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糖酵解关键酶PKM2的O-GlcNAc糖基化修饰研究

已有 3567 次阅读 2022-10-14 15:57 |系统分类:论文交流

    O-GlcNAc糖基化修饰 (O-GlcNAcylation)是一种发生在丝氨酸或苏氨酸羟基上的单糖修饰。因该修饰的供体底物UDP-GlcNAc的合成与四种生物大分子的代谢密切相关,这种动态的单糖修饰被视为 一种“营养状态的感受器” (nutrient sensor)。近期研究也表明O-GlcNAc糖基化有可能积极的直接参与调控细胞代谢过程。在癌症中,O-GlcNAc修饰水平明显升高,异常的O-GlcNAc糖基化与癌细胞的增殖、侵袭以及转移密切相关。

    丙酮酸激酶(PK)使磷酸烯醇式丙酮酸和ADP变为ATP和丙酮酸,是糖酵解过程中的主要限速酶之一,有M型和L型两种同工酶,M型又有M1及M2亚型。M1分布于心肌、骨骼肌和脑组织;M2分布于脑及肝脏等组织。L型同工酶主要存在于肝、肾及红细胞内。许多正常细胞能产生PKM1,肿瘤细胞则表达更多活性低的PKM2,通过阻断PKM2可能具有阻断肿瘤细胞增殖的作用。

    东北师范大学魏民/冯云鹏课题组致力于蛋白质的O-GlcNAc修饰的研究。该课题组前期的研究发现( Wang et al., PNAS, 2017 ), O-GlcNAc糖基化修饰可以通过调节糖酵解途径的最后一个限速酶丙酮酸激酶M2 (PKM2) 的活性和功能,直接影响肿瘤细胞的增殖。【1】

PKM2.jpg

    O-GlcNAc修饰大量存在于肿瘤细胞中PKM2蛋白α螺旋Loop环上的Thr405和Ser406上。这两个位点的O-GlcNAc修饰直接破坏了稳定PKM2四聚体的关键作用力,并导致四聚体解聚,二聚体和单体大量形成。这种变化,一方面下调了细胞质中PKM2的丙酮酸激酶活性,引起的代谢重编程更好地满足了肿瘤细胞对生物大分子合成的需求,使得肿瘤细胞葡萄糖消耗以及乳酸产量增加,并且增加了脂质以及DNA合成水平,说明PKM2的O-GlcNAc糖基化能促进“瓦博格效应”;另一方面,四聚体的解聚有利于PKM2的核转运,调节GLUT1和LDHA等代谢相关基因的表达。该研究表明中O-GlcNAc糖基化是调控肿瘤细胞PKM2的一种机制,并且作为PKM2和“瓦博格效应”代谢重塑特征的连接桥梁。

    论文第一单位为东北师范大学,我院博士研究生王杨、刘佳和金鑫为共同第一作者,通讯作者为魏民教授。

    意义:Cancer cells are characterized by a high rate of glycolysis even under normal oxygen availability to meet the demand of biomass production during rapid proliferation. An isoform of pyruvate kinase (PK), PKM2, preferentially expressed in cancers, was recently shown to be critical for this metabolic reprogramming with adjustable activity and dynamic cellular relocalization. However, specific molecular mechanisms mediating PKM2’s role in cancer-specific metabolism remain largely elusive. We demonstrate that O-GlcNAcylation of PKM2 on threonine/serine encoded by an alternatively spliced exon disrupts the intersubunit interactions in the active PKM2 tetramer. This causes the tetramer disassembly, reduced PK activity, and its nuclear translocation to facilitate cell proliferation. Thus, our findings furnish a key piece in the puzzle of aerobic glycolysis in cancer.

    摘要:The Warburg effect, characterized by increased glucose uptake and lactate production, is a well-known universal across cancer cells and other proliferating cells. PKM2, a splice isoform of the pyruvate kinase (PK) specifically expressed in these cells, serves as a major regulator of this metabolic reprogramming with an adjustable activity subjected to numerous allosteric effectors and posttranslational modifications. Here, we have identified a posttranslational modification on PKM2, O-GlcNAcylation, which specifically targets Thr405 and Ser406, residues of the region encoded by the alternatively spliced exon 10 in cancer cells. We show that PKM2 O-GlcNAcylation is up-regulated in various types of human tumor cells and patient tumor tissues. The modification destabilized the active tetrameric PKM2, reduced PK activity, and led to nuclear translocation of PKM2. We also observed that the modification was associated with an increased glucose consumption and lactate production and enhanced level of lipid and DNA synthesis, indicating that O-GlcNAcylation promotes the Warburg effect. In vivo experiments showed that blocking PKM2 O-GlcNAcylation attenuated tumor growth. Thus, we demonstrate that O-GlcNAcylation is a regulatory mechanism for PKM2 in cancer cells and serves as a bridge between PKM2 and metabolic reprogramming typical of the Warburg effect.

