Warburg效应促进癌细胞增殖的作用主要体现在三个方面：1）糖酵解能快速地为细胞提供ATP；2）糖酵解的中间代谢物能作为其它生物大分子合成的前体；3）糖酵解能减少细胞的氧化自由基浓度，维持氧化还原平衡。 在糖酵解途径中，约2-5%的葡萄糖会经由支路已糖胺生物合成途径（HBP）生成UDP-乙酰氨基葡糖胺（UDP-GlcNAc），用于蛋白质的糖基化修饰（例如：O-GlcNAc修饰）。 

    O-GlcNAc糖基化修饰是通过N-乙酰葡糖胺以β-糖苷键形式共价连接到蛋白质的丝氨酸（Ser，S）或苏氨酸（Thr，T）羟基上的一种翻译后修饰。该修饰是一种高度动态的修饰方式，会随着细胞内的营养状态和细胞外的刺激发生变化。O-GlcNAc修饰基团的添加和去除分别是由O-GlcNAc糖基转移酶 （O-GlcNAc transferase，OGT）和N-乙酰氨基葡萄糖苷酶（O-GlcNAcase，OGA）一对酶来完成，供体GlcNAc是以尿嘧啶-5’-二磷酸-N-GlcNAc的形式存在。

    在经代谢物修饰后，蛋白质的化学结构特性发生改变，继而通过影响蛋白质的定位、构象、稳定性、原有修饰类型以及蛋白质互作，调控蛋白质自身功能。O-连接的β--N-乙酰氨基葡萄糖 (O-GlcNAc) 是最常见的糖基化修饰，可修饰细胞核、细胞质、细胞膜和线粒体中数千种蛋白质。例如：Hippo信号主要的效应分子YAP/TAZ，在发生磷酸化后，可与细胞浆中的14-3-3蛋白相互作用形成复合物，从而抑制YAP/TAZ入核进行转录激活，最终抑制细胞增殖。而当YAP的109位丝氨酸 (S109) 发生O-GlcNAc糖基化修饰后，YAP则无法被磷酸化，从而进入细胞核启动转录促进细胞增殖。

    O-GlcNAc糖基化修饰广泛发生在细胞内的蛋白上，并调控基因转录、信号传导、蛋白合成和代谢重编程等重要的生物学过程; 但细胞中O-GlcNAc修饰的阅读器“reader”还并不清楚。此外，在绝大多数肿瘤组织中，O-GlcNAc修饰的整体水平都有显著上调，因此鉴定其中O-GlcNAc修饰的重要底物与相应的功能机制研究显得尤为关键。

    2021年3月3日凌晨，裴华东团队与秦伟捷团队以及贺福初院士团队等合作再次在Molecular Cell杂志上发表了题为“Posttranscriptional Regulation of De Novo Lipogenesis by Glucose-Induced O-GlcNAcylation”的研究论文。 该研究报道了O-GlcNAc糖基化修饰通过增强SRPK2的细胞核内定位、在转录后水平上调控肿瘤细胞的脂质从头合成水平、进而促进肿瘤生长的新机制；这一调控依赖于importin α/β入核转运系统，且进一步研究提示importin α蛋白可能是O-GlcNAc修饰的“reader”。

    在细胞中，importin α/β系统是调控蛋白入核的经典途径，其中importin α蛋白的N端可以结合importin β蛋白，C端可以结合货物蛋白的核定位序列（Nuclear-localization signal, NLS），从而形成三元复合物通过核孔将蛋白运入细胞核内。研究人员结合自有数据和已发表的数据库信息，发现在三百多个鼠源或者人源蛋白的NLS里面或附近都存在糖基化修饰位点。从这些蛋白中，研究人员选择了RELA与Sp1进行了实验验证，结果证实了这些蛋白的糖基化修饰可以显著增强其与相应importin α蛋白的相互作用，进而促进其在细胞核内的定位。这就说明糖基化修饰促进蛋白入核是细胞内普遍存在的一个重要机制，而importin α蛋白本身很可能是一个O-GlcNAc修饰的“reader”。

