细胞信号转导中 mTOR、 AMPK 与氨基己糖生物合成途径的相互作用

0 Comments

Real Talk: The Inter-play Between the mTOR, AMPK, and Hexosamine Biosynthetic Pathways in Cell Signaling

Gentry K. Cork 金特里 · k · 科克1,2Jeffrey Thompson 杰弗里 · 汤普森3 and 及Chad Slawson 查德 · 斯劳森1*

  • 1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, United States 堪萨斯大学生物化学与分子生物学系
  • 2Department of Pathology, University of Kansas Medical Center, Kansas City, KS, United States 美国堪萨斯州堪萨斯市堪萨斯大学医学中心病理学系
  • 3Department of Biostatistics, University of Kansas Medical Center, Kansas City, KS, United States 美国堪萨斯州堪萨斯市堪萨斯大学医学中心生物统计部

O-linked N-acetylglucosamine, better known as O-GlcNAc, is a sugar post-translational modification participating in a diverse range of cell functions. Disruptions in the cycling of O-GlcNAc mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, is a driving force for aberrant cell signaling in disease pathologies, such as diabetes, obesity, Alzheimer’s disease, and cancer. Production of UDP-GlcNAc, the metabolic substrate for OGT, by the Hexosamine Biosynthetic Pathway (HBP) is controlled by the input of amino acids, fats, and nucleic acids, making O-GlcNAc a key nutrient-sensor for fluctuations in these macromolecules. The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) pathways also participate in nutrient-sensing as a means of controlling cell activity and are significant factors in a variety of pathologies. Research into the individual nutrient-sensitivities of the HBP, AMPK, and mTOR pathways has revealed a complex regulatory dynamic, where their unique responses to macromolecule levels coordinate cell behavior. Importantly, cross-talk between these pathways fine-tunes the cellular response to nutrients. Strong evidence demonstrates that AMPK negatively regulates the mTOR pathway, but O-GlcNAcylation of AMPK lowers enzymatic activity and promotes growth. On the other hand, AMPK can phosphorylate OGT leading to changes in OGT function. Complex sets of interactions between the HBP, AMPK, and mTOR pathways integrate nutritional signals to respond to changes in the environment. In particular, examining these relationships using systems biology approaches might prove a useful method of exploring the complex nature of cell signaling. Overall, understanding the complex interactions of these nutrient pathways will provide novel mechanistic information into how nutrients influence health and disease.

O 连锁的 n- 乙酰氨基葡萄糖,更好地被称为 O-GlcNAc,是一种糖翻译后修饰参与多种细胞功能。O-GlcNAc 转移酶(OGT)和 O-GlcNAc 酶(OGA)介导的 O-GlcNAc 循环的中断是疾病病理中异常细胞信号转导的驱动力,如糖尿病、肥胖症、阿尔茨海默病和癌症。氨基己糖生物合成途径(Hexosamine Biosynthetic Pathway,HBP)通过输入氨基酸、脂肪和核酸控制产生 OGT 的代谢底物 UDP-GlcNAc,使 O-GlcNAc 成为反映这些大分子波动的关键营养传感器。哺乳动物的雷帕霉素靶蛋白(mTOR)和 AMP活化蛋白激酶蛋白激酶(AMPK)途径也参与营养传感作为控制细胞活性的一种手段,是多种疾病的重要因素。对 HBP、 AMPK 和 mTOR 途径的个体营养敏感性的研究揭示了一个复杂的调控动态,它们对大分子水平的独特反应协调了细胞行为。重要的是,这些通路之间的串扰微调了细胞对营养物质的反应。强有力的证据表明 AMPK 负向调节 mTOR 途径,而 AMPK 的 o- 谷氨酰胺酰基化降低酶活性,促进生长。另一方面,AMPK 可以磷酸化 OGT 导致 OGT 功能的改变。HBP、 AMPK 和 mTOR 通路之间复杂的相互作用集合了营养信号以应对环境的变化。特别是,使用系统生物学方法检查这些关系可能被证明是探索细胞信号的复杂性质的一个有用的方法。总的来说,了解这些营养通路的复杂相互作用将为营养素如何影响健康和疾病提供新颖的机械信息。

Introduction

引言

At its core, pathology is largely a matter of cell signaling gone awry. Think of it as a game of “Rumors,” where a group of players are passing a message down the line by whispering it to each other, but when it reaches the last person and they announce it, the message is entirely different from the original. Now, imagine the people as proteins, and when each one receives the message, they perform some function that will then signal the next protein in the pathway to perform a function until the endgame target is reached. If one of the components in a signaling pathway deviates from typical behavior or conditions, the resulting “message” can be exceedingly different from the original signal, altering cell behavior. Diseases like cancer, diabetes, and Alzheimer’s are often rooted in aberrant signal transduction and abnormal pathway regulation. Nutrient-sensitivity is one of several key factors that impact a pathway’s activity. Over- or under-nutrition can heighten or inhibit activity through alterations in post-translational modifications (PTM); any miscommunication in these modifications can push a cell toward pathology. A PTM can propagate a signal cascade to change the function of a final target, act as a sensor for regulators of a pathway, or alter the interactions of specific proteins in the pathway. To understand why these changes are occurring, a greater understanding of the complexity and fine-tuning of a pathway by a PTM is needed.

从本质上讲,病理学主要是细胞信号失调的问题。可以把它想象成一个“谣言”游戏,一群玩家通过互相窃窃私语来传递信息,但是当信息到达最后一个人时,他们宣布了这个信息,这个信息和最初的信息完全不同。现在,把这些人想象成蛋白质,当每个人接收到信息时,他们执行一些功能,然后信号通路中的下一个蛋白质执行一项功能,直到最终目标达到。如果信号通路中的一个成分偏离了典型的行为或条件,由此产生的“信息”可能与原始信号大相径庭,改变细胞的行为。像癌症、糖尿病和阿尔茨海默氏症这样的疾病往往根源于异常的信号转导和异常的通路调节。营养敏感性是影响通路活性的几个关键因素之一。营养过剩或营养不足可以通过翻译后修饰(PTM)的改变提高或抑制活性,这些修饰中的任何错误沟通都可以推动细胞走向病理。PTM 可以通过传播信号级联来改变最终目标的功能,充当路径调节器的传感器,或者改变路径中特定蛋白质的相互作用。为了理解为什么会发生这些变化,需要更好地理解 PTM 的复杂性和路径的微调。

Along with phosphorylation, acetylation, and methylation; glycosylation is one of the most important protein modifications. O-linked N-Acetylglucosamine (O-GlcNAc) is one of the many kinds of glycosidic PTMs found in eukaryotic cells, but what makes it different from other sugar additions is that it exists purely as an intracellular molecule and does form oligomers (1). The addition and removal of O-GlcNAc is facilitated solely by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) (2), so the coordinated activity of these two enzymes creates a versatile signaling dynamic that can quickly alter signaling pathways (3). Therefore, a wide scope of cellular processes is controlled and fine-tuned by O-GlcNAc, including apoptosis, mitochondrial function, proliferation, and gene transcription (49). Because O-GlcNAc plays such a crucial and diverse role in eukaryotic cells, atypical O-GlcNAcylation can be a driving force in a variety of pathologies. Alzheimer’s disease (10), diabetes, and several types of cancers have been linked to abnormal levels or behavior of O-GlcNAc, OGT, and OGA (5916). Research into the underlying reasons behind these physiological aberrations has yielded a plethora of new insights into cell signaling mechanisms and the role of O-GlcNAc in overall cellular function, particularly nutrient-sensing. As a nutrient-sensor, O-GlcNAc reacts to fluctuations in specific macromolecule levels in order to direct cellular response in an appropriate manner. However, nutrient-sensing is a vital aspect of many different pathways, including the mammalian target of rapamycin (mTOR) and AMP-activated protein kinase pathways (AMPK). By understanding how these pathways react to nutrient levels and how that impacts their interactions between one another, we can begin to ascertain how nutrient sensing impacts overall cell activity (12).

