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1 2 3 Implications of the mitochondrial interactome of mammalian thioredoxin 2 for normal 4 cellular function and disease 5 6 7 8 9 1 2 3 10 Christos T. Chasapis , Manousos Makridakis , Anastassios E. Damdimopoulos , Jerome 11 Zoidakis2, Vassiliki Lygirou2, Manolis Mavroidis2, Antonia Vlahou2, Antonio Miranda- 12 13 Vizuete4, Giannis Spyrou5, Alexios Vlamis-Gardikas6* 14 15 16 1 Institute of Chemical Engineering Sciences (ICE-HT), Foundation for Research and 17 18 Technology, Hellas (FORTH), Platani 26504, Greece 19 20 For Review Only 21 2 22 Biomedical Research Foundation, Academy of Athens (BRFAA), Athens, Greece 23 24 25 3 Department of Biosciences and Nutrition, Center for Innovative Medicine (CIMED), 26 27 Karolinska Institutet, Huddinge, Sweden 28 29 30 4 Redox Homeostasis Group, Instituto de Biomedicina de Sevilla (IBIS), Hospital 31 32 Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013 Sevilla, Spain 33 34 35 5 Department of Clinical and Experimental Medicine, Division of Clinical Chemistry, 36 37 Linköping University, S-581 85 Linköping, Sweden 38 39 40 6 41 Department of Chemistry, University of Patras, Rion 26504, Greece 42 43 44 45 46 47 48 *Corresponding author. Email: [email protected] 49 50 51 52 53 54 Running title: The thioredoxin 2 interactome 55 56 57 58 59 60

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1 2 3 Abstract 4 5 6 Multiple thioredoxin isoforms exist in all living cells. To explore the possible functions of 7 8 mammalian mitochondrial thioredoxin 2 (Trx2), an interactome of mouse Trx2 was initially 9 10 created using (i) a monothiol mouse Trx2 species for capturing partners from different 11 12 organs and (ii) yeast two hybrid screens on human liver and rat brain cDNA libraries. The 13 resulting interactome consisted of 195 (Trx2 included) plus the mitochondrial 16S 14 15 RNA. 48 of these proteins were classified as mitochondrial (MitoCarta2.0 human inventory). 16 17 In a second step, the mouse interactome was combined with the current four-membered 18 mitochondrial sub-network of human Trx2 (BioGRID) to give a 53-membered human Trx2 19 20 mitochondrial interactome (52 interactor proteins plus the mitochondrial 16S RNA). Although 21 For Review Only 22 thioredoxins are thiol-employing disulfide , approximately half of the 23 24 detected interactions were not due to covalent disulfide bonds. This finding reinstates the 25 extended role of thioredoxins as moderators of protein function by specific non-covalent, 26 27 protein-protein interactions. Analysis of the mitochondrial interactome suggested that human 28 29 Trx2 was involved potentially in mitochondrial integrity, formation of iron sulfur clusters, 30 31 detoxification of aldehydes, mitoribosome assembly and protein synthesis, protein folding, 32 ADP ribosylation, amino acid and lipid metabolism, glycolysis, the TCA cycle and the 33 34 electron transport chain. The functions of Trx2 were verified by its detected 35 36 interactions with mitochondrial peroxiredoxins and methionine sulfoxide reductase. 37 Parkinson’s disease, triosephosphate deficiency, combined oxidative 38 39 phosphorylation deficiency, and lactate dehydrogenase b deficiency are some of the diseases 40 41 where the proposed mitochondrial network of Trx2 may be implicated. 42 43 44 45 46 Key words: redox, disulfide, interactome, interactor, thiol-disulfide interchange, thioredoxin, 47 48 49 50 51 52 53 Abbreviations 54 55 2D: two dimensional; NADPH: reduced nicotinamide adenine dinucleotide phosphate; Trx: 56 thioredoxin; TrxR: ; Y2H: yeast two hybrid. 57 58 59 60

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1 2 3 Introduction 4 5 6 Thioredoxin and glutaredoxin are proteins that maintain the reduction of cytosolic disulfides 7 8 in most living cells and are implicated in many different functions [1]. Their first discovered 9 10 pivotal role was their ability to donate electrons to ribonucleotide reductases for the reduction 11 12 of ribonucleotides to deoxyribonucleotides, the precursors of DNA synthesis [2, 3]. While 13 reducing substrates, thioredoxins and glutaredoxins get oxidized to be in turn reduced by 14 15 thioredoxin reductase (TrxR) and glutathione respectively. The ultimate electron donor for 16 17 these systems is NADPH. Their ability to donate electrons places thioredoxins and 18 glutaredoxins in the general category of ‘antioxidants’ whose specific functions are 19 20 determined by the particular molecule that receives their electrons. Reduction of methionine 21 For Review Only 22 sulfoxide reductase by thioredoxins and glutaredoxins is a well known example of such 23 24 antioxidant function [4, 5]. Thioredoxins and glutaredoxins may have common substrates with 25 the reduction of glutathione mixed disulfides being exclusive for glutaredoxins [6]. In some 26 27 cases, thioredoxins (and glutaredoxins) may bind non-covalently to other proteins for 28 29 gain/loss of function. In the case of Escherichia coli (E. coli), binding of reduced thioredoxin 30 31 1 (EcoTrx1) to 5 protein of phage T7 gives rise to a functional T7 DNA polymerase [7]. 32 Reduced cytosolic human thioredoxin 1 (Trx1) may bind to apoptosis signaling kinase 1 33 34 (ASK1) to keep the kinase inactive. Removal of Trx1 from ASK1 by oxidation of Trx1 or by 35 36 the thioredoxin-interacting protein (Txnip) promotes apoptosis ([8] and references therein). 37 Reduced mitochondrial thioredoxin 2 (Trx2) will also normally bind to mitochondrial ASK1 38 39 and prevent its activation and concomitant apoptosis [9, 10]. Therefore, thioredoxins (and 40 41 glutaredoxins) may associate with a number of proteins that constitute their interactomes and 42 43 affect their functions decisively. The thioredoxin and glutaredoxin interactomes, however, are 44 far from complete. 45 46 Mammalian cells have two distinct thioredoxin types; one cytosolic (Trx1) and one 47 48 mitochondrial (Trx2), each protein being reduced by its specific TrxR. Trx1 is reduced by 49 50 cytosolic TrxR isoforms [11], whereas Trx2 is reduced by a mitochondrial TrxR (thioredoxin 51 reductase 2 or TrxR2) [12]. Although Trx1 is the prominent thioredoxin species in most 52 53 tissues, it is barely detectable in heart and skeletal muscle where Trx2 is highly expressed 54 55 [13]. Trx1 and Trx2 may respond differently to external stimuli affecting the redox state of 56 the cell. In the case of increased reactive species (ROS) via epidermal growth factor 57 58 addition, selective oxidation of cytoplasmic Trx1 but not Trx2 or the glutathione pools [14] 59 60

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1 2 3 occurred. On the other hand, tumor necrosis factor α (TNFα) caused preferential oxidation of 4 Trx2 whose overexpression inhibited the TNFα-induced, NF-κB translocation to the nucleus 5 6 (activation) [15]. Overexpresion of Trx1 or Trx2 may have opposing effets: in the regulation 7 8 of transcription of hypoxia-inducible factor-1α (Hif-1α) [16], Trx2 overexpression in HEK 9 10 cells lowered Hif-1α protein levels, whereas Trx1 raised them. Since the mRNA levels of Hif- 11 1α were unaltered, results were explained in view of the activation of transcription factors that 12 13 regulated cap-dependent translation and by the production of ROS. Overexpression of Trx2 14 15 increased the production of mitochondrial ROS and decreased translation of Hif-1α [16]. 16 Thioredoxins (and dithiol glutaredoxins) have an composed of two vicinal thiols 17 18 (CxxC) that is reversibly oxidized/reduced during the catalytic cycle of the . The 19 20 electron transfer of the thioredoxin thiols to the disulfide starts with a nucleophilic 21 For Review Only 22 attack from the thiolate of the N-terminal thiol (catalytic) of reduced thioredoxin. A following 23 attack from the second non-catalytic, resolving thiol to the sulfur of the catalytic that 24 25 participates in the temporary disulfide with the substrate leads to an oxidized thioredoxin and 26 27 a reduced substrate. This mechanism has been employed to trap thioredoxin-substrates by 28 29 using mutant monothiol thioredoxins with the second “resolving” thiol of the active site being 30 changed to Ser (mutant active site: CxxS). The mutated monothiol thioredoxins can attack and 31 32 bind to their dithiol substrates but cannot be released from them as they lack the second non- 33 34 catalytic cysteine. Monothiol thioredoxin mutants of EcoTrx1 [17, 18], plant thioredoxins 35 [19], Plasmodium falciparum [20] and Entamoeba histolytica [21] have been used (among 36 37 others) to identify their potential substrates. Monothiol mutant glutaredoxins have served the 38 39 same purpose (e.g. monothiol human glutaredoxin 2 [22]). 40 41 Monothiol mouse Trx1 has been used in combination with isotope-coded affinity tags to 42 identify transnitrosylation targets [23] and protein networks [24], while expression of 43 44 monothiol Trx1 in whole mice has provided the interacting proteome of cytosolic Trx1 [25]. 45 46 The current experiment-based interactome of human Trx1 is composed of 147 proteins [26] 47 48 compared to the 59 interactors described for Trx2 [26], with only four of the latter being 49 mitochondrial (MitoCarta [27]). Respective interactomes for the rat and mouse Trx2 are 50 51 currently not available ([26]). To create an interactome for mouse Trx2, we employed an 52 53 immobilized monothiol mouse Trx2 lacking its mitochondrial signal sequence, for the 54 detection of strong non-covalent and covalent (disulfides) interactions with proteins from 55 56 different mouse tissues. In addition, yeast two hybrid (Y2H) screens with rat and human 57 58 cDNA libraries were used to identify weaker non-covalent interactions. The novel results (194 59 60 proteins plus the mitochondrial 16S RNA) were integrated with the described 59 interactors

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1 2 3 (BioGRID) of human Trx2 to give an updated human interactome of 253 proteins (excluding 4 Trx2). 52 of these proteins were mitochondrial (four from BioGRID, plus 48 novel hits), 5 6 while the mitochondrial 16S RNA was identified as an additional interactor by Y2H screens. 7 8 The possible novel cellular functions of Trx2 and its association with human diseases are 9 10 discussed within the context of its mitochondrial interactome. 11 12 13 14 15 16 17 18 19 20 21 For Review Only 22 23 24 25 26 27 28 29 30 31 32 33 34 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

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1 2 3 Materials and Methods 4 5 6 Construction of plasmid for the overexpression of ΔTrx2C93S. 7 8 A plasmid (pET15b) containing the gene encoding mouse Trx2 [13] was used as a template to 9 10 initially generate the 5’ and 3; parts of the TXN2 gene, each coding for the C93S mutation and 11 12 containing some common sequence. The 5’ part was amplified with primers F-NdeI (CGA 13 GGA TCC ATA TGA CAA CCT TTA ATA TCC AGG) and C93S-RC (CAG GAT CTT 14 15 GCT GGG TCC ACA CC) as forward and reverse complementary respectively, while the 3’ 16 17 part was amplified using F-C93S (GGT GTG GAC CCA GCA AGA TCC TG) and EcoRI- 18 RC (CCC TTA GAA TTC CCA ATC AGC TTC TTC AGG) as forward and reverse 19 20 complementary respectively. The 5’ and 3’ PCR fragments of the gene containing the C93S 21 For Review Only 22 mutation were gel-purified, mixed and used as a template for the F-NdeI and EcoRI-RC 23 24 primers to receive a PCR encoding Trx2 without its 1-59 mitochondrial targeting 25 signal (now starting as (M)TTF...) and bearing the C93S mutation (ΔTrx2C93S). Insertion of 26 27 the product in the NdeI and EcoRI of vector pET24a+ gave rise to plasmid 28 29 pET24a+ΔTrx2C93S coding for ΔTrx2C93S comprised of 107 amino acids. The mouse Trx2 30 31 lacking its mitochondrial targeting sequence is identical to the enzyme from rat and differs in 32 only two amino acids from human Trx2. 33 34 35 36 Purification of ΔTrx2C93S 37 E. coli BL21(DE3) cells were transformed with plasmid pET24a+ΔTrx2C93S and were 38 39 grown in LB medium with kanamycin (50 mg/ml), in 1 l cultures in 2 l flasks at 37 ºC, 140 40 41 rpm. At A600 of ~0.5, IPTG was added (0.5 mM), temperature was set to 25 ºC and cells were 42 43 further grown for 6 hours after which they were harvested and resuspended in 20 mM Tris 44 (non-buffered) as 100 A600/ml. EDTA (1 mM), lysozyme (0.1 mg/ml) and 2 mercaptoethanol 45 46 (50 mM) were added and the resuspension was frozen and thawed for three times. The sample 47 48 was sonicated thoroughly and centrifuged at 10,000 g for 30 min. The occurring total lysate 49 50 supernatant (TCLS) was filtered through a 2 μm filter and loaded on a DEAE FF column 51 equilibrated with 20 mM Bis-Tris-HCl, pH 6.7. ΔTrx2C93S was eluted in a 0 to 1 M NaCl 52 53 gradient at ~15 mS/cm (supplementary figures 2 and 3). Fractions containing ΔTrx2C93S (by 54 55 SDS-PAGE) were concentrated, made up to 5 mM DTT, stood for 30 min at RT and 56 chromatographed on Sephadex G-50 Superfine in 20 mM Hepes-KOH, 1 mM EDTA, 100 57 58 mM NaCl, pH 7.0, at 0.1 ml/min. ΔTrx2C93S was obtained in a homogeneous stage after this 59 60

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1 2 3 purification step. The extinction coefficient ε280 of purified ΔTrx2C93S was assumed as 4 6990·M-1·cm-1 (https://web.expasy.org/protparam/). 5 6 7 8 Immobilization of ΔTrx2C93S on solid phase 9 10 Homogeneous ΔTrx2C93S (estimated pI of 4.9) was coupled to Affi-Gel 15 according to the 11 protocol of the manufacturer (Bio-Rad). In experiments concerning interaction of ΔTrx2C93S 12 13 with crude lysates, ~10 mg ΔTrx2C93S was immobilized/ml of Affi-Gel beads. When 14 15 mitochondrial extracts were obtained, 1 ml of beads contained ~1 mg ΔTrx2C93S. The 16 conjugated beads were used for a maximum of two affinity experiments after which they were 17 18 discarded. 19 20 21 For Review Only 22 Preparation of tissue extracts 23 Μale mice of C57BL/6 genetic background, 5-6 months old, were used. The procedures for 24 25 the care and treatment of animals were approved by the Animal Care Committee of East 26 27 Attica County, Athens, Greece, and followed the guidelines of the Association for the 28 29 Assessment and Accreditation of Laboratory Animal Care and the recommendations of the 30 Federation of European Laboratory Animal Science Association. 31 32 Lung, kidney and skeletal muscle were isolated immediately after sacrifice, inserted in liquid 33 34 N2 and stored at -80 ºC. Frozen tissue was initially crushed and homogenized by pestle in a 35 liquid N cooled mortar to a fine powder which was transferred to ice cold 50 mM Tris-HCl, 1 36 2 37 mM EDTA, 0.2 M KCl, pH 8.0 with a freshly added cocktail of protease inhibitors (1 mM 38 v 39 PMSF, 0.5 mM Na3VO4, 5 mM NaF, 1:25 /v complete (EDTA free) protease inhibitor 40 41 cocktail in PBS from Roche (catalogue number: 11873580001). The sample was sonicated on 42 v ice thoroughly. Triton X-100 (1 % /v) was added and the homogenate was centrifuged at 43 44 14.000 g for 45 min to obtain the total cell lysate supernatant (TCLS) fraction. In the case of 45 46 kidney lysate preparations for example, to 0.647 gr of pulverized kidney tissue, 5 ml of 50 47 48 mM Tris-HCl, 1 mM EDTA, 0.2 M KCl, pH 8.0 were added. Typical protein concentrations 49 of the TCLS were in the order of ~10 mg/ml. 50 51 52 53 Preparation of mitochondrial extracts 54 Heart and brain (and skeletal muscle in one case: supplementary table 1.13) mitochondrial 55 56 fractions were prepared according to [28]. In more detail, animals were perfused with ice-cold 57 58 PBS, and hearts and quadriceps muscle were isolated from six mice. Freshly isolated tissue 59 60 (1-2 gr muscle/heart, or seven whole brains without their cerebella) was minced in 10 ml ice-

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1 2 3 cold MSE (225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 1 mM Tris–HCl pH 8.0, 0.5 4 mM Na VO , 5 mM NaF, 1 mM PMSF and and protease inhibitors (Sigma Protease Inhibitor 5 3 4 6 Cocktail P8340). The minced tissue was further homogenized by up to 15 strokes of Teflon 7 8 head at 1500 rpm on ice using a Glas-Col homogenizer. The homogenate was centrifuged at 9 10 800 g for 10 min and the pellet was re-extracted with 6 ml MSE and centrifuged again at 800 11 g for 10 min at 4 °C. The two resulting supernatants were combined and centrifuged at 12,000 12 13 g for 30 min at 4 °C to give crude mitochondria at the pellet and cytoplasm in the supernatant. 14 15 Crude mitochondria were suspended in 1 ml MSE and placed on the top of a sucrose step 16 gradient (60, 50, 40, 30 and 20 %, 2 ml each) in which were centrifuged at 200,000 g for 30 17 18 min at 4 °C using a TH641 (Sorvall) swing out rotor. 0.5 ml fractions were collected from the 19 20 40 % and the 50 % sucrose steps. The selected fractions were subjected to Western blotting to 21 For Review Only 22 ensure the presence of mitochondria. The adenine nucleotide translocator 1 (ANT 1) was 23 considered as a positive mitochondrial marker, while LAMP-1 protein and calsequestrin were 24 25 considered as markers for late endosomes-lysosomes and endoplasmic reticulum respectively. 26 27 Representative Western blots with antibodies against ANT 1 (sc 9300, Santa Cruz 28 29 Biotechnology, 1:300 dilution), LAMP-1 (NB1952 from Novus Biologicals antibody at 1:400 30 dilution) and calsequestrin (sc 28274, Santa Cruz Biotechnology, 1:400 dilution) are shown in 31 32 supplementary figure 3 for samples derived from cardiac muscle. Fractions positive for ANT 33 34 1 but negative for LAMP-1 and calsequestrin were considered as mitochondrial fractions 35 (supplementary figure 3). This rationale and method were followed to isolate mitochondrial 36 37 fractions from brain, heart and quadriceps muscle. 38 39 40 41 Purification of ΔTrx2C93S interacting substrates 42 Prior to adding to cell lysates, beads-ΔTrx2C93S (1 ml) were reduced with DTT 5 mM for 30 43 44 min at RT in 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2 M KCl. Thereafter beads were 45 46 transferred to a column and washed extensively in 50 mM Tris-HCl, 1 mM EDTA, 0.2 M 47 48 KCl, pH 8.0 to remove the DTT. 5 ml of the lysate (~10 mg/ml protein) in 50 mM Tris-HCl, 1 49 mM EDTA, 0.2 M KCl, pH 8.0 was added to solid phase (1 ml) with bound ΔTrx2C93S and 50 51 the slurry was left (in 15 ml tubes) on a rocking platform at 4 °C overnight. To complete all 52 53 nucleophilic attacks of any remaining thiolates of ΔTrx2C93S to possible substrates, selenite 54 (0.1 mM) was added the next day and the suspension was mildly rotated at room temperature 55 56 for 1 hour after which it was transferred on a glass column. The flow through was discarded 57 58 and the beads were washed extensively with (2x13 ml) 50 mM Tris-HCl, 1 mM EDTA, 0.2 M 59 60 KCl, pH 8.0 (high salt wash), (13 ml) 20 mM Hepes-KOH pH 8.0 (low salt wash), 50 mM

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1 2 3 Tris-HCl, 1 mM EDTA, 0.2 M KCl, pH 8.0 1 % v/v Triton X-100 (13 ml) (high salt wash 4 with detergent) and finally with 20 mM Hepes-KOH pH 8.0 (4x13 ml). At this stage, the first 5 6 elution of non-covalently bound material (via hydrophobic interactions) was performed by 7 8 adding 0.1 M acetic acid-formic acid pH 2.1 (2x1 ml). Immediately after, 1 M Tris-HCl pH 9 10 9.0 (0.4 ml) plus 20 mM Hepes-KOH pH 8.0 (1 ml) were sequentially added. The column was 11 further washed with 20 mM Hepes-KOH pH 8.0 (4 ml) and the column temporarily gravity 12 13 dried. All the above elutions were polled in the “acid wash” fraction (about 7.5 ml) to which 14 15 KOH 10 M (60 μl) were added to a final pH of ~8. The slightly dried column beads- 16 ΔTrx2C93S after the acid elution were transferred to a new tube (15 ml) where 20 mM 17 18 Hepes-KOH pH 8.0 with 10 mM DTT (3 ml) were added. The slurry stood at RT for 1 h after 19 20 which it was reloaded on a glass column and washed with 20 mM Hepes-KOH pH 8.0 (2x4 21 For Review Only 22 ml). The whole 11 ml constituted the “DTT fraction” corresponding to covalently bound 23 material (via disulfide bonds) freed from the column by DTT reduction. Acid and DTT 24 w 25 fractions were kept on ice. Their proteins were precipitated with TCA (6 % /v) and 26 27 deoxycholate (125 μg/ml) [29], centrifuged (10.000 g, 30 min, 4 °C), the resulting pellets 28 29 were washed with acetone, methanol, resuspended in isoelectrofocusing buffer (IEF) and 30 subjected to electrophoresis. All fractions resulting from the different elutions prior and after 31 32 TCA precipitation were kept on ice. 33 34 35 Electrophoretic separation of fractions 36 37 Two dimensional separation of the purified fractions took place as follows: Samples were 38 39 resuspended in IEF sample buffer (7 M urea, 2 M thiourea, 50 mM Tris-HCl, 2 % CHAPS, 40 41 0.4 % DTE, 0.1 % bromophenol blue, pH 7.5) including 3.6 % v/v of protease inhibitors mix 42 (Roche, Cat No: 11873580001) and loaded onto 7 cm strips pH 3-10 (Biorad) using cups. For 43 44 the first dimensional separation a voltage of 4000 V was applied for a total run of 12,500 VH. 45 46 Strips were then incubated for 15 min at room temperature with equilibration solution I (6 M 47 48 urea, 50 mM Tris-HCl, pH 8.8, 30 % glycerol, 2.0 % SDS, 30 mM DTE) followed by a 15 49 min incubation with equilibration solution II (same composition as in solution I but with 230 50 51 mM iodoacetamide instead of DTE) for reduction and alkylation of the proteins. Strips were 52 53 then applied on the top of 12.5 % polyacrylamide mini gels, by the use of 0.5 % agarose in 54 TGS buffer and SDS-PAGE run at 40 V for 15 min followed by 80 V for 2 hours 55 56 approximately. 2D gels were stained with colloidal Coomassie blue overnight and destained 57 58 with ultra pure water. 59 60