 亮点Highlights：

•Interfering with O-GlcNAcylation disrupts de novo lipogenesis

•SRPK2 is O-GlcNAcylated at a nuclear localization signal

•Importin α proteins recognize O-GlcNAcylated nuclear localization signals

•SRPK2 O-GlcNAcylation promotes breast cancer cell growth in culture and in vivo

摘要Summary：

O-linked β-N-acetyl glucosamine (O-GlcNAc) is attached to proteins under glucose-replete conditions; this posttranslational modification results in molecular and physiological changes that affect cell fate. Here we show that posttranslational modification of serine/arginine-rich protein kinase 2 (SRPK2) by O-GlcNAc regulates de novo lipogenesis by regulating pre-mRNA splicing. We found that O-GlcNAc transferase O-GlcNAcylated SRPK2 at a nuclear localization signal (NLS), which triggers binding of SRPK2 to importin α. Consequently, O-GlcNAcylated SRPK2 was imported into the nucleus, where it phosphorylated serine/arginine-rich proteins and promoted splicing of lipogenic pre-mRNAs. We determined that protein nuclear import by O-GlcNAcylation-dependent binding of cargo protein to importin α might be a general mechanism in cells. This work reveals a role of O-GlcNAc in posttranscriptional regulation of de novo lipogenesis, and our findings indicate that importin α is a “reader” of an O-GlcNAcylated NLS.

原文：https://doi.org/10.1016/j.molcel.2021.02.009 

    此外，2018年Duke大学Michael Boyce教授在PNAS上发表了题为“Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins”论文，利用糖肽鉴定到了多个与O-GlcNAc特异结合的蛋白，其中包括哺乳动物14-3-3蛋白。

   研究人员首先对已知O-GlcNAc糖基化修饰肽段进行序列分析，找到了一些共同特征：即ProValSer的保守序列，于是他们合成了这样的肽段，用OGT连接上O-GlcNAc修饰，作为“鱼饵”钓出潜在的O-GlcNAc修饰的 reader。在293T等三种人源体细胞的细胞核与细胞质中共鉴定到数十个蛋白，在此数据集中，他们挑选并验证到三个能与O-GlcNAc直接修饰的蛋白：α烯醇酶、EBP1和14-3-3。另外，为了鉴定这些潜在O-GlcNAc reader是否能与含O-GlcNAc的底物蛋白相互作用，他们又用含diazirine的GlcNAc类似物代谢标记蛋白并照紫外交联，在分子量大于100kD的区域寻找这些O-GlcNAc修饰 reader以及它们的底物，证明了在体内这些蛋白确实能识别O-GlcNAc修饰。

    研究者通过对14-3-3蛋白结合O-GlcNAc底物的复合物进行结构研究，阐释了14-3-3是如何识别并结合O-GlcNAc底物的。14-3-3有多种同源蛋白，包括本文鉴定到的α、β和γ三种，能与特定序列上的多种翻译后修饰结合，包括pSer. XRD结构表面，肽段上的糖基化修饰可以嵌入14-3-3两亲性凹槽，与周围氨基酸形成氢键。对关键位点氨基酸进行点突变可以增强或减弱该蛋白对O-GlcNAc修饰的识别。根据结构和点突变信息，他们解释了不同14-3-3蛋白能在细胞内分布和信号通路的关系：WT 14-3-3γ能同时识别磷酸化和O-GlcNAc修饰，在胞质与核内均有分布；某些突变型的14-3-3γ只能识别O-GlcNAc修饰，它们只分布于核内。

Significance：

O-GlcNAc is an abundant, reversible posttranslational modification (PTM) of nuclear and cytoplasmic proteins in animals and plants. O-GlcNAc regulates a wide range of biological processes, and aberrant O-GlcNAcylation is implicated in numerous human diseases. However, key aspects of O-GlcNAc signaling remain poorly understood. For example, it is not known whether “reader” proteins exist to recognize and bind to O-GlcNAc, as is true for many other PTMs. We used a biochemical method to identify candidate human O-GlcNAc reader proteins, and then characterized them at the biochemical and biophysical levels. Our results address a significant gap in the cell signaling field by revealing the biochemical and structural basis for the recognition of O-GlcNAc by conserved human proteins.

