丝氨酸消旋酶在中枢神经系统中的研究进展

中枢神经系统中,丝氨酸消旋酶是5'吡哆醛依赖性酶,通过合成调控D型丝氨酸,参与N-甲基-D-天冬氨酸受体介导的神经发生、突触可塑性及学习记忆的调节。丝氨酸消旋酶表达与活性可以通过转录、翻译、翻译后修饰,小分子配基与蛋白相互作用,亚细胞分布多种方式调节。丝氨酸消旋酶失调影响了精神分裂症、脑损伤及神经退行性疾病等多种中枢神经系统疾病。本文简要介绍丝氨酸消旋酶的结构、分布、调节因素和在中枢神经系统中的生理病理功能,为神经及精神疾病的治疗和药物开发提供了新的思路。     

小鼠丝氨酸消旋酶的克隆、表达与纯化

丝氨酸消旋酶(Serine Racemase,SR)是一种磷酸吡多醛依赖酶,在ATP和Mg2+的辅助下,催化L型丝氨酸转变为D型丝氨酸,D-丝氨酸通过结合在NMDA受体的Gly结合位点,调节其生理功能。本文将小鼠来源的丝氨酸消旋酶基因克隆至原核表达载体pMAL-C2上,构建重组质粒pMAL-C2-SR。将重组质粒转入E.coil BL21(DE3)宿主菌中,经IPTG诱导,获得重组表达。重组蛋白带有MBP标签,经Amylose亲合柱和Sephacryl S-200纯化,获得电泳纯的目的蛋白。     

D构象丝氨酸在中枢神经系统中的功能研究进展

哺乳动物中枢神经系统中D构象丝氨酸的区域性高浓度分布与N-甲基-D-天冬氨酸(NMDA)受体相一致.它主要由丝氨酸消旋酶将L丝氨酸直接消旋而来,也可能通过肠道菌群产生后吸收至体内,最终被D构象氨基酸氧化酶氧化.这种从胶质细胞而非神经元来源的“异常”构象氨基酸作为一种新型神经递质,不仅更新了传统“神经递质”的定义,而且为许多与NMDA受体过度兴奋或表达下调相关的神经系统疾病治疗提出了新的线索.     

以DL-丝氨酸为原料制备D-丝氨酸的新方法

以DL-丝氨酸为原料经过酯化、拆分和水解制备D-丝氨酸。使L-2,3-二苯甲酰酒石酸(L-DBTA)与DL-丝氨酸甲酯在无水乙醇中于60℃反应形成非对映体盐,冷却到0℃,D-丝氨酸甲酯.L-DBTA二盐析出,过滤后再经水解,得到D-丝氨酸,总收率为74.8%,旋光纯度达到98%以上。     

燕麦生物碱A通过调控丝氨酸羟甲基转移酶2重塑丝氨酸代谢抑制结肠癌生长

流行病学研究表明，燕麦类全谷食品可有效降低结肠癌的发病风险。丝氨酸/甘氨酸代谢途径对于维持肿瘤生长十分重要，能为肿瘤细胞核苷酸等生物大分子的合成提供底物，并保障肿瘤细胞免受氧化损伤。我们的前期研究表明，燕麦生物碱A(avenanthramide A, AVN A)具有抑制小鼠原位结肠癌生长的作用。本研究通过对氧化偶氮甲烷/葡聚糖硫酸钠(azoxymethane/dextran sulphate sulfate, AOM/DSS)小鼠结肠癌模型进行血液代谢组学研究，发现AVN A处理后，丝氨酸/甘氨酸代谢途径的变化最为显著(P<0.01)。丝氨酸、甘氨酸饥饿条件下，AVN A对结肠癌的杀伤效果显著增强(P<0.05),表明AVN A靶向丝氨酸代谢从而发挥抗结肠癌作用。随后，对丝氨酸代谢关键酶SHMT1、SHMT2、PHGDH、PSAT1及PSPH的mRNA及蛋白质表达水平进行检测，发现AVN A明显抑制SHMT2的表达(P<0.01或P<0.001)。过表达SHMT2后，由AVN A介导的对结肠癌细胞的生长、增殖、GSH/GSSG以及NADPH/NADP^...     

glyA基因及其编码的丝氨酸羟甲基转移酶

glyA基因广泛存在于生物体中 ,其编码的丝氨酸羟甲基转移酶 (serinehydroxymethyltransferase,SHMT)催化丝氨酸和甘氨酸之间的相互转化 ,转化反应产生的 5,1 0 亚甲基四氢叶酸 (M THF)提供细胞新陈代谢—碳单位 ,此反应在细胞新陈代谢中处于重要地位。因此 ,研究 glyA基因及其编码的丝氨酸羟甲基转移酶具有重要的意义。介绍了 glyA基因的克隆、序列分析、调控组分和丝氨酸羟甲基转移酶的部分性质。     