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1 2 3 Protein identification by MALDI-TOF-MS (Peptide Mass Fingerprinting) 4 Following this procedure gels were scanned, spots were detected and excised. In gel-trypsin 5 6 digestion was performed according to standard protocols. In brief, destaining of the excised 7 8 spots took place by 30 min incubation in 150 μl of destaining solution (50 mM ammonium 9 10 hydrogen carbonate, 30 % acetonitrile). Protein spots were then washed with 150 μl ultra pure 11 water twice and dried with the help of a vacuum centrifuge. Dried protein spots were 12 13 trypsinized with the addition of 30 ng trypsin (diluted in 10 mM ammonium hydrogen 14 15 carbonate pH 8.0) and overnight incubation at room temperature. Finally tryptic digests were 16 extracted by the addition of 10 μl extraction solution (50 % v/ acetonitrile, 0.1 % v/ TFA) 17 v v 18 and 15 min incubation at room temperature. Peptide mixture (1 μl) was applied on an anchor 19 v v 20 chip MALDI plate with 1 μl matrix solution (50 % /v acetonitrile, 0.1 % /v TFA, 0.025 % α- 21 For Review Only 22 cyano-4-hydroxycinnamicacid) containing the peptides des-Arg-bradykinin (Sigma, 904.4681 23 Da) and adrenocorticotropic hormone fragment 18–39 (Sigma, 2465.1989 Da) as internal 24 25 standards. Samples were analysed in a TOF mass spectrometer (Ultraflex, Bruker Daltonics, 26 27 Bremen, Germany). Peak list was created with Flexanalysis v2.2 software by Bruker by 28 29 summarizing 400 laser shots at intensity between 40 and 60 %. Smoothing was applied with 30 Savitzky-Golay algorithm (width 0.2 m/z, cycle number 1). Signal to noise was calculated by 31 32 SNAP algorithm and a threshold ratio of 2.5 was allowed. Peptide matching and protein 33 34 searches were performed automatically with Mascot Server 2 (Matrix Science). The peptide 35 masses were compared to the theoretical peptide masses of all available proteins from Mus 36 37 musculus in the Swissprot database. Monoisotopic masses were used, 1 miscleavage site was 38 39 calculated for trypsin proteolytic products, carbamidomethylation of cysteine was calculated 40 41 as fixed and oxidation of methionine as variable modifications and finally a mass tolerance of 42 25 ppm was allowed. 43 44 45 46 Isolation of thioredoxin interacting clones using the yeast two hybrid system 47 48 cDNA libraries from human liver and rat brain were cloned into plasmid pGAD10 [LEU2, 49 GAL4 (768–881)] (Clontech). All cDNA clones were therefore expressed as fusion proteins 50 51 with the GAL4 activation domain (AD). The human Trx2 gene (without its mitochondrial 52 53 signal, Trx2 starting as TTF) was cloned into the pGBT9y (Clontech) and expressed as a 54 fusion protein with the DNA binding domain (BD) of the GAL4 transcriptional activator. The 55 56 fusion site was verified by sequencing (not shown). pGBT9y also contained the gene for Amp 57 58 resistance but in the place of the Leu gene (as in pGAD10) it contained the gene coding for 59 60 tryptophan (Trp). The Trx2 gene was also cloned into the pAS2 vector which is similar to

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1 2 3 pGBT9y but with the ability to express higher amounts of GAL4-Trx2, allowing thus, the 4 detection of weak protein interactions with Trx2. The pGBT9y/Trx2 construct was 5 6 transformed into yeast HF7C cells. HF7C-pGBT9y/Trx2 cells surviving on plates without Trp 7 8 were in addition transformed with the human liver or the rat brain library in pGAD10 plasmid 9 10 using PEG/ lithium acetate. The yeast colonies growing on plates lacking Leu/Trp/His were 11 numbered, picked with an inoculating loop and replated on plates lacking Leu/Trp/His. After 12 13 three days at 30 °C, growing (positive) colonies were collected and used for preparation of 14 15 plasmid DNA which was electroporated into the HB 101 bacterial strain. Selection was on M9 16 Amp-containing plates lacking Leu. Plasmids from distinct growing colonies were isolated 17 18 and transformed separately into yeast strain Y187 which grown on synthetic defined medium 19 20 (SD) plates lacking Leu so that only cells containing the same pGAD10/cDNA clone would 21 For Review Only 22 grow. Growing colonies from each plate were mated with pAS2/Trx2, HF7C yeast cells and 23 spread on SD plates lacking Leu/Trp. All cells that grew contained the same pGAD10/cDNA 24 25 clone and the pAS2/Trx2 vector. cDNA inserts from the growing clones were sequenced and 26 27 provided the presented in supplementary tables 2.2 and 2.3. 28 29 30 Construction of the Trx2 interactome and diseasome. 31 32 Human interactors of Trx2 were mined from the BioGRID web resource [26]. The 33 34 mitochondrial topology of a protein species was according to Human MitoCarta2.0 (1158 35 mitochondrial genes [27]). The disease network (diseasome) uncovering disease-disease 36 37 relationships was reconstructed by the Gene Prioritization and Evidence Collection (GPEC) 38 39 plug-in of Cytoscape v.3.0.0 [30] taking into consideration the interactions between the 40 41 mitochondrial Trx2 targets (MitoCarta and BioGRID) and the gene-disease associations 42 retrieved from OMIM [31]. GO annotation of human proteins was performed by DAVID tool 43 44 [32] and the ClueGO v.2.0.0 plug-in of Cytoscape as described previously [33, 34]. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Results 4 5 6 1. Construction of the interactome of human Trx2 7 8 9 10 Isolation and analysis of Trx2-interacting proteins by proteomics 11 12 To isolate Trx2 interacting proteins, mouse total protein extracts from lung, kidney or skeletal 13 muscle and mitochondrial extracts from mouse brain were initially left to interact overnight 14 15 with Affi-Gel 15 coupled to ΔTrx2C93S. Next, the Affi-Gel slurry was loaded onto a column 16 17 and washed with a succession of low and high salt washes. Two types of elutions followed. 18 The first elution (“acid wash” by acetic/formic acid) aimed to the isolation of proteins bound 19 20 strongly but in a non-covalent manner to Trx2. The second elution by DTT aimed at the 21 For Review Only 22 proteins bound exclusively by a disulfide bond. The eluted proteins were visualized by 2D 23 24 electrophoresis and picked and analysed by MALDI-TOF. Mouse mitochondrial extracts from 25 cardiac and skeletal muscle were also used but analysed by 1D gels. The proteins identified 26 27 using the two different elution methods from the different tissues and mitochondrial fractions 28 29 are presented in supplementary tables 1.1-1.13. A synopsis of the proteomics results 30 31 (supplementary tables 1.1-1.13) is presented in supplementary table 2.1 (176 protein species). 32 33 34 Trx2-interacting proteins as revealed by Y2H screens 35 36 A human liver cDNA library was used in Y2H screens to isolate proteins interacting with 37 Trx2. Approximately 16x106 clones were examined. 110 positive clones (growing on 38 39 Leu/Trp/His) were analyzed. The Y2H screen of the human liver library gave 11 clones, all 40 41 novel interacting partners for Trx2 (supplementary table 2.2). Another Y2H screen using a 42 43 cDNA library from rat brain, gave 7 novel interactors (supplementary table 2.3). 44 45 46 Integration of the mouse, rat and published data for the construction of a probable 47 48 interactome of human Trx2 49 50 The information obtained from the mouse proteomics analysis and the human and rat Y2H 51 experiments were combined to construct an interactome for human Trx2. This is most likely a 52 53 valid approach as mouse, rat and human Trx2 are practically identical (supplementary figure 54 55 1). The resulting combined interactome consisted of 195 proteins (Trx2 itself, plus 176 56 interactors from the proteomics approach, plus 11+7 protein from the Y2H screens, 57 58 supplementary tables 2.1, 2.2, and 2.3), plus the mitochondrial 16S RNA. Inclusion of the 59 60

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1 2 3 known (BioGRID) human Trx2 interactome of 59 proteins (supplementary table 2.4) raised 4 the interactome of human Trx2 to 254 proteins (including Trx2), plus the mitochondrial 16S 5 6 RNA. Not a single protein species was common between the current and published data. 7 8 9 10 Selection of Trx2-specific mitochondrial protein interactors 11 As Trx2 is considered a mitochondrial protein, its potential interactors would be expected to 12 13 be mitochondrial too. In the protocols used for the isolation of Trx2 interactors, the total cell 14 15 lysates used contained non-mitochondrial proteins, thus, a number of cytosolic proteins 16 appeared interacting with Trx2 (supplementary table 2.1). The same applied for the Y2H 17 18 screens (supplementary tables 2.2, 2.3). To overcome these cytosolic interactions, we 19 20 examined whether the proteomics approach would be more specific if only mitochondrial 21 For Review Only 22 fractions were examined as the source of Trx2 interactors (supplementary tables 1.9-1.13). 23 However, this approach did not achieve exclusive selectivity for mitochondrial interactors: in 24 25 the case of the mitochondrial fractions isolated from brain (supplementary tables 1.9, 1.10, 2.1 26 27 combined acid and DTT elutions) for example, 16 out of a total of 26 protein species were 28 29 mitochondrial according to the MitoCarta repository of proteins with strong evidence for 30 mitochondrial localization (MitoCarta2.0 [27]). To overcome the apparently false cytosolic 31 32 positives, all potential interactors of Trx2 had their localization validated by MitoCarta. 33 34 According to this approach, the novel mouse, human and rat mitochondrial Trx2 interactors 35 were 48 proteins, plus the mitochondrial 16S RNA (supplementary tables 2.1, 2.2, and 2.3; 36 37 mitochondrial species in green background). Integration of these 48 proteins with the 4 known 38 39 human mitochondrial interactors of Trx2 (combination of BioGRID [26] and MitoCarta2.0 40 41 [27]) raised the number of human mitochondrial protein species interacting with Trx2 to 52 42 (table 1). The herein identified mitochondrial interactors included expected hits 43 44 (peroxiredoxins, methionine sulfoxide reductase) but also proteins participating in the 45 46 formation of iron sulfur clusters, protein folding in the mitochondrion, and proteins of lipid 47 48 and carbohydrate metabolism. The novel findings regarding mitochondrial interactors and 49 their potential significance to normal cellular function and disease are further elaborated in the 50 51 “Discussion” section. 52 53 54 General observations on the proteins that associated with Trx2 55 56 A general outcome of the presented data (Fig. 1) is that almost the same number of protein 57 58 species interacted covalently or non-covalently with Trx2. 19 mitochondrial interactors, were 59 60 from non-covalent interactions (16 acid-eluted, 3 from Y2H screens), 21 were of the covalent

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1 2 3 type, while 7 were common in covalent and non-covalent interactions. Most mitochondrial- 4 specific interactions were derived from the proteomics experiments (46/182) while 3/19 were 5 6 derived from the Y2H screens and 4/59 from BioGRID. The highest number of interacting 7 8 proteins was detected in lung lysates, a finding that may correlate with the higher levels of 9 10 mRNA Trx2 in the tissue [35]. 11 12 13 2. The diseasome of human Trx2 14 15 Any diseasome (disease network) links the phenotypic features of a disease (human disease 16 phenome) to common molecular mechanisms such as genetic associations and/or protein 17 18 interactions. Clustering of disease phenotypes is likely due to underlying common 19 20 pathophysiology mechanisms [36]. Diseasome analysis by the GPEC plug-in of Cytoscape for 21 For Review Only 22 the mitochondrial interactome of Trx2 showed that the genes encoding Trx2 and its 23 interactors were directly associated with ten different diseases forming four cliques (Figure 2, 24 25 supplementary table 2.5). 26 27 28 29 30 31 32 33 34 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

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1 2 3 Discussion 4 5 6 The acceptors of the flow of electrons via thioredoxins are per se the interacting partners of 7 8 thioredoxins and constitute the “classical” thiol-reacting substrates such as ribonucleotide 9 10 reductases, methionine sulfoxide reductases etc. These interactions are fast covalent 11 12 exchanges of electrons via the active site thiols of thioredoxins and should in principle be 13 detected by a monothiol thioredoxin species. As thioredoxins may associate non-covalently 14 15 with other proteins, Y2H approaches should be able to detect this type of interactions. Up till 16 17 now, 59 interactors for human Trx2 are described in the BioGRID interaction repository, with 18 only four of them being mitochondrial (MitoCarta2.0). Using a proteomics approach 19 20 combined with Y2H screens, the potential mitochondrial interactome of Trx2 was increased 21 For Review Only 22 herein by 49 additional interactors. 23 24 25 Novelty of the method 26 27 While the general principle of the monothiol trapping method has been used for other 28 29 thioredoxins/glutaredoxins, the current approach differed in subtle aspects. First, 30 31 immobilization of Trx2 was not directly on activated Sepharose beads but on Affi-Gel resin 32 via a 15 carbon spacer to reduce steric hindrance preventing the interaction of thioredoxin 33 34 with ligands: binding of EcoTrx1 directly on dextran via N-hydroxy succinimide esters, 35 36 greatly limited its interaction with gene 5 protein of phage T7 [37]. By adding a spacer of six 37 extra carbons (6-amino-n-hexanoic acid), the hindrance was eliminated [37]. Second, the 38 39 method detected proteins interacting strongly with Trx2 in a non-covalent manner that were 40 41 removed from Trx2 prior to the reducing step (the latter detecting covalent interactions with 42 43 Trx2) by a succession of low and high salt washes, finally followed by an acid wash to detect 44 strong hydrophobic interactions. Such washing protocols are used for the affinity purification 45 46 of antibodies (KDs in the low nM range) and in the present case gave a significant number of 47 48 potential interactors (almost 50 % of total interactome), that might have been overlooked or 49 50 misinterpreted by a disulfide reducing protocols aiming at the specific detection of covalent 51 redox (S-S) interactions. In addition, acid treatment before reduction increased the specificity 52 53 of the method for the following detection of S-S covalent interactions. Third, selenite was 54 55 employed to force the oxidative interaction of thioredoxin and ensure the nucleophilic attack 56 to its potential substrates as the compound is a fast oxidant of the thioredoxin system [38]. 57 58 Fourth, the amounts of protein used in the interaction experiments were generally high for 59 60

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1 2 3 immobilised Trx2 and lysates to enhance interactions. Finally, material from different tissues 4 was examined, to include the possible tissue-specific expression patterns of potential 5 6 interactors of Trx2. Apart from the suggested novel interactors (table 1), the greater emerging 7 8 concept for the Trx2 interactome is that the number of non-covalent interactors is as much if 9 10 not greater than the number of the covalent ones (figure 1). Assuming some degree of 11 complementarity between the shapes of Trx2 and its interactors, we propose three major types 12 13 of interactions (Fig. 3): (i) somewhat weak ionic (Fig. 3A), mostly not detected here due to 14 15 the preceding relatively high salt washes; (ii) strong hydrophobic as revealed by the acidic 16 elutions (Fig. 3B); (iii) covalent (SS bonds, Fig. 3C) due to the thiol oxidoreductase activity 17 18 of Trx2. The hypothesis should not exclude mixed types of interactions between the contact 19 20 areas (e.g. mostly hydrophobic and ionic to a lesser extent etc). 21 For Review Only 22 23 On the specificity of the detected interactions 24 25 The immediate concern of the applied approach is whether the monothiol motif within the 26 27 substrate contact area of Trx2 could select for specific interactors. In the case of EcoTrx1 with 28 29 3′-phosphoadenosine 5′-phosphosulfate reductase, the interaction was not dependent on the 30 exact active site sequence (CxxC) of thioredoxin but on complementarities in geometry and 31 32 charges in the contact areas of the two molecules [39]. This finding showed that the very strict 33 34 characteristics of thioredoxin structure dictate themselves the interaction with specific 35 substrates. Fine details on the catalytic mechanism of thioredoxins revealed that it differed 36 37 among these oxidoreductases [40]: while all thioredoxins apparently use the Michaelis- 38 39 Menten mechanism, the eukaryotic species (mithochondrial Trx2 excluded) may also apply 40 41 single-electron transfer reactions (SET) [40]. Prokaryotic thioredoxins and mitochondrial 42 Trx2 reduce their substrates by nucleophilic substitution (SN2) and SET reactions [40]. The 43 44 difference in the reductions mechanism were explained in view of the deeper substrate 45 46 binding groove of eukaryotic thioredoxins compared to the more shallow one of the 47 48 prokaryotic and Trx2 [40]. 49 Under the previous considerations, the overall structure of monothiol Trx2 used herein for 50 51 monothiol trapping should have been able to recognize specific interactors. However, the 52 53 mitochondrial interactors identified by the proteomics approach were 46 out of 176. In 54 BioGRID, the common interactors between Trx1 and 2 (147 and 59 interactors respectivly) 55 56 were eight proteins, suggesting increased specificity for each thioredoxin species. Perhaps, the 57 58 considered non-specific cytosolic interactors of Trx2 (supplementary table 2.1) could be 59 60 envisaged as a bycatch of the contact area of a mitochondrial protein exposed to the cytosol

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1 2 3 and interacting with complementary surfaces of cytosolic proteins. In case that Trx2 may have 4 hitherto unidentified splicing variants without the mitochondrial targeting sequence, these 5 6 interactions could be significant: the splice isoforms the TXN2 gene have not been analysed 7 8 for all types of cells. In any case, the monothiol method picked bona fide interactors of Trxs 9 10 (e.g. mitochondrial peroxiredoxins, mitochondrial methionine sulfoxide reductase), while 11 classic substrates of Trx1 such as ribonucleotide reductase and cytosolic methionine sulfoxide 12 13 reductase, were not selected. The latter could reflect the afore-mentioned structural 14 15 characteristics conferring exclusive specificity on Trx2, or the reducing action of alternative 16 disulfide reductants (the glutaredoxin system or the action of cytosolic Trx1), preventing the 17 18 formation of the complexes in total cell lysates. Considering the likelihood that the high 19 20 amounts of Trx2 immobilized on the affinity columns should compete with interferences from 21 For Review Only 22 other redoxins, the mentioned subtleties in thioredoxin specificity, and the currently believed 23 mitochondrial localisation of Trx2, it could well be that at least the mitochondrial interactors 24 25 of table 1 represent specific findings of functional significance. 26 27 28 29 Implications on the possible cellular functions of Trx2 in view of the proposed specific 30 interactions 31 32 33 34 1. Mitochondrial integrity 35 MICOS complex subunit Mic60 (IMMT) is one of the major subunits of the mitochondrial 36 37 intermembrane space bridging complex that is involved in the invaginative folding of inner 38 39 mitochondrial membranes into cristae [41]. The possible interaction of Trx2 with IMMT 40 41 suggests the involvement of Trx2 in maintaining the inner mitochondrial membrane structure. 42 Mitochondrial inorganic pyrophosphatase 2 (PPA2) hydrolyzes inorganic pyrophosphate to 43 44 pyrophosphate, an essential activity for the regulation of a proper mitochondrial membrane 45 46 potential, organization and function. PPA2 mutations are pathogenic and can be related to 47 48 cardiomyopathy [42]. Overexpression of Trx1 [43], or Trx2 [44, 45] prevent oxidative 49 damage to the heart, while ablation of the TrxR2 in mice resulted in fatal dilated 50 51 cardiomyopathy [46]. Our data suggest that Trx2 could be the linked to cardiomyopathy via 52 53 TrxR2 and PPA2. 54 55 56 2. Formation of iron-sulfur clusters 57 58 A number of proteins participating in iron-sulfur cluster formation were associated with Trx2. 59 60 The mitochondrial Stress-70 protein (HSPA9, Uniprot ID: P38646), is a chaperone involved

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1 2 3 with iron-sulfur cluster (ISC) biogenesis that occurs exclusively in mitochondria [47]. It 4 interacts with and stabilizes ISC cluster assembly proteins frataxin (FXN, the prokaryotic 5 6 CyaY, an iron donor), NFU1 (maturation of lipoate synthase and succinate dehydrogenase, 7 8 prokaryotic equivalents are the C-terminal domains of NifU, NfuA, both scaffold proteins), 9 10 cysteine desulfurase NFS1 (the equivalent of IscS, a sulfur donor) and ISCU (a scaffold 11 protein for Fe/S cluster synthesis) [47, 48]. Therefore, HSP9 is a central protein in ISC 12 13 biogenesis whose depletion causes a deficit in mitochondrial ISCs ultimately affecting heme 14 15 synthesis and differentiation of red blood cells [48, 49]. Its herein detected interaction with 16 Trx2 raises the possibility that the activity of HSP9, hence Fe/S cluster biogenesis (and heme 17 18 synthesis) in mitochondria may be affected by Trx2. Apart from HSPA9, Trx2 interacted with 19 20 the Fe/S cluster containing proteins NDUFS2, NDUFS3, and NDUFV1. In the super 21 For Review Only 22 molecular assembly of the human mitochondrial respiratory mega complex [50], NDUFS3 23 and NDUFV1 appear accessible to solvent and thus could in principle interact with Trx2. It is 24 25 tempting to assume that the interaction of Trx2 with these proteins would result in the 26 27 reduction of their cysteines in a step related to protein folding or the formation/assembly or 28 29 rearrangement of their iron-sulfur clusters. In fission yeast, the mitochondrial Trx2 system 30 could affect the activity of Fe-S proteins (such as mitochondrial aconitase and cytosolic sulfite 31 32 reductase but not succinate dehydrogenase) by lowering the levels of free cellular iron [51]. 33 34 EcoTrx1 along with TrxR were able to supply iron specifically to IscA, the protein (with 35 NFS1 as the eukaryotic homologue) that may provide iron for normal Fe/S cluster assembly 36 37 on IscU [52]. The mitochondrial Trx2 system could thus affect iron sulfur cluster biogenesis 38 39 by (i) affecting the activity of HSPA9 (ii) affecting the redox state of cysteines in specific Fe- 40 41 S enzymes and indirectly by (iii) having an impact on free Fe supply [51]. The proposed 42 interaction of HSPA9 with Trx2 implicates the latter with sideroblastic anemia, the inability 43 44 to insert iron in the heme of hemoglobin resulting in the characteristic altered phenotype of 45 46 red blood cells [53]. 47 48 49 3. Oxidative stress 50 51 Superoxide generated from stray electrons of the electron transport chain may be reduced by 52 53 superoxide dismutases (SODs) to generate hydrogen peroxide (H2O2) that may promote 54 oxidative stress via formation of hydroxyl radicals by the Fenton reaction ([54] and references 55 56 therein). Trx2 could itself reduce H2O2 to H2O at low rates but is considered as the main 57 58 electron donor to peroxiredoxins (Prx) 3 and 5 who together with glutathione peroxidases 1 59 60 and 4 are on top of the hierarchy for the reduction of H2O2 in cells [55]. The central role of

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1 2 3 Prx3 in detoxifying H2O2 has been demonstrated for cell lines [56] and transgenic mice [57]. 4 Trx2 is the main reductant of Prx3 in mitochondria [58] while human glutaredoxin 2 (hGrx2) 5 6 may also reduce Prx3 but with lower (double Km) catalytic efficiency. Trx2 (but not hGrx2) 7 8 may also reduce Prx5 [59], although the complex of a mutated Trx2C93S did not form stable 9 10 complexes with Prx5 as it did with Prx3 [60]. Cytosolic human Trx1 can reduce Prx3 and 11 Prx5 with better kinetics than Trx2 [59] but this interaction is not likely to happen due to the 12 13 different compartmentalization of the two thioredoxins. Levels of H2O2 were elevated when 14 15 auranofin, an inhibitor of TrxRs was applied in mouse mitochondrial extracts concomitant 16 with increased levels of oxidized Trx2, Prx3 and ROS (as detected by MitoSOX) [61]. Thus, 17 18 an intact mitochondrial TrxR2 was essential for the reduction of H2O2. The participation of 19 20 Trx2 is not limited to the elimination of H2O2 via Prx3 but also to inhibition of apoptosis: 21 For Review Only 22 knockdown of Trx2 in lung epithelial cells growing in hyperoxia increased the 23 phosphorylation of ASK1 due to lack of reduced Trx2 binding to it and preventing the 24 25 serine/threonine kinase activity of ASK1 [62]. In the current work, all peroxiredoxins were 26 27 picked up as interactors of Trx2. This finding, as well as the interactions with mitochondrial 28 29 methionine sulfoxide reductase (MSRA) validates the method used. In addition, we present 30 the possible interaction of Trx2 with the peroxisomal bifunctional enzyme (EHHADH) that 31 32 normally participates in fatty acid β-oxidation by NAD+. Absence of EHHADH is related to 33 34 peroxisomal disorders as the enzyme may affect catalase activity and thus the elimination of 35 H O produced by the β-oxidation of fatty acids [63]. 36 2 2 37 Trx2 is considered interacting with SOD1 (BioGRID) implicating further Trx2 with reactions 38 39 involving H2O2 and amyotrophic lateral sclerosis [64]. More than a hundred mutations in the 40 41 human sod1 have been associated with familial ALS [65]. Reduction of mutant SOD1s by 42 Trx2 1was followed by the formation of aggregates via interaction of the reduced Cys57- 43 44 Cys146 thiols of SOD1s with the Cys6 and Cys111 thiols of neighbouring SOD1s [66]. The 45 46 toxic effects of mutant SOD1s are considered as a consequence of formation of soluble or 47 48 insoluble oligomers [67]. In full agreement with the antioxidant functions of Trx2, increased 49 oxidative stress was evident in mitochondria of haploid mice for TXN2 [68]. 50 51 52 53 4. Detoxification from aldehydes 54 Trx2 interacted with two detoxifying enzymes: carbonyl reductase [NADPH] 3 (CBR3) and 55 56 2 (ALDH2). Aldehyde dehydrogenases catalyse the reduction of 57 58 aldehydes to carboxylic acids and participate in different biological processes including 59 60 detoxification. The herein identified partner of Trx2 ALDH2, is crucial for the oxidation of