糖基化是与磷酸化、乙酰化和甲基化一样重要的蛋白质修饰之一。O 连锁的 n- 乙酰氨基葡萄糖(O-GlcNAc)是真核细胞中发现的多种糖苷 PTMs 中的一种,但与其他糖加成物不同的是,它完全以细胞内分子的形式存在,并形成寡聚体(1)。O ー glcnac 的加入和去除仅仅是由 o ー glcnac 转移酶(OGT)和 o ー glcnacase (OGA)(2)促进的,所以这两种酶的协调活动产生了一种多功能的信号动力,可以快速改变信号通路(3)。因此,许多细胞过程都受到 O-GlcNAc 的调控,包括细胞凋亡、线粒体功能、增殖和基因转录(4-9)。由于 O-GlcNAc 在真核细胞中起着至关重要和多样化的作用,非典型的 O-GlcNAc 化可能是各种疾病的驱动力。阿尔茨海默氏病(10)、糖尿病和几种类型的癌症都与 O-GlcNAc、 OGT 和 OGA (5,9-16)的异常水平或行为有关。对这些生理异常背后的深层原因的研究已经产生了对细胞信号机制和 O-GlcNAc 在整个细胞功能,特别是营养感知中的作用的过多的新见解。作为一种营养传感器,O-GlcNAc 对特定大分子水平的波动作出反应,以便以适当的方式引导细胞的反应。然而,营养物感知是许多不同途径的一个重要方面,包括哺乳动物雷帕霉素靶蛋白(mTOR)和 AMP活化蛋白激酶途径(AMPK)。通过了解这些途径对营养水平的反应以及它们之间的相互作用,我们可以开始确定营养感知是如何影响整个细胞活动的。

The Hexosamine Biosynthetic Pathway, mTOR Pathway, and AMPK Pathway Participate in Nutrient Sensing

氨基己糖生物合成途径、 mTOR 途径和 AMPK 途径参与营养传感

The Hexosamine Biosynthetic Pathway

氨基己糖生物合成途径

The Hexosamine Biosynthetic Pathway (HBP) utilizes approximately 3-5% of cellular glucose as well as glutamine, acetyl-Coenzyme A (CoA), and uridine, to generate the OGT substrate, UDP-GlcNAc (1217). This process begins with the conversion of a single glucose molecule to glucose-6-phosphate, which is then converted to fructose-6-phosphate. Glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme in the HBP, utilizes a glutamine amino acid and fructose-6-phosphate to create glucosamine-6-phosphate (1417). Acetyltransferase EMEG2 then generates N-acetylglucosamine-6-phosphate using acetyl-Coenzyme A (CoA) before it is finally converted to uridine 5′-diphospho- N-acetylglucosamine (UDP-GlcNAc) using uridine-5′-triphosphate (17). While glucose is the initial input, HBP’s sensitivities extend to fats, amino acids, and nucleotides, as well (Figure 1).

氨基己糖生物合成途径(HBP)利用大约3-5% 的细胞葡萄糖以及谷氨酰胺、乙酰辅酶 a (CoA)和尿苷生成 OGT 底物 UDP-GlcNAc (12,17)。这个过程开始于一个葡萄糖分子转化为葡萄糖-6-磷酸,然后转化为果糖 -6- 磷酸。谷氨酰胺: 果糖 -6- 磷酸酰胺转移酶(GFAT)是 HBP 中的限速酶,利用谷氨酰胺氨基酸和果糖 -6- 磷酸合成葡萄糖 -6- 磷酸盐(14,17)。乙酰基转移酶 EMEG2然后使用乙酰辅酶 a 生成 n- 乙酰氨基葡萄糖 -6- 磷酸盐,最后使用尿苷 -5′-三磷酸盐(17)将其转化为尿苷 -5′-二磷酸 n- 乙酰氨基葡萄糖(UDP-GlcNAc)。虽然葡萄糖是初始输入,HBP 的敏感性扩展到脂肪、氨基酸和核苷酸,以及(图1)。FIGURE 1 图1

Figure 1. The shared components between the HBP, AMPK, and mTOR pathways allow them to work in a synchronized manner to direct cell activity, but perturbations in these interactions can also drive pathology. O-GlcNAcylation of AMPK inhibits its ability to phosphorylate TSC1/2 and repress mTORC1 activation. This, in turn, can lead to unchecked cell proliferation, a hallmark of cancer and other diseases. The balance of nutrient intake also plays a pivotal role in guiding the interactions between these pathways. Increased glucose levels can bolster ATP production, both of which are necessary components for the HBP. Along with heightened UDP-GlcNAc levels, the ATP:AMP ratio shifts, thus hindering AMPK activation. Increasing amino acid intake contributes to AMPK suppression and direct/indirect mTORC1 activation, though the exact mechanisms behind these phenomenon are not entirely understood.

图1。HBP、 AMPK 和 mTOR 途径之间的共享成分允许它们以同步的方式指导细胞活动,但是这些相互作用中的扰动也可以驱动病理学。AMPK 的 o- 谷氨酰基化抑制其磷酸化 TSC1/2和抑制 mTORC1活性。这反过来又会导致未加抑制的细胞增殖,这是癌症和其他疾病的标志。营养摄入的平衡也在指导这些途径之间的相互作用方面起着关键作用。葡萄糖水平的提高可以促进 ATP 的产生,这两者都是 HBP 的必要组成部分。随着 UDP-GlcNAc 水平的提高,ATP: AMP 比率发生变化,从而阻碍 AMPK 的激活。增加氨基酸的摄入有助于 AMPK 的抑制和 mTORC1的直接/间接激活,尽管这些现象背后的确切机制尚不完全清楚。

Fluctuations in the levels of the macromolecules that feed into the HBP alter the output of this pathway, making it a diverse and responsive nutrient sensor (12). For instance, over-expression of the glucose transporter, GLUT1, in the skeletal muscle of transgenic mice resulted in a 2-to-3-fold increase in UDP-GlcNAc concentration (14). However, GLUT1 over-expression did not increase expression of the rate-limiting enzyme in the HBP, glutamine: fructose-6-phosphate amidotransferase (GFAT), suggesting that glucose intake, as opposed to GFAT levels, could account for the rise in UDP-GlcNAc (14).

进入 HBP 的大分子水平的波动改变了这一通路的输出,使其成为一种多样性和反应灵敏的营养传感器(12)。例如,在转基因小鼠的骨骼肌中过度表达葡萄糖转运蛋白 GLUT1,导致 UDP-GlcNAc 浓度增加2到3倍(14)。但是,GLUT1过表达并没有增加 HBP 中限速酶谷氨酰胺: 果糖 -6- 磷酸酰胺转移酶(GFAT)的表达,提示葡萄糖摄入,而不是 GFAT 水平,可以解释 UDP-GlcNAc (14)的上升。

While nutrient concentrations in vivo can alter the output of the HBP, OGT activity also plays a role in nutrient-sensing. Transgenic mice with skeletal muscle overexpression of OGT demonstrated increased levels of serum insulin, indicating hyperinsulinemia, which is characteristic in type II diabetes (16). Furthermore, in glucose-deprived HepG2 cells, OGA transcription was suppressed and OGT expression was up-regulated coinciding with a dramatic amplification in global O-GlcNAcylation (12) and decreased activity for glycogen synthase (GS), the enzyme responsible for constructing glycogen from individual glucose molecules (12). These data suggest that OGT plays an active role in energy conservation during starvation by inhibiting pathways that store glucose (12). However, there is a dark side to this mechanism. Overfeeding of cells with glucose or glucosamine resulted in significant impairment of insulin activation of glycogen synthase (13). In normal cell physiology, insulin signaling results in activation of glycogen synthase via dephosphorylation by phosphatases and inactivation of glycogen synthase kinase-3 (13). However, the heightened nutrient conditions increased overall O-GlcNAc levels, including O-GlcNAcylation of phosphorylation sites on glycogen synthase resulting in reduced glycogen synthase activity. Since the phosphorylation-dephosphorylation dynamic of glycogen synthase renders it sensitive to insulin signaling, O-GlcNAcylation disturbs this process and instigates insulin resistance (13).

