The crosstalk of NAD, ROS and autophagy in cellular health and ageing

0 Comments

NAD、 ROS 和自噬在细胞健康和衰老中的相互作用

Abstract

摘要

Cellular adaptation to various types of stress requires a complex network of steps that altogether lead to reconstitution of redox balance, degradation of damaged macromolecules and restoration of cellular metabolism. Advances in our understanding of the interplay between cellular signalling and signal translation paint a complex picture of multi-layered paths of regulation. In this review we explore the link between cellular adaptation to metabolic and oxidative stresses by activation of autophagy, a crucial cellular catabolic pathway. Metabolic stress can lead to changes in the redox state of nicotinamide adenine dinucleotide (NAD), a co-factor in a variety of enzymatic reactions and thus trigger autophagy that acts to sequester intracellular components for recycling to support cellular growth. Likewise, autophagy is activated by oxidative stress to selectively recycle damaged macromolecules and organelles and thus maintain cellular viability. Multiple proteins that help regulate or execute autophagy are targets of post-translational modifications (PTMs) that have an effect on their localization, binding affinity or enzymatic activity. These PTMs include acetylation, a reversible enzymatic modification of a protein’s lysine residues, and oxidation, a set of reversible and irreversible modifications by free radicals. Here we highlight the latest findings and outstanding questions on the interplay of autophagy with metabolic stress, presenting as changes in NAD levels, and oxidative stress, with a focus on autophagy proteins that are regulated by both, oxidation and acetylation. We further explore the relevance of this multi-layered signalling to healthy human ageing and their potential role in human disease.

细胞对各种压力的适应需要复杂的步骤网络,这些步骤共同导致氧化还原平衡的重建、受损大分子的降解和细胞新陈代谢的恢复。在我们对细胞信号和信号转换之间相互作用的理解上的进展,描绘了一幅多层次调节路径的复杂图景。在这篇综述中,我们探讨了细胞适应代谢和氧化应激激活自噬之间的联系,一个重要的细胞分解代谢途径。新陈代谢的压力可以导致烟酰胺腺嘌呤二核苷酸的氧化还原状态的改变,这是一种在各种酶反应中的共同因子,因此触发自噬作用,将细胞内的成分固定起来,以支持细胞的生长。同样地,自噬被氧化应激激活,以选择性地回收受损的大分子和细胞器,从而维持细胞的活力。有助于调节或执行自噬的多种蛋白质是翻译后修饰(PTMs)的目标,这些翻译后修饰对它们的定位、结合亲和力或酶活性有影响。这些 PTMs 包括乙酰化,一种蛋白质赖氨酸残基的可逆的酶修饰,和氧化,一系列可逆的和不可逆的自由基修饰。在这里,我们强调了自噬与代谢应激相互作用的最新发现和突出问题,表现为 NAD 水平的变化,以及氧化应激,重点是由氧化和乙酰化共同调节的自噬蛋白。我们进一步探讨这种多层次的信号与健康人类老龄化的相关性及其在人类疾病中的潜在作用。

Introduction

引言

NAD depletion, oxidative stress and loss of macroautophagy (from herein referred to as autophagy) efficiency have all been linked to healthy, pathological and premature ageing (Kubben and Misteli 2017; López-Otín et al. 20132016). Individually, these alterations may underlie seven of the nine outlined hallmarks of ageing including genomic instability (all), telomere attrition (oxidative stress), epigenetic alterations (NAD), loss of proteostasis (autophagy), de-regulated nutrient sensing (NAD), cellular senescence (all) and mitochondrial dysfunction (all) (López-Otín et al. 20132016). Moreover, it is becoming increasingly clear that a significant degree of crossover and interdependence between the three phenomena occur in ageing cells and tissues. Specifically, increased reactive oxygen species (ROS) and depletion of NAD can impact autophagy by influencing post-translational modifications (PTMs) of autophagy proteins (Filomeni et al. 2015; Sedlackova et al. 2020; Zhang et al. 2016a). Furthermore, autophagy impairment may lead to the failure to reconstitute cellular metabolism and detoxify oxidised substrates (Li et al. 2015; Morishita and Mizushima 2019).

NAD 损耗、氧化应激和巨噬细胞自噬效率的丧失都与健康、病理和早衰有关(Kubben 和 Misteli,2017; López-Otín 等人,2013,2016)。单独来看,这些改变可能是老化的9个标志中的7个标志的基础,包括基因组不稳定(全部) ,端粒磨损(氧化应激) ,表观遗传改变(NAD) ,蛋白质平衡(自噬)的丢失,去调节营养传感(NAD) ,细胞衰老(全部)和线粒体功能障碍(全部)。此外,日益明显的是,这三种现象在衰老的细胞和组织中发生了很大程度的交叉和相互依存。具体来说,活性氧类的增加和 NAD 的缺失可以通过影响自噬蛋白的翻译后修饰(PTMs)来影响自噬。此外,自噬损伤可能导致细胞代谢重组和解毒氧化底物的失败(Li 等人,2015; 森下和水岛,2019)。

Nicotinamide adenine dinucleotide (NAD)

烟酰胺腺嘌呤二核苷酸

NAD is an essential metabolite that participates in cellular energy generation and signalling. When plentiful, the redox balance and availability of NAD aid cellular adaptation to metabolic stress and help maintain genomic stability, mitochondrial function, detoxification of ROS and cell survival (Fang et al. 2017). Due to its ability to accept or donate electrons, NAD in its reduced (NADH) or oxidised (NAD+) form assists energy metabolism in the cytosol and within mitochondria (Canto et al. 2015). In addition, NAD+ is cleaved into ADP-ribose (ADPR) and nicotinamide (NAM) by three classes of enzymes: sirtuins (SIRTs), poly(ADPR) polymerases (PARPs) and cyclic ADPR synthases (CD38 and CD157) (Fig. 1), which require ADPR for their enzymatic activity (Canto et al. 2015; Fang et al. 2017). Crucially, although SIRT activity depends on NAD+ availability and cannot contribute to uncontrolled NAD+ cleavage, PARPs and CD38 are known for their indiscriminate NAD+ consumption and their role in age- and disease-related NAD depletion (Canto et al. 2015). Homeostasis of intracellular NAD pools is maintained by either local synthesis from NAD+ precursors (nicotinamide (NAM), nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN)) or centralised de novo synthesis from nicotinic acid or L-tryptophan (Canto et al. 2015). Therefore, it is the balance between NAD+ cleavage and synthesis that dictates the total intracellular NAD pool, and by extension, cellular metabolism and protein acetylation status (Strømland et al. 2019).

NAD 是一种重要的代谢产物,参与细胞能量的产生和信号传递。在充足时,NAD 的氧化还原平衡和可用性有助于细胞适应代谢压力,并有助于维持基因组稳定性、线粒体功能、活性氧解毒和细胞存活(Fang 等人,2017年)。由于它能够接受或捐赠电子,NAD 的还原形式(NADH)或氧化形式(NAD +)有助于细胞质和线粒体内的能量代谢(Canto 等人,2015年)。此外,NAD + 被三类酶切割成 adp- 核糖(ADPR)和烟酰胺(NAM) : sirtuins (SIRTs) ,poly (ADPR)聚合酶(PARPs)和环状 ADPR 合酶(CD38和 CD157)(图1) ,这些酶的酶活性需要 ADPR (Canto 等人,2015; Fang 等人,2017)。至关重要的是,虽然 SIRT 的活性取决于 NAD + 的可获得性,并且不能导致 NAD + 无控制的分裂,但是 PARPs 和 CD38因其不分青红皂白的 NAD + 消费及其在与年龄和疾病相关的 NAD 损耗中的作用而为人所知(Canto 等人,2015年)。细胞内 NAD 池的稳态可以通过以下两种途径来维持: 从 NAD + 前体(尼古丁酰胺(NAM)、尼古丁酰胺核苷(NR)或尼古丁酰胺单核苷酸(NMN))局部合成,或从尼古丁酸或 l- 色氨酸集中合成从头合成(Canto et al. 2015)。因此,正是 NAD + 裂解和合成之间的平衡决定了细胞内 NAD 池的总量,进而决定了细胞代谢和蛋白质乙酰化状态(Strømland 等人,2019年)。

figure1
Fig. 1 图一

Reactive oxygen species (ROS)

活性氧类

ROS are highly reactive molecules of oxygen, which harbour one unpaired electron (superoxide anion (O2•−), hydroxyl radical (OH)) or an additional electron pair (H2O2) on its valence orbital (Halliwell and Gutteridge 2015). The increased electron content in oxygen molecules makes them more reactive and more likely to participate in one-electron oxidative transfer reactions that lead to macromolecule modification and/or damage (Halliwell and Gutteridge 2015). ROS can also interact with nitric oxide to generate reactive nitrogen species (RNS), including peroxynitrite (ONOO) (Bartesaghi and Radi 2018). Electrons from ROS/RNS interact with amino acid residues incorporated in proteins and thus translate the cellular redox state into protein activating or inhibiting signals by the modulation of protein enzymatic activity, binding affinity or structural conformation. Particularly sensitive to ROS-mediated redox regulation are cysteine (Cys) residues (Bischoff and Schlüter 2012). Cys is one of the least represented, and yet often highly conserved amino acids that participates in protein structural integrity by formation of covalent disulphide-bridges between two cysteine residues, or protein enzymatic activity, i.e. by thioester bond formation or co-factor stabilisation (Bak et al. 2019; Marino and Gladyshev 2010).

活性氧是高活性氧分子,在其价轨道上含有一个不成对电子(超氧阴离子(O2• -)、羟基自由基(OH •)或一个额外的电子对(H2O2)。氧分子中电子含量的增加使它们更活泼,更有可能参与单电子氧化转移反应,从而导致高分子修饰和/或损伤(哈利维尔和古特里奇,2015年)。ROS 也可以与一氧化氮相互作用产生活性氮,包括过氧亚硝基阴离子(ONOO -)(Bartesaghi 和 Radi 2018)。ROS/RNS 的电子与蛋白质中的氨基酸残基相互作用,通过调节蛋白质的酶活性、结合亲和力或结构构象,将细胞的氧化还原状态转化为蛋白质激活或抑制信号。对 ros 介导的氧化还原调节特别敏感的是半胱氨酸(Cys)残基(Bischoff 和 Schlüter 2012)。半胱氨酸是最少代表的,但是通常是高度保守的氨基酸,通过在两个半胱氨酸残基之间形成共价二硫键或蛋白质酶活性,即通过硫酯键形成或共因子稳定作用,参与蛋白质结构的完整性(Bak 等人,2019; Marino 和 gladys,2010)。

Autophagy

自噬作用

Autophagy is a cytosolic pathway of dynamic membrane rearrangement and cargo sequestration that is assisted and executed by a set of highly conserved autophagy (ATG) proteins (Dikic and Elazar 2018). Autophagy is a catabolic process responsible for cargo recognition, its engulfment in a double membraned vesicle called autophagosome and delivery to the lysosomal lumen for degradation. The subsequent release of amino acids, lipids and nucleosides reconstitutes cellular homeostasis and sustains viability in times of stress (Morishita and Mizushima 2019). The molecular execution of autophagy initiation is mediated by ATG protein association into functional complexes known as the Unc-51-like kinase 1 (ULK1) complex, the class III phosphatidylinositol 3 kinase (PI(3)K) complex, the ATG9-membrane complex, an ATG2–WIPI (WD-repeat protein interacting with phosphoinositides) complex and two conjugation systems consisting of the ATG3-ATG8/LC3 and the ATG5-ATG12:ATG16L complex (Table 1) (Suzuki et al. 2017). The combined action of these complexes is responsible for ER localization of all autophagy components and for the formation and maturation of the autophagic membrane. In addition, a group of autophagy receptors, e.g. sequestosome 1 (SQSTM1/p62), is then responsible for spatially linking the ubiquitylated cargo, including long-lived or aggregated proteins, pathogens and organelles, to the growing autophagosome (Dikic and Elazar 2018; Johansen and Lamark 2019).

自噬是一种动态膜重排和货物隔离的胞浆通路,由一组高度保守的自噬蛋白(ATG)辅助和执行(Dikic 和 Elazar 2018)。自噬是一个分解代谢过程,负责货物识别,它吞噬在一个叫做自噬小体的双膜泡中,并传递到溶酶体腔进行降解。随后释放的氨基酸,脂类和核苷重新构成细胞内稳态和维持活力时的压力(森下和水岛2019年)。自噬启动的分子执行是通过 ATG 蛋白的结合介导的功能复合物称为 Unc-51-like kinase 1(ULK1)复合物,第 III 类磷脂酰肌醇激酶3(PI (3) k)复合物,atg9-膜复合物,ATG2-WIPI (WD-repeat 蛋白与磷酸肌醇相互作用)复合物和两个连接系统组成的 ATG3-ATG8/LC3和 ATG5-ATG12: ATG16L 复合物(表1)(Suzuki et al. 2017)。这些复合物的联合作用负责所有自噬组分的 ER 定位和自噬膜的形成和成熟。此外,一组自噬受体,例如序列体1(SQSTM1/p62) ,负责在空间上将无处不在的化合物(包括长寿命或聚集的蛋白质、病原体和细胞器)与正在生长的自噬体联系起来(Dikic 和 Elazar 2018; Johansen 和 Lamark 2019)。Table 1 Acetylation-sensitive proteins in autophagy 表1自噬中乙酰化敏感蛋白Full size table 全尺寸表

The canonical pathway of starvation-induced autophagy was long thought to rely on phosphorylation cascades that are triggered by the loss of nutrient signalling and converge on a small number of regulating kinase complexes (Beurel et al. 2015; Rabanal-Ruiz et al. 2017; Tamargo-Gómez and Mariño 2018). These regulators then either lose function and thus release downstream autophagy components from an inhibitory state, or become activated and promote autophagy initiation. In addition, multiple layers of regulation involved in autophagy initiation, cargo sequestration and degradation, incorporate various stress signals and often improve the efficiency of autophagic flux via PTMs of autophagy proteins or their upstream regulators (Filomeni et al. 2015; Montagna et al. 2016; Sedlackova et al. 2020; Zhang et al. 2016a).

长期以来,人们一直认为饥饿诱导的自噬的典型途径依赖于磷酸化级联,这种级联由营养信号的丢失触发,并在少数调节激酶复合物上汇聚(Beurel 等人,2015年; Rabanal-Ruiz 等人,2017年; Tamargo-Gómez 和 Mariño 2018年)。这些调节因子要么失去功能,从而从抑制状态释放下游自噬组分,要么被激活,促进自噬启动。此外,涉及自噬启动、货物固存和降解的多层次调节,包含各种应激信号,通过自噬蛋白的 PTMs 或其上游调节因子常常提高自噬通量的效率(Filomeni 等人,2015年; Montagna 等人,2016年; Sedlackova 等人,2020年; Zhang 等人,2016a 年)。

In this review, we explore the current knowledge of how two types of PTMs, lysine (Lys) acetylation and cysteine (Cys) oxidation, regulate the abundance and activity of ATG proteins, and highlight which Lys modifications are subject to NAD+ availability. We then summarize the main concepts of autophagy regulation by oxidative stress and discuss the implications and consequences of age-related changes to NAD+ availability and an increase in oxidative stress on the efficiency of autophagy. We further explore whether autophagy directly influences the homeostasis of cellular NAD levels and outline how aberrations in either of the three phenomena could lead to dysfunction observed in physiological and pathological ageing.

本文综述了赖氨酸(Lys)乙酰化和半胱氨酸(Cys)氧化两种类型的 PTMs 如何调节 ATG 蛋白的丰度和活性,并着重介绍了哪些 Lys 修饰受 NAD + 的影响。然后,我们总结了氧化应激的自噬调节的主要概念,并讨论了年龄相关变化对 NAD + 可用性的影响和后果,以及提高氧化应激对自噬效率的影响。我们进一步探讨了自噬是否直接影响细胞 NAD 水平的稳态,并概述了这三种现象中的任何一种畸变是如何导致生理和病理衰老中观察到的功能障碍的。

Targets of acetylation in autophagy

自噬中的乙酰化作用靶点

Lysine acetylation is a major reversible PTM in eukaryotes that arises by donation of the acetyl moiety from acetyl coenzyme A (Ac-CoA) via its re-direction from mitochondrial energy generation (Drazic et al. 2016). Protein acetylation status is balanced by the activity of multiple lysine acetyl transferases (KATs, historically known as histone acetyl transferases HATs) and lysine deacetylases (KDAC, or HDACs) (Narita et al. 2019). KATs catalyse acetyl moiety transfer from Ac–CoA to a lysine residue of the target protein, while KDACs cleave and release the acetyl moiety (KDAC, classes I, II and IV) or catalyse transfer of the acetyl moiety onto ADPR, a product of NAD+ cleavage (class III KDACs, sirtuins (SIRTs) (Fig. 1). Acetylation status of autophagy proteins is largely controlled by p300 (KAT3B) and 60 kDa Tat-interactive protein (TIP60/KAT5) KATs and SIRT1-3 and HDAC2/6 KDACs (summarized in Table 1). In the next section, we explore how protein acetylation status, generally high in conditions of nutrient abundance and low under nutrient starvation, regulates the activity and localisation of TFs, proteins and receptors involved in autophagy.

赖氨酸乙酰化是真核生物中一种主要的可逆 PTM,通过线粒体产生的能量重新导向乙酰辅酶A 乙酰基部分(Ac-CoA)而产生。蛋白质的乙酰化状态由多个赖氨酸乙酰转移酶(KATs,历史上称为组蛋白乙酰转移酶 HATs)和赖氨酸脱乙酰酶(KDAC,或 HDACs)的活性来平衡(成田等人,2019年)。KATs 催化乙酰基从 Ac-CoA 转移到目标蛋白的赖氨酸残基,而 KDAC 裂解并释放乙酰基(KDAC,i 类,II 类和 IV 类)或催化乙酰基转移到 NAD + 裂解产物 ADPR (III 类 KDAC,SIRTs)上(图1)。自噬蛋白的乙酰化状态主要受 p300(KAT3B)和60kda tat 相互作用蛋白(TIP60/KAT5) KATs、 SIRT1-3和 hdac2/6kdac 控制(见表1)。在下一节,我们将探讨在营养丰富和营养缺乏的情况下,蛋白质的乙酰化状态如何调节自噬相关的转录因子、蛋白质和受体的活性和定位。

Regulation of transcription factors involved in autophagy gene transcription

自噬基因转录相关转录因子的调控

The loss of lysine acetylation triggers stimulation of several TFs involved in the transcription of ATG genes (Fig. 2a) (Füllgrabe et al. 2016). The strongest link between TF deacetylation and autophagy stimulation comes from studies of transcription factor EB (TFEB), a member of the microphthalmia family of bHLH-LZ transcription factors (Mit/TFE), a group of TFs that stimulate lysosomal biogenesis and expression of autophagy proteins (Yang et al. 2018). Specifically, TFEB is responsible for transcription of multiple autophagy genes (ATG4, ATG9B, MAP1LC3B (LC3B), UVRAG (UV radiation resistance associated gene), WIPI (WD repeat domain phosphoinositide-interacting protein 1), and SQSTM1 (p62)) (Füllgrabe et al. 2016; Settembre et al. 2011). Acetylation of a conserved lysine residue Lys116 was independently identified in three studies as a modifier of TFEB activity in microglia (Bao et al. 2016) and in cancer cells (Wang et al. 2019b; Zhang et al. 2018). In microglia, Lys116 was directly deacetylated by SIRT1 which promoted degradation of fibrillar amyloid β (Bao et al. 2016). In cultured cells, treatment with a KDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), increased the transcriptional activity of TFEB and influenced acetylation of four lysine residues (Lys91, Lys103, Lys116 and Lys430) (Zhang et al. 2018). In addition, authors of this study identified acetyl-coenzyme A acetyltransferase 1 (ACAT1) and HDAC2 as modulators of the overall TFEB acetylation status. Furthermore, a study in a model of chronic kidney disease identified HDAC6 as another KDAC involved in the regulation of TFEB activity (Brijmohan et al. 2018). Importantly, authors of neither of the studies demonstrated a direct interaction between TFEB and HDAC2 or HDAC6, respectively (Brijmohan et al. 2018; Zhang et al. 2018). Overexpression of another KAT, the general control non-repressed protein 5 (GCN5/KAT2A), but not TIP60, p300 or CREB-binding protein (CBP), led to increased TFEB acetylation of Lys116, Lys274 and Lys279 residues (Wang et al. 2019b). Authors further demonstrated that TFEB acetylation at Lys274 and Lys279 mechanistically disrupts TFEB dimerization and its ability to bind DNA, and thus negatively regulates expression of lysosomal and autophagy genes (Fig. 2A) (Wang et al. 2019b). Crucially, Lys116 of TFEB is not conserved in Drosophila melanogaster and Caenorhabditis elegans or in other members of the Mit-TFE family (Wang et al. 2019b), thus SIRT1 and HDAC regulation of TFEB activity is likely to be unique to vertebrates.

