Department of Biochemistry, Weill Cornell Medical College, New York, New York, USA.
Department of Biochemistry, Weill Cornell Medical College, New York, New York, USA.
J Biol Chem. 2022 Sep;298(9):102239. doi: 10.1016/j.jbc.2022.102239. Epub 2022 Jul 6.
Alpha-synuclein (a-Syn) is a presynaptic protein, the misfolding of which is associated with Parkinson's disease. Rab GTPases are small guanine nucleotide binding proteins that play key roles in vesicle trafficking and have been associated with a-Syn function and dysfunction. a-Syn is enriched on synaptic vesicles, where it has been reported to interact with GTP-bound Rab3a, a master regulator of synaptic vesicle trafficking. a-Syn is known to bind weakly to Rab8a in solution via a positively charged patch, but the physiological implications of such interactions have not been explored. Here, we investigate direct interactions between a-Syn and Rab3a in solution and on lipid membranes using NMR spectroscopy. We find that the C terminus of a-Syn interacts with Rab3a in a manner similar to its previously reported interaction with Rab8a. While weak in solution, we demonstrate that this interaction becomes stronger when the proteins are bound to a membrane surface. The Rab3a binding site for a-Syn is similar to the surface that contacts the Rab3a effector rabphilin-3A, which modulates the enzymatic activity of Rab3a. Accordingly, we show that a-Syn inhibits GTP hydrolysis by Rab3a and that inhibition is more potent on the membrane surface, suggesting that their interaction may be functionally relevant. Finally, we show that phosphorylation of a-Syn residue Ser 129, a modification associated with Parkinson's disease pathology, enhances its interactions with Rab3a and increases its ability to inhibit Rab3a GTP hydrolysis. These results represent the first observation of a functional role for synuclein-Rab interactions and for a-Syn Ser 129 phosphorylation.
α-突触核蛋白（a-Syn）是一种突触前蛋白，其错误折叠与帕金森病有关。Rab GTP 酶是小分子鸟嘌呤核苷酸结合蛋白，在囊泡运输中发挥关键作用，并与 a-Syn 的功能和功能障碍有关。a-Syn 在突触小泡中丰富，据报道它与 GTP 结合的 Rab3a 相互作用，Rab3a 是突触小泡运输的主要调节因子。已知 a-Syn 通过带正电荷的斑块在溶液中与 Rab8a 弱结合，但尚未探索这种相互作用的生理意义。在这里，我们使用 NMR 光谱法研究了 a-Syn 和 Rab3a 在溶液中和脂质膜上的直接相互作用。我们发现，a-Syn 的 C 端以类似于其先前报道的与 Rab8a 相互作用的方式与 Rab3a 相互作用。虽然在溶液中较弱，但我们证明当蛋白质结合到膜表面时，这种相互作用会变得更强。a-Syn 与 Rab3a 的结合位点类似于与 Rab3a 效应物 rabphilin-3A 接触的表面，rabphilin-3A 调节 Rab3a 的酶活性。因此，我们表明 a-Syn 抑制 Rab3a 的 GTP 水解，并且在膜表面上抑制作用更强，这表明它们的相互作用可能具有功能相关性。最后，我们表明，与帕金森病病理学相关的修饰物 a-Syn 残基 Ser129 的磷酸化增强了它与 Rab3a 的相互作用，并增加了它抑制 Rab3a GTP 水解的能力。这些结果代表了首次观察到突触核蛋白-Rab 相互作用和 a-Syn Ser129 磷酸化的功能作用。
