Elsevier

Redox Biology  氧化还原生物学

Volume 55, September 2022, 102410
第 55 卷，2022 年 9 月，102410
Redox Biology

Transfer of H2O2 from Mitochondria to the endoplasmic reticulum via Aquaporin-11
H 2 O 2通过 Aquaporin-11 从线粒体转移到内质网

https://doi.org/10.1016/j.redox.2022.102410 IF: 10.7 Q1 Get rights and content  获取权利和内容
Under a Creative Commons license
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Highlights  亮点

  • •
    Silencing Ero1α causes a paradoxical increase of H2O2 in the ER.
    沉默Ero1α会导致 ER 中 H 2 O 2的反常增加。
  • •
    Mitochondrial ETC complex III is the main source of these molecules.
    线粒体 ETC复合体 III是这些分子的主要来源。
  • •
    H2O2 from mitochondria reaches the ER via AQP11.
    来自线粒体的H 2 O 2通过 AQP11 到达 ER。

Abstract  抽象的

Some aquaporins (AQPs) can transport H2O2 across membranes, allowing redox signals to proceed in and between cells. Unlike other peroxiporins, human AQP11 is an endoplasmic reticulum (ER)-resident that can conduit H2O2 to the cytosol. Here, we show that silencing Ero1α, an ER flavoenzyme that generates abundant H2O2 during oxidative folding, causes a paradoxical increase in luminal H2O2 levels. The simultaneous AQP11 downregulation prevents this increase, implying that H2O2 reaches the ER from an external source(s). Pharmacological inhibition of the electron transport chain reveals that Ero1α downregulation activates superoxide production by complex III. In the intermembrane space, superoxide dismutase 1 generates H2O2 that enters the ER channeled by AQP11. Meanwhile, the number of ER-mitochondria contact sites increases as well, irrespective of AQP11 expression. Taken together, our findings identify a novel interorganellar redox response that is activated upon Ero1α downregulation and transfers H2O2 from mitochondria to the ER via AQP11.
一些水通道蛋白(AQP) 可以跨膜运输 H 2 O 2 ，​​从而允许氧化还原信号在细胞内和细胞之间进行。与其他过氧孔蛋白不同，人 AQP11 是一种内质网(ER) 驻留物，可以将 H 2 O 2引导至细胞质。在这里，我们发现沉默 Ero1α（一种在氧化折叠过程中产生大量H 2 O 2 的内质网黄素酶）会导致管腔 H 2 O 2水平的反常增加。同时 AQP11下调阻止了这种增加，这意味着 H 2 O 2从外部来源到达 ER。电子传递链的药理学抑制表明 Ero1α下调可激活复合物 III 产生超氧化物。在膜间隙中，超氧化物歧化酶 1产生 H 2 O 2 ，通过 AQP11 进入内质网。同时，无论 AQP11 表达如何，ER-线粒体接触位点的数量也会增加。总而言之，我们的研究结果确定了一种新型细胞器间氧化还原反应，该反应在 Ero1α 下调时被激活，并通过 AQP11 将 H 2 O 2从线粒体转移到内质网。

Keywords  关键词

Hydrogen peroxide
Redox homeostasis
Interorganellar crosstalk/ peroxiporin
Complex III
Mitochondrial-associated membranes

过氧化氢
氧化还原稳态
细胞器间串扰/过氧孔蛋白
复合物III
线粒体相关膜

Abbreviations  缩写

AQP
aquaporin
ER
Endoplasmic Reticulum
Ero1α
ER oxidoreductin 1- α
H2O2
Hydrogen Peroxide
MAM
mitochondria-associated ER membranes
NOX
NADPH oxidase
Mfn2
mitofusin-2
PDI
protein disulfide isomerase
SOD1
Superoxide Dismutase 1
VAPB
vesicle-associated membrane protein-associated protein B

空气质量蛋白
水通道蛋白
急诊室
内质网
埃罗1α
ER氧化还原素1-α
H 2 O 2
过氧化氢
MAM
线粒体相关内质网膜
氮氧化物
NADPH氧化酶
MFn2
丝裂霉素-2
PDI
蛋白质二硫键异构酶
超氧化物歧化酶1
超氧化物歧化酶1
VAPB
囊泡相关膜蛋白相关蛋白B

1. Introduction  一、简介

The endoplasmic reticulum (ER) is a multifunctional organelle that acts as the cradle for many proteins and lipids, a calcium store, and a central signaling hub. All these systems must act coordinately to preserve functional integrity. A demanding task is to generate and maintain optimal redox conditions for the formation and rearrangement of disulfide bonds in proteins destined to the extracellular space [1,2]. Key in these processes is the ER oxidoreductin 1- protein disulfide isomerase (Ero1-PDI) pathway. Oxidized PDI donates disulfide bonds to newly made cargo proteins and is recharged by the oxidases Ero1α or Ero1β [3,4]. These flavoenzymes can transfer electrons directly to molecular oxygen, generating H2O2 as secondary product [[5], [6], [7]]. Besides protein relays capable of precisely targeting redox reactions [8,9], small non-protein thiols and oxidants participate in oxidative folding [10], including H2O2 itself [11]. Therefore, not only does the ER environment sustain oxidative folding, but disulfide bond formation itself generates reactive by-products, defining a mutual interdependence [12,13].
内质网(ER) 是一种多功能细胞器，充当许多蛋白质和脂质的摇篮、钙储存库和中央信号中枢。所有这些系统必须协调行动以保持功能完整性。一项艰巨的任务是生成并维持最佳的氧化还原条件，以便在前往细胞外空间的蛋白质中形成和重排二硫键[ 1 , 2 ]。这些过程的关键是 ER 氧化还原素 1-蛋白二硫键异构酶(Ero1-PDI) 途径。氧化的 PDI 向新制造的货物蛋白提供二硫键，并由氧化酶 Ero1α 或 Ero1β 重新充电 [ 3 , 4 ]。这些黄素酶可以将电子直接转移到分子氧，产生H 2 O 2作为次级产物[ [5] , [6] , [7] ]。 除了能够精确靶向氧化还原反应的蛋白质中继[ 8 , 9 ]之外，小的非蛋白质硫醇和氧化剂也参与氧化折叠[ 10 ]，包括H 2 O 2本身[ 11 ]。因此，内质网环境不仅维持氧化折叠，而且二硫键形成本身也会产生反应性副产物，从而定义了相互依赖性[ 12 , 13 ]。
Redundant mechanisms allow higher eukaryotes to maintain a cysteine-rich proteome without severe proteostatic problems. For instance, while Ero1 knock-out is lethal in yeast and worms, rather minor phenotypes hallmark mammalian cells and mice [14,15]. Indeed, Prdx4, Gpx7 and Gpx8 are ER-resident enzymes that can use H2O2 to oxidize PDI and fuel disulfide bond formation upon Ero1α depletion [13,[16], [17], [18], [19]]. Also NADPH oxidase 4 (NOX4) [20] and VKOR are alternative H2O2 producers that could vicariate Ero1α [21]. H2O2 molecules produced during oxidative folding -particularly abundant in cells with a robust secretory profile-can be also used as signals, exploiting the steep ER-cytosol gradient [22]. As the ER membrane acts as a barrier against passive diffusion of H2O2 [23], channels are needed. One is AQP11, a resident peroxiporin that constitutively transports H2O2 outside the ER [22].
冗余机制允许高等真核生物维持富含半胱氨酸的蛋白质组，而不会出现严重的蛋白质抑制问题。例如，虽然 Ero1 敲除对于酵母和蠕虫来说是致命的，但哺乳动物细胞和小鼠的标志性表型却相当轻微 [ 14 , 15 ]。事实上，Prdx4、Gpx7 和 Gpx8 是内质网驻留酶，可以使用 H 2 O 2氧化 PDI 并在 Ero1α 耗尽时促进二硫键形成 [ 13 、 [16] 、 [17] 、 [18] 、 [19] ]。 NADPH 氧化酶4 (NOX4) [ 20 ] 和 VKOR 也是替代 Ero1α [ 21 ] 的替代 H 2 O 2产生者。氧化折叠过程中产生的 H 2 O 2分子（在具有强大分泌特性的细胞中尤其丰富）也可以利用陡峭的 ER-胞质梯度作为信号使用 [ 22 ]。由于 ER 膜充当 H 2 O 2被动扩散的屏障[ 23 ]，因此需要通道。 其中之一是 AQP11，一种常驻过氧孔蛋白，可将 H 2 O 2持续转运到 ER 之外 [ 22 ]。
Which are the source(s) of the H2O2 molecules that reach the cytosol via AQP11? To answer this question, we focused on two main suspects: NOX4 and Ero1α. Silencing the former slightly decreased H2O2 levels in the ER, albeit non significatively. In contrast, a paradoxical increase was observed upon Ero1α downregulation. Our results identify complex III coupled to the SOD1 fraction residing in the mitochondria intermembrane space, as the main source of H2O2 molecules that eventually enter the ER via AQP11, facilitated also by tightening of the ER-mitochondria contacts.
通过 AQP11 到达细胞质的 H 2 O 2分子的来源是什么？为了回答这个问题，我们关注两个主要嫌疑人：NOX4 和 Ero1α。沉默前者会稍微降低ER 中的H 2 O 2水平，尽管不显着。相反，在 Ero1α下调时观察到矛盾的增加。我们的结果确定了与位于线粒体膜间隙中的SOD1部分偶联的复合物 III ，作为 H 2 O 2分子的主要来源，最终通过 AQP11 进入内质网，这也通过加强内质网与线粒体接触来促进。

