葡萄糖-6-磷酸脱氢酶的糖基化在肿瘤代谢中的作用

发布时间：2018-05-21 12:55

本文选题：戊糖磷酸途径 + 葡萄糖-6-磷酸脱氢酶　；参考：《浙江大学》2016年博士论文

【摘要】：肿瘤细胞为满足自身营养和能量需求,要消耗大量的葡萄糖,而且即使在有充足氧气条件下,糖酵解过程也会明显增加,大量的葡萄糖转换成乳酸,即所谓的瓦伯格效应(the Warburg effect)。瓦伯格效应是肿瘤细胞代谢重编程的一种体现,而肿瘤细胞的代谢重编程会发生在许多代谢通路中,包括葡萄糖转运、糖酵解、戊糖磷酸途径、谷胺酰胺代谢、三羧酸循环、电子传递链等过程。戊糖磷酸途径在快速增殖的细胞中,扮演着大分子生物合成和维持细胞氧化还原状态的作用。尤其是在许多肿瘤细胞中,戊糖磷酸途径都是上调的。然而,戊糖磷酸途径是如何上调以满足肿瘤细胞生长优势这一点还没完全弄清楚。葡萄糖-6-磷酸脱氢酶(G6PD)是戊糖磷酸途径的第一个酶,也是该途径的限速酶,催化来自糖酵解通路中的葡萄糖-6-磷酸,进行氧化脱氢,生成6-磷酸葡萄糖酸-δ-内脂和提供还原氢的NADPH。本研究用化学酶标法及质谱的方法,鉴定出G6PD的S84位被O-糖苷键共价连接的N-乙酰氨基葡萄糖(O-G1cNAc)糖基化修饰；将84位上的丝氨酸S突变成缬氨酸V,将不能检测到糖基化。S84位上的糖基化提升了G6PD的酶活性；而且通过戊二醛交联反应证实,高糖基化水平的G6PD更趋向于形成有活性的二聚体和四聚体。前人的研究已经发现有1000多种核质蛋白被O-G1cNAc修饰,并且这种糖基化修饰作为一种感受器,是细胞对外界营养和压力的动态反应。本研究通过缺氧处理、高糖刺激和血清刺激,发现G6PD对这些外界刺激非常敏感,其糖基化水平会随着缺氧处理时间的延长、葡萄糖浓度和血清浓度的增加而显著升高。同时,研究也证实高糖基化的G6PD,会提高戊糖磷酸途径中葡萄糖的摄入量,生成更多中间代谢产物,促进核苷酸和脂质的生物合并提升抗氧化防御能力,以此为细胞提供生长优势,促进细胞增殖。更具生理意义的是,异位裸鼠成瘤实验证实G6PD的糖基化有利于肿瘤的生长,而阻止G6PD的糖基化,显著降低细胞增殖速度,减慢肿瘤生长；对39例临床肿瘤样本的研究发现,除了有8例由于低表达G6PD而无法可靠地研究糖基化外,其余31例中有16例,G6PD糖基化水平在人肺癌组织中比癌旁组织中高,并且31例中有20例,糖基转移酶(OGT)的表达水平在癌组织中比癌旁组织中高,表明肺癌组织中G6PD的糖基化升高与OGT的高表达相关。在许多疾病中,特别是溶血性贫血中有大量G6PD的突变,而在肿瘤中虽然对G6PD的作用研究也很广泛,但也是集中在G6PD的突变上,很少研究蛋白质翻译后修饰对其酶功能的影响。本研究工作揭示了O-G1cNAc糖基化是如何直接调控戊糖磷酸途径进而为肿瘤细胞提供生长优势这一机制,为以抑制G6PD糖基化为靶点的肿瘤治疗提供了新的思路。
[Abstract]:In order to meet their own nutritional and energy needs, tumor cells consume a large amount of glucose, and even when there is sufficient oxygen, the glycolysis process increases significantly, and a large amount of glucose is converted to lactic acid, which is called the Warburg effect and the Warburg effect. The Warburg effect is a manifestation of the metabolic reprogramming of tumor cells, which occurs in many metabolic pathways, including glucose transport, glycolysis, pentose phosphoric acid pathway, glutamine metabolism, and tricarboxylic acid cycle. Electron transfer chain, etc. The pentose phosphoric acid pathway plays the role of macromolecular biosynthesis and redox state in rapidly proliferating cells. Especially in many tumor cells, pentose phosphoric acid pathway is up-regulated. However, it is not entirely clear how the pentose phosphoric acid pathway is up-regulated to satisfy the growth advantage of tumor cells. Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphoric acid pathway, which catalyzes the oxidative dehydrogenation of glucose-6-phosphoric acid from the glycolysis pathway. 6-phosphogluconic acid-未 -internal lipids and NADPHs providing reductive hydrogen were obtained. In this study, chemical enzyme labeling and mass spectrometry were used to identify the glycosylation modification of G6PD S84 by N-acetylglucosamine O-G1cNAc. which is covalently linked with O-glucosamine. By mutating serine S from 84 to valine V, the glycosylation at S84 could not be detected, and the enzyme activity of G6PD was enhanced by glutaraldehyde crosslinking reaction. G6PD with high glycosylation level tends to form active dimer and tetramer. Previous studies have found that more than 1000 nucleoplasmic proteins have been modified by O-G1cNAc, and this glycosylation as a receptor is a dynamic response of cells to external nutrition and stress. In this study, we found that G6PD was very sensitive to these external stimuli by hypoxia treatment, high glucose stimulation and serum stimulation. The glycosylation level of G6PD increased significantly with the prolongation of hypoxia treatment time and the increase of glucose concentration and serum concentration. At the same time, the study also confirmed that high glycosylation of G6PDcould increase glucose intake in the pentose phosphoric acid pathway, produce more intermediate metabolites, and promote the combination of nucleotides and lipids to enhance antioxidant defense. In order to provide growth advantages for cells, promote cell proliferation. More physiologically, the heterotopic nude mice tumorigenesis experiment confirmed that the glycosylation of G6PD was beneficial to the growth of tumor, but to prevent the glycosylation of G6PD, significantly reduce the speed of cell proliferation and slow down the growth of tumor. Except for 8 cases whose glycosylation could not be studied reliably because of low expression of G6PD, 16 out of 31 cases had higher levels of G6PD glycosylation in human lung cancer tissues than in adjacent tissues, and 20 out of 31 cases had higher glycosylation levels. The expression level of glycosyltransferase (OGT) in cancer tissues was higher than that in adjacent tissues, indicating that the increased glycosylation of G6PD in lung cancer tissues was correlated with the high expression of OGT. In many diseases, especially in hemolytic anemia, there are a large number of mutations of G6PD. Although the role of G6PD in tumor is also widely studied, but also concentrated on the mutation of G6PD, the effect of protein post-translational modification on its enzyme function is seldom studied. This study revealed how O-G1cNAc glycosylation directly regulates the pentose phosphoric acid pathway and provides a growth advantage for tumor cells, and provides a new idea for tumor therapy with inhibition of G6PD glycosylation as its target.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.2

【相似文献】