Review  回顾

Role of peroxisomes in ROS/RNS-metabolism: Implications for human disease☆
过氧化物酶体在 ROS/RNS 代谢中的作用：对人类疾病的影响 ☆

Peroxisomes contain various enzymes that produce H₂O₂ as part of their normal catalytic cycle. These enzymes, which are mainly flavoproteins, include acyl-CoA oxidases, urate oxidase, d-amino acid oxidase, d-aspartate oxidase, l-pipecolic acid oxidase, l-α-hydroxyacid oxidase, polyamine oxidase, and xanthine oxidase. For a detailed description of these enzymes, we refer to an excellent recent review covering this topic [31].
过氧化物酶体含有多种酶，这些酶会产生 H₂O₂ 作为其正常催化循环的一部分。这些酶主要是黄素蛋白，包括酰基辅酶 A 氧化酶、尿酸盐氧化酶、d-氨基酸氧化酶、d-天冬氨酸氧化酶、l-哌哥酸氧化酶、l-α-羟基酸氧化酶、多胺氧化酶和黄嘌呤氧化酶。有关这些酶的详细描述，我们参考了最近一篇关于该主题的优秀综述 [31]。

Peroxisomes contain two potential enzymatic sources of O₂⁻ and NO, namely xanthine oxidase and the inducible form of nitric oxide synthase. Xanthine oxidase (XDH) is an enzyme that produces H₂O₂ (see Section 2.1.1.) and O₂⁻ as byproducts of its catalytic cycle [32]. In addition, this enzyme can also reduce nitrates and nitrites to NO [33]. The inducible form of nitric oxide synthase (NOS2) is a homodimeric enzyme that catalyzes the oxidation of l-arginine to NO and citrulline in a complex reaction requiring O₂, NADPH, tetrahydrobiopterin (BH4), FMN, and FAD [16]. Interestingly, in the absence of adequate substrate or when in its monomeric form, the enzyme can also produce significant amounts of O₂⁻ [34]. The precise subcellular localization of NOS2 remains enigmatic and can vary depending on the cell type and the environment of the cell [35]. Localization studies of NOS2 have shown that this protein displays a dual cytosolic-peroxisomal localization in hepatocytes [35], [36]. Interestingly, although the molecular mechanisms underlying NOS2 targeting to peroxisomes remain to be determined, it has been shown that the peroxisomal pool of NOS2 mainly consists of ‘inactive’ monomers while the cytosolic pool is composed of both monomers and ‘active’ homodimers [36]. These findings, in combination with the observation that monomeric NOS2 can generate O₂⁻, led to the hypothesis that NOS2 might be sequestered by peroxisomes to protect the larger cellular environment from monomeric NOS2-generated superoxide [36]. Nevertheless, at the moment, it cannot be rigorously excluded that – under certain circumstances – peroxisomal NOS2 may also actively produce NO and function as a source of RNS signaling molecules.
过氧化物酶体含有两种潜在的酶源 O₂ ⁻ 和 NO ，即黄嘌呤氧化酶和诱导型一氧化氮合酶。黄嘌呤氧化酶（XDH）是一种酶，可产生 H₂O₂（见第 2.1.1 节 ）和 O₂ ⁻ 作为其催化循环的副产物 [32]。 此外，这种酶还可以将硝酸盐和亚硝酸盐还原为 NO [33]。 一氧化氮合酶（notric oxide synthase， NOS2）的诱导型是一种同源二聚体酶，在需要 _O2、NADPH、四氢生物蝶呤（tetrahydrobiopterin， BH4）、FMN 和 FAD 的复杂反应中，催化 l-精氨酸氧化为 NO 和瓜氨酸[16]。 有趣的是，在没有足够的底物或单体形式的情况下，该酶也可以产生大量的 O₂ ⁻[34]。NOS2 的精确亚细胞定位仍然是一个谜，并且可能因细胞类型和细胞环境而异 [35]。NOS2 的定位研究表明，该蛋白在肝细胞中表现出双胞质-过氧化物酶体定位 [35]、[36]。 有趣的是，尽管 NOS2 靶向过氧化物酶体的分子机制仍有待确定，但已经表明，NOS2 的过氧化物酶体库主要由“非活性”单体组成，而胞质库由单体和“活性”同源二聚体组成 [36]。 这些发现，结合单体 NOS2 可以产生 _O2 ⁻ 的观察结果，得出了 NOS2 可能被过氧化物酶体隔离的假设，以保护更大的细胞环境免受单体 NOS2 产生的超氧化物的影响[36]。 然而，目前不能严格排除在某些情况下，过氧化物酶体 NOS2 也可能主动产生 NO 并充当 RNS 信号分子的来源。

Currently, there is no evidence that mammalian peroxisomes contain enzymes that produce OH or ONOO⁻. However, as already mentioned in the Introduction, H₂O₂ inside peroxisomes may give rise to OH through the Fenton reaction. In addition, as (i) these organelles contain enzymatic sources of O₂⁻ and NO (see Section 2.2.2.), and (ii) the reaction of NO with O₂⁻ to form ONOO⁻ is kinetically and thermodynamically favored [37], it is very likely that peroxisomes also generate ONOO⁻.
目前，没有证据表明哺乳动物过氧化物酶体含有产生 OH 或 ^ONOO− 的酶。然而，正如引言中已经提到的，过氧化物酶体内的 H₂O₂ 可能通过芬顿反应产生 OH。此外，由于（i）这些细胞器含有 O₂ ⁻ 和 NO 的酶源 （见第 2.2.2 节 ），以及（ii）NO 与 O₂ ⁻ 反应形成 ONOO⁻ 在动力学和热力学上是有利的[37]，因此过氧化物酶体很可能也会产生 ^ONOO−。