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1 2 3 toxic biogenic aldehydes (e.g. acetaldehyde, catecholaminergic metabolites including DOPAL 4 and DOPEGAL and the principal product of lipid peroxidation 4-HNE) to harmless products 5 6 [69]. Mutations compromising the catalytic activity of mitochondrial ALDH2 are responsible 7 8 for increased susceptibility to alcohol intoxication and increased cancer risk due to alcohol 9 10 consumption [70]. Because of its central role as a phase 1 detoxifying enzyme, dysfunction of 11 ALDH2 has been related to many human diseases and pathologies [71]. The activity of the 12 13 enzyme may be inhibited by high concentrations of aldehydes such as those occurring at 14 15 Parkinson’s disease [72]. The detoxification of 4-HNE, which is at the beginning of signaling 16 cascades leading to Alzheimer’s disease, is dependent on ALDH2 [69]. Activation of ALDH2 17 18 has even been proposed as a therapeutic approach for Parkinson’s disease [69]. The activity of 19 20 ALDH2 is redox sensitive: 6-methoxy-2-naphthylaldehyde oxidation by ALDH2 was 21 For Review Only 22 stimulated by addition of DTT either/or TrxR1 and Trx1 [73]. As TrxR1 and Trx1 are 23 normally cytosolic, these findings most likely reflect the interaction of the mitochondrial 24 25 thioredoxin system with ALDH2. 26 27 CBR3 belongs to the category of monomeric carbonyl reductases that catalyze the reduction 28 29 of carbonyls in endogenous and xenobiotic compounds, including steroids and prostaglandins 30 [74]. The latter activities are specific for the abundant human CBR1 but not for the highly 31 32 homologous, less transcribed CBR3 that remains an enigmatic enzyme in terms of substrate 33 34 specificity and cellular function [74]. CBR3 is somewhat abundant in lung, liver, spleen, 35 pancreas, intestine, and more abundant in the ovary [74]. Trx2 that is known to be inhibited 36 37 by cyclopentenone-type prostaglandins [75] interacted in the present work covalently with 38 39 CBR3 from lung tissue preparations. The significance of the proposed interaction between 40 41 Trx2 and CBR3 is not obvious. 42 43 44 5. Protein synthesis 45 46 Trx2 is likely to participate in protein synthesis via its detected interactions with the 47 48 mitochondrial 16S ribosomal RNA gene (identified here by a Y2H screen), the 40S ribosomal 49 protein S18 (RPS18 gene product) and the mitochondrial elongation factor Tu (mEF-Tu, 50 51 TUFM gene product). The mitochondrial 16S ribosomal RNA gene is essential for protein 52 53 synthesis. RPS18 (40S ribosomal protein S18) is a protein of the small ribosomal subunit [76] 54 that has also been identified as an mRNA binding protein [77]. The interaction of the two 55 56 proteins with Trx2 raises the possibility of the participation of the latter in mitoribosomal 57 58 assembly. There has been a possible lingering association between thioredoxins and EF-Tu: 59 60 (i) EcoTrx1 and EF-Tu belong to the same osmotically-sensitive compartment [78]; (ii) EF-

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1 2 3 Tu has thioredoxin/protein-disulfide isomerase activity (reduction of insulin) that is dependent 4 on DTT [79]; (iii) Inactive oxidised EF-Tu is reactivated after reduction by thioredoxin [80]. 5 6 In line to previous publications, this work implies the reduction and therefore concomitant 7 8 activation of mEF-Tu by Trx2. 9 10 11 6. Protein folding in the mitochondrion 12 13 The mitochondrial 60 kDa heat shock protein (HSPD1) together with HSP10 (HSPE1) are 14 15 implicated in the import, folding and assembly of proteins in the mitochondrial matrix 16 (Uniprot ID: P10809). Hsp60 forms heptameric rings [81] in which protein folding may occur 17 18 [82]. Hsp60 and Hsp10 are the structural and functional equivalents of prokaryotic GroEL 19 20 [83] and GroES [84] respectively. HSPD1 rings are properly folded in the mitochondria by 21 For Review Only 22 the concerted action of the HSP9-Hsp10 complex that binds to the unassembled Hsp60 23 precursor to promote its assembly into mature Hsp60 complexes [85]. The association of Trx2 24 25 with this cytosolic pathway (and PDI or P07237, table 1) points to the direction of Trx2 being 26 27 involved in the folding of mitochondrial proteins. 28 29 30 7. ADP-ribosylation 31 32 Trx2 associated with O-acetyl-ADP-ribose deacetylase MACROD1 (also known as LRP16) 33 34 that confers post translational modification of the ADP-ribosylation type. The latter is a 35 reversible post-translational modification with wide-ranging biological implications [86]. 36 37 LRP16/ MACROD1 may hydrolyse the ester bonds forming between glutamate residues in 38 39 proteins [87] or phosphorylated double-stranded DNA ends and a single ADP-ribose moiety 40 41 [88]. MACROD1 may interact with estrogen receptor alpha (ERα) [89] and integrate into the 42 NF-κB transcriptional complex and activate it [89]. Trx2 may also interact directly with the 43 44 p65 subunit of NF-κB [90], while Trx1 may associate with DNA-bound ERα [91]. These 45 46 observations bring into proximity MACROD1 and Trx2 and suggest that the covalent 47 48 interaction observed in this work may be of physiological significance and implicate Trx2 in 49 ADP-ribosylation. 50 51 52 53 8. Glycolysis 54 Trx2 was associated with triosephosphate isomerase (TPI), glyceraldehyde 3-phosphate 55 56 dehydrogenase (GAPDH) and lactate dehydrogenases (LDHs) of the glycolytic cycle. TPI 57 58 catalyzes the reversible interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 59 60 3-phosphate in glycolysis [92]. Several studies show that TPI is a target for different plant

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1 2 3 thioredoxins and EcoTrx1 ([93] and references therein), making it highly possible that the 4 protein may indeed interact with mammalian Trx1 or Trx2. TPI is of mitochondrial 5 6 localization in 14 tissues reported in the Human MitoCarta2.0 [27], while cytosolic TPI may 7 8 have evolved from the mitochondrial form [94]. Therefore, the presently reported covalent 9 10 and non-covalent interaction of TPI with Trx2 could well mean that TPI may interact in vivo 11 with Trx2. The potential interactions of Trx2 with TPI introduces a potential role of Trx2 in 12 13 the pathogenesis of triosephosphate isomerase deficiency [95] (Fig. 2). 14 15 16 17 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), had high prevalence of interaction 18 19 with Trx2 in different tissues (4 hits, supplementary table 2.1). The cytosolic thioredoxin of 20 Plasmodium falciparum [96], EcoTrx1 and plant thioredoxins are known to interact with 21 For Review Only 22 GAPDH ([93] and references therein). In the case of Arabidopsis thaliana cytosolic GAPDH 23 24 thioredoxin/glutaredoxin could reduce a mixed disulfide forming between GSH and an 25 149 26 exposed catalytic cysteine (Cys ), crucial for enzyme function [97]. Trx1 could denitrosylate 27 the active site Cys152 of human GAPDH and thus control the pool of free heme [98]. Human 28 29 Trx1 could also act as an S-desulfhydrase on human GAPDH with the activity residing on the 30 32 31 attacking Cys of Trx1 [99]. Trx1 alone or in combination with TrxR1 and α crystallin were 32 able to revive the activity of GADPH in the cortex and nucleus from cataract lenses [100], 33 34 although the exact mechanism is not known. These observations may validate (via Trx1) our 35 36 data regarding the interaction of Trx2 with GAPDH. Both GAPDH and α crystalline B chain 37 38 were identified as potential targets of Trx2 suggesting that redox interactions between Trx2 39 with α crystallin and GAPDH may occur simultaneously. 40 41 Lactate dehydrogenases (LDHs) A (cytosolic) and B (cytosolic and mitochondrial 42 43 localization) are glycolytic enzymes that catalyze the reversible oxidation of lactate to 44 + 45 pyruvate coupled to the concomitant reduction of NAD to NADH. In the case of LDHD 46 (exclusive mitochondrial localization Uniprot ID: Q86WU2) the electrons for the reduction 47 48 are derived from ferricytochrome c that is oxidized to ferrocytochrome. The balance of 49 50 lactate/pyruvate levels may affect levels of intracellular NADH and thus the activity of 51 NADPH oxidase (production of superoxide when higher levels of NADH and lactate) [101]. 52 53 Higher pyruvate levels can thus promote a reduced intracellular environment [102]. In 54 55 addition, lactate can enhance hydroxyl radical formation by the Fenton reaction [103]. High 56 57 LDH levels are thus considered cytotoxic [104]. Higher lactate levels lead to elevation of 58 Trx1 resulting in HIF-1 increase and synthesis of HIF-1-dependent growth factors [105]. The 59 60

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1 2 3 high reactivity (inhibition) of ebselen with LDH from heart and liver homogenates [106] 4 suggests that LDHs may react with nucleophiles such as thiols. This work presented evidence 5 6 of possible interactions of the three LDHs with Trx2 (LDHA: non-covalent, LDHB: non- 7 8 covalent, LDHD: covalent). Considering the promotion of an oxidative environment by 9 10 LDHs, such interactions would seem biologically logical as a means of interfering directly 11 with the activity of LDHs. In addition, the proposed interaction of Trx2 with LDHB raises the 12 13 possibility of Trx2 being involved in LDHB deficiency although the condition appears non 14 15 pathogenic (OMIM entry 614128). 16 17 18 9. Pyruvate dehydrogenase activity 19 20 The enzyme complex of pyruvate dehydrogenase catalyzes the decarboxylation of pyruvate to 21 For Review Only 22 acetyl-CoA [107, 108], linking the glycolytic pathway to the tricarboxylic cycle [109]. It 23 consists of the E1 (pyruvate dehydrogenase activity), the E2 (dihydrolipoamide 24 25 acetyltransferase) and the E3 (lipoamide dehydrogenase) components ([109], 26 27 https://www.ebi.ac.uk/interpro/entry/IPR027110). The human E1 component is a 28 29 heterotetramer of the α2β2 type (α = PDHA1 and β = PDHB) with two catalytic sites [107]. It 30 contains thiamine pyrophosphate as a co factor and catalyses the rate limiting step of the 31 32 whole reaction [110]. To our knowledge, there is no reported direct interaction of any 33 34 component of the thioredoxin system with any member of the pyruvate dehydrogenase 35 complex. EcoTrx1 could reduce in vitro the disulfides of insulin after receiving electrons from 36 37 reduced lipoamide [111], a of the E2 component. However, suppressor mutants of 38 39 the lipoamide dehydrogenase gene in E. coli showed that glutaredoxin 1 (and not EcoTrx1) 40 41 was able to transfer electrons from oxidized lipoamide to ribonucleotide reductase [112]. 42 Herein, Trx2 was associated to the E1 component subunits PDHA1 and PDHB in a non- 43 44 covalent and covalent manner respectively. The active site loops of PDHA1 (E1 component 45 46 subunit alpha) and PDH (E1 component subunit beta) are flexible and may affect the activity 47 48 of E1 [107, 108, 113]. The detected interaction of Trx2 could perhaps modify their flexibility 49 resulting in alterations of the active sites of E1, raising the possibility of regulation of the 50 51 whole activity of eukaryotic pyruvate dehydrogenase by the thioredoxin system. The two 52 53 systems are interconnected in terms of electron transfer as the pyruvate dehydrogenase 54 complex may provide electrons to mitochondrial TrxR via the mitochondrial membrane 55 56 nicotinamide nucleotide transhydrogenase [114]. The proposed interactions of Trx2 with 57 58 PDHA1 and PDHB implicate the protein with pyruvate dehydrogenase complex deficiencies 59 60 (entries 300502 and 179060 respectively in the OMIM data base: www.omim.org).

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1 2 3 Trx2 interacted covalently with nucleoside diphosphate-linked moiety X motif 19 (NUDT19) 4 that may hydrolyze in vitro the diphosphate bond of free CoA and acyl-CoAs to form 3',5'- 5 6 ADP and 4'-(acyl)phosphopantetheine, while it contributes to the regulation of CoA levels in 7 8 the kidney in vivo [115]. The interaction of Trx2 with NUDT19 implicates the thioredoxin 9 10 system in pathways utilizing CoA (the proposed relations of Trx2 with lipid metabolism will 11 be commented later). 12 13 14 15 10. TCA cycle 16 Trx2 interacted with mitochondrial aconitate hydratase (aconitase, ACO2) that catalyzes the 17 18 stereo-specific isomerization of citrate to isocitrate via cis-aconitate in the subpathway 19 20 synthesizing isocitrate from oxaloacetate in the citric acid cycle. Aconitase contains [4Fe-4S] 21 For Review Only 22 clusters that do not participate in electron flow but may reversibly loose one Fe atom to obtain 23 a [3Fe-4S] cluster (apo aconitase). In E. coli, the cluster is delivered by IscU [116]. In 24 25 conditions of low iron, apo aconitase may bind to the mRNA of iron responsive elements 26 27 [117] associated with iron metabolism [118]. In E. coli, the respective enzyme binds to its 28 29 own mRNA and stabilizes it [119]. Mutations in the human gene may result in vision defects 30 [120], while loss of Fe in wild type enzyme due to smoking related oxidants may result in 31 32 increased iron deposition in arterial cells [121]. In fission yeast, overexpression of Trx2 (but 33 34 not cytosolic Trx1) was able to restore aconitase activity in depleted 35 cells, most likely by regulating the levels of free iron [51]. The detected interaction of Trx2 36 37 with mitochondrial aconitase suggests that Trx2 may reduce a disulfide forming between the 38 39 three cysteine residues comprising the coordination site for the Fe-S cluster, allowing thus 40 41 normal coordination of the cluster. 42 43 44 Trx2 interacted non-covalently with human isocitrate dehydrogenases 1, 2 and 3 (IDH1, 2, 45 46 3G) and covalently with IDH3A. The enzymes catalyze the oxidative decarboxylation of 47 48 isocitrate to alpha-ketoglutarate in the citric acid cycle. In the reaction, IDH1 and IDH2 49 reduce NADP+ to NADPH, whereas IDH3 reduces NAD+ to NADH [122, 123]. IDH1 is 50 51 regarded as cytosolic (although also described with mitochondrial localization [27]), whereas 52 53 IDH2 and 3 are considered strictly mitochondrial. Due to its role in replenishing NADPH 54 pools, IDH2 may supply electrons for the reduction of mitochondrial TrxR and oxidized 55 56 glutathione, affecting thus the activity of glutathione peroxidase [123]. Null mutants for Idh2 57 58 had increased loss of hair cells and neurons in the cochlea of male mice due to oxidative stress 59 60 resulting from a defective mitochondrial thioredoxin system [122]. The interaction of Trx2

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1 2 3 with IDH2 could affect the activity of the latter. The possible implication is a feedback 4 between the thioredoxin system and IDH2. 5 6 IDH3G may form part of the heterotetramer of IDH3 which has an α2βγ composition with α 7 8 being IDH3A, β IDH3B and γ IDH3G, the allosteric activator of the complex [124]. In this 9 10 work, Trx2 could interact with IDH3G non- covalently introducing the possibility of an 11 additional, redox regulation of the allosteric activator component IDH3G of IDH3. In E. coli, 12 13 EcoTrx1 is known to associate with isocitrate dehydrogenase (ICD) [17]. ICD shows an 14 15 approximately 30 % identity to subunits α (IDH3A) and β (IDH3B) and 24 % to IDH3G. 16 Apart from its interactions with the E1 component subunits PDHA1 and PDHB of pyruvate 17 18 dehydrogenase, Trx2 interacted non-covalently with IDH1. Since mutant IDH1 perturbs the 19 20 normal metabolism of pyruvate in glioblastoma cells [125], Trx2 could be affecting pyruvate 21 For Review Only 22 metabolism by IDHs, PDHA1 and PDHB. 23 24 25 11. Electron transport chain 26 27 28 29 Complex I 30 Complex I is a large L-shaped protein complex (almost 1000 kDa [126]), composed of three 31 32 domains: a dehydrogenase domain (N module) and a hydrogenase domain (Q module) both 33 34 located at the mitochondrial matrix, and a transmembrane part (P module) located on the inner 35 mitochondrial membrane [126, 127]. Electrons from NADH (produced from the Krebs cycle) 36 37 are transferred to the dehydrogenase domain (N module), then to the hydrogenase domain (Q 38 39 module) where reduction of coenzyme Q10 (CoQH2) is performed. The reduction is followed 40 41 by translocation of protons from the mitochondrial matrix to the intramembrane space by the 42 transmembrane part (P module), resulting in formation of a proton gradient. 43 44 Trx2 bound non-covalently to NDUFS1, 2, 3 and NDUFV1, all iron-sulfur cluster containing 45 46 proteins of the Q module [126]. NDUFS3 along with NDUFS2 [128] initiate the in vivo 47 48 assembly of Complex-I subunits in the mitochondrial matrix [129]. The detected interactions 49 with NDUFS1, 2 and 3 imply a possible contribution of Trx2 to the functional and structural 50 51 integrity of Complex I. 52 53 54 Complex II 55 56 Trx2 interacted non-covalently with SDHA, the flavoprotein (FP) subunit of the enzyme 57 58 complex of succinate dehydrogenase (SDH), or respiratory Complex II. SDHA is located at 59 60 the mitochondrial matrix and catalyses the oxidation of succinate to fumarate. The electrons

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1 2 3 abstracted, travel via the iron sulfur clusters of adjacent SDHB, to the inner mitochondrial 4 membrane located SDHC and SDHD that reduce CoQ to CoQH (exemplified for the enzyme 5 2 6 from E. coli [130]). The result is an increase of the CoQH2 pool that transfers electrons to the 7 8 cytochrome bc1 complex (complex III) that in succession relocates electrons to complex IV 9 10 for the reduction of molecular oxygen to water [131]. A defective SDHA resulted in elevated 11 succinate levels that activated mitochondrial TrxR2 [132]. This finding implicates further the 12 13 potential interaction of Trx2 with SDHA for the reduction of CoQ in mitochondria and 14 15 pinpoints TrxR2 as a possible target for the modulation of the electron chain: inhibitors of 16 TrxR2 could affect the normal function of the electron chain at the levels of complex II, 17 18 possibly via Trx2. 19 20 21 For Review Only 22 Complex III 23 Trx2 apparently interacted with UQCRC1 (covalently) and UQCRC2 (non-covalently), both 24 25 core subunit proteins of the mitochondrial cytochrome b-c1 complex (complex III) of the 26 27 electron transport chain that catalyzes the reduction of cytochrome c by oxidation of CoQH2 28 29 to CoQ with simultaneous transfer of protons from the mitochondrial matrix to the 30 intermembrane space [133]. Complex III contains 3 additional respiratory subunits 31 32 (cytochrome b, cytochrome c1 and Rieske/UQCRFS1) and 6 low-molecular weight proteins. 33 34 UQCRC1 may mediate formation of the complex between cytochromes c and c1 (Uniprot ID: 35 P31930, [134]), while its normal function is required for spermatogenesis [135]. 36 37 Overexpression of UQCRC1 may affect mitochondrial morphology/physiology and may lead 38 39 to development of obesity [136]. UQCRC2 contributes to the assembly of complex III 40 41 (Uniprot ID: P22695) and may be involved in carcinogenesis [137-139]. 42 Trx2 interacted covalently and non-covalently in brain extracts with UQCRFS1, whose 43 44 incorporation is the penultimate step in the biogenesis of complex III [140]. Dysfunction of 45 46 the latter is related to neurological diseases. 47 48 49 Complex IV 50 51 Trx2 interacted with cytochrome c oxidase (complex IV) subunits MT-CO1 (cytochrome c 52 53 oxidase subunit 1 or COX1, data from Y2H) and NDUFA4 (non-covalent binding, proteomics 54 data) of complex IV. MT-CO1 is one of the three core components, (the other two being, 55 56 COX2, and COX3) that form the core of cytochrome oxidase. COX1 is the nucleus for the 57 58 biogenesis process of complex IV to which imported subunits (including COX2 and COX3), 59 60 associate and assemble in a sequential manner [141, 142]. NDUFA4 is required for normal

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1 2 3 activity of complex IV [143]. Mutant forms of NDUFA4 may give the neurological 4 phenotype of the Leigh Syndrome [144]. 5 6 We did not detect any binding of Trx2 to cytochrome c as has been observed in 7 8 immunoprecipitations [145]. In a previous work, cytochrome c was a substrate for TrxR2 that 9 10 reduced the protein in vitro with similar efficiency in the presence or absence of Trx2 [146] 11 suggesting that Trx2 may not reduce cytochrome c. 12 13 14 15 ATP synthase and Trx2 16 The most hits for Trx2 interactions from different tissues were for the β subunit of 17 18 mitochondrial ATP synthase (ATP5B), while the α subunit (ATP5A) was in the second 19 20 position (with peroxiredoxin 6). These subunits form a structure within the F1 domain located 21 For Review Only 22 in the mitochondrial matrix that binds to ADP to form ATP from the proton gradient that 23 generates a torque on the F0 domain where the transmembrane rotating c-ring is located ([147] 24 25 and references therein). Trx2 interacted also covalently with the subunit d of ATP synthase 26 27 that is part of the peripheral stalk connecting the F1 and F0 domains [147]. Although we found 28 29 reports for redox regulation of the gamma subunit of the F1 domain [148-150], no data were 30 available for such a mechanism for the alpha and beta subunits. The evidence presented 31 32 herein, raises this possibility which is likely to be validated by previous work in which 33 34 overexpression of Trx2 in HEK293 cells was specifically affecting the activity of ATP 35 synthase and therefore, the potential of the mitochondrial membrane [35]. 36 37 Another possible association of Trx2 with ATP levels is via S-type mitochondrial creatine 38 39 kinase (CKMT2) with which Trx2 was covalently associated. CKMT2 reversibly catalyzes 40 41 the transfer of phosphate between ATP and various phosphogens (e.g. creatine phosphate). 42 CKMT2 isoenzymes are involved in energy transduction in tissues with large and fluctuating 43 44 energy demands (e.g. skeletal muscle, heart, brain, spermatozoa, Uniprot ID: P17540). 45 46 Overall, interactors of Trx2 were found in all complexes of the electron transport chain. It is 47 48 not therefore surprising that the protein is related to combined oxidative phosphorylation 49 deficiency. Decreased activity of the transport chain has been observed in haploid mutants for 50 51 the gene encoding Trx2 [68]. A possibility of therapeutic interference could be the use of 52 53 TrxR2 inhibitors, expected to modify the redox state and thus the interactions of Trx2 with its 54 related binding partners within the electron transport chain. 55 56 57 58 59 60

