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Abstract：
RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.
摘要：
RASGTPases与生物膜结合，它们作为分子开关调节细胞生长。最近的研究表明RAS蛋白在膜上寡聚化，破坏这些组件代表了另一种治疗策略。然而,关于RAS程序集的相互冲突的报告，大小从二聚体到纳米簇，已经提出了关于影响低聚的化学计量和参数的关键问题。这里,我们探索RAS的三种亚型[Kirsten大鼠肉瘤病毒癌基因（KRAS），哈维大鼠肉瘤病毒癌基因（HRAS），和神经母细胞瘤癌基因（NRAS）]使用质谱直接来自膜。我们表明，非活性状态（结合GDP）的膜上的KRAS是单体的，但在活性状态（结合GTP）下形成二聚体。我们证明小分子BI2852可以诱导KRAS的二聚化，而效应蛋白的结合破坏了二聚化。我们还表明RAS二聚化依赖于脂质组成，并揭示NRAS的寡聚化受到棕榈酰化的调节。通过监测RAS的固有GTP酶活性，我们在膜上捕获了含有混合核苷酸或GDP的二聚体的出现。我们发现，RAS与Sevenless的Son（SOScat）的催化结构域的相互作用受膜组成的影响。我们还通过SOScat捕获KRAS的活化和单体到二聚体的转化。这些结果不仅揭示了膜上RAS组件的化学计量，而且揭示了关键因素对低聚的影响。包括核苷酸的调节，脂质，和棕榈酰化。
