在酿酒酵母中研究人类mOGT基质导向序列的功能

人类线粒体N-乙酰氨基葡萄糖转移酶(mitochondrial O-GlcNAc transferse,m OGT)通过其N端基质导向序列(matrix targeting sequence,MTS)定位于线粒体内膜上,m OGT的过表达将导致细胞的凋亡。蛋白质的O-Glc NAc修饰存在于绝大多数真核生物中,而酿酒酵母唯独缺乏该修饰途径。对人体m OGT过表达引起酿酒酵母生长缺陷的机制进行分析,期望利用该模型研究m OGT导致的细胞凋亡调控机制。在酿酒酵母细胞中表达人体m OGT及其截短序列发现:m OGT对酿酒酵母的生长抑制及细胞中的定位依赖于N端全长的MTS序列; MTS序列在酿酒酵母中共表达导致线粒体融合。MTS序列过表达的酿酒酵母菌可以作为研究m OGT引起细胞凋亡的模式细胞。     

蛋白质O-GlcNAc修饰的检测技术

O-连接的N-乙酰葡糖胺（O-GlcNAc）修饰是位于细胞浆和细胞核蛋白质的丝氨酸或苏氨酸上的一种翻译后修饰,在高等真核生物细胞中广泛存在.越来越多的研究表明,O-GlcNAc修饰在代谢调控、压力应激、细胞周期、凋亡、糖尿病、心血管疾病和癌症等多种生理和病理过程中发挥重要作用,因此, O-GlcNAc修饰已受到众多生命科学领域研究人员的关注.然而,由于O-GlcNAc修饰与传统的N聚糖和O聚糖修饰有所不同,常规糖基化修饰的检测方法并不适用于O-GlcNAc.本文对O-GlcNAc修饰的检测及其修饰位点的确定方法进行了综述,并分析了各种方法的优缺点.     

蛋白质O-GlcNAc糖基化修饰对tau蛋白磷酸化修饰的影响

蛋白质的O位N-乙酰葡萄糖胺(O-GlcNAc)糖基化修饰是一种新近发现的广泛存在于细胞核蛋白与细胞浆蛋白的蛋白质翻译后修饰.其性质与经典的膜蛋白和分泌蛋白的糖基化修饰不同,而与蛋白质磷酸化修饰更相似.O-GlcNAc糖基化和磷酸化均修饰tau蛋白的丝氨酸和苏氨酸残基,通过改变O-GlcNAc糖基化供体底物浓度以及其关键酶活性等方法,改变分化后成神经细胞样的PC12细胞中的蛋白质O-GlcNAc糖基化修饰水平,然后用特异性识别不同位点磷酸化的tau蛋白抗体,进行蛋白质印迹分析来检测tau蛋白磷酸化水平的变化.结果发现细胞内蛋白质O-GlcNAc糖基化对tau蛋白磷酸化的影响,在不同的磷酸化位点其影响不同.增加蛋白质O-GlcNAc糖基化修饰导致tau蛋白大多数磷酸位点的磷酸化水平降低,反之亦然.这些结果说明,tau磷酸化在大多数位点受到O-GlcNAc糖基化修饰的负性调节.这一研究为阐明调节tau蛋白磷酸化水平的机理和阿尔茨海默病脑中tau异常过度磷酸化的分子机制提供了新的线索.     

O-连接的N-乙酰葡糖胺糖蛋白的研究进展

O-连接的N-乙酰葡糖胺(O-GlcNAc)修饰是普遍存在的翻译后修饰.已有许多的蛋白被发现是O-GlcNAc蛋白.目前,许多分析方法可以检测O-GlcNAc,将其从内膜系统的多种糖基化中区分出来.O-GlcNAc修饰在细胞事件中发挥着重要的功能,O-GlcNAc的调控异常可能会引起某些人类疾病,如癌症、阿尔茨海默病和II型糖尿病.杆状病毒GP41蛋白也是糖蛋白,它介导芽殖型病毒粒子(budded virus,BV)的核衣壳通过胞核.O-GlcNAc的调控研究为探讨GP41蛋白O-GlcNAc的调控作用提供了参考模式.     

