Molecular mechanism of citrate transporter NaCT and its mutations that cause pediatric epilepsies

摘要 癫痫脑病是一组不同类型的严重癫痫发作障碍，约占 25%的儿童癫痫。神经元柠檬酸转运蛋白SLC13A5/NACT基因突变使其丧失 功能性活动，导致早发性癫痫脑病(EOEE)。患者表现为癫痫发作。 生命的最初几个小时，并经历发育迟缓。钠离子驱动的柠檬酸转运蛋白(NACT)位于 神经元的质膜。作为柠檬酸循环的中间体，柠檬酸对能量至关重要。 在细胞中产生，它作为合成脂质和神经递质的前体。中断 因此，这种柠檬酸盐输入过程对神经元的伤害是可以理解的。根据它们的细胞表达 导致EOEE的模式、突变被分为两种类型。而I型(蛋白质位于 ER)降低蛋白质稳定性，II型(位于质膜中的蛋白质)直接影响底物和 钠结合部位或阻止运输所需的构象变化。基于同调模型 从我们的细菌琥珀酸转运蛋白VcINDY的晶体结构中构建的Nact，我们假设每个 突变类型改变了NACT结构，阻碍了运输活动。我们将描述这些机制的特征 以及使用生化、生物物理和结构方法相结合的结构变化。结果是 将帮助设计小分子增强剂和折叠校正器，有可能帮助年轻人 病人。

SUMMARY Epileptic encephalopathies are a heterogeneous group of severe seizure disorders accounting for approximately 25% of childhood epilepsies. Mutations in the neuronal citrate transporter SLC13A5/NaCT gene abolish its functional activity, causing early onset epileptic encephalopathy (EOEE). The patients display seizures in the first hours of life, and experience developmental delays. The Na+-driven citrate transporter (NaCT) is located in the plasma membrane of neurons. As an intermediate of the citric acid cycle, citrate is critical for energy production in the cell, and it acts as a precursor for the synthesis of lipids and neurotransmitters. Disruption of this citrate import process is therefore understandably harmful to neurons. According to their cellular expression patterns, mutations causing EOEE have been classified as two types. Whereas Type I (protein located in the ER) reduces protein stability, Type II (protein located in the plasma membrane) directly affects the substrate and sodium binding sites or blocks the conformational changes necessary for transport. Based on a homology model of NaCT built from our crystal structure of the bacterial succinate transporter VcINDY, we postulate how each type of mutation alters the NaCT structure and hinders transport activity. We will characterize the mechanisms and structural changes using a combination of biochemical, biophysical and structural approaches. The results will aid in the design of small molecule potentiators and folding correctors with the potential to help the young patients.