Catalase (CAT) is a homotetrameric heme-containing enzyme that can remove H₂O₂ in a catalatic (2 H₂O₂ → 2 H₂O + O₂) or peroxidatic (H₂O₂ + AH₂ → A + 2 H₂O) manner. Potential hydrogen donors (AH₂) include – among others – alcohols, formate, and nitrite [38]. Mammalian catalase can also act as an oxidase, using O₂ when H₂O₂ is absent [39]. Among the substrates identified for this oxidase action are indole, β-phenylethylamine, and various carcinogens and anticarcinogens [38]. Catalase is one of the most abundant peroxisomal proteins within mammalian cells. It contains a non-canonical C-terminal peroxisomal targeting signal (PTS1) and is targeted to the organelle by PEX5, the PTS1-import receptor [40]. However, in certain cell types or under certain conditions, catalase may also be (partially) localized to the cytosol and the nucleus [41], [42]. The exact physiological function of catalase is not yet completely understood. Its predominant role is most likely to prevent the accumulation of toxic levels of H₂O₂. This may in turn prevent the formation of hydroxyl radicals by the Fenton reaction. The oxidatic and peroxidatic activities of catalase may serve to metabolize and/or detoxify small molecular weight electron donors. For example, it is well-established that in brain, the catalase-mediated oxidation of ethanol is an important source of acetaldehyde, a compound involved in the behavioral and neurotoxic effects of ethanol in humans [43], [44]. Intriguingly, despite these apparently important functions, catalase activity is not essential for life. Indeed, catalase null mice develop normally and do not display any gross physical or behavioral abnormalities [45]. Nevertheless, tissues from these mice show a differential sensitivity to oxidant injury and exhibit a retarded rate in consuming extracellular H₂O₂ [45].
过氧化氢酶 （CAT） 是一种含同源四聚体血红素的酶，可以过氧化物 （2 H₂O₂ → 2 H₂O + O₂） 或过氧化物 （H₂O₂ + AH₂ → A + 2 H₂O） 方式去除 H₂O₂。潜在的氢供体（AH₂）包括醇类、甲酸盐和亚硝酸盐[38]。 哺乳动物过氧化氢酶也可以作为氧化酶，当 H₂O₂ 不存在时使用 O₂ [39]。 这种氧化酶作用的底物包括吲哚、β-苯乙胺以及各种致癌物和抗癌物 [38]。 过氧化氢酶是哺乳动物细胞中最丰富的过氧化物酶体蛋白之一。它含有非典型 C 端过氧化物酶体靶向信号（PTS1），并通过 PTS1 导入受体 PEX5 靶向细胞器 [40]。 然而，在某些细胞类型或某些条件下，过氧化氢酶也可能（部分）定位于胞质和细胞核[41]、[42]。 过氧化氢酶的确切生理功能尚不完全清楚。它的主要作用最有可能防止 H₂O₂ 毒性水平的积累。这反过来又可以防止芬顿反应形成羟基自由基。过氧化氢酶的氧化和过氧化活性可能有助于代谢和/或解毒小分子量电子供体。 例如，众所周知，在大脑中，过氧化氢酶介导的乙醇氧化是乙醛的重要来源，乙醛是一种参与乙醇对人类行为和神经毒性作用的化合物 [43]，[44]。 有趣的是，尽管这些功能显然很重要，但过氧化氢酶活性对生命来说并不是必需的。事实上，过氧化氢酶无效小鼠发育正常，没有表现出任何严重的身体或行为异常 [45]。 然而，这些小鼠的组织对氧化剂损伤表现出不同的敏感性，并且在消耗细胞外 H₂O₂ 方面表现出迟缓的速率 [45]。

Superoxide dismutase 1 (SOD1) is a homodimeric enzyme that can convert O₂⁻ to O₂ and H₂O₂ (2 O₂⁻ + 2 H⁺ → O₂ + H₂O₂). The protein, which can also be found in the cytosol, the nucleus, and mitochondria, is imported into peroxisomes via a piggyback mechanism in complex with ‘copper chaperone for SOD1’, a PTS1-containing physiological interaction partner [46]. Currently, it is postulated that SOD1 and catalase comprise a short metabolic route protecting cells from damage by ROS [31]. In addition, as O₂⁻ can rapidly react with NO to form ONOO⁻, SOD1 has been suggested to play an important role in the reduction of nitrosative stress [47]. Interestingly, mutations in the SOD1 gene account for approximately 20–25% of the patients with familial amyotrophic lateral sclerosis [48]. It has also been shown that transgenic mice containing an extra copy of the human SOD1 gene display a similar phenotype to Down syndrome, including neurological defects and premature aging [49].
超氧化物歧化酶 1 （SOD1） 是一种同源二聚体酶，可以将 O₂ ⁻ 转化为 O₂ 和 H₂O₂ （2 O₂ ⁻ + 2 H⁺ → O₂ + H₂O₂）。这种蛋白质也存在于细胞质、细胞核和线粒体中，通过与含有 PTS1 的生理相互作用伙伴“SOD1 的铜伴侣”复合的背负机制导入过氧化物酶体[46]。 目前，假设 SOD1 和过氧化氢酶是保护细胞免受 ROS 损伤的短代谢途径 [31]。 此外，由于 O₂ ⁻ 可以与 NO 快速反应 形成 ^ONOO−，因此 SOD1 在减少亚硝化应激方面发挥着重要作用 [47]。 有趣的是，SOD1 基因突变约占家族性肌萎缩侧索硬化症患者的 20-25%[48]。 研究还表明，含有额外人 SOD1 基因拷贝的转基因小鼠表现出与唐氏综合征相似的表型，包括神经缺陷和过早衰老[49]。