植物蛋白质O-糖基化修饰的研究进展

糖基化是最主要的蛋白质翻译后修饰方式之一,主要有N-糖基化、O-糖基化和糖基磷脂酰肌醇锚定修饰三种类型。在植物细胞中, O-糖基化修饰广泛发生,它不仅参与蛋白质转录调节、信号转导,还与细胞壁合成等生物学过程紧密相关。在多种O-糖基化修饰类型中, O-N-乙酰氨基葡萄糖(O-GlcNAc)糖基化修饰结构独特、易于检测和表征,因此已经有许多相关技术实现了对其的表征。然而,其他类型O-糖基化修饰蛋白的结构和功能仍有待更全面的研究。该文综述了植物蛋白中不同类型O-糖基化修饰的相关研究进展,总结了植物O-糖基化修饰蛋白检测技术的优缺点,最后展望了这些技术在植物蛋白质O-糖基化修饰研究中的应用前景。     

O-GlcNAc修饰与肿瘤发生及转移

O-连接的β-N-乙酰葡糖胺（O-GlcNAc）修饰是一种广泛存在于细胞浆和细胞核蛋白质丝/苏氨酸上的动态、可逆的翻译后修饰. 这种修饰与经典的糖基化不同而类似于磷酸化修饰,它在生命过程中发挥重要的调节作用. O-GlcNAc修饰作为潜在的营养感受器,可以调节转录、代谢等众多细胞进程,并与癌症等人类重大疾病密切相关. 本文主要综述了O-GlcNAc修饰与肿瘤形成和转移的关系,并对O-GlcNAc促进肿瘤形成与转移的潜在分子机制进行了探讨.     

O-GlcNAc修饰蛋白质的生理功能和研究方法

氧连N-乙酰葡糖胺（O-GlcNAc）修饰是与磷酸化相类似的蛋白质翻译后修饰方式,它主要发生在细胞核和细胞质中的蛋白质上．与细胞信号通路密切相关,成为近年来的研究热点。该文主要从O-GlcNAc修饰蛋白质的生理功能和研究方法两方面介绍该领域近年来的研究成果。     

N-乙酰氨基葡萄糖化在信号转导中的作用

蛋白质磷酸化在生命活动以及信号转导过程中的重要作用已经被研究证实,但不少研究发现在大多数核,胞液蛋白质上不仅存在磷酸化动态修饰,还存在广泛的动态N－乙酰氨基葡萄糖修饰,N－乙酰氨基葡萄糖基转移酶和N－乙酰氨基葡萄糖基酶以类似于蛋白质激酶和磷酸酶的方式调节蛋白质是否发生N－乙酰氨基葡萄糖化。N－乙酰氨基葡萄糖化蛋白质主要分布在细胞核与胞液,其生理功能涉及细胞基本生命活动和调节信号传递。N－乙酰氨基葡萄糖的作用基础与阻断或影响蛋白质的磷酸化有关。     

O-乙酰氨基葡萄糖(O-GlcNAc)修饰及其生物学功能研究进展

O-GlcNAc修饰系发生在蛋白质丝氨酸、苏氨酸羟基末端连接的乙酰氨基葡萄糖上的单糖基修饰。自1984年以来,针对O-GlcNAc糖基化修饰的研究日益升温。O-GlcNAc修饰是动态变化、可调控的,满足蛋白质翻译后修饰参与信号通路的必要条件。在多数情况下,O-GlcNAc修饰与磷酸化修饰发生在蛋白质的相同氨基酸残基上,故两种修饰之间常存在竞争性抑制,亦被称之为"阴阳"制衡。O-GlcNAc修饰参与细胞内多种信号通路的调控,调节着生长、增殖、激素响应等过程,在糖尿病、神经退行性疾病和肿瘤等代谢性疾病中扮演重要角色。探究O-GlcNAc修饰及其在生理、病理状态中的作用具有极为重要的意义。     