虽然体内营养浓度可以改变 HBP 的输出,但 OGT 活性也在营养传感中起作用。转基因小鼠骨骼肌 OGT 过度表达显示血清胰岛素水平升高,提示高胰岛素血症,这是 II 型糖尿病的特征(16)。此外,在葡萄糖缺乏的 HepG2细胞中,OGA 转录被抑制,OGT 表达上调,与全球葡萄糖苷酰化(12)和糖原合成酶(GS)活性下降相一致,后者负责从单个葡萄糖分子(12)中构建糖原。这些数据表明,OGT 通过抑制存储葡萄糖的途径在饥饿期间发挥了积极的节能作用(12)。然而,这种机制也有黑暗的一面。细胞过量摄入葡萄糖或葡萄糖胺导致糖原合成酶(13)的胰岛素活化明显减弱。在正常细胞生理中,胰岛素信号通过磷酸酶去磷酸化和糖原合成酶激酶 -3(13)失活来激活糖原合成酶。然而,营养条件的提高增加了整体的葡萄糖苷合成酶的水平,包括葡萄糖苷合成酶的磷酸化位点的葡萄糖苷酰基化导致降低糖原合成酶的活性。由于糖原合成酶的磷酸化-去磷酸化动力学使其对胰岛素信号变得敏感,o- 谷氨酰胺酰基化扰乱了这一过程并引起胰岛素抵抗(13)。

HBP flux regulates multiple steps of the insulin signaling pathway (18). Skeletal muscle of mice containing an OGT-KO showed heightened glucose uptake in response to insulin as opposed to wild type counterparts, suggesting a link between insulin sensitivity and O-GlcNAc levels (19). When looking into the molecular mechanism behind this phenomenon, insulin receptor substrate 1 (IRS-1), a protein phosphorylated by the insulin receptor (IR) tyrosine kinase after it binds extracellular insulin, has multiple O-GlcNAcylation sites (20). Moreover O-GlcNAcylation of IRS-1 also correlates with a dramatic decrease in phosphorylation of this protein (18), which is a necessary step in activating downstream pathways such as AKT signaling and vesicular trafficking of GLUT4 transporters. Importantly, insulin signaling sees the redistribution of OGT to the plasma membrane within 20–30 min post-insulin induction and suggests that OGT then phosphorylates IRS and IRS2 leading to decreased signaling (2122). Overall, increased O-GlcNAc appears to foster insulin insensitivity and hinder cellular glucose uptake making the HBP a novel therapeutic target in type II diabetes research.

HBP 通量调节胰岛素信号通路的多个步骤(18)。与野生型相比,含有 ogt ー ko 基因的小鼠骨骼肌对胰岛素的反应显示了更高的葡萄糖摄取,这表明胰岛素敏感性和 o ー glcnac 水平之间存在联系(19)。当研究这种现象背后的分子机制时,胰岛素受体底物1(IRS-1) ,一种在与细胞外胰岛素结合后被胰岛素受体酪氨酸激酶磷酸化的蛋白质,有多个 o- 谷氨酰胺酰化位点(20)。此外,IRS-1的 o- 谷氨酰基化也与该蛋白(18)的磷酸化程度急剧下降有关,这是激活 AKT 信号和 GLUT4转运蛋白水泡运输等下游途径的必要步骤。重要的是,胰岛素信号通路可以在胰岛素诱导后20-30分钟内将 OGT 重新分布到质膜上,这表明 OGT 可以磷酸化 IRS 和 IRS2,从而导致信号通路的减少(21,22)。总的来说,O-GlcNAc 增加似乎促进胰岛素敏感性和阻碍细胞葡萄糖摄取,使 HBP 成为 II 型糖尿病研究中的一个新的治疗靶点。

The mTOR Pathway

mTOR 路径

While the HBP is a nutrient-sensor for several major macromolecules (121416172325), the mTOR pathway also shares several of these sensitivities. The mTOR pathway is a well-characterized signal transduction pathway that is a focal point in studies for diseases like cancer, diabetes, and Alzheimer’s. The mTOR protein is a serine/threonine protein kinase that functions as a component in two unique multi-protein complexes, mTORC1 and mTORC2, directing cell activities distinct from one another (2627). While mTORC2 regulates cell survival and cytoskeletal organization (2627), mTORC1 participates in directing proliferation and biosynthetic pathways (262829). Regulation of mTORC2 remains underdeveloped when compared to mTORC1, whose sensitivities to cellular energy levels and amino acids direct its activity (26).

虽然 HBP 是几个主要大分子(12-14,16,17,23-25)的营养传感器,mTOR 途径也具有几个这些敏感性。mTOR 通路是一种特征明显的信号转导通路,是癌症、糖尿病和阿尔茨海默病等疾病研究的焦点。mTOR 蛋白是一种丝氨酸/苏氨酸蛋白激酶,作为两种独特的多蛋白复合物(mTORC1和 mTORC2)的组成部分发挥作用,引导细胞活动彼此不同(26,27)。mTORC2调节细胞存活和细胞骨架组织(26,27) ,mTORC1参与指导增殖和生物合成途径(26,28,29)。与 mTORC1相比,mTORC2的调节仍然不发达,mTORC1对细胞能量水平和氨基酸的敏感性直接影响其活性(26)。

Activation of the mTOR pathway occurs when RAS homolog enriched in brain (Rheb) binds GTP to activate mTORC1 (3032). Amino acid withdrawal will decrease phosphorylation of the p70S6 kinase, a key mTORC1 target, but overexpression of Rheb rescues p70S6K activation (31), drawing a clear connection between mTORC1 activity and Rheb. However, a protein complex referred to as the Ragulator must first recruit Rheb and mTORC1 to the surface of a lysosome in order for activation of mTORC1 to occur; for cells lacking Ragulator components, mTORC1 activity was undetected, indicating that lysosomal localization was a necessary step in the mTOR pathway (32). Tuberous sclerosis 1 (TSC1) and TSC2 are upstream inhibitors of mTORC1 that act as GTPase-activating proteins (GAPs) for Rheb (26). Active Rheb (GTP-Rheb) facilitates mTORC1 activation once localized to the surface of a lysosome, while TSC1/2 stimulates Rheb to hydrolyze GTP to GDP, mTORC1 activation becomes repressed (26) (Figure 1).

mTORC1(30-32)在大脑中富含 RAS 同源物(Rheb)结合 GTP 激活 mTORC1(30-32)时,mTOR 通路被激活。氨基酸的去除会降低 mTORC1关键靶点 p70S6激酶的磷酸化水平,但是 Rheb 过度表达则会破坏 p70S6K 激活(31) ,从而明确了 mTORC1活性与 Rheb 之间的关系。然而,被称为 Ragulator 的蛋白复合体必须首先将 Rheb 和 mTORC1补充到溶酶体表面,以激活 mTORC1; 对于缺乏 Ragulator 成分的细胞,mTORC1活性未被发现,这表明溶酶体定位是 mTOR 途径中的必要步骤(32)。结节性硬化症1(TSC1)和 TSC2是 mTORC1的上游抑制剂,作为 rhib (26)的 gtpase 激活蛋白(gap)。活性 Rheb (GTP-Rheb)一旦定位于溶酶体表面,mTORC1的激活就会增强,而 TSC1/2则促使 GTP 水解为 GDP,mTORC1的激活受到抑制(26)(图1)。

Like the HBP, the mTOR pathway also participates in insulin signaling. Inhibition of TSC1/2 occurs via phosphorylation by AKT (33). First, the IRS proteins gather the components for a class III phosphatidylinositol 3-kinase (PI3K), which converts phosphatidylinositol 2-phosphate (PIP2) to PIP3; the increase in PIP3 signals AKT to localize to the plasma membrane for activation (34). Interestingly, the PI3K protein, Vps34, has demonstrated amino acid-sensitive regulation of mTORC1 activation (34). Cells cultured in high levels of amino acids demonstrated increased Ca2+ uptake, which is necessary for calmodulin to bind to Vps34 and facilitate activation (34). In turn, Vps34 generates higher levels of PIP3, which has been speculated to recruit protein domains necessary for the conformational changes in the mTORC1 signalsome that lead to mTORC1 activation (34).

与 HBP 一样,mTOR 途径也参与胰岛素信号转导。TSC1/2的抑制通过 AKT (33)的磷酸化发生。首先,IRS 蛋白质收集了一种 III 类磷脂酰肌醇3激酶(PI3K)的成分,这种激酶能够将2- 磷酸磷脂酰肌醇(PIP2)转化为 PIP3; 增加的 PIP3信号 AKT 定位到质膜上激活(34)。有趣的是,PI3K 蛋白 Vps34已经证明了 mTORC1活化的氨基酸敏感性调节(34)。在高氨基酸水平培养的细胞表现出钙摄取增加,这是钙调素结合 Vps34和促进活化所必需的(34)。反过来,Vps34产生更高水平的 PIP3,这已被推测为补充必要的蛋白质结构域的 mTORC1信号体的构象变化,导致 mTORC1激活(34)。

However, amino acids also have the capacity to activate mTORC1 in a TSC1/2 independent manner. For TSC2-null cells that underwent amino acid starvation, phosphorylation of the mTORC1 target, S6K1, was not rescued, indicating the necessity of amino acids for mTORC1 activation (35). Further examination of this phenomenon revealed that amino acid withdrawal prevented mTORC1 localization to the lysosome, a crucial step in mTORC1 activation (32). However, evidence suggests that amino acids alone are not responsible for mTORC1 localization, but are instead mediated by the trimeric Ragulator complex, which localizes to lysosomes in high amino acid conditions (32). For cells lacking Ragulator components, amino acid treatment could not stimulate mTORC1 activation while control cells demonstrated an increase in phosphorylated mTORC1 targets (32). While the exact mechanism is not entirely understood, there has been speculation that amino acids signal Ragulator to bind to the surface of a lysosome and act as a docking scaffold to facilitate mTORC1 activation (3236).

