Modulation of D-serine levels via ubiquitin-dependent proteasomal degradation of serine racemase
Modulation of D-serine levels via ubiquitin-dependent proteasomal degradation of serine racemase
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DOI：
10.1074/jbc.m601971200
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发表时间：
2006-07-21
影响因子：
4.8
通讯作者：
Wolosker, Herman
中科院分区：
文献类型：
--
作者：
Dumin, Elena;Bendikov, Inna;Wolosker, Herman
哺乳动物的丝氨酸消旋酶是一种富含大脑的酶，在神经系统中将L-转化为D-丝氨酸。D-丝氨酸是受体/通道开放所必需的N-甲基D-天冬氨酸(NMDA)受体“甘氨酸位点”的内源性协同激动剂。调节D-丝氨酸合成的因素对NMDA受体的传递有影响，但对影响丝氨酸消旋酶水平的信号和事件知之甚少。我们在酵母双杂交筛选中发现丝氨酸外消旋酶与Golgin亚家族A成员3(Golga3)蛋白相互作用。通过脑组织匀浆的免疫共沉淀实验，证实了重组蛋白在体外与共转染HEK293细胞中的相互作用。在原代培养中，高尔基体3和丝氨酸消旋酶共同定位于胞浆、核周高尔基体区以及神经元和神经胶质细胞突起。在共转基因HEK293细胞和原代培养的星形胶质细胞中，GolGa3显著增加丝氨酸消旋酶的稳定水平。这一观察结果使我们研究了调节丝氨酸外消旋酶水平的机制。我们发现，丝氨酸消旋酶是通过泛素-蛋白酶体系统以Golga3调节的方式降解的。在体外和体内实验中，GolGa3都降低了丝氨酸消旋酶的泛素化，并显著延长了脉冲追逐实验中的蛋白质半衰期。我们的结果表明，泛素系统是丝氨酸外消旋酶和D-丝氨酸水平的主要调节因子。调节丝氨酸外消旋酶的降解，例如由GolGa3促进的外消旋酶的降解，为调节大脑D-丝氨酸水平和NMDA受体活性提供了新的机制。
Mammalian serine racemase is a brain-enriched enzyme that converts L-into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the "glycine site" of N-methyl D-as-partate (NMDA) receptors that is required for the receptor/ channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine racemase co- localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels. We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine racemase and D-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain D-serine levels and NMDA receptor activity.