2. Materials and methods  2 材料与方法

2.1. Cell culture and generation of HeLa polyclonal stable cell lines
2.1. HeLa 多克隆稳定细胞系的细胞培养和生成

Stable HeLa transfectants expressing HyPer1 [24] in the ER lumen (HyPer ER Lumen) or in the mitochondrial matrix (HyPer Mito) were generated as previously described in Refs. [[22], [23], [24], [25]]. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) + GlutaMAXTM-I medium (Gibco, ThermoFisher) complemented with 10% fetal bovine serum (FBS; EuroClone) and 5 mg/ml penicillin-streptomycin (Lonza).
如之前参考文献中所述，生成在内质网腔 ( HyPer ER Lumen ) 或线粒体基质( HyPer Mito ) 中表达 HyPer1 [ 24 ]的稳定HeLa转染子。 [ [22] 、 [23] 、 [24] 、 [25] ]。将细胞维持在补充有10%胎牛血清（FBS；EuroClone）和5mg/ml青霉素-链霉素（Lonza）的Dulbecco改良Eagle培养基（DMEM）+GlutaMAXTM-I培养基（Gibco，ThermoFisher）中。

2.2. Plasmids, siRNAs, and transfection procedures
2.2.质粒、siRNA 和转染程序

The plasmid to express a myc-tagged ER-targeted catalase (ER-CAT) was a generous gift of Dr. E. Avezov (University of Cambridge, UK). The HyPer ER Lumen plasmid, kindly donated by Drs. E. Margittai and M. Geistz (Semmelweis University, Budapest, Hungary), served as template to generate the H2O2-insensitive HyPer vector (SyPher ER Lumen) using the primers Fw: AGATGGTCACTCTTTGCGCGAT and Rv: ATCGCGCAAAGAGTGACCATCT and validated by sequencing.
表达 myc 标签的 ER 靶向过氧化氢酶(ER-CAT) 的质粒是 E. Avezov 博士（英国剑桥大学）的慷慨捐赠。 HyPer ER Lumen质粒，由 Drs 捐赠。 E. Margittai 和 M. Geistz（Semmelweis 大学，布达佩斯，匈牙利）作为模板，使用引物 Fw：AGATGGTCACTCTTTGCGCGAT 和 Rv：ATCGCGCAAAGAGTGACCATCT 生成 H 2 O 2不敏感的 HyPer 载体（ SyPher ER Lumen ），并通过测序进行验证。
Transient transfections were performed onto 2 × 105 plated HeLa cells using JetPei (Polyplus) and cultured for further 48 h before either immunofluorescence or HyPer imaging confocal laser scanning.
使用JetPei (Polyplus)对2×10 5铺板的HeLa细胞进行瞬时转染，并在免疫荧光或HyPer成像共聚焦激光扫描之前进一步培养48小时。
The reagents to silence AQP11 (Custom siRNA, 5′-GAGCUUCGCUUGCAAGAAU-3), NOX4 (Custom siRNA, 5'-GCAAGACCUGGUCAGUAUA-3') and Mfn2 (Predesigned siRNA #19262) were purchased from Ambion (Life Technologies), while Ero1α-specific siRNA oligonucleotides (5’-CUGUUUUAAGCCACAGACA-3’) and VAPB [26] were obtained from Merck. For silencing experiments 8 × 104 cells were grown in 6-well plates and transfected with 30 pmol of each siRNA for 2 days, using RNAiMAX lipofectamine (Invitrogen) according to the manufacturer's instructions. Silencing efficiency was monitored by real-time PCR as detailed in Ref. [27] using the following primers: AQP11- Fw 5'-TAGCTTGCAGGAATCCCATC-3' and Rv 5'-CTCCTGCATAGGCCAAAAAG-3'; Ero1α- Fw 5'-GTGTGGCTGCTCAGCTCG-3' and Rv 5'-TCAATGGTTTCAAACATCACAGG-3'; NOX4- Fw 5'-AAGACTCCGAAATTCTGCCC-3' and Rv 5'-AACCAACGGAAGGACTGGA-3'; Mfn2- Fw 5’-ATTGCAGAGGCGGTTCGACTCA-3’ and Rv 5’-TTCAGTCGGTCTTGCCGCTCTT-3’; VAPB- Fw 5’-AGGTTA TGGAAGAATGTAAGAGGC-3’ and Rv 5’-GTTGCTCTGCACTGTCTTCCTC-3’.
用于沉默 AQP11（定制siRNA ，5'-GAGCUUCGCUUGCAAGAAU-3）、 NOX4 （定制 siRNA，5'-GCAAGACCUGGUCAGUAUA-3'）和Mfn2 （预设计 siRNA #19262）的试剂购自 Ambion (Life Technologies)，而 Ero1α-特异性 siRNA寡核苷酸(5'-CUGUUUUAAGCCACAGACA-3') 和VAPB [ 26 ] 获自 Merck。对于沉默实验，8×10 4 个细胞在6孔板中生长，并根据制造商的说明使用RNAiMAX lipofectamine (Invitrogen)用30 pmol的每种siRNA转染2天。沉默效率通过实时 PCR 进行监测，详细信息请参见参考文献 1。 [ 27 ]使用以下引物：AQP11-Fw 5'-TAGCTTGCAGGAATCCCATC-3'和Rv 5'-CTCCTGCATAGGCCAAAAAG-3'； Ero1α- Fw 5'-GTGTGGCTGCTCAGCTCG-3' 和 Rv 5'-TCAATGGTTTCAAACATCACAGG-3'； NOX4-Fw 5'-AAGACTCCGAAATTCTGCCC-3' 和 Rv 5'-AACCAACGGAAGGACTGGA-3'； Mfn2- Fw 5'-ATTGCAGAGGCGGTTCGACTCA-3' 和 Rv 5'-TTCAGTCGGTCTTGCCGCTCTT-3'； VAPB-Fw 5'-AGGTTA TGGAAGAATGTAAGAGGC-3' 和 Rv 5'-GTTGCTCTGCACTGTCTTCCTC-3'。

