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首页> 外文期刊>Science Signaling >Proximity biotinylation provides insight into themolecular composition of focal adhesions at thenanometer scale
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Proximity biotinylation provides insight into themolecular composition of focal adhesions at thenanometer scale

机译：邻近生物素化技术可洞察纳米级粘着斑的分子组成

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摘要

Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through theintegrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referredto as the “adhesome.” We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells tolabel proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, whichdirectly binds b integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only sevenof these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. Theproteins in proximity to b integrin included 15 of the adhesion proteins identified in the paxillin BioID dataset. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller dataset, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin b1 andEFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesomebased on both data sets suggests that focal adhesions contain fewer components than previouslysuspected and that paxillin lies away from the plasma membrane. These data not only illustrate thepower of using BioID and stable isotope–labeled mass spectrometry to define macromolecularcomplexes but also enable the correct identification of therapeutic targets within the adhesome.
机译：粘着斑是通过后跨膜蛋白整联蛋白家族将后生细胞连接到细胞外基质的蛋白复合物。整合素将许多蛋白质募集到这些复合物中，称为“ adhesome”。我们在U2OS骨肉瘤细胞中使用了邻近依赖性生物素化（BioID）来标记Paxillin在15至25 nm内的蛋白，一种胞浆粘着蛋白和kindlin-2，后者直接结合b整合素。通过对生物素化蛋白的质谱分析，我们鉴定出27种已知的脂质体蛋白和8种先前未知的成分（接近于paxillin）。但是，这些蛋白中只有7种直接与paxillin相互作用，其中之一是衔接蛋白Kank2。 b整联蛋白附近的蛋白质包括在paxillin BioID数据集中鉴定的15种粘附蛋白。 BioID还正确地将kindlin-2建立为细胞-细胞连接蛋白。通过关注这个较小的数据集，发现了kindlin-2的新伙伴，即内吞作用促进蛋白脂蛋白b1和EFR3A，但与以前的报道相反，不是纤维蛋白结合蛋白migfilin。基于这两个数据集的模型，表明粘着斑包含的成分比以前怀疑的要少，并且帕西林远离质膜。这些数据不仅说明了使用BioID和稳定同位素标记的质谱法定义大分子复合物的能力，而且还能够正确识别胶体中的治疗靶标。

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