    原文:https://doi.org/10.1073/pnas.1704145115 


    该课题组近日的研究成果显示 ( Wang et al., JBC, 2022 ), 肿瘤细胞中PKM2的Thr405/Ser406的O-GlcNAc修饰不但对于葡萄糖及谷氨酰胺等营养物质的波动较为敏感,而且可以直接受到生长因子EGF的调控,导致PKM2的四聚体解聚,酶活性随之下调。【2】

gr7.jpg

    究其机制,EGF促进PKM2与O-GlcNAc糖基转移酶 (OGT) 的结合,增加PKM2的O-GlcNAc修饰。已知PKM2是一种酪氨酸磷酸化结合蛋白,研究者推测作为磷酸化经典调控信号的EGF很可能通过调节OGT的酪氨酸磷酸化修饰促进其与PKM2的结合。据报道,OGT具有Y384、Y976、Y844和Y989四个酪氨酸磷酸化位点,进一步的结果显示,通过单点突变破坏OGT Y976位的磷酸化削弱其与PKM2的结合,突变其他三个位点几乎不改变二者结合 ,且质谱结果表明Y976位磷酸化确实响应EGF信号。

    同时研究者制备了OGT Y976特异性位点的磷酸化抗体 (OGT pY976),免疫印记结果显示EGF确实通过诱导OGT Y976位的磷酸化促进其与PKM2的结合,进一步促进PKM2的O-GlcNAc修饰, 但Y976磷酸化位点本身并不影响OGT的活性 。另一方面,虽然目前已鉴定的O-GlcNAc修饰的蛋白超过1000种,但是负责催化的酶只有OGT一种,研究者发现EGF引起的OGT Y976磷酸化可增加其与包含PKM2在内的“磷酸化酪氨酸结合蛋白家族”与OGT的结合并提高该家族O-GlcNAc修饰的水平,如STAT1、STAT3、STAT5、PKCδ和p85等。

    此项研究一方面探究了EGF调控O-GlcNAc修饰发生的新机制,另一方面为理解OGT的底物选择规律提供了新思路。东北师范大学博士后王杨为第一作者,魏民教授和冯云鹏副教授为通讯作者。

    摘要:Epidermal growth factor (EGF) is one of the most well-characterized growth factors and plays a crucial role in cell proliferation and differentiation. Its receptor EGFR has been extensively explored as a therapeutic target against multiple types of cancers, such as lung cancer and glioblastoma. Recent studies have established a connection between deregulated EGF signaling and metabolic reprogramming, especially rewiring in aerobic glycolysis, which is also known as the Warburg effect and recognized as a hallmark in cancer. Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme controlling the final step of glycolysis and serves as a major regulator of the Warburg effect. We previously showed that PKM2 T405/S406 O-GlcNAcylation, a critical mark important for PKM2 detetramerization and activity, was markedly upregulated by EGF. However, the mechanism by which EGF regulates PKM2 O-GlcNAcylation still remains uncharacterized. Here, we demonstrated that EGF promoted O-GlcNAc transferase (OGT) binding to PKM2 by stimulating OGT Y976 phosphorylation. As a consequence, we found PKM2 O-GlcNAcylation and detetramerization were upregulated, leading to a significant decrease in PKM2 activity. Moreover, distinct from PKM2, we observed that the association of additional phosphotyrosine-binding proteins with OGT was also enhanced when Y976 was phosphorylated. These proteins included STAT1, STAT3, STAT5, PKCδ, and p85, which are reported to be O-GlcNAcylated. Together, we show EGF-dependent Y976 phosphorylation is critical for OGT-PKM2 interaction and propose that this posttranslational modification might be important for substrate selection by OGT.

    关键词: O-GlcNAc transferase; O-GlcNAcylation; epidermal growth factor; phosphorylation; phosphotyrosine-binding protein; pyruvate kinase M2.