2.3. HyPer confocal laser scanning
2.3.超共焦激光扫描

To perform imaging assays, 8 × 104 HeLa cells stably expressing either HyPer ER Lumen or HyPer Mito were silenced and/or transfected on 23 mm glass coverslips for 48 h as described above. For analyzing the mitochondrial source of the flux, cells were further treated for the indicated times with 33 μM S3QEL [28] from Sigma, 33 μM S1QEL [29] from Cayman Chemical C. or 2 μM LCS-1 [30] from EMD Millipore, all prepared in DMSO. Time 0 in these experiments corresponds with cells treated only with the vehicle.
为了进行成像测定，将稳定表达HyPer ER Lumen或HyPer Mito的 8 × 10 4 HeLa 细胞在 23 mm 玻璃盖玻片上沉默和/或转染 48 小时，如上所述。为了分析通量的线粒体来源，使用来自 Sigma 的 33 μM S3QEL [ 28 ]、来自Cayman Chemical C. 的33 μM S1QEL [ 29 ] 或来自 EMD Millipore 的2 μM LCS-1 [ 30 ] 进一步处理细胞指定的时间。 ，全部在DMSO中制备。这些实验中的时间 0 对应于仅用载体处理的细胞。
After 48 h, cells on coverslips were equilibrated in Ringer buffer (RB: 140 mM NaCl, 2 mM CaCl2, 1 mM MgSO4, 1.5 mM K2HPO4, 10 mM Glucose, pH 7.4) for 10min at room temperature before acquisition. Confocal images were collected every 2sec for 1min by dual excitation with 488-nm argon and 405-nm violet diode lasers using an Ultraview confocal laser scanning microscope equipped with an EC Plan - Neofluar 20X (NA 0.45) Dry (Carl Zeiss). To determine the response of a completely reduced probe (Fig. S1A), cells were treated with 5 mM DTT (Sigma) for 5min, recorded for a further minute and then challenged with 50 μM H2O2 (Sigma). Each biological condition is represented after averaging 3 technical replicates. For each technical sample, the 488/405-nm ratios were calculated for ≥25 cells using ImageJ, and averaged excluding the initial 20sec of acquisition to allow for laser equilibration. The results are showed as the mean fold change in the basal ratio with respect to controls ± SEM. From 2 to 10 experiments were conducted for each condition assayed.
48小时后，盖玻片上的细胞在室温下在林格缓冲液（RB：140 mM NaCl、2 mM CaCl 2 、1 mM MgSO 4 、1.5 mM K 2 HPO 4 、10 mM 葡萄糖，pH 7.4）中平衡10分钟，然后采集。使用配备 EC Plan - Neofluar 20X (NA 0.45) Dry (Carl Zeiss) 的 Ultraview 共焦激光扫描显微镜，通过 488 nm 氩气和 405 nm 紫色二极管激光器的双重激发，每 2 秒收集一次共焦图像，持续 1 分钟。为了确定完全还原的探针的响应（图S1A ），将细胞用5 mM DTT (Sigma)处理5分钟，再记录一分钟，然后用50 μM H 2 O 2 (Sigma)攻击。每种生物状况均在 3 次技术重复的平均值后表示。对于每个技术样品，使用 ImageJ 计算 ≥25 个细胞的 488/405 nm 比率，并计算平均值，排除最初的 20 秒采集以实现激光平衡。结果显示为相对于对照±SEM 的基础比率的平均倍数变化。对每个分析条件进行 2 至 10 次实验。
Representative images of the basal ratio of HyPer ER Lumen or HyPer Mito were acquired using a GE Healthcare DeltaVisionTM Ultra microscope equipped with a Plan - Apo 60 X (NA 1 0.42) oil objective lens. To this end, 8 × 104 HyPer ER Lumen- or Mito-expressing HeLa cells were silenced and/or transfected onto coverslips as described above and equilibrated 10min in FluoroBrite DMEM medium (Gibco) with 10% FCS before acquisition.
使用配备 Plan - Apo 60 X (NA 1 0.42) 油物镜的 GE Healthcare DeltaVisionTM Ultra 显微镜获取HyPer ER Lumen或HyPer Mito基础比率的代表性图像。为此，如上所述，将表达 8 × 10 4 HyPer ER Lumen或Mito的 HeLa 细胞沉默和/或转染到盖玻片上，并在采集前在具有 10% FCS 的 FluoroBrite DMEM 培养基（Gibco）中平衡 10 分钟。

2.4. Immunofluorescence analyses
2.4.免疫荧光分析

To assess the correct localization of the myc-tagged ER-targeted catalase, 8 × 104 Hela cells were plated on 13 mm coverslips placed in 6-well plates and transfected for 48 h. The recombinant protein was detected using in-house generated mouse monoclonal antibodies (9E10, PBS 5% FCS). Rabbit anti-calnexin antibodies (#ADI-SPA-860F, 1 Enzo Life Sciences) were used to decorate the ER. Briefly, cells were fixed with 4%PFA for 20min at room temperature and permeabilized with 0.1% Triton X100 before incubation with the indicated antibodies. Suitable anti-mouse or anti-rabbit secondary antibodies Alexa Fluor-488 and Alexa Fluor-546 (Molecular Probes) were then used, and fluorescent images acquired by a GE Healthcare DeltaVisionTM. Images were processed with ImageJ.
为了评估 myc 标记的 ER 靶向过氧化氢酶的正确定位，将 8 × 10 4 Hela 细胞铺在 6 孔板中的 13 mm 盖玻片上并转染 48 小时。使用内部生成的小鼠单克隆抗体（9E10，PBS 5% FCS）检测重组蛋白。使用兔抗钙连接蛋白抗体（#ADI-SPA-860F，1 Enzo Life Sciences）来装饰 ER。简而言之，细胞在室温下用 4% PFA 固定 20 分钟，并用 0.1% Triton X100 透化，然后与指定抗体一起孵育。然后使用合适的抗小鼠或抗兔二抗 Alexa Fluor-488 和 Alexa Fluor-546（分子探针），并通过 GE Healthcare DeltaVisionTM 获取荧光图像。图像使用 ImageJ 进行处理。

2.5. Transmission electron microscopy
2.5.透射电子显微镜

HeLa cells were fixed with 2.5% glutaraldehyde in 0,1 M cacodylate buffer (pH 7.4), washed thoroughly in cacodylate buffer and post-fixed in 1% osmium tetroxide (OsO4), 1.5% potassium ferricyanide (K4 [Fe(CN)6]), 0.1 M Na-Cacodylate buffer for 1 h on ice, washed with distilled water (dH2O) and enbloc stained with 0.5% uranyl acetate in dH2O overnight at 4 °C in the dark. Finally, samples were rinsed in dH2O, dehydrated with increasing concentrations of ethanol, embedded in Epon, and cured in an oven at 60 °C for 48 h. Ultrathin sections (70–90 nm) were obtained using an ultramicrotome (UC7, Leica microsystem), collected, stained with uranyl acetate and Sato’s lead solutions, and observed in a Transmission Electron Microscope Talos L120C (FEI, Thermo Fisher Scientific) operating at 120 kV. Images were acquired with a Ceta CCD camera (FEI, Thermo Fisher Scientific). For morphometric analyses of mitochondria and MAMs, the areas and the perimeters (ROIs) of at least 200 mitochondria per sample were drawn using the freehand selections of ImageJ (n = 3). All ROIs were then extended with the enlarge function and the ER cisternae or tubules present within 30 nm or less from the mitochondria selected were scored as a MAM. GraphPad Prism was used to represent the number of contacts.
HeLa 细胞用 0.1 M二甲胂酸盐缓冲液（pH 7.4）中的 2.5%戊二醛固定，在二甲胂酸盐缓冲液中彻底清洗，并在 1%四氧化锇(OsO 4 )、1.5%铁氰化钾(K 4 [Fe(CN)) 中后固定。 ) 6 ]), 0.1 M 二甲胂酸钠缓冲液在冰上放置 1 小时，用蒸馏水洗涤水 (dH 2 O) 和0.5%乙酸铀酰的 dH 2 O 溶液进行整体染色，4 °C 避光过夜。最后，将样品在 dH 2 O 中冲洗，用浓度增加的乙醇脱水，包埋在 Epon 中，并在 60 °C 的烘箱中固化 48 小时。使用超薄切片机（UC7，Leica 显微系统）获得超薄切片（70-90 nm），收集并用醋酸双氧铀和 Sato 铅溶液染色，并在 120 ℃ 下运行的透射电子显微镜 Talos L120C（FEI，Thermo Fisher Scientific）中观察。千伏。使用 Ceta CCD 相机（FEI，Thermo Fisher Scientific）获取图像。对于线粒体和 MAM 的形态计量分析，使用 ImageJ 徒手选择绘制每个样本至少 200 个线粒体的面积和周长 (ROI) (n = 3)。然后使用放大功能扩展所有 ROI，并将所选线粒体 30 nm 或更短范围内存在的 ER 池或小管评分为 MAM。 GraphPad Prism 用于表示触点数量。

