Ledesma Maria Dolores, Da Silva Jorge Santos, Schevchenko Anna, Wilm Matthias, Dotti Carlos G
Cavalieri Ottolenghi Scientific Institute, Universita degli Studi di Torino, Ospedale San Luigi, Regione Gonzole 10, 10043 Turin, Orbassano, Italy.
Brain Res. 2003 Oct 10;987(1):107-16. doi: 10.1016/s0006-8993(03)03296-7.
Sorting of certain membrane proteins requires a mechanism involving rafts, protein-lipid complexes enriched in glycosphingolipids and cholesterol. These microdomains remain at the plasma membrane of different cell types and play a role in signal transduction. Although recent reports have begun to describe molecules associated with rafts, their protein composition remains largely unknown, especially in neuronal cells. To address this question, we have purified detergent-insoluble raft fractions (DRMs) from primary cultures of hippocampal neurons. Bidimensional gel analysis and pharmacological raft lipid manipulation allowed the identification of neuronal raft proteins and their characterisation by MALDI-TOF analysis. Enolases were found among the proteins identified and functional studies demonstrate their participation in plasminogen binding. We also show the specific enrichment in rafts of several other plasminogen binding molecules and the exclusive activation of plasminogen to the protease plasmin in these microdomains. These observations suggest that neuronal rafts may play, in addition to intracellular signaling, a role in extracellular/membrane protein proteolysis.
某些膜蛋白的分选需要一种涉及脂筏的机制，脂筏是富含糖鞘脂和胆固醇的蛋白质 - 脂质复合物。这些微结构域存在于不同细胞类型的质膜上，并在信号转导中发挥作用。尽管最近的报道已开始描述与脂筏相关的分子，但其蛋白质组成在很大程度上仍然未知，尤其是在神经元细胞中。为了解决这个问题，我们从海马神经元的原代培养物中纯化了去污剂不溶性脂筏组分（DRM）。二维凝胶分析和药理学脂筏脂质操作使我们能够鉴定神经元脂筏蛋白，并通过基质辅助激光解吸电离飞行时间（MALDI - TOF）分析对其进行表征。在所鉴定的蛋白质中发现了烯醇化酶，功能研究表明它们参与纤溶酶原结合。我们还展示了其他几种纤溶酶原结合分子在脂筏中的特异性富集，以及在这些微结构域中纤溶酶原向蛋白酶纤溶酶的特异性激活。这些观察结果表明，神经元脂筏除了在细胞内信号传导中发挥作用外，可能还在细胞外/膜蛋白蛋白水解中发挥作用。