丝氨酸生产菌的筛选及静息细胞培养系统中L-丝氨酸的合成

从广西隆安县沼气池里的残渣中筛选到一株能以甲醇为唯一碳源生长的MB200菌株。根据常规形态特征、生理生化性状及16S rDNA基因序列分析将其鉴定为甲基杆菌属(M ethylobacteriumsp.)。其最佳生长条件为:温度32℃、pH值8.0、甲醇体积分数1.25%。建立了MB200生成L-丝氨酸的静息细胞培养系统。确定静息细胞培养的条件为:甘氨酸质量浓度为10 g.L-1,甲醇50 g.L-1,菌体质量浓度为30 g.L-1,pH8.9,于摇床250 r.m in-1,32℃静息培养48 h,L-丝氨酸产量为7.2 g.L-1。     

shPLCε通过YAP抑制前列腺癌细胞的丝氨酸/甘氨酸代谢和增殖 *

目的 该研究旨在探讨磷脂酰肌醇特异性磷脂酶C epsilon(phospholipase C epsilon, PLCε)对前列腺癌细胞丝氨酸/甘氨酸代谢及细胞增殖的影响。方法 慢病毒及质粒转染LNCAP、PC3细胞,q-PCR、Western blot分别检测LNCAP、PC3细胞中 PLCε、Yes相关蛋白(yes associated protein,YAP)、丝氨酸/甘氨酸生成酶[包括磷酸丝氨酸转氨酶1(phosphoserine aminotransferase1,PSAT1)、磷酸丝氨酸磷酸酶(phosphoserine phosphatase,PSPH)、丝氨酸羟甲基转移酶2(serine hydroxymethyltransferase2,SHMT2)及增殖相关基因细胞周期蛋白D1(Cyclin D1)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)]的表达情况;克隆形成实验及MTT实验检测细胞的克隆形成率及增殖活性。结果 (1)感染LV-shPLCε可显著下调前列腺癌细胞LNCAP、PC3中的PLCε、YAP、PSAT1、PSPH、SHMT2及增殖相关基因的mRNA及蛋白质水平,同时抑制细胞的克隆形成能力和增殖活性;(2)在shPLCε组细胞中加入过表达YAP质粒后,能明显逆转YAP、PSAT1、PSPH、SHMT2及增殖相关基因的下调,但加入干扰YAP质粒后结果相反。结论 shPLCε可通过下调YAP的表达抑制前列腺癌细胞的丝氨酸/甘氨酸生成,从而抑制细胞的增殖。     

SERPIN超家族的结构,功能及活性调节

丝氨酸蛋白酶抑制剂ＳＥＲＰＩＮ）超家族在体内生理及病理过程中起重要的调节作用,了是研究蛋白质结构与功能关系的良好模型,其独特的构象特征、作用机制、广泛的生物不肥及与疾病发生的联系等,已引起人们极大的关注。     

QscR基因的调控对菌株M.sp.SDM11产L-丝氨酸的影响

M.sp.SDM11是一株能以甲醇为唯一碳源生长的细菌,初步发酵检测发现能转化甘氨酸为L-丝氨酸。QscR基因产物是甲基营养菌中丝氨酸循环的一个转录调控关键因子,根据在GenBank中已报道的QscR基因序列(登录号:NC_012988.1)设计引物,以M.sp.SDM11的染色体DNA为模板,利用PCR扩增得到一大小为987 bp的QscR基因,将该基因克隆到广泛宿主载体pLAFR3上,在帮助质粒pRK2013的介导下,利用三亲本结合使其导入到菌株SDM11中构建重组菌株SDM12。对SDM12进一步研究发现,重组菌株中与L-丝氨酸合成相关的关键酶丝氨酸羟甲基还原酶(SHMT)的酶活比野生型菌株SDM11要低,约为野生型菌株的70%左右,另一个酶——羟基丙酮酸还原酶(HPR)的酶活力也只有野生型的75%。进一步将菌株进行产L-丝氨酸研究,结果表明,重组菌的产L-丝氨酸能力也明显降低,约为野生型菌株的67%左右。     

Neurobiology through the looking-glass: D-serine as a new glial-derived transmitter