乙酰氨基葡萄糖转移酶Ⅴ在肿瘤转移中的作用及其分子机理

目前研究表明N-乙酰氨基葡萄糖转移酶Ⅴ在肿瘤转移中有重要作用.在恶性肿瘤中, N-乙酰氨基葡萄糖转移酶Ⅴ活性增高,其催化产物β1,6分支也增加,β1,6分支与肿 瘤的侵袭转移密切相关.本文综述了N-乙酰氨基葡萄糖转移酶Ⅴ催化形成N-糖链 β1,6分支的特点以及在N-糖链生物合成中的重要作用;还介绍了N-乙酰氨基葡萄糖转移酶Ⅴ基因组成和参与其基因调控的转录因子Ets-1,及基因表达组织特异性;着重综述了近年来N-乙酰氨基葡萄糖转移酶Ⅴ与肿瘤侵袭转移相关的分子机理的最新研究进展,包括了粘附分子钙粘蛋白(cadherin)和整合素α5β1的作用,修饰表皮生长因子受体调节信号 转导,及通过对上皮衍生的细胞表面丝氨酸蛋白酶matriptase的β1,6分支修饰促进仲瘤的 侵袭等方面.提示有效抑制N-乙酰氨基葡萄糖转移酶Ⅴ参与作用的位点,为设计抗肿瘤新药提供潜在的治疗靶点.     

Hsp90 regulates O-linked β-N-acetylglucosamine transferase: a novel mechanism of modulation of protein O-linked β-N-acetylglucosamine modification in endothelial cells

O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins is involved in many important cellular processes. Increased O-GlcNAc has been implicated in major diseases, such as diabetes and its complications and cardiovascular and neurodegenerative diseases. Recently, we reported that O-GlcNAc modification occurs in the proteasome and serves to inhibit proteasome function by blocking the ATPase activity in the 19S regulatory cap, explaining, at least in part, the adverse effects of O-GlcNAc modification and suggesting that downregulating O-GlcNAc might be important in the treatment of human diseases. In this study, we report on a novel mechanism to modulate cellular O-GlcNAc modification, namely through heat shock protein 90 (Hsp90) inhibition. We observed that O-linked β-N-acetylglucosamine transferase (OGT) interacts with the tetratricopeptide repeat binding site of Hsp90. Inhibition of Hsp90 by its specific inhibitors, radicicol or 17-N-allylamino-17-demethoxygeldanamycin, destabilized OGT in primary endothelial cell cultures and enhanced its degradation by the proteasome. Furthermore, Hsp90 inhibition downregulated O-GlcNAc protein modifications and attenuated the high glucose-induced increase in O-GlcNAc protein modification, including high glucose-induced increase in endothelial or type 3 isoform of nitric oxide synthase (eNOS) O-GlcNAcylation. These results suggest that Hsp90 is involved in the regulation of OGT and O-GlcNAc modification and that Hsp90 inhibitors might be used to modulate O-GlcNAc modification and reverse its adverse effects in human diseases.     

New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro

O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3′,5,5′-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.     

Molecular mechanisms of O-GlcNAcylation

Protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification of serines/threonines on metazoan proteins and occurring with similar time scales, dynamics and stoichiometry as protein phosphorylation. Levels of this modification are regulated by two enzymes-O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Although the biochemistry of these enzymes and functional implications of O-GlcNAc have been studied extensively, until recently the structures and molecular mechanisms of OGT/OGA were not understood. This review covers a body of recent work that has led to an understanding of the structure of OGA, its catalytic mechanism and the development of a plethora of different inhibitors that are finding their use in cell biological studies towards the functional implications of O-GlcNAc. Furthermore, the very recent structure determination of a bacterial OGT orthologue has given the first insights into the contribution of the tetratricopeptide repeats (TPRs) to the active site and the role of some residues in catalysis and substrate binding.     