然而,氨基酸也具有以 TSC1/2独立方式激活 mTORC1的能力。对于经历氨基酸饥饿的 TSC2-null 细胞来说,mTORC1靶点 S6K1的磷酸化并没有被挽救,这表明 mTORC1活化需要氨基酸(35)。对这一现象的进一步研究表明,氨基酸的退出阻止了 mTORC1定位于溶酶体,这是 mTORC1激活的关键步骤(32)。然而,有证据表明,单独的氨基酸并不是 mTORC1定位的原因,而是通过三聚体 Ragulator 复合物介导的,这种复合物在高氨基酸条件下定位于溶酶体(32)。对于缺乏 Ragulator 成分的细胞,氨基酸处理不能刺激 mTORC1的活化,而对照细胞显示出磷酸化 mTORC1靶点的增加(32)。虽然确切的机制尚不完全清楚,但有推测认为氨基酸信号 Ragulator 与溶酶体表面结合,并作为一个对接支架促进 mTORC1的活化(32,36)。

Nutrient-sensing in the mTOR pathway also extends to ATP, allowing it to direct cell activity according to energy levels. This sensitivity is evident in mTORC1 regulation of superoxide dismutase 1 (SOD1) (37), which converts reactive oxygen species (ROS) into H2O2. Several cell lines treated with a major mTORC1 inhibitor, Rapamycin, demonstrated amplifications in SOD1 activity and decreased phosphorylated SOD1, which was similar to the outcome for glucose starved cells (37). Because glucose starvation halts cytosolic ATP production by glycolysis and mTORC1 is dependent upon these specific ATP reserves (38), mTORC1 inhibition of SOD1 is hinged by ATP availability. This sensitivity even extends to fluctuations in nucleic acids, as demonstrated by mTORC1 suppression in tissue culture cells treated purine synthesis inhibitors, lometrexol (LTX), and methotrexate (MTX). However, when exposed to exogenous nucleosides, only adenosine was able to revive mTORC1 activity, indicating that derivatives of ATP can regulate mTORC1.

mTOR 途径中的营养物感知也延伸到 ATP,允许它根据能量水平指导细胞活动。这种敏感性在 mTORC1对超氧化物歧化酶1(SOD1)(37)的调节中表现得很明显,该调节将活性氧类(ROS)转化为 H2O2。一些细胞株经 mTORC1抑制剂雷帕霉素处理后,其 SOD1活性增强,磷酸化 SOD1减少,这与葡萄糖饥饿细胞的结果相似(37)。由于葡萄糖饥饿抑制了糖酵解产生细胞内 ATP,mTORC1依赖于这些 ATP 特异性储备(38) ,因此 mTORC1对 SOD1的抑制与 ATP 的可利用性有关。这种敏感性甚至延伸到核酸的波动,正如 mTORC1抑制在组织培养细胞处理嘌呤合成抑制剂,乐米曲索(LTX) ,甲氨蝶呤(MTX)证明。但是,当外源性核苷暴露时,只有腺苷能够恢复 mTORC1的活性,这表明 ATP 衍生物能够调节 mTORC1。

The AMPK Pathway

AMPK 路径

AMP-activated protein kinase (AMPK), another nutrient sensing pathway, senses shifts in the AMP: ATP dynamic and responds inversely to energy levels in relation to mTORC1 (3940). While there is not a completed structure for AMPK as of yet, current data suggests that it is a heterotrimeric complex with a catalytic kinase domain and two regulatory regions (40). When energy intake falls and ATP consumption produces large amounts of cytosolic AMP, and in turn, Liver Kinase B1 (LKB1) facilitates the activation of AMPK by utilizing AMP to phosphorylate AMPK (41). For MEFs treated with the AMPK stimulator, 5-aminoimidazole-4-carboxylamine ribonucleotide (AICAR), LKB1-null cells failed to produce AMPK phosphorylated at Thr-172, while wild-type MEFS demonstrated a dramatic surge in phosphorylated AMPK levels when compared with non-treated cells (41) (Figure 1).

另一种营养传感途径—- AMP活化蛋白激酶,感知 AMP: ATP 的动态变化,并对 mTORC1的能量水平作出相反的反应。虽然 AMPK 还没有一个完整的结构,但是目前的数据表明它是一个具有催化激酶结构域和两个调控区(40)的异三聚体复合体。当能量摄入下降,ATP 消耗产生大量的胞浆 AMP,反过来,肝激酶 B1(LKB1)利用 AMP 磷酸化 AMPK (41)促进 AMPK 的激活。对于使用 AMPK 刺激剂治疗的 MEFs,5- 氨基咪唑 -4- 羧基胺核糖核苷酸(AICAR) ,LKB1-null 细胞在 Thr-172磷酸化的 AMPK 未能产生,而野生型 MEFs 与未治疗细胞(41)相比,磷酸化的 AMPK 水平显著升高(图1)。

Once activated, AMPK participates in a wide scope of pathways and cellular processes, including lipid metabolism (42). Acetyl-Coenzyme A carboxylase (ACC), the enzyme responsible for catalyzing the conversion of acetyl-CoA to malonyl-CoA, a precursor in fatty acid synthesis, is a known target of AMPK inhibition (4243). While there are three phosphorylation sites on ACC2 and it is a target for three different kinases, AMPK modification of this enzyme most potently decreased the VMAX (43), which is significant when we consider the spectrum of acuteness in regulating enzyme activity. AMPK phosphorylates Ser79 for ACC1 and Ser212 for ACC2, so a double alanine knock-in of both sites in mice revealed a decline in fatty acid oxidation and increased lipogenesis (44), demonstrating that AMPK plays a critical role in fatty acid metabolism. Muscle cells treated with Compound C, an AMPK inhibitor, demonstrated a marked decrease in phosphorylated AMPK in conjunction with heightened triglyceride levels (45). Combining these data, there appears to be a clear role for AMPK in repressing lipid production.

一旦激活,AMPK 参与广泛的通路和细胞过程,包括脂质代谢。乙酰辅酶 a 羧化酶(ACC)是一种催化乙酰辅酶 a 转化为丙二酰辅酶 a 的酶,是脂肪酸合成的前体,是 AMPK 抑制剂的已知目标(42,43)。ACC2上有三个磷酸化位点,它是三种不同激酶的作用靶点,而 AMPK 修饰对 VMAX (43)的降低作用最为显著。AMPK 将 Ser79磷酸化为 ACC1,将 Ser212磷酸化为 ACC2,因此小鼠两个位点的双丙氨酸敲入显示脂肪酸氧化下降和脂肪生成增加(44) ,表明 AMPK 在脂肪酸代谢中起关键作用。经 AMPK 抑制剂化合物 c 处理的肌肉细胞,显示出磷酸化 AMPK 明显减少,与甘油三酯水平升高有关(45)。综合这些数据,AMPK 在抑制脂质生成方面似乎有明显的作用。

Another AMPK target is the mTORC1 pathway, which accounts for the inverse response of both pathways in respect to AMP:ATP ratios. In poor nutrient conditions, ATP depletion causes the ratio to shift toward AMP bolstering AMPK activation in turn, AMPK phosphorylates TSC1/2 in order to stimulate GAP activity toward Rheb, thus suppressing mTORC1. However, it was also revealed that AMPK inhibits mTORC1 by phosphorylating its scaffold protein, raptor (39). Therefore, AMPK has the capacity to both directly and indirectly regulate mTORC1.