赖氨酸乙酰化缺失触发了几个参与 ATG 基因转录的转录因子(图2a)(Füllgrabe 等人,2016)。TF 脱乙酰化和自噬刺激之间最强有力的联系来自对转录因子 EB (TFEB)的研究,这种转录因子属于小眼睛 bHLH-LZ 转录因子家族(Mit/TFE) ,是一组促进溶酶体生成和自噬蛋白表达的 TFs (Yang et al. 2018)。具体来说,TFEB 负责多种自噬基因的转录(ATG4,ATG9B,MAP1LC3B (LC3B) ,UVRAG (UV 辐射抗性相关基因) ,WIPI (WD 重复结构域磷酸肌醇-相互作用蛋白1) ,和 SQSTM1(p62))(Füllgrabe 等人,Settembre 等人,2011)。保守赖氨酸残基 Lys116的乙酰化在三项研究中被独立鉴定为小胶质细胞(Bao 等人,2016年)和癌细胞(Wang 等人,2019b; Zhang 等人,2018年)中 TFEB 活性的修饰剂。在小胶质细胞中,Lys116被 SIRT1直接去乙酰化,促进了淀粉样蛋白的降解(Bao 等人,2016)。在培养的细胞中,使用 KDAC 抑制剂伏立诺他(SAHA)增加 TFEB 的转录活性并影响4个赖氨酸残基(Lys91,Lys103,Lys116和 Lys430)的乙酰化。此外,本研究作者确定乙酰辅酶 a 乙酰转移酶1(ACAT1)和 HDAC2作为调节整个 tmb 乙酰化状态。此外,在一个慢性肾脏疾病模型中的研究确定 HDAC6是另一个参与调节 TFEB 活性的 KDAC (Brijmohan 等人,2018)。重要的是,这两项研究的作者都没有证明 TFEB 和 HDAC2或 HDAC6之间存在直接的相互作用。另一个 KAT 蛋白,一般对照的非抑制蛋白5(GCN5/KAT2A)的过表达,而 TIP60、 p300或 creb 结合蛋白(CBP)的过表达,导致 Lys116、 Lys274和 Lys279残基 TFEB 的增加(Wang 等,2019b)。作者进一步证明,Lys274和 Lys279的 tbb 乙酰化作用可以机械地干扰 tmb 的二聚化及其与 DNA 的结合能力,从而对溶酶体和自噬基因的表达产生负性调节(图2A)(Wang 等人,2019b)。至关重要的是,TFEB 的 Lys116在黑腹果蝇和秀丽隐桿线虫或 Mit-TFE 家族的其他成员中并不保守,因此 SIRT1和 HDAC 对 TFEB 活性的调节可能是脊椎动物所独有的。

figure2
Fig. 2 图二

Additionally, two members of the forkhead box class O (FoxO) TF family, FoxO1 and FoxO3a, recognized for their role in autophagy/mitophagy (ATG4, ATG5, ATG12, ATG14, BECN1(beclin 1), BNIP3 (BCL2 interacting protein 3), LC3B, ULK1, VPS34 (vacuolar protein sorting 34) GABARAPL1 (gamma-aminobutyric acid receptor-associated protein-like1), and PARK6/PINK1 (PTEN-induced kinase 1)) gene transcription, are regulated by acetylation PTMs (Fang et al. 2019; Füllgrabe et al. 2016; Requejo-Aguilar et al. 2015). It was first demonstrated that FoxO1 acetylation on Lys242, Lys245 and Lys262 residues (in mice) by CBP is opposed by SIRT1 in response to serum (Daitoku et al. 2004) and glucose starvation (Hariharan et al. 2010). Mechanistically, acetylation of the three Lys residues within FoxO1 interferes with its DNA binding and inhibits its transcriptional activity (Matsuzaki et al. 2005). Furthermore, FoxO1 acetylation permits access for upstream kinases to phosphorylate its Ser253 residue that is otherwise shielded by FoxO1-DNA complex formation (Matsuzaki et al. 2005). FoxO1 phosphorylation sites have since became known to act as docking or shielding sites for 14–3-3 protein binding, and the heterodimer exit into and retention within the cytoplasm (Brunet et al. 1999; Saline et al. 2019).

此外,两个成员的叉头盒类 o (FoxO) TF 家族,FoxO1和 FoxO3a,认为其作用于自噬/吞噬(ATG4,ATG5,ATG12,ATG14,BECN1(beclin 1) ,BNIP3(BCL2相互作用蛋白3)(BCL2相互作用蛋白3) ,lC3B、 ULK1、 VPS34(液泡蛋白分类34) GABARAPL1(γ-氨基丁酸受体相关蛋白1)和 PARK6/PINK1(pten 诱导的激酶1)基因转录受乙酰化 PTMs 调控(Fang 等人,2019; Füllgrabe 等人,2016; Requejo-Aguilar 等人,2015)。首次证明 CBP 在小鼠体内对 Lys242、 Lys245和 Lys262残基的 FoxO1乙酰化反应受到血清(Daitoku 等人,2004年)和葡萄糖饥饿的影响(Hariharan 等人,2010年)。机制上,FoxO1中三个赖氨酸残基的乙酰化干扰其 DNA 结合并抑制其转录活性(松崎等人,2005)。此外,FoxO1乙酰化允许上游激酶磷酸化其被 FoxO1-dna 复合体屏蔽的 Ser253残基(Matsuzaki 等人,2005年)。FoxO1磷酸化位点后来被认为是14-3-3蛋白结合的对接或屏蔽位点,异二聚体进入并保留在细胞质中(Brunet al. 1999; Saline et al. 2019)。

Similarly to FoxO1, the transcriptional activity of FoxO3 is modulated by SIRT1-3 deacetylases, though the Lys residues susceptible to acetylation remain unknown. First, caloric restriction and oxidative stress increase SIRT2 expression, decrease FoxO3 acetylation and improve gene transcription (Wang et al. 2007). In mitochondria, SIRT3 mediated deacetylation of the mitochondrial FoxO3 pool and led to cellular detoxification of oxidative stress by increased expression of the mitochondrial superoxide dismutase (Jacobs et al. 2008). However, some controversy exists in the perceived outcome of FoxO acetylation. A few articles published in early 2000s reported entirely opposite findings, demonstrating that FoxO acetylation improves transcription of its target genes (Motta et al. 2004; Yang et al. 2005). The pitfall of the majority of FoxO studies centres on the lack of distinction between CBP-mediated acetylation of FoxO, which may increase its DNA binding, and acetylation of histones, which would relax the chromatin condensation and promote gene transcription (discussed in detail in (Daitoku et al. 2011)).

与 FoxO1相似,FoxO3的转录活性也受到 SIRT1-3去乙酰化酶的调控,但赖氨酸残基对乙酰化的敏感性尚不清楚。首先,限制热量摄入和氧化应激可以增加 SIRT2的表达,降低 FoxO3乙酰化水平,提高基因转录水平。在线粒体中,SIRT3介导了线粒体 FoxO3库的去乙酰化,并通过增加线粒体超氧化物歧化酶蛋白的表达导致细胞解毒(Jacobs et al. 2008)。然而,对于 FoxO 乙酰化的感知结果还存在一些争议。2000年代初发表的一些文章报道了完全相反的发现,表明 FoxO 乙酰化改善了其靶基因的转录(Motta 等,2004; Yang 等,2005)。大多数 FoxO 研究的缺陷在于缺乏区分 cbp 介导的 FoxO 的乙酰化和组蛋白的乙酰化,前者可能增加其 DNA 结合,后者可能放松染色质凝聚和促进基因转录(详见 Daitoku 等人2011年)。

Overall, transcriptional activity of TFEB, a member of the Mit/TFE family, and two FoxO isoforms, FoxO1 and FoxO3, is regulated by acetylation. Three classes of KDACs, SIRT1, HDAC2/6 and GCN5, are thought to deacetylate multiple and variable Lys residues in TFEB, of which the SIRT1 target, Lys116, is unique to vertebrates, and Lys274 and Lys279 deacetylation regulates TFEB-DNA complex formation (Wang et al. 2019b). In the FoxO family, FoxO1 is the better studied isoform, with known target Lys residues in the mouse (Table 1), though both FoxO1 and FoxO3a are likely activated by SIRT-mediated deacetylation. Thus, transcriptional regulation of autophagy/mitophagy genes by TFEB and FoxO1/3a is, at least in part, responsive to intracellular NAD+ levels that influence SIRT activity.

总的来说,Mit/TFE 家族成员 TFEB 和 FoxO1和 FoxO3两种同源异构体的转录活性受乙酰化调控。目前认为 TFEB 中有三类 kdac,SIRT1、 HDAC2/6和 GCN5,其中 SIRT1的作用靶点 Lys116是脊椎动物特有的,Lys274和 Lys279脱乙酰基调节 TFEB-dna 复合物的形成(Wang 等人,2019b)。在 FoxO 家族中,FoxO1是更好的研究异构体,在小鼠中有已知的目标赖氨酸残基(表1) ,尽管 FoxO1和 FoxO3a 都可能被 sirt 介导的脱乙酰化活化。因此,tbb 和 FoxO1/3a 的自噬/噬转录调控基因至少部分地对影响 SIRT 活性的细胞内 NAD + 水平有反应。

Regulation of autophagy protein complexes

自噬蛋白复合物的调节

Several autophagy proteins involved in autophagosome formation, growth and maturation may be modified by acetylation (Table 1). Studies from the last decade, summarized below, identify lysine residues sensitive to acetylation in members of the ULK1 kinase complex, the class III PI(3)K kinase complex and the two conjugation systems, ATG12 (ATG7, ATG10, ATG5) and LC3 (ATG4, ATG7, ATG3), as well as ATG12 and LC3 themselves (Fig. 2B).

一些参与自噬体形成、生长和成熟的自噬蛋白可能通过乙酰化修饰(表1)。近十年来的研究表明,ULK1激酶复合物、 III 类 PI (3) k 激酶复合物和两个接合系统 ATG12(ATG7、 ATG10、 ATG5)和 LC3(ATG4、 ATG7、 ATG3)中的赖氨酸残基对乙酰化敏感,以及 ATG12和 LC3本身(图2B)。

In the ULK1 complex, ULK1 itself is a target of acetylation by TIP60 (Lin et al. 2012). In serum starved cells, ULK1 was shown to be a target of GSK3-dependent and TIP60-mediated acetylation of two crucial residues, Lys162 and Lys606 (in mouse; likely Lys162 and Lys607 in human), that together stimulate its kinase activity and promote autophagy initiation (Lin et al. 2012). Furthermore, oxidative stress that induces ER stress, was also shown to stimulate ULK1 acetylation by a GSK3-TIP60-dependent mechanism (Nie et al. 2016). These studies together support the idea that ULK1 kinase activity can be modulated by oxidative and metabolic stress via an upstream signalling cascade that results in TIP60 activation and ULK1 acetylation.

在 ULK1复合物中,ULK1本身就是 TIP60的乙酰化靶点(Lin 等人,2012)。在血清饥饿的细胞中,ULK1被证明是 gsk3依赖和 tip60介导的两个关键残基 Lys162和 Lys606的乙酰化靶点(在小鼠中; 在人类中可能是 Lys162和 Lys607) ,这两个残基共同刺激其激酶活性并促进自噬启动(Lin 等人,2012)。此外,引起内质网应力的氧化应激,也表明通过 gsk3-tip60依赖机制刺激 ULK1乙酰化。这些研究共同支持的想法,ULK1激酶的活性可以调节氧化和代谢应激通过上游信号级联,导致 TIP60活化和 ULK1乙酰化。

Within the Class III PI(3)K complex, VPS34 kinase acetylation by p300 occurs on residues Lys29, Lys771 and Lys781 and inhibits its lipid kinase activity and PI(3)P production (Su et al. 2017). It was further determined that acetylation of Lys29 residue prevents VPS34 association with Beclin 1 that is required for the formation of a complex involved in autophagy progression. Another layer of VPS34 activity regulation occurs upon acetylation of the Lys771residue located within its catalytic site. In a manner similar to the level of regulation at the Lys29 residue, acetylation of Lys771 disrupts binding between VPS34 and its substrate, PI (Su et al. 2017). However, the KDAC responsible for Lys29 and Lys771 deacetylation remains unknown. In addition to VPS34, Beclin 1 of the Class III PI(3)K complex is also a target of inhibitory acetylation on Lys430 and Lys437 residues by p300 (Sun et al. 2015). Beclin 1 acetylation was demonstrated to promote its binding to Rubicon, and thus shown to result in the loss of autophagosome maturation (Ohashi et al. 2019; Sun et al. 2015). Furthermore, in vitro acetylation analysis revealed that SIRT1 is preferentially responsible for Beclin 1 deacetylation (Sun et al. 2015).

在 III 类 PI (3) k 复合物中,VPS34激酶的 p300乙酰化发生在 Lys29、 Lys771和 Lys781的残基上,并抑制其脂质激酶活性和 PI (3) p 的产生(Su 等人,2017年)。进一步确定,Lys29残基的乙酰化可以阻止 VPS34与 Beclin 1的结合,而后者是形成一个参与自噬进程的复合物所必需的。另一层 VPS34的活性调控发生在 Lys771残基的乙酰化过程中,该残基位于催化位点。与 Lys29残基的调控水平相似,Lys771的乙酰化破坏了 VPS34与其底物之间的结合,PI (Su 等人,2017)。然而,负责 Lys29和 Lys771脱乙酰基的 KDAC 仍然是未知的。除 VPS34外,p300还将 III 类 PI (3) k 复合物的 Beclin 1作为抑制 Lys430和 Lys437残基乙酰化的靶标(Sun 等人,2015年)。研究表明,Beclin 1乙酰化可促进其与 Rubicon 的结合,从而导致自噬体成熟的丧失(Ohashi 等人,2019; Sun 等人,2015)。此外,体外乙酰化分析显示 SIRT1优先负责 Beclin 1脱乙酰化(Sun 等人,2015)。

Next, SIRT1-mediated deacetylation of nuclear LC3 at Lys49 and Lys51 residues initiates LC3 translocation to the cytoplasm via a diabetes and obesity regulated (DOR/TP53INP2)-dependent interaction with deacetylated LC3 (Huang et al. 2015). DOR then further assists in LC3 localization to nascent autophagosomes thanks to its ATG7-binding affinity (You et al. 2019b). Furthermore, DOR also contains a ubiquitin-interacting motif and is thus likely to promote LC3-ATG7 formation in the vicinity of ubiquitylated cargo (Xu and Wan 2019; You et al. 2019b). Upon relocation to the cytoplasm, LC3 Lys49 and Lys51 acetylation, that is lost upon nutrient starvation, was recently shown to completely abolish p62 binding (Song et al. 2019). Due to the location and conservation of the two critical lysine residues in the hydrophobic binding grooves of LC3 (Huang et al. 2015; Song et al. 2019), it stands to reason that Lys49 and Lys51 acetylation could disrupt LC3 interaction with multiple binding partners including, but not limited to DOR and p62. Altogether, LC3 deacetylation in response to nutrient starvation not only promotes its exit from the nucleus, but also determines substrate binding specificity of protein partners via their LC3-interacting regions (LIRs).

其次,sirt1介导的 Lys49和 Lys51基因残基上的 LC3去乙酰化启动了 LC3通过糖尿病和肥胖调节(DOR/TP53INP2)依赖的与去乙酰化 LC3的相互作用到细胞质的转位(Huang 等人,2015)。DOR 然后进一步协助 LC3定位到初生的自噬体,这要归功于其 atg7结合的亲和力(You 等人,2019b)。此外,DOR 还包含泛素相互作用基序,因此可能促进泛素化货物附近的 LC3-ATG7的形成(Xu and Wan 2019; You et al. 2019b)。在重新定位到细胞质后,在营养缺乏时丢失的 LC3 Lys49和 Lys51乙酰化,最近被证明完全消除了 p62结合(Song 等人,2019年)。由于两个关键赖氨酸残基在 LC3的疏水结合槽中的位置和守恒(Huang 等人,2015; Song 等人,2019) ,可以推断 Lys49和 Lys51乙酰化可能破坏 LC3与多个结合伙伴的相互作用,包括但不限于 DOR 和 p62。总之,在营养缺乏的条件下,LC3脱乙酰化不仅促进了其从细胞核中的脱出,而且通过其 LC3相互作用区域决定了蛋白质伙伴的底物结合特异性。

Cytoplasmic LC3 targeting to and docking on the nascent autophagosomes requires covalent conjugation of LC3 to phosphatidylethanolamine (PE). In a ubiquitin-like conjugation system, ATG7 (and E1-like enzyme), ATG3 (an E2-like enzyme) and an ATG5-ATG12:ATG16L complex (an E3-like enzyme) assist LC3 conjugation to PE (Dikic and Elazar 2018). Nutrient starvation in yeast was first reported to decrease or not change acetylation levels of ATG proteins, with the notable exception of ATG3, in which Lys19, Lys48 and Lys183 acetylation increased (Yi et al. 2012). Authors of this study had further shown that while acetylation of Lys183 is crucial for the enzymatic activity of ATG3, Lys19 and Lys48 acetylation was crucial for autophagy progression by improving interaction between ATG3 and ATG8 (LC3 in mammals), and was regulated by the opposing activities of the yeast histone acetyltransferase Esa1 (TIP60/KAT5 orthologue)) and a histone deacetylase Rpd3 (HDAC1/2 orthologue) enzymes. Furthermore, ATG3 acetylation on Lys19 and Lys48 was shown to enhance its ER membrane localization and binding in vitro (Li et al. 2017).

细胞质 LC3靶向并与新生的自噬体对接需要 LC3与磷脂酰乙醇胺的共价结合。在泛素样结合系统中,ATG7(和 e1样酶)、 ATG3(一种 e2样酶)和 ATG5-ATG12: ATG16L 复合物(一种 e3样酶)协助 LC3结合 PE (Dikic 和 Elazar 2018)。首次报道酵母营养缺乏可以降低或不改变 ATG 蛋白的乙酰化水平,但 ATG3除外,其中 Lys19、 Lys48和 Lys183的乙酰化水平升高(Yi 等,2012年)。作者进一步指出,虽然 Lys183的乙酰化是至关重要的 ATG3,Lys19和 Lys48乙酰化的酶活性是至关重要的自噬进展,改善 ATG3和 ATG8(LC3在哺乳动物中)之间的相互作用,并调节相反的活动酵母组蛋白乙酰基转移酶 Esa1(TIP60/KAT5的直系亲属)和组蛋白脱乙酰酶 Rpd3(HDAC1/2直系亲属)的酶。此外,在 Lys19和 Lys48上的 ATG3乙酰化被证明能增强其 ER 膜的定位和体外结合(Li 等人,2017)。

Other members of the ubiquitin-like conjugation system, ATG7, ATG5 and ATG12, are targets of p300-mediated acetylation (Lee and Finkel 2009) and SIRT1-dependent deacetylation (Lee et al. 2008). In direct contrast to ULK1 and ATG3, acetylation of these ATG proteins generally inhibits their function. However, the specific residues, their location and effect of acetylation on the structure or function of ATG proteins remains unknown. Structural studies of the ATG12-ATG5:ATG16 complex (Otomo et al. 2013) and the nature of interaction between ATG12 and ATG3 (Metlagel et al. 2013) point towards several key lysine residues that could be targets of acetylation in ATG12. First, lysine residues 60, 69, 71 and 128 located on the surface of ATG12 (Metlagel et al. 2013) could contribute to binding affinity between ATG12 (E3-like) and ATG3 (E2-like) that is required for the spatiotemporal regulation of LC3 lipidation. Furthermore, ATG5 contains multiple lysine residues, of which Lys53, Lys130, Lys171 are conserved (Matsushita et al. 2007). Although Lys130 is the known catalytic site for conjugation between ATG5 and ATG12 (Mizushima et al. 1998), the function and acetylation-sensitivity of Lys53 and Lys171 remain unknown. Lastly, no published study followed-up reports of ATG7 acetylation-sensitivity (Lee et al. 2008; Lee and Finkel 2009). However, a high resolution mass spectrometry study of global protein acetylation identified Lys306 of the human ATG7 protein as a residue that might be relevant for further study (Choudhary et al. 2009). Thus, although ATG5, ATG7 and ATG12 have been known substrates of p300 and SIRT for almost a decade, the lysine residues sensitive to acetylation, or indeed the nature of protein inhibition by acetylation have not been elucidated.

泛素样结合系统的其他成员 ATG7、 ATG5和 ATG12是 p300介导的乙酰化(Lee 和 Finkel,2009)和 sirt1依赖的去乙酰化(Lee 等人,2008)的靶点。与 ULK1和 ATG3相反,这些 ATG 蛋白的乙酰化通常会抑制它们的功能。然而,ATG 蛋白的特异性残基、乙酰化位置及其对 ATG 蛋白结构和功能的影响尚不清楚。ATG12-ATG5: ATG16复合物的结构研究(Otomo 等人,2013年)和 ATG12与 ATG3之间相互作用的性质(Metlagel 等人,2013年)表明 ATG12中几个关键的赖氨酸残基可能是其乙酰化的目标。首先,位于 ATG12表面的赖氨酸残基60、69、71和128(Metlagel 等人,2013年)可能有助于 ATG12(类 e3)和 ATG3(类 e2)之间的结合亲和力,这是 LC3脂肪化的时空调节所必需的。此外,ATG5还含有多个赖氨酸残基,其中 Lys53、 Lys130、 Lys171是保守的(Matsushita et al. 2007)。虽然 Lys130是 ATG5和 ATG12(Mizushima et al. 1998)结合的已知催化位点,但 Lys53和 Lys171的功能和乙酰化敏感性仍不清楚。最后,没有发表 ATG7乙酰化敏感性的随访报告(Lee 等人,2008年; Lee 和 Finkel,2009年)。然而,一项高分辨率的质谱法全球蛋白质乙酰化研究证实人类 ATG7蛋白的 Lys306是一个残基,可能与进一步的研究有关(Choudhary 等人,2009年)。因此,虽然 ATG5、 ATG7和 ATG12作为 p300和 SIRT 的底物已有近10年的历史,但其赖氨酸残基对乙酰化的敏感性,或者说对蛋白质的乙酰化抑制作用的本质尚未阐明。

Selective cargo recognition

选择性货物识别

Autophagy receptors modulate the selectivity and specificity of cargo recognition in the autophagy pathway. Although the current knowledge of about PTMs that affect the structure, function and localisation of the canonical autophagy receptors is fairly limited, phosphorylation and ubiquitylation sites were identified in all canonical receptors (THANATOS, https://thanatos.biocuckoo.org) (Deng et al. 2018). The best characterization of acetylation-dependent regulation of autophagy receptors concerns the p62 protein and its affinity for ubiquitin (Fig. 2c). Binding between ubiquitin and p62 to spatially link cargo to the forming autophagosome is, in fed condition, restricted due to the low binding activity of the ubiquitin associated (UBA) domain of p62 and further restricted by UBA homodimerisation (Long et al. 2010). Briefly, Lys420 monoubiquitylation (Lee et al. 2017; Peng et al. 2017), and Ser403 and Ser407 (in humans; Ser405 and Ser409 in mice) phosphorylation (Matsumoto et al. 2015) strengthen the interaction and binding affinity between p62 and ubiquitin. In addition, acetylation of p62 Lys420 and Lys435 residues, regulated by TIP60 and opposed by HDAC6 upon serum and amino acid starvation, interferes with UBA dimerization (Lys420 and Lys435) and enhances ubiquitin-binding affinity (Lys435) (You et al. 2019a). Moreover, spatial proximity between p62 and HDAC6 at sites of protein aggregation promotes their interaction and regulation of HDAC6 deacetylase activity and, by extension, protein aggregate recycling by p62 (Yan et al. 2013).