2.6. Statistical analyses
2.6。统计分析

We used the one-way ANOVA method for multiple samples and the Tukey's HSD post-hoc test to find out which groups were significantly different from others. In all cases, statistical significance was defined as p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***).
我们对多个样本使用单向方差分析方法，并使用 Tukey 的 HSD 事后检验来找出哪些组与其他组显着不同。在所有情况下，统计显着性定义为 p < 0.05 (*)、p < 0.01 (**) 或 p < 0.001 (***)。

3. Results  3. 结果

3.1. H2O2 increases inside the ER upon Ero1α silencing
3.1. Ero1α 沉默后，ER 内的 H 2 O 2增加

Stable HeLa transfectants expressing targeted ratiometric HyPer probes [31] allow to reproducibly measure variations in the H2O2 levels of the ER lumen despite the high oxidative environment of this organelle (Fig. S1A; [22]). As previously reported [22], AQP11 silencing increased the basal oxidation of HyPer ER lumen, likely reflecting the trapping of locally generated H2O2 molecules inside the ER. To investigate their main sources, we silenced the expression of Ero1α and NOX4 (Fig. S1B), two ER-resident enzymes known to release H2O2 into the lumen [32,33]. NOX4 downregulation caused only a statistically non-significant decrease in the basal H2O2 levels (Fig. 1A, green column). In contrast, the results obtained upon Ero1α silencing were most surprising, in that lowering the levels of an oxidase strongly increased the oxidation state of the HyPer ER lumen probe (Fig. 1A, red column, and 1B upper panels). Cells stably expressing a pH-insensitive sensor (SHyPer ER lumen) did not undergo significant fluorescent shifts, excluding changes in luminal pH (Fig. S1C).
表达靶向比率HyPer探针的稳定 HeLa 转染子 [ 31 ] 允许重复测量 ER 腔 H 2 O 2水平的变化，尽管该细胞器处于高氧化环境（图 S1A ；[ 22 ]）。正如之前报道的[ 22 ]，AQP11 沉默增加了HyPer ER 管腔的基础氧化，可能反映了 ER 内局部产生的 H 2 O 2分子的捕获。为了研究它们的主要来源，我们沉默了 Ero1α 和 NOX4 的表达（图 S1B ），这两种内质网驻留酶已知可将 H 2 O 2释放到管腔中 [ 32 , 33 ]。 NOX4 下调仅导致基础 H 2 O 2水平出现统计学上不显着的下降（图 1 A，绿柱）。相比之下，Ero1α 沉默后获得的结果是最令人惊讶的，因为降低氧化酶的水平会强烈增加HyPer ER 内腔探针的氧化态（图 1A ，红柱和 1B 上图）。 稳定表达pH不敏感传感器（ SHyPer ER lumen ）的细胞没有经历显着的荧光变化，排除腔内pH的变化（图S1C ）。
Fig. 1
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Fig. 1. Ero1α silencing causes a paradoxical increase in the levels of H2O2 inside the ER lumen.
图1 . Ero1α 沉默会导致 ER 腔内 H 2 O 2水平反常增加。

A) The graph summarizes the changes induced by the indicated treatments in the basal ratio (488/405) of HyPer ER Lumen, expressed in fold change with respect to untreated cells. As previously reported [22], the silencing of AQP11 (orange column) caused an increase in probe oxidation. Downregulation of NOX4, alone or in combination with (green and green-orange striped columns) had effects below the significance levels. In contrast, Ero1α downregulation caused a higher increase in basal HyPer ER Lumen oxidation (red column) than AQP11 silencing alone. This increase was abolished when both AQP11 and Ero1α were downregulated (red-orange striped column). Bars represent the average of ≥5 independent experiments ± SEM. P-Value *<0.05 ** < 0.01. B) Expression of catalase in the ER lumen (ER-CAT) abolished the differences observed upon AQP11, Ero1α or NOX4 silencing, confirming that the observed results reflected rises in the luminal concentration of H2O2. Average of ≥2 independent experiments ± SEM. P-Value *<0.05 ** < 0.01. The top panels show representative images of HeLa cells expressing Hyper ER Lumen under the indicated silencing conditions. The four bottom panels show cells co-expressing ER-catalase. Scale Bar = 20 μm.. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
A) 该图总结了所示处理引起的HyPer ER Lumen基础比率 (488/405) 的变化，以相对于未处理细胞的倍数变化表示。正如之前报道的[ 22 ]，AQP11（橙色柱）的沉默导致探针氧化增加。 NOX4 的下调，单独或与（绿色和绿橙色条纹柱）组合，具有低于显着性水平的效果。相比之下，Ero1α 下调导致基础HyPer ER 管腔氧化（红柱）比单独 AQP11 沉默更高。当 AQP11 和 Ero1α 均下调时，这种增加被消除（红橙色条纹柱）。条形代表≥5个独立实验的平均值±SEM。 P 值 *<0.05 ** < 0.01。 B) ER 腔中过氧化氢酶的表达 (ER-CAT) 消除了 AQP11、Ero1α 或 NOX4 沉默时观察到的差异，证实观察到的结果反映了 H 2 O 2腔浓度的上升。 ≥2 次独立实验的平均值 ± SEM。 P 值 *<0.05 ** < 0.01。上图显示了在指定沉默条件下表达Hyper ER Lumen的 HeLa 细胞的代表性图像。底部的四个图显示了共表达 ER-过氧化氢酶的细胞。比例尺 = 20 μm..（为了解释该图例中对颜色的引用，读者可以参考本文的网络版本。）
Importantly, simultaneous AQP11 silencing prevented the increase in luminal H2O2 observed in cells deprived of Ero1α (Fig. 1A, red and orange-striped column, and 1B upper panels), implying a role for AQP11 in importing H2O2 from sources external to the ER. The simultaneous silencing of NOX4 and AQP11 increased the oxidation levels of HyPer with respect to single-silencing of NOX4, albeit in a non-significant manner. These results point at Ero1α as the main source of luminal H2O2 in untreated HeLa cells (Fig. 1A, green and orange-striped column, and 1B upper panels).
重要的是，同时 AQP11 沉默阻止了在缺乏 Ero1α 的细胞中观察到的管腔 H 2 O 2的增加（图 1 A，红色和橙色条纹柱，以及 1B 上图），这意味着 AQP11 在导入 H 2 O 2 方面发挥了作用。来自急诊室外部的来源。相对于单一沉默 NOX4，同时沉默 NOX4 和 AQP11 增加了HyPer的氧化水平，尽管程度不显着。这些结果表明，Ero1α 是未经处理的 HeLa 细胞中腔内 H 2 O 2的主要来源（图 1 A，绿色和橙色条纹柱，以及 1B 上图）。
To prove that indeed H2O2 accumulates in the ER upon Ero1α or AQP11 downregulation, we co-expressed an ER-targeted catalase (Fig. S1D). As shown in panel B of Fig. 1, the presence of a powerful H2O2 scavenger counteracted the increases observed in HyPer ER lumen activation, further confirming that the sensor faithfully reports on the H2O2 levels in the ER lumen.
为了证明 Ero1α 或 AQP11 下调时 H 2 O 2确实在ER 中积累，我们共表达了一种针对 ER 的过氧化氢酶（图 S1D ）。如图1的图B所示，强大的H 2 O 2清除剂的存在抵消了在HyPer ER腔激活中观察到的增加，进一步证实了传感器如实地报告了ER腔中的H 2 O 2水平。
The observation that H2O2 levels do not increase upon simultaneous AQP11 and Ero1α knockdown confirm that Ero1α is a key source of the H2O2 molecules that eventually reach the cytosol via AQP11 [22]. They also imply that –unexpectedly- abundant H2O2 molecules enter the ER lumen of cells lacking Ero1α. Thus, an external source is activated upon silencing Ero1α, which generates H2O2 molecules that can reach the ER lumen via AQP11.
同时敲低 AQP11 和 Ero1α 后 H 2 O 2水平不会增加的观察结果证实，Ero1α 是 H 2 O 2分子的关键来源，最终通过 AQP11 到达细胞质 [ 22 ]。它们还意味着，出乎意料地大量的 H 2 O 2分子进入了缺乏 Ero1α 的细胞的 ER 腔。因此，外部来源在 Ero1α 沉默后被激活，产生 H 2 O 2分子，可通过 AQP11 到达 ER 腔。