AMPK 的另一个靶点是 mTORC1通路,该通路负责两条通路对 AMP: ATP 比值的反向响应。在营养条件较差的情况下,ATP 的耗竭导致该比值向 AMP 转移,促进 AMPK 活化,AMPK 磷酸化 TSC1/2,以刺激 GAP 活性向 Rheb 转移,从而抑制 mTORC1。然而,研究也表明 AMPK 通过磷酸化 mTORC1的骨架蛋白,raptor (39)来抑制 mTORC1。因此 AMPK 具有直接和间接调控 mTORC1的能力。

While energy levels drive the dynamic between AMPK and mTORC1, amino acid concentrations also influence this relationship (46). For instance, pancreatic β-cells treated with high doses of glucose, leucine, and glutamine experienced a significant increase in mTORC1 activity, while phosphorylated AMPK diminished (46), indicating a contrasting reaction for AMPK and mTORC1 in terms of amino acid exposure.

虽然能量水平驱动 AMPK 和 mTORC1之间的动态关系,氨基酸浓度也影响这种关系(46)。例如,高剂量葡萄糖、亮氨酸和谷氨酰胺处理的胰腺细胞 mTORC1活性显著增加,而磷酸化 AMPK 减少(46) ,表明 AMPK 和 mTORC1在氨基酸暴露方面的反应不同。

Nutrient-sensing is a complex activity that directs the behavior of individual pathways, so when we consider pathways affected by similar macromolecules, we must explore how fluctuations in nutrient concentrations can impact the way these pathways interact and coordinate in cell signaling. For the HBP, mTOR, and AMPK pathways, there is a complex inter-play in regards to their responses to nutrient levels (Figure 1). However, in order to truly understand how O-GlcNAc impacts cell signaling in specific pathologies, we must further analyze the interactions of the HBP with AMPK and mTORC1, respectively.

营养感知是一种复杂的活动,指导个体通路的行为,所以当我们考虑通路受到类似的大分子影响,我们必须探索营养浓度的波动如何影响这些通路相互作用和协调细胞信号的方式。对于 HBP、 mTOR 和 AMPK 途径,它们对营养水平的反应存在复杂的相互作用(图1)。然而,为了真正了解 O-GlcNAc 如何在特定的病理过程中影响细胞信号传导,我们必须进一步分析 HBP 与 AMPK 和 mTORC1的相互作用。

The Cross-Talk Between the HBP and AMPK Pathways

HBP 与 AMPK 通路之间的相互作用

In cell signaling, cross-talk between pathways is a relationship based on a mutual capacity to regulate one another, so the discovery of a cross-talk relationship between OGT and AMPK opened a whole new avenue for exploring inter-pathway dynamics and their impact on cell function (21). The potential for crosstalk between O-GlcNAc and AMPK was first suggested with studies using glucosamine (GlcN) as a supplement. Mice treated with high concentrations of GlcN quickly increase O-GlcNAc levels since GlcN supplementation by-passes GFAT regulation of the HBP (242547), but GlcN treatment negatively impaired insulin signaling quickly leading to elevated blood glucose levels. Compounding with the loss of insulin sensitivity, high levels of GlcN treatment rapidly lowers ATP levels due to the actions of hexokinase phosphorylating GlcN (23). Hence, rapid unregulated flux through the HBP increases AMP levels and could activate AMPK (48). Furthermore, low levels of sustained GlcN treatment transiently activates AMPK activity, lowers oxidative phosphorylation, and increases lifespan in C. elegans and mice (49). The effect of the GlcN treatment is mediated by changes in O-GlcNAcylation since sustained treatment with OGA inhibitor Thiamet-G (TMG) in mice and cell lines also reduces oxidative phosphorylation, lowers ATP production, and reprograms the transcriptome (7). Together, these data would argue that changes in HBP flux influence AMPK activation via increased cellular O-GlcNAcylation. Of note, AMPK activity is higher in OGT KO mice skeletal muscle cells suggesting loss of OGT activates AMPK (50).

在细胞信号传导中,通路间的串扰是一种基于相互调节能力的关系,因此 OGT 和 AMPK 之间串扰关系的发现为探索通路间动力学及其对细胞功能的影响开辟了一条全新的途径。用葡萄糖胺(GlcN)作为补充剂,首次提出了 O-GlcNAc 与 AMPK 之间存在串扰的可能性。由于 GlcN 补充剂通过 GFAT 调节 HBP (24,25,47) ,高浓度 GlcN 处理的小鼠迅速增加 O-GlcNAc 水平,但 GlcN 治疗负面影响胰岛素信号迅速导致血糖水平升高。伴随着胰岛素敏感性的丧失,由于己糖激酶磷酸化 GlcN (23)的作用,高水平的 GlcN 治疗迅速降低 ATP 水平。因此,通过 HBP 的快速非常规通量增加 AMP 水平,并可激活 AMPK (48)。此外,低水平的持续 GlcN 治疗短暂地激活 AMPK 活性,降低氧化磷酸化,并延长线虫和老鼠的寿命(49)。GlcN 治疗的效果是通过 O-GlcNAcylation 的变化而介导的,因为在小鼠和细胞系中持续使用 OGA 抑制剂 TMG 也会降低氧化磷酸化,降低 ATP 产量,并重新编码转录组(7)。总之,这些数据可以证明 HBP 通量的变化通过增加细胞的 o- 谷氨酰基化作用影响 AMPK 的激活。值得注意的是,在 OGT KO 小鼠骨骼肌细胞中 AMPK 活性更高,这表明 OGT 的缺失激活了 AMPK (50)。

Finally, studies on glucose deprivation revealed (5051) distinct, tissue-dependent relationships between O-GlcNAc and AMPK. HepG2 and Neuro-2a cells treated with AMPK inhibitors and subjected to glucose starvation experienced significantly lower global O-GlcNAcylation when compared to cells that were only glucose starved (51), inferring an AMPK-dependent mechanism for heightened O-GlcNAcylation in glucose starvation. However, A459 carcinoma lung cells responded differently than the previously observation; while glucose starvation did result in dramatic increases in O-GlcNAcylation, it did not coincide with any major changes in AMPK activation (50). Treatment with Compound C, an AMPK inhibitor, in conjunction with glucose starvation still resulted in an increase in global O-GlcNAcylation, though the effects were somewhat less pronounced than cells only undergoing glucose starvation (50). AMPK inhibition also resulted in a significant decrease in glycogen synthase, a key enzyme in the process of converting glucose molecules into glycogen, but did not impact the expression of glycogen phosphorylase (GP), an enzyme responsible for catalyzing glycogen degradation (50). Further investigation revealed that inhibition of GP in conjunction with glucose starvation dramatically repressed the starvation-induced increase in global O-GlcNAcylation (50), suggesting that AMPK and GP are responsible for coordinating a shift in glycogen metabolism under starvation periods, possibly to generate large pools of glucose for input into the HBP. Overall, while the impact of AMPK activity on starvation-induced O-GlcNAcylation appears to vary between tissue types, the observed evidence indicates a clear niche for AMPK in regulating metabolism in response to nutrient deprivation stress. However, complicating these conclusions is that glucose starvation induced increases in O-GlcNAcylation is dependent on AMPK activity (51). Hence, these data suggest manipulation of O-GlcNAcylation through HBP flux alters cellular energy usage and could influence AMPK activity although these changes could be tissue specific.

最后,对葡萄糖剥夺的研究显示(50,51) O-GlcNAc 和 AMPK 之间有明显的组织依赖关系。经 AMPK 抑制剂处理的 HepG2和 Neuro-2a 细胞在经受葡萄糖饥饿处理后,其全球 o- 谷氨酰基化水平明显低于只经受葡萄糖饥饿处理的 HepG2和 Neuro-2a 细胞(51) ,推测在葡萄糖饥饿条件下,AMPK 依赖的机制增强了 o- 谷氨酰基化水平。然而,A459肺癌细胞的反应不同于以前的观察,虽然葡萄糖饥饿确实导致了巨大的葡萄糖酰化增加,它并不符合任何主要的变化 AMPK 活化(50)。使用 AMPK 抑制剂化合物 c 与葡萄糖饥饿联合治疗仍然导致全球 o- 葡萄糖基酰化增加,尽管这种效果比只经历葡萄糖饥饿的细胞有所减弱(50)。AMPK 抑制也导致糖原合成酶显著减少,糖原合成酶是葡萄糖分子转化为糖原过程中的关键酶,但并不影响糖原磷酸化酶的表达,糖蛋白是一种催化糖原降解的酶。进一步的研究表明,GP 与葡萄糖饥饿联合抑制饥饿诱导的全球葡萄糖基酰化(50)的增加,提示 AMPK 和 GP 负责协调饥饿时期糖原代谢的变化,可能产生大量葡萄糖进入 HBP。总的来说,虽然 AMPK 活性对饥饿诱导的 o- 谷氨酰胺酰胺化作用的影响在不同组织类型之间似乎有所不同,但观察到的证据表明,AMPK 在应对营养剥夺胁迫调节代谢方面具有明确的生态位。然而,使这些结论复杂化的是,葡萄糖饥饿诱导的 o- 谷氨酰基化增加依赖于 AMPK 活性(51)。因此,这些数据表明通过 HBP 通量操纵 o- 谷氨酰基化改变细胞能量的使用,并可能影响 AMPK 活性,虽然这些改变可能是组织特异性的。