自噬受体调节自噬途径中货物识别的选择性和特异性。虽然目前关于影响典型自噬受体结构、功能和定位的 PTMs 的知识相当有限,但是在所有典型受体(THANATOS, https://THANATOS.biocuckoo.org )中都发现了磷酸化和泛素化位点(Deng et al. 2018)。自噬受体的乙酰化依赖性调节的最佳角色塑造是 p62蛋白及其对泛素的亲和力(图2 c)。在食物条件下,泛素和 p62与形成的自噬体之间的空间连接受到限制,这是因为 p62泛素相关(UBA)结构域的结合活性较低,而且进一步受到非洲大陆泛素同二聚体的限制(Long 等人,2010年)。简而言之,Lys420单基化(Lee 等人,2017年; Peng 等人,2017年)和 Ser403和 Ser407(人类; Ser405和 Ser409小鼠)磷酸化(松本等人,2015年)增强 p62和泛素之间的相互作用和结合亲和力。此外,p62 Lys420和 Lys435残基的乙酰化受 TIP60调节,而 HDAC6对血清和氨基酸饥饿产生反作用,干扰了非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性聚合。此外,p62和 HDAC6在蛋白质聚集位点的空间接近促进了它们之间的相互作用和 HDAC6脱乙酰酶活性的调节,进而促进了 p62蛋白质聚集再循环(Yan 等人,2013年)。

Moreover, ubiquitin (Ub) itself is a target of lysine acetylation (Ohtake et al. 2015). Formation of stable polyubiquitin chains by covalent linkages of single Ub moieties via homotypic Lys63linkage (also known as K63) promotes autophagy (Grumati and Dikic 2018). Although the KAT(s) and KDAC(s) involved and the physiological relevance of Ub acetylation remain unknown, acetylation of Lys6 and Lys48 residues was shown to interfere with poly-Ub chain formation (Lys11-, Lys48– and Lys63-linked) in vitro (Ohtake et al. 2015).

此外,泛素(Ub)本身就是赖氨酸乙酰化的靶标(Ohtake 等人,2015)。通过同型 Lys63连锁(也称为 K63)的 Ub 单体共价连锁形成稳定的多聚泛素链促进自噬(Grumati 和 Dikic 2018)。虽然 KAT (s)和 KDAC (s)参与和生理相关性的 Ub 乙酰化仍然是未知的,Lys6和 Lys48残基的乙酰化已被证明干扰聚 Ub 链的形成(Lys11-,Lys48-和 lys63-连接)在体外(Ohtake 等人,2015年)。

Demystification of the acetylation riddle in autophagy regulation

自噬调控中乙酰化之谜的揭秘

Recent advances in our understanding of which KATs and KDACs are involved in the regulation of autophagy protein acetylation highlight a few interesting phenomena. Overall, autophagy protein acetylation status is mainly regulated by p300, CREB binding protein (CBP) and TIP60 KATs, and HDAC2/6 and SIRT1 KDACs (Table 1). Upon a closer look, targets of p300-mediated acetylation are generally opposed by SIRT1-dependent deacetylation, while the targets of TIP60 may be opposed by HDACs but remain largely unknown. Following this train of thought, targets of p300/SIRT are activated by the loss of acetylation, whereas it is the addition of acetyl group to TIP60 targets that triggers their activation (summarized in Table 1, shown in Fig. 2).

近年来,我们对 KATs 和 kdac 参与自噬蛋白乙酰化调控的研究取得了一些新进展。总的来说,自噬蛋白的乙酰化状态主要受 p300,CREB结合蛋白(CBP)和 TIP60 KATs,以及 HDAC2/6和 SIRT1 kdac 的调节(表1)。近距离观察发现,p300介导的乙酰化作用靶点一般与 sirt1相关的脱乙酰化作用相反,TIP60的靶点可能与 HDACs 相反,但仍不清楚。根据这一思路,p300/SIRT 的靶基因因乙酰化缺失而被激活,而 TIP60的靶基因因乙酰化缺失而被激活(见表1,如图2所示)。

Autophagy is stimulated by the depletion of key nutrients including amino acids, growth factors and glucose. Recognition of nutrient availability by multiple intracellular sensors converges on a handful of regulators that integrate nutrient signals into several key responses. These include mammalian target of rapamycin complex 1 (mTORC1) (Rabanal-Ruiz et al. 2017), glycogen synthase kinase 3 (GSK3) (Mancinelli et al. 2017), and adenine monophosphate-activated protein kinase (AMPK) (Tamargo-Gómez and Mariño 2018). Perhaps unsurprisingly, these three kinases have also been directly linked to the regulation of KATs and KDACs that influence the acetylation status of autophagy proteins. mTORC1 was recently shown to activate the acetyl-transferase activity of p300 by serine phosphorylation that was lost upon amino acid starvation (Wan et al. 2017). Activation of GSK3β by the loss of growth factor signalling is known to phosphorylate and thus activate TIP60 (Lin et al. 2012). Finally, AMPK activation releases SIRT1 inhibition in a GAPDH-dependent manner in response to glucose starvation (Chang et al. 2015). Thus, three potential axes regulate autophagy stimulation in response to nutrient stress by (a) loss of FoxO, VPS34, Beclin1, ATG7, ATG5 and ATG12 acetylation (amino acids/growth factors-mTORC1-p300), (b) increased ULK1 and possible ATG3 and p62 acetylation (serum/ER stress-GSK3β-TIP60) (Lin et al. 2012; Nie et al. 2016; Yi et al. 2012; You et al. 2019a), and (c) FoxO, Beclin1, ATG7, ATG5 and ATG12 deacetylation (glucose–AMPK–GAPDH–SIRT1) that could explain the conundrum of the varied nature of autophagy protein acetylation status upon nutrient starvation and its link to autophagy stimulation.

自噬是由包括氨基酸、生长因子和葡萄糖在内的关键营养物质的耗竭而引起的。通过多个细胞内传感器识别营养物质的可利用性汇聚在一些调节器上,这些调节器将营养信号整合到几个关键反应中。这些包括哺乳动物靶蛋白雷帕霉素复合物1(mTORC1)(Rabanal-Ruiz 等人2017年) ,糖原合成酶激酶3(GSK3)(Mancinelli 等人2017年) ,腺嘌呤单磷酸激活蛋白激酶(AMPK)(Tamargo-Gómez 和 Mariño 2018年)。也许不足为奇的是,这三种激酶也直接与影响自噬蛋白乙酰化状态的 KATs 和 kdac 的调节有关。mTORC1最近被证明通过丝氨酸磷酸化激活 p300的乙酰基转移酶活性,而丝氨酸磷酸化在氨基酸饥饿时失去了这种活性(Wan 等人,2017年)。由于生长因子信号的丢失而激活的 gsk3被认为是磷酸化的,因此激活 TIP60(Lin 等人,2012)。最后,AMPK 的激活以 gapdhh 依赖的方式释放 SIRT1的抑制作用以应对葡萄糖饥饿(Chang et al. 2015)。因此,三个潜在的轴通过(a) FoxO、 VPS34、 Beclin1、 ATG7、 ATG5和 ATG12乙酰化(氨基酸/生长因子 -mtorc1-p300)的缺失,调节对营养胁迫的自噬刺激,(b)增加 ULK1和可能的 ATG3和 p62乙酰化(serum/ER stress-gsk3-tip60)(Lin et al. 2012; Nie et al. 2016; Yi et al. 2012; You et al. 2019a) ,(c) FoxO,Beclin1,ATG7,ATG5和 ATG12 deacetylation (glucose-AMPK-GAPDH-SIRT1)难题可以解释在营养缺乏刺激下自噬蛋白乙酰化状态的不同性质及其与自噬缺乏的联系。

Targets of cysteine oxidative PTMs in autophagy

半胱氨酸氧化 PTMs 在自噬中的作用

Protein modification by ROS and RNS constitutes a covalent modification of amino acid residues by the reactive species directly, or as a secondary interaction in an oxidative relay. Briefly, irreversible (carbonylation, nitration) oxidative modifications affect a variety of amino acids including cysteine (Cys), threonine and tyrosine (Ahmad et al. 2017; Cai and Yan 2013; Xie et al. 2018). In contrast, reversible amino acid oxidation involves modification of the thiol group (-SH) of Cys protein residues that are first modified to sulfenic acid (–SOH) (Cai and Yan 2013). Sulfenic acid can then undergo nitrosylation (-SNO) by reacting with RNS, or disulphide bond formation (R–S–S–R) by intra-/inter-molecular bond formation between two cysteine residues. A specialised form of disulphide bond formation, glutathionylation (R–S–S–G) arises as a mixed disulphide bond formation between a target protein Cys residue and the non-enzymatic antioxidant, glutathione (GSH) (Cai and Yan 2013). Further oxidation of –SOH results in an irreversible Cys oxidation by the formation of sulfinic (–SO2H) and sulfonic (–SO3H) acids (Ahmad et al. 2017; Cai and Yan 2013; Murray and Van Eyk 2012). Autophagy regulation by ROS is linked to the reversible oxidative Cys modification of (a) transcription factors (TFs) that regulate expression of proteins involved in the autophagy process, (b) upstream regulators of autophagy initiation, (c) autophagy proteins themselves and (d) receptors that mediate autophagy substrate selectivity (Filomeni et al. 2015; Montagna et al. 2016; Sedlackova et al. 2020).

活性氧和 RNS 对蛋白质的修饰是由反应物直接对氨基酸残基进行共价修饰,或者作为氧化继电器中的二次相互作用。简而言之,不可逆(羰基化,硝化)氧化修饰影响各种氨基酸,包括半胱氨酸(Cys) ,苏氨酸和酪氨酸(Ahmad et al. 2017; Cai and Yan 2013; Xie et al. 2018)。相比之下,可逆的氨基酸氧化涉及到胱氨酸蛋白质残基的硫醇基(- SH)的修饰,该基因首先被修饰为磺胺酸(- SOH)(Cai 和 Yan,2013)。亚砜酸通过与 RNS 反应发生亚硝基化(- SNO) ,或通过两个半胱氨酸残基之间的分子内/分子间键形成二硫化物键(r-s-s-r)。谷胱甘肽(r-s-s-g)是二硫键形成的一种特殊形式,它是目标蛋白 Cys 残基和非抗氧化酶谷胱甘肽(GSH)之间的二硫键形成的混合物。- SOH 的进一步氧化导致不可逆的 Cys 氧化,形成亚硫酸盐(- SO2H)和磺酸盐(- SO3H)酸(Ahmad 等人,2017; Cai 和 Yan,2013; Murray 和 Van Eyk,2012)。ROS 的自噬调节与(a)转录因子(tf)的可逆氧化性 Cys 修饰有关,tf 调节自噬过程中蛋白质的表达,(b)自噬启动的上游调节因子,(c)自噬蛋白本身和(d)调节自噬底物选择性的受体(Filomeni et al. 2015; Montagna et al. 2016; lackova et al. 2020)。

The most substantial link between ROS and autophagy TF activation was established in the studies of the Mit/TFE family of transcription factors (Yang et al. 2018). Three members of the Mit/TFE protein family were recently shown to contain redox sensitive Cys residues (TFEB Cys212, TFE3 Cys322, MITF Cys281) that mediate a rapid response to increased intracellular oxidative stress by promoting their nuclear translocation (Wang et al. 2019a). Another layer of regulation by oxidative stress was previously uncovered for TFEB that regulates expression of several autophagy proteins including, ATG4, ATG9, LC3B and p62 (Settembre et al. 2011). Increased intracellular oxidative stress is sensed by the lysosomal cation channel, mucolipin 1 (MCOLN1/TRPML1) in a manner that is not yet understood (Zhang et al. 2016c). What is known is that MCOLN1 oxidation promotes channel opening, Ca2+ release from the lysosomal lumen and activation of a Ca2+ dependent phosphatase, calcineurin (Medina et al. 2015; Zhang et al. 2016c). Calcineurin-dependent TFEB phosphorylation then promotes TFEB translocation to the nucleus and autophagy stimulation (Fig. 2A).

在对 Mit/TFE 转录因子家族的研究中建立了活性氧和自噬 TF 激活之间最实质性的联系(Yang 等人,2018)。最近发现 Mit/TFE 蛋白家族的3个成员含有氧化还原敏感的 Cys 残基(TFEB Cys212,TFE3 Cys322,MITF Cys281) ,这些残基通过促进细胞核移位对增加的细胞内氧化应激做出快速反应。另外一层由氧化应激基因组调控的 TFEB 蛋白,包括 ATG4,ATG9,LC3B 和 p62蛋白的表达。增加的细胞内氧化应激通过溶酶体阳离子通道—- 粘液脂蛋白1(MCOLN1/TRPML1)检测到,但这种方式尚不清楚(Zhang et al. 2016c)。已知的是,MCOLN1氧化促进通道开放,从溶酶体腔释放 Ca2 + 和激活一个 Ca2 + 依赖性磷酸酶,钙调神经磷酸酶(Medina 等人,2015; Zhang 等人,2016 c)。钙调神经磷酸酶依赖的 tmb 磷酸化促进 tmb 转运到细胞核和自噬刺激(图2A)。

At the stage of autophagy execution, redox-sensitive Cys residues were identified in proteins involved in LC3 processing (ATG4B) and LC3-PE conjugation (ATG7 and ATG3). ATG4B is a Cys-dependent protease that cleaves pro-LC3 at a C-terminal glycine residue prior to LC3-PE conjugation (Kirisako et al. 2000). Its protease activity is also involved in correcting the amount of LC3–PE formation on non-autophagic membranes by the hydrolysis of the LC3–PE bond, and presumably on the outer membrane leaflet of the growing autophagosome. In human cells, the hydrolysing (deconjugating) activity of ATG4B is inhibited by the oxidation of one of two Cys residues (Cys74 or Cys78) and leads to improved stability of LC3–PE and increased formation of autophagosomes (Scherz‐Shouval et al. 2007). Similarly, oxidation of the catalytic thiols in ATG3 (Cys264) and ATG7 (Cys572) inhibits their activity in LC3–PE conjugation and results in the loss of autophagic flux (Fig. 2b) (Frudd et al. 2018). Interestingly, oxidation of these Cys residues can only occur when the thiols are not shielded by their interaction with LC3.

在自噬执行阶段,在参与 LC3加工(ATG4B)和 LC3-pe 接合(ATG7和 ATG3)的蛋白质中发现了氧化还原敏感的 Cys 残基。ATG4B 是一种依赖于胱氨酸的蛋白酶,在 LC3-PE 接合之前在 c 末端甘氨酸残基上分离 pro-LC3(Kirisako 等人,2000)。它的蛋白酶活性也参与了通过 LC3-PE 键的水解纠正非自噬膜上 LC3-PE 的形成量,推测是在生长中的自噬体的外膜小叶上。在人类细胞中,ATG4B 的水解(去聚集)活性被两个 Cys74或 Cys78中的一个残基的氧化所抑制,从而提高了 LC3-PE 的稳定性并增加了自噬体的形成(Scherz-Shouval 等人,2007年)。同样,ATG3(Cys264)和 ATG7(Cys572)中催化硫醇的氧化抑制了它们在 LC3-PE 共轭中的活性,导致自噬通量的损失(图2b)(Frudd 等人,2018)。有趣的是,只有当硫醇与 LC3的相互作用没有屏蔽时,这些 Cys 残基才会发生氧化。

Oxidative stress influences the selectivity of the autophagic process via p62, a known redox sensitive autophagy receptor protein (Fig. 2c). Intermolecular disulphide formation in p62 was first observed in studies of its involvement in the N-end rule pathway of substrate degradation, where Cys113-dependent oligomerisation promoted substrate clearance via autophagy (Cha-Molstad et al. 2017). Subsequently, we have demonstrated that elevated ROS levels promote the formation of disulphide-linked conjugates, intermolecular Cys bonds, that assist p62 oligomer assembly (Carroll et al. 2018). Crucially, we have identified two Cys residues (Cys105and Cys113) located within the regulatory linker region of the p62 protein, that are necessary and sufficient for the activation of pro-survival autophagy triggered by increased ROS (Carroll et al. 2018).

氧化应激通过 p62影响自噬过程的选择性,p62是一种已知的氧化还原敏感性自噬受体蛋白(图2 c)。P62分子间二硫化物的形成首先是在研究其参与底物降解的 n 端规则途径时观察到的,其中 cys113依赖的寡聚化通过自噬促进底物清除(Cha-Molstad 等人,2017年)。随后,我们证明,活性氧水平升高促进形成二硫化物连接复合物,分子间 Cys 键,这有助于 p62低聚物组装(Carroll 等人,2018年)。至关重要的是,我们已经确定了两个 Cys 残基(Cys105和 Cys113)位于 p62蛋白的调节连接器区域,这对于激活活性氧增加引发的有利于存活的自噬是必要和充分的(Carroll 等人,2018)。

Reversible oxidation of Cys residues in redox-sensitive autophagy proteins thus appears to have a dual role of pathway stimulation by autophagy gene expression (TFEB), increased autophagosome formation (ATG4B) and substrate selectivity (p62), and autophagy inhibition upon depletion of available LC3 substrate (ATG3, ATG7). However, due to the novelty of these findings, the physiological role of ATG3 and ATG7 inhibition and possible downstream signalling events remain unknown. We propose a regulatory feedback loop whereby sensing depletion of local LC3 pools results in inactivation of ATG3 and ATG7 that serves to prevent indiscriminate autophagy activation. We envision that this inactivation would persist until such a time that the antioxidant defences decrease the oxidative stress load and resolve the ATG3-ATG7 heterodimer, and the expression of autophagy genes restores the available pools of ATG proteins to sustain further autophagy.

因此,氧化还原敏感性自噬蛋白中 Cys 残基的可逆氧化似乎具有通过自噬基因表达(TFEB)刺激、增加自噬体形成(ATG4B)和底物选择性(p62)以及通过消耗可利用的 LC3底物(ATG3,ATG7)抑制自噬的双重作用。然而,由于这些发现的新颖性,ATG3和 ATG7的生理作用抑制和可能的下游信号事件仍然是未知的。我们提出了一个监管反馈回路,通过感知当地 LC3池的耗尽导致 ATG3和 ATG7的失活,从而防止不分青红皂白的自噬激活。我们设想,这种失活将持续到抗氧化防御降低氧化应激负荷,解决 ATG3-ATG7异二聚体,自噬基因的表达恢复可用的 ATG 蛋白池,以维持进一步的自噬。

The interrelatedness of target oxidation and acetylation in autophagy

自噬过程中靶向氧化与乙酰化的相互关系

Protein deacetylation and oxidation appear to be individually sufficient to regulate the initiation, promotion, efficiency and selectivity of autophagy. However, an interesting crosstalk between oxidative and acetyl-linked PTMs of autophagy proteins arises due to the dual control of several proteins including TFEB, ATG3, ATG7 and p62, which appear to be regulated by both, oxidation and acetylation status (Fig. 2a–c). First, upstream oxidation of MCOLN1 regulates TFEB localization by calcineurin-dependent dephosphorylation (Medina et al. 2015; Zhang et al. 2016c) and direct oxidation of its Cys212 residue (Wang et al. 2019a). Further, TFEB deacetylation at residues Lys274 and Lys279 promotes its dimerization and increases its binding affinity for DNA (Wang et al. 2019b). It would be interesting to study whether oxidation and acetylation PTMs act in concert to establish the optimal TFEB activity and whether TFEB oxidation promotes rapid expression of its target genes in the absence of Lys residue deacetylation.

蛋白质的脱乙酰化和氧化作用足以调节自噬的启动、促进、效率和选择性。然而,由于 TFEB、 ATG3、 ATG7和 p62等蛋白质的双重调控,自噬蛋白的氧化和乙酰连接的 PTMs 之间出现了一个有趣的串扰,它们似乎都受到氧化和乙酰化状态的调控(图2a-c)。首先,MCOLN1的上游氧化通过依赖于钙调神经磷酸酶的脱磷酸化(Medina 等人,2015; Zhang 等人,2016c)和其 Cys212残基的直接氧化(Wang 等人,2019a)来调节 tmb 的定位。另外,在 Lys274和 Lys279残基上的 tbb 脱乙酰化促进了它的二聚化并增加了它与 DNA 的结合亲和力(Wang 等人,2019b)。研究在没有赖氨酸残基脱乙酰化的情况下,氧化和乙酰化 PTMs 是否协同作用,以确定最佳的 tbb 活性,以及 tbb 氧化是否促进其靶基因的快速表达,将会引起人们的重视。

Second, ATG3 acetylation at residues Lys19 and Lys48 by TIP60, increased in conditions of nutrient starvation, improves interaction between ATG3 and LC3 and promotes autophagy (Yi et al. 2012). Not much is known regarding the functional effect of deacetylation in ATG7, except that it promotes autophagy and Lys306 residue may be the target (Choudhary et al. 2009). In contrast to TFEB, a recently published study suggests that upon loss of LC3 binding, oxidation of ATG3 (Cys264) and ATG7 (Cys572) catalytic cysteine residues inhibits their enzymatic activity and blocks their further interaction with LC3 (Frudd et al. 2018).

其次,TIP60在 Lys19和 Lys48残基上的 ATG3乙酰化,在营养缺乏条件下增加,改善 ATG3和 LC3之间的相互作用,促进自噬(Yi 等人,2012)。目前对 ATG7中脱乙酰基的功能作用还知之甚少,除了它促进了自噬,Lys306残基可能是其作用目标(Choudhary 等人,2009年)。与 TFEB 相反,最近发表的一项研究表明,ATG3(Cys264)和 ATG7(Cys572)催化半胱氨酸残基的氧化会抑制它们的酶活性,阻碍它们与 LC3的进一步相互作用(Frudd 等人,2018年)。

Lastly, oxidation and acetylation of p62 could act in concert to achieve optimal selectivity of its interaction with cargo and oligomerization to stimulate autophagy. First, TIP60-dependent acetylation of Lys420 and Lys435 within the UBA domain interferes with its inter-protein dimerization and enhances the ubiquitin binding affinity of p62 upon serum and amino acid starvation (You et al. 2019a). Second, oxidation of Cys105 and Cys113 residues within the regulatory linker region promotes intermolecular p62 disulphide bond formation and thus assist in autophagy stimulation (Carroll et al. 2018).