Abstract：

O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by “reader” proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3β/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein–protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.
原文：https://doi.org/10.1073/pnas.1722437115 

Cell signaling through O-GlcNAc reader proteins

Boyce, Michael S.   

Duke University, Durham, NC, United States

O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification in mammals, decorating thousands of nuclear, cytoplasmic and mitochondrial proteins. O-GlcNAc cycling is an essential regulator of cell metabolism, cell cycle progression and cell death, and is dysregulated in numerous human diseases, such as cancer, diabetes and cardiac arrhythmia. Despite its broad pathophysiological significance, major aspects of O-GlcNAc signaling remain obscure, including how O-GlcNAc transduces biological information inside the cell. Recently, several studies showed that O-GlcNAcylation induces protein-protein interactions in processes as diverse as chromatin remodeling, deubiquitination and nuclear envelope assembly, suggesting that O-GlcNAc may signal through conserved modes of protein-protein interaction. However, little is known about either the structure or function of these intracellular glycoprotein-protein complexes. We hypothesized that mammalian reader proteins might exist and recognize O-GlcNAc moieties for signaling purposes. By analogy to reader proteins for other post-translational modifications, we reasoned that dedicated O-GlcNAc readers - or protein domains common to many readers - might relay glycosylation-encoded signals. In preliminary studies, we used a new biochemical approach to identify several proteins that bind specifically and directly to O-GlcNAcylated (but not unglycosylated) peptides and proteins in vitro and in cells. Interestingly, one group of putative O-GlcNAc reader proteins also binds to phosphoproteins, suggesting that they may be signal integrators, mediating the previously described crosstalk between O-GlcNAc and O- phosphate. This result may have wide-ranging implications for our understanding of cell signaling through post- translational modifications. The objective of this project is to characterize the biochemical and cell biological functions of the O- GlcNAc reader proteins that we identified. We will accomplish this goal through three specific aims.

In Aim 1, we will define the biochemical scope of O-GlcNAc binding by the candidate readers.

In Aim 2, we will identify the endogenous glycoprotein binding partners of O-GlcNAc reader proteins, and establish the role of these interactions in cell signaling.

In Aim 3, we will determine the biophysical basis of an O-GlcNAc-mediated protein- protein interaction by solving the crystal structure of a reader protein bound to a glycosylated ligand. Our work will significantly advance the field of cell signaling by characterizing O-GlcNAc reader proteins at the cell biological, biochemical and atomic levels.

Public Health Relevance

Human cells regulate the functions of proteins by decorating them with sugar molecules, a process known as glycosylation. Our project aims to understand how glycosylation controls multiprotein complexes that drive cell growth and proliferation. This work may reveal new therapeutic opportunities to treat diseases in which glycosylation is dysregulated, including cancer, diabetes and cardiac arrhythmia.

原文： https://grantome.com/grant/NIH/R01-GM118847-02 

主要参考文献：

1.  Tan, Wei, et al. "Posttranscriptional regulation of de novo lipogenesis by glucose-induced O-GlcNAcylation." Molecular Cell 81.9 (2021): 1890-1904.
   https://www.sciencedirect.com/science/article/pii/S1097276521000952 
   

2. Toleman, Clifford A., et al. "Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins." PNAS 115.23 (2018): 5956-5961.
   https://www.pnas.org/doi/abs/10.1073/pnas.1722437115