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1 2 3 12. Lipid metabolism 4 Trx2 interacted non-covalently with subunit alpha of the trifunctional protein/enzyme TFP. 5 6 TFP is an octameric complex composed of four α-subunits (HADHA), each composed of a 7 8 long-chain enoyl-CoA hydratase and a long-chain L-3-hydroxyacyl-CoA dehydrogenase 9 10 domain and four β-subunits (HADHB) encoding a long-chain 3-ketoacyl-CoA thiolase 11 domain [151]. The enzyme catalyzes the last three steps of the of long-chain fatty acids β- 12 13 oxidation. Defects in TFP may result in hepatic steatosis and insulin resistance [152]. So far 14 15 there has not been any report correlating thioredoxin proteins to TFP. Regulation of fatty acid 16 biosynthesis has been described for Txnip not via thioredoxin but a specific regulatory 17 18 microRNA (miR-33a), affecting HADHB activity [153]. 19 20 Trx2 interacted covalently with mitochondrial acetyl-coenzyme A synthetase 2-like (ACSS1) 21 For Review Only 22 that converts acetate to acetyl-CoA to be oxidized via the tricarboxylic cycle to produce ATP 23 and CO2. In view of its similarity to ACSL1, ACSS1 is likely to be involved in energy 24 25 homeostasis and in maintaining normal body temperature during fasting. Deacetylation of 26 27 ACSS1 by SIRT3 activates the acetyl-CoA synthetase activity of ACSS1 [154]. The 28 29 interaction with Trx2 reported here raises another potential level of regulation. 30 31 32 13. Amino acid metabolism 33 34 The non-covalent interaction of Trx2 with the β chain of mitochondrial methylcrotonoyl-CoA 35 carboxylase (MCCC2), potentially links the thioredoxin system to amino acid metabolism. 36 37 MCCC2 is the carboxyltransferase subunit of the biotin requiring mitochondrial enzyme 3- 38 39 methylcrotonyl-CoA carboxylase, that is involved in the catabolism of leucine and isovaleric 40 41 acid. Deficiency of the holoenzyme leads to an autosomal inherited disease that has a variable 42 phenotype ranging from asymptomatic to death in infancy from neurological disorders [155] 43 44 [156]. Analysis of the transcriptome of a deficient human skin fibroblast cell line revealed that 45 46 its mitochondria were dysfunctional (e.g. downregulation of transcripts concerning respiratory 47 48 complex proteins) leading to oxidative stress [156]. It were the Trx1 transcripts, however, that 49 were upregulated in that study and not the Trx2 ones [156]. 50 51 Trx2 reacted covalently with cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) 52 53 an abundant enzyme that catalyses the reductive (via NADPH) decarboxylation of 10- 54 formyltetrahydrofolate to tetrahydrofolate and CO . Although a mitochondrial form of the 55 2 56 enzyme exists (ALDH1L2 with 74 % identity to ALDH1L1 [157]), ALDH1L1 is also 57 58 considered mitochondrial [27]. Folate participates in one-carbon metabolism and comprises a 59 60 network of interconnected folate-dependent metabolic pathways that are involved in serine

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1 2 3 and glycine interconversion, homocysteine remethylation to methionine and de novo 4 biosynthesis of purine an thymidylate [158]. It is not known how the reported interaction 5 6 between ALDH1L1 and Trx2 may affect the activity of either enzyme. 7 8 9 10 14. Protein/nucleic acid deglycase DJ-1 and Parkinson’s disease 11 DJ-1/PARK7 is a 2x20 kDa protein localized in the cytosol, nucleus and mitochondria. 12 13 Autosomal recessive mutations in the DJ-1 gene causing loss of function, lead to an early start 14 15 of Parkinson’s disease [159]. In this work, Trx2 was able to interact directly with DJ-1. As 16 was the case for human Trx1, it is likely that mitochondrial Trx2 will be able to reduce Cys53 17 18 of DJ-1 [24, 160]. While the particular residue may help the dimerisation of DJ-1, the redox 19 106 20 state of Cys that is responsible for the delivery of the antioxidant effects of DJ-1, is affected 21 For Review Only 22 by peroxiredoxin 2 [160] herein, another interactor of Trx2. In an epidemiological study, 23 Parkinson’s development after exposure to pesticides was related to defects in Complex I 24 25 [161]. In this work, Trx2 interacted with four components of the Complex I. Another potential 26 27 Trx2 interactor, lactate dehydrogenase b was down regulated in midbrain-derived 28 29 dopaminergic neuronal cells when exposed to 1-methyl-4-phenyl-1, 2, 3, 6- 30 tetrahydropyridine (MPTP) known to promote a Parkinson’s phenotype [162]. A 16-year-old 31 32 adolescent with infantile-onset neurodegenerative disorder had an homozygous stop mutation 33 34 in the gene encoding Trx2, demonstrating the importance of the protein for neuronal integrity 35 [163]. Taken together these observations provide evidence that Trx2 participates in protein 36 37 networks with neuroprotective roles. 38 39 40 41 Prospects 42 This work aimed at the identification of mainly strong non-covalent and covalent interactions 43 44 of Trx2 in the context of the mitochondrion. Novel partners were identified and thus increased 45 46 the spectrum of putative functions for Trx2. The number of interactor species (and 47 48 concomitant functions) is expected to increase even more with the use of more sensitive 49 detection methods that allow for better resolution and more certain identification of protein 50 51 species (e.g. Orbitrap LC-MS). Equally relevant are the weaker non-covalent interactions that 52 53 could have been detected by Y2H screens but here gave only three novel interactors. It is 54 obvious that the “weak” interactome of Trx2 was by no means properly explored. Use of 55 56 immobilized Trx2, interaction with tissue lysates and elutions by increasing ionic strengths, is 57 58 likely to reveal more interactions, some of them already known (e.g. ASK1, Txnip) but not 59 60 detected within the context of this work. Finally, in vitro and in vivo validation of all these

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1 2 3 putative interactors is expected to clarify the effective functional interactome of Trx2 and 4 perhaps provide fine points of therapeutic interference. 5 6 7 8 Acknowledgements 9 10 This investigation was supported by grants from the Greek General Secretariat of Research 11 and Technology in collaboration with the European Union (program Enter, 01EP104). 12 13 14 15 16 17 18 19 20 21 For Review Only 22 23 24 25 26 27 28 29 30 31 32 33 34 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

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1 2 3 III subunits identifies UQCRC1 as biomarker in clear cell renal cell carcinoma, Oncotarget. 7, 86490- 4 86499. 5 140. Bottani, E., Cerutti, R., Harbour, M. E., Ravaglia, S., Dogan, S. A., Giordano, C., Fearnley, I. M., 6 D'Amati, G., Viscomi, C., Fernandez-Vizarra, E. & Zeviani, M. (2017) TTC19 Plays a Husbandry Role on 7 UQCRFS1 Turnover in the Biogenesis of Mitochondrial Respiratory Complex III, Molecular cell. 67, 96- 8 105 e4. 9 141. Dennerlein, S., Oeljeklaus, S., Jans, D., Hellwig, C., Bareth, B., Jakobs, S., Deckers, M., Warscheid, 10 B. & Rehling, P. (2015) MITRAC7 Acts as a COX1-Specific Chaperone and Reveals a Checkpoint during 11 12 Cytochrome c Oxidase Assembly, Cell reports. 12, 1644-55. 13 142. Dennerlein, S. & Rehling, P. (2015) Human mitochondrial COX1 assembly into cytochrome c 14 oxidase at a glance, Journal of cell science. 128, 833-7. 15 143. Balsa, E., Marco, R., Perales-Clemente, E., Szklarczyk, R., Calvo, E., Landazuri, M. O. & Enriquez, 16 J. A. (2012) NDUFA4 is a subunit of complex IV of the mammalian electron transport chain, Cell 17 metabolism. 16, 378-86. 18 144. Pitceathly, R. D., Rahman, S., Wedatilake, Y., Polke, J. M., Cirak, S., Foley, A. R., Sailer, A., Hurles, 19 M. E., Stalker, J., Hargreaves, I., Woodward, C. E., Sweeney, M. G., Muntoni, F., Houlden, H., 20 21 Taanman, J. W., Hanna, M.For G. & Consortium, Review U. K. (2013) Only NDUFA4 mutations underlie dysfunction of 22 a cytochrome c oxidase subunit linked to human neurological disease, Cell reports. 3, 1795-805. 23 145. Tanaka, T., Hosoi, F., Yamaguchi-Iwai, Y., Nakamura, H., Masutani, H., Ueda, S., Nishiyama, A., 24 Takeda, S., Wada, H., Spyrou, G. & Yodoi, J. (2002) Thioredoxin-2 (TRX-2) is an essential gene 25 regulating mitochondria-dependent apoptosis, The EMBO journal. 21, 1695-703. 26 146. Nalvarte, I., Damdimopoulos, A. E. & Spyrou, G. (2004) Human mitochondrial thioredoxin 27 reductase reduces cytochrome c and confers resistance to complex III inhibition, Free radical biology 28 & medicine. 36, 1270-8. 29 147. Jonckheere, A. I., Smeitink, J. A. & Rodenburg, R. J. (2012) Mitochondrial ATP synthase: 30 31 architecture, function and pathology, Journal of inherited metabolic disease. 35, 211-25. 32 148. Buchert, F., Konno, H. & Hisabori, T. (2015) Redox regulation of CF1-ATPase involves interplay 33 between the gamma-subunit neck region and the turn region of the betaDELSEED-loop, Biochimica et 34 biophysica acta. 1847, 441-450. 35 149. Kim, Y., Konno, H., Sugano, Y. & Hisabori, T. (2011) Redox regulation of rotation of the 36 cyanobacterial F1-ATPase containing thiol regulation switch, The Journal of biological chemistry. 286, 37 9071-8. 38 150. Belogrudov, G. I. & Hatefi, Y. (2002) Factor B and the mitochondrial ATP synthase complex, The 39 Journal of biological chemistry. 40 277, 6097-103. 41 151. Spiekerkoetter, U., Khuchua, Z., Yue, Z., Bennett, M. J. & Strauss, A. W. (2004) General 42 mitochondrial trifunctional protein (TFP) deficiency as a result of either alpha- or beta-subunit 43 mutations exhibits similar phenotypes because mutations in either subunit alter TFP complex 44 expression and subunit turnover, Pediatric research. 55, 190-6. 45 152. Ibdah, J. A., Perlegas, P., Zhao, Y., Angdisen, J., Borgerink, H., Shadoan, M. K., Wagner, J. D., 46 Matern, D., Rinaldo, P. & Cline, J. M. (2005) Mice heterozygous for a defect in mitochondrial 47 trifunctional protein develop hepatic steatosis and insulin resistance, Gastroenterology. 128, 1381- 48 90. 49 50 153. Chen, J., Young, M. E., Chatham, J. C., Crossman, D. K., Dell'Italia, L. J. & Shalev, A. (2016) TXNIP 51 regulates myocardial fatty acid oxidation via miR-33a signaling, American journal of physiology Heart 52 and circulatory physiology. 311, H64-75. 53 154. Schwer, B., Bunkenborg, J., Verdin, R. O., Andersen, J. S. & Verdin, E. (2006) Reversible lysine 54 acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2, Proceedings of 55 the National Academy of Sciences of the United States of America. 103, 10224-10229. 56 155. Forsyth, R., Vockley, C. W., Edick, M. J., Cameron, C. A., Hiner, S. J., Berry, S. A., Vockley, J., 57 Arnold, G. L. & Inborn Errors of Metabolism, C. (2016) Outcomes of cases with 3-methylcrotonyl-CoA 58 59 carboxylase (3-MCC) deficiency - Report from the Inborn Errors of Metabolism Information System, 60 Molecular genetics and metabolism. 118, 15-20.

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1 2 3 156. Zandberg, L., van Dyk, H. C., van der Westhuizen, F. H. & van Dijk, A. A. (2016) A 3- 4 methylcrotonyl-CoA carboxylase deficient human skin fibroblast transcriptome reveals underlying 5 mitochondrial dysfunction and oxidative stress, The international journal of biochemistry & cell 6 biology. 78, 116-129. 7 157. Strickland, K. C., Krupenko, N. I., Dubard, M. E., Hu, C. J., Tsybovsky, Y. & Krupenko, S. A. (2011) 8 Enzymatic properties of ALDH1L2, a mitochondrial 10-formyltetrahydrofolate dehydrogenase, 9 Chemico-biological interactions. 191, 129-36. 10 158. Lan, X., Field, M. S. & Stover, P. J. (2018) Cell cycle regulation of folate-mediated one-carbon 11 12 metabolism, Wiley interdisciplinary reviews Systems biology and medicine, e1426. 13 159. Bonifati, V., Rizzu, P., van Baren, M. J., Schaap, O., Breedveld, G. J., Krieger, E., Dekker, M. C., 14 Squitieri, F., Ibanez, P., Joosse, M., van Dongen, J. W., Vanacore, N., van Swieten, J. C., Brice, A., 15 Meco, G., van Duijn, C. M., Oostra, B. A. & Heutink, P. (2003) Mutations in the DJ-1 gene associated 16 with autosomal recessive early-onset parkinsonism, Science. 299, 256-9. 17 160. Fernandez-Caggiano, M., Schroder, E., Cho, H. J., Burgoyne, J., Barallobre-Barreiro, J., Mayr, M. 18 & Eaton, P. (2016) Oxidant-induced Interprotein Disulfide Formation in Cardiac Protein DJ-1 Occurs 19 via an Interaction with Peroxiredoxin 2, The Journal of biological chemistry. 291, 10399-410. 20 21 161. Terron, A., Bal-Price, A.,For Paini, A.,Review Monnet-Tschudi, F.,Only Bennekou, S. H., Members, E. W. E., Leist, 22 M. & Schildknecht, S. (2018) An adverse outcome pathway for parkinsonian motor deficits associated 23 with mitochondrial complex I inhibition, Archives of toxicology. 92, 41-82. 24 162. Wang, J., Duhart, H. M., Xu, Z., Patterson, T. A., Newport, G. D. & Ali, S. F. (2008) Comparison of 25 the time courses of selective gene expression and dopaminergic depletion induced by MPP+ in MN9D 26 cells, Neurochemistry international. 52, 1037-43. 27 163. Holzerova, E., Danhauser, K., Haack, T. B., Kremer, L. S., Melcher, M., Ingold, I., Kobayashi, S., 28 Terrile, C., Wolf, P., Schaper, J., Mayatepek, E., Baertling, F., Friedmann Angeli, J. P., Conrad, M., 29 Strom, T. M., Meitinger, T., Prokisch, H. & Distelmaier, F. (2016) Human thioredoxin 2 deficiency 30 31 impairs mitochondrial redox homeostasis and causes early-onset neurodegeneration, Brain : a 32 journal of neurology. 139, 346-54. 33 164. Havugimana, P. C., Hart, G. T., Nepusz, T., Yang, H., Turinsky, A. L., Li, Z., Wang, P. I., Boutz, D. 34 R., Fong, V., Phanse, S., Babu, M., Craig, S. A., Hu, P., Wan, C., Vlasblom, J., Dar, V. U., Bezginov, A., 35 Clark, G. W., Wu, G. C., Wodak, S. J., Tillier, E. R., Paccanaro, A., Marcotte, E. M. & Emili, A. (2012) A 36 census of human soluble protein complexes, Cell. 150, 1068-81. 37 165. Rolland, T., Tasan, M., Charloteaux, B., Pevzner, S. J., Zhong, Q., Sahni, N., Yi, S., Lemmens, I., 38 Fontanillo, C., Mosca, R., Kamburov, A., Ghiassian, S. D., Yang, X., Ghamsari, L., Balcha, D., Begg, B. E., 39 40 Braun, P., Brehme, M., Broly, M. P., Carvunis, A. R., Convery-Zupan, D., Corominas, R., Coulombe- 41 Huntington, J., Dann, E., Dreze, M., Dricot, A., Fan, C., Franzosa, E., Gebreab, F., Gutierrez, B. J., 42 Hardy, M. F., Jin, M., Kang, S., Kiros, R., Lin, G. N., Luck, K., MacWilliams, A., Menche, J., Murray, R. R., 43 Palagi, A., Poulin, M. M., Rambout, X., Rasla, J., Reichert, P., Romero, V., Ruyssinck, E., Sahalie, J. M., 44 Scholz, A., Shah, A. A., Sharma, A., Shen, Y., Spirohn, K., Tam, S., Tejeda, A. O., Trigg, S. A., Twizere, J. 45 C., Vega, K., Walsh, J., Cusick, M. E., Xia, Y., Barabasi, A. L., Iakoucheva, L. M., Aloy, P., De Las Rivas, J., 46 Tavernier, J., Calderwood, M. A., Hill, D. E., Hao, T., Roth, F. P. & Vidal, M. (2014) A proteome-scale 47 map of the human interactome network, Cell. 159, 1212-1226. 48 166. Wan, C., Borgeson, B., Phanse, S., Tu, F., Drew, K., Clark, G., Xiong, X., Kagan, O., Kwan, J., 49 50 Bezginov, A., Chessman, K., Pal, S., Cromar, G., Papoulas, O., Ni, Z., Boutz, D. R., Stoilova, S., 51 Havugimana, P. C., Guo, X., Malty, R. H., Sarov, M., Greenblatt, J., Babu, M., Derry, W. B., Tillier, E. R., 52 Wallingford, J. B., Parkinson, J., Marcotte, E. M. & Emili, A. (2015) Panorama of ancient metazoan 53 macromolecular complexes, Nature. 525, 339-44. 54 55 56 57 58 59 60

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1 2 3 Table 1. Human mitochondrial proteins interacting with Trx2 as revealed by 4 proteomics, Y2H screens and BioGRID. NC corresponds to non-covalent interaction; C to 5 6 covalent. The four protein species in gray background correspond to mitochondrial proteins 7 8 (combination of MitoCarta2.0 and BioGRID) described as interacting with Trx2. 9 10 11 Interaction Type of 12 UniProt # Protein species Gene confirmed interaction 13 KB 14 by (NC or C) 15 1 Q99798 Aconitate hydratase, mitochondrial, ACO2 DTT C 16 Acetyl-coenzyme A synthetase 2-like, 2 Q9NUB1 ACSS1 DTT C 17 mitochondrial 18 19 3 O75891 Cytosolic 10-formyltetrahydrofolate dehydrogenase ALDH1L1 DTT C 20 4 P05091 Aldehyde dehydrogenase, mitochondrial ALDH2 Acid NC 21 5 P25705 ATP synthaseFor subunit Review alpha, mitochondrial Only ATP5F1A Acid, DTT NC, C 22 6 P06576 ATP synthase subunit beta, mitochondrial ATP5F1B Acid, DTT NC, C 23 24 7 O75947 ATP synthase subunit d, mitochondrial ATP5PD Acid, DTT NC, C 25 8 O75828 Carbonyl reductase [NADPH] 3 CBR3 DTT C 26 9 P17540 Creatine kinase S-type, mitochondrial CKMT2 DTT C 27 Cytochrome b-c1 complex subunit Rieske, 28 10 P47985 UQCRFS1 Acid, DTT NC, C 29 mitochondrial 30 11 Q08426 Peroxisomal bifunctional enzyme EHHADH DTT C 31 12 P04406 Glyceraldehyde-3-phosphate dehydrogenase GAPDH Acid, DTT NC, C 32 13 P40939 Trifunctional enzyme subunit alpha, mitochondrial HADHA Acid NC 33 34 14 P38646 Stress-70 protein, mitochondrial HSPA9 DTT C 35 15 P10809 60 kDa heat shock protein, mitochondrial HSPD1 DTT C 36 16 O75874 Isocitrate dehydrogenase [NADP] cytoplasmic IDH1 Acid NC 37 38 17 P48735 Isocitrate dehydrogenase [NADP], mitochondrial IDH2 Acid NC Isocitrate dehydrogenase [NAD] subunit alpha, 39 18 P50213 IDH3A DTT C 40 mitochondrial 41 Isocitrate dehydrogenase [NAD] subunit gamma, 19 P51553 IDH3G Acid NC 42 mitochondrial 43 44 20 Q16891 MICOS complex subunit MIC60 IMMT DTT C 45 21 P07195 L-lactate dehydrogenase B chain, LDH-B LDHB Acid NC 46 22 Q86WU2 Probable D-lactate dehydrogenase, mitochondrial LDHD DTT C 47 MACROD 48 23 Q9BQ69 O-acetyl-ADP-ribose deacetylase MACROD1 DTT C 49 1 Methylcrotonoyl-CoA carboxylase beta chain, 50 24 Q9HCC0 MCCC2 Acid NC 51 mitochondrial, 52 25 Mitochondrially encoded 16S RNA MT-RNR2 Y2H rat NC 53 Mitochondrial peptide methionine sulfoxide 54 26 Q9UJ68 MSRA DTT C 55 reductase 56 27 P00395 Cytochrome c oxidase subunit 1 MT-CO1 Y2H rat NC 57 28 O00483 Cytochrome c oxidase subunit NDUFA4 NDUFA4 Acid NC 58 NADH-ubiquinone oxidoreductase 75 kDa subunit, 29 P28331 NDUFS1 Acid NC 59 mitochondrial 60

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1 2 NADH dehydrogenase [ubiquinone] iron-sulfur 3 30 O75306 NDUFS2 Acid NC 4 protein 2, mitochondrial 5 NADH dehydrogenase [ubiquinone] iron-sulfur 31 O75489 NDUFS3 Acid NC 6 protein 3, mitochondrial 7 NADH dehydrogenase [ubiquinone] flavoprotein 1, 8 32 P49821 NDUFV1 Acid NC 9 mitochondrial 10 33 A8MXV4 Nucleoside diphosphate-linked moiety X motif 19 NUDT19 DTT C 11 Y2H 34 P07237 Protein disulfide-isomerase P4HB NC 12 human 13 35 Q99497 Protein/nucleic acid deglycase DJ-1 PARK7 DTT C 14 Pyruvate dehydrogenase E1 component subunit 15 36 P08559 PDHA1 Acid NC 16 alpha, somatic form, mitochondrial 17 Pyruvate dehydrogenase E1 component subunit 37 P11177 PDHB DTT C 18 beta, mitochondrial 19 38 Q9H2U2 Inorganic pyrophosphatase 2, mitochondrial PPA2 DTT C 20 21 39 P32119 ForPeroxiredoxin Review-2 Only PRDX2 Acid, DTT NC, C 22 Thioredoxin-dependent peroxide reductase, 40 P30048 PRDX3 DTT C 23 mitochondrial (Peroxiredoxin-3) 24 41 Q13162 Peroxiredoxin-4 PRDX4 Acid, DTT NC, C 25 26 42 P30044 Peroxiredoxin-5 PRDX5 Acid, DTT NC, C 27 43 P30041 Peroxiredoxin-6 PRDX6 DTT C 28 44 P62269 40S ribosomal protein S18 RPS18 Acid NC 29 Succinate dehydrogenase [ubiquinone] 30 45 P31040 SDHA Acid NC 31 flavoprotein subunit 32 46 P60174 Triosephosphate isomerase TPI1 Acid, DTT NC, C 33 47 P49411 Elongation factor Tu, mitochondrial, EF-Tu TUFM DTT C 34 35 48 P31930 Cytochrome b-c1 complex subunit 1, mitochondrial UQCRC1 DTT C 36 49 P22695 Cytochrome b-c1 complex subunit 2, mitochondrial UQCRC2 Acid NC 37 Co fractionation/BioGRID 50 Q16540 Mitochondrial ribosomal protein L23 MRPL23 38 [164] 39 40 51 P49247 Ribose 5-phosphate isomerase A RPIA BioGRID [165] Co fractionation/BioGRID 41 52 P00441 Superoxide dismutase 1, soluble SOD1 42 [166] 43 of outer mitochondrial membrane 22 Co fractionation/BioGRID 53 Q9NS69 TOMM22 44 homolog [164] 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Review Only 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Figure 1: The human Trx2 mitochondrial interactome. Proteins are presented by their 44 45 primary gene names (table 1). In summary, 21 proteins were eluted by DTT (yellow), 16 by 46 47 acid (red), and 9 proteins were eluted by both acid and DTT (black). The blue slice represents 48 49 data from Y2H screens where the mitochondrial 16S RNA gene is in red letters. The white 50 slice contains the four mitochondrial interactors of Trx2 from the BioGRID data base [26]. 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 Clique 1 Clique 2 12 13 14 15 16 17 18 19 20 21 For Review Only 22 23 24 25 26 27 Clique 3 28 29 Clique 4 30 31 32 33 34 35 36 Fig 2: Four cliques of the diseasome of Trx2. The diseasome is based on the Trx2 37 38 mitochondrial interactome. The disease-related genes/proteins are shown with capital letters 39 40 within the circular nodes of each clique (Unipot IDs are provided on supplementary table 2.5). 41 42 The lines connecting different nodes (genes) indicate the herein supported interactions for 43 Trx2 and the interactions among the interactors of Trx2 (data from BioGRID). 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Review Only 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Fig. 3. Proposed interactions of Trx2 with potential substrates. 3A: weak ionic 41 interactions; 3B: strong hydrophobic interactions (interacting hydrophobic areas in black and 42 43 yellow); 3C: covalent interactions via a disulfide between ΔTrx2C93S and interactor. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Supplementary figures 4 5 6 7 Supplementary figure 1. Alignment of mouse, rat and human Trx2 lacking their 8 9 mitochondrial targeting sequene. All different amino acids are highlighted in yellow 10 background. Alignment was performed using the CLUSTAL O(1.2.4) multiple sequence 11 12 alignment tool (https://www.ebi.ac.uk/Tools/msa/clustalo/). 13 14 15 mouse Trx2 TTFNVQDGPDFQDRVVNSETPVVVDFHAQWCGPCKILGPRLEKMVAKQHGKVVMAKVDID 60 16 rat Trx2 TTFNVQDGPDFQDRVVNSETPVVVDFHAQWCGPCKILGPRLEKMVAKQHGKVVMAKVDID 60 17 human Trx2 TTFNIQDGPDFQDRVVNSETPVVVDFHAQWCGPCKILGPRLEKMVAKQHGKVVMAKVDID 60 18 ****:******************************************************* 19 For Review Only 20 mouse Trx2 DHTDLAIEYEVSAVPTVLAIKNGDVVDKFVGIKDEDQLEAFLKKLIG 107 21 rat Trx2 DHTDLAIEYEVSAVPTVLAIKNGDVVDKFVGIKDEDQLEAFLKKLIG 107 22 human Trx2 DHTDLAIEYEVSAVPTVLAMKNGDVVDKFVGIKDEDQLEAFLKKLIG 107 23 *******************:*************************** 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 59 60 Page 47 of 105 The FEBS Journal