    原文:https://doi.org/10.1016/j.jbc.2022.102340 


    此外,2020年Oncogene上的研究论文“ O-GlcNAcase targets pyruvate kinase M2 to regulate tumor growth”也报道了PKM2的糖基化修饰【3】。 研究者Xiaoyong Yang认为,癌细胞对营养物质有着无限的需求,但在机体很多部位,尤其是对于实体瘤而言,营养物质和氧气通常是有限的,因此细胞就会决定生长还是生存,文章中我们揭示了癌细胞如何适应这种微环境,并检测营养物质的可用性从而做出下一步决定。研究者阐明了O-GlcNAc蛋白修饰过程在癌症代谢中所扮演的关键角色,O-GlcNAc蛋白修饰能通过吸附特定类型的唐分子来改变蛋白质的功能,其被认为在细胞中扮演着营养传感器的角色,研究人员对该修饰过程非常感兴趣,因为其是很多类型癌症中的一种共有特征。

    文章中,研究者对多种类型的癌症组织样本进行分析来检测O-GlcNAc修饰的迹象,包括OGT和OGA酶类表达的水平等,他们发现,相比正常组织,OGT和OGA会在许多癌症中高水平表达,OGA能促进癌细胞生长及其代谢的重编程作用,同时OGA还能改变PKM2(细胞代谢的重要成员)蛋白的乙酰化修饰,该活动会随着细胞中葡萄糖可用水平的增加而增加;随后OGT则能通过O-GlcNAc蛋白修饰来抑制PKM2的活性,从而驱动代谢重编程并促进肿瘤生长。

    此前研究人员认为,OGT和OGA能够直接发生相互作用,人们总认为其是敌人,而本文研究中研究者发现OGT和OGA实际上是朋友关系,其在营养丰富的环境中能够协同发挥作用,从而驱动癌细胞生长和繁殖。研究者希望能够开发出新型药物来误导OGA/OGT通路,以便癌细胞无法决定开启有氧糖酵解,即使在营养物质自由的时候癌细胞也无法进行生长。

    OGA/OGT生物性信号通路在正常细胞中的水平较低,因此所有的药物都是减少其水平而并不是完全阻断该信号通路,目前研究人员正在通力合作来调查能够靶向作用OGT和OGA酶类表达的特殊化合物,他们希望最终能够阐明是否这些药物能同其它药物有效结合来治疗多种类型的癌症。

    摘要:Cancer cells are known to adopt aerobic glycolysis in order to fuel tumor growth, but the molecular basis of this metabolic shift remains largely undefined. O-GlcNAcase (OGA) is an enzyme harboring O-linked β-N-acetylglucosamine (O-GlcNAc) hydrolase and cryptic lysine acetyltransferase activities. Here, we report that OGA is upregulated in a wide range of human cancers and drives aerobic glycolysis and tumor growth by inhibiting pyruvate kinase M2 (PKM2). PKM2 is dynamically O-GlcNAcylated in response to changes in glucose availability. Under high glucose conditions, PKM2 is a target of OGA-associated acetyltransferase activity, which facilitates O-GlcNAcylation of PKM2 by O-GlcNAc transferase (OGT). O-GlcNAcylation inhibits PKM2 catalytic activity and thereby promotes aerobic glycolysis and tumor growth. These studies define a causative role for OGA in tumor progression and reveal PKM2 O-GlcNAcylation as a metabolic rheostat that mediates exquisite control of aerobic glycolysis.

    原文:https://doi.org/10.1038/s41388-019-0975-3 


主要参考文献

1. Y. Wang, J. Liu, X. Jin, D. Zhang, D. Li, F. Hao, Y. Feng, S. Gu, F. Meng, M. Tian, Y. Zheng, L. Xin, X. Zhang, X. Han, L. Aravind, M. Wei, O-GlcNAcylation destabilizes the active tetrameric PKM2 to promote the Warburg effect, PNAS, 2017, 114, 13732-13737.
https://www.pnas.org/doi/10.1073/pnas.1704145115 

2. Y. Wang, H. Shu, J. Liu, X. Jin, L. Wang, Y. Qu, M. Xia, P. Peng, Y. Feng, M. Wei, EGF promotes PKM2 O-GlcNAcylation by stimulating O-GlcNAc transferase phosphorylation at Y976 and their subsequent association, The Journal of biological chemistry, 2022, 298, 102340.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9436816/ 

3. Singh J P, Qian K, Lee J S, et al. O-GlcNAcase targets pyruvate kinase M2 to regulate tumor growth . Oncogene, 2020, 39(3): 560-573. 

https://www.nature.com/articles/s41388-019-0975-3 

 



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