3.2. H2O2 increases also in mitochondria upon Ero1α silencing
3.2. Ero1α 沉默后线粒体中的 H 2 O 2也会增加

In search of the generator(s) of the H2O2 detected with HyPer ER Lumen when Ero1α was silenced, our suspects fell first on mitochondria. These organelles contain at least 11 different ROS sources [29], establish close contacts with the ER, and are known to exchange other diffusible molecules such as calcium with it [34,35]. Moreover, both AQP11 [22] and Ero1α [36] have been reported to be partially localized in mitochondrial-associated membranes (MAMs).
在寻找当 Ero1α 沉默时用HyPer ER Lumen检测到的 H 2 O 2发生器时，我们的嫌疑人首先落在线粒体上。这些细胞器含有至少 11 种不同的 ROS 来源 [ 29 ]，与 ER 建立密切联系，并且已知可以与其交换其他可扩散分子，例如钙 [ 34 , 35 ]。此外，据报道，AQP11 [ 22 ] 和 Ero1α [ 36 ] 部分定位于线粒体相关膜 (MAM)。
To explore this possibility, we analyzed HeLa S3 transfectants that stably express HyPer in the mitochondrial matrix (HyPer Mito). Clearly, also the mitochondrial sensor was oxidized upon Ero1α downregulation (Fig. 2A, red column), mirroring the increase detected in the ER (Fig. 2B, compare middle images). Remarkably, HyPer Mito basal oxidation levels did not increase when only AQP11 was silenced, while HyPer ER lumen did [22]. Moreover, H2O2 levels increased in the mitochondrial matrix also upon combined downregulation of AQP11 and Ero1α (Fig. 2A, red and orange column, and Fig. 2B, right panels). Thus, the peroxiporin activity of AQP11 is not required for HyPer Mito oxidation in cells with low Ero1α levels.
为了探索这种可能性，我们分析了在线粒体基质中稳定表达HyPer ( HyPer Mito ) 的 HeLa S3 转染子。显然，线粒体传感器在 Ero1α 下调时也被氧化（图 2A ，红色柱），反映了 ER 中检测到的增加（图 2B ，比较中间图像）。值得注意的是，当仅沉默 AQP11 时， HyPer Mito基础氧化水平并未增加，而HyPer ER 管腔却增加了 [ 22 ]。此外，AQP11 和 Ero1α 联合下调后，线粒体基质中的 H 2 O 2水平也增加（图2A，红色和橙色柱，图 2B ，右图）。因此，在 Ero1α 水平较低的细胞中， HyPer Mito氧化不需要 AQP11 的过氧孔蛋白活性。
Fig. 2
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Fig. 2. The H2O2 levels increase also in mitochondria upon Ero1α silencing.
图2 . Ero1α 沉默后，线粒体中的 H 2 O 2水平也会增加。

A) Cells expressing HyPer in the mitochondrial matrix were silenced with Ero1α-specific oligos alone or in combination with AQP11-specific reagents, as indicated. Variations in the basal oxidative level of HyPer Mito are expressed as fold change relative to untreated cells. Downregulation of Ero1α (siEro1α) favors HyPer Mito oxidation (red column) also in cells with low AQP11 activity (red-orange striped column). Bars represent the average of ≥5 independent experiments ± SEM. P-Value **<0.01; n.s. non significant. B) Representative images of HyPer Mito and HyPer ER Lumen basal fluorescence after the knock-down of Ero1α or Ero1α/AQP11 simultaneously. Scale Bar = 20 μm.. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
A) 如图所示，单独使用 Ero1α 特异性寡核苷酸或与 AQP11 特异性试剂组合沉默线粒体基质中表达HyPer 的细胞。 HyPer Mito基础氧化水平的变化表示为相对于未处理细胞的倍数变化。在 AQP11 活性较低的细胞（红橙色条纹柱）中，Ero1α (siEro1α) 的下调也有利于HyPer Mito氧化（红色柱）。条形代表≥5个独立实验的平均值±SEM。 P值**<0.01； ns 不显着。 B) 同时敲低 Ero1α 或 Ero1α/AQP11 后HyPer Mito和HyPer ER Lumen基础荧光的代表性图像。比例尺 = 20 μm..（为了解释该图例中对颜色的引用，读者可以参考本文的网络版本。）

3.3. Complex III is activated upon silencing of Ero1α
3.3. Ero1α 沉默后复合物 III 被激活

Amongst the numerous potential H2O2 source(s) in mitochondria, site IQ in complex I and site IIIQ0 in complex III stand out for their capacity to produce redox equivalents [37]. Their topology determines the side of the mitochondrial inner membrane (IMM) in which the molecules are produced [38]. Site IQ site produces superoxide (O2•-) and H2O2 inside the matrix (MM, Fig. 3A), the former being transformed into the latter by a matrix superoxide dismutase (MnSOD or SOD2). In contrast, complex III site IIIQ0 releases O2•- into either side of the IMM (Fig. 3A). In the intermembrane space (IMS), H2O2 is generated by cytosolic Cu/ZnSOD or SOD1. To identify the source of H2O2 activated in mitochondria upon Ero1α silencing, we selectively blocked electron leakage from site IQ or site IIIQ0 using a new generation of specific inhibitors [39,40]. Unlike classical compounds such as rotenone or antimycin A, these drugs neither completely interrupt electron flow nor do they alter metabolite consumption. Consequently, they induce fewer compensatory mechanisms from upstream and downstream sites and conserve the membrane potential and ATP production rate, limiting cell toxicity and other potential artefacts [41]. Clearly, the inhibition of site IIIQ0 in complex III by S3qel caused a time-dependent decrease in the levels of H2O2 in the ER lumen (Fig. 3B, pink columns). In contrast, inhibiting complex I activity did not significantly affect the luminal levels of H2O2 (Fig. 3B, yellow columns). Both compounds lowered the H2O2 levels sensed in the mitochondrial matrix by HyPer Mito, though to different extents (Fig. S2). Altogether, the above data confirm that lowering the Ero1α levels in the ER induces a higher electron leakage in mitochondria and identifies mitochondrial complex III as the main source of H2O2 molecules entering the ER.
在线粒体中众多潜在的 H 2 O 2来源中，复合物 I 中的位点 IQ 和复合物 III 中的位点 IIIQ 0因其产生氧化还原当量的能力而脱颖而出 [ 37 ]。它们的拓扑结构决定了产生分子的线粒体内膜（IMM）的一侧[ 38 ]。位点IQ位点在基质内产生超氧化物(O 2 •- )和H 2 O 2 (MM，图3A )，前者通过基质超氧化物歧化酶(MnSOD或SOD2)转化为后者。相反，复合体III位点IIIQ 0将O 2 •-释放到IMM的任一侧（图3A ）。在膜间隙(IMS) 中，H 2 O 2由胞质 Cu/ZnSOD 或SOD1产生。为了确定 Ero1α 沉默后线粒体中激活的 H 2 O 2的来源，我们使用新一代特异性抑制剂选择性地阻断了 IQ 或 IIIQ 0位点的电子泄漏 [ 39 , 40 ]。与鱼藤酮或抗霉素 A等经典化合物不同，这些药物既不会完全中断电子流，也不会改变代谢物的消耗。 因此，它们从上游和下游位点诱导较少的补偿机制，并保留膜电位和 ATP 生产率，限制细胞毒性和其他潜在的人为因素 [ 41 ]。显然，S3qel 对复合物 III 中位点 IIIQ 0的抑制导致 ER 腔中 H 2 O 2水平随时间依赖性下降（图 3 B，粉色柱）。相反，抑制复合物I活性并没有显着影响H 2 O 2的管腔水平（图3B ，黄色柱）。两种化合物均降低了HyPer Mito在线粒体基质中检测到的 H 2 O 2水平，但程度不同（图 S2 ）。总之，上述数据证实，降低内质网中的Ero1α水平会导致线粒体中更高的电子泄漏，并确定线粒体复合物III是进入内质网的H 2 O 2分子的主要来源。
Fig. 3
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Fig. 3. Complex III is activated upon silencing of Ero1α.
图3 . Ero1α 沉默后复合物 III 被激活。