1 2 3 4 5 Supplementary figure 2. Elution profile of ΔTrx2C93S on a DEAE FF column. 6 7 Chromatography was carried out in 20 mM Bis-Tris-HCl, pH 6,7 and a 0-1 M NaCl gradient 8 9 (light green line). The other shown lines correspond to: conductivity (brown line); A280 (blue); 10 A254 (magenta); A310 (red). ΔTrx2C93S was eluted at ~15 mS/cm (black arrow). (i) whole 11 12 chromatogram, (ii) partial chromatogram. 13 14 15 16 17 18 19 For Review Only 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Supplementary figure 2 (i). 50 51 52 53 54 55 56 57 58 2 59 60 The FEBS Journal Page 48 of 105

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Review Only 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Supplementary figure 2 (ii). 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 3 59 60 Page 49 of 105 The FEBS Journal

1 2 3 4 5 Supplementary figure 3. Western blots of fractions derived by centrifugation gradient from 6 7 cardiac muscle. 8 9 10 11 12 13 14 15 16 17 18 19 For Review Only 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Analysis of sucrose gradient fractions, for mitochondrial (ANT1), lysosomal (LAMP-1) and ER 37 38 (calsequestrin) proteins by Western blot in 10 % SDS-PAGE. Lane 1: molecular weight protein 39 40 markers, lanes 2-8: fractions from sucrose gradient. The respective antibody together with an 41 42 arrow indicting the detected antigen (ANT 1, LAMP1, calsequestrin) are shown at the right side 43 of each Western blot. Fractions corresponding to lanes 3, 4, and 6 were pooled as 44 45 “mitochondrial” fractions and chromatographed on the ΔTrx2C93S affinity columns. The same 46 47 criteria were set for the selection of mitochondrial fractions from brain, skeletal muscle. 48 49 50 51 52 53 54 55 56 57 58 4 59 60 The FEBS Journal Page 50 of 105

1 2 3 Supplementary tables 1. Mass spec analysis of individual spots obtained from 2D (and 1D) 4 5 electrophoresis 6 7 8 9 MS data for the interactors of Trx2 in different mouse tissues. 10 Proteins were isolated and identified from 2D gels by MALDI-TOF-MS analysis, using the 11 12 parameters described under Materials and Methods: % coverage represents the percentage of 13 14 total protein sequence by the identified peptides. The probability of a false identity was usually 15 lower than 10-5. Spot numbering (Spot #, left column) corresponds to the different spots/bands 16 17 identified per experiment. The numbering is according to the alphabetical order of the protein 18 19 species identified. The proteinFor species Review identified according Only to the set criteria are numbered in the 20 21 right column (# of species) and are coloured in red. The individual experiments and their 22 corresponding results are presented in the following tables. 23 24 25 Table 1.1: mass spec analysis of a 2D gel, of an acid-eluted fraction from a lysate derived 26 from mouse lungs. 27 28 Spot Mascot % Protein Uniprot # of 29 Protein species Accession # Score Coverage MW ID species 30 12 days embryo spinal 31 ganglion cDNA, RIKEN full- 32 1 27 13 33405.48 Q8BJI0_MOUSE Q8BJI0 length enriched library, 33 clone:D130020I06 product:2 34 16 days embryo head cDNA, 35 RIKEN full-length enriched 2 42 26 26055.66 Q542P5_MOUSE Q542P5 36 library, clone:C130079C10 37 product:carbonyl red 38 40S ribosomal protein S4, X 39 3 isoform - Mus musculus 106 49 29807.14 RS4X_MOUSE P62702 1 40 (Mouse) 41 40S ribosomal protein SA - 4 151 54 32931.48 RSSA_MOUSE P14206 2 42 Mus musculus (Mouse) 43 , cytoplasmic 1 - Mus 5 160 63 42051.86 ACTB_MOUSE P60710 3 44 musculus (Mouse) 45 Adult male testis cDNA, 46 RIKEN full-length enriched 6 47 21 61098.16 Q9D5K8_MOUSE Q9D5K8 47 library, clone:4930427I11 48 product:similar to ZIN 49 Aldehyde dehydrogenase, 50 7 mitochondrial precursor - Mus 247 50 57014.94 ALDH2_MOUSE musculus (Mouse) 51 P47738 4 52 Aldehyde dehydrogenase, 53 8 mitochondrial precursor - Mus 194 48 57014.94 ALDH2_MOUSE 54 musculus (Mouse) Alpha crystallin B chain - Mus 55 9 109 54 20056.41 CRYAB_MOUSE P23927 5 56 musculus (Mouse) 57 58 1 59 60 Page 51 of 105 The FEBS Journal

1 2 3 Alpha-centractin - Mus 10 107 45 42700.95 ACTZ_MOUSE P61164 6 4 musculus (Mouse) 5 Alpha-enolase - Mus 11 167 63 47453.34 ENOA_MOUSE P17182 7 6 musculus (Mouse) 7 ATP synthase subunit alpha, 8 12 mitochondrial precursor - Mus 152 45 59829.63 ATPA_MOUSE Q03265 8 9 musculus (Mouse) 10 ATP synthase subunit beta, 11 13 mitochondrial precursor - Mus 144 56 56265.46 ATPB_MOUSE P56480 9 12 musculus (Mouse) 13 Dihydropyrimidinase-related 14 14 protein 2 - Mus musculus 189 59 62637.74 DPYL2_MOUSE 15 (Mouse) 16 Dihydropyrimidinase-related 17 15 protein 2 - Mus musculus 201 57 62637.74 DPYL2_MOUSE Q62188 10 18 (Mouse) 19 DihydropyrimidinaseFor-related Review Only 20 16 protein 3 - Mus musculus 221 64 62296.23 DPYL3_MOUSE 21 (Mouse) 22 F-actin capping protein 23 17 subunit alpha-1 - Mus 103 63 33090.37 CAZA1_MOUSE P47753 11 24 musculus (Mouse) 25 F-actin capping protein 26 18 subunit alpha-2 - Mus 136 82 33117.68 CAZA2_MOUSE musculus (Mouse) 27 P47754 12 28 F-actin capping protein 29 19 subunit alpha-2 - Mus 166 76 33117.68 CAZA2_MOUSE musculus (Mouse) 30 precursor - Mus 31 20 118 34 86287.23 GELS_MOUSE musculus (Mouse) 32 P13020 13 Gelsolin precursor - Mus 33 21 134 35 86287.23 GELS_MOUSE musculus (Mouse) 34 Glyceraldehyde-3-phosphate 35 22 dehydrogenase - Mus 68 34 36072.32 G3P_MOUSE P16858 14 36 musculus (Mouse) 37 GTP-binding nuclear protein 23 159 50 24578.68 RAN_MOUSE P62827 15 38 Ran - Mus musculus (Mouse) 39 Hist1h2bj protein - Mus 24 43 42 13570.43 A0JLV3_MOUSE A0JLV3 40 musculus (Mouse) 41 Indolethylamine N- 42 25 methyltransferase - Mus 125 58 30068.05 INMT_MOUSE 43 musculus (Mouse) 44 Indolethylamine N- 45 26 methyltransferase - Mus 89 50 30068.05 INMT_MOUSE P40936 17 46 musculus (Mouse) 47 Indolethylamine N- 48 27 methyltransferase - Mus 119 52 30068.05 INMT_MOUSE 49 musculus (Mouse) 50 Isocitrate dehydrogenase 51 28 [NADP] cytoplasmic - Mus 131 38 47029.56 IDHC_MOUSE P70404 18 52 musculus (Mouse) 53 Isocitrate dehydrogenase [NADP], mitochondrial 54 29 123 38 59419.05 IDHP_MOUSE O88844 19 55 precursor - Mus musculus 56 (Mouse) 57 58 2 59 60 The FEBS Journal Page 52 of 105

1 2 3 , type II cytoskeletal 8 - 30 149 48 54531.46 K2C8_MOUSE 4 Mus musculus (Mouse) P11679 20 5 Keratin, type II cytoskeletal 8 - 31 148 48 54531.46 K2C8_MOUSE 6 Mus musculus (Mouse) 7 Lung carbonyl reductase 8 32 [NADPH] - Mus musculus 96 55 26055.66 CBR2_MOUSE Q8K354 21 9 (Mouse) 10 M-calpain large subunit - Mus 33 33 45 4173.06 Q920R9_MOUSE Q920R9 11 musculus (Mouse) 12 Methylcrotonoyl-CoA 13 carboxylase beta chain, 34 166 48 61910.42 MCCC2_MOUSE Q3ULD5 22 14 mitochondrial precursor - Mus 15 musculus (Mouse) 16 -9 - Mus musculus 35 160 25 227414.02 MYH9_MOUSE Q8VDD5 23 17 (Mouse) Myosin-Ib - Mus musculus 18 36 93 23 129473.30 MYO1B_MOUSE P46735 24 19 (Mouse) For Review Only Myosin-Ic - Mus musculus 20 37 218 31 118880.08 MYO1C_MOUSE Q9WTI7 25 21 (Mouse) Peroxiredoxin-2 - Mus 22 38 104 51 21936.12 PRDX2_MOUSE Q61171 26 23 musculus (Mouse) Peroxiredoxin-4 - Mus 24 39 118 53 31261.23 PRDX4_MOUSE O08807 27 25 musculus (Mouse) Plastin-3 - Mus musculus 26 40 131 40 70835.27 PLST_MOUSE (Mouse) 27 Q99K51 28 Plastin-3 - Mus musculus 28 41 171 39 70835.27 PLST_MOUSE 29 (Mouse) Ras suppressor protein 1 - 30 42 71 25 31530.74 RSU1_MOUSE Q01730 29 31 Mus musculus (Mouse) Retinal dehydrogenase 1 - 32 43 156 32 55060.08 AL1A1_MOUSE P24549 30 33 Mus musculus (Mouse) Ribosomal protein L26 - Mus 34 44 46 24 17247.53 Q5SWS8_MOUSE Q5SWS8 musculus (Mouse) 35 Ribosomal protein S3 - Mus 36 45 45 30 26828.48 Q5YLW3_MOUSE Q5YLW3 musculus (Mouse) 37 RuvB-like 2 - Mus musculus 38 46 147 53 51251.59 RUVB2_MOUSE Q9WTM5 31 (Mouse) 39 Selenium-binding protein 1 - 40 47 149 54 53050.58 SBP1_MOUSE Q63836 32 Mus musculus (Mouse) 41 Septin-11 - Mus musculus 48 128 42 50005.44 SEP11_MOUSE Q8C1B7 33 42 (Mouse) 43 Septin-7 - Mus musculus 49 69 37 50860.03 SEPT7_MOUSE O55131 34 44 (Mouse) 45 Serum albumin precursor - 50 182 46 70700.48 ALBU_MOUSE 46 Mus musculus (Mouse) P07724 35 47 Serum albumin precursor - 51 261 54 70700.48 ALBU_MOUSE 48 Mus musculus (Mouse) 49 Trifunctional enzyme subunit 52 84 22 83275.66 ECHA_MOUSE Q8BMS1 16 50 alpha, mitochondrial 51 beta-5 chain - Mus 53 233 69 50095.14 TBB5_MOUSE Q7TMM9 36 52 musculus (Mouse) 53 Ubiquinol-cytochrome-c 54 reductase complex core 54 60 30 48262.03 UQCR2_MOUSE Q9DB77 37 55 protein 2, mitochondrial 56 precursor - Mus musculus (Mo 57 58 3 59 60 Page 53 of 105 The FEBS Journal

1 2 3 Vacuolar ATP synthase 4 55 catalytic subunit A - Mus 153 47 68624.68 VATA_MOUSE P50516 38 5 musculus (Mouse) 6 - Mus musculus 56 163 52 53712.08 VIME_MOUSE P20152 39 7 (Mouse) 8 14-3-3 protein zeta/delta - 57 139 62 27924.81 1433Z_MOUSE P62259 40 9 Mus musculus (Mouse) 10 11 12 13 14 Table 1.2: mass spec analysis of a 2D gel of a DTT-eluted fraction from a lysate derived 15 from mouse lungs. The analysis is for the proteins remaining on the affinity column after acidic 16 elution has taken place. 17 18 Spot Mascot % Protein Uniprot # of Protein species Accession 19 # For ReviewScore Coverage Only MW ID species 20 0 day neonate cerebellum 21 cDNA, RIKEN full-length 22 1 enriched library, 50 19 47099.10 Q8BX68_MOUSE Q8BX68 23 clone:C230064E07 24 product:VIGILIN 25 0 day neonate eyeball cDNA, 26 RIKEN full-length enriched 2 35 16 44425.81 Q3UPU8_MOUSE Q3UPU8 27 library, clone:E130303L10 28 product:acetyl-Coe 29 11 days embryo whole body 30 cDNA, RIKEN full-length 31 3 enriched library, 46 10 42224.17 Q3V274_MOUSE Q3V274 32 clone:2700001D14 33 product:Hypoth 34 12 days embryo female mullerian duct includes 35 4 40 20 37475.98 Q3UXL1_MOUSE Q3UXL1 36 surrounding region cDNA, 37 RIKEN full-length enriched l 38 13 days embryo head cDNA, RIKEN full-length enriched 39 5 39 27 18775.76 Q544Y7_MOUSE Q544Y7 library, clone:3110001F06 40 product:cofilin 1, n 41 16 days embryo heart cDNA, 42 RIKEN full-length enriched 6 32 18 18811.88 Q3UHW9_MOUSE Q3UHW9 43 library, clone:I920194A06 44 product:cofilin 2, 45 16 days neonate cerebellum 46 cDNA, RIKEN full-length 47 7 enriched library, 29 20 17699.11 Q8BZ86_MOUSE Q8BZ86 48 clone:9630058I18 49 product:weakl 50 182 kDa tankyrase 1-binding 51 8 protein - Mus musculus 316 36 183025.90 TB182_MOUSE P58871 1 52 (Mouse) 53 26S proteasome non-ATPase 54 9 regulatory subunit 4 - Mus 89 36 40906.24 PSMD4_MOUSE O35226 2 55 musculus (Mouse) 56 10 26S proteasome non-ATPase 64 28 24875.63 PSMD9_MOUSE Q9CR00 3 57 58 4 59 60 The FEBS Journal Page 54 of 105

1 2 3 regulatory subunit 9 - Mus 4 musculus (Mouse) 5 3 days neonate thymus 6 cDNA, RIKEN full-length 7 11 enriched library, 30 9 64928.87 Q8BY99_MOUSE Q8BY99 8 clone:A630013A09 9 product: SU 10 8 days embryo whole body 11 cDNA, RIKEN full-length 12 12 enriched library, 41 23 27313.68 Q9CYD1_MOUSE Q9CYD1 13 clone:5730531E12 14 product:CHLORID 15 9.5 days embryo 16 parthenogenote cDNA, 17 13 RIKEN full-length enriched 44 18 54104.92 Q8BRB8_MOUSE 18 library, clone:B130044C16 19 product:h For Review Only 20 9.5 days embryo 21 parthenogenote cDNA, 22 14 RIKEN full-length enriched 46 22 54104.92 Q8BRB8_MOUSE Q8BRB8 23 library, clone:B130044C16 24 product:h 25 9.5 days embryo parthenogenote cDNA, 26 15 RIKEN full-length enriched 60 15 54104.92 Q8BRB8_MOUSE 27 library, clone:B130044C16 28 product:h 29 9.5 days embryo 30 parthenogenote cDNA, 31 16 RIKEN full-length enriched 64 24 54104.92 Q8BRB8_MOUSE 32 library, clone:B130044C16 33 product:h Q8BRB8 4 34 9.5 days embryo 35 parthenogenote cDNA, 36 17 RIKEN full-length enriched 82 18 54104.92 Q8BRB8_MOUSE 37 library, clone:B130044C16 38 product:h 39 Acid phosphatase 1, soluble - 18 39 24 18636.05 Q4VAI2_MOUSE Q4VAI2_ 40 Mus musculus (Mouse) 41 Adult male kidney cDNA, 42 RIKEN full-length enriched 19 48 31 14904.63 Q6ZWZ6_MOUSE Q6ZWZ6 43 library, clone:0610039M20 44 product:ribosomal prot 45 Adult male testis cDNA, RIKEN full-length enriched 46 20 50 12 47508.65 Q3TTN2_MOUSE Q3TTN2 47 library, clone:4922505E06 48 product:calpastatin, f 49 Adult male testis cDNA, RIKEN full-length enriched 50 21 27 17 29785.00 Q8CDS7_MOUSE Q8CDS7 51 library, clone:4930429C23 52 product:hypothetical p Akap8l protein - Mus 53 22 36 18 39279.30 Q80XE9_MOUSE Q80XE9 54 musculus (Mouse) A-kinase anchor protein 2 - 55 23 237 37 99089.83 AKAP2_MOUSE O54931 5 56 Mus musculus (Mouse) 57 58 5 59 60 Page 55 of 105 The FEBS Journal

1 2 3 A-kinase anchor protein 2 - 24 203 34 99089.83 AKAP2_MOUSE 4 Mus musculus (Mouse) 5 A-kinase anchor protein 2 - 25 154 24 99089.83 AKAP2_MOUSE 6 Mus musculus (Mouse) 7 A-kinase anchor protein 2 - 26 139 25 99089.83 AKAP2_MOUSE 8 Mus musculus (Mouse) 9 A-kinase anchor protein 2 - 27 129 20 99089.83 AKAP2_MOUSE 10 Mus musculus (Mouse) 11 Aldh1a3 protein - Mus 28 34 28 18949.69 Q810V9_MOUSE 12 musculus (Mouse) 13 Aldh1a3 protein - Mus 29 37 28 18949.69 Q810V9_MOUSE 14 musculus (Mouse) 15 Aldh1a3 protein - Mus 30 38 28 18949.69 Q810V9_MOUSE 16 musculus (Mouse) 17 Aldh1a3 protein - Mus 31 30 28 18949.69 Q810V9_MOUSE 18 musculus (Mouse) 19 Aldh1a3 protein - Mus 32 For Review32 28 Only18949.69 Q810V9_MOUSE 20 musculus (Mouse) Aldh1a3 protein - Mus 21 33 31 28 18949.69 Q810V9_MOUSE 22 musculus (Mouse) Aldh1a3 protein - Mus 23 34 30 28 18949.69 Q810V9_MOUSE musculus (Mouse) 24 Q810V9 Aldh1a3 protein - Mus 25 35 27 28 18949.69 Q810V9_MOUSE 26 musculus (Mouse) Aldh1a3 protein - Mus 27 36 32 28 18949.69 Q810V9_MOUSE 28 musculus (Mouse) Aldh1a3 protein - Mus 29 37 39 30 18949.69 Q810V9_MOUSE 30 musculus (Mouse) Aldh1a3 protein - Mus 31 38 39 30 18949.69 Q810V9_MOUSE 32 musculus (Mouse) Aldh1a3 protein - Mus 33 39 37 28 18949.69 Q810V9_MOUSE 34 musculus (Mouse) Aldh1a3 protein - Mus 35 40 38 33 18949.69 Q810V9_MOUSE 36 musculus (Mouse) Aldh1a3 protein - Mus 37 41 32 28 18949.69 Q810V9_MOUSE musculus (Mouse) 38 Alpha-enolase - Mus 39 42 147 41 47453.34 ENOA_MOUSE musculus (Mouse) 40 P17182 6 Alpha-enolase - Mus 41 43 65 23 47453.34 ENOA_MOUSE musculus (Mouse) 42 Annexin A2 - Mus musculus 43 44 151 48 38936.95 ANXA2_MOUSE (Mouse) 44 Annexin A2 - Mus musculus 45 127 46 38936.95 ANXA2_MOUSE 45 (Mouse) 46 Annexin A2 - Mus musculus 46 154 46 38936.95 ANXA2_MOUSE P07356 7 47 (Mouse) 48 Annexin A2 - Mus musculus 47 134 47 38936.95 ANXA2_MOUSE 49 (Mouse) 50 Annexin A2 - Mus musculus 48 164 52 38936.95 ANXA2_MOUSE 51 (Mouse) 52 Caldesmon 1 - Mus musculus 49 67 20 60530.80 Q8VCQ8_MOUSE 53 (Mouse) Q8VCQ8 8 54 Caldesmon 1 - Mus musculus 50 114 23 60530.80 Q8VCQ8_MOUSE 55 (Mouse) 56 57 58 6 59 60 The FEBS Journal Page 56 of 105