D-Amino acids have been known to be present in bacteria for more than 50 years, but only recently they were identified in mammals. The occurrence of D-amino acids in mammals challenge classic concepts in biology in which only L-amino acids would be present or thought to play important roles. Recent discoveries uncovered a role of endogenous D-serine as a putative glial-derived transmitter that regulates glutamatergic neurotransmission in mammalian brain. Free D-serine levels in the brain are about one third of L-serine values and its extracellular concentration is higher than many common L-amino acids. D-Serine occurs in protoplasmic astrocytes, a class of glial cells that ensheath the synapses and modulate neuronal activity. Biochemical and electrophysiological studies suggest that endogenous D-serine is a physiological modulator at the co-agonist site of NMDA-type of glutamate receptors. We previously showed that D-serine is synthesized by a glial serine racemase, a novel enzyme converting L- to D-serine in mammalian brain. The enzyme requires pyridoxal 5'-phosphate and it was the first racemase to be cloned from eucaryotes. Inhibitors of serine racemase have therapeutic implications for pathological processes in which over-stimulation of NMDA receptors takes place, such as stroke and neurodegenerative diseases. Here, we review the role of endogenous D-serine in modulating NMDA neurotransmission, its biosynthetic apparatus and the potential usefulness of serine racemase inhibitors as a novel neuroprotective strategy to decrease glutamate/NMDA excitotoxicity.     

D-amino acids in the brain: D-serine in neurotransmission and neurodegeneration

The mammalian brain contains unusually high levels of D-serine, a D-amino acid previously thought to be restricted to some bacteria and insects. In the last few years, studies from several groups have demonstrated that D-serine is a physiological co-agonist of the N-methyl D-aspartate (NMDA) type of glutamate receptor -- a key excitatory neurotransmitter receptor in the brain. D-Serine binds with high affinity to a co-agonist site at the NMDA receptors and, along with glutamate, mediates several important physiological and pathological processes, including NMDA receptor transmission, synaptic plasticity and neurotoxicity. In recent years, biosynthetic, degradative and release pathways for D-serine have been identified, indicating that D-serine may function as a transmitter. At first, D-serine was described in astrocytes, a class of glial cells that ensheathes neurons and release several transmitters that modulate neurotransmission. This led to the notion that D-serine is a glia-derived transmitter (or gliotransmitter). However, recent data indicate that serine racemase, the D-serine biosynthetic enzyme, is widely expressed in neurons of the brain, suggesting that D-serine also has a neuronal origin. We now review these findings, focusing on recent questions regarding the roles of glia versus neurons in d-serine signaling.     

Protein kinase C activity regulates D-serine availability in the brain

D-serine is a co-agonist of NMDA receptor (NMDAR) and plays important roles in synaptic plasticity mechanisms. Serine racemase (SR) is a brain-enriched enzyme that converts L-serine to D-serine. SR interacts with the protein interacting with C-kinase 1 (PICK1), which is known to direct protein kinase C (PKC) to its targets in cells. Here, we investigated whether PKC activity regulates SR activity and D-serine availability in the brain. In vitro, PKC phosphorylated SR and decreased its activity. PKC activation increased SR phosphorylation in serine residues and reduced D-serine levels in astrocyte and neuronal cultures. Conversely, PKC inhibition decreased basal SR phosphorylation and increased cellular D-serine levels. In vivo modulation of PKC activity regulated both SR phosphorylation and D-serine levels in rat frontal cortex. Finally, rats that completed an object recognition task showed decreased SR phosphorylation and increased D-serine/total serine ratios, which was markedly correlated with decreased PKC activity in both cortex and hippocampus. Results indicate that PKC phosphorylates SR in serine residues and regulates D-serine availability in the brain. This interaction may be relevant for the regulation of physiological and pathological mechanisms linked to NMDAR function.     

AMPA receptor mediated D-serine release from retinal glial cells

The N-methyl-D-aspartate receptor (NMDAR) co-agonist D-serine is important in a number of different processes in the CNS, ranging from synaptic plasticity to disease states, including schizophrenia. D-serine appears to be the major co-agonist acting on retinal ganglion cell NMDA receptors, but the cell type from which it originates and whether its release can be modulated by activity are unknown. In this study, we utilized a mutant mouse line with elevated d-serine to investigate this question. Direct measurements of extracellular D-serine using capillary electrophoresis demonstrate that D-serine can be released from the intact mouse retina through an α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) dependent mechanism. α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-evoked D-serine release persisted in the presence of a cocktail of neural inhibitors but was abolished after administration of a glial toxin. These findings provide the first evidence that extracellular D-serine levels in the retina can be modulated, and that such modulation is contingent upon glial cell activity.     