Peroxiredoxin 5 (PRDX5) is a thiol-dependent monomeric peroxidase that can reduce H₂O₂ to H₂O, alkyl hydroperoxides (ROOH) to their respective alcohols (ROH), and ONOO⁻ to nitrite (ONO⁻) [50]. The reducing equivalents needed for these reactions are thought to be provided by thioredoxins (TRXs), a group of small, multifunctional proteins that act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange [51]. Interestingly, human PRDX5 has been shown to react much faster with ONOO⁻ (~ 10⁷ M^− 1 s^− 1) and ROOH (~ 10⁶ M^− 1 s^− 1) than with H₂O₂ (~ 10⁵ M^− 1 s^− 1) [52]. In addition, this enzyme has been found in different subcellular compartments including the cytosol, the nucleus, mitochondria and peroxisomes [53]. Currently, it is well-established that mammalian PRDX5 is targeted to peroxisomes by virtue of a PTS1 targeting signal [54]. The exact function of this protein inside peroxisomes is not yet known. On one hand, it is thought that PRDX5 may assist catalase in the removal of H₂O₂. However, based on the measured rate constants, it is more likely that the enzyme functions in the detoxification process of peroxynitrite and lipid peroxides [50]. Finally, it should be noted that the physiological electron donor for PRDX5 in the peroxisomal matrix remains to be identified.
过氧化还原蛋白 5（peroxiredoxin 5， PRDX5）是一种硫醇依赖性单体过氧化物酶，可将 H₂O₂ 还原为 H₂O，烷基氢过氧化物（ROOH）可还原为各自的醇（ROH），将 ^ONOO− 还原为亚硝酸盐（ONO⁻）[50]。 这些反应所需的还原当量被认为是由硫氧还蛋白（TRX）提供的，TRX 是一组小型的多功能蛋白质，通过半蛋白硫醇-二硫键交换促进其他蛋白质的还原，从而充当抗氧化剂[51]。 有趣的是，人类 PRDX5 与 ONOO⁻（~ 10⁷ M^− 1 s^− 1）和 ROOH（~ 10⁶ M^− 1 s^− 1）的反应速度比与 H₂O₂（~ 10⁵ M^− 1 s^− 1）的反应要快得多 [52].此外，这种酶还存在于不同的亚细胞区室中，包括细胞质、细胞核、线粒体和过氧化物酶体[53]。 目前，哺乳动物 PRDX5 凭借 PTS1 靶向信号靶向过氧化物酶体已经得到充分证实 [54]。这种蛋白质在过氧化物酶体内的确切功能尚不清楚。一方面，人们认为 PRDX5 可能有助于过氧化氢酶去除 H₂O₂。然而，根据测量的速率常数，该酶更有可能在过氧亚硝酸盐和脂质过氧化物的解毒过程中发挥作用 [50]。 最后，应该注意的是，过氧化物酶体基质中 PRDX5 的生理电子供体仍有待鉴定。

Glutathione S-transferases (GST) are a superfamily of enzymes that catalyze the conjugation of xenobiotics and potentially damaging oxidative metabolites with glutathione [55]. Currently, there is some evidence that both glutathione S-transferase kappa (GSTK1) and ‘microsomal’ glutathione S-transferase 1 (MGST1) are at least partially located in peroxisomes [56], [57]. Both enzymes have also been found in mitochondria, and – as can be deduced from its name – MGST1 is also present in the ER [58], [59]. The peroxisomal localization of GSTK1, which is a soluble homodimeric enzyme, depends on a PTS1 sequence [56]. How the membrane-bound homotrimeric protein MGST1 is targeted to peroxisomes is not yet known [57]. The exact functions of GSTK1 and MGST1 within peroxisomes remain to be established. However, based on their in vitro activities, it has been suggested that these proteins may play an important role in the detoxification of xenobiotic compounds and lipid peroxide products [31], [57].
谷胱甘肽 S-转移酶（GST）是一种酶超家族，可催化异生素与谷胱甘肽偶联，并可能破坏氧化代谢物 [55]。 目前，有一些证据表明，谷胱甘肽 S-转移酶κ（glutathione S-transferase kappa， GSTK1）和“微粒体”谷胱甘肽 S-转移酶 1（mgst1）都至少部分位于过氧化物酶体中 [56]， [57]。 这两种酶也存在于线粒体中，并且从其名称中可以推断出，MGST1 也存在于内质网中 [58]、[59]。GSTK1 是一种可溶性同源二聚体酶，其过氧化物酶体定位取决于 PTS1 序列[56]。 膜结合同源三聚体蛋白 MGST1 如何靶向过氧化物酶体尚不清楚 [57]。GSTK1 和 MGST1 在过氧化物酶体内的确切功能仍有待确定。然而，基于它们的体外活性，有人认为这些蛋白质可能在异源化合物和脂质过氧化物产物的解毒中发挥重要作用 [31]，[57]。