A) Schematic representation of the main electron transport chain components present in the inner mitochondrial membrane (IMM). Both complex I and complex III (highlighted in yellow and pink, respectively) produce superoxide. Unlike complex I, however, complex III releases superoxide also in the mitochondrial intermembrane space (IMS). Here, superoxide dismutase 1 (SOD1, in blue) can generate H2O2. The boxes indicate the drugs used to specifically inhibit the three activities. B) Time-dependent effects of the S3qel, S1qel and LCS-1 on the basal oxidative level of HyPer ER Lumen before (left panel) or after (right panel) Ero1α silencing. The “0” column represents cells treated with the drug vehicle (DMSO). S3qel and S1qel inhibit complex III and complex I (pink and yellow columns, respectively); LCS-1 (blue columns) inhibits SOD1. Average of ≥5 independent experiments ± SEM. P-Value with respect to control is indicated at the top of the significant columns, while statistically significant differences among groups are highlighted with a linker. In all cases P-Value *<0.05 ** < 0.01.. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
A)线粒体内膜 (IMM) 中主要电子传递链成分的示意图。复合物 I 和复合物 III（分别以黄色和粉色突出显示）都会产生超氧化物。然而，与复合物 I 不同的是，复合物 III 也在线粒体膜间隙(IMS) 中释放超氧化物。在这里，超氧化物歧化酶1（SOD1，蓝色）可以产生H 2 O 2 。方框表示用于特异性抑制这三种活性的药物。 B) Ero1α 沉默之前（左图）或之后（右图）S3qel、S1qel 和 LCS-1 对HyPer ER Lumen基础氧化水平的时间依赖性影响。 “0”列代表用药物载体（DMSO）处理的细胞。 S3qel 和 S1qel 抑制复合物 III 和复合物 I（分别为粉色和黄色柱）； LCS-1（蓝色柱）抑制 SOD1。 ≥5 次独立实验的平均值 ± SEM。相对于对照的 P 值显示在显着列的顶部，而组间的统计显着差异则用链接器突出显示。在所有情况下，P 值 *<0.05 ** < 0.01..（为了解释该图例中对颜色的引用，读者可以参阅本文的网络版本。）
To further confirm the above conclusions, we devised a strategy based on the notion that AQPs are not able to transport charged solutes. As stated above, complex III only produces O2•-. Owing to its charged nature, O2•- cannot be transported across peroxiporins [42]. Therefore, we reasoned that if peroxiporin were needed to mediate H2O2 entry into the ER, blockade of SOD1 should prevent HyPer ER Lumen oxidation upon Ero1α silencing (see the scheme in Fig. 3A). Accordingly, a pyridazin-3-one derivative (LCS-1), known to effectively inhibit SOD1 activity [43], prevented the H2O2 increase in the ER lumen, confirming the requirement of O2•- transformation into a suitable peroxiporin substrate.
为了进一步证实上述结论，我们基于AQP不能运输带电溶质的概念设计了一种策略。如上所述，络合物III仅产生O 2 ·- 。由于其带电性质，O 2 •-不能通过过氧孔蛋白运输[ 42 ]。因此，我们推断，如果需要过氧孔蛋白介导H 2 O 2进入ER，则阻断SOD1应防止Ero1α沉默后HyPer ER管腔氧化（参见图3 A中的方案）。因此，已知能有效抑制 SOD1 活性的哒嗪-3-酮衍生物 (LCS-1) [ 43 ] 可防止 ER 腔中 H 2 O 2的增加，证实了 O 2 •-转化为合适的过氧孔蛋白的需要基材。

3.4. Reorganization of ER-mitochondria contact sites promotes H2O2 transfer upon Ero1α silencing
3.4. ER-线粒体接触位点重组促进 Ero1α 沉默后 H 2 O 2转移

As effective communication between cellular compartments is facilitated by the vicinity of the organelles involved [44], physical contacts between the ER and mitochondria are emerging as key signaling hubs [[35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45]]. In the case of H2O2, vicinity is of paramount relevance, as the reactivity of this compound and the abundance of scavenger antioxidants in the cytosol would limit signal diffusion [46,47]. We and others have shown previously that key redox modulators including AQP11 and Ero1α accumulate partly in MAMs [[22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34],48,49]. Therefore, we reasoned that the entry of H2O2 molecules generated in mitochondria upon Ero1α silencing, would be facilitated by tightening of the ER-mitochondria physical links. To visualize MAMs, we performed systematic morphometric analyses of transmission electron microscopy images (Fig. 4A and B). As there is still controversy on minimal length of the interorganellar interface that can define a MAM from casual contacts between organelles in crowded cells, we selected a stringent gap width threshold for discrimination (≤30 nm) [50]. The results of this endeavor are summarized in panel B of Fig. 4. Clearly, the number of ER-mitochondria contacts was dramatically increased upon Ero1α silencing. Importantly, these were not due to an expansion of mitochondrial dimensions as Ero1α silencing slightly reduced their area (Fig. S3). Simultaneous downregulation of AQP11 neither prevented nor inhibited the effects of Ero1α silencing. Thus, like complex III activation, MAM remodeling can occur also without efficient H2O2 transport across the ER membrane. Numerous proteins are thought to dynamically control MAMs [[51], [52], [53]]. To prove that the tightening of the ER-mitochondria contacts observed above was important for H2O2 transfer, we silenced the vesicle-associated membrane protein-associated protein B (VAPB), and mitofusin-2 (Mfn2), two proteins known to be essential for correct juxtaposition of the two organelles, despite in different manner [26–54],[55], [56], [57]]. Clearly, neither VAPB nor Mfn2 silencing impacted the ER H2O2 basal levels (Fig. 4C columns pink and purple). However, the increase in H2O2 normally observed upon Ero1α downregulation was no longer detectable in cells devoid of VAPB or Mfn2 (Fig. 4C red-pink and red-purple striped columns). These results confirm that the flux of H2O2 from mitochondria to ER depends also on the architecture of MAMs.
由于相关细胞器附近促进了细胞区室之间的有效通讯[ 44 ]，内质网和线粒体之间的物理接触正在成为关键的信号传导中枢[ [35] 、 [36] 、 [37] 、 [38] 、 [ 39] 、 [40] 、 [41] 、 [42] 、 [43] 、 [44] 、 [45] ]。就 H 2 O 2而言，附近至关重要，因为该化合物的反应性和细胞质中丰富的清除抗氧化剂会限制信号扩散[ 46 , 47 ]。 我们和其他人之前已经证明，包括 AQP11 和 Ero1α 在内的关键氧化还原调节剂部分积聚在 MAM 中 [ [22] 、 [ 23 ] 、 [24] 、 [ 25] 、 [ 26] 、 [27] 、 [28] 、 [29] , [30] , [31] , [32] , [33] , [34] , 48 , 49 ]。因此，我们推断 Ero1α 沉默后线粒体中产生的 H 2 O 2分子的进入将通过加强 ER-线粒体物理联系而促进。为了可视化 MAM，我们对透射电子显微镜图像进行了系统的形态分析（图 4 A 和 B）。由于对于可以根据拥挤细胞中细胞器之间的偶然接触来定义 MAM 的细胞间界面的最小长度仍然存在争议，因此我们选择了严格的间隙宽度阈值进行区分（≤30 nm）[ 50 ]。这一努力的结果总结在图4的B组中。 显然，Ero1α 沉默后 ER-线粒体接触的数量显着增加。重要的是，这并不是由于线粒体尺寸的扩大，因为 Ero1α 沉默略微减少了线粒体面积（图 S3 ）。同时下调 A​​QP11 既不能阻止也不能抑制 Ero1α 沉默的作用。因此，与复合物 III 激活一样，MAM 重塑也可能在没有有效的 H 2 O 2跨内质网转运的情况下发生。许多蛋白质被认为可以动态控制 MAM [ [51] 、 [52] 、 [53] ]。为了证明上述观察到的 ER-线粒体接触的收紧对于 H 2 O 2转移很重要，我们沉默了囊泡相关膜蛋白相关蛋白 B (VAPB) 和线粒体融合蛋白-2 (Mfn2)，这两种蛋白已知尽管方式不同，但对于两个细胞器的正确并置至关重要[26-54]， [55] ， [56] ， [57] ]。显然，VAPB 和 Mfn2 沉默均不影响 ER H 2 O 2基础水平（图 4 C 柱粉色和紫色）​​。然而，在缺乏 VAPB 或 Mfn2 的细胞中，不再检测到 Ero1α 下调时通常观察到的 H 2 O 2增加（图 1）。 4 C 红粉色和红紫色条纹柱）。这些结果证实，H 2 O 2从线粒体到内质网的通量也取决于 MAM 的结构。
Fig. 4
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Fig. 4. MAM’s reorganization allows H2O2 transfer from mitochondria when Ero1α is silenced.
图4 .当 Ero1α 沉默时，MAM 的重组允许 H 2 O 2从线粒体转移。