1 2 3 Caldesmon 1 - Mus musculus 51 101 19 60530.80 Q8VCQ8_MOUSE 4 (Mouse) 5 Calpastatin - Mus musculus 52 74 17 85098.73 ICAL_MOUSE 6 (Mouse) P51125 9 7 Calpastatin - Mus musculus 53 161 31 85098.73 ICAL_MOUSE 8 (Mouse) 9 Calponin-2 - Mus musculus 54 62 27 33590.21 CNN2_MOUSE 10 (Mouse) 11 Calponin-2 - Mus musculus 55 105 49 33590.21 CNN2_MOUSE 12 (Mouse) Q08093 10 13 Calponin-2 - Mus musculus 56 63 41 33590.21 CNN2_MOUSE 14 (Mouse) 15 Calponin-2 - Mus musculus 57 79 41 33590.21 CNN2_MOUSE 16 (Mouse) 17 Calponin-3 - Mus musculus 58 113 39 36576.90 CNN3_MOUSE 18 (Mouse) 19 Calponin-3 - Mus musculus 59 For Review96 35 Only36576.90 CNN3_MOUSE Q9DAW9 11 20 (Mouse) Calponin-3 - Mus musculus 21 60 59 21 36576.90 CNN3_MOUSE 22 (Mouse) Capping protein - Mus 23 61 61 19 39029.78 Q99LB4_MOUSE Q99LB4 24 musculus (Mouse) Carbonyl reductase 3 - Mus 25 62 78 33 31333.07 Q8K354_MOUSE Q8K354 12 26 musculus (Mouse) 27 Cellular nucleic acid-binding 28 63 protein - Mus musculus 67 40 20833.08 CNBP_MOUSE (Mouse) 29 P53996 13 30 Cellular nucleic acid-binding 31 64 protein - Mus musculus 62 33 20833.08 CNBP_MOUSE 32 (Mouse) Clathrin light polypeptide - 33 65 29 17 23521.36 A2AJ56_MOUSE A2AJ56 34 Mus musculus (Mouse) Cofilin-1 - Mus musculus 35 66 138 65 18775.76 COF1_MOUSE (Mouse) 36 Cofilin-1 - Mus musculus 37 67 87 48 18775.76 COF1_MOUSE (Mouse) 38 P18760 14 Cofilin-1 - Mus musculus 39 68 107 48 18775.76 COF1_MOUSE (Mouse) 40 Cofilin-1 - Mus musculus 41 69 73 26 18775.76 COF1_MOUSE (Mouse) 42 CRL-1722 L5178Y-R cDNA, 43 RIKEN full-length enriched 70 28 15 37056.85 Q3THA4_MOUSE Q3THA4 44 library, clone:I730098K12 45 product:deoxynucleotid 46 - Mus musculus 71 68 32 18851.61 DEST_MOUSE 47 (Mouse) Q9R0P5 15 48 Destrin - Mus musculus 72 59 35 18851.61 DEST_MOUSE 49 (Mouse) 50 Dihydropyrimidinase-related 51 73 protein 2 - Mus musculus 159 35 62637.74 DPYL2_MOUSE 52 (Mouse) 53 Dihydropyrimidinase-related O08553 16 54 74 protein 2 - Mus musculus 204 48 62637.74 DPYL2_MOUSE 55 (Mouse) 56 75 Dihydropyrimidinase-related 85 39 62637.74 DPYL2_MOUSE 57 58 7 59 60 Page 57 of 105 The FEBS Journal

1 2 3 protein 2 - Mus musculus 4 (Mouse) 5 Dstn protein - Mus musculus 76 48 38 18851.61 Q4FK36_MOUSE 6 (Mouse) Q4FK36 7 Dstn protein - Mus musculus 77 32 28 18851.61 Q4FK36_MOUSE 8 (Mouse) 9 Dual specificity protein 10 78 phosphatase 3 - Mus 64 40 20687.29 DUS3_MOUSE Q9D7X3 17 11 musculus (Mouse) 12 Extracellular superoxide 13 79 dismutase [Cu-Zn] precursor - 58 29 27716.81 SODE_MOUSE O09164 18 14 Mus musculus (Mouse) 15 -A - Mus musculus 80 58 7 283868.91 FLNA_MOUSE Q8BTM8 19 16 (Mouse) 17 Galectin-1 - Mus musculus 81 71 45 15198.33 LEG1_MOUSE P16045 20 18 (Mouse) 19 Glucocorticoid modulatoryFor Review Only 20 82 element binding protein 2 - 36 22 23370.57 A2AS07_MOUSE A2AS07 21 Mus musculus (Mouse) Glycine N-methyltransferase - 22 83 32 16 33070.48 Q5I0T9_MOUSE Q5I0T9 23 Mus musculus (Mouse) H2-K1 protein - Mus musculus 24 84 33 19 20819.69 Q66JU6_MOUSE Q66JU6 25 (Mouse) Hemoglobin subunit beta-1 - 26 85 71 58 15944.19 HBB1_MOUSE P02088 21 27 Mus musculus (Mouse) 28 Heterogeneous nuclear 29 86 ribonucleoprotein D0 - Mus 73 27 38501.35 HNRPD_MOUSE Q60668 22 30 musculus (Mouse) 31 Heterogeneous nuclear 32 87 ribonucleoprotein F - Mus 138 42 46043.04 HNRPF_MOUSE musculus (Mouse) 33 Q9Z2X1 23 Heterogeneous nuclear 34 88 ribonucleoprotein F - Mus 131 42 46043.04 HNRPF_MOUSE 35 musculus (Mouse) 36 Heterogeneous nuclear 37 89 ribonucleoprotein H - Mus 86 26 49453.50 HNRH1_MOUSE O35737 24 38 musculus (Mouse) 39 Heterogeneous nuclear 40 90 ribonucleoprotein L - Mus 72 16 60712.34 HNRPL_MOUSE Q8R081 25 41 musculus (Mouse) 42 Immunoglobulin-binding 43 91 protein 1 - Mus musculus 71 23 39117.65 IGBP1_MOUSE Q61249 26 44 (Mouse) 45 In vitro fertilized eggs cDNA, 46 RIKEN full-length enriched 92 37 13 19898.71 Q3UXC8_MOUSE Q3UXC8 47 library, clone:7420408C23 48 product:heterog 49 Inorganic pyrophosphatase - 93 91 35 33102.33 IPYR_MOUSE Q91VM9 27 50 Mus musculus (Mouse) 51 Leukocyte elastase inhibitor A 94 138 35 42718.83 ILEUA_MOUSE Q9D154 28 52 - Mus musculus (Mouse) 53 LIM and SH3 domain protein 95 62 29 30374.46 LASP1_MOUSE 54 1 - Mus musculus (Mouse) Q61792 29 55 LIM and SH3 domain protein 96 127 39 30374.46 LASP1_MOUSE 56 1 - Mus musculus (Mouse) 57 58 8 59 60 The FEBS Journal Page 58 of 105

1 2 3 Lymphocyte-specific protein 1 97 106 48 36805.87 LSP1_MOUSE 30 4 - Mus musculus (Mouse) 5 Melanocyte cDNA, RIKEN 6 full-length enriched library, 98 36 15 44189.84 Q3UGC3_MOUSE Q3UGC3 7 clone:G270138C06 8 product:caldesmon 1, full ins 9 MFLJ00400 protein - Mus 99 28 4 68989.33 Q571H8_MOUSE Q571H8 10 musculus (Mouse) 11 Mszf77 - Mus musculus 100 33 62 3417.61 O88227_MOUSE O88227 12 (Mouse) 13 Myl6 protein - Mus musculus 101 55 22 17121.20 Q642K0_MOUSE Q642K0 14 (Mouse) 15 Myosin regulatory light chain 16 102 2, atrial isoform - Mus 76 55 19608.63 MLRA_MOUSE Q9QVP4 31 17 musculus (Mouse) 18 Myosin, heavy polypeptide 1, 19 103 skeletal muscle, adultFor - Mus Review29 4 Only224115.73 Q32P18_MOUSE Q32P18 20 musculus (Mouse) 21 Na(+)/H(+) exchange 22 104 regulatory cofactor NHE-RF2 96 32 37721.99 NHRF2_MOUSE 23 - Mus musculus (Mouse) 24 Na(+)/H(+) exchange 25 105 regulatory cofactor NHE-RF2 173 43 37721.99 NHRF2_MOUSE 26 - Mus musculus (Mouse) 27 Na(+)/H(+) exchange 28 106 regulatory cofactor NHE-RF2 214 58 37721.99 NHRF2_MOUSE - Mus musculus (Mouse) 29 Q9JHL1 32 30 Na(+)/H(+) exchange 107 regulatory cofactor NHE-RF2 269 66 37721.99 NHRF2_MOUSE 31 - Mus musculus (Mouse) 32 Na(+)/H(+) exchange 33 108 regulatory cofactor NHE-RF2 244 61 37721.99 NHRF2_MOUSE 34 - Mus musculus (Mouse) 35 Na(+)/H(+) exchange 36 109 regulatory cofactor NHE-RF2 174 49 37721.99 NHRF2_MOUSE 37 - Mus musculus (Mouse) 38 Novel protein putative 39 orthologue to human 110 28 21 21020.77 A2AF35_MOUSE A2AF35 40 mitochondrial carrier triple 41 repeat 6 - Mus musculus (Mo 42 Nucleoredoxin - Mus 111 38 82 3538.83 Q5H8T6_MOUSE Q5H8T6 43 musculus (Mouse) 44 Osteoclast-like cell cDNA, 45 RIKEN full-length enriched 112 53 16 23274.25 Q3TWZ9_MOUSE Q3TWZ9 46 library, clone:I420020N05 47 product:clathrin, l 48 Peroxiredoxin 1 - Mus 113 36 22 22390.44 A2AP16_MOUSE A2AP16 49 musculus (Mouse) 50 Peroxiredoxin-1 - Mus 114 145 57 22390.44 PRDX1_MOUSE P35700 51 musculus (Mouse) 52 Peroxiredoxin-1 - Mus 115 75 49 22390.44 PRDX1_MOUSE 53 musculus (Mouse) Peroxiredoxin-1 - Mus 54 116 158 63 22390.44 PRDX1_MOUSE P35700 33 55 musculus (Mouse) 56 117 Peroxiredoxin-1 - Mus 96 44 22390.44 PRDX1_MOUSE 57 58 9 59 60 Page 59 of 105 The FEBS Journal

1 2 3 musculus (Mouse) 4 Peroxiredoxin-2 - Mus 118 73 40 21936.12 PRDX2_MOUSE Q61171 34 5 musculus (Mouse) 6 Peroxiredoxin-4 - Mus 119 61 40 31261.23 PRDX4_MOUSE 7 musculus (Mouse) O08807 35 8 Peroxiredoxin-4 - Mus 120 58 25 31261.23 PRDX4_MOUSE 9 musculus (Mouse) 10 Peroxiredoxin-6 - Mus 121 67 40 24969.02 PRDX6_MOUSE 11 musculus (Mouse) 12 Peroxiredoxin-6 - Mus 122 149 66 24969.02 PRDX6_MOUSE 13 musculus (Mouse) 14 Peroxiredoxin-6 - Mus 123 79 56 24969.02 PRDX6_MOUSE 15 musculus (Mouse) O08709 36 16 Peroxiredoxin-6 - Mus 124 172 76 24969.02 PRDX6_MOUSE 17 musculus (Mouse) 18 Peroxiredoxin-6 - Mus 125 96 45 24969.02 PRDX6_MOUSE 19 musculus (Mouse)For Review Only 20 Peroxiredoxin-6 - Mus 126 87 50 24969.02 PRDX6_MOUSE 21 musculus (Mouse) 22 Poly(rC)-binding protein 1 - 127 134 59 37987.14 PCBP1_MOUSE 23 Mus musculus (Mouse) P60335 37 24 Poly(rC)-binding protein 1 - 128 110 43 37987.14 PCBP1_MOUSE 25 Mus musculus (Mouse) 26 Predicted - Mus musculus 129 56 14 47453.34 Q5FW97_MOUSE 27 (Mouse) Selenium-binding protein 1 - 28 130 80 20 53050.58 SBP1_MOUSE 29 Mus musculus (Mouse) Selenium-binding protein 1 - 30 131 98 40 53050.58 SBP1_MOUSE 31 Mus musculus (Mouse) Selenium-binding protein 1 - 32 132 136 50 53050.58 SBP1_MOUSE 33 Mus musculus (Mouse) Selenium-binding protein 1 - 34 133 288 82 53050.58 SBP1_MOUSE 35 Mus musculus (Mouse) Selenium-binding protein 1 - 36 134 181 54 53050.58 SBP1_MOUSE 37 Mus musculus (Mouse) Selenium-binding protein 1 - 38 135 284 75 53050.58 SBP1_MOUSE 39 Mus musculus (Mouse) Selenium-binding protein 1 - 40 136 290 61 53050.58 SBP1_MOUSE Mus musculus (Mouse) 41 P17563 38 Selenium-binding protein 1 - 42 137 309 87 53050.58 SBP1_MOUSE Mus musculus (Mouse) 43 Selenium-binding protein 1 - 44 138 249 73 53050.58 SBP1_MOUSE Mus musculus (Mouse) 45 Selenium-binding protein 1 - 46 139 145 60 53050.58 SBP1_MOUSE Mus musculus (Mouse) 47 Selenium-binding protein 1 - 48 140 192 48 53050.58 SBP1_MOUSE Mus musculus (Mouse) 49 Selenium-binding protein 1 - 141 189 50 53050.58 SBP1_MOUSE 50 Mus musculus (Mouse) 51 Selenium-binding protein 1 - 142 155 43 53050.58 SBP1_MOUSE 52 Mus musculus (Mouse) 53 Selenium-binding protein 1 - 143 58 13 53050.58 SBP1_MOUSE 54 Mus musculus (Mouse) 55 144 Serum albumin precursor - 113 33 70700.48 ALBU_MOUSE P07724 39 56 57 58 10 59 60 The FEBS Journal Page 60 of 105

1 2 3 Mus musculus (Mouse) 4 Serum albumin precursor - 145 78 23 70700.48 ALBU_MOUSE 5 Mus musculus (Mouse) 6 Serum albumin precursor - 146 225 46 70700.48 ALBU_MOUSE 7 Mus musculus (Mouse) 8 Serum albumin precursor - 147 221 42 70700.48 ALBU_MOUSE 9 Mus musculus (Mouse) 10 Serum albumin precursor - 148 113 25 70700.48 ALBU_MOUSE 11 Mus musculus (Mouse) 12 Serum albumin precursor - 149 255 52 70700.48 ALBU_MOUSE 13 Mus musculus (Mouse) 14 Serum albumin precursor - 150 170 43 70700.48 ALBU_MOUSE 15 Mus musculus (Mouse) 16 Src substrate cortactin - Mus 151 219 43 61393.52 SRC8_MOUSE 17 musculus (Mouse) Q60598 40 18 Src substrate cortactin - Mus 152 63 18 61393.52 SRC8_MOUSE 19 musculus (Mouse)For Review Only 20 Stress-induced- 21 153 phosphoprotein 1 - Mus 97 32 63169.63 STIP1_MOUSE Q60864 41 22 musculus (Mouse) 23 Synapse-associated protein 1 154 67 17 41381.51 SYAP1_MOUSE Q9D5V6 42 24 - Mus musculus (Mouse) 25 -1 - Mus musculus 155 114 17 271831.92 TLN1_MOUSE P26039 43 26 (Mouse) Thioredoxin 1 - Mus musculus 27 156 44 42 12009.77 A2AV97_MOUSE 28 (Mouse) 29 Uncharacterized protein 30 157 C1orf198 homolog - Mus 95 31 35408.68 CA198_MOUSE Q8C3W1 44 31 musculus (Mouse) Uridine phosphorylase 2 - 32 158 30 14 36623.21 A2AIG1_MOUSE 33 Mus musculus (Mouse) Vigilin - Mus musculus 34 159 64 8 142225.48 VIGLN_MOUSE Q8VDJ3 45 35 (Mouse) Vimentin - Mus musculus 36 160 111 42 53712.08 VIME_MOUSE P20152 46 37 (Mouse) Zinc finger protein 248 - Mus 38 161 36 10 68338.20 Q640N4_MOUSE musculus (Mouse) 39 Zinc finger protein 248 - Mus 40 162 41 13 68338.20 Q640N4_MOUSE musculus (Mouse) 41 42 43 44 45 Table 1.3: mass spec analysis of a 2D gel, of an acid-eluted fraction from lysate 1 derived 46 from mouse skeletal muscles. 47 48 Spot Mascot % Protein Uniprot # of 49 Protein species Accession # Score Coverage MW ID species 50 10 days neonate cerebellum 51 cDNA, RIKEN full-length 52 1 enriched library, 30 25 12035.14 Q8C5H4_MOUSE 53 clone:6530402D11 54 product:hypot 55 13 days embryo male testis 2 37 15 40784.43 Q8CD30_MOUSE 56 cDNA, RIKEN full-length 57 58 11 59 60 Page 61 of 105 The FEBS Journal

1 2 3 enriched library, 4 clone:6030456D20 5 product:heat 6 14 days embryo liver cDNA, 7 RIKEN full-length enriched 3 41 70 2544.24 Q8CEE9_MOUSE 8 library, clone:4432406E13 9 product:zinc finger 10 15 days embryo head cDNA, 11 RIKEN full-length enriched 4 34 7 89621.14 Q8BUA6_MOUSE 12 library, clone:D930027M19 13 product:NUCLEOPORIN 14 16 days embryo head cDNA, RIKEN full-length enriched 15 5 44 37 11570.81 Q8C4P8_MOUSE 16 library, clone:C130026O10 17 product:weakly simil 18 16 days neonate cerebellum 19 cDNA, RIKEN fullFor-length Review Only 20 6 enriched library, 27 14 17873.27 Q3V3R0_MOUSE 21 clone:9630059H04 22 product:neure 23 18-day embryo whole body 24 cDNA, RIKEN full-length 25 7 enriched library, 46 26 41740.31 Q9D102_MOUSE clone:1110036D22 26 product:hypothe 27 18-day embryo whole body 28 cDNA, RIKEN full-length 29 8 enriched library, 34 13 33672.90 Q545S0_MOUSE 30 clone:1110029E05 31 product:thiosul 32 9830124H08Rik protein - Mus 9 41 24 27961.68 Q6PDL1_MOUSE 33 musculus (Mouse) 34 Actin, alpha cardiac muscle 1 - 10 75 23 42334.01 ACTC_MOUSE 35 Mus musculus (Mouse) P68033 1 36 Actin, alpha cardiac muscle 1 - 11 65 23 42334.01 ACTC_MOUSE 37 Mus musculus (Mouse) 38 Actin, alpha skeletal muscle - 12 125 46 42365.98 ACTS_MOUSE P68134 2 39 Mus musculus (Mouse) 40 Actin, aortic smooth muscle - 13 61 15 42380.96 ACTA_MOUSE P62737 3 41 Mus musculus (Mouse) 42 Activated spleen cDNA, 43 RIKEN full-length enriched 14 37 27 25090.58 Q3U046_MOUSE 44 library, clone:F830207J03 45 product:yes-associated 46 Activated spleen cDNA, 47 RIKEN full-length enriched 15 36 27 25090.58 Q3U046_MOUSE 48 library, clone:F830207J03 49 product:yes-associated 50 Activated spleen cDNA, RIKEN full-length enriched 51 16 32 27 25090.58 Q3U046_MOUSE 52 library, clone:F830207J03 53 product:yes-associated 54 Adult male cerebellum cDNA, 55 17 RIKEN full-length enriched 31 29 18159.84 Q8CEI4_MOUSE 56 library, clone:1520402A20 57 58 12 59 60 The FEBS Journal Page 62 of 105

1 2 3 product:hypothetic 4 Adult male testis cDNA, 5 RIKEN full-length enriched 18 31 18 29785.00 Q8CDS7_MOUSE 6 library, clone:4930429C23 7 product:hypothetical p 8 Adult male thymus cDNA, 9 RIKEN full-length enriched 19 38 11 58684.92 Q3UZ80_MOUSE 10 library, clone:5830406G23 11 product:deoxynucleotid 12 Adult male tongue cDNA, 13 RIKEN full-length enriched 20 42 21 16703.17 Q545G5_MOUSE 14 library, clone:2310051N24 15 product:myosin light c 16 Adult retina cDNA, RIKEN full- 17 length enriched library, 21 38 34 17208.90 Q8C8P3_MOUSE 18 clone:A930038E24 19 product:hypotheticalFor protei Review Only Ahdc1 protein - Mus musculus 20 22 26 13 26700.08 Q8VCU7_MOUSE 21 (Mouse) Aldh1a3 protein - Mus 22 23 29 28 18949.69 Q810V9_MOUSE 23 musculus (Mouse) Aldh1a3 protein - Mus 24 24 33 28 18949.69 Q810V9_MOUSE 25 musculus (Mouse) Aldh1a3 protein - Mus 26 25 34 28 18949.69 Q810V9_MOUSE 27 musculus (Mouse) Aldh1a3 protein - Mus 28 26 26 28 18949.69 Q810V9_MOUSE 29 musculus (Mouse) Aldh1a3 protein - Mus 30 27 31 28 18949.69 Q810V9_MOUSE 31 musculus (Mouse) Aldh1a3 protein - Mus 32 28 29 28 18949.69 Q810V9_MOUSE 33 musculus (Mouse) Aldh1a3 protein - Mus 34 29 30 28 18949.69 Q810V9_MOUSE musculus (Mouse) 35 Aldh1a3 protein - Mus 36 30 37 28 18949.69 Q810V9_MOUSE musculus (Mouse) 37 Aldh1a3 protein - Mus 38 31 32 28 18949.69 Q810V9_MOUSE musculus (Mouse) 39 Aldolase 1, A isoform - Mus 40 32 50 25 39787.45 Q5FWB7_MOUSE musculus (Mouse) 41 Aldolase 1, A isoform - Mus 33 32 11 39743.43 Q6NY00_MOUSE 42 musculus (Mouse) 43 Alpha crystallin B chain - Mus 34 90 50 20056.41 CRYAB_MOUSE 44 musculus (Mouse) P23927 4 45 Alpha crystallin B chain - Mus 35 80 48 20056.41 CRYAB_MOUSE 46 musculus (Mouse) 47 Alpha-actin - Mus musculus 36 52 24 39453.86 Q61275_MOUSE 48 (Mouse) 49 ATP synthase subunit alpha, 50 37 mitochondrial precursor - Mus 59 16 59829.63 ATPA_MOUSE 51 musculus (Mouse) Q03265 5 52 ATP synthase subunit alpha, 53 38 mitochondrial precursor - Mus 84 16 59829.63 ATPA_MOUSE 54 musculus (Mouse) 55 ATP synthase subunit beta, 39 78 25 56265.46 ATPB_MOUSE P56480 6 56 mitochondrial precursor - Mus 57 58 13 59 60 Page 63 of 105 The FEBS Journal

1 2 3 musculus (Mouse) 4 ATP synthase subunit beta, 5 40 mitochondrial precursor - Mus 60 22 56265.46 ATPB_MOUSE 6 musculus (Mouse) 7 ATP synthase subunit beta, 8 41 mitochondrial precursor - Mus 191 42 56265.46 ATPB_MOUSE 9 musculus (Mouse) 10 Beta-enolase - Mus musculus 42 62 13 47337.43 ENOB_MOUSE 11 (Mouse) 12 Beta-enolase - Mus musculus 43 103 29 47337.43 ENOB_MOUSE P21550 7 13 (Mouse) 14 Beta-enolase - Mus musculus 44 128 30 47337.43 ENOB_MOUSE 15 (Mouse) 16 Bone marrow macrophage 17 cDNA, RIKEN full-length 18 45 enriched library, 36 21 22358.46 Q3U9J9_MOUSE 19 clone:I830036L09For Review Only 20 product:peroxired 21 Creatine kinase M-type - Mus 46 61 25 43245.88 KCRM_MOUSE P07310 8 22 musculus (Mouse) 23 Fructose-bisphosphate 24 47 aldolase A - Mus musculus 70 21 39787.45 ALDOA_MOUSE 9 (Mouse) 25 P05064 26 Fructose-bisphosphate 27 48 aldolase A - Mus musculus 108 51 39787.45 ALDOA_MOUSE 28 (Mouse) 29 G patch domain and KOW 30 49 motifs - Mus musculus 32 12 54083.43 A2AEU7_MOUSE 31 (Mouse) 32 Glyceraldehyde-3-phosphate 33 50 dehydrogenase - Mus 90 34 36072.32 G3P_MOUSE musculus (Mouse) 34 Glyceraldehyde-3-phosphate 35 51 dehydrogenase - Mus 93 41 36072.32 G3P_MOUSE P16858 10 36 musculus (Mouse) 37 Glyceraldehyde-3-phosphate 38 52 dehydrogenase - Mus 96 41 36072.32 G3P_MOUSE 39 musculus (Mouse) 40 acidic 5 - Mus 53 32 6 48983.58 Q9Z2T8_MOUSE 41 musculus (Mouse) 42 Heat-shock protein beta-1 - 54 89 42 23056.74 HSPB1_MOUSE P14602 11 43 Mus musculus (Mouse) 44 Keratin complex 1, acidic, 45 55 gene 10 - Mus musculus 49 16 57177.78 A2A513_MOUSE 46 (Mouse) 47 LATS2B - Mus musculus 56 42 20 19476.01 Q8VHE2_MOUSE 48 (Mouse) 49 L-lactate dehydrogenase A 57 87 27 36817.35 LDHA_MOUSE P06151 12 50 chain - Mus musculus (Mouse) 51 Low-voltage-activated calcium 52 58 channel alpha13.2 subunit - 31 32 12283.98 Q99P43_MOUSE 53 Mus musculus (Mouse) 54 NADH dehydrogenase 55 59 [ubiquinone] flavoprotein 1, 73 14 51485.97 NDUV1_MOUSE Q91YT0 13 56 mitochondrial precursor - Mus 57 58 14 59 60 The FEBS Journal Page 64 of 105