D-serine: a new word in the glutamatergic neuro-glial language

Summary. Gliotransmission is a process in which astrocytes are dynamic elements that influence synaptic transmission and synaptogenesis. The best-known gliotransmitters are glutamate and ATP. However, in the past decade, it has been demonstrated that D-serine, a D-amino acid, acts as a gliotransmitter in glutamatergic synapses. The physiological relevance of D-serine is sustained by the way in which it modulates the action of glutamatergic neurotransmission, neuronal migration and long-term potentiation (LTP). In addition, the synthesis and degradation mechanisms of D-serine have been proposed as potential therapeutic targets for the treatment of Alzheimer’s disease, schizophrenia and related disorders. In the present review, detailed information is provided about the physiological and physiopathological relevance of D-serine, including metabolic and regulation aspects.     

D-Amino Acids as Putative Neurotransmitters: Focus on D-Serine

Of the twenty amino acids in the mammalian body, only serine and aspartate occur in D-configuration as well as L-configuration in significant amount. D-serine is selectively concentrated in the brain, localized to protoplasmic astrocytes that ensheath synapses and distributed similarly to N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. D-serine has been found to function as an endogenous ligand for the glycine site of the NMDA receptor. Evidences for this include the greater potency of D-serine to activate this site than glycine, and D-amino acid oxidase, which degrades D-serine as well as other neutral D-amino acids, markedly attenuates NMDA neurotransmission. D-serine is also formed by serine racemase, a recently cloned enzyme that converts L-serine to D-serine. Thus, in many ways D-serine fulfills criteria for defining its functionality as a neurotransmitter and challenges the dogma relating to neurotransmission, for it is the unnatural isomeric form of an amino acid derived from glia rather than neurons.     

Glia-derived D-serine controls NMDA receptor activity and synaptic memory

The NMDA receptor is a key player in excitatory transmission and synaptic plasticity in the central nervous system. Its activation requires the binding of both glutamate and a co-agonist like D-serine to its glycine site. As D-serine is released exclusively by astrocytes, we studied the physiological impact of the glial environment on NMDA receptor-dependent activity and plasticity. To this end, we took advantage of the changing astrocytic ensheathing of neurons occurring in the supraoptic nucleus during lactation. We provide direct evidence that in this hypothalamic structure the endogenous co-agonist of NMDA receptors is D-serine and not glycine. Consequently, the degree of astrocytic coverage of neurons governs the level of glycine site occupancy on the NMDA receptor, thereby affecting their availability for activation and thus the activity dependence of long-term synaptic changes. Such a contribution of astrocytes to synaptic metaplasticity fuels the emerging concept that astrocytes are dynamic partners of brain signaling.     

Microglia and Microglia-Like Cell Differentiated from DC Inhibit CD4 T Cell Proliferation

The central nervous system (CNS) is generally regarded as a site of immune privilege, whether the antigen presenting cells (APCs) are involved in the immune homeostasis of the CNS is largely unknown. Microglia and DCs are major APCs in physiological and pathological conditions, respectively. In this work, primary microglia and microglia-like cells obtained by co-culturing mature dendritic cells with CNS endothelial cells in vitro were functional evaluated. We found that microglia not only cannot prime CD4 T cells but also inhibit mature DCs (maDCs) initiated CD4 T cells proliferation. More importantly, endothelia from the CNS can differentiate maDCs into microglia-like cells (MLCs), which possess similar phenotype and immune inhibitory function as microglia. Soluble factors including NO lie behind the suppression of CD4 T cell proliferation induced by both microglia and MLCs. All the data indicate that under physiological conditions, microglia play important roles in maintaining immune homeostasis of the CNS, whereas in a pathological situation, the infiltrated DCs can be educated by the local microenvironment and differentiate into MLCs with inhibitory function.     

Protective roles of melatonin in central nervous system diseases by regulation of neural stem cells

Neural stem cells (NSCs) are immature precursors of the central nervous system (CNS), with self‐renewal and multipotential differentiation abilities. Their proliferation and differentiation are dynamically regulated by hormonal and local factors. Alteration in neurogenesis is associated with many neurological disorders. Increasing evidence suggests that modulation of NSCs can be a promising therapeutic approach for neural injury and neurodegenerative disorders. Melatonin, a pineal gland‐derived hormone, regulates the neuroimmuno‐endocrine axis and is functionally important to the circadian rhythm, tumour suppression and immunity. In the CNS, melatonin exerts neuroprotective effects in many diseases, such as Parkinson's disease, Alzheimer's disease and ischaemic brain injury. Emerging evidence suggests that it might also mediate such protective action by influencing proliferation and differentiation of NSCs. In this article, we review the current literature concerned with effects of melatonin on NSCs in different physiological and pathological conditions.     

Neuronal Chemokines: Versatile Messengers In Central Nervous System Cell Interaction

Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron–astrocyte, neuron–microglia, and neuron–neuron interaction.