Epoxide hydrolase 2 (EPHX2) is a homodimeric enzyme that can bind epoxides and convert them to the corresponding dihydrothiols [60]. The protein, found in both the cytosol and peroxisomes, contains a relatively weak PTS1 targeting signal [61]. Currently, it is thought that the main physiological role of EPHX2 is to detoxify fatty-acid derived epoxides [62]. Interestingly, certain polymorphisms in the human gene are associated with an increased risk of atherosclerosis and cardiovascular diseases [63].
环氧化物水解酶 2（EPHX2）是一种同源二聚体酶，可以结合环氧化物并将其转化为相应的二氢硫醇[60]。 这种蛋白质存在于细胞质和过氧化物酶体中，含有相对较弱的 PTS1 靶向信号 [61]。 目前，人们认为 EPHX2 的主要生理作用是解毒脂肪酸衍生的环氧化物 [62]。 有趣的是，人类基因中的某些多态性与动脉粥样硬化和心血管疾病的风险增加有关 [63]。

Free radicals can also be scavenged by non-enzymatic low molecular weight antioxidants. These compounds, which are either water- or lipid-soluble, are synthesized within the body or supplemented by diet [64]. The low molecular weight antioxidants that will be discussed in more detail below include glutathione, ascorbic acid, and plasmalogens.
自由基也可以被非酶低分子量抗氧化剂清除。这些化合物可溶于水或脂溶性，可在体内合成或通过饮食补充 [64]。 下面将更详细讨论的低分子量抗氧化剂包括谷胱甘肽、抗坏血酸和缩醛磷脂。

Glutathione, a cysteinyl tripeptide, is one of the most prevalent and important redox buffers of the cell [65]. The molecule can exist in either a reduced (GSH) or oxidized (GSSG) state. GSH, the biologically active form, can participate in numerous redox reactions and is oxidized to GSSG during oxidative and nitrosative stress conditions [7]. GSSG is reduced back to GSH by an NADPH-dependent glutathione reductase (GR, EC 1.8.1.7). Currently, it is widely accepted that the GSH/GSSG ratio reflects the redox capacity of a cell [66]. Nevertheless, it is also clear that the GSH/GSSG redox couple functions in conjunction with redox proteins such as TRXs and glutaredoxins (GRXs) [5]. GSH is exclusively synthesized in the cytosol, from where it is transferred into other cellular compartments [65]. Whether or not peroxisomes contain a functional glutathione redox system is not yet clear. For example, there is some evidence that GSH may freely penetrate the peroxisomal membrane through PXMP2, a non-selective pore-forming protein with an upper molecular size limit of 300–600 Da [67]. In addition, it has been shown that roGFP2, a genetically-encoded probe that senses changes in the glutathione redox potential via GRX [68], responds quickly and reversibly to redox changes in the peroxisomal matrix (for more details, see Section 3.1.1.) [69]. However, it remains to be investigated how oxidized roGFP2 and GSSG are reduced inside the peroxisomal matrix, and – in case the organelles would completely lack glutathione reductase activity – how GSSG is exported back into the cytosol.
谷胱甘肽是一种半胱氨酰三肽，是细胞中最普遍和最重要的氧化还原缓冲液之一[65]。 该分子可以以还原 （GSH） 或氧化 （GSSG） 状态存在。GSH 是一种生物活性形式，可以参与多种氧化还原反应，并在氧化和亚硝化应激条件下被氧化为 GSSG[7]。GSSG 通过 NADPH 依赖性谷胱甘肽还原酶（GR，EC 1.8.1.7）还原回 GSH。目前，人们普遍认为 GSH/GSSG 比值反映了电池的氧化还原能力 [66]。 然而，同样明显的是，GSH/GSSG 氧化还原偶与 TRX 和谷氨酸（GRX）等氧化还原蛋白共同发挥作用 [5]。GSH 仅在细胞质中合成，从细胞质中转移到其他细胞区室中[65]。 过氧化物酶体是否含有功能性谷胱甘肽氧化还原系统尚不清楚。例如，有一些证据表明 GSH 可以通过 PXMP2 自由穿透过氧化物酶体膜，PXMP2 是一种非选择性成孔蛋白，分子大小上限为 300-600 Da[67]。 此外，研究表明，roGFP2 是一种基因编码的探针，可通过 GRX 感知谷胱甘肽氧化还原电位的变化[68]，对过氧化物酶体基质中的氧化还原变化做出快速、可逆的反应（有关更多详细信息，请参见第 3.1.1 节 ）。[69]。 然而，氧化的 roGFP2 和 GSSG 如何在过氧化物酶体基质内还原，以及 GSSG 如何输出回细胞质，仍有待研究。

Ascorbic acid (vitamin C) is a water-soluble dietary supplement that can act as cofactor and antioxidant [70]. In peroxisomes, the compound has been reported to be necessary for maximal activity of phytanoyl-CoA 2-hydroxylase, a peroxisomal α-oxidation enzyme that catalyzes the 2-hydroxylation of 3-methyl-branched acyl-CoAs [71]. Whether or not ascorbic acid also has an antioxidant function inside mammalian peroxisomes is less clear. Indeed, it has recently been demonstrated that cultivating mammalian cells in the presence of ascorbic acid actually results in an increased redox state of the peroxisomal matrix [69]. This may be explained by the combined findings that (i) peroxisomes contain relatively large amounts of heme- and non-heme iron-containing enzymes [72], and (ii) ascorbic acid can reduce transition metals, and this may in turn lead to the generation of free radicals through the Fenton reaction [70].
抗坏血酸（维生素 C）是一种水溶性膳食补充剂，可作为辅助因子和抗氧化剂[70]。 据报道，在过氧化物酶体中，该化合物是植烷酰辅酶 A-2-羟化酶的最大活性所必需的，植烷酰辅酶 A-2-羟化酶是一种过氧化物酶体α氧化酶，可催化 3-甲基支链酰基辅酶 A 的 2-羟基化[71]。 抗坏血酸在哺乳动物过氧化物酶体内是否也具有抗氧化功能尚不清楚。事实上，最近有研究表明，在抗坏血酸存在下培养哺乳动物细胞实际上会导致过氧化物酶体基质的氧化还原状态增加 [69]。 这可以通过以下综合发现来解释：（i）过氧化物酶体含有相对大量的含血红素和非血红素铁的酶 [72]，以及（ii）抗坏血酸可以减少过渡金属，这反过来又可能导致通过 Fenton 反应产生自由基 [70]。