最后,p62的氧化和乙酰化可以协同作用,实现其与载体相互作用的最佳选择性和促进自噬的齐聚作用。首先,tip60依赖于 Lys420和 Lys435的乙酰化作用,干扰了其蛋白间二聚化,增强了 p62与血清和氨基酸饥饿的泛素结合亲和力(You 等,2019a)。其次,调节连接物区域内 Cys105和 Cys113残基的氧化促进了分子间 p62二硫键的形成,从而有助于自噬刺激(Carroll 等人,2018年)。

In addition, activity of NAD+-dependent KDACs, or SIRTs, is directly or indirectly regulated by both, oxidative and metabolic stress stimuli. First, a shift in the NAD redox balance towards oxidation, suggestive of metabolic stress, leads to an increased pool of available NAD+ and thus stimulates SIRT activity (Imai and Guarente 2016). Second, SIRT regulation by oxidative stress was demonstrated in multiple cell culture experiments (reviewed in (Santos et al. 2016)), in which a mild oxidative environment promotes SIRT1 expression and activation by upstream kinases. In contrast, study of SIRT1 oxidation, specifically nitrosylation (–SNO+), suggests that this reversible oxidative PTM of Cys371, Cys374, Cys395 and Cys398 residues within a tetrathiolate formation results in loss of Zn2+ binding, structural destabilization and loss of NAD+ and acetyl-lysine binding ability (Kalous et al. 2016). Thus, SIRT1 activity can be stimulated by both, nutrient starvation, and oxidative stress. However, persistent ROS release may lead to SIRT1 destabilization, loss of its deacetylase activity and might contribute to its degradation by the proteasome (Caito et al. 2010).

此外,NAD + 依赖的 kdac 或 SIRTs 的活性直接或间接地受到氧化和代谢应激刺激的调节。首先,NAD 的氧化还原平衡转向氧化,暗示代谢应激,导致可用 NAD + 池增加,因此刺激 SIRT 活性(今井和瓜伦特2016年)。其次,在多次细胞培养实验中证实了氧化应激对 SIRT 的调节作用(见 Santos 等人2016年的综述) ,在这些实验中,温和的氧化环境通过上游激酶促进 SIRT1的表达和活化。与此相反,SIRT1氧化特异性硝基化(- SNO +)的研究表明,Cys371、 Cys374、 Cys395和 Cys398残基在四硫醇盐形成过程中的可逆氧化性 PTM 导致 Zn2 + 结合的丧失、 NAD + 结构的不稳定性和乙酰赖氨酸结合能力的丧失(Kalous et al. 2016)。因此,SIRT1的活性可以同时受到营养缺乏和氧化应激的刺激。然而,持续释放 ROS 可能导致 SIRT1的不稳定,失去其去乙酰化酶活性,并可能有助于其降解的蛋白酶体(Caito 等人。2010年)。

NAD depletion, oxidative stress, and autophagy in physiological and pathological ageing

生理和病理衰老过程中的 NAD 损耗、氧化应激和自噬

The NAD nucleotide is an important redox molecule required for fundamental molecular processes of energy generation via glycolysis, tricarboxylic acid cycle, oxidative phosphorylation and β-oxidation, and a co-factor to enzymes involved in cellular signalling and longevity. Age-related depletion of available NAD+ pools was, in human disease, animal models and in vitro studies, reported as a result of increased PARP activity due to an elevation in oxidative stress and levels of DNA damage (Pacher and Szabo 2008) and increased CD38 expression and activity (Camacho-Pereira et al. 2016; Polzonetti et al. 2012). Combined with the age-dependent reduction in the enzymatic activity of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAM-based NAD+salvage pathway (Stein and Imai 2014), these conditions perpetuate the perfect storm of total NAD depletion, loss of NAM recycling and reduction in SIRT activity in physiological ageing.

NAD 核苷酸是一种重要的氧化还原分子,它是通过糖酵解、三羧酸循环、氧化磷酸化和氧化等基本分子过程产生能量的必需物质,也是参与细胞信号传递和延长寿命的酶的辅助因子。在人类疾病、动物模型和体外研究中,与年龄相关的 NAD + 池损耗被报道为由于氧化应激升高和 DNA 损伤水平增加而导致 PARP 活性增加(Pacher 和 Szabo,2008年)和 CD38表达和活性增加(Camacho-Pereira 等人,2016年; Polzonetti 等人,2012年)。结合年龄依赖性烟酰胺磷酸核糖基转移酶(NAMPT)酶活性的降低,这些条件延续了生理衰老过程中 NAD 总耗竭、 NAM 循环丢失和 SIRT 活性降低的完美风暴。

Study of human skin tissue from volunteers of different ages partially supports these findings (Massudi et al. 2012). In this study, an age-dependent increase in DNA damage correlated with an increase in PARP activity, NAD+ depletion and, in the elderly, a reduction in SIRT1 activity. Notably, these associations with age were strong only in the male participants and it would be interesting to see whether these findings can be reproduced in females and other accessible human tissues, including muscle or post-mortem brain tissues. In a more recent study carried out on human skeletal muscle samples, authors demonstrate that levels of NAMPT negatively correlate with age, body mass index and body fat percentage (de Guia et al. 2019). Another study utilised the power of magnetic resonance-based non-invasive in vivo imaging of the human brain and revealed an age-dependent decrease in total NAD levels, concomitant with an increase in NADH/NAD+ ratio, indicative of metabolic dysfunction (Zhu et al. 2015). While these studies were carried out on healthy human volunteers and suggest that a decline in NAD levels occurs in physiological ageing, multiple studies of accelerated human ageing (progeria) syndromes and patients suffering from metabolic and neurodegenerative diseases strongly link NAD decline to age-related pathology (Kubben and Misteli 2017; Lautrup et al. 2019; Okabe et al. 2019).

对不同年龄的志愿者皮肤组织的研究部分支持了这些发现(Massudi et al. 2012)。在这项研究中,DNA 损伤的年龄依赖性增加与 PARP 活性的增加、 NAD + 损耗以及老年人 SIRT1活性的减少相关。值得注意的是,这些与年龄的联系只在男性参与者中很强烈,看看这些发现能否在女性和其他可获得的人体组织中重现,包括肌肉组织或死后的脑组织,将是很有意思的。在最近对人类骨骼肌样本的研究中,作者证明 NAMPT 水平与年龄、身体质量指数和身体脂肪百分比呈负相关(de Guia 等人,2019年)。另一项研究利用了基于磁共振的人脑非侵入性体内成像技术,揭示了 NAD 总水平随年龄增长而下降,同时 nadh/NAD + 比值增加,表明存在代谢功能障碍(Zhu 等人,2015年)。虽然这些研究是在健康的人类志愿者身上进行的,并表明 NAD 水平的下降发生在生理性衰老中,但对人类加速衰老(早衰症)综合征和患有代谢和神经退行性疾病的患者进行的多项研究强烈地将 NAD 的下降与年龄相关的病理学联系起来(Kubben 和 Misteli,2017年; Lautrup 等人,2019年; Okabe 等人,2019年)。

Furthermore, studies of two age-related conditions, sarcopenia and frailty, as well as a variety of progeria, neurodegenerative, metabolic and cardiac diseases, demonstrate a strong link between pathology and increased oxidative stress (Derbré et al. 2014; Inglés et al. 2014; Kubben and Misteli 2017; Liguori et al. 2018; Massudi et al. 2012; Soysal et al. 2017). Not only does lipid peroxidation, a proxy measurement for increased oxidative stress, correlate with age (Massudi et al. 2012), a systematic review of available literature suggests that long lived humans (centenarians) have lower levels of oxidative protein damage and lipid peroxidation compared to other elderly individuals (Belenguer-Varea et al. 2019). Given the number and severity of clinical conditions related to healthy ageing, and age-related diseases that are associated with an increase in oxidative stress, it is necessary to design interventions that prevent production of free radicals, boost cellular antioxidant systems, or understand and target the processes downstream of ROS-mediated protein, lipid or nucleotide damage.

此外,与年龄相关的条件,骨骼肌减少和脆弱,以及各种早衰症,神经退行性,新陈代谢和心脏疾病的研究表明,病理学和氧化应激增加之间有很强的联系(derbré 等人2014; Inglés 等人2014; Kubben 和 Misteli 2017; Liguori 等人2018; Massudi 等人2012; Soysal 等人2017)。脂质过氧化不仅是氧化应激增加的代理测量,与年龄相关(Massudi 等人2012年) ,一系统综述可用的文献表明,长寿的人(百岁老人)与其他老年人相比,有较低的氧化蛋白质损伤和脂质过氧化。鉴于与健康老龄化相关的临床疾病的数量和严重程度,以及与氧化应激增加相关的与年龄相关的疾病,有必要设计干预措施,以防止自由基的产生,增强细胞抗氧化系统,或了解和瞄准 ros 介导的蛋白质、脂质或核苷酸损伤的下游过程。

Importantly, molecular studies of free radical generation, NAD+-dependent enzymatic processes and disease pathology suggest a link between ROS accumulation, NAD depletion and compromised mitochondrial recycling by autophagy, mitophagy. Mitochondria are energy-generating organelles that act as hubs of pro-survival or pro-apoptotic signalling (Sedlackova and Korolchuk 2019). Although mitochondrial health is maintained by a complex net of quality control mechanisms, whole organelle recycling of damaged and ROS-producing mitochondria is only achieved by selective autophagy. A causal link between NAD+ depletion and mitochondrial dysfunction due to loss of mitophagy was established in studies of premature ageing syndromes including Xeroderma Pigmentosum, Cockayne syndrome and Ataxia-telangiectasia (Fang et al. 20162014; Scheibye-Knudsen et al. 20142012; Valentin-Vega et al. 2012). In these studies, loss of SIRT activity and autophagy abnormalities occur as a result of PARP1 hyperactivation due to unresolved DNA damage. In addition to SIRT inactivation, uncontrolled NAD+ cleavage and protein PAR-ylation by PARPs results in loss of ATP availability and, if persistent, in cell death (Andrabi et al. 2014; Bai et al. 2011; Fouquerel et al. 2014; Pillai et al. 2005). Persistent NAD+ depletion was thus shown to compromise mitochondrial function due to loss of energy generation, impairment in mitochondrial recycling through lack of autophagy/mitophagy stimulation, and to initiate cellular death due to energy collapse. An alternative outcome to cell death upon PARP1 activation was linked to autophagy initiation in independent cell culture experiments (Jiang et al. 2018; Muñoz-Gámez et al. 2009). In the earlier study, authors demonstrated that PARP-dependent stimulation of autophagy due to short-lived energy crisis had a cytoprotective effect as genetic or pharmacological inhibition of autophagy led to increased level of necrotic death (Muñoz-Gámez et al. 2009). In the latter study, authors aimed to mimic constant ROS production in vivo by glucose oxidase (GO) treatment, which led to PARP-induced cell death, parthanatos (Jiang et al. 2018). In this study, inhibition of autophagy led to a significant collapse in mitochondrial polarization and an approximately 50% increase in cell death within four hours of GO treatment. Taken together with the role of SIRT-mediated autophagy stimulation, we wonder whether convergence of these signalling pathways on autophagy suggests a conserved role of this catabolic pathway in healthy ageing by preservation of cellular NAD pools.

重要的是,对自由基产生、 NAD + 依赖的酶过程和疾病病理学的分子研究表明,ROS 的积累、 NAD 的耗竭和线粒体自噬、噬细胞的损伤循环之间存在联系。线粒体是能量产生的细胞器,充当促生存或促凋亡信号的枢纽(Sedlackova 和 Korolchuk 2019)。虽然线粒体的健康是通过一个复杂的质量控制机制网来维持的,但是受损和产生 ros 的线粒体的整个细胞器的再循环只能通过选择性自噬来实现。在对早衰综合症的研究中,包括着色性干皮症、柯凯因氏症候群和共济失调-毛细血管扩张症,确立了 NAD + 耗竭与线粒体功能障碍之间的因果关系。在这些研究中,由于未解决的 DNA 损伤导致 PARP1过度激活,引起 SIRT 活性丧失和自噬异常。除 SIRT 失活外,PARPs 不受控制的 NAD + 分裂和蛋白质 PAR-ylation 导致 ATP 供应的丧失,如果持续存在,则导致细胞死亡(Andrabi 等人,2014年; Bai 等人,2011年; Fouquerel 等人,2014年; Pillai 等人,2005年)。因此,研究表明,NAD + 持续耗竭会损害线粒体功能,原因是能量代谢的丧失,缺乏自噬/吞噬刺激导致线粒体循环受损,以及能量崩溃导致细胞死亡。在独立的细胞培养实验中,PARP1激活导致细胞死亡的另一个结果与自噬启动有关(Jiang 等人,2018; Muñoz-Gámez 等人,2009)。在早期的研究中,作者证明,由于短暂的能量危机引起的 parp 依赖性自噬的刺激具有细胞保护作用,因为对自噬的遗传或药理抑制导致坏死死亡水平的增加(Muñoz-Gámez 等人,2009年)。在后一项研究中,作者的目标是通过葡萄糖氧化酶(GO)处理在体内模拟稳定的活性氧产生,这导致了 parp 诱导的细胞死亡(parthanatos)(Jiang 等人,2018年)。在这项研究中,抑制自噬导致线粒体极化明显崩溃,并在 GO 治疗4小时内增加约50% 的细胞死亡。结合 sirt 介导的自噬刺激的作用,我们想知道这些自噬信号通路的聚合是否表明这种分解代谢途径通过保存细胞 NAD 池在健康老龄化过程中发挥了保守的作用。

An exciting development in the field of ageing and NAD metabolism is the ‘druggability’ of NAD metabolism by exogenous addition of natural, or synthetic, bioavailable NAD+precursors. This universal approach of NAD+ precursor supplementation is known to increase NAD biosynthesis and alleviate the symptoms of pathological states including metabolic, cardiac and neurodegenerative disorders (Kane and Sinclair 2018; Lautrup et al. 2019). Additionally, evidence from NAD+ supplementation studies in cell culture and in animal models suggests that boosting NAD levels is sufficient to not only improve mitochondrial function, but also stimulate SIRT-dependent mitochondrial recycling via increased TFEB- and FoxO-dependent expression of autophagy/mitophagy genes and PTMs of autophagy proteins, and thus promote clearance of dysfunctional organelles and protein aggregates (Fang et al. 20192016; Hou et al. 2018; Schöndorf et al. 2018; Vannini et al. 2019; Zhang et al. 2016b). Altogether, this ‘silver bullet’ approach might serve as an intervention to the vicious cycle of damage and NAD depletion and thus not only combat the depletion itself, but also support resolution of the underlying stresses and promote long-term cellular health.

在衰老和 NAD 代谢领域的一个令人兴奋的发展是通过外源添加天然的或合成的生物可利用的 NAD + 前体的 NAD 代谢的‘药物可利用性’。众所周知,补充 NAD + 前体的这种普遍方法可以增加 NAD 的生物合成,减轻包括代谢、心脏和神经退行性疾病在内的病理状态的症状(Kane 和 Sinclair,2018年; Lautrup 等人,2019年)。此外,在细胞培养和动物模型中补充 NAD + 的研究证据表明,提高 NAD 水平不仅足以改善线粒体功能,而且通过增加 TFEB-和 foxo 依赖的自噬/细胞吞噬基因和自噬蛋白 PTMs 的表达,刺激依赖 sirt 的线粒体循环,从而促进清除功能失调的细胞器和蛋白质聚集体(Fang et al. 2019,2016; Hou et al. 2018; Schöndorf et al. 2018; Vannini et al. 2019; Zhang et al. 2016 b)。总之,这种”银弹”办法可以作为对损害和 NAD 耗竭的恶性循环的干预,因此不仅可以对付耗竭本身,而且还可以解决潜在的压力,促进长期的细胞健康。

Following the success of NAD+-boosting strategies in cell and animal models, NAD+precursors, and predominantly nicotinamide riboside (NR), are now subjects of multiple clinical trials. Precursors have so far been reported as safe, well tolerated and capable of increasing NAD levels in healthy volunteers (Conze et al. 2019; Martens et al. 2018; Minto et al. 2017; Stea et al. 2017). However, challenges remain in translation of laboratory findings into the design of clinical trials (Gilmour et al. 2020). While some early success was found in disease outcomes of amyotrophic lateral sclerosis (ALS) (NCT03489200) (de la Rubia et al. 2019), others found no benefit in patients with Alzheimer’s disease (NCT00580931) (Phelan et al. 2017), or studies of metabolic disorders or mitochondrial bioenergetics in men (NCT02303483) (Dollerup et al. 20182019a2019b). Although only a limited number of trials testing NAM, NMN and NR have been recently completed (> 10) or are currently ongoing (− 3), many are actively recruiting (− 21) (https://clinicaltrials.gov/) (Lautrup et al. 2019) and it will be interesting to see what lessons can be learned about precursor dosage, NAD+/NADH detection methods and bioavailability in the coming years. Considering that the pathological role of NAD depletion in many metabolic and neurodegenerative diseases is not yet firmly established, reporting of relevant disease outcomes is eagerly awaited as they will inform about the feasibility of translating success from the laboratory to human age- and disease-related interventions.

随着 NAD + 增强策略在细胞和动物模型中的成功,NAD + 前体,以及主要是烟酰胺核糖苷(NR) ,现在已经成为多种临床试验的对象。迄今为止,前体被报告为安全、耐受性良好并能够提高健康志愿者的 NAD 水平(Conze 等人,2019年; Martens 等人,2018年; Minto 等人,2017年; Stea 等人,2017年)。然而,在将实验室发现转化为临床试验设计方面仍然存在挑战(Gilmour et al. 2020)。虽然一些早期的成功被发现在肌萎缩性嵴髓侧索硬化症的疾病结果(NCT03489200)(de la Rubia 等人,2019年) ,其他人发现在阿尔茨海默病患者(NCT00580931)(Phelan 等人,2017年) ,或在男性代谢紊乱或线粒体生物能量学的研究(NCT02303483)(erup 等人,2018年,2019年 a,2019年 b)。虽然只有有限数量的试验测试 NAM,NMN 和 NR 最近已经完成(> 10)或目前正在进行(- 3) ,许多正在积极招募(- 21)(Lautrup 等人2019年)和它将是有趣的,看看什么教训可以学习前体剂量,NAD +/NADH 检测方法和生物利用度在未来几年。考虑到 NAD 耗竭在许多新陈代谢和神经退行性疾病中的病理作用尚未确定,人们迫切期待相关疾病结果的报告,因为它们将告知将实验室的成功转化为与人类年龄和疾病有关的干预措施的可行性。

Concluding Remarks

结语

Autophagy is a highly conserved catabolic process that is controlled by multiple nutritional and stress-related cues by reversible protein PTMs. In this review, we first explored the latest findings on how two PTMs, Lys acetylation and Cys oxidation, regulate the localization and function of autophagy proteins. Collectively, novel findings published in 2015–2020 identify TFEB, ULK1, VPS34, ATG3, LC3 and p62 as targets of acetylation PTMs which, in response to metabolic cues, stimulate the expression and enzymatic activity of autophagy proteins and improve pathway selectivity. Furthermore, Mit/TFE family of TFs (including TFEB), ATG3, ATG7 and p62 are also known to contain redox-sensitive Cys residues the oxidation of which influences autophagy outcomes. The dual control of protein localization/enzymatic activity by acetylation and oxidation links the efficiency of autophagy outcomes to nutrient loss and metabolic dysfunction and thus contributes to cellular homeostasis and healthy ageing.

自噬是一个高度保守的分解代谢过程,受多种营养和应激相关线索的可逆蛋白质 PTMs 控制。本文首先综述了赖氨酸乙酰化和赖氨酸氧化两种 PTMs 对自噬蛋白定位和功能调控的最新研究进展。总的来说,2015-2020年发表的新发现确定 TFEB,ULK1,VPS34,ATG3,LC3和 p62作为乙酰化 PTMs 的目标,在代谢的线索,刺激表达和自噬蛋白的酶活性,并改善通路的选择性。此外,Mit/TFE 家族的 TFs (包括 TFEB)、 ATG3、 ATG7和 p62也含有对氧化还原敏感的 Cys 残基,其氧化影响自吞噬结果。通过乙酰化和氧化双重控制蛋白质定位/酶活性,将自噬结果的效率与营养损失和代谢功能障碍联系起来,从而有助于细胞内稳态和健康老化。

Crucially, studies of the molecular mechanisms of NAD function in cellular physiology and ageing suggest a central role of autophagy in first, preventing increases in DNA damage and NAD+ consumption via mitochondrial recycling and second, by alleviating nutritional crisis via recycling amino acids, lipids and nucleosides. Autophagy thus appears to be necessary in supporting cellular survival upon either nutritional stress that changes NAD redox ratio towards the oxidised form (NAD+) and stimulates SIRTs, or upon DNA damage followed by NAD+ depletion due to PARP1 hyperactivation. Thus, although short term insults to cellular heath are resolved by autophagy stimulation and cellular detoxification, we wonder whether persistent oxidation and NAD+ loss in aged tissues result in stalled autophagy, and due to lack of stress resolution, ultimately in loss of cell viability and tissue dysfunction.