O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic beta cells

O-GlcNAc transferase (OGT)-mediated modification of protein Ser/Thr residues with O-GlcNAc influences protein activity, similar to the effects of phosphorylation. The anti-apoptotic Akt1 is both activated by phosphorylation and modified with O-GlcNAc. However, the nature and significance of the Akt1 O-GlcNAc modification is unknown. The relationship of O-GlcNAc modification and phosphorylation at Akt1 Ser473 was examined with respect to apoptosis of murine beta-pancreatic cells. Glucosamine treatment induced apoptosis, which correlated with enhanced O-GlcNAc modification of Akt1 and concomitant reduction in Ser473 phosphorylation. Pharmacological inhibition of OGT or O-GlcNAcase revealed an inverse correlation between O-GlcNAc modification and Ser473 phosphorylation of Akt1. MALDI-TOF/TOF mass spectrometry analysis of Akt1 immunoprecipitates from glucosamine-treated cells, but not untreated controls, showed a peptide containing S473/T479 that was presumably modified with O-GlcNAc. Furthermore, in vitro O-GlcNAc-modification analysis of wildtype and mutant Akt1 revealed that S473 was targeted by recombinant OGT. A S473A Akt1 mutant demonstrated reduced basal and glucosamine-induced Akt1 O-GlcNAc modification compared with wildtype Akt1. Furthermore, wildtype Akt1, but not the S473A mutant, appeared to be associated with OGT following glucosamine treatment. Together, these observations suggest that Akt1 Ser473 may undergo both phosphorylation and O-GlcNAc modification, and the balance between these may regulate murine beta-pancreatic cell fate.     

Increased O-GlcNAc levels correlate with decreased O-GlcNAcase levels in Alzheimer disease brain

The potential role of the posttranslational modification of proteins with O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) in the pathogenesis of Alzheimer disease (AD) has been studied extensively, yet the exact function of O-GlcNAc in AD remains elusive. O-GlcNAc cycling is facilitated by only two highly conserved enzymes: O-GlcNAc transferase (OGT) catalyzes the addition, while O-GlcNAcase (OGA) catalyzes the removal of GlcNAc from proteins. Studies analyzing global O-GlcNAc levels in AD brain have produced inconsistent results and the reasons for altered O-GlcNAcylation in AD are still poorly understood. In this study, we show a 1.2-fold increase in cytosolic protein O-GlcNAc modification in AD brain when compared to age-matched controls. Interestingly, O-GlcNAc changes seem to be attributable to differential modification of a few individual proteins. While our finding of augmented O-GlcNAcylation concurs with some reports, it is contrary to others demonstrating decreased O-GlcNAc levels in AD brain. These conflicting results emphasize the need for further studies providing conclusive evidence on the subject of O-GlcNAcylation in AD. We further demonstrate that, while OGT protein levels are unaffected in AD, OGA protein levels are significantly decreased to 75% of those in control samples. In addition, augmented protein O-GlcNAc modification correlates to decreased OGA protein levels in AD subjects. While OGA inhibitors are already being tested for AD treatment, our results provide a strong indication that the general subject of O-GlcNAcylation and specifically its regulation by OGA and OGT in AD need further investigation to conclusively elucidate its potential role in AD pathogenesis and treatment.     

Developmental Regulation of Protein O-GlcNAcylation, O-GlcNAc Transferase, and O-GlcNAcase in Mammalian Brain

O-GlcNAcylation is a common posttranslational modification of nucleocytoplasmic proteins by β-N-acetylglucosamine (GlcNAc). The dynamic addition and removal of O-GlcNAc groups to and from proteins are catalyzed by O-linked N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and β-N-acetylglucosaminidase (O-GlcNAcase, OGA), respectively. O-GlcNAcylation often modulates protein phosphorylation and regulates several cellular signaling and functions, especially in the brain. However, its developmental regulation is not well known. Here, we studied protein O-GlcNAcylation, OGT, and OGA in the rat brain at various ages from embryonic day 15 to the age of 2 years. We found a gradual decline of global protein O-GlcNAcylation during developmental stages and adulthood. This decline correlated positively to the total protein phosphorylation at serine residues, but not at threonine residues. The expression of OGT and OGA isoforms was regulated differently at various ages. Immunohistochemical studies revealed ubiquitous distribution of O-GlcNAcylation at all ages. Strong immunostaining of O-GlcNAc, OGT, and OGA was observed mostly in neuronal cell bodies and processes, further suggesting the role of O-GlcNAc modification of neuronal proteins in the brain. These studies provide fundamental knowledge of age-dependent protein modification by O-GlcNAc and will help guide future studies on the role of O-GlcNAcylation in the mammalian brain.