1 2 3 musculus (Mouse) 4 NALP12 - Mus musculus 60 38 8 83710.93 Q6UQE6_MOUSE 5 (Mouse) 6 NOD-derived CD11c +ve 7 dendritic cells cDNA, RIKEN 61 32 12 42883.29 Q3U3L3_MOUSE 8 full-length enriched library, 9 clone:F630103L03 pr 10 NOD-derived CD11c +ve 11 dendritic cells cDNA, RIKEN 62 27 9 24492.13 Q3U317_MOUSE 12 full-length enriched library, 13 clone:F630113L23 pr 14 Puromycin-sensitive 15 63 aminopeptidase - Mus 30 23 6408.20 A2A9T3_MOUSE 16 musculus (Mouse) 17 Serine proteinase inhibitor 18 64 mBM17 - Mus musculus 46 39 16994.47 O08799_MOUSE 19 (Mouse) For Review Only 20 Succinate dehydrogenase [ubiquinone] flavoprotein 21 65 114 24 73622.58 DHSA_MOUSE Q8K2B3 14 22 subunit, mitochondrial 23 precursor - Mus musculus ( Synaptobrevin-like protein - 24 66 32 13 25293.13 P70280_MOUSE 25 Mus musculus (Mouse) Telokin - Mus musculus 26 67 39 49 7968.95 Q8CIY6_MOUSE 27 (Mouse) Triosephosphate isomerase - 28 68 131 53 27037.89 TPIS_MOUSE P17751 15 29 Mus musculus (Mouse) KA22 - Mus 30 69 38 21 50444.02 Q6IFX2_MOUSE 31 musculus (Mouse) Upf3b protein - Mus musculus 32 70 36 16 29749.13 Q149I6_MOUSE 33 (Mouse) ZFP421 - Mus musculus 34 71 44 22 50248.87 Q8CG23_MOUSE (Mouse) 35 36 37 38 39 Table 1.4: mass spec analysis of a 2D gel of a DTT-eluted fraction from lysate 1 derived 40 from mouse skeletal muscles. The analysis is for the proteins remaining on the affinity column 41 after acidic elution has occurred. 42 43 44 45 Spot Mascot % Protein Uniprot # of Protein species Accession 46 # Score Coverage MW ID species 47 18-day embryo whole body 48 cDNA, RIKEN full-length 49 1 enriched library, 82 32 24405.14 Q8BK84_MOUSE Q8BK84 1 50 clone:1110056B09 51 product:weakly 52 60 kDa heat shock protein, 53 2 mitochondrial precursor - Mus 59 22 61088.45 CH60_MOUSE P63038 2 54 musculus (Mouse) Adenylate kinase isoenzyme 1 55 3 68 52 21640.16 KAD1_MOUSE Q9R0Y5 3 56 - Mus musculus (Mouse) 57 58 15 59 60 Page 65 of 105 The FEBS Journal

1 2 3 Adenylate kinase isoenzyme 1 4 130 56 21640.16 KAD1_MOUSE 4 - Mus musculus (Mouse) 5 Aldh1a3 protein - Mus 5 28 28 18949.69 Q810V9_MOUSE 6 musculus (Mouse)

7 Aldh1a3 protein - Mus 6 37 30 18949.69 Q810V9_MOUSE 8 musculus (Mouse) 9 Carbonic anhydrase 3 - Mus 7 60 31 29632.75 CAH3_MOUSE P16015 4 10 musculus (Mouse) 11 Creatine kinase M-type - Mus 8 74 22 43245.88 KCRM_MOUSE 12 musculus (Mouse) P07310 5 13 Creatine kinase M-type - Mus 9 120 34 43245.88 KCRM_MOUSE 14 musculus (Mouse) 15 Creatine kinase, sarcomeric 16 10 mitochondrial precursor - Mus 158 38 47899.49 KCRS_MOUSE 17 musculus (Mouse) Q6P8J7 6 18 Creatine kinase, sarcomeric 19 11 mitochondrial precursorFor - Mus Review156 38 Only47899.49 KCRS_MOUSE 20 musculus (Mouse) Crip2 protein - Mus musculus 21 12 53 40 23510.36 Q4FJU3_MOUSE 22 (Mouse) Fbxo33 protein - Mus 23 13 37 17 31480.00 A0JLR4_MOUSE 24 musculus (Mouse) Galectin-1 - Mus musculus 25 14 70 58 15198.33 LEG1_MOUSE (Mouse) 26 P16045 7 Galectin-1 - Mus musculus 27 15 65 45 15198.33 LEG1_MOUSE 28 (Mouse) 29 Glyceraldehyde-3-phosphate 30 16 dehydrogenase - Mus 93 48 36072.32 G3P_MOUSE 31 musculus (Mouse) 32 Glyceraldehyde-3-phosphate 33 17 dehydrogenase - Mus 98 51 36072.32 G3P_MOUSE musculus (Mouse) 34 Glyceraldehyde-3-phosphate 35 18 dehydrogenase - Mus 124 51 36072.32 G3P_MOUSE 36 musculus (Mouse) 37 Glyceraldehyde-3-phosphate 38 19 dehydrogenase - Mus 58 40 36072.32 G3P_MOUSE 39 musculus (Mouse) 40 Glyceraldehyde-3-phosphate 41 20 dehydrogenase - Mus 100 57 36072.32 G3P_MOUSE 42 musculus (Mouse) P16858 8 43 Glyceraldehyde-3-phosphate 44 21 dehydrogenase - Mus 106 47 36072.32 G3P_MOUSE 45 musculus (Mouse) 46 Glyceraldehyde-3-phosphate 47 22 dehydrogenase - Mus 81 40 36072.32 G3P_MOUSE 48 musculus (Mouse) 49 Glyceraldehyde-3-phosphate 50 23 dehydrogenase - Mus 83 36 36072.32 G3P_MOUSE 51 musculus (Mouse) 52 Glyceraldehyde-3-phosphate 53 24 dehydrogenase - Mus 92 45 36072.32 G3P_MOUSE 54 musculus (Mouse) 55 Glyceraldehyde-3-phosphate 25 74 48 36072.32 G3P_MOUSE 56 dehydrogenase - Mus 57 58 16 59 60 The FEBS Journal Page 66 of 105

1 2 3 musculus (Mouse) 4 Glyceraldehyde-3-phosphate 5 26 dehydrogenase - Mus 117 49 36072.32 G3P_MOUSE 6 musculus (Mouse) 7 Glyceraldehyde-3-phosphate 8 27 dehydrogenase - Mus 101 51 36072.32 G3P_MOUSE 9 musculus (Mouse) 10 Glyceraldehyde-3-phosphate 11 28 dehydrogenase - Mus 76 42 36072.32 G3P_MOUSE 12 musculus (Mouse) 13 Glyceraldehyde-3-phosphate 14 29 dehydrogenase - Mus 67 28 36072.32 G3P_MOUSE 15 musculus (Mouse) 16 Glyceraldehyde-3-phosphate 17 30 dehydrogenase - Mus 88 45 36072.32 G3P_MOUSE 18 musculus (Mouse) 19 Glyceraldehyde-3-phosphateFor Review Only 20 31 dehydrogenase - Mus 104 45 36072.32 G3P_MOUSE 21 musculus (Mouse) 22 Glyceraldehyde-3-phosphate 23 32 dehydrogenase - Mus 97 51 36072.32 G3P_MOUSE 24 musculus (Mouse) 25 Glyceraldehyde-3-phosphate 26 33 dehydrogenase - Mus 95 51 36072.32 G3P_MOUSE 27 musculus (Mouse) 28 Glyceraldehyde-3-phosphate 29 34 dehydrogenase - Mus 69 30 36072.32 G3P_MOUSE 30 musculus (Mouse) 31 Glyceraldehyde-3-phosphate 32 35 dehydrogenase - Mus 67 34 36072.32 G3P_MOUSE musculus (Mouse) 33 Glyceraldehyde-3-phosphate 34 36 dehydrogenase - Mus 85 52 36072.32 G3P_MOUSE 35 musculus (Mouse) 36 Glyceraldehyde-3-phosphate 37 37 dehydrogenase - Mus 103 51 36072.32 G3P_MOUSE 38 musculus (Mouse) 39 Glyceraldehyde-3-phosphate 40 38 dehydrogenase - Mus 119 42 36072.32 G3P_MOUSE 41 musculus (Mouse) 42 Glyceraldehyde-3-phosphate 43 39 dehydrogenase - Mus 103 45 36072.32 G3P_MOUSE 44 musculus (Mouse) 45 Glyceraldehyde-3-phosphate 46 40 dehydrogenase - Mus 89 45 36072.32 G3P_MOUSE 47 musculus (Mouse) 48 Glyceraldehyde-3-phosphate 49 41 dehydrogenase - Mus 102 48 36072.32 G3P_MOUSE 50 musculus (Mouse) 51 Glyceraldehyde-3-phosphate 52 42 dehydrogenase - Mus 93 45 36072.32 G3P_MOUSE 53 musculus (Mouse) 54 Glyceraldehyde-3-phosphate 55 43 dehydrogenase - Mus 76 45 36072.32 G3P_MOUSE 56 musculus (Mouse) 57 58 17 59 60 Page 67 of 105 The FEBS Journal

1 2 3 Glyceraldehyde-3-phosphate 4 44 dehydrogenase - Mus 90 48 36072.32 G3P_MOUSE 5 musculus (Mouse) 6 Glyceraldehyde-3-phosphate 7 45 dehydrogenase - Mus 90 41 36072.32 G3P_MOUSE 8 musculus (Mouse) 9 Glyceraldehyde-3-phosphate 10 46 dehydrogenase - Mus 105 51 36072.32 G3P_MOUSE 11 musculus (Mouse) 12 Glyceraldehyde-3-phosphate 13 47 dehydrogenase - Mus 106 51 36072.32 G3P_MOUSE 14 musculus (Mouse) 15 Glyceraldehyde-3-phosphate 16 48 dehydrogenase - Mus 58 25 36072.32 G3P_MOUSE 17 musculus (Mouse) 18 Glyceraldehyde-3-phosphate 19 49 dehydrogenase For- Mus Review70 34 Only36072.32 G3P_MOUSE 20 musculus (Mouse) 21 Glyceraldehyde-3-phosphate 22 50 dehydrogenase - Mus 108 45 36072.32 G3P_MOUSE 23 musculus (Mouse) M-calpain large subunit - Mus 24 51 30 45 4173.06 Q920R9_MOUSE musculus (Mouse) 25 M-calpain large subunit - Mus 26 52 33 45 4173.06 Q920R9_MOUSE 27 musculus (Mouse) Myomesin-1 - Mus musculus 28 53 100 14 186470.45 MYOM1_MOUSE Q62234 9 29 (Mouse) 30 1, skeletal 54 muscle isoform - Mus 105 63 20695.45 MLE1_MOUSE P05977 10 31 musculus (Mouse) 32 Myosin regulatory light chain 33 55 2, skeletal muscle isoform - 133 70 19057.39 MLRS_MOUSE P97457 11 34 Mus musculus (Mouse) 35 Myosin-binding protein C, fast- 56 79 19 128127.87 MYPC2_MOUSE 36 type - Mus musculus (Mouse) 37 Myosin-binding protein C, fast- 57 114 19 128127.87 MYPC2_MOUSE 38 type - Mus musculus (Mouse) 39 Myosin-binding protein C, fast- 58 291 38 128127.87 MYPC2_MOUSE Q5XKE0 12 40 type - Mus musculus (Mouse) 41 Myosin-binding protein C, fast- 59 193 34 128127.87 MYPC2_MOUSE 42 type - Mus musculus (Mouse) 43 Myosin-binding protein C, fast- 60 82 13 128127.87 MYPC2_MOUSE 44 type - Mus musculus (Mouse) 45 Myosin-binding protein H - 61 115 38 53068.82 MYBPH_MOUSE 46 Mus musculus (Mouse) 47 Myosin-binding protein H - 62 146 49 53068.82 MYBPH_MOUSE 48 Mus musculus (Mouse) P70402 13 49 Myosin-binding protein H - 63 115 49 53068.82 MYBPH_MOUSE 50 Mus musculus (Mouse) 51 Myosin-binding protein H - 64 136 49 53068.82 MYBPH_MOUSE 52 Mus musculus (Mouse) 53 Myotilin - Mus musculus 65 173 43 55738.03 MYOTI_MOUSE Q9JIF9 14 54 (Mouse) 55 Peroxiredoxin-1 - Mus 66 112 47 22390.44 PRDX1_MOUSE P35700 15 56 musculus (Mouse) 57 58 18 59 60 The FEBS Journal Page 68 of 105

1 2 3 Peroxiredoxin-1 - Mus 67 160 63 22390.44 PRDX1_MOUSE 4 musculus (Mouse) 5 Peroxiredoxin-1 - Mus 68 83 35 22390.44 PRDX1_MOUSE 6 musculus (Mouse) 7 Peroxiredoxin-1 - Mus 69 133 58 22390.44 PRDX1_MOUSE 8 musculus (Mouse) 9 Peroxiredoxin-1 - Mus 70 161 63 22390.44 PRDX1_MOUSE 10 musculus (Mouse) 11 Peroxiredoxin-1 - Mus 71 165 63 22390.44 PRDX1_MOUSE 12 musculus (Mouse) 13 Peroxiredoxin-1 - Mus 72 130 53 22390.44 PRDX1_MOUSE 14 musculus (Mouse) 15 Peroxiredoxin-1 - Mus 73 121 53 22390.44 PRDX1_MOUSE 16 musculus (Mouse) 17 Peroxiredoxin-2 - Mus 74 95 51 21936.12 PRDX2_MOUSE Q61171 16 18 musculus (Mouse) 19 Peroxiredoxin-6 - Mus 75 For Review153 73 Only24969.02 PRDX6_MOUSE 20 musculus (Mouse) Peroxiredoxin-6 - Mus 21 76 132 57 24969.02 PRDX6_MOUSE O08709 17 22 musculus (Mouse) Peroxiredoxin-6 - Mus 23 77 149 73 24969.02 PRDX6_MOUSE 24 musculus (Mouse) Protein DJ-1 - Mus musculus 25 78 88 56 20236.47 PARK7_MOUSE Q99LX0 18 26 (Mouse) 27 Thioredoxin-dependent peroxide reductase, 28 79 77 36 28337.49 PRDX3_MOUSE P20108 19 29 mitochondrial precursor - Mus 30 musculus (Mouse) 31 32 33 34 Table 1.5: mass spec analysis of a 2D gel of a DTT-eluted fraction from a lysate 2 derived 35 from mouse skeletal muscles. The analysis is for the proteins remaining on the affinity column 36 37 after acidic elution. 38 39 40 Spot Mascot % Protein Uniprot # of Protein species Accession 41 # Score Coverage MW ID species 42 1 cell embryo 1 cell cDNA, RIKEN full-length enriched 43 1 32 21 15783.77 Q3TLD8_MOUSE 44 library, clone:I0C0026C19 45 product:hypothetica 46 13 days embryo male testis 47 cDNA, RIKEN full-length 48 2 enriched library, 31 14 32360.93 Q8BJ62_MOUSE 49 clone:6030400N17 product:CGMP- 50 51 13 days embryo male testis cDNA, RIKEN full-length 52 3 enriched library, 30 14 32360.93 Q8BJ62_MOUSE 53 clone:6030400N17 54 product:CGMP- 55 4 15 days embryo head cDNA, 31 22 15219.59 Q3TNY2_MOUSE 56 57 58 19 59 60 Page 69 of 105 The FEBS Journal

1 2 3 RIKEN full-length enriched 4 library, clone:D930049J08 5 product: alp 6 17 days pregnant adult female 7 amnion cDNA, RIKEN full- 5 47 12 57406.91 Q3TGS0_MOUSE 8 length enriched library, 9 clone:I920038I15 pro 10 26S proteasome non-ATPase 11 6 regulatory subunit 4 - Mus 75 19 40906.24 PSMD4_MOUSE O35226 1 12 musculus (Mouse) 13 4833405L16Rik protein - Mus 7 33 19 26899.60 Q4FZF0_MOUSE 14 musculus (Mouse) 15 60 kDa heat shock protein, 16 8 mitochondrial precursor - Mus 61 21 61088.45 CH60_MOUSE 17 musculus (Mouse) 18 60 kDa heat shock protein, 19 9 mitochondrial precursorFor - Mus Review131 32 Only61088.45 CH60_MOUSE P63038 2 20 musculus (Mouse) 21 60 kDa heat shock protein, 22 10 mitochondrial precursor - Mus 74 22 61088.45 CH60_MOUSE 23 musculus (Mouse) 24 6-phosphofructokinase, 25 11 muscle type - Mus musculus 71 12 86069.88 K6PF_MOUSE P47857 3 26 (Mouse) 27 Aconitate hydratase, 28 12 mitochondrial precursor - Mus 67 15 86151.30 ACON_MOUSE musculus (Mouse) 29 Aconitate hydratase, 30 13 mitochondrial precursor - Mus 108 20 86151.30 ACON_MOUSE 31 musculus (Mouse) 32 Aconitate hydratase, 33 14 mitochondrial precursor - Mus 136 27 86151.30 ACON_MOUSE 34 musculus (Mouse) 35 Aconitate hydratase, 36 15 mitochondrial precursor - Mus 127 20 86151.30 ACON_MOUSE Q99KI0 4 37 musculus (Mouse) 38 Aconitate hydratase, 39 16 mitochondrial precursor - Mus 130 25 86151.30 ACON_MOUSE 40 musculus (Mouse) 41 Aconitate hydratase, 42 17 mitochondrial precursor - Mus 180 40 86151.30 ACON_MOUSE 43 musculus (Mouse) 44 Aconitate hydratase, 45 18 mitochondrial precursor - Mus 151 28 86151.30 ACON_MOUSE 46 musculus (Mouse) 47 Actin, aortic smooth muscle - 19 64 15 42380.96 ACTA_MOUSE P62737 5 48 Mus musculus (Mouse) 49 Activated spleen cDNA, 50 RIKEN full-length enriched 20 32 20 25090.58 Q3U046_MOUSE 51 library, clone:F830207J03 52 product:yes-associated 53 Adult male testis cDNA, RIKEN full-length enriched 54 21 34 41 12324.27 Q9D556_MOUSE 55 library, clone:4930512B01 56 product:hypothetical p 57 58 20 59 60 The FEBS Journal Page 70 of 105

1 2 3 Aldh1a3 protein - Mus 22 30 28 18949.69 Q810V9_MOUSE 4 musculus (Mouse) 5 Aldh1a3 protein - Mus 23 30 28 18949.69 Q810V9_MOUSE 6 musculus (Mouse)

7 Aldh1a3 protein - Mus 24 35 28 18949.69 Q810V9_MOUSE 8 musculus (Mouse) 9 Aldh1a3 protein - Mus 25 35 28 18949.69 Q810V9_MOUSE 10 musculus (Mouse) 11 ATP synthase subunit beta, 12 26 mitochondrial precursor - Mus 167 46 56265.46 ATPB_MOUSE P56480 6 13 musculus (Mouse) 14 Beta-enolase - Mus musculus 27 93 26 47337.43 ENOB_MOUSE 15 (Mouse) 16 Beta-enolase - Mus musculus 28 186 61 47337.43 ENOB_MOUSE P21550 7 17 (Mouse) 18 Beta-enolase - Mus musculus 29 211 64 47337.43 ENOB_MOUSE 19 (Mouse) For Review Only Beta-taxilin - Mus musculus 20 30 58 13 77729.63 TXLNB_MOUSE Q8VBT1 8 21 (Mouse) 22 Bone marrow macrophage 23 cDNA, RIKEN full-length 24 31 enriched library, 62 17 47204.99 Q3UD36_MOUSE 25 clone:I830001B16 26 product:vimentin, 27 cAMP-dependent protein kinase type I-alpha regulatory 28 32 67 21 43443.14 KAP0_MOUSE Q9DBC7 9 29 subunit - Mus musculus 30 (Mouse) Creatine kinase M-type - Mus 31 33 170 53 43245.88 KCRM_MOUSE P07310 10 musculus (Mouse) 32 Creatine kinase, sarcomeric 33 34 mitochondrial precursor - Mus 92 25 47899.49 KCRS_MOUSE 34 musculus (Mouse) 35 Creatine kinase, sarcomeric 36 35 mitochondrial precursor - Mus 101 28 47899.49 KCRS_MOUSE 37 musculus (Mouse) 38 Creatine kinase, sarcomeric 39 36 mitochondrial precursor - Mus 172 55 47899.49 KCRS_MOUSE 40 musculus (Mouse) 41 Creatine kinase, sarcomeric 42 37 mitochondrial precursor - Mus 211 64 47899.49 KCRS_MOUSE Q6P8J7 11 43 musculus (Mouse) 44 Creatine kinase, sarcomeric 45 38 mitochondrial precursor - Mus 101 28 47899.49 KCRS_MOUSE 46 musculus (Mouse) 47 Creatine kinase, sarcomeric 48 39 mitochondrial precursor - Mus 129 43 47899.49 KCRS_MOUSE 49 musculus (Mouse) 50 Creatine kinase, sarcomeric 51 40 mitochondrial precursor - Mus 121 32 47899.49 KCRS_MOUSE 52 musculus (Mouse) 53 Dehydrogenase/reductase - 41 38 18 35504.86 Q148Q4_MOUSE 54 Mus musculus (Mouse) Glyceraldehyde-3-phosphate 55 42 69 34 36072.32 G3P_MOUSE P16858 12 56 dehydrogenase - Mus 57 58 21 59 60 Page 71 of 105 The FEBS Journal