关键的是,对 NAD 在细胞生理和衰老中作用的分子机制的研究表明,自噬首先起着核心作用,通过线粒体循环防止 DNA 损伤和 NAD + 消耗的增加,其次通过氨基酸、脂类和核苷的循环缓解营养危机。因此,自噬在支持细胞存活方面似乎是必要的,无论是在营养应激改变 NAD 的氧化还原比率(NAD +)并刺激 SIRTs,还是在 DNA 损伤后由于 PARP1过度激活而 NAD + 耗尽。因此,尽管对细胞健康的短期损害可以通过自噬刺激和细胞解毒来解决,我们想知道是否在老化组织中持续的氧化和 NAD + 丢失会导致停滞的自噬,并且由于缺乏应力解决,最终导致细胞活力的丧失和组织功能障碍。

References

The crosstalk of NAD, ROS and autophagy in cellular health and ageing

NAD、 ROS 和自噬在细胞健康和衰老中的相互作用

Abstract

摘要

Cellular adaptation to various types of stress requires a complex network of steps that altogether lead to reconstitution of redox balance, degradation of damaged macromolecules and restoration of cellular metabolism. Advances in our understanding of the interplay between cellular signalling and signal translation paint a complex picture of multi-layered paths of regulation. In this review we explore the link between cellular adaptation to metabolic and oxidative stresses by activation of autophagy, a crucial cellular catabolic pathway. Metabolic stress can lead to changes in the redox state of nicotinamide adenine dinucleotide (NAD), a co-factor in a variety of enzymatic reactions and thus trigger autophagy that acts to sequester intracellular components for recycling to support cellular growth. Likewise, autophagy is activated by oxidative stress to selectively recycle damaged macromolecules and organelles and thus maintain cellular viability. Multiple proteins that help regulate or execute autophagy are targets of post-translational modifications (PTMs) that have an effect on their localization, binding affinity or enzymatic activity. These PTMs include acetylation, a reversible enzymatic modification of a protein’s lysine residues, and oxidation, a set of reversible and irreversible modifications by free radicals. Here we highlight the latest findings and outstanding questions on the interplay of autophagy with metabolic stress, presenting as changes in NAD levels, and oxidative stress, with a focus on autophagy proteins that are regulated by both, oxidation and acetylation. We further explore the relevance of this multi-layered signalling to healthy human ageing and their potential role in human disease.

细胞对各种压力的适应需要复杂的步骤网络,这些步骤共同导致氧化还原平衡的重建、受损大分子的降解和细胞新陈代谢的恢复。在我们对细胞信号和信号转换之间相互作用的理解上的进展,描绘了一幅多层次调节路径的复杂图景。在这篇综述中,我们探讨了细胞适应代谢和氧化应激激活自噬之间的联系,一个重要的细胞分解代谢途径。新陈代谢的压力可以导致烟酰胺腺嘌呤二核苷酸的氧化还原状态的改变,这是一种在各种酶反应中的共同因子,因此触发自噬作用,将细胞内的成分固定起来,以支持细胞的生长。同样地,自噬被氧化应激激活,以选择性地回收受损的大分子和细胞器,从而维持细胞的活力。有助于调节或执行自噬的多种蛋白质是翻译后修饰(PTMs)的目标,这些翻译后修饰对它们的定位、结合亲和力或酶活性有影响。这些 PTMs 包括乙酰化,一种蛋白质赖氨酸残基的可逆的酶修饰,和氧化,一系列可逆的和不可逆的自由基修饰。在这里,我们强调了自噬与代谢应激相互作用的最新发现和突出问题,表现为 NAD 水平的变化,以及氧化应激,重点是由氧化和乙酰化共同调节的自噬蛋白。我们进一步探讨这种多层次的信号与健康人类老龄化的相关性及其在人类疾病中的潜在作用。

Introduction

引言

NAD depletion, oxidative stress and loss of macroautophagy (from herein referred to as autophagy) efficiency have all been linked to healthy, pathological and premature ageing (Kubben and Misteli 2017; López-Otín et al. 20132016). Individually, these alterations may underlie seven of the nine outlined hallmarks of ageing including genomic instability (all), telomere attrition (oxidative stress), epigenetic alterations (NAD), loss of proteostasis (autophagy), de-regulated nutrient sensing (NAD), cellular senescence (all) and mitochondrial dysfunction (all) (López-Otín et al. 20132016). Moreover, it is becoming increasingly clear that a significant degree of crossover and interdependence between the three phenomena occur in ageing cells and tissues. Specifically, increased reactive oxygen species (ROS) and depletion of NAD can impact autophagy by influencing post-translational modifications (PTMs) of autophagy proteins (Filomeni et al. 2015; Sedlackova et al. 2020; Zhang et al. 2016a). Furthermore, autophagy impairment may lead to the failure to reconstitute cellular metabolism and detoxify oxidised substrates (Li et al. 2015; Morishita and Mizushima 2019).

NAD 损耗、氧化应激和巨噬细胞自噬效率的丧失都与健康、病理和早衰有关(Kubben 和 Misteli,2017; López-Otín 等人,2013,2016)。单独来看,这些改变可能是老化的9个标志中的7个标志的基础,包括基因组不稳定(全部) ,端粒磨损(氧化应激) ,表观遗传改变(NAD) ,蛋白质平衡(自噬)的丢失,去调节营养传感(NAD) ,细胞衰老(全部)和线粒体功能障碍(全部)。此外,日益明显的是,这三种现象在衰老的细胞和组织中发生了很大程度的交叉和相互依存。具体来说,活性氧类的增加和 NAD 的缺失可以通过影响自噬蛋白的翻译后修饰(PTMs)来影响自噬。此外,自噬损伤可能导致细胞代谢重组和解毒氧化底物的失败(Li 等人,2015; 森下和水岛,2019)。

Nicotinamide adenine dinucleotide (NAD)

烟酰胺腺嘌呤二核苷酸

NAD is an essential metabolite that participates in cellular energy generation and signalling. When plentiful, the redox balance and availability of NAD aid cellular adaptation to metabolic stress and help maintain genomic stability, mitochondrial function, detoxification of ROS and cell survival (Fang et al. 2017). Due to its ability to accept or donate electrons, NAD in its reduced (NADH) or oxidised (NAD+) form assists energy metabolism in the cytosol and within mitochondria (Canto et al. 2015). In addition, NAD+ is cleaved into ADP-ribose (ADPR) and nicotinamide (NAM) by three classes of enzymes: sirtuins (SIRTs), poly(ADPR) polymerases (PARPs) and cyclic ADPR synthases (CD38 and CD157) (Fig. 1), which require ADPR for their enzymatic activity (Canto et al. 2015; Fang et al. 2017). Crucially, although SIRT activity depends on NAD+ availability and cannot contribute to uncontrolled NAD+ cleavage, PARPs and CD38 are known for their indiscriminate NAD+ consumption and their role in age- and disease-related NAD depletion (Canto et al. 2015). Homeostasis of intracellular NAD pools is maintained by either local synthesis from NAD+ precursors (nicotinamide (NAM), nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN)) or centralised de novo synthesis from nicotinic acid or L-tryptophan (Canto et al. 2015). Therefore, it is the balance between NAD+ cleavage and synthesis that dictates the total intracellular NAD pool, and by extension, cellular metabolism and protein acetylation status (Strømland et al. 2019).

NAD 是一种重要的代谢产物,参与细胞能量的产生和信号传递。在充足时,NAD 的氧化还原平衡和可用性有助于细胞适应代谢压力,并有助于维持基因组稳定性、线粒体功能、活性氧解毒和细胞存活(Fang 等人,2017年)。由于它能够接受或捐赠电子,NAD 的还原形式(NADH)或氧化形式(NAD +)有助于细胞质和线粒体内的能量代谢(Canto 等人,2015年)。此外,NAD + 被三类酶切割成 adp- 核糖(ADPR)和烟酰胺(NAM) : sirtuins (SIRTs) ,poly (ADPR)聚合酶(PARPs)和环状 ADPR 合酶(CD38和 CD157)(图1) ,这些酶的酶活性需要 ADPR (Canto 等人,2015; Fang 等人,2017)。至关重要的是,虽然 SIRT 的活性取决于 NAD + 的可获得性,并且不能导致 NAD + 无控制的分裂,但是 PARPs 和 CD38因其不分青红皂白的 NAD + 消费及其在与年龄和疾病相关的 NAD 损耗中的作用而为人所知(Canto 等人,2015年)。细胞内 NAD 池的稳态可以通过以下两种途径来维持: 从 NAD + 前体(尼古丁酰胺(NAM)、尼古丁酰胺核苷(NR)或尼古丁酰胺单核苷酸(NMN))局部合成,或从尼古丁酸或 l- 色氨酸集中合成从头合成(Canto et al. 2015)。因此,正是 NAD + 裂解和合成之间的平衡决定了细胞内 NAD 池的总量,进而决定了细胞代谢和蛋白质乙酰化状态(Strømland 等人,2019年)。

figure1
Fig. 1 图一

Reactive oxygen species (ROS)

活性氧类

ROS are highly reactive molecules of oxygen, which harbour one unpaired electron (superoxide anion (O2•−), hydroxyl radical (OH)) or an additional electron pair (H2O2) on its valence orbital (Halliwell and Gutteridge 2015). The increased electron content in oxygen molecules makes them more reactive and more likely to participate in one-electron oxidative transfer reactions that lead to macromolecule modification and/or damage (Halliwell and Gutteridge 2015). ROS can also interact with nitric oxide to generate reactive nitrogen species (RNS), including peroxynitrite (ONOO) (Bartesaghi and Radi 2018). Electrons from ROS/RNS interact with amino acid residues incorporated in proteins and thus translate the cellular redox state into protein activating or inhibiting signals by the modulation of protein enzymatic activity, binding affinity or structural conformation. Particularly sensitive to ROS-mediated redox regulation are cysteine (Cys) residues (Bischoff and Schlüter 2012). Cys is one of the least represented, and yet often highly conserved amino acids that participates in protein structural integrity by formation of covalent disulphide-bridges between two cysteine residues, or protein enzymatic activity, i.e. by thioester bond formation or co-factor stabilisation (Bak et al. 2019; Marino and Gladyshev 2010).

活性氧是高活性氧分子,在其价轨道上含有一个不成对电子(超氧阴离子(O2• -)、羟基自由基(OH •)或一个额外的电子对(H2O2)。氧分子中电子含量的增加使它们更活泼,更有可能参与单电子氧化转移反应,从而导致高分子修饰和/或损伤(哈利维尔和古特里奇,2015年)。ROS 也可以与一氧化氮相互作用产生活性氮,包括过氧亚硝基阴离子(ONOO -)(Bartesaghi 和 Radi 2018)。ROS/RNS 的电子与蛋白质中的氨基酸残基相互作用,通过调节蛋白质的酶活性、结合亲和力或结构构象,将细胞的氧化还原状态转化为蛋白质激活或抑制信号。对 ros 介导的氧化还原调节特别敏感的是半胱氨酸(Cys)残基(Bischoff 和 Schlüter 2012)。半胱氨酸是最少代表的,但是通常是高度保守的氨基酸,通过在两个半胱氨酸残基之间形成共价二硫键或蛋白质酶活性,即通过硫酯键形成或共因子稳定作用,参与蛋白质结构的完整性(Bak 等人,2019; Marino 和 gladys,2010)。

Autophagy

自噬作用

Autophagy is a cytosolic pathway of dynamic membrane rearrangement and cargo sequestration that is assisted and executed by a set of highly conserved autophagy (ATG) proteins (Dikic and Elazar 2018). Autophagy is a catabolic process responsible for cargo recognition, its engulfment in a double membraned vesicle called autophagosome and delivery to the lysosomal lumen for degradation. The subsequent release of amino acids, lipids and nucleosides reconstitutes cellular homeostasis and sustains viability in times of stress (Morishita and Mizushima 2019). The molecular execution of autophagy initiation is mediated by ATG protein association into functional complexes known as the Unc-51-like kinase 1 (ULK1) complex, the class III phosphatidylinositol 3 kinase (PI(3)K) complex, the ATG9-membrane complex, an ATG2–WIPI (WD-repeat protein interacting with phosphoinositides) complex and two conjugation systems consisting of the ATG3-ATG8/LC3 and the ATG5-ATG12:ATG16L complex (Table 1) (Suzuki et al. 2017). The combined action of these complexes is responsible for ER localization of all autophagy components and for the formation and maturation of the autophagic membrane. In addition, a group of autophagy receptors, e.g. sequestosome 1 (SQSTM1/p62), is then responsible for spatially linking the ubiquitylated cargo, including long-lived or aggregated proteins, pathogens and organelles, to the growing autophagosome (Dikic and Elazar 2018; Johansen and Lamark 2019).

自噬是一种动态膜重排和货物隔离的胞浆通路,由一组高度保守的自噬蛋白(ATG)辅助和执行(Dikic 和 Elazar 2018)。自噬是一个分解代谢过程,负责货物识别,它吞噬在一个叫做自噬小体的双膜泡中,并传递到溶酶体腔进行降解。随后释放的氨基酸,脂类和核苷重新构成细胞内稳态和维持活力时的压力(森下和水岛2019年)。自噬启动的分子执行是通过 ATG 蛋白的结合介导的功能复合物称为 Unc-51-like kinase 1(ULK1)复合物,第 III 类磷脂酰肌醇激酶3(PI (3) k)复合物,atg9-膜复合物,ATG2-WIPI (WD-repeat 蛋白与磷酸肌醇相互作用)复合物和两个连接系统组成的 ATG3-ATG8/LC3和 ATG5-ATG12: ATG16L 复合物(表1)(Suzuki et al. 2017)。这些复合物的联合作用负责所有自噬组分的 ER 定位和自噬膜的形成和成熟。此外,一组自噬受体,例如序列体1(SQSTM1/p62) ,负责在空间上将无处不在的化合物(包括长寿命或聚集的蛋白质、病原体和细胞器)与正在生长的自噬体联系起来(Dikic 和 Elazar 2018; Johansen 和 Lamark 2019)。Table 1 Acetylation-sensitive proteins in autophagy 表1自噬中乙酰化敏感蛋白Full size table 全尺寸表

The canonical pathway of starvation-induced autophagy was long thought to rely on phosphorylation cascades that are triggered by the loss of nutrient signalling and converge on a small number of regulating kinase complexes (Beurel et al. 2015; Rabanal-Ruiz et al. 2017; Tamargo-Gómez and Mariño 2018). These regulators then either lose function and thus release downstream autophagy components from an inhibitory state, or become activated and promote autophagy initiation. In addition, multiple layers of regulation involved in autophagy initiation, cargo sequestration and degradation, incorporate various stress signals and often improve the efficiency of autophagic flux via PTMs of autophagy proteins or their upstream regulators (Filomeni et al. 2015; Montagna et al. 2016; Sedlackova et al. 2020; Zhang et al. 2016a).

长期以来,人们一直认为饥饿诱导的自噬的典型途径依赖于磷酸化级联,这种级联由营养信号的丢失触发,并在少数调节激酶复合物上汇聚(Beurel 等人,2015年; Rabanal-Ruiz 等人,2017年; Tamargo-Gómez 和 Mariño 2018年)。这些调节因子要么失去功能,从而从抑制状态释放下游自噬组分,要么被激活,促进自噬启动。此外,涉及自噬启动、货物固存和降解的多层次调节,包含各种应激信号,通过自噬蛋白的 PTMs 或其上游调节因子常常提高自噬通量的效率(Filomeni 等人,2015年; Montagna 等人,2016年; Sedlackova 等人,2020年; Zhang 等人,2016a 年)。

In this review, we explore the current knowledge of how two types of PTMs, lysine (Lys) acetylation and cysteine (Cys) oxidation, regulate the abundance and activity of ATG proteins, and highlight which Lys modifications are subject to NAD+ availability. We then summarize the main concepts of autophagy regulation by oxidative stress and discuss the implications and consequences of age-related changes to NAD+ availability and an increase in oxidative stress on the efficiency of autophagy. We further explore whether autophagy directly influences the homeostasis of cellular NAD levels and outline how aberrations in either of the three phenomena could lead to dysfunction observed in physiological and pathological ageing.

本文综述了赖氨酸(Lys)乙酰化和半胱氨酸(Cys)氧化两种类型的 PTMs 如何调节 ATG 蛋白的丰度和活性,并着重介绍了哪些 Lys 修饰受 NAD + 的影响。然后,我们总结了氧化应激的自噬调节的主要概念,并讨论了年龄相关变化对 NAD + 可用性的影响和后果,以及提高氧化应激对自噬效率的影响。我们进一步探讨了自噬是否直接影响细胞 NAD 水平的稳态,并概述了这三种现象中的任何一种畸变是如何导致生理和病理衰老中观察到的功能障碍的。

Targets of acetylation in autophagy

自噬中的乙酰化作用靶点

Lysine acetylation is a major reversible PTM in eukaryotes that arises by donation of the acetyl moiety from acetyl coenzyme A (Ac-CoA) via its re-direction from mitochondrial energy generation (Drazic et al. 2016). Protein acetylation status is balanced by the activity of multiple lysine acetyl transferases (KATs, historically known as histone acetyl transferases HATs) and lysine deacetylases (KDAC, or HDACs) (Narita et al. 2019). KATs catalyse acetyl moiety transfer from Ac–CoA to a lysine residue of the target protein, while KDACs cleave and release the acetyl moiety (KDAC, classes I, II and IV) or catalyse transfer of the acetyl moiety onto ADPR, a product of NAD+ cleavage (class III KDACs, sirtuins (SIRTs) (Fig. 1). Acetylation status of autophagy proteins is largely controlled by p300 (KAT3B) and 60 kDa Tat-interactive protein (TIP60/KAT5) KATs and SIRT1-3 and HDAC2/6 KDACs (summarized in Table 1). In the next section, we explore how protein acetylation status, generally high in conditions of nutrient abundance and low under nutrient starvation, regulates the activity and localisation of TFs, proteins and receptors involved in autophagy.

赖氨酸乙酰化是真核生物中一种主要的可逆 PTM,通过线粒体产生的能量重新导向乙酰辅酶A 乙酰基部分(Ac-CoA)而产生。蛋白质的乙酰化状态由多个赖氨酸乙酰转移酶(KATs,历史上称为组蛋白乙酰转移酶 HATs)和赖氨酸脱乙酰酶(KDAC,或 HDACs)的活性来平衡(成田等人,2019年)。KATs 催化乙酰基从 Ac-CoA 转移到目标蛋白的赖氨酸残基,而 KDAC 裂解并释放乙酰基(KDAC,i 类,II 类和 IV 类)或催化乙酰基转移到 NAD + 裂解产物 ADPR (III 类 KDAC,SIRTs)上(图1)。自噬蛋白的乙酰化状态主要受 p300(KAT3B)和60kda tat 相互作用蛋白(TIP60/KAT5) KATs、 SIRT1-3和 hdac2/6kdac 控制(见表1)。在下一节,我们将探讨在营养丰富和营养缺乏的情况下,蛋白质的乙酰化状态如何调节自噬相关的转录因子、蛋白质和受体的活性和定位。

Regulation of transcription factors involved in autophagy gene transcription

自噬基因转录相关转录因子的调控

The loss of lysine acetylation triggers stimulation of several TFs involved in the transcription of ATG genes (Fig. 2a) (Füllgrabe et al. 2016). The strongest link between TF deacetylation and autophagy stimulation comes from studies of transcription factor EB (TFEB), a member of the microphthalmia family of bHLH-LZ transcription factors (Mit/TFE), a group of TFs that stimulate lysosomal biogenesis and expression of autophagy proteins (Yang et al. 2018). Specifically, TFEB is responsible for transcription of multiple autophagy genes (ATG4, ATG9B, MAP1LC3B (LC3B), UVRAG (UV radiation resistance associated gene), WIPI (WD repeat domain phosphoinositide-interacting protein 1), and SQSTM1 (p62)) (Füllgrabe et al. 2016; Settembre et al. 2011). Acetylation of a conserved lysine residue Lys116 was independently identified in three studies as a modifier of TFEB activity in microglia (Bao et al. 2016) and in cancer cells (Wang et al. 2019b; Zhang et al. 2018). In microglia, Lys116 was directly deacetylated by SIRT1 which promoted degradation of fibrillar amyloid β (Bao et al. 2016). In cultured cells, treatment with a KDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), increased the transcriptional activity of TFEB and influenced acetylation of four lysine residues (Lys91, Lys103, Lys116 and Lys430) (Zhang et al. 2018). In addition, authors of this study identified acetyl-coenzyme A acetyltransferase 1 (ACAT1) and HDAC2 as modulators of the overall TFEB acetylation status. Furthermore, a study in a model of chronic kidney disease identified HDAC6 as another KDAC involved in the regulation of TFEB activity (Brijmohan et al. 2018). Importantly, authors of neither of the studies demonstrated a direct interaction between TFEB and HDAC2 or HDAC6, respectively (Brijmohan et al. 2018; Zhang et al. 2018). Overexpression of another KAT, the general control non-repressed protein 5 (GCN5/KAT2A), but not TIP60, p300 or CREB-binding protein (CBP), led to increased TFEB acetylation of Lys116, Lys274 and Lys279 residues (Wang et al. 2019b). Authors further demonstrated that TFEB acetylation at Lys274 and Lys279 mechanistically disrupts TFEB dimerization and its ability to bind DNA, and thus negatively regulates expression of lysosomal and autophagy genes (Fig. 2A) (Wang et al. 2019b). Crucially, Lys116 of TFEB is not conserved in Drosophila melanogaster and Caenorhabditis elegans or in other members of the Mit-TFE family (Wang et al. 2019b), thus SIRT1 and HDAC regulation of TFEB activity is likely to be unique to vertebrates.

