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Proteome analysis of SYT-SSX protein complexes in synovial sarcomas.

滑膜肉瘤中 SYT-SSX 蛋白复合物的蛋白质组分析。

基本信息

批准号：
18591632
负责人：
OUCHIDA Mamoru
金额：
$ 2.39万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591632/
关键词：
Synovial Sarcoma Chromosomal translocation SYT gene SSX gene 相互作用

项目摘要

In order to understand the molecular mechanism underlying the onset of synovial sarcomas, we analyzed fit SYT-SSX protein complex in synovial sarcoma cells by protecmics methods.Detection of the unknown proteins the SYT-SSX protein complex;We male the SYT, SSX, or SYT-SSX cDNA expression plasmid to produce FLAG-lag fusion protein and GST-tag fusion protein. These plasmids were transfected into HEK293 cells. The cellular proteins were prepared, and the fusion proteins were pulled dorm by anti-tag antibody-beads, and analyzed by western blotting. We could detect tie FLAG-tagged piths, but The yeti of FLAG-tagged SYT-SSX protein was low. The almost sane result was observed in tie experiment with GST-tagged *smiths, suggesting that the ION yeti was die to tie cellular localization of SYT-SSX fusion proteins within nucleus. So, we isolated the nucleus and the nuclear proteins were eluted in high cancer &fled salt condition. The eluted nuclear proteins were mixed will cytoplasmic proteins to make total cellular protein. FLAG-lagged SYT-,SSX protein was purified from the total cellular proteins, and the puffed FLAG-tagged SYT-SSX protein was incubated and re-constructed to make the SYT-SSX protein complex. Then the complex was puled by FLAG-beads, and the proteins were applied on SDS-PAGE. We analyzed sane bands of the complex by LC-MS.Cellular localization analysis of SYT-SSX protein in the presence of some inhibitors. We made the SYT, SSX, and SYT-SSX cDNA expression plasmids to produce EGFP-fusion proteins. The plasmids were reverse-transfected into SYO-1 that is a synivial cell line we established. We found that the SYTT-SSX proteins localize in nucleus with speckled form. We analyzed the effect of some inhibitors on the localization of the SYT-SSX proteins.We established an inducible cell line that can express the SYT-SSX gene in the presence of doxicyclin, and the gene expression pattern in the induced/non-induced cells were analyzed on cDNA microarray analysis.

为了了解滑膜肉瘤发病的分子机制，我们用保护性方法对滑膜肉瘤细胞中的Fit SYT-SSX蛋白复合体进行分析，检测未知蛋白质的SYT-SSX蛋白复合体；将SYT、SSX或SYT-SSX基因表达载体克隆到融合表达载体中，产生FLAG-LAG融合蛋白和GST-TAG融合蛋白。将其导入HEK293细胞。制备细胞蛋白，用抗Tag抗体珠拉取融合蛋白，进行Western blotting分析。我们可以检测到带有标志的pITS，但带有标志的SYT-SSX蛋白的yeti很低。在GST标记的Smiths的Tie实验中观察到几乎相同的结果，表明离子yeti与SYT-SSX融合蛋白在细胞核内的细胞定位有关。因此，我们分离了细胞核，并在高癌逃盐条件下洗脱了核蛋白。将洗脱出的核蛋白与胞浆蛋白混合制成细胞总蛋白。从细胞总蛋白中提纯FLAG标记的SYT-、SSX蛋白，并将膨化的FLAG标记的SYT-SSX蛋白孵育和重构，形成SYT-SSX蛋白复合体。然后用标志小球打浆，并将表达的蛋白质进行SDS-PAGE分析。我们用LC-MS分析了该复合体的同源性条带。在某些抑制剂存在的情况下，对SYT-SSX蛋白进行了细胞定位分析。构建了SYT、SSX和SYT-SSX基因表达载体，构建了融合蛋白的表达载体。将构建的重组表达载体反转染入我们建立的融合细胞系SYO-1。我们发现SYTT-SSX蛋白以斑点状存在于细胞核中。我们分析了几种抑制剂对SYT-SSX蛋白定位的影响，建立了在多西环素存在下可诱导表达SYT-SSX基因的细胞系，并利用基因芯片技术分析了诱导和未诱导细胞中的基因表达谱。