A) Representative transmission electron microscopy images of HeLa cells treated for ≤48 h with the indicated silencing reagents. Scale Bar = 1 μm. Insets are enlarged 3.8 times to better illustrate ER-mitochondria contacts (see red arrows). B) The table shows the average number of contacts that a single mitochondrion establishes with the ER (mitochondria with 0, 1, 2, ≥3 contacts). Mean of three independent experiments, in which at least 200 mitochondria were counted for each condition. The graphs summarize the increase in the number of contacts that mitochondria establish with ER membranes. P-Value *<0.05 ** < 0.01. C) Disturbing the architecture of ER-mitochondria contact sites inhibits H2O2 transfer. HeLa cells stably expressing HyPer ER Lumen were treated with the indicated oligos to downregulate VAPB (pink column) or Mfn2 (purple column) alone or in combination with Ero1α (red-pink and red-purple striped columns). Clearly, both VAPB and Mfn2 are required for efficient H2O2 transfer from mitochondria to the ER lumen in cells with low Ero1α levels (red column). Average of ≥3 independent experiments ± SEM. P-Value *<0.05 ** < 0.01.. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
A) 用所示沉默试剂处理 ≤48 小时的 HeLa 细胞的代表性透射电子显微镜图像。比例尺 = 1 μm。插图放大了 3.8 倍，以更好地说明 ER-线粒体接触（参见红色箭头）。 B) 该表显示单个线粒体与 ER 建立的平均接触数（线粒体有 0、1、2、≥3 个接触）。三个独立实验的平均值，其中每种条件至少计数 200 个线粒体。这些图总结了线粒体与内质网膜建立的接触数量的增加。 P 值 *<0.05 ** < 0.01。 C) 扰乱 ER-线粒体接触位点的结构会抑制 H 2 O 2转移。用指定的寡核苷酸处理稳定表达HyPer ER Lumen的 HeLa 细胞，以单独下调 VAPB（粉色柱）或 Mfn2（紫色柱）或与 Ero1α（红粉色和红紫色条纹柱）组合。显然，在 Ero1α 水平较低的细胞中（红柱），VAPB 和 Mfn2 都是 H 2 O 2从线粒体有效转移到内质网腔所必需的。 ≥3 次独立实验的平均值 ± SEM。 P 值 *<0.05 ** < 0.01..（为了解释该图例中对颜色的引用，读者可以参考本文的网络版本。）
Taken together, our results show that not only mitochondrial complex III is induced to produce more H2O2 in the absence of Ero1α, but MAMs are remodeled to allow efficient ER delivery.
总而言之，我们的结果表明，在没有 Ero1α 的情况下，不仅线粒体复合物 III 被诱导产生更多的 H 2 O 2 ，而且 MAM 也被重塑以允许有效的 ER 递送。

4. Discussion  4. 讨论

Functional specialization is important for achieving competence in complex societies, but proficiency cannot be reached without distributing responsibilities and emergency plans. Hence, collaborative relationships constitute the basis of successful cohabitation. Clearly, this concept also applies to cells. Compartmentalization of biochemical reactions in membrane-bound organelles has paved the thriving of eukaryotic organisms. Enclosed generation of energy in mitochondria, achievement of intricate protein folds inside the ER [58] and efficient transmission of signals across the cytosol are relevant examples, all sustained by physical isolation of the processes using lipid bilayers. Still, maintenance of cellular homeostasis involves redundancies and interorganellar cooperation via sensors and effector elements that allow survival and adaptation. Our paradoxical finding that inhibiting a powerful source of H2O2, Ero1α, increases the H2O2 levels in the ER lumen is to be seen in this context, as adequate safeguarding responses must be readily available when key players are compromised. Gradients must be generated and maintained across membranes [[59], [60], [61]] and channels gated in a timely and spatially regulated manner [25–46],[61,62].
职能专业化对于在复杂社会中获得能力非常重要，但如果不分配责任和应急计划，就无法达到熟练程度。因此，合作关系构成了成功同居的基础。显然，这个概念也适用于细胞。膜结合细胞器中生化反应的区室化为真核生物的繁荣铺平了道路。线粒体中封闭的能量产生、内质网内复杂蛋白质折叠的实现以及跨细胞质的信号有效传输都是相关的例子，所有这些都是通过使用脂质双层的过程的物理隔离来维持的。尽管如此，细胞稳态的维持涉及通过传感器和效应元件进行的冗余和细胞间合作，以实现生存和适应。我们的矛盾发现是，抑制 H 2 O 2的强大来源Ero1α 会增加 ER 腔中的 H 2 O 2水平，这一发现是在这种情况下看到的，因为当关键参与者受到损害时，必须随时提供足够的保护反应。必须以及时和空间调节的方式跨膜[ [59] 、 [60] 、 [61] ]和通道产生和维持梯度[25-46]、[ 61、62 ]。
The notion that Ero1α generates H2O2 during disulfide bond formation links oxidative folding to redox signaling. Thus, H2O2 molecules flow from the ER to the cytosol via the peroxiporin AQP11 [22], conceivably yielding information on the rate of protein biogenesis within the secretory compartment. Our experiments reveal that NOX4 is a minor source of luminal H2O2, a role played by Ero1α also in non-professional secretory cells like HeLa. Not only does Ero1α provide most luminal H2O2, it also ensures that H2O2 levels be restored in its absence, asking and obtaining help from mitochondrial complex III (Fig. 5).
Ero1α在二硫键形成过程中产生 H 2 O 2的概念将氧化折叠与氧化还原信号联系起来。因此，H 2 O 2分子通过过氧孔蛋白 AQP11 [ 22 ] 从内质网流到胞质溶胶，可以想象得到有关分泌室内蛋白质生物合成速率的信息。我们的实验表明，NOX4 是腔内 H 2 O 2的次要来源，Ero1α 在 HeLa 等非专业分泌细胞中也发挥着作用。 Ero1α不仅提供大部分管腔H 2 O 2 ，​​还确保H 2 O 2水平在没有H 2 O 2 的情况下得到恢复，向线粒体复合物III寻求帮助（图5 ）。
Fig. 5
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Fig. 5. Model of the Ero1α-AQP11 signaling pathway.
图5 。 Ero1α-AQP11信号通路模型。