赖氨酸乙酰化缺失触发了几个参与 ATG 基因转录的转录因子(图2a)(Füllgrabe 等人,2016)。TF 脱乙酰化和自噬刺激之间最强有力的联系来自对转录因子 EB (TFEB)的研究,这种转录因子属于小眼睛 bHLH-LZ 转录因子家族(Mit/TFE) ,是一组促进溶酶体生成和自噬蛋白表达的 TFs (Yang et al. 2018)。具体来说,TFEB 负责多种自噬基因的转录(ATG4,ATG9B,MAP1LC3B (LC3B) ,UVRAG (UV 辐射抗性相关基因) ,WIPI (WD 重复结构域磷酸肌醇-相互作用蛋白1) ,和 SQSTM1(p62))(Füllgrabe 等人,Settembre 等人,2011)。保守赖氨酸残基 Lys116的乙酰化在三项研究中被独立鉴定为小胶质细胞(Bao 等人,2016年)和癌细胞(Wang 等人,2019b; Zhang 等人,2018年)中 TFEB 活性的修饰剂。在小胶质细胞中,Lys116被 SIRT1直接去乙酰化,促进了淀粉样蛋白的降解(Bao 等人,2016)。在培养的细胞中,使用 KDAC 抑制剂伏立诺他(SAHA)增加 TFEB 的转录活性并影响4个赖氨酸残基(Lys91,Lys103,Lys116和 Lys430)的乙酰化。此外,本研究作者确定乙酰辅酶 a 乙酰转移酶1(ACAT1)和 HDAC2作为调节整个 tmb 乙酰化状态。此外,在一个慢性肾脏疾病模型中的研究确定 HDAC6是另一个参与调节 TFEB 活性的 KDAC (Brijmohan 等人,2018)。重要的是,这两项研究的作者都没有证明 TFEB 和 HDAC2或 HDAC6之间存在直接的相互作用。另一个 KAT 蛋白,一般对照的非抑制蛋白5(GCN5/KAT2A)的过表达,而 TIP60、 p300或 creb 结合蛋白(CBP)的过表达,导致 Lys116、 Lys274和 Lys279残基 TFEB 的增加(Wang 等,2019b)。作者进一步证明,Lys274和 Lys279的 tbb 乙酰化作用可以机械地干扰 tmb 的二聚化及其与 DNA 的结合能力,从而对溶酶体和自噬基因的表达产生负性调节(图2A)(Wang 等人,2019b)。至关重要的是,TFEB 的 Lys116在黑腹果蝇和秀丽隐桿线虫或 Mit-TFE 家族的其他成员中并不保守,因此 SIRT1和 HDAC 对 TFEB 活性的调节可能是脊椎动物所独有的。

figure2
Fig. 2 图二

Additionally, two members of the forkhead box class O (FoxO) TF family, FoxO1 and FoxO3a, recognized for their role in autophagy/mitophagy (ATG4, ATG5, ATG12, ATG14, BECN1(beclin 1), BNIP3 (BCL2 interacting protein 3), LC3B, ULK1, VPS34 (vacuolar protein sorting 34) GABARAPL1 (gamma-aminobutyric acid receptor-associated protein-like1), and PARK6/PINK1 (PTEN-induced kinase 1)) gene transcription, are regulated by acetylation PTMs (Fang et al. 2019; Füllgrabe et al. 2016; Requejo-Aguilar et al. 2015). It was first demonstrated that FoxO1 acetylation on Lys242, Lys245 and Lys262 residues (in mice) by CBP is opposed by SIRT1 in response to serum (Daitoku et al. 2004) and glucose starvation (Hariharan et al. 2010). Mechanistically, acetylation of the three Lys residues within FoxO1 interferes with its DNA binding and inhibits its transcriptional activity (Matsuzaki et al. 2005). Furthermore, FoxO1 acetylation permits access for upstream kinases to phosphorylate its Ser253 residue that is otherwise shielded by FoxO1-DNA complex formation (Matsuzaki et al. 2005). FoxO1 phosphorylation sites have since became known to act as docking or shielding sites for 14–3-3 protein binding, and the heterodimer exit into and retention within the cytoplasm (Brunet et al. 1999; Saline et al. 2019).

此外,两个成员的叉头盒类 o (FoxO) TF 家族,FoxO1和 FoxO3a,认为其作用于自噬/吞噬(ATG4,ATG5,ATG12,ATG14,BECN1(beclin 1) ,BNIP3(BCL2相互作用蛋白3)(BCL2相互作用蛋白3) ,lC3B、 ULK1、 VPS34(液泡蛋白分类34) GABARAPL1(γ-氨基丁酸受体相关蛋白1)和 PARK6/PINK1(pten 诱导的激酶1)基因转录受乙酰化 PTMs 调控(Fang 等人,2019; Füllgrabe 等人,2016; Requejo-Aguilar 等人,2015)。首次证明 CBP 在小鼠体内对 Lys242、 Lys245和 Lys262残基的 FoxO1乙酰化反应受到血清(Daitoku 等人,2004年)和葡萄糖饥饿的影响(Hariharan 等人,2010年)。机制上,FoxO1中三个赖氨酸残基的乙酰化干扰其 DNA 结合并抑制其转录活性(松崎等人,2005)。此外,FoxO1乙酰化允许上游激酶磷酸化其被 FoxO1-dna 复合体屏蔽的 Ser253残基(Matsuzaki 等人,2005年)。FoxO1磷酸化位点后来被认为是14-3-3蛋白结合的对接或屏蔽位点,异二聚体进入并保留在细胞质中(Brunet al. 1999; Saline et al. 2019)。

Similarly to FoxO1, the transcriptional activity of FoxO3 is modulated by SIRT1-3 deacetylases, though the Lys residues susceptible to acetylation remain unknown. First, caloric restriction and oxidative stress increase SIRT2 expression, decrease FoxO3 acetylation and improve gene transcription (Wang et al. 2007). In mitochondria, SIRT3 mediated deacetylation of the mitochondrial FoxO3 pool and led to cellular detoxification of oxidative stress by increased expression of the mitochondrial superoxide dismutase (Jacobs et al. 2008). However, some controversy exists in the perceived outcome of FoxO acetylation. A few articles published in early 2000s reported entirely opposite findings, demonstrating that FoxO acetylation improves transcription of its target genes (Motta et al. 2004; Yang et al. 2005). The pitfall of the majority of FoxO studies centres on the lack of distinction between CBP-mediated acetylation of FoxO, which may increase its DNA binding, and acetylation of histones, which would relax the chromatin condensation and promote gene transcription (discussed in detail in (Daitoku et al. 2011)).

与 FoxO1相似,FoxO3的转录活性也受到 SIRT1-3去乙酰化酶的调控,但赖氨酸残基对乙酰化的敏感性尚不清楚。首先,限制热量摄入和氧化应激可以增加 SIRT2的表达,降低 FoxO3乙酰化水平,提高基因转录水平。在线粒体中,SIRT3介导了线粒体 FoxO3库的去乙酰化,并通过增加线粒体超氧化物歧化酶蛋白的表达导致细胞解毒(Jacobs et al. 2008)。然而,对于 FoxO 乙酰化的感知结果还存在一些争议。2000年代初发表的一些文章报道了完全相反的发现,表明 FoxO 乙酰化改善了其靶基因的转录(Motta 等,2004; Yang 等,2005)。大多数 FoxO 研究的缺陷在于缺乏区分 cbp 介导的 FoxO 的乙酰化和组蛋白的乙酰化,前者可能增加其 DNA 结合,后者可能放松染色质凝聚和促进基因转录(详见 Daitoku 等人2011年)。

Overall, transcriptional activity of TFEB, a member of the Mit/TFE family, and two FoxO isoforms, FoxO1 and FoxO3, is regulated by acetylation. Three classes of KDACs, SIRT1, HDAC2/6 and GCN5, are thought to deacetylate multiple and variable Lys residues in TFEB, of which the SIRT1 target, Lys116, is unique to vertebrates, and Lys274 and Lys279 deacetylation regulates TFEB-DNA complex formation (Wang et al. 2019b). In the FoxO family, FoxO1 is the better studied isoform, with known target Lys residues in the mouse (Table 1), though both FoxO1 and FoxO3a are likely activated by SIRT-mediated deacetylation. Thus, transcriptional regulation of autophagy/mitophagy genes by TFEB and FoxO1/3a is, at least in part, responsive to intracellular NAD+ levels that influence SIRT activity.

总的来说,Mit/TFE 家族成员 TFEB 和 FoxO1和 FoxO3两种同源异构体的转录活性受乙酰化调控。目前认为 TFEB 中有三类 kdac,SIRT1、 HDAC2/6和 GCN5,其中 SIRT1的作用靶点 Lys116是脊椎动物特有的,Lys274和 Lys279脱乙酰基调节 TFEB-dna 复合物的形成(Wang 等人,2019b)。在 FoxO 家族中,FoxO1是更好的研究异构体,在小鼠中有已知的目标赖氨酸残基(表1) ,尽管 FoxO1和 FoxO3a 都可能被 sirt 介导的脱乙酰化活化。因此,tbb 和 FoxO1/3a 的自噬/噬转录调控基因至少部分地对影响 SIRT 活性的细胞内 NAD + 水平有反应。

Regulation of autophagy protein complexes

自噬蛋白复合物的调节

Several autophagy proteins involved in autophagosome formation, growth and maturation may be modified by acetylation (Table 1). Studies from the last decade, summarized below, identify lysine residues sensitive to acetylation in members of the ULK1 kinase complex, the class III PI(3)K kinase complex and the two conjugation systems, ATG12 (ATG7, ATG10, ATG5) and LC3 (ATG4, ATG7, ATG3), as well as ATG12 and LC3 themselves (Fig. 2B).

一些参与自噬体形成、生长和成熟的自噬蛋白可能通过乙酰化修饰(表1)。近十年来的研究表明,ULK1激酶复合物、 III 类 PI (3) k 激酶复合物和两个接合系统 ATG12(ATG7、 ATG10、 ATG5)和 LC3(ATG4、 ATG7、 ATG3)中的赖氨酸残基对乙酰化敏感,以及 ATG12和 LC3本身(图2B)。

In the ULK1 complex, ULK1 itself is a target of acetylation by TIP60 (Lin et al. 2012). In serum starved cells, ULK1 was shown to be a target of GSK3-dependent and TIP60-mediated acetylation of two crucial residues, Lys162 and Lys606 (in mouse; likely Lys162 and Lys607 in human), that together stimulate its kinase activity and promote autophagy initiation (Lin et al. 2012). Furthermore, oxidative stress that induces ER stress, was also shown to stimulate ULK1 acetylation by a GSK3-TIP60-dependent mechanism (Nie et al. 2016). These studies together support the idea that ULK1 kinase activity can be modulated by oxidative and metabolic stress via an upstream signalling cascade that results in TIP60 activation and ULK1 acetylation.

在 ULK1复合物中,ULK1本身就是 TIP60的乙酰化靶点(Lin 等人,2012)。在血清饥饿的细胞中,ULK1被证明是 gsk3依赖和 tip60介导的两个关键残基 Lys162和 Lys606的乙酰化靶点(在小鼠中; 在人类中可能是 Lys162和 Lys607) ,这两个残基共同刺激其激酶活性并促进自噬启动(Lin 等人,2012)。此外,引起内质网应力的氧化应激,也表明通过 gsk3-tip60依赖机制刺激 ULK1乙酰化。这些研究共同支持的想法,ULK1激酶的活性可以调节氧化和代谢应激通过上游信号级联,导致 TIP60活化和 ULK1乙酰化。

Within the Class III PI(3)K complex, VPS34 kinase acetylation by p300 occurs on residues Lys29, Lys771 and Lys781 and inhibits its lipid kinase activity and PI(3)P production (Su et al. 2017). It was further determined that acetylation of Lys29 residue prevents VPS34 association with Beclin 1 that is required for the formation of a complex involved in autophagy progression. Another layer of VPS34 activity regulation occurs upon acetylation of the Lys771residue located within its catalytic site. In a manner similar to the level of regulation at the Lys29 residue, acetylation of Lys771 disrupts binding between VPS34 and its substrate, PI (Su et al. 2017). However, the KDAC responsible for Lys29 and Lys771 deacetylation remains unknown. In addition to VPS34, Beclin 1 of the Class III PI(3)K complex is also a target of inhibitory acetylation on Lys430 and Lys437 residues by p300 (Sun et al. 2015). Beclin 1 acetylation was demonstrated to promote its binding to Rubicon, and thus shown to result in the loss of autophagosome maturation (Ohashi et al. 2019; Sun et al. 2015). Furthermore, in vitro acetylation analysis revealed that SIRT1 is preferentially responsible for Beclin 1 deacetylation (Sun et al. 2015).

在 III 类 PI (3) k 复合物中,VPS34激酶的 p300乙酰化发生在 Lys29、 Lys771和 Lys781的残基上,并抑制其脂质激酶活性和 PI (3) p 的产生(Su 等人,2017年)。进一步确定,Lys29残基的乙酰化可以阻止 VPS34与 Beclin 1的结合,而后者是形成一个参与自噬进程的复合物所必需的。另一层 VPS34的活性调控发生在 Lys771残基的乙酰化过程中,该残基位于催化位点。与 Lys29残基的调控水平相似,Lys771的乙酰化破坏了 VPS34与其底物之间的结合,PI (Su 等人,2017)。然而,负责 Lys29和 Lys771脱乙酰基的 KDAC 仍然是未知的。除 VPS34外,p300还将 III 类 PI (3) k 复合物的 Beclin 1作为抑制 Lys430和 Lys437残基乙酰化的靶标(Sun 等人,2015年)。研究表明,Beclin 1乙酰化可促进其与 Rubicon 的结合,从而导致自噬体成熟的丧失(Ohashi 等人,2019; Sun 等人,2015)。此外,体外乙酰化分析显示 SIRT1优先负责 Beclin 1脱乙酰化(Sun 等人,2015)。

Next, SIRT1-mediated deacetylation of nuclear LC3 at Lys49 and Lys51 residues initiates LC3 translocation to the cytoplasm via a diabetes and obesity regulated (DOR/TP53INP2)-dependent interaction with deacetylated LC3 (Huang et al. 2015). DOR then further assists in LC3 localization to nascent autophagosomes thanks to its ATG7-binding affinity (You et al. 2019b). Furthermore, DOR also contains a ubiquitin-interacting motif and is thus likely to promote LC3-ATG7 formation in the vicinity of ubiquitylated cargo (Xu and Wan 2019; You et al. 2019b). Upon relocation to the cytoplasm, LC3 Lys49 and Lys51 acetylation, that is lost upon nutrient starvation, was recently shown to completely abolish p62 binding (Song et al. 2019). Due to the location and conservation of the two critical lysine residues in the hydrophobic binding grooves of LC3 (Huang et al. 2015; Song et al. 2019), it stands to reason that Lys49 and Lys51 acetylation could disrupt LC3 interaction with multiple binding partners including, but not limited to DOR and p62. Altogether, LC3 deacetylation in response to nutrient starvation not only promotes its exit from the nucleus, but also determines substrate binding specificity of protein partners via their LC3-interacting regions (LIRs).

其次,sirt1介导的 Lys49和 Lys51基因残基上的 LC3去乙酰化启动了 LC3通过糖尿病和肥胖调节(DOR/TP53INP2)依赖的与去乙酰化 LC3的相互作用到细胞质的转位(Huang 等人,2015)。DOR 然后进一步协助 LC3定位到初生的自噬体,这要归功于其 atg7结合的亲和力(You 等人,2019b)。此外,DOR 还包含泛素相互作用基序,因此可能促进泛素化货物附近的 LC3-ATG7的形成(Xu and Wan 2019; You et al. 2019b)。在重新定位到细胞质后,在营养缺乏时丢失的 LC3 Lys49和 Lys51乙酰化,最近被证明完全消除了 p62结合(Song 等人,2019年)。由于两个关键赖氨酸残基在 LC3的疏水结合槽中的位置和守恒(Huang 等人,2015; Song 等人,2019) ,可以推断 Lys49和 Lys51乙酰化可能破坏 LC3与多个结合伙伴的相互作用,包括但不限于 DOR 和 p62。总之,在营养缺乏的条件下,LC3脱乙酰化不仅促进了其从细胞核中的脱出,而且通过其 LC3相互作用区域决定了蛋白质伙伴的底物结合特异性。

Cytoplasmic LC3 targeting to and docking on the nascent autophagosomes requires covalent conjugation of LC3 to phosphatidylethanolamine (PE). In a ubiquitin-like conjugation system, ATG7 (and E1-like enzyme), ATG3 (an E2-like enzyme) and an ATG5-ATG12:ATG16L complex (an E3-like enzyme) assist LC3 conjugation to PE (Dikic and Elazar 2018). Nutrient starvation in yeast was first reported to decrease or not change acetylation levels of ATG proteins, with the notable exception of ATG3, in which Lys19, Lys48 and Lys183 acetylation increased (Yi et al. 2012). Authors of this study had further shown that while acetylation of Lys183 is crucial for the enzymatic activity of ATG3, Lys19 and Lys48 acetylation was crucial for autophagy progression by improving interaction between ATG3 and ATG8 (LC3 in mammals), and was regulated by the opposing activities of the yeast histone acetyltransferase Esa1 (TIP60/KAT5 orthologue)) and a histone deacetylase Rpd3 (HDAC1/2 orthologue) enzymes. Furthermore, ATG3 acetylation on Lys19 and Lys48 was shown to enhance its ER membrane localization and binding in vitro (Li et al. 2017).

细胞质 LC3靶向并与新生的自噬体对接需要 LC3与磷脂酰乙醇胺的共价结合。在泛素样结合系统中,ATG7(和 e1样酶)、 ATG3(一种 e2样酶)和 ATG5-ATG12: ATG16L 复合物(一种 e3样酶)协助 LC3结合 PE (Dikic 和 Elazar 2018)。首次报道酵母营养缺乏可以降低或不改变 ATG 蛋白的乙酰化水平,但 ATG3除外,其中 Lys19、 Lys48和 Lys183的乙酰化水平升高(Yi 等,2012年)。作者进一步指出,虽然 Lys183的乙酰化是至关重要的 ATG3,Lys19和 Lys48乙酰化的酶活性是至关重要的自噬进展,改善 ATG3和 ATG8(LC3在哺乳动物中)之间的相互作用,并调节相反的活动酵母组蛋白乙酰基转移酶 Esa1(TIP60/KAT5的直系亲属)和组蛋白脱乙酰酶 Rpd3(HDAC1/2直系亲属)的酶。此外,在 Lys19和 Lys48上的 ATG3乙酰化被证明能增强其 ER 膜的定位和体外结合(Li 等人,2017)。

Other members of the ubiquitin-like conjugation system, ATG7, ATG5 and ATG12, are targets of p300-mediated acetylation (Lee and Finkel 2009) and SIRT1-dependent deacetylation (Lee et al. 2008). In direct contrast to ULK1 and ATG3, acetylation of these ATG proteins generally inhibits their function. However, the specific residues, their location and effect of acetylation on the structure or function of ATG proteins remains unknown. Structural studies of the ATG12-ATG5:ATG16 complex (Otomo et al. 2013) and the nature of interaction between ATG12 and ATG3 (Metlagel et al. 2013) point towards several key lysine residues that could be targets of acetylation in ATG12. First, lysine residues 60, 69, 71 and 128 located on the surface of ATG12 (Metlagel et al. 2013) could contribute to binding affinity between ATG12 (E3-like) and ATG3 (E2-like) that is required for the spatiotemporal regulation of LC3 lipidation. Furthermore, ATG5 contains multiple lysine residues, of which Lys53, Lys130, Lys171 are conserved (Matsushita et al. 2007). Although Lys130 is the known catalytic site for conjugation between ATG5 and ATG12 (Mizushima et al. 1998), the function and acetylation-sensitivity of Lys53 and Lys171 remain unknown. Lastly, no published study followed-up reports of ATG7 acetylation-sensitivity (Lee et al. 2008; Lee and Finkel 2009). However, a high resolution mass spectrometry study of global protein acetylation identified Lys306 of the human ATG7 protein as a residue that might be relevant for further study (Choudhary et al. 2009). Thus, although ATG5, ATG7 and ATG12 have been known substrates of p300 and SIRT for almost a decade, the lysine residues sensitive to acetylation, or indeed the nature of protein inhibition by acetylation have not been elucidated.

泛素样结合系统的其他成员 ATG7、 ATG5和 ATG12是 p300介导的乙酰化(Lee 和 Finkel,2009)和 sirt1依赖的去乙酰化(Lee 等人,2008)的靶点。与 ULK1和 ATG3相反,这些 ATG 蛋白的乙酰化通常会抑制它们的功能。然而,ATG 蛋白的特异性残基、乙酰化位置及其对 ATG 蛋白结构和功能的影响尚不清楚。ATG12-ATG5: ATG16复合物的结构研究(Otomo 等人,2013年)和 ATG12与 ATG3之间相互作用的性质(Metlagel 等人,2013年)表明 ATG12中几个关键的赖氨酸残基可能是其乙酰化的目标。首先,位于 ATG12表面的赖氨酸残基60、69、71和128(Metlagel 等人,2013年)可能有助于 ATG12(类 e3)和 ATG3(类 e2)之间的结合亲和力,这是 LC3脂肪化的时空调节所必需的。此外,ATG5还含有多个赖氨酸残基,其中 Lys53、 Lys130、 Lys171是保守的(Matsushita et al. 2007)。虽然 Lys130是 ATG5和 ATG12(Mizushima et al. 1998)结合的已知催化位点,但 Lys53和 Lys171的功能和乙酰化敏感性仍不清楚。最后,没有发表 ATG7乙酰化敏感性的随访报告(Lee 等人,2008年; Lee 和 Finkel,2009年)。然而,一项高分辨率的质谱法全球蛋白质乙酰化研究证实人类 ATG7蛋白的 Lys306是一个残基,可能与进一步的研究有关(Choudhary 等人,2009年)。因此,虽然 ATG5、 ATG7和 ATG12作为 p300和 SIRT 的底物已有近10年的历史,但其赖氨酸残基对乙酰化的敏感性,或者说对蛋白质的乙酰化抑制作用的本质尚未阐明。

Selective cargo recognition

选择性货物识别

Autophagy receptors modulate the selectivity and specificity of cargo recognition in the autophagy pathway. Although the current knowledge of about PTMs that affect the structure, function and localisation of the canonical autophagy receptors is fairly limited, phosphorylation and ubiquitylation sites were identified in all canonical receptors (THANATOS, https://thanatos.biocuckoo.org) (Deng et al. 2018). The best characterization of acetylation-dependent regulation of autophagy receptors concerns the p62 protein and its affinity for ubiquitin (Fig. 2c). Binding between ubiquitin and p62 to spatially link cargo to the forming autophagosome is, in fed condition, restricted due to the low binding activity of the ubiquitin associated (UBA) domain of p62 and further restricted by UBA homodimerisation (Long et al. 2010). Briefly, Lys420 monoubiquitylation (Lee et al. 2017; Peng et al. 2017), and Ser403 and Ser407 (in humans; Ser405 and Ser409 in mice) phosphorylation (Matsumoto et al. 2015) strengthen the interaction and binding affinity between p62 and ubiquitin. In addition, acetylation of p62 Lys420 and Lys435 residues, regulated by TIP60 and opposed by HDAC6 upon serum and amino acid starvation, interferes with UBA dimerization (Lys420 and Lys435) and enhances ubiquitin-binding affinity (Lys435) (You et al. 2019a). Moreover, spatial proximity between p62 and HDAC6 at sites of protein aggregation promotes their interaction and regulation of HDAC6 deacetylase activity and, by extension, protein aggregate recycling by p62 (Yan et al. 2013).