Recently, a study demonstrated that AMPK and OGT are substrates for each other and regulate each other’s activity (4). In HEK293T kidney cells treated with OGA inhibitors GlcNAc Thiazoline (GT) or TMG, several AMPK subunits were O-GlcNAcylated leading to decreased AMPK activating phosphorylation. These data clearly demonstrate a regulatory role for OGT in AMPK activity (21). On the other hand, AMPK phosphorylation of Thr44 on OGT increases OGT nuclear localization, increases nuclear O-GlcNAcylation, increases histone H3K9 acetylation (4), while O-GlcNAcylation of the H2B histone is lower (22), revealing AMPK as a regulator of O-GlcNAc mediated epigenetic modifications. Furthermore, AMPK regulation of the HBP is not limited to interactions with OGT. GFAT is an AMPK target for phosphorylation at Ser243 (23), and cardiomyocytes treated with the AMPK activator, A769662, have demonstrated a marked decrease in overall O-GlcNAcylation of proteins as well as increased phosphorylation of GFAT (24), hence UDP-GlcNAc production can be directed by AMPK.

最近,一项研究表明,AMPK 和 OGT 是彼此的底物,并调节彼此的活性(4)。在用 OGA 抑制剂 GlcNAc 噻唑啉(GT)或 TMG 处理的 HEK293T 细胞中,几个 AMPK 亚基被 o- 糖基化,导致 AMPK 活化磷酸化下降。这些数据清楚地表明了 OGT 在 AMPK 活性中的调节作用(21)。另一方面,Thr44在 OGT 上的磷酸化增加了 OGT 的核定位,增加了 oglcnacylation,增加了组蛋白 H3K9乙酰化(4) ,而 H2B 组蛋白的 oglcnacylation 较低(22) ,表明 AMPK 是 oglcnnac 介导的表观遗传修饰的调控因子。此外,AMPK 对 HBP 的调节并不局限于与 OGT 的相互作用。在 Ser243(23)位点,GFAT 是磷酸化的 AMPK 靶点,用 AMPK 激活剂 A769662处理的心肌细胞,表现出蛋白质的 o- 谷氨酰胺化明显减少,而 GFAT (24)的磷酸化增加,因此,UDP-GlcNAc 的产生可以被 AMPK 控制。

OGT appears to be a clear regulator of AMPK activity in cancer development. In several breast cancer cell lines, either knockdown of OGT expression by shRNA or pharmacological inhibition led to increased LKB1 phosphorylation and activation of AMPK (52). In turn, loss of OGT activity and increased AMPK activity reduced cancer cell growth, impaired HIF-1α activation, and increased SIRT1 activity (5253). In LoVo colon cancer cells treated with TMG a marked increase in growth and proliferation occurred (5). Interestingly, TMG-treated cells also demonstrated increased levels of O-GlcNAcylated AMPK, as well as a decrease in phosphorylated AMPK (5) coupled with an increase in phosphorylated p70S6K (Ribosomal Protein S6 Kinase), an mTORC1 target. In these experiments, total cellular levels of O-GlcNAc regulated AMPK activity, with high levels of O-GlcNAc reducing activation while low levels increased activation. These data agree with the previous data showing AMPK O-GlcNAcylation inhibits kinase function (4). Overall, sustained O-GlcNAcylation is linked to suppression of AMPK activation, which could increase mTORC1 activity and heightened cell proliferation rates. Hence, O-GlcNAcylation can influence mTORC1 activity indirectly through AMPK, but is there crosstalk between mTOR and OGT that would influence activity of each pathway?

OGT 似乎是癌症发展中 AMPK 活性的明确调节因子。在一些乳腺癌细胞系中,无论是通过 shRNA 抑制 OGT 的表达,还是药物抑制导致 LKB1磷酸化和 AMPK (52)的激活增加。反过来,OGT 活性的丧失和 AMPK 活性的增加降低了癌细胞的生长,阻碍了 hif-1的激活,增加了 SIRT1的活性(52,53)。TMG 处理的 LoVo 结肠癌细胞生长和增殖明显增加(5)。有趣的是,tmg 处理的细胞也表现出 o- 谷氨酰胺化 AMPK 水平的增加,以及磷酸化 AMPK (5)的减少,同时增加磷酸化 p70S6K (核糖体蛋白质 S6激酶) ,一个 mTORC1的靶点。在这些实验中,细胞总水平的葡萄糖苷钠调节 AMPK 活性,高水平的葡萄糖苷钠降低活性,而低水平的活性增加。这些数据与先前显示 AMPK o- 谷氨酰基化抑制激酶功能的数据一致(4)。总的来说,持续的 o- 谷氨酰基化与抑制 AMPK 的活化有关,这可以增加 mTORC1的活性和提高细胞增殖率。因此,o- 谷氨酰胺酰化可以通过 AMPK 间接影响 mTORC1的活性,但是 mTOR 和 OGT 之间是否存在交叉作用,从而影响各个途径的活性?

Sustained O-GlcNAcylation Correlates With Increased Phosphorylation of mTOR Targets

维持 o- 谷氨酰胺酰化与 mTOR 靶点磷酸化增加的关系

Interactions between the HBP and AMPK appear to result in inverse responses between the two, not unlike the opposing dynamic between the AMPK and mTOR pathways. The HBP and mTOR pathway share sensitivities for specific macromolecules and similar responses to fluctuations in nutrition, (121416172325323637), leaving a wide door for exploring possible interactions. Recent evidence has suggested that mTOR and O-GlcNAc coordinate together to direct autophagy (5455). Autophagy is the process of degrading and recycling organelles, as well as other cellular components, which is significant in maintaining cellular function (54). Treatment with mTOR inhibitors Torin1 and PP242 resulted in induced autophagy and a drop in global O-GlcNAcylation, which occurred with increased OGA and decreased OGT protein expression (54). While it appears that mTOR inhibition couples with active autophagy, another study (55) demonstrated that the acuity of mTOR suppression impacts this dynamic; moderate mTOR suppression resulted in a significant increase in autophagy, but was attenuated with severe mTOR inhibition. What’s more, an inter-play between phosphorylation and O-GlcNAcylation of the autophagy regulator, Beclin1, demonstrated that moderate mTOR suppression promoted the former modification, while severe inhibition increased the latter (55). While the exact synergy between O-GlcNAc and mTOR has not been detected in autophagy, there appears to be a clear dynamic that is hinged on the spectrum of mTOR repression.

HBP 和 AMPK 之间的相互作用似乎导致了两者之间的反向反应,不像 AMPK 和 mTOR 之间的反向动力学。HBP 和 mTOR 通路对特定大分子具有共同的敏感性,对营养波动的反应也相似,(12-14,16,17,23-25,32,36,37) ,为探索可能的相互作用留下了一扇大门。最近的证据表明 mTOR 和 O-GlcNAc 共同协调指导自噬(54,55)。自噬是细胞器以及其他细胞成分的降解和循环过程,在维持细胞功能方面起着重要作用。mTOR 抑制剂 Torin1和 PP242的作用导致诱导自噬和全球 o- 谷氨酰基化下降,这与 OGA 增加和 OGT 蛋白表达减少有关(54)。虽然 mTOR 抑制与活跃的自噬有关,但另一项研究(55)表明 mTOR 抑制的灵敏度会影响这种动力; 中度的 mTOR 抑制会导致自噬的显著增加,但是严重的 mTOR 抑制会减弱。另外,自噬调节因子 Beclin1的磷酸化和 o- 谷氨酰基化之间的相互作用表明,适度的 mTOR 抑制促进了前者的修饰,而严重的抑制增加了后者的修饰(55)。虽然在自噬过程中没有发现 O-GlcNAc 和 mTOR 之间的确切协同作用,但似乎存在一种明显的动力,这种动力与 mTOR 的抑制频谱有关。

Obesity is another area of research that has revealed a dynamic between mTOR and O-GlcNAc. When analyzing normal mice and Ob/Ob type mice, there were increased levels of OGT expression and mTOR phosphorylation (56). A corresponding in vitro experiment using colon cancer cells also demonstrated higher OGT expression and phosphorylated mTOR, as well as higher levels of O-GlcNAcylation. Treatment of these cell lines with an mTOR activator (MHY1485) showed a slight increase in phosphorylated mTOR, OGT, and O-GlcNAcylation and a significant amplification in phosphorylated p70S6K (56). On the other hand, treatment with an mTOR inhibitor, rapamycin, caused distinct decreases in phosphorylated mTOR, OGT, O-GlcNAcylation and complete inhibition of p70S6K phosphorylation (56).

