广谱抗癫痫药丙戊酸诱导裂殖酵母蛋白糖基化缺陷的机制研究

VPA is used to treat a variety of psychiatric diseases such as seizure disorders, bipolar disorder and migraine, that is supposed by targeting GABA transaminase, succinate semialdehyde dehydrogenase, and alpha-ketoglutarate dehydrogenase and Na+ channels. Histone deacetylases (HDACs) were identified as direct targets of VPA in 2001. Although histone proteins were the first and the most important targets of HDACs, increasing evidence indicates that HDACs also deacetylate other nonhistone proteins such as α-tubulin, β-catenin, protein 53, 90-kDa heat shock protein, and et al. Our previous study indicated that the effects of VPA are likely to be much broader and more complicated than originally envisioned. Despite the progress that has been made in elucidating the biological and biochemical action of VPA, including VPA-induced defects in glycosylation, the exact mechanisms underlying their therapeutic effects have not been fully established. To better understand the molecular mechanisms of VPA action, we performed in fission yeast a genetic screen for mutants that show hypersensitivity to VPA. Here, we identified vrg4+ that encodes Golgi GDP-mannose transmembrane transporter Vrg4, which is essential protein for glycosylation. In Homo sapiens, SLC35D1 is orthologs of vrg4+, which encodes UDP-GlcA/ UDP-GalNAc transporter SLC35D1, which is critical to chondroitin sulfate synthesis in cartilage and skeletal development. Loss-of-function mutations of SLC35D1 induced Schneckenbecken dysplasia, but not with other severe spondylodysplastic dysplasias group diseases. In the present study, we supposed to find out the molecular mechanisms of VPA-induced defects in glycosylation via GDP-mannose trafficking pathway.

丙戊酸（VPA）是一种临床应用广、疗效高和安全性好的广谱抗癫痫药，首选用于治疗失神发作、肌阵挛发作，也可用于全身强直一阵挛性发作。对复杂部分性发作、单纯部分性发作的也有效果。但是其作用机理尚未完全阐明。我们前期实验提示VPA不仅作为组蛋白去乙酰化酶（HDAC）抑制剂参与调节各项生理机能，其作用机制可能存在多个靶点，比预期的更为广泛与复杂。同时VPA还可以诱导细胞内的糖基化作用缺陷。为了进一步阐明VPA的作用机制，我们将通过对模式生物裂殖酵母vrg4+基因（编码参与糖基化作用的必须蛋白—GDP-甘露糖运输体）进行功能解析，探讨VPA诱导细胞内蛋白糖基化缺陷的机制，为进一步阐明VPA的作用机制及研究由人类细胞内核苷酸糖（主要为甘露糖）的输送受损而导致的罕见致死性软骨发育不良（Schneckenbecken发育不良）提供新的依据。

丙戊酸(valproic acid, VPA)是一种临床广泛应用、疗效高和安全性好的广谱抗癫痫药物。目前普遍认为VPA能通过增加细胞内抑制性神经递质γ-氨基丁酸(GABA)的浓度，来降低神经元的兴奋性以及抑制钠离子通道从而抑制癫痫的发作，但其作用机制尚未完全阐明。组蛋白去乙酰化酶(histone deacetylases, HDACs)是第一个被报道的VPA直接作用靶点。前期的实验证明，裂殖酵母(Schizosaccharomyces pombe)许多基因缺失的突变株表现出对VPA敏感，而对其它的HDAC抑制剂sodium butyrate (SB)或Trichostatin A （TSA）不敏感，这提示了VPA存在多个作用靶点。本研究利用筛选裂殖酵母VPA感突(valproic acid sensitive, vas)变株，来进一步阐明VPA的作用机制。通过遗传互补实验克隆了vas4-1中VPA作用靶点基因vas4+/vrg4+。vas4+/vrg4+，编码高尔基体定位的GDP-甘露糖转运蛋白(Golgi GDP-mannose transporter), 参与调控蛋白糖基化修饰。序列比对发现SpVrg4p与酿酒酵母S. cerevisiae ScVrg4p高度同源。SpVrg4p是一个10跨膜蛋白，测序分析发现在vas4-1中SpVrg4p携带S263A和A271V两个突变位点，且都位于保守的GDP-mannose结合序列中。温度胁迫和细胞壁完整性(Cell wall integrity, CWI)抑制剂处理提示，SpVrg4p参与温度胁迫条件下维持酵母细胞壁完整性的生理过程。免疫共沉淀结果提示，SpVrg4p可能通过SpGot1p存在的COPII小泡在内质网与高尔基体之间转运，而S263A，A271V以及双突变均导致此过程的紊乱。研究结果提示vas4+/vrg4+是一个新的VPA靶点。VPA可能通过调控SpVrg4p的功能活性和蛋白转运来调控酵母细胞的糖基化及细胞壁合成等生理过程。