自噬受体调节自噬途径中货物识别的选择性和特异性。虽然目前关于影响典型自噬受体结构、功能和定位的 PTMs 的知识相当有限,但是在所有典型受体(THANATOS, https://THANATOS.biocuckoo.org )中都发现了磷酸化和泛素化位点(Deng et al. 2018)。自噬受体的乙酰化依赖性调节的最佳角色塑造是 p62蛋白及其对泛素的亲和力(图2 c)。在食物条件下,泛素和 p62与形成的自噬体之间的空间连接受到限制,这是因为 p62泛素相关(UBA)结构域的结合活性较低,而且进一步受到非洲大陆泛素同二聚体的限制(Long 等人,2010年)。简而言之,Lys420单基化(Lee 等人,2017年; Peng 等人,2017年)和 Ser403和 Ser407(人类; Ser405和 Ser409小鼠)磷酸化(松本等人,2015年)增强 p62和泛素之间的相互作用和结合亲和力。此外,p62 Lys420和 Lys435残基的乙酰化受 TIP60调节,而 HDAC6对血清和氨基酸饥饿产生反作用,干扰了非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性非特异性聚合。此外,p62和 HDAC6在蛋白质聚集位点的空间接近促进了它们之间的相互作用和 HDAC6脱乙酰酶活性的调节,进而促进了 p62蛋白质聚集再循环(Yan 等人,2013年)。

Moreover, ubiquitin (Ub) itself is a target of lysine acetylation (Ohtake et al. 2015). Formation of stable polyubiquitin chains by covalent linkages of single Ub moieties via homotypic Lys63linkage (also known as K63) promotes autophagy (Grumati and Dikic 2018). Although the KAT(s) and KDAC(s) involved and the physiological relevance of Ub acetylation remain unknown, acetylation of Lys6 and Lys48 residues was shown to interfere with poly-Ub chain formation (Lys11-, Lys48– and Lys63-linked) in vitro (Ohtake et al. 2015).

此外,泛素(Ub)本身就是赖氨酸乙酰化的靶标(Ohtake 等人,2015)。通过同型 Lys63连锁(也称为 K63)的 Ub 单体共价连锁形成稳定的多聚泛素链促进自噬(Grumati 和 Dikic 2018)。虽然 KAT (s)和 KDAC (s)参与和生理相关性的 Ub 乙酰化仍然是未知的,Lys6和 Lys48残基的乙酰化已被证明干扰聚 Ub 链的形成(Lys11-,Lys48-和 lys63-连接)在体外(Ohtake 等人,2015年)。

Demystification of the acetylation riddle in autophagy regulation

自噬调控中乙酰化之谜的揭秘

Recent advances in our understanding of which KATs and KDACs are involved in the regulation of autophagy protein acetylation highlight a few interesting phenomena. Overall, autophagy protein acetylation status is mainly regulated by p300, CREB binding protein (CBP) and TIP60 KATs, and HDAC2/6 and SIRT1 KDACs (Table 1). Upon a closer look, targets of p300-mediated acetylation are generally opposed by SIRT1-dependent deacetylation, while the targets of TIP60 may be opposed by HDACs but remain largely unknown. Following this train of thought, targets of p300/SIRT are activated by the loss of acetylation, whereas it is the addition of acetyl group to TIP60 targets that triggers their activation (summarized in Table 1, shown in Fig. 2).

近年来,我们对 KATs 和 kdac 参与自噬蛋白乙酰化调控的研究取得了一些新进展。总的来说,自噬蛋白的乙酰化状态主要受 p300,CREB结合蛋白(CBP)和 TIP60 KATs,以及 HDAC2/6和 SIRT1 kdac 的调节(表1)。近距离观察发现,p300介导的乙酰化作用靶点一般与 sirt1相关的脱乙酰化作用相反,TIP60的靶点可能与 HDACs 相反,但仍不清楚。根据这一思路,p300/SIRT 的靶基因因乙酰化缺失而被激活,而 TIP60的靶基因因乙酰化缺失而被激活(见表1,如图2所示)。

Autophagy is stimulated by the depletion of key nutrients including amino acids, growth factors and glucose. Recognition of nutrient availability by multiple intracellular sensors converges on a handful of regulators that integrate nutrient signals into several key responses. These include mammalian target of rapamycin complex 1 (mTORC1) (Rabanal-Ruiz et al. 2017), glycogen synthase kinase 3 (GSK3) (Mancinelli et al. 2017), and adenine monophosphate-activated protein kinase (AMPK) (Tamargo-Gómez and Mariño 2018). Perhaps unsurprisingly, these three kinases have also been directly linked to the regulation of KATs and KDACs that influence the acetylation status of autophagy proteins. mTORC1 was recently shown to activate the acetyl-transferase activity of p300 by serine phosphorylation that was lost upon amino acid starvation (Wan et al. 2017). Activation of GSK3β by the loss of growth factor signalling is known to phosphorylate and thus activate TIP60 (Lin et al. 2012). Finally, AMPK activation releases SIRT1 inhibition in a GAPDH-dependent manner in response to glucose starvation (Chang et al. 2015). Thus, three potential axes regulate autophagy stimulation in response to nutrient stress by (a) loss of FoxO, VPS34, Beclin1, ATG7, ATG5 and ATG12 acetylation (amino acids/growth factors-mTORC1-p300), (b) increased ULK1 and possible ATG3 and p62 acetylation (serum/ER stress-GSK3β-TIP60) (Lin et al. 2012; Nie et al. 2016; Yi et al. 2012; You et al. 2019a), and (c) FoxO, Beclin1, ATG7, ATG5 and ATG12 deacetylation (glucose–AMPK–GAPDH–SIRT1) that could explain the conundrum of the varied nature of autophagy protein acetylation status upon nutrient starvation and its link to autophagy stimulation.

自噬是由包括氨基酸、生长因子和葡萄糖在内的关键营养物质的耗竭而引起的。通过多个细胞内传感器识别营养物质的可利用性汇聚在一些调节器上,这些调节器将营养信号整合到几个关键反应中。这些包括哺乳动物靶蛋白雷帕霉素复合物1(mTORC1)(Rabanal-Ruiz 等人2017年) ,糖原合成酶激酶3(GSK3)(Mancinelli 等人2017年) ,腺嘌呤单磷酸激活蛋白激酶(AMPK)(Tamargo-Gómez 和 Mariño 2018年)。也许不足为奇的是,这三种激酶也直接与影响自噬蛋白乙酰化状态的 KATs 和 kdac 的调节有关。mTORC1最近被证明通过丝氨酸磷酸化激活 p300的乙酰基转移酶活性,而丝氨酸磷酸化在氨基酸饥饿时失去了这种活性(Wan 等人,2017年)。由于生长因子信号的丢失而激活的 gsk3被认为是磷酸化的,因此激活 TIP60(Lin 等人,2012)。最后,AMPK 的激活以 gapdhh 依赖的方式释放 SIRT1的抑制作用以应对葡萄糖饥饿(Chang et al. 2015)。因此,三个潜在的轴通过(a) FoxO、 VPS34、 Beclin1、 ATG7、 ATG5和 ATG12乙酰化(氨基酸/生长因子 -mtorc1-p300)的缺失,调节对营养胁迫的自噬刺激,(b)增加 ULK1和可能的 ATG3和 p62乙酰化(serum/ER stress-gsk3-tip60)(Lin et al. 2012; Nie et al. 2016; Yi et al. 2012; You et al. 2019a) ,(c) FoxO,Beclin1,ATG7,ATG5和 ATG12 deacetylation (glucose-AMPK-GAPDH-SIRT1)难题可以解释在营养缺乏刺激下自噬蛋白乙酰化状态的不同性质及其与自噬缺乏的联系。

Targets of cysteine oxidative PTMs in autophagy

半胱氨酸氧化 PTMs 在自噬中的作用

Protein modification by ROS and RNS constitutes a covalent modification of amino acid residues by the reactive species directly, or as a secondary interaction in an oxidative relay. Briefly, irreversible (carbonylation, nitration) oxidative modifications affect a variety of amino acids including cysteine (Cys), threonine and tyrosine (Ahmad et al. 2017; Cai and Yan 2013; Xie et al. 2018). In contrast, reversible amino acid oxidation involves modification of the thiol group (-SH) of Cys protein residues that are first modified to sulfenic acid (–SOH) (Cai and Yan 2013). Sulfenic acid can then undergo nitrosylation (-SNO) by reacting with RNS, or disulphide bond formation (R–S–S–R) by intra-/inter-molecular bond formation between two cysteine residues. A specialised form of disulphide bond formation, glutathionylation (R–S–S–G) arises as a mixed disulphide bond formation between a target protein Cys residue and the non-enzymatic antioxidant, glutathione (GSH) (Cai and Yan 2013). Further oxidation of –SOH results in an irreversible Cys oxidation by the formation of sulfinic (–SO2H) and sulfonic (–SO3H) acids (Ahmad et al. 2017; Cai and Yan 2013; Murray and Van Eyk 2012). Autophagy regulation by ROS is linked to the reversible oxidative Cys modification of (a) transcription factors (TFs) that regulate expression of proteins involved in the autophagy process, (b) upstream regulators of autophagy initiation, (c) autophagy proteins themselves and (d) receptors that mediate autophagy substrate selectivity (Filomeni et al. 2015; Montagna et al. 2016; Sedlackova et al. 2020).

活性氧和 RNS 对蛋白质的修饰是由反应物直接对氨基酸残基进行共价修饰,或者作为氧化继电器中的二次相互作用。简而言之,不可逆(羰基化,硝化)氧化修饰影响各种氨基酸,包括半胱氨酸(Cys) ,苏氨酸和酪氨酸(Ahmad et al. 2017; Cai and Yan 2013; Xie et al. 2018)。相比之下,可逆的氨基酸氧化涉及到胱氨酸蛋白质残基的硫醇基(- SH)的修饰,该基因首先被修饰为磺胺酸(- SOH)(Cai 和 Yan,2013)。亚砜酸通过与 RNS 反应发生亚硝基化(- SNO) ,或通过两个半胱氨酸残基之间的分子内/分子间键形成二硫化物键(r-s-s-r)。谷胱甘肽(r-s-s-g)是二硫键形成的一种特殊形式,它是目标蛋白 Cys 残基和非抗氧化酶谷胱甘肽(GSH)之间的二硫键形成的混合物。- SOH 的进一步氧化导致不可逆的 Cys 氧化,形成亚硫酸盐(- SO2H)和磺酸盐(- SO3H)酸(Ahmad 等人,2017; Cai 和 Yan,2013; Murray 和 Van Eyk,2012)。ROS 的自噬调节与(a)转录因子(tf)的可逆氧化性 Cys 修饰有关,tf 调节自噬过程中蛋白质的表达,(b)自噬启动的上游调节因子,(c)自噬蛋白本身和(d)调节自噬底物选择性的受体(Filomeni et al. 2015; Montagna et al. 2016; lackova et al. 2020)。

The most substantial link between ROS and autophagy TF activation was established in the studies of the Mit/TFE family of transcription factors (Yang et al. 2018). Three members of the Mit/TFE protein family were recently shown to contain redox sensitive Cys residues (TFEB Cys212, TFE3 Cys322, MITF Cys281) that mediate a rapid response to increased intracellular oxidative stress by promoting their nuclear translocation (Wang et al. 2019a). Another layer of regulation by oxidative stress was previously uncovered for TFEB that regulates expression of several autophagy proteins including, ATG4, ATG9, LC3B and p62 (Settembre et al. 2011). Increased intracellular oxidative stress is sensed by the lysosomal cation channel, mucolipin 1 (MCOLN1/TRPML1) in a manner that is not yet understood (Zhang et al. 2016c). What is known is that MCOLN1 oxidation promotes channel opening, Ca2+ release from the lysosomal lumen and activation of a Ca2+ dependent phosphatase, calcineurin (Medina et al. 2015; Zhang et al. 2016c). Calcineurin-dependent TFEB phosphorylation then promotes TFEB translocation to the nucleus and autophagy stimulation (Fig. 2A).

在对 Mit/TFE 转录因子家族的研究中建立了活性氧和自噬 TF 激活之间最实质性的联系(Yang 等人,2018)。最近发现 Mit/TFE 蛋白家族的3个成员含有氧化还原敏感的 Cys 残基(TFEB Cys212,TFE3 Cys322,MITF Cys281) ,这些残基通过促进细胞核移位对增加的细胞内氧化应激做出快速反应。另外一层由氧化应激基因组调控的 TFEB 蛋白,包括 ATG4,ATG9,LC3B 和 p62蛋白的表达。增加的细胞内氧化应激通过溶酶体阳离子通道—- 粘液脂蛋白1(MCOLN1/TRPML1)检测到,但这种方式尚不清楚(Zhang et al. 2016c)。已知的是,MCOLN1氧化促进通道开放,从溶酶体腔释放 Ca2 + 和激活一个 Ca2 + 依赖性磷酸酶,钙调神经磷酸酶(Medina 等人,2015; Zhang 等人,2016 c)。钙调神经磷酸酶依赖的 tmb 磷酸化促进 tmb 转运到细胞核和自噬刺激(图2A)。

At the stage of autophagy execution, redox-sensitive Cys residues were identified in proteins involved in LC3 processing (ATG4B) and LC3-PE conjugation (ATG7 and ATG3). ATG4B is a Cys-dependent protease that cleaves pro-LC3 at a C-terminal glycine residue prior to LC3-PE conjugation (Kirisako et al. 2000). Its protease activity is also involved in correcting the amount of LC3–PE formation on non-autophagic membranes by the hydrolysis of the LC3–PE bond, and presumably on the outer membrane leaflet of the growing autophagosome. In human cells, the hydrolysing (deconjugating) activity of ATG4B is inhibited by the oxidation of one of two Cys residues (Cys74 or Cys78) and leads to improved stability of LC3–PE and increased formation of autophagosomes (Scherz‐Shouval et al. 2007). Similarly, oxidation of the catalytic thiols in ATG3 (Cys264) and ATG7 (Cys572) inhibits their activity in LC3–PE conjugation and results in the loss of autophagic flux (Fig. 2b) (Frudd et al. 2018). Interestingly, oxidation of these Cys residues can only occur when the thiols are not shielded by their interaction with LC3.

在自噬执行阶段,在参与 LC3加工(ATG4B)和 LC3-pe 接合(ATG7和 ATG3)的蛋白质中发现了氧化还原敏感的 Cys 残基。ATG4B 是一种依赖于胱氨酸的蛋白酶,在 LC3-PE 接合之前在 c 末端甘氨酸残基上分离 pro-LC3(Kirisako 等人,2000)。它的蛋白酶活性也参与了通过 LC3-PE 键的水解纠正非自噬膜上 LC3-PE 的形成量,推测是在生长中的自噬体的外膜小叶上。在人类细胞中,ATG4B 的水解(去聚集)活性被两个 Cys74或 Cys78中的一个残基的氧化所抑制,从而提高了 LC3-PE 的稳定性并增加了自噬体的形成(Scherz-Shouval 等人,2007年)。同样,ATG3(Cys264)和 ATG7(Cys572)中催化硫醇的氧化抑制了它们在 LC3-PE 共轭中的活性,导致自噬通量的损失(图2b)(Frudd 等人,2018)。有趣的是,只有当硫醇与 LC3的相互作用没有屏蔽时,这些 Cys 残基才会发生氧化。

Oxidative stress influences the selectivity of the autophagic process via p62, a known redox sensitive autophagy receptor protein (Fig. 2c). Intermolecular disulphide formation in p62 was first observed in studies of its involvement in the N-end rule pathway of substrate degradation, where Cys113-dependent oligomerisation promoted substrate clearance via autophagy (Cha-Molstad et al. 2017). Subsequently, we have demonstrated that elevated ROS levels promote the formation of disulphide-linked conjugates, intermolecular Cys bonds, that assist p62 oligomer assembly (Carroll et al. 2018). Crucially, we have identified two Cys residues (Cys105and Cys113) located within the regulatory linker region of the p62 protein, that are necessary and sufficient for the activation of pro-survival autophagy triggered by increased ROS (Carroll et al. 2018).

氧化应激通过 p62影响自噬过程的选择性,p62是一种已知的氧化还原敏感性自噬受体蛋白(图2 c)。P62分子间二硫化物的形成首先是在研究其参与底物降解的 n 端规则途径时观察到的,其中 cys113依赖的寡聚化通过自噬促进底物清除(Cha-Molstad 等人,2017年)。随后,我们证明,活性氧水平升高促进形成二硫化物连接复合物,分子间 Cys 键,这有助于 p62低聚物组装(Carroll 等人,2018年)。至关重要的是,我们已经确定了两个 Cys 残基(Cys105和 Cys113)位于 p62蛋白的调节连接器区域,这对于激活活性氧增加引发的有利于存活的自噬是必要和充分的(Carroll 等人,2018)。

Reversible oxidation of Cys residues in redox-sensitive autophagy proteins thus appears to have a dual role of pathway stimulation by autophagy gene expression (TFEB), increased autophagosome formation (ATG4B) and substrate selectivity (p62), and autophagy inhibition upon depletion of available LC3 substrate (ATG3, ATG7). However, due to the novelty of these findings, the physiological role of ATG3 and ATG7 inhibition and possible downstream signalling events remain unknown. We propose a regulatory feedback loop whereby sensing depletion of local LC3 pools results in inactivation of ATG3 and ATG7 that serves to prevent indiscriminate autophagy activation. We envision that this inactivation would persist until such a time that the antioxidant defences decrease the oxidative stress load and resolve the ATG3-ATG7 heterodimer, and the expression of autophagy genes restores the available pools of ATG proteins to sustain further autophagy.

因此,氧化还原敏感性自噬蛋白中 Cys 残基的可逆氧化似乎具有通过自噬基因表达(TFEB)刺激、增加自噬体形成(ATG4B)和底物选择性(p62)以及通过消耗可利用的 LC3底物(ATG3,ATG7)抑制自噬的双重作用。然而,由于这些发现的新颖性,ATG3和 ATG7的生理作用抑制和可能的下游信号事件仍然是未知的。我们提出了一个监管反馈回路,通过感知当地 LC3池的耗尽导致 ATG3和 ATG7的失活,从而防止不分青红皂白的自噬激活。我们设想,这种失活将持续到抗氧化防御降低氧化应激负荷,解决 ATG3-ATG7异二聚体,自噬基因的表达恢复可用的 ATG 蛋白池,以维持进一步的自噬。

The interrelatedness of target oxidation and acetylation in autophagy

自噬过程中靶向氧化与乙酰化的相互关系

Protein deacetylation and oxidation appear to be individually sufficient to regulate the initiation, promotion, efficiency and selectivity of autophagy. However, an interesting crosstalk between oxidative and acetyl-linked PTMs of autophagy proteins arises due to the dual control of several proteins including TFEB, ATG3, ATG7 and p62, which appear to be regulated by both, oxidation and acetylation status (Fig. 2a–c). First, upstream oxidation of MCOLN1 regulates TFEB localization by calcineurin-dependent dephosphorylation (Medina et al. 2015; Zhang et al. 2016c) and direct oxidation of its Cys212 residue (Wang et al. 2019a). Further, TFEB deacetylation at residues Lys274 and Lys279 promotes its dimerization and increases its binding affinity for DNA (Wang et al. 2019b). It would be interesting to study whether oxidation and acetylation PTMs act in concert to establish the optimal TFEB activity and whether TFEB oxidation promotes rapid expression of its target genes in the absence of Lys residue deacetylation.

蛋白质的脱乙酰化和氧化作用足以调节自噬的启动、促进、效率和选择性。然而,由于 TFEB、 ATG3、 ATG7和 p62等蛋白质的双重调控,自噬蛋白的氧化和乙酰连接的 PTMs 之间出现了一个有趣的串扰,它们似乎都受到氧化和乙酰化状态的调控(图2a-c)。首先,MCOLN1的上游氧化通过依赖于钙调神经磷酸酶的脱磷酸化(Medina 等人,2015; Zhang 等人,2016c)和其 Cys212残基的直接氧化(Wang 等人,2019a)来调节 tmb 的定位。另外,在 Lys274和 Lys279残基上的 tbb 脱乙酰化促进了它的二聚化并增加了它与 DNA 的结合亲和力(Wang 等人,2019b)。研究在没有赖氨酸残基脱乙酰化的情况下,氧化和乙酰化 PTMs 是否协同作用,以确定最佳的 tbb 活性,以及 tbb 氧化是否促进其靶基因的快速表达,将会引起人们的重视。

Second, ATG3 acetylation at residues Lys19 and Lys48 by TIP60, increased in conditions of nutrient starvation, improves interaction between ATG3 and LC3 and promotes autophagy (Yi et al. 2012). Not much is known regarding the functional effect of deacetylation in ATG7, except that it promotes autophagy and Lys306 residue may be the target (Choudhary et al. 2009). In contrast to TFEB, a recently published study suggests that upon loss of LC3 binding, oxidation of ATG3 (Cys264) and ATG7 (Cys572) catalytic cysteine residues inhibits their enzymatic activity and blocks their further interaction with LC3 (Frudd et al. 2018).

其次,TIP60在 Lys19和 Lys48残基上的 ATG3乙酰化,在营养缺乏条件下增加,改善 ATG3和 LC3之间的相互作用,促进自噬(Yi 等人,2012)。目前对 ATG7中脱乙酰基的功能作用还知之甚少,除了它促进了自噬,Lys306残基可能是其作用目标(Choudhary 等人,2009年)。与 TFEB 相反,最近发表的一项研究表明,ATG3(Cys264)和 ATG7(Cys572)催化半胱氨酸残基的氧化会抑制它们的酶活性,阻碍它们与 LC3的进一步相互作用(Frudd 等人,2018年)。

Lastly, oxidation and acetylation of p62 could act in concert to achieve optimal selectivity of its interaction with cargo and oligomerization to stimulate autophagy. First, TIP60-dependent acetylation of Lys420 and Lys435 within the UBA domain interferes with its inter-protein dimerization and enhances the ubiquitin binding affinity of p62 upon serum and amino acid starvation (You et al. 2019a). Second, oxidation of Cys105 and Cys113 residues within the regulatory linker region promotes intermolecular p62 disulphide bond formation and thus assist in autophagy stimulation (Carroll et al. 2018).