In resting HeLa cells (left panel), H2O2 leaves the ER and reaches the cytosol via AQP11 [22]. Upon Ero1α silencing, complex III (pink) produces more superoxide in the IMS, which is converted by SOD1. H2O2 eventually enters the ER via AQP11, presumably due to the augmented contacts with the ER (right panel). The different green intensity in mitochondrial matrix and ER lumen summarizes the observed changes in HyPer basal oxidation states. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
在静息的 HeLa 细胞中（左图），H 2 O 2离开内质网并通过 AQP11 到达细胞质 [ 22 ]。 Ero1α 沉默后，复合物 III（粉色）在 IMS 中产生更多的超氧化物，并被 SO​​D1 转化。 H 2 O 2最终通过 AQP11 进入 ER，可能是由于与 ER 的接触增强（右图）。线粒体基质和 ER 腔中不同的绿色强度总结了观察到的 HyPer 基础氧化态的变化。 。 （为了解释该图例中对颜色的引用，读者可以参考本文的网络版本。）
In all likelihood, the process we describe here is important in maintaining redox homeostasis in an ER deprived of a key player. The teleology of having closer contacts between mitochondria and the ER is therefore clear, especially considering the abundance of antioxidants in the cytosol. It would be of great interest to identify the mechanisms that tether the two organelles and regulate the intervening distances. That Ero1α is involved in tight relationships with mitochondria does not come as a surprise. In fact, we and others have previously shown that its levels impact calcium fluxes [[35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64]]. How can the absence of a soluble enzyme in the ER lumen be perceived by complex III in the inner mitochondrial membrane? Since the HyPer Mito sensor is activated upon Ero1α silencing also when H2O2 export via AQP11 is inhibited, H2O2 is unlikely to be involved. The nature of the messages remains to be established, calcium ions [48–49][63], and oxygen molecules [[65], [66], [67], [68]] being reasonable candidates.
很可能，我们在此描述的过程对于维持失去关键参与者的 ER 中的氧化还原稳态非常重要。因此，线粒体和内质网之间更密切接触的目的是明确的，特别是考虑到细胞质中丰富的抗氧化剂。确定连接两个细胞器并调节间隔距离的机制将非常有意义。 Ero1α 与线粒体有着密切的关系，这一点并不令人意外。 事实上，我们和其他人之前已经证明它的水平会影响钙通量 [ [35] 、 [36] 、 [37] 、 [38] 、 [39] 、 [40] 、 [41] 、 [42] 、 [43 ] 、 [44] 、 [45] 、 [46] 、 [47] 、 [48 ]、[49] 、[ 50] 、 [51] 、 [52] 、 [53] 、 [54] 、 [55] 、 [56]、[57 ]、[ 58 ]、[59] 、 [60] 、 [61] 、 [62] 、 [63] 、 [ 64] ]。 线粒体内膜中的复合物 III 如何感知内质网腔内缺乏可溶性酶？由于HyPer Mito传感器在Ero1α 沉默时被激活，并且当通过AQP11 的H 2 O 2输出被抑制时，H 2 O 2不太可能参与其中。信息的性质仍有待确定，钙离子[48-49][ 63 ]和氧分子[ [65] 、 [66] 、 [67] 、 [68] ]是合理的候选者。
As it is often the case with novel findings, our study raises more questions than it answers. The notion that Ero1α levels impact the activity of complex III in mitochondria as well as the anatomy of the organelles involved highlights once more how all the ingredients in our cells operate in a synergic fashion to guarantee survival also in dire conditions. Dissecting the underlying mechanisms is bound to identify key targets in a wide spectrum of pathophysiologic conditions.
正如新发现经常出现的情况一样，我们的研究提出的问题多于它给出的答案。 Ero1α 水平影响线粒体中复合物 III 的活性以及相关细胞器的解剖结构，这一概念再次强调了我们细胞中的所有成分如何以协同方式发挥作用，以保证在恶劣条件下的生存。剖析潜在机制必将确定广泛病理生理条件下的关键靶标。

Funding  资金

This work was supported through grants from the Associazione Italiana Ricerca sul Cancro (IG 2019–23285 to R.S.) and Ministero dell'Istruzione, dell’Università e della Ricerca (MIUR)-PRIN (grant no. 2017XA5J5N). I.M.-F. was supported by the Madrid Government (Comunidad de Madrid) under the Multiannual Agreement with UC3M in the line of "Research Funds for Beatriz Galindo Fellowships" (REDOXSKIN-CM-UC3M), and in the context of the V PRICIT (Regional Programme of Research and Technological Innovation", and by “Proyectos de I + D + I” (PID2020-114230 GA-I00 to I.M.-F.) funded by MCIN/AEI/10.13039/501100011033/ . MG was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 754432 and the Polish Ministry of Science and Higher Education, from financial resources for science in 2018–2023 granted for the implementation of an international co-financed project.
这项工作得到了（RS）和（拨款号）的资助。国际货币基金组织。根据与 UC3M 的多年协议，在“Beatriz Galindo 奖学金研究基金”(REDOXSKIN-CM-UC3M) 的范围内，并在 和 (PID2020-114230 GA-I00 至 IM-F。）资助者MCIN/AEI/10.13039/501100011033/ 。 MG 得到了资助协议 No 和 的支持，其中科学财政资源用于实施国际共同资助项目。

Author contributions  作者贡献

I.M-F., I.S. and R.S. designed the strategy of the study. I.S. performed most experiments, while M.G. contributed in the initial ratio analyses and RT-PCR assays. Andrea Raimondi from the Alembic Facilities performed EM images while analyses were made by IS. All authors contributed in interpreting the data. I.S., R.S. and I.M.-F. wrote the manuscript.
IM-F.、IS 和 RS 设计了本研究的策略。 IS 进行了大部分实验，而 MG 则参与了初始比率分析和 RT-PCR 测定。 Alembic 设施的 Andrea Raimondi 拍摄了 EM 图像，同时由 IS 进行分析。所有作者都对数据的解释做出了贡献。 IS、RS 和 IM-F。写了手稿。

Data and materials availability
数据和材料的可用性

Most data needed to evaluate the conclusions in the study are present in the text, figures and supplementary materials. Raw data may be obtained upon request.
评估研究结论所需的大部分数据都存在于文本、图表和补充材料中。原始数据可根据要求获取。

Declaration of competing interest
竞争利益声明

The authors declare no competing interests.
作者声明没有竞争利益。

Acknowledgements  致谢

In addition to all members of our laboratories, we thank L. Rampoldi, A. Rubartelli, E. van Anken and L. Cassina (San Raffaele Scientific Institute, Milan, Italy), Paola Pizzo (University of Padova, Italy), G.P. Bienert (Technical University of Munich, Germany) and S. Bestetti and T. Simmen (University of Alberta, Canada) for useful suggestions, exciting discussions, and constructive criticisms. IS thanks V. Belousov for being her most helpful external PhD supervisor. Part of this work was carried out in the Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC) of San Raffaele Scientific Institute and Vita-Salute University to which staff -particularly Dr. A. Raimondi who performed the electron microscopy assays-we are profoundly grateful.
除了我们实验室的所有成员之外，我们还感谢 L. Rampoldi、A. Rubartelli、E. van Anken 和 L. Cassina（意大利米兰圣拉斐尔科学研究所）、Paola Pizzo（意大利帕多瓦大学）、GP Bienert （德国慕尼黑工业大学）以及 S. Bestetti 和 T. Simmen（加拿大阿尔伯塔大学）提供了有用的建议、激动人心的讨论和建设性的批评。 IS 感谢 V. Belousov 成为她最有帮助的外部博士生导师。这项工作的一部分是在圣拉斐尔科学研究所和生命健康大学的先进光学和电子显微镜生物成像中心 (ALEMBIC) 进行的，我们对工作人员，特别是进行电子显微镜检测的 A. Raimondi 博士深表感谢。
We are indebted with Drs. E. Avezov (University of Cambridge, UK), V. Belousov (Institute of Bioorganic Chemistry, Moscow, Russia), E. Margittai and M. Geistz (Semmelweis University, Budapest, Hungary), L. Cassina (San Raffaele Scientific Institute, Milan, Italy) for providing plasmids and reagents. Because of space limitations, we apologize to all those colleagues and researchers in the field whose work is not directly cited here.
我们感谢博士。 E. Avezov（英国剑桥大学）、V. Belousov（俄罗斯莫斯科生物有机化学研究所）、E. Margittai 和 M. Geistz（匈牙利布达佩斯 Semmelweis 大学）、L. Cassina（圣拉斐尔科学研究所，意大利米兰）提供质粒和试剂。由于篇幅限制，我们向该领域所有未直接引用其工作的同事和研究人员表示歉意。

Appendix A. Supplementary data
附录 A 补充数据

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Multimedia component 1.


以下是本文的补充数据：下载：下载 Acrobat PDF 文件 (163KB)

多媒体组件 1 。

Data availability  数据可用性

The data that has been used is confidential.
已使用的数据是保密的。

References

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