Another interesting group of non-enzymatic antioxidants may be plasmalogens. Indeed, albeit the physiological function of this specific class of glycerolipids is still poorly understood, there is currently good evidence that these molecules – which are produced from peroxisome-derived intermediates – may act as radical scavengers [26]. For example, it has been shown that plasmalogens are able to protect unsaturated membrane lipids against oxidation by singlet oxygen without producing oxidation products that are excessively toxic [73]. In addition, cells deficient in peroxisome assembly and/or plasmalogen biosynthesis are several orders of magnitude more sensitive to UV-induced ROS production than control cells [27].
另一组有趣的非酶促抗氧化剂可能是缩醛磷脂。事实上，尽管人们对这一类特定甘油脂的生理功能知之甚少，但目前有充分的证据表明，这些分子（由过氧化物酶体衍生的中间体产生）可能充当自由基清除剂 [26]。 例如，已经表明，缩醛磷脂能够保护不饱和膜脂免受单线态氧的氧化，而不会产生毒性过大的氧化产物 [73]。 此外，缺乏过氧化物酶体组装和/或缩醛磷脂生物合成的细胞对紫外线诱导的 ROS 产生的敏感性比对照细胞高几个数量级[27]。

Redox-sensitive green fluorescent proteins (roGFPs) are currently the most commonly used probes for monitoring the organellar redox state in living cells. Members of this class of proteins contain engineered cysteine residues on adjacent surface-exposed β-strands that form a disulfide bond under oxidizing conditions [78]. As oxidation of the dithiol pair causes reciprocal changes in emission intensity when excited at ~ 400 and ~ 490 nm, the ratio of roGFP emissions (at ~ 510 nm) can provide a non-destructive read-out of the redox environment of the fluorophore [77], [78]. RoGFPs preferentially interact with GRXs, indicating that these probes most likely equilibrate with the local glutathione redox potential [79]. As distinct subcellular compartments often have different redox environments, various roGFPs with different reduction potentials of the disulphide have been developed [68], [80]. We have recently shown that roGFP2 is a suitable probe to monitor redox changes in the peroxisomal matrix in living cells [69].
氧化还原敏感绿色荧光蛋白 （roGFP） 是目前最常用的探针，用于监测活细胞中细胞器的氧化还原状态。这类蛋白质的成员在相邻的表面暴露β链上含有工程半胱氨酸残基，这些残基在氧化条件下形成二硫键 [78]。 由于二硫醇对的氧化在~ 400 和~ 490 nm 激发时会导致发射强度发生相互变化，因此 roGFP 发射比（在~ 510 nm 处）可以提供荧光团氧化还原环境的无损读数 [77]、[78]。RoGFP 优先与 GRX 相互作用，表明这些探针很可能与局部谷胱甘肽氧化还原电位平衡[79]。 由于不同的亚细胞区室通常具有不同的氧化还原环境，因此已经开发出具有不同二硫化物还原电位的各种 roGFPs[68]、[80]。 我们最近表明，roGFP2 是监测活细胞中过氧化物酶体基质氧化还原变化的合适探针 [69]。

HyPer is a ratiometric probe specifically developed to detect H₂O₂ [81]. The protein, which consists of a circularly permutated yellow fluorescent protein inserted into the regulatory domain of a prokaryotic H₂O₂-sensing protein, contains two cysteine residues that are specifically oxidized by H₂O₂, but not by O₂⁻, ONOO⁻, and GSSG [82], [83]. HyPer exhibits two excitation peaks at ~ 420 and ~ 500 nm and one emission peak at ~ 516 nm. Rise in H₂O₂ concentration results in a decrease in the 420 nm excitation peak and a proportional increase in the 500 nm excitation peak [81]. Interestingly, the half maximal effective concentration of HyPer for H₂O₂ is ± 8 μM, which is almost 25 times less than that of roGFP [83]. A potential disadvantage of this probe is that it is rather pH-sensitive [81]. Recently, HyPer has been used to demonstrate that H₂O₂ produced inside peroxisomes is an important mediator of lipotoxicity in insulin-producing cells [84].
HyPer 是专门为检测 H₂O₂ 而开发的比例探头 [81]。 该蛋白由插入原核 H₂O₂ 感应蛋白的调控结构域中的环状排列的黄色荧光蛋白组成，含有两个半胱氨酸残基，它们被 H₂O₂ 特异性氧化，但不被 O₂ ⁻、^ONOO− 和 GSSG 氧化[82]， [83].HyPer 在~ 420 和~ 500 nm 处表现出两个激发峰，在~ 516 nm 处表现出一个发射峰。H₂O₂ 浓度的升高导致 420 nm 激发峰降低，500 nm 激发峰成比例增加[81]。 有趣的是，HyPer 对 H₂O₂ 的半最大有效浓度为± 8 μM，几乎是 roGFP 的 25 倍 [83]。 该探针的一个潜在缺点是它对 pH 值相当敏感 [81]。 最近，HyPer 已被用于证明过氧化物酶体内产生的 H₂O₂ 是胰岛素产生细胞中脂毒性的重要介质 [84]。