最后,p62的氧化和乙酰化可以协同作用,实现其与载体相互作用的最佳选择性和促进自噬的齐聚作用。首先,tip60依赖于 Lys420和 Lys435的乙酰化作用,干扰了其蛋白间二聚化,增强了 p62与血清和氨基酸饥饿的泛素结合亲和力(You 等,2019a)。其次,调节连接物区域内 Cys105和 Cys113残基的氧化促进了分子间 p62二硫键的形成,从而有助于自噬刺激(Carroll 等人,2018年)。

In addition, activity of NAD+-dependent KDACs, or SIRTs, is directly or indirectly regulated by both, oxidative and metabolic stress stimuli. First, a shift in the NAD redox balance towards oxidation, suggestive of metabolic stress, leads to an increased pool of available NAD+ and thus stimulates SIRT activity (Imai and Guarente 2016). Second, SIRT regulation by oxidative stress was demonstrated in multiple cell culture experiments (reviewed in (Santos et al. 2016)), in which a mild oxidative environment promotes SIRT1 expression and activation by upstream kinases. In contrast, study of SIRT1 oxidation, specifically nitrosylation (–SNO+), suggests that this reversible oxidative PTM of Cys371, Cys374, Cys395 and Cys398 residues within a tetrathiolate formation results in loss of Zn2+ binding, structural destabilization and loss of NAD+ and acetyl-lysine binding ability (Kalous et al. 2016). Thus, SIRT1 activity can be stimulated by both, nutrient starvation, and oxidative stress. However, persistent ROS release may lead to SIRT1 destabilization, loss of its deacetylase activity and might contribute to its degradation by the proteasome (Caito et al. 2010).

此外,NAD + 依赖的 kdac 或 SIRTs 的活性直接或间接地受到氧化和代谢应激刺激的调节。首先,NAD 的氧化还原平衡转向氧化,暗示代谢应激,导致可用 NAD + 池增加,因此刺激 SIRT 活性(今井和瓜伦特2016年)。其次,在多次细胞培养实验中证实了氧化应激对 SIRT 的调节作用(见 Santos 等人2016年的综述) ,在这些实验中,温和的氧化环境通过上游激酶促进 SIRT1的表达和活化。与此相反,SIRT1氧化特异性硝基化(- SNO +)的研究表明,Cys371、 Cys374、 Cys395和 Cys398残基在四硫醇盐形成过程中的可逆氧化性 PTM 导致 Zn2 + 结合的丧失、 NAD + 结构的不稳定性和乙酰赖氨酸结合能力的丧失(Kalous et al. 2016)。因此,SIRT1的活性可以同时受到营养缺乏和氧化应激的刺激。然而,持续释放 ROS 可能导致 SIRT1的不稳定,失去其去乙酰化酶活性,并可能有助于其降解的蛋白酶体(Caito 等人。2010年)。

NAD depletion, oxidative stress, and autophagy in physiological and pathological ageing

生理和病理衰老过程中的 NAD 损耗、氧化应激和自噬

The NAD nucleotide is an important redox molecule required for fundamental molecular processes of energy generation via glycolysis, tricarboxylic acid cycle, oxidative phosphorylation and β-oxidation, and a co-factor to enzymes involved in cellular signalling and longevity. Age-related depletion of available NAD+ pools was, in human disease, animal models and in vitro studies, reported as a result of increased PARP activity due to an elevation in oxidative stress and levels of DNA damage (Pacher and Szabo 2008) and increased CD38 expression and activity (Camacho-Pereira et al. 2016; Polzonetti et al. 2012). Combined with the age-dependent reduction in the enzymatic activity of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAM-based NAD+salvage pathway (Stein and Imai 2014), these conditions perpetuate the perfect storm of total NAD depletion, loss of NAM recycling and reduction in SIRT activity in physiological ageing.

NAD 核苷酸是一种重要的氧化还原分子,它是通过糖酵解、三羧酸循环、氧化磷酸化和氧化等基本分子过程产生能量的必需物质,也是参与细胞信号传递和延长寿命的酶的辅助因子。在人类疾病、动物模型和体外研究中,与年龄相关的 NAD + 池损耗被报道为由于氧化应激升高和 DNA 损伤水平增加而导致 PARP 活性增加(Pacher 和 Szabo,2008年)和 CD38表达和活性增加(Camacho-Pereira 等人,2016年; Polzonetti 等人,2012年)。结合年龄依赖性烟酰胺磷酸核糖基转移酶(NAMPT)酶活性的降低,这些条件延续了生理衰老过程中 NAD 总耗竭、 NAM 循环丢失和 SIRT 活性降低的完美风暴。

Study of human skin tissue from volunteers of different ages partially supports these findings (Massudi et al. 2012). In this study, an age-dependent increase in DNA damage correlated with an increase in PARP activity, NAD+ depletion and, in the elderly, a reduction in SIRT1 activity. Notably, these associations with age were strong only in the male participants and it would be interesting to see whether these findings can be reproduced in females and other accessible human tissues, including muscle or post-mortem brain tissues. In a more recent study carried out on human skeletal muscle samples, authors demonstrate that levels of NAMPT negatively correlate with age, body mass index and body fat percentage (de Guia et al. 2019). Another study utilised the power of magnetic resonance-based non-invasive in vivo imaging of the human brain and revealed an age-dependent decrease in total NAD levels, concomitant with an increase in NADH/NAD+ ratio, indicative of metabolic dysfunction (Zhu et al. 2015). While these studies were carried out on healthy human volunteers and suggest that a decline in NAD levels occurs in physiological ageing, multiple studies of accelerated human ageing (progeria) syndromes and patients suffering from metabolic and neurodegenerative diseases strongly link NAD decline to age-related pathology (Kubben and Misteli 2017; Lautrup et al. 2019; Okabe et al. 2019).

对不同年龄的志愿者皮肤组织的研究部分支持了这些发现(Massudi et al. 2012)。在这项研究中,DNA 损伤的年龄依赖性增加与 PARP 活性的增加、 NAD + 损耗以及老年人 SIRT1活性的减少相关。值得注意的是,这些与年龄的联系只在男性参与者中很强烈,看看这些发现能否在女性和其他可获得的人体组织中重现,包括肌肉组织或死后的脑组织,将是很有意思的。在最近对人类骨骼肌样本的研究中,作者证明 NAMPT 水平与年龄、身体质量指数和身体脂肪百分比呈负相关(de Guia 等人,2019年)。另一项研究利用了基于磁共振的人脑非侵入性体内成像技术,揭示了 NAD 总水平随年龄增长而下降,同时 nadh/NAD + 比值增加,表明存在代谢功能障碍(Zhu 等人,2015年)。虽然这些研究是在健康的人类志愿者身上进行的,并表明 NAD 水平的下降发生在生理性衰老中,但对人类加速衰老(早衰症)综合征和患有代谢和神经退行性疾病的患者进行的多项研究强烈地将 NAD 的下降与年龄相关的病理学联系起来(Kubben 和 Misteli,2017年; Lautrup 等人,2019年; Okabe 等人,2019年)。

Furthermore, studies of two age-related conditions, sarcopenia and frailty, as well as a variety of progeria, neurodegenerative, metabolic and cardiac diseases, demonstrate a strong link between pathology and increased oxidative stress (Derbré et al. 2014; Inglés et al. 2014; Kubben and Misteli 2017; Liguori et al. 2018; Massudi et al. 2012; Soysal et al. 2017). Not only does lipid peroxidation, a proxy measurement for increased oxidative stress, correlate with age (Massudi et al. 2012), a systematic review of available literature suggests that long lived humans (centenarians) have lower levels of oxidative protein damage and lipid peroxidation compared to other elderly individuals (Belenguer-Varea et al. 2019). Given the number and severity of clinical conditions related to healthy ageing, and age-related diseases that are associated with an increase in oxidative stress, it is necessary to design interventions that prevent production of free radicals, boost cellular antioxidant systems, or understand and target the processes downstream of ROS-mediated protein, lipid or nucleotide damage.

此外,与年龄相关的条件,骨骼肌减少和脆弱,以及各种早衰症,神经退行性,新陈代谢和心脏疾病的研究表明,病理学和氧化应激增加之间有很强的联系(derbré 等人2014; Inglés 等人2014; Kubben 和 Misteli 2017; Liguori 等人2018; Massudi 等人2012; Soysal 等人2017)。脂质过氧化不仅是氧化应激增加的代理测量,与年龄相关(Massudi 等人2012年) ,一系统综述可用的文献表明,长寿的人(百岁老人)与其他老年人相比,有较低的氧化蛋白质损伤和脂质过氧化。鉴于与健康老龄化相关的临床疾病的数量和严重程度,以及与氧化应激增加相关的与年龄相关的疾病,有必要设计干预措施,以防止自由基的产生,增强细胞抗氧化系统,或了解和瞄准 ros 介导的蛋白质、脂质或核苷酸损伤的下游过程。

Importantly, molecular studies of free radical generation, NAD+-dependent enzymatic processes and disease pathology suggest a link between ROS accumulation, NAD depletion and compromised mitochondrial recycling by autophagy, mitophagy. Mitochondria are energy-generating organelles that act as hubs of pro-survival or pro-apoptotic signalling (Sedlackova and Korolchuk 2019). Although mitochondrial health is maintained by a complex net of quality control mechanisms, whole organelle recycling of damaged and ROS-producing mitochondria is only achieved by selective autophagy. A causal link between NAD+ depletion and mitochondrial dysfunction due to loss of mitophagy was established in studies of premature ageing syndromes including Xeroderma Pigmentosum, Cockayne syndrome and Ataxia-telangiectasia (Fang et al. 20162014; Scheibye-Knudsen et al. 20142012; Valentin-Vega et al. 2012). In these studies, loss of SIRT activity and autophagy abnormalities occur as a result of PARP1 hyperactivation due to unresolved DNA damage. In addition to SIRT inactivation, uncontrolled NAD+ cleavage and protein PAR-ylation by PARPs results in loss of ATP availability and, if persistent, in cell death (Andrabi et al. 2014; Bai et al. 2011; Fouquerel et al. 2014; Pillai et al. 2005). Persistent NAD+ depletion was thus shown to compromise mitochondrial function due to loss of energy generation, impairment in mitochondrial recycling through lack of autophagy/mitophagy stimulation, and to initiate cellular death due to energy collapse. An alternative outcome to cell death upon PARP1 activation was linked to autophagy initiation in independent cell culture experiments (Jiang et al. 2018; Muñoz-Gámez et al. 2009). In the earlier study, authors demonstrated that PARP-dependent stimulation of autophagy due to short-lived energy crisis had a cytoprotective effect as genetic or pharmacological inhibition of autophagy led to increased level of necrotic death (Muñoz-Gámez et al. 2009). In the latter study, authors aimed to mimic constant ROS production in vivo by glucose oxidase (GO) treatment, which led to PARP-induced cell death, parthanatos (Jiang et al. 2018). In this study, inhibition of autophagy led to a significant collapse in mitochondrial polarization and an approximately 50% increase in cell death within four hours of GO treatment. Taken together with the role of SIRT-mediated autophagy stimulation, we wonder whether convergence of these signalling pathways on autophagy suggests a conserved role of this catabolic pathway in healthy ageing by preservation of cellular NAD pools.

重要的是,对自由基产生、 NAD + 依赖的酶过程和疾病病理学的分子研究表明,ROS 的积累、 NAD 的耗竭和线粒体自噬、噬细胞的损伤循环之间存在联系。线粒体是能量产生的细胞器,充当促生存或促凋亡信号的枢纽(Sedlackova 和 Korolchuk 2019)。虽然线粒体的健康是通过一个复杂的质量控制机制网来维持的,但是受损和产生 ros 的线粒体的整个细胞器的再循环只能通过选择性自噬来实现。在对早衰综合症的研究中,包括着色性干皮症、柯凯因氏症候群和共济失调-毛细血管扩张症,确立了 NAD + 耗竭与线粒体功能障碍之间的因果关系。在这些研究中,由于未解决的 DNA 损伤导致 PARP1过度激活,引起 SIRT 活性丧失和自噬异常。除 SIRT 失活外,PARPs 不受控制的 NAD + 分裂和蛋白质 PAR-ylation 导致 ATP 供应的丧失,如果持续存在,则导致细胞死亡(Andrabi 等人,2014年; Bai 等人,2011年; Fouquerel 等人,2014年; Pillai 等人,2005年)。因此,研究表明,NAD + 持续耗竭会损害线粒体功能,原因是能量代谢的丧失,缺乏自噬/吞噬刺激导致线粒体循环受损,以及能量崩溃导致细胞死亡。在独立的细胞培养实验中,PARP1激活导致细胞死亡的另一个结果与自噬启动有关(Jiang 等人,2018; Muñoz-Gámez 等人,2009)。在早期的研究中,作者证明,由于短暂的能量危机引起的 parp 依赖性自噬的刺激具有细胞保护作用,因为对自噬的遗传或药理抑制导致坏死死亡水平的增加(Muñoz-Gámez 等人,2009年)。在后一项研究中,作者的目标是通过葡萄糖氧化酶(GO)处理在体内模拟稳定的活性氧产生,这导致了 parp 诱导的细胞死亡(parthanatos)(Jiang 等人,2018年)。在这项研究中,抑制自噬导致线粒体极化明显崩溃,并在 GO 治疗4小时内增加约50% 的细胞死亡。结合 sirt 介导的自噬刺激的作用,我们想知道这些自噬信号通路的聚合是否表明这种分解代谢途径通过保存细胞 NAD 池在健康老龄化过程中发挥了保守的作用。

An exciting development in the field of ageing and NAD metabolism is the ‘druggability’ of NAD metabolism by exogenous addition of natural, or synthetic, bioavailable NAD+precursors. This universal approach of NAD+ precursor supplementation is known to increase NAD biosynthesis and alleviate the symptoms of pathological states including metabolic, cardiac and neurodegenerative disorders (Kane and Sinclair 2018; Lautrup et al. 2019). Additionally, evidence from NAD+ supplementation studies in cell culture and in animal models suggests that boosting NAD levels is sufficient to not only improve mitochondrial function, but also stimulate SIRT-dependent mitochondrial recycling via increased TFEB- and FoxO-dependent expression of autophagy/mitophagy genes and PTMs of autophagy proteins, and thus promote clearance of dysfunctional organelles and protein aggregates (Fang et al. 20192016; Hou et al. 2018; Schöndorf et al. 2018; Vannini et al. 2019; Zhang et al. 2016b). Altogether, this ‘silver bullet’ approach might serve as an intervention to the vicious cycle of damage and NAD depletion and thus not only combat the depletion itself, but also support resolution of the underlying stresses and promote long-term cellular health.

在衰老和 NAD 代谢领域的一个令人兴奋的发展是通过外源添加天然的或合成的生物可利用的 NAD + 前体的 NAD 代谢的‘药物可利用性’。众所周知,补充 NAD + 前体的这种普遍方法可以增加 NAD 的生物合成,减轻包括代谢、心脏和神经退行性疾病在内的病理状态的症状(Kane 和 Sinclair,2018年; Lautrup 等人,2019年)。此外,在细胞培养和动物模型中补充 NAD + 的研究证据表明,提高 NAD 水平不仅足以改善线粒体功能,而且通过增加 TFEB-和 foxo 依赖的自噬/细胞吞噬基因和自噬蛋白 PTMs 的表达,刺激依赖 sirt 的线粒体循环,从而促进清除功能失调的细胞器和蛋白质聚集体(Fang et al. 2019,2016; Hou et al. 2018; Schöndorf et al. 2018; Vannini et al. 2019; Zhang et al. 2016 b)。总之,这种”银弹”办法可以作为对损害和 NAD 耗竭的恶性循环的干预,因此不仅可以对付耗竭本身,而且还可以解决潜在的压力,促进长期的细胞健康。

Following the success of NAD+-boosting strategies in cell and animal models, NAD+precursors, and predominantly nicotinamide riboside (NR), are now subjects of multiple clinical trials. Precursors have so far been reported as safe, well tolerated and capable of increasing NAD levels in healthy volunteers (Conze et al. 2019; Martens et al. 2018; Minto et al. 2017; Stea et al. 2017). However, challenges remain in translation of laboratory findings into the design of clinical trials (Gilmour et al. 2020). While some early success was found in disease outcomes of amyotrophic lateral sclerosis (ALS) (NCT03489200) (de la Rubia et al. 2019), others found no benefit in patients with Alzheimer’s disease (NCT00580931) (Phelan et al. 2017), or studies of metabolic disorders or mitochondrial bioenergetics in men (NCT02303483) (Dollerup et al. 20182019a2019b). Although only a limited number of trials testing NAM, NMN and NR have been recently completed (> 10) or are currently ongoing (− 3), many are actively recruiting (− 21) (https://clinicaltrials.gov/) (Lautrup et al. 2019) and it will be interesting to see what lessons can be learned about precursor dosage, NAD+/NADH detection methods and bioavailability in the coming years. Considering that the pathological role of NAD depletion in many metabolic and neurodegenerative diseases is not yet firmly established, reporting of relevant disease outcomes is eagerly awaited as they will inform about the feasibility of translating success from the laboratory to human age- and disease-related interventions.

随着 NAD + 增强策略在细胞和动物模型中的成功,NAD + 前体,以及主要是烟酰胺核糖苷(NR) ,现在已经成为多种临床试验的对象。迄今为止,前体被报告为安全、耐受性良好并能够提高健康志愿者的 NAD 水平(Conze 等人,2019年; Martens 等人,2018年; Minto 等人,2017年; Stea 等人,2017年)。然而,在将实验室发现转化为临床试验设计方面仍然存在挑战(Gilmour et al. 2020)。虽然一些早期的成功被发现在肌萎缩性嵴髓侧索硬化症的疾病结果(NCT03489200)(de la Rubia 等人,2019年) ,其他人发现在阿尔茨海默病患者(NCT00580931)(Phelan 等人,2017年) ,或在男性代谢紊乱或线粒体生物能量学的研究(NCT02303483)(erup 等人,2018年,2019年 a,2019年 b)。虽然只有有限数量的试验测试 NAM,NMN 和 NR 最近已经完成(> 10)或目前正在进行(- 3) ,许多正在积极招募(- 21)(Lautrup 等人2019年)和它将是有趣的,看看什么教训可以学习前体剂量,NAD +/NADH 检测方法和生物利用度在未来几年。考虑到 NAD 耗竭在许多新陈代谢和神经退行性疾病中的病理作用尚未确定,人们迫切期待相关疾病结果的报告,因为它们将告知将实验室的成功转化为与人类年龄和疾病有关的干预措施的可行性。

Concluding Remarks

结语

Autophagy is a highly conserved catabolic process that is controlled by multiple nutritional and stress-related cues by reversible protein PTMs. In this review, we first explored the latest findings on how two PTMs, Lys acetylation and Cys oxidation, regulate the localization and function of autophagy proteins. Collectively, novel findings published in 2015–2020 identify TFEB, ULK1, VPS34, ATG3, LC3 and p62 as targets of acetylation PTMs which, in response to metabolic cues, stimulate the expression and enzymatic activity of autophagy proteins and improve pathway selectivity. Furthermore, Mit/TFE family of TFs (including TFEB), ATG3, ATG7 and p62 are also known to contain redox-sensitive Cys residues the oxidation of which influences autophagy outcomes. The dual control of protein localization/enzymatic activity by acetylation and oxidation links the efficiency of autophagy outcomes to nutrient loss and metabolic dysfunction and thus contributes to cellular homeostasis and healthy ageing.

自噬是一个高度保守的分解代谢过程,受多种营养和应激相关线索的可逆蛋白质 PTMs 控制。本文首先综述了赖氨酸乙酰化和赖氨酸氧化两种 PTMs 对自噬蛋白定位和功能调控的最新研究进展。总的来说,2015-2020年发表的新发现确定 TFEB,ULK1,VPS34,ATG3,LC3和 p62作为乙酰化 PTMs 的目标,在代谢的线索,刺激表达和自噬蛋白的酶活性,并改善通路的选择性。此外,Mit/TFE 家族的 TFs (包括 TFEB)、 ATG3、 ATG7和 p62也含有对氧化还原敏感的 Cys 残基,其氧化影响自吞噬结果。通过乙酰化和氧化双重控制蛋白质定位/酶活性,将自噬结果的效率与营养损失和代谢功能障碍联系起来,从而有助于细胞内稳态和健康老化。

Crucially, studies of the molecular mechanisms of NAD function in cellular physiology and ageing suggest a central role of autophagy in first, preventing increases in DNA damage and NAD+ consumption via mitochondrial recycling and second, by alleviating nutritional crisis via recycling amino acids, lipids and nucleosides. Autophagy thus appears to be necessary in supporting cellular survival upon either nutritional stress that changes NAD redox ratio towards the oxidised form (NAD+) and stimulates SIRTs, or upon DNA damage followed by NAD+ depletion due to PARP1 hyperactivation. Thus, although short term insults to cellular heath are resolved by autophagy stimulation and cellular detoxification, we wonder whether persistent oxidation and NAD+ loss in aged tissues result in stalled autophagy, and due to lack of stress resolution, ultimately in loss of cell viability and tissue dysfunction.

关键的是,对 NAD 在细胞生理和衰老中作用的分子机制的研究表明,自噬首先起着核心作用,通过线粒体循环防止 DNA 损伤和 NAD + 消耗的增加,其次通过氨基酸、脂类和核苷的循环缓解营养危机。因此,自噬在支持细胞存活方面似乎是必要的,无论是在营养应激改变 NAD 的氧化还原比率(NAD +)并刺激 SIRTs,还是在 DNA 损伤后由于 PARP1过度激活而 NAD + 耗尽。因此,尽管对细胞健康的短期损害可以通过自噬刺激和细胞解毒来解决,我们想知道是否在老化组织中持续的氧化和 NAD + 丢失会导致停滞的自噬,并且由于缺乏应力解决,最终导致细胞活力的丧失和组织功能障碍。

发表评论

您的电子邮箱地址不会被公开。 必填项已用*标注