肥胖是另一个研究领域,它揭示了 mTOR 和 O-GlcNAc 之间的动态关系。在正常小鼠和 Ob/Ob 型小鼠中,OGT 表达水平和 mTOR 磷酸化水平均有所增加(56)。相应的结肠癌细胞体外实验也显示了更高的 OGT 表达和磷酸化 mTOR,以及更高水平的 o- 谷氨酰胺酰基化。用 mTOR 激活剂(MHY1485)处理这些细胞系时,磷酸化的 mTOR、 OGT 和 o- 谷氨酰基化略有增加,磷酸化的 p70S6K (56)有显著的扩增。另一方面,使用 mTOR 抑制剂雷帕霉素处理后,磷酸化的 mTOR、 OGT、 o- 谷氨酰胺酰化明显减少,完全抑制 p70S6K 磷酸化(56)。

Interestingly, downstream targets in the mTOR pathway also appear to respond to O-GlcNAcylation. Ribosomal protein S6 (RPS6) is a phosphorylation target of p70S6K, but is also one of many ribosomal components that can be O-GlcNAcylated. Of note, the O-GlcNAc modification on S6 does not appear to be influenced by glucose starvation (57). While the purpose of this is not yet known, it was hypothesized (57) that this phenomenon might play a role regulating translation of proteins associated with glucose metabolism. What’s more, O-GlcNAc also impacts mRNA selectivity and translation rates in diabetes via another mTORC1 target, 4E-binding protein 1 (4E-BP1). Hypophosphorylated 4E-BP1 binds to eIF4E, a ribosomal component that binds to the 5′ cap of mRNA, which blocks selective translation and alters the normal pattern of mRNA translation. Examination of the liver tissue of diabetic mice revealed that 4E-BP1 binding of eIF4E was not only dramatically increased, but O-GlcNAcylation of 4E-BP1 was elevated almost 2-fold while 4E-BP1 phosphorylation declined significantly (58), suggesting that O-GlcNAc plays a role in cap-independent translation in diabetes signaling.

有趣的是,mTOR 途径的下游靶点似乎也对 o- 谷氨酰胺酰基化作用有反应。核糖体蛋白质 S6(RPS6)是 p70S6K 磷酸化的靶点,但也是许多核糖体组分之一,可以 o- 谷氨酰基化。值得注意的是,葡萄糖饥饿(57)对 S6的 O-GlcNAc 修饰似乎没有影响。虽然其目的尚不清楚,但有假设认为这种现象可能在调节与葡萄糖代谢有关的蛋白质的翻译中起作用。另外,O-GlcNAc 还通过另一个 mTORC1靶点4E-binding protein 1(4E-BP1)影响糖尿病的 mRNA 选择性和翻译率。低磷酸化的4E-BP1与 eIF4E 结合,eIF4E 是一种与 mRNA 5′帽结合的核糖体成分,它阻止了选择性翻译,改变了 mRNA 翻译的正常模式。对糖尿病小鼠肝组织的检测显示,4E-BP1与 eIF4E 的结合不仅显著增加,而且4E-BP1的 o- 谷氨酰胺酰化几乎增加了2倍,而4E-BP1磷酸化明显下降(58) ,提示 o- 谷氨酰胺在糖尿病信号转导中起着非 cap 依赖的翻译作用。

While there is not a large amount of literature detailing the interactions between mTORC1 and the HBP, current documentation does reveal an intricate, fine-tuned dynamic between the two pathways. With diabetes in particular, this relationship appears to play a substantial role, which makes it a prime focal point for future research into these diseases. However, when considering the web of interactions occurring between the HBP and the mTOR pathway, as well as their relationships with the AMPK pathway, it is important to understand the complexity of cell behavior and sheer amount of variables required for signaling events. Therefore, exploring the dynamics between these pathways using a systems biology approach, alongside conventional laboratory techniques might be the best approach for future investigations.

虽然没有大量的文献详细描述 mTORC1和 HBP 之间的相互作用,但目前的文献确实揭示了这两个通路之间错综复杂、微调的动态关系。尤其是糖尿病,这种关系似乎发挥了重要作用,这使它成为未来研究这些疾病的主要焦点。然而,当考虑到 HBP 和 mTOR 通路之间的相互作用网络,以及它们与 AMPK 通路的关系时,了解细胞行为的复杂性和信号事件所需的大量变量是很重要的。因此,使用系统生物学方法和传统的实验室技术探索这些途径之间的动态关系可能是未来研究的最佳方法。

Systems Biology Approaches will Improve Understanding of Signaling Cross-Talk by Nutrient Sensing

系统生物学方法将通过营养传感提高对信号串扰的理解

While the evidence discussed provides a possible framework of interactions between the HBP, mTOR, and AMPK pathways, the intrinsic complexity of cellular function must be considered when formulating our understanding of these relationships. Ultimately, the true story of nutrient sensing and cellular response cannot be reduced to simple interactions, such as the repression of mTORC1 by AMPK. The maintenance of cellular homeostasis is a vital process, which must be finely tuned. Therefore, a more accurate picture would be like the mixing board used in a recording studio or a concert, rather than the volume knob on the radio in a car. There are many inputs, which interact in complex and dynamic ways. Nutritional sensing pathways, such as AMPK, mTORC1, and O-GlcNAc are replete with feedback loops (5960), which allow them to self-regulate and accomplish a fine degree of control. Unfortunately, this has substantially complicated efforts to understand these pathways, because they exercise a robust control over many perturbations.

虽然所讨论的证据提供了 HBP、 mTOR 和 AMPK 通路之间相互作用的可能框架,但在阐述我们对这些关系的理解时,必须考虑到细胞功能的内在复杂性。最终,营养感知和细胞反应的真实故事不能简单地归结为相互作用,比如 AMPK 对 mTORC1的抑制作用。维持细胞内稳态是一个重要的过程,必须进行精细的调整。因此,更准确的图像应该是录音棚或音乐会中使用的混音板,而不是汽车收音机上的音量旋钮。有许多输入,它们以复杂和动态的方式相互作用。像 AMPK、 mTORC1和 O-GlcNAc 这样的营养传感途径充满了反馈回路(59,60) ,使它们能够自我调节并完成一定程度的控制。不幸的是,这大大增加了理解这些路径的难度,因为它们对许多扰动都有强有力的控制。

An additional challenge in understanding the complexities of nutrient sensing is biological “noise” in a cell. Biological pathways are frequently represented as an orderly set of protein-protein interactions (PPIs), frequently in the form of a directed graph (61). Naturally, this is an oversimplification of reality. In fact, for any given interaction, an individual protein may have a large number of competitors for its receptor. However, these alternate ligands may not induce the same effect (e.g. conformational change) in the receptor. From one perspective, this “promiscuity” of proteins involved in PPIs is a major contributor of biological noise in a pathway, but it is important to note that noise is a matter of perspective. In some cases, it would be more accurate to consider these proteins as competing signals. Partly, this arrangement is likely the byproduct of evolution, and the mechanism through which new functions can be introduced (i.e., evolution works with what is already there, so new proteins will have similar binding domains to previous domains, especially early on) (6263). Thus, a more specific protein may have to compete with a large number of less specific alternatives. This has contributed to a view that signal transduction, such as that involved in nutrient sensing, is sometimes more probabilistic than deterministic (6465).