Redoxfluor is a recently developed fluorescence resonance energy transfer (FRET)-based probe that can sense the redox potential of glutathione via its internal disulfide bonds [74]. The protein contains a tandem repeat of a partial region within the carboxy-terminal cysteine-rich domain of Yap1, a transcription factor crucial for oxidative stress response in Saccharomyces cerevisiae, and mediates FRET between cerulean, a variant of the cyan fluorescent protein (CFP), and citrine, a yellow fluorescent protein (YFP) derivative [85]. Upon oxidation, CFP emission is enhanced at the expense of YFP emission, thereby decreasing the yellow-to-cyan emission ratio (527/476 nm). Recently, this probe has been successfully used to develop an efficient screening system for redox modulators that can restore the redox status in mammalian cell lines with defective peroxisome assembly without affecting the redox status of normal cells [74].
氧化还原荧光是最近开发的一种基于荧光共振能量转移（FRET）的探针，可以通过谷胱甘肽的内部二硫键感知谷胱甘肽的氧化还原电位[74]。 该蛋白含有 Yap1 羧基末端富含半胱氨酸结构域内部分区域的串联重复序列，Yap1 是酿酒酵母氧化应激反应的关键转录因子，可介导青色荧光蛋白（CFP）的变体天蓝色和黄水晶（黄色荧光蛋白（YFP）衍生物）之间的 FRET[85].氧化后，CFP 发射以牺牲 YFP 发射为代价增强，从而降低黄青色发射比（527/476 nm）。最近，该探针已成功用于开发一种有效的氧化还原调节剂筛选系统，该系统可以在不影响正常细胞的氧化还原状态的情况下恢复过氧化物酶体组装缺陷的哺乳动物细胞系的氧化还原状态[74]。

Already more than a decade ago, several studies reported that the peroxisomal redox balance can be disturbed by the selective activation of peroxisomal metabolism. For example, after Reddy and colleagues discovered that the administration of peroxisome proliferators to rodents can induce liver cancer [86], they also studied the effect of such a treatment on oxidative stress and found a disproportionate increase in H₂O₂-producing enzymes and catalase [22], [87]. Other studies showed that (i) mammalian cells stably overexpressing peroxisomal fatty acyl-CoA oxidase (FAO) became neoplastic upon exposure to a fatty acid substrate for 2–6 weeks [88], [89], (ii) a transient overexpression of peroxisomal FAO in Cos-1 cells led to nuclear NFKB1 DNA binding activity in a dose-dependent manner [90], and (iii) a chronic exposure of pancreatic β-cells to long-chain non-esterified fatty acids increased peroxisomal H₂O₂-production, ultimately leading to β-cell death [91]. In summary, these findings demonstrate that it is technically possible to induce peroxisomal oxidative stress in intact cells by stimulating the organelle's metabolism.
早在十多年前，几项研究就报告说，过氧化物酶体氧化还原平衡会因过氧化物酶体代谢的选择性激活而受到干扰。例如，在 Reddy 及其同事发现对啮齿动物施用过氧化物酶体增殖剂可诱发肝癌后[86]，他们还研究了这种治疗对氧化应激的影响，发现产生 H₂O₂ 的酶和过氧化氢酶不成比例地增加 [22]，[87].其他研究表明，（i）稳定过表达过氧化物酶体脂肪酰基辅酶 A 氧化酶（FAO）的哺乳动物细胞在暴露于脂肪酸底物 2-6 周后变成肿瘤[88]，[89]，（ii）过氧化物酶体 FAO 在 Cos-1 细胞中的短暂过表达导致核 NFKB1 DNA 以剂量依赖性方式结合活性 [90]，以及（iii）胰腺β细胞长期暴露于长链非酯化脂肪酸会增加过氧化物酶体 _H2O2 的产生，最终导致β细胞死亡[91]。 总之，这些发现表明，在技术上可以通过刺激细胞器的代谢在完整细胞中诱导过氧化物酶体氧化应激。

As catalase is the most abundant antioxidant enzyme in mammalian peroxisomes, it represents an attractive target to modulate the organelle's antioxidative stress system in intact cells. Several completely different approaches have already been used for this purpose. Perhaps the most straightforward way is to incubate the cells with 3-amino-1,2,4-triazole (3-AT), a well-characterized irreversible inhibitor of catalase [92]. At non-cytotoxic dosages, such a treatment may decrease catalase activity by ± 70% [93]. Alternatively, one may employ genetically modified cells (e.g. from transgenic or knockout mice) [45], [94], vector-driven expression systems [69], [95], or even cell-penetrating catalase derivatives [96], [97]. In this context, it is interesting to mention that both reducing and increasing catalase activity may make cells more vulnerable to different types of oxidative stress [94], [96]. These findings indicate that nonlethal concentrations of H₂O₂ may exert net beneficial effects on the cell, and that catalase overexpression may interfere with the potential signaling mechanisms of H₂O₂ [94]. The effects of altered catalase activities on human health will be discussed in Section 6.
由于过氧化氢酶是哺乳动物过氧化物酶体中最丰富的抗氧化酶，因此它是调节完整细胞中细胞器抗氧化应激系统的有吸引力的靶点。为此，已经使用了几种完全不同的方法。也许最直接的方法是将细胞与 3-氨基-1,2,4-三唑（3-AT）一起孵育，3-AT 是一种特征明确的过氧化氢酶不可逆抑制剂 [92]。 在非细胞毒性剂量下，这种治疗可能会使过氧化氢酶活性降低± 70%[93]。 或者，可以使用转基因细胞（例如来自转基因或基因敲除小鼠）[45]、[94]、载体驱动的表达系统 [69]、[95]，甚至细胞穿透过氧化氢酶衍生物 [96]、[97].在这种情况下，有趣的是，降低和增加过氧化氢酶活性都可能使细胞更容易受到不同类型的氧化应激的影响[94]、[96]。 这些发现表明，非致死浓度的 H₂O₂ 可能对细胞产生净有益作用，过氧化氢酶过表达可能会干扰 H₂O₂ 的潜在信号机制 [94]。 过氧化氢酶活性改变对人类健康的影响将在第 6 节  中讨论。

