爱泼斯坦-巴尔病毒核抗原5（EBNA5）是初次感染EBV后最早检测到的病毒蛋白之一，已被证明是B淋巴细胞有效转化所必需的。EBNA5是一种具有多种假定功能的蛋白质，但其潜在生物学机制仍有待阐明。为了进一步深入了解多功能EBNA5的生物学作用，我们使用改良的串联亲和纯化（TAP）方法分离了含EBNA5的蛋白质复合物，并通过在LTQ-FT-ICR质谱仪上对胰蛋白酶消化产物进行LC-MS/MS分析来鉴定蛋白质成分。改良的TAP标签包含一个蛋白A结构域和一个StrepTagII序列，两者由两个烟草蚀纹病毒蛋白酶切割位点隔开，并与EBNA5的C末端融合。我们的结果证实了这种两步亲和纯化策略在哺乳动物细胞中纯化蛋白质复合物的广泛适用性。总共鉴定出147个新的假定EBNA5相互作用伙伴，其中37个在裂标签实验或从HEK293细胞提取物的共免疫沉淀中通过LC-MS/MS得到验证。这个亚组包括参与蛋白质折叠和复性的Bcl2相关抗凋亡基因2（BAG2）共伴侣蛋白、参与泛素/蛋白酶体蛋白降解调控的26S蛋白酶体亚基2以及参与前体mRNA加工的不均一核糖核蛋白M（hnRNP M）。这些EBNA5相互作用蛋白通过从三个EBV阳性淋巴母细胞系的细胞提取物中进行共免疫沉淀进一步得到验证。Hsp70、Hsc70、BAG2和26S蛋白酶体亚基2相互作用蛋白的组合表明，EBNA5可能与识别结构异常蛋白质并将其重新折叠为正常构象或使其降解的蛋白质质量控制系统存在功能关系。我们的研究还证实了先前鉴定的相互作用蛋白，包括HA95、Hsp70、Hsc70、Hsp27、HAX-1、脯氨酰4-羟化酶、S3a以及α-和β-微管蛋白。