理解营养传感的复杂性的另一个挑战是细胞中的生物“噪音”。生物途径经常被表示为一组有序的蛋白质-蛋白质相互作用(ppi) ,经常以有向图的形式出现。当然,这是对现实的过度简化。事实上,对于任何给定的相互作用,一个单独的蛋白质可能有大量的竞争对手为其受体。然而,这些替代配体可能不会在受体中诱导同样的效应(例如构象改变)。从一个角度来看,质子泵抑制剂中蛋白质的“杂乱”是通路中生物噪声的主要贡献者,但重要的是要注意,噪声是一个透视问题。在某些情况下,将这些蛋白质视为竞争信号更为准确。在一定程度上,这种安排可能是进化的副产品,以及引入新功能的机制(例如,进化与已经存在的功能协同工作,因此新的蛋白质将有类似的结合域与以前的领域,特别是早期)(62,63)。因此,一个更具体的蛋白质可能不得不与大量不太具体的替代品竞争。这促成了一种观点,即信号转导,例如涉及营养传感,有时更具概率性而非确定性。

Nevertheless, a cell must have some method for fine-tuning signals to maintain homeostasis in a noisy environment, which is where we see PTMs come into play (64). One such way that O-GlcNAcylation functions to polish these signaling mechanism is to impede protein degradation (6668), thus increasing the probability of signal transduction, increase or decrease binding affinity for a particular interaction (6971), or competetion against other PTMs that might change a protein’s function in different ways (72). The rapid cycling of O-GlcNAc may allow for much more granular control of nutrient response (73), so taking a probabilistic view of nutrient sensing, O-GlcNAcylation might be understood as one way a cell has of weighting the dice toward a required response.

然而,细胞必须有某种方法来微调信号,以便在嘈杂的环境中保持内环境稳定,这就是我们看到 ptm 发挥作用的地方(64)。O-GlcNAcylation 功能完善这些信号机制的一种方式是阻止蛋白质降解(66-68) ,从而增加信号转导的可能性,增加或减少对特定相互作用的结合亲和力(69-71) ,或与其他 PTMs 竞争,可能以不同的方式改变一个蛋白质的功能(72)。O-GlcNAc 的快速循环可能允许更多的颗粒对营养反应进行控制(73) ,因此从营养感知的概率角度来看,O-GlcNAc 化可能被理解为细胞对骰子进行加权以获得所需反应的一种方式。

The complex and dynamic nature of maintaining glucose homeostasis suggests the need for the more holistic view that can be provided by genomic or even multi-omic methods. However, due to the reactive nature of these pathways, this view requires more than a simple “snapshot” of the cell. Rather, approaches that can paint a picture of cellular dynamics are called for. Such studies are not only expensive, but they require new methods to understand the collected data. Nevertheless, early attempts to tackle this problem show promise (7374). A recent computational method was developed after collecting transcriptional data over time, while introducing a change in nitrogen sources for Saccharomyces cerevisiae (65) in order to study the effect of metabolic signals on transcriptome perturbations mediated by TORC1. A key requirement in unraveling these complexities is to demonstrate a causal relationship between a change in metabolites and transcription, by ensuring that the metabolite change proceeds the transcriptional change temporally, so a quality dataset is required. Using these data, a probabilistic model was built, which provided evidence that glutamine availability is a regulator of TORC1 and through TORC1 is able to drive changes in other metabolites, such as Inosine monophosphate (IMP) and adenosine (the latter of which may suggest another feedback mechanism for TORC1). The accumulation of IMP, downstream of TORC1 activation is interesting, and may point to one mechanism through which the inverse relationship of TORC1 and AMPK is maintained. AMP is deaminated to IMP by Amd1, which may be regulated by TORC1. Further work was built on this approach, demonstrating that TORC1 target Sch9 may be the kinase responsible phosphorylating Amd1 (75), pointing to yet another regulatory loop that may tie TORC1 and AMPK together. Naturally, these results are in yeast, but Sch9 functions similarly to S6K1 (76), which is a known target of mTORC1. Such insights would not be possible without a closely linked computational and experimental approach, such as those used in these studies. Future work could extend these concepts to mammalian organisms and incorporate a more robust range of PTMs, such as O-GlcNAc, to construct a more accurate picture of cell signaling and the fine-tuning that regulates these mechanisms.

维持葡萄糖稳态的复杂和动态性质表明需要更全面的观点,可以通过基因组甚至多体方法提供。然而,由于这些通路的反应性质,这种观点需要的不仅仅是一个简单的细胞“快照”。相反,我们需要的是能够描绘细胞动力学图景的方法。这样的研究不仅昂贵,而且需要新的方法来理解收集到的数据。尽管如此,早期解决这个问题的尝试还是有希望的(73,74)。一个最近的计算方法是在收集了一段时间的转录数据之后发展起来的,同时引入了酿酒酵母的氮源变化,以便研究代谢信号对 TORC1介导的转录组扰动的影响。解开这些复杂性的一个关键要求是通过确保代谢产物的变化进行暂时的转录变化,来证明代谢产物变化和转录之间的因果关系,因此需要一个高质量的数据集。利用这些数据,建立了一个概率模型,该模型提供了证据,证明谷氨酰胺的可用性是 TORC1的调节器,并通过 TORC1能够驱动其他代谢物的变化,如肌苷酸(IMP)和腺苷(腺苷后者可能提示 TORC1的另一种反馈机制)。TORC1激活下游 IMP 的积累是一个有趣的现象,并且可能指向一个维持 TORC1和 AMPK 逆相关关系的机制。Amd1对 AMP 进行脱氨基作用,可能受 TORC1调节。进一步的工作建立在这种方法上,证明 TORC1靶点 Sch9可能是磷酸化 Amd1(75)的激酶,指向另一个可能将 TORC1和 AMPK 联系在一起的调节环。当然,这些结果是在酵母中得到的,但 Sch9的功能与 S6K1(76)相似,S6K1是 mTORC1的已知靶点。如果没有密切联系的计算和实验方法,例如在这些研究中使用的方法,这样的见解是不可能的。今后的工作可以将这些概念扩展到哺乳动物,并纳入更健壮的 ptm 范围,如 O-GlcNAc,以构建更准确的细胞信号图像和微调控这些机制。

Summary

摘要

The HBP, AMPK, and mTOR pathways all present a unique niche in cellular function, with respect to nutrient-sensing. However, it is the crosstalk between these pathways that fosters a particularly interesting but unexplored spotlight in cell signaling. While reduced energy levels trigger AMPK inhibition of mTORC1, these conditions can also trigger conservation of energy via increased global O-GlcNAcylation. However, increased glucose and glucosamine uptake bolsters cytosolic ATP production and triggers deviations in cellular behavior, such as insulin resistance, while repressing AMPK and allowing the mTOR pathway to encourage proliferation. Amino acid uptake facilitates mTORC1 activation and can up-regulate the HBP in a fine-tuned manner, while diminishing AMPK activity. Cross-talk between OGT and AMPK presents a mutually inhibitory relationship between the two enzymes based on nutrient availability and stimulation, while the mTOR pathway and O-GlcNAc coordinate with one another to direct autophagy.

HBP、 AMPK 和 mTOR 途径在营养传感方面都呈现出细胞功能的独特生态位。然而,正是这些途径之间的串扰促成了细胞信号中一个特别有趣但尚未探索的聚光灯。虽然降低能量水平触发 AMPK 抑制 mTORC1,这些条件也可以触发能量守恒通过增加全球葡萄糖基化。然而,葡萄糖和葡萄糖摄取的增加会促进细胞内 ATP 的产生,并引发细胞行为的改变,如胰岛素抵抗,同时抑制 AMPK 并允许 mTOR 通路促进细胞增殖。氨基酸摄取促进 mTORC1的活化,可以上调 HBP 的方式,同时减少 AMPK 活性。OGT 和 AMPK 之间的串扰基于营养物质的有效性和刺激性呈现出相互抑制的关系,而 mTOR 通路和 O-GlcNAc 之间相互协调,直接导致自噬。

Overall, there appears to be an intricate dynamic between the three pathways, where deviations in their communication lend to various pathologies. Future research should elucidate more connections between these pathways using both computational methods and traditional bench work, with the hopes that we’ll understand this “real talk” and how to counteract it when it leads to disease-related miscommunication.

总的来说,在这三条路径之间似乎存在着一种错综复杂的动态关系,在这种关系中,沟通的偏差导致了各种各样的疾病。未来的研究应该使用计算方法和传统的实验室工作来阐明这些通路之间的更多联系,希望我们能够理解这种“真实的对话” ,以及当它导致与疾病相关的错误沟通时如何消除它。

发表评论

邮箱地址不会被公开。 必填项已用*标注