Another strategy that has already been successfully employed to selectively produce oxidative stress inside peroxisomes is the use of KillerRed, a genetically-encoded photosensitizer which produces radicals and H₂O₂ upon green light illumination [98], [99]. By using a peroxisomal variant of this protein, we recently found that excessive ROS production inside peroxisomes may disturb the mitochondrial redox balance, and this can in turn lead to mitochondrial fragmentation [69]. An alternative method that may be better suited for biochemical investigations are transgenic cells overexpressing d-amino acid oxidase, a bona fide peroxisomal matrix protein [100]. Indeed, a nuclear targeted version of this protein has already successfully been used to study the effects of nuclear localized oxidative stress [101]. In addition, it is most likely easier to control the production of H₂O₂ in a d-amino acid-regulatable system than in a peroxisome proliferator- or free fatty acid-dependent system. Finally, one may consider altering the activities of other peroxisomal antioxidant enzymes. However, as most of these enzymes are located in multiple cellular compartments (see Section 2.2.), it is virtually impossible to study the peroxisome-specific effects on the cellular phenotype by knocking down their expression (e.g. through RNA interference). Nevertheless, one may solve this problem, at least partially, by artificially targeting these enzymes to peroxisomes by the use of a strong PTS1 (see Section 4.3.2).
另一种已经成功用于在过氧化物酶体内选择性产生氧化应激的策略是使用 KillerRed，这是一种基因编码的光敏剂，在绿光照射下产生自由基和 H₂O₂[98]，[99]。 通过使用这种蛋白质的过氧化物酶体变体，我们最近发现过氧化物酶体内过多的 ROS 产生可能会扰乱线粒体氧化还原平衡，进而导致线粒体碎片化 [69]。 另一种可能更适合生化研究的方法是过表达 d-氨基酸氧化酶的转基因细胞，d-氨基酸氧化酶是一种真正的过氧化物酶体基质蛋白[100]。 事实上，这种蛋白质的核靶向版本已经成功地用于研究核局部氧化应激的影响 [101]。 此外，在 d-氨基酸调节系统中控制 H₂O₂ 的产生很可能比在过氧化物酶体增殖物或游离脂肪酸依赖性系统中更容易。最后，可以考虑改变其他过氧化物酶体抗氧化酶的活性。然而，由于这些酶中的大多数位于多个细胞区室中（见第 2.2 节 ），因此几乎不可能通过敲低过氧化物酶体的表达（例如通过 RNA 干扰）来研究过氧化物酶体对细胞表型的特异性影响。然而，通过使用强 PTS1 人为地将这些酶靶向过氧化物酶体，可以至少部分解决这个问题（见第 4.3.2 节 ）。

