Xue Ting, Zhang Yan, Zhang Luofu, Yao Ling, Hu Xiaofang, Xu Lisa X
School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University , Shanghai, China.
J Proteome Res. 2013 Apr 5;12(4):1754-63. doi: 10.1021/pr301100r. Epub 2013 Mar 14.
Metastasis is the main cause for death of breast cancer patients. However, the underlying mechanism is still poorly understood. Plasma membrane (PM) proteins play a key role in various biological processes, especially for cell migration. In this study, we used a set of well-characterized mammary mouse cell lines, 67NR, 168FARN, 4T1, representing the metastatic progression, to study the differentially expressed membrane proteins. These proteins were analyzed by a linear ion trap tandem mass spectrometry (LTQ-MS/MS) following cell surface biotinylation and streptavidin purification. A total of 1667 membrane proteins were identified, out of which 472 were characterized as differentially expressed with at least 2-fold change and p-value < 0.01. Functional clustering of the 472 proteins revealed that 178 of them were metabolic proteins. Finally, we focused on two metabolic proteins, fatty acid synthase (FASN) and NAD(P)H steroid dehydrogenase-like protein (NSDHL), which were validated by Western blot and immunofluorescence. We found that FASN and NSDHL translocated to the plasma membrane from the intracellular compartment, and their expressions increased from 67NR to 4T1. This alteration of localization along with differential expressions might be necessary for metastasis development. Potentially, FASN and NSDHL could serve as drug targets in new antimetastasis therapy.
转移是乳腺癌患者死亡的主要原因。然而，其潜在机制仍知之甚少。质膜（PM）蛋白在各种生物学过程中起着关键作用，尤其是在细胞迁移方面。在本研究中，我们使用了一组特征明确的乳腺小鼠细胞系，67NR、168FARN、4T1，它们代表了转移进程，以研究差异表达的膜蛋白。在细胞表面生物素化和链霉亲和素纯化后，通过线性离子阱串联质谱（LTQ-MS/MS）对这些蛋白进行分析。总共鉴定出1667种膜蛋白，其中472种被鉴定为差异表达，变化倍数至少为2倍且p值<0.01。对这472种蛋白的功能聚类分析表明，其中178种是代谢蛋白。最后，我们聚焦于两种代谢蛋白，脂肪酸合酶（FASN）和NAD（P）H类固醇脱氢酶样蛋白（NSDHL），通过蛋白质免疫印迹和免疫荧光进行验证。我们发现FASN和NSDHL从细胞内区室转运到质膜，并且它们的表达从67NR到4T1逐渐增加。这种定位的改变以及差异表达可能是转移发展所必需的。潜在地，FASN和NSDHL可作为新的抗转移治疗中的药物靶点。
