Abstract：
Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat kinase 2 (LRRK2) that is implicated in both familial and sporadic forms of Parkinson\'s disease (PD), and an abnormal increase in Rab3A phosphorylation has been proposed as a cause of PD. Despite the potential importance of Rab3A in PD pathogenesis, its structural information is limited and the effects of bound nucleotides on its biophysical and biochemical properties remain unclear. Here, we show that GDP-bound Rab3A is preferentially phosphorylated by LRRK2 compared with GTP-bound Rab3A. The secondary structure of Rab3A, measured by circular dichroism (CD) spectroscopy, revealed that Rab3A is resistant to heat-induced denaturation at pH 7.4 or 9.0 regardless of the nucleotides bound. In contrast, Rab3A underwent heat-induced denaturation at pH 5.0 at a lower temperature in its GDP-bound form than in its GTP-bound form. The unfolding temperature of Rab3A was studied by differential scanning fluorimetry, which showed a significantly higher unfolding temperature in GTP-bound Rab3A than in GDP-bound Rab3A, with the highest at pH 7.4. These results suggest that Rab3A has unusual thermal stability under physiologically relevant conditions and that bound nucleotides influence both thermal stability and phosphorylation by LRRK2.
摘要：
Rab3A是参与突触小泡运输的RabGTP酶家族的成员。最近的证据表明，Rab3A被富含亮氨酸的重复激酶2（LRRK2）磷酸化，该激酶与家族性和散发性帕金森病（PD）有关。Rab3A磷酸化的异常增加已被认为是PD的原因。尽管Rab3A在PD发病机制中具有潜在的重要性，其结构信息有限，结合核苷酸对其生物物理和生化特性的影响尚不清楚。这里,我们表明，与GTP结合的Rab3A相比，与GDP结合的Rab3A优先被LRRK2磷酸化。Rab3A的二级结构,通过圆二色性（CD）光谱测量，显示Rab3A在pH7.4或9.0时对热诱导的变性具有抗性，而与结合的核苷酸无关。相比之下,Rab3A在其GDP结合形式的温度低于GTP结合形式的温度下，在pH5.0下进行了热诱导的变性。用差示扫描荧光分析法研究了Rab3A的展开温度,GTP结合的Rab3A的展开温度明显高于GDP结合的Rab3A，在pH7.4时最高。这些结果表明Rab3A在生理相关条件下具有不寻常的热稳定性,并且结合的核苷酸影响热稳定性和LRRK2的磷酸化。