The intracellular localization and activity of numerous signaling proteins and transcription factors are, directly or indirectly, controlled by the oxidation of thiol groups of redox-sensitive cysteine residues [9], [110]. For example, it has been shown that some transcription factors (e.g. nuclear factor (erythroid-derived 2)-like 2) possess reversibly oxidizable cysteines in their active site, and that – upon exposure to ROS – their sulfhydryl groups are oxidatively modified, which may cause their nuclear translocation and lead to enhanced cytoprotective gene expression [111], [112]. Currently, there is strong evidence that peroxisomal H₂O₂ may function as an important modulator of NFKB1, a pleiotropic transcription factor that is involved in many biological processes, including inflammation, immunity, differentiation, cell growth, tumorigenesis, and apoptosis [113]. For example, it has been reported that a transient overexpression of the H₂O₂-producing peroxisomal enzyme FAO resulted in a concentration-dependent DNA binding activity of NFKB1 DNA in Cos-1 cells, and that this phenomenon could be counteracted by overexpression of catalase [90]. In addition, it has been shown that a genetic or pharmacological inactivation of catalase in mouse neutrophils inhibited the nuclear accumulation of NFKB1 and the production of the proinflammatory cytokines TNF-α and MIP-2 [114]. Note that, to make sense of these apparently conflicting data, it is important to know that – depending on the cell type and the experimental conditions – H₂O₂ may stimulate or inhibit the NFKB1 activation pathway [115]. Despite this, these data clearly show that the production of H₂O₂ inside peroxisomes can affect nuclear gene expression. As the peroxisomal localization of NOS2 has been associated with a reduced expression level of catalase in hepatocytes [35], the same might be true for peroxisomal NO production. However, note that – as (i) only a fraction of NOS2 is localized in peroxisomes (see Section 2.1.2.), and (ii) it is not yet clear whether or not peroxisomal NOS2 effectively produces NO (see Section 2.2.2.) – this remains to be investigated in more detail.
许多信号蛋白和转录因子的细胞内定位和活性直接或间接受到氧化还原敏感半胱氨酸残基的硫醇基团的氧化控制[9]，[110]。 例如，已经表明，一些转录因子（例如核因子（红系衍生的 2）样 2）在其活性位点具有可逆的可氧化半胱氨酸，并且在暴露于 ROS 后，它们的巯基被氧化修饰，这可能导致它们的核易位并导致细胞保护基因表达增强[111]、[112]。 目前，有强有力的证据表明，过氧化物酶体 H₂O₂ 可能作为 NFKB1 的重要调节因子，NFKB1 是一种多效性转录因子，参与许多生物学过程，包括炎症、免疫、分化、细胞生长、肿瘤发生和细胞凋亡[113]。 例如，据报道，产生 H₂O₂ 的过氧化物酶 FAO 的瞬时过表达导致 NFKB1 DNA 在 Cos-1 细胞中具有浓度依赖性 DNA 结合活性，并且这种现象可以通过过氧化氢酶的过表达来抵消 [90]。 此外，研究表明，小鼠中性粒细胞中过氧化氢酶的遗传或药理学失活抑制了 NFKB1 的核积累以及促炎细胞因子 TNF-α和 MIP-2 的产生[114]。 请注意，为了理解这些明显相互矛盾的数据，重要的是要知道，根据细胞类型和实验条件，H₂O₂ 可能会刺激或抑制 NFKB1 激活途径 [115]。 尽管如此，这些数据清楚地表明，过氧化物酶体内 H₂O₂ 的产生会影响核基因表达。由于 NOS2 的过氧化物酶体定位与肝细胞中过氧化氢酶表达水平降低有关 [35]，因此过氧化物酶体 NO 的产生可能也是如此。然而，请注意，由于 （i） 只有一小部分 NOS2 位于过氧化物酶体中（见第 2.1.2 节 ），以及 （ii） 尚不清楚过氧化物酶体 NOS2 是否有效产生 NO （见第 2.2.2 节 ）——这仍有待更详细地研究。

Peroxisomes and mitochondria exhibit a functional interplay that continues to emerge [116], [117]. Over the past years, it has become clear that these organelles also share a redox-sensitive relationship. For example, it has been shown that catalase-SKL, a catalase derivative with enhanced peroxisome targeting, can efficiently repolarize mitochondria in late passage cells [118]. In addition, we recently found that the mitochondrial redox balance is disturbed in cells lacking catalase or functional peroxisomes, and upon the generation of excess ROS inside peroxisomes [69; Apanasets and Fransen, unpublished results]. On the other hand, peroxisomes were found to resist oxidative stress generated inside mitochondria [69]. In summary, these findings suggest that peroxisome-derived oxidative stress may trigger signaling/communication events that ultimately result in increased mitochondrial stress. The molecular mechanisms underlying this phenomenon remain unclear. A number of molecules may participate in linking these organelles. For example, peroxisomes initiate oxidative metabolism of specific fatty acids which are then trafficked to mitochondria where processing is completed [119]. The organelles also communicate via anaplerotic metabolism [17]. It can be envisioned that as peroxisomal metabolism is slowed down, critical metabolic intermediates (e.g. acetyl-CoA) are not properly produced and trafficked to mitochondria [69]. If mitochondrial metabolism is similarly disrupted, an increase in uncoupled reactions and ROS production can certainly be anticipated. There also exists the mitochondrial retrograde signaling pathway, a process involving multiple factors that sense mitochondrial dysfunction and transmit signals to the nucleus to affect gene expression [120]. The protein products of these genes promote, among other processes, peroxisomal β-oxidation and peroxisome proliferation [17]. Finally, the organelles may also interact via reactive oxygen species.
过氧化物酶体和线粒体表现出持续出现的功能相互作用 [116]、[117]。 在过去的几年里，很明显这些细胞器也具有氧化还原敏感关系。例如，研究表明，过氧化氢酶-SKL 是一种具有增强过氧化物酶体靶向的过氧化氢酶衍生物，可以有效地使晚传代细胞中的线粒体重极化[118]。 此外，我们最近发现，在缺乏过氧化氢酶或功能性过氧化物酶体的细胞中，线粒体氧化还原平衡受到干扰，并且在过氧化物酶体内产生过量的 ROS 时[69;Apanasets 和 Fransen，未发表的结果]。另一方面，研究发现过氧化物酶体可以抵抗线粒体内产生的氧化应激 [69]。 总之，这些发现表明过氧化物酶体衍生的氧化应激可能会触发信号传导/通讯事件，最终导致线粒体应激增加。这种现象背后的分子机制尚不清楚。许多分子可能参与连接这些细胞器。例如，过氧化物酶体启动特定脂肪酸的氧化代谢，然后被转运到线粒体，在那里完成加工[119]。 细胞器还通过同步代谢进行交流 [17]。 可以想象，随着过氧化物酶体代谢减慢，关键代谢中间体（例如乙酰辅酶 A）无法正常产生并转运到线粒体[69]。 如果线粒体代谢同样受到破坏，则当然可以预期不偶联反应和 ROS 产生的增加。 还存在线粒体逆行信号通路，这是一个涉及多种因素的过程，这些因素感知线粒体功能障碍并将信号传递到细胞核以影响基因表达[120]。 这些基因的蛋白质产物促进过氧化物酶体β氧化和过氧化物酶体增殖等过程[17]。 最后，细胞器还可以通过活性氧相互作用。