1 2 3 musculus (Mouse) 4 Glyceraldehyde-3-phosphate 5 43 dehydrogenase - Mus 138 55 36072.32 G3P_MOUSE 6 musculus (Mouse) 7 Glyceraldehyde-3-phosphate 8 44 dehydrogenase - Mus 128 54 36072.32 G3P_MOUSE 9 musculus (Mouse) 10 Lethal(3)malignant brain 11 45 tumor-like protein - Mus 28 56 5938.85 Q3V555_MOUSE 12 musculus (Mouse) 13 Low-voltage-activated calcium 14 46 channel alpha13.2 subunit - 36 41 12283.98 Q99P43_MOUSE 15 Mus musculus (Mouse) 16 Myomesin-1 - Mus musculus 47 193 24 186470.45 MYOM1_MOUSE 17 (Mouse) 18 Myomesin-1 - Mus musculus 48 315 34 186470.45 MYOM1_MOUSE 19 (Mouse) For Review Only 20 Myomesin-1 - Mus musculus 49 306 35 186470.45 MYOM1_MOUSE 21 (Mouse) 22 Myomesin-1 - Mus musculus 50 251 33 186470.45 MYOM1_MOUSE (Mouse) 23 Q62234 13 Myomesin-1 - Mus musculus 24 51 326 36 186470.45 MYOM1_MOUSE 25 (Mouse) Myomesin-1 - Mus musculus 26 52 245 28 186470.45 MYOM1_MOUSE 27 (Mouse) Myomesin-1 - Mus musculus 28 53 219 30 186470.45 MYOM1_MOUSE 29 (Mouse) Myomesin-1 - Mus musculus 30 54 91 14 186470.45 MYOM1_MOUSE 31 (Mouse) Myosin heavy chain IIB - Mus 32 55 84 18 61050.65 Q9JHR4_MOUSE Q9JHR4 14 33 musculus (Mouse) Myosin-binding protein C, fast- 34 56 217 33 128127.87 MYPC2_MOUSE 35 type - Mus musculus (Mouse) Myosin-binding protein C, fast- 36 57 275 42 128127.87 MYPC2_MOUSE 37 type - Mus musculus (Mouse) Myosin-binding protein C, fast- 38 58 150 31 128127.87 MYPC2_MOUSE Q5XKE0 type - Mus musculus (Mouse) 39 Myosin-binding protein C, fast- 40 59 178 33 128127.87 MYPC2_MOUSE type - Mus musculus (Mouse) 41 Myosin-binding protein C, fast- 42 60 293 39 128127.87 MYPC2_MOUSE 15 type - Mus musculus (Mouse) 43 Myosin-binding protein H - 44 61 81 29 53068.82 MYBPH_MOUSE Mus musculus (Mouse) 45 Myosin-binding protein H - 62 71 14 53068.82 MYBPH_MOUSE 46 Mus musculus (Mouse) P70402 47 Myosin-binding protein H - 63 68 14 53068.82 MYBPH_MOUSE 48 Mus musculus (Mouse) 49 Myosin-binding protein H - 64 79 28 53068.82 MYBPH_MOUSE 50 Mus musculus (Mouse) 51 Otc protein - Mus musculus 65 30 12 39511.45 Q8R1A8_MOUSE 52 (Mouse) 53 RAS-like family 11 member A 66 31 7 27221.18 Q6IMB1_MOUSE 54 - Mus musculus (Mouse) 55 Stress-70 protein, 67 71 18 73767.84 GRP75_MOUSE P38647 16 56 mitochondrial precursor - Mus 57 58 22 59 60 The FEBS Journal Page 72 of 105

1 2 3 musculus (Mouse) 4 Stress-induced- 5 68 phosphoprotein 1 - Mus 132 28 63169.63 STIP1_MOUSE 6 musculus (Mouse) 7 Stress-induced- 8 69 phosphoprotein 1 - Mus 212 43 63169.63 STIP1_MOUSE 9 musculus (Mouse) Q60864 17 10 Stress-induced- 11 70 phosphoprotein 1 - Mus 177 37 63169.63 STIP1_MOUSE 12 musculus (Mouse) 13 Stress-induced- 14 71 phosphoprotein 1 - Mus 97 19 63169.63 STIP1_MOUSE 15 musculus (Mouse) 16 Thioredoxin-like 2 - Mus 72 33 15 38039.48 Q5I0V8_MOUSE 17 musculus (Mouse) 18 Zinc finger protein 52 - Mus 73 37 6 83721.30 Q8VDL3_MOUSE 19 musculus (Mouse)For Review Only 20 21 22 23 24 Table 1.6: mass spec analysis of a 2D gel of a DTT-eluted fraction from lysate 2 (continued) 25 derived from mouse skeletal muscles. The analysis is for the proteins remaining on the affinity 26 column after acidic elution. 27 28 29 Spot Mascot % Protein Uniprot # Protein species Accession 30 # Score Coverage MW ID species 31 0 day neonate cerebellum 32 cDNA, RIKEN full-length 33 1 enriched library, 43 28 14380.01 Q8C4A2_MOUSE 34 clone:C230089I12 35 product:hypothe 36 10 days neonate skin cDNA, 37 RIKEN full-length enriched 2 62 20 47977.68 Q8C139_MOUSE 38 library, clone:4732482K20 39 product:, HEAR 40 13 days embryo head cDNA, 41 RIKEN full-length enriched 3 30 21 18775.76 Q544Y7_MOUSE 42 library, clone:3110001F06 43 product:cofilin 1, n 44 6430706D22Rik protein - Mus 4 38 54 8161.08 Q99KB1_MOUSE 45 musculus (Mouse) 46 Adenylate kinase isoenzyme 1 5 76 39 21640.16 KAD1_MOUSE 47 - Mus musculus (Mouse) 48 Adenylate kinase isoenzyme 1 6 87 60 21640.16 KAD1_MOUSE 49 - Mus musculus (Mouse) 50 Adenylate kinase isoenzyme 1 7 83 55 21640.16 KAD1_MOUSE Q9R0Y5 1 51 - Mus musculus (Mouse) 52 Adenylate kinase isoenzyme 1 8 63 24 21640.16 KAD1_MOUSE 53 - Mus musculus (Mouse) 54 Adenylate kinase isoenzyme 1 9 65 30 21640.16 KAD1_MOUSE 55 - Mus musculus (Mouse) 56 10 Adult male diencephalon 26 21 11469.66 Q3TRY6_MOUSE 57 58 23 59 60 Page 73 of 105 The FEBS Journal

1 2 3 cDNA, RIKEN full-length 4 enriched library, 5 clone:9330101M15 6 product:hypothet 7 Adult male hypothalamus 8 cDNA, RIKEN full-length 9 11 enriched library, 30 7 22094.09 Q8CAH4_MOUSE 10 clone:A230060C20 11 product:ubiquiti 12 Adult male urinary bladder 13 cDNA, RIKEN full-length 14 12 enriched library, 36 7 133509.27 Q8CBV3_MOUSE 15 clone:9530003A05 16 product:hypot Aldh1a3 protein - Mus 17 13 31 28 18949.69 Q810V9_MOUSE 18 musculus (Mouse) Aldh1a3 protein - Mus 19 14 For Review28 28 Only18949.69 Q810V9_MOUSE 20 musculus (Mouse) Aldh1a3 protein - Mus 21 15 30 28 18949.69 Q810V9_MOUSE 22 musculus (Mouse) Aldh1a3 protein - Mus 23 16 30 28 18949.69 Q810V9_MOUSE 24 musculus (Mouse) Aldh1a3 protein - Mus 25 17 45 30 18949.69 Q810V9_MOUSE musculus (Mouse) 26 Aldh1a3 protein - Mus 27 18 40 30 18949.69 Q810V9_MOUSE 28 musculus (Mouse) Aldh1a3 protein - Mus 29 19 31 28 18949.69 Q810V9_MOUSE musculus (Mouse) 30 Aldh1a3 protein - Mus 31 20 29 28 18949.69 Q810V9_MOUSE musculus (Mouse) 32 Aldh1a3 protein - Mus 33 21 30 28 18949.69 Q810V9_MOUSE musculus (Mouse) 34 Aldh3b2 protein - Mus 35 22 29 23 20242.26 Q497U8_MOUSE musculus (Mouse) 36 Aldolase 1, A isoform - Mus 23 54 36 39787.45 Q5FWB7_MOUSE 37 musculus (Mouse) 38 Blastocyst blastocyst cDNA, 39 RIKEN full-length enriched 24 27 17 26557.13 Q3TKL5_MOUSE 40 library, clone:I1C0024B03 41 product:UPF0183 pr 42 Bone marrow macrophage 43 cDNA, RIKEN full-length 44 25 enriched library, 37 22 19983.72 Q3U935_MOUSE 45 clone:I830044A19 46 product:cellular 47 Carbonic anhydrase 3 - Mus 26 59 40 29632.75 CAH3_MOUSE 48 musculus (Mouse) 49 Carbonic anhydrase 3 - Mus 27 106 41 29632.75 CAH3_MOUSE P16015 2 50 musculus (Mouse) 51 Carbonic anhydrase 3 - Mus 28 81 45 29632.75 CAH3_MOUSE 52 musculus (Mouse) 53 Cellular nucleic acid-binding 54 29 protein - Mus musculus 76 40 20833.08 CNBP_MOUSE P53996 3 55 (Mouse) 56 30 Cpsf4 protein - Mus musculus 32 30 4626.31 Q8R3L9_MOUSE 57 58 24 59 60 The FEBS Journal Page 74 of 105

1 2 3 (Mouse) 4 Dual specificity protein 5 31 phosphatase 3 - Mus 87 47 20687.29 DUS3_MOUSE Q9D7X3 4 6 musculus (Mouse) 7 ES cells cDNA, RIKEN full- 8 length enriched library, 9 32 clone:2410014J02 50 31 19057.39 Q545G1_MOUSE 10 product:myosin light chain, 11 pho

12 ES cells cDNA, RIKEN full- 13 length enriched library, 14 33 clone:2410014J02 39 21 19057.39 Q545G1_MOUSE 15 product:myosin light chain, 16 pho 17 FERM domain containing 4A - 34 35 28 21080.99 A3KGJ1_MOUSE 18 Mus musculus (Mouse) 19 Galectin-1 - Mus musculus 35 For Review80 52 Only15198.33 LEG1_MOUSE (Mouse) 20 P16045 5 Galectin-1 - Mus musculus 21 36 72 45 15198.33 LEG1_MOUSE 22 (Mouse) 23 Glyceraldehyde-3-phosphate 24 37 dehydrogenase - Mus 99 48 36072.32 G3P_MOUSE 25 musculus (Mouse) 26 Glyceraldehyde-3-phosphate 27 38 dehydrogenase - Mus 62 18 36072.32 G3P_MOUSE 28 musculus (Mouse) 29 Glyceraldehyde-3-phosphate 30 39 dehydrogenase - Mus 58 17 36072.32 G3P_MOUSE 31 musculus (Mouse) 32 Glyceraldehyde-3-phosphate dehydrogenase - Mus 92 41 36072.32 G3P_MOUSE 33 40 musculus (Mouse) 34 Glyceraldehyde-3-phosphate 35 41 dehydrogenase - Mus 85 44 36072.32 G3P_MOUSE 36 musculus (Mouse) 37 Glyceraldehyde-3-phosphate 38 42 dehydrogenase - Mus 105 54 36072.32 G3P_MOUSE P16858 6 39 musculus (Mouse) 40 Glyceraldehyde-3-phosphate 41 43 dehydrogenase - Mus 72 41 36072.32 G3P_MOUSE 42 musculus (Mouse) 43 Glyceraldehyde-3-phosphate 44 44 dehydrogenase - Mus 96 37 36072.32 G3P_MOUSE 45 musculus (Mouse) 46 Glyceraldehyde-3-phosphate 47 45 dehydrogenase - Mus 95 42 36072.32 G3P_MOUSE 48 musculus (Mouse) 49 Glyceraldehyde-3-phosphate 50 46 dehydrogenase - Mus 91 38 36072.32 G3P_MOUSE 51 musculus (Mouse) 52 Glyceraldehyde-3-phosphate 53 47 dehydrogenase - Mus 82 35 36072.32 G3P_MOUSE 54 musculus (Mouse) 55 Glycerol-3-phosphate 48 140 44 38175.66 GPDA_MOUSE P13707 7 56 dehydrogenase [NAD+], 57 58 25 59 60 Page 75 of 105 The FEBS Journal

1 2 3 cytoplasmic - Mus musculus 4 (Mouse) 5 Hemoglobin subunit beta-1 - 49 72 40 15944.19 HBB1_MOUSE P02088 8 6 Mus musculus (Mouse) 7 - Mus musculus 50 48 18 51973.48 Q3SYP5_MOUSE 8 (Mouse) 9 Mercaptopyruvate 10 51 sulfurtransferase - Mus 29 11 33246.61 Q505N7_MOUSE 11 musculus (Mouse) 12 Myomesin-1 - Mus musculus 52 160 18 186470.45 MYOM1_MOUSE 13 (Mouse) Q62234 9 14 Myomesin-1 - Mus musculus 53 159 22 186470.45 MYOM1_MOUSE 15 (Mouse) 16 Myosin light chain 1, skeletal 17 54 muscle isoform - Mus 119 75 20695.45 MLE1_MOUSE P05977 10 18 musculus (Mouse) 19 Myosin regulatory lightFor chain Review Only 20 55 2, skeletal muscle isoform - 115 55 19057.39 MLRS_MOUSE Mus musculus (Mouse) 21 P97457 11 22 Myosin regulatory light chain 23 56 2, skeletal muscle isoform - 58 40 19057.39 MLRS_MOUSE 24 Mus musculus (Mouse) 25 Myosin, heavy polypeptide 1, 26 57 skeletal muscle, adult - Mus 31 5 224115.73 Q32P18_MOUSE 27 musculus (Mouse) Myosin-binding protein C, fast- 28 58 97 28 128127.87 MYPC2_MOUSE Q5XKE0 12 29 type - Mus musculus (Mouse) Peroxiredoxin-1 - Mus 30 59 86 41 22390.44 PRDX1_MOUSE musculus (Mouse) 31 P35700 13 Peroxiredoxin-1 - Mus 32 60 158 63 22390.44 PRDX1_MOUSE musculus (Mouse) 33 Polymerase kappa isoform 1 - 34 61 34 22 60850.15 Q5Q9H8_MOUSE Mus musculus (Mouse) 35 Protein DJ-1 - Mus musculus 36 62 96 56 20236.47 PARK7_MOUSE Q99LX0 14 (Mouse) 37 Protein LRP16 - Mus 38 63 72 26 35842.59 LRP16_MOUSE Q922B1 15 musculus (Mouse) 39 Triosephosphate isomerase - 64 128 53 27037.89 TPIS_MOUSE 40 Mus musculus (Mouse) 41 Triosephosphate isomerase - 65 108 53 27037.89 TPIS_MOUSE 42 Mus musculus (Mouse) P17751 16 43 Triosephosphate isomerase - 66 118 61 27037.89 TPIS_MOUSE 44 Mus musculus (Mouse) 45 Triosephosphate isomerase - 67 104 46 27037.89 TPIS_MOUSE 46 Mus musculus (Mouse) 47 C, skeletal muscle - 68 109 50 18155.38 TNNC2_MOUSE 48 Mus musculus (Mouse) P20801 17 49 , skeletal muscle - 69 81 40 18155.38 TNNC2_MOUSE 50 Mus musculus (Mouse) 51 Ttn protein - Mus musculus 70 78 24 39353.95 Q8R112_MOUSE Q8R112 18 52 (Mouse) 53 Zinc finger protein 248 - Mus 71 38 17 68338.20 Q640N4_MOUSE 54 musculus (Mouse) 55 56 57 58 26 59 60 The FEBS Journal Page 76 of 105

1 2 3 4 5 Table 1.7: mass spec analysis of a 2D gel of an acid-eluted fraction from a lysate derived 6 from mouse kidneys. 7 8 Spot % Protein Uniprot # of Protein species Score Accession 9 # Coverage MW ID species 14-3-3 protein epsilon - Mus 10 1 77 34 29326.48 1433E_MOUSE P62259 1 11 musculus (Mouse) 2210023G05Rik protein - 12 2 28 73 3312.91 Q8K197_MOUSE 13 Mus musculus (Mouse) 40S ribosomal protein S18 - 14 3 63 40 17707.86 RS18_MOUSE P62270 2 15 Mus musculus (Mouse) 16 6 days neonate head cDNA, RIKEN full-length enriched 17 4 48 14 85514.92 Q8C0L4_MOUSE 18 library, clone:5430417M15 19 product:hypotheticalFor Review Only 20 Activated spleen cDNA, RIKEN full-length enriched 21 5 35 24 25090.58 Q3U046_MOUSE library, clone:F830207J03 22 product:yes-associated 23 Aldehyde dehydrogenase, 24 6 mitochondrial precursor - Mus 145 39 57014.94 ALDH2_MOUSE 3 25 musculus (Mouse) P47738 26 Aldehyde dehydrogenase, 27 7 mitochondrial precursor - Mus 87 20 57014.94 ALDH2_MOUSE 28 musculus (Mouse) 29 Aldh1a3 protein - Mus 8 33 32 18949.69 Q810V9_MOUSE 30 musculus (Mouse)

31 Aldh1a3 protein - Mus 9 34 32 18949.69 Q810V9_MOUSE 32 musculus (Mouse) 33 Alpha--4 - Mus 10 65 12 105367.59 ACTN4_MOUSE P57780 4 34 musculus (Mouse) 35 Argininosuccinate synthase - 11 91 34 46840.12 ASSY_MOUSE 36 Mus musculus (Mouse) P16460 5 37 Argininosuccinate synthase - 12 69 15 46840.12 ASSY_MOUSE 38 Mus musculus (Mouse) 39 ARL-6 interacting protein-2 - 13 28 9 16442.39 Q9WUG4_MOUSE 40 Mus musculus (Mouse) 41 Aspartoacylase-2 - Mus 14 74 38 35719.76 ACY3_MOUSE Q91XE4 6 42 musculus (Mouse) 43 ATP synthase subunit beta, 44 15 mitochondrial precursor - Mus 77 25 56265.46 ATPB_MOUSE 45 musculus (Mouse) P56480 7 46 ATP synthase subunit beta, 47 16 mitochondrial precursor - Mus 166 45 56265.46 ATPB_MOUSE 48 musculus (Mouse) 49 ATP synthase, H+ transporting, mitochondrial F0 50 17 56 41 18794.61 A2A8X4_MOUSE 51 complex, subunit d - Mus 52 musculus (Mouse) Cytidine deaminase - Mus 53 18 85 73 16520.10 CDD_MOUSE P56389 8 54 musculus (Mouse) Dihydropyrimidinase-related 55 19 130 31 62637.74 DPYL2_MOUSE O08553 9 56 protein 2 - Mus musculus 57 58 27 59 60 Page 77 of 105 The FEBS Journal

1 2 3 (Mouse) 4 Dihydropyrimidinase-related 5 20 protein 2 - Mus musculus 164 54 62637.74 DPYL2_MOUSE 6 (Mouse) 7 Eukaryotic translation 8 21 initiation factor 3 subunit 2 - 83 28 36836.74 IF32_MOUSE Q9QZD9 10 9 Mus musculus (Mouse) 10 F-actin capping protein 11 22 subunit alpha-1 - Mus 92 51 33090.37 CAZA1_MOUSE P47753 11 12 musculus (Mouse) 13 Histone cluster 1, H2bh - Mus 23 43 51 13911.57 A2RTP6_MOUSE 14 musculus (Mouse)

15 Histone cluster 1, H2bh - Mus 24 34 35 13911.57 A2RTP6_MOUSE 16 musculus (Mouse) 17 Hypothetical protein - Mus 25 34 25 12290.56 Q8R177_MOUSE 18 musculus (Mouse) 19 M-calpain large subunit - Mus 26 For Review30 45 Only4173.06 Q920R9_MOUSE 20 musculus (Mouse) 21 M-calpain large subunit - Mus 27 34 45 4173.06 Q920R9_MOUSE 22 musculus (Mouse) 23 M-calpain large subunit - Mus 28 29 45 4173.06 Q920R9_MOUSE 24 musculus (Mouse) M-calpain large subunit - Mus 25 29 35 45 4173.06 Q920R9_MOUSE 26 musculus (Mouse) M-calpain large subunit - Mus 27 30 35 45 4173.06 Q920R9_MOUSE 28 musculus (Mouse) M-calpain large subunit - Mus 29 31 38 45 4173.06 Q920R9_MOUSE 30 musculus (Mouse) M-calpain large subunit - Mus 31 32 34 45 4173.06 Q920R9_MOUSE 32 musculus (Mouse) Meprin 1 alpha - Mus 33 33 40 11 86693.65 Q91WH9_MOUSE 34 musculus (Mouse) 35 Methylcrotonoyl-CoA carboxylase beta chain, 36 34 176 46 61910.42 MCCC2_MOUSE 37 mitochondrial precursor - Mus musculus (Mouse) 38 Methylcrotonoyl-CoA 39 carboxylase beta chain, 40 35 116 38 61910.42 MCCC2_MOUSE Q3ULD5 12 mitochondrial precursor - Mus 41 musculus (Mouse) 42 Methylcrotonoyl-CoA 43 carboxylase beta chain, 36 151 48 61910.42 MCCC2_MOUSE 44 mitochondrial precursor - Mus 45 musculus (Mouse) 46 Myosin-9 - Mus musculus 37 74 10 227414.02 MYH9_MOUSE Q8VDD5 13 47 (Mouse) 48 NADH dehydrogenase 49 [ubiquinone] iron-sulfur 50 38 protein 2, mitochondrial 82 18 52990.80 NDUS2_MOUSE Q91WD5 14 51 precursor - Mus musculus 52 (Mous 53 NADH-ubiquinone 54 oxidoreductase 75 kDa 39 101 17 80723.93 NDUS1_MOUSE Q91VD9 15 55 subunit, mitochondrial 56 precursor - Mus musculus 57 58 28 59 60 The FEBS Journal Page 78 of 105

1 2 3 (Mouse) 4 Oxysterol binding protein 2 - 40 41 92 3507.68 Q5F210_MOUSE 5 Mus musculus (Mouse) 6 Peroxiredoxin-2 - Mus 41 67 31 21936.12 PRDX2_MOUSE Q61171 16 7 musculus (Mouse) 8 Peroxiredoxin-5, 9 42 mitochondrial precursor - Mus 62 42 22225.67 PRDX5_MOUSE 10 musculus (Mouse) 11 Peroxiredoxin-5, 12 43 mitochondrial precursor - Mus 108 50 22225.67 PRDX5_MOUSE 13 musculus (Mouse) 14 Peroxiredoxin-5, 15 44 mitochondrial precursor - Mus 115 55 22225.67 PRDX5_MOUSE 16 musculus (Mouse) 17 Peroxiredoxin-5, 18 45 mitochondrial precursor - Mus 108 45 22225.67 PRDX5_MOUSE 19 musculus (Mouse) For Review Only P99029 17 20 Peroxiredoxin-5, 21 46 mitochondrial precursor - Mus 164 45 22225.67 PRDX5_MOUSE 22 musculus (Mouse) 23 Peroxiredoxin-5, 24 47 mitochondrial precursor - Mus 160 56 22225.67 PRDX5_MOUSE 25 musculus (Mouse) 26 Peroxiredoxin-5, 27 48 mitochondrial precursor - Mus 157 67 22225.67 PRDX5_MOUSE 28 musculus (Mouse) 29 Peroxiredoxin-5, 30 49 mitochondrial precursor - Mus 146 55 22225.67 PRDX5_MOUSE 31 musculus (Mouse) Ppnx protein - Mus musculus 32 50 28 11 33504.96 Q8K3S0_MOUSE 33 (Mouse) RAS-like family 11 member A 34 51 34 7 27221.18 Q6IMB1_MOUSE - Mus musculus (Mouse) 35 RAS-like family 11 member A 36 52 31 7 27221.18 Q6IMB1_MOUSE - Mus musculus (Mouse) 37 RAS-like family 11 member A 38 53 29 7 27221.18 Q6IMB1_MOUSE - Mus musculus (Mouse) 39 RAS-like family 11 member A 40 54 27 7 27221.18 Q6IMB1_MOUSE - Mus musculus (Mouse) 41 RAS-like family 11 member A 55 34 7 27221.18 Q6IMB1_MOUSE 42 - Mus musculus (Mouse) 43 RAS-like family 11 member A 56 31 7 27221.18 Q6IMB1_MOUSE 44 - Mus musculus (Mouse) 45 Septin-2 - Mus musculus 57 58 26 41727.36 SEPT2_MOUSE P42208 18 46 (Mouse) 47 Spectrin alpha chain, brain - 58 248 16 285220.58 SPTA2_MOUSE P16546 19 48 Mus musculus (Mouse) 49 Succinate dehydrogenase 50 [ubiquinone] flavoprotein 59 243 58 73622.58 DHSA_MOUSE 51 subunit, mitochondrial 52 precursor - Mus musculus ( Q8K2B3 20 53 Succinate dehydrogenase 54 [ubiquinone] flavoprotein 60 225 52 73622.58 DHSA_MOUSE 55 subunit, mitochondrial 56 precursor - Mus musculus ( 57 58 29 59 60 Page 79 of 105 The FEBS Journal

1 2 3 TRAV9D-3 - Mus musculus 61 39 24 12795.42 Q5R1G4_MOUSE 4 (Mouse) 5 Vacuolar ATP synthase 6 62 catalytic subunit A - Mus 205 53 68624.68 VATA_MOUSE P50516 21 7 musculus (Mouse) 8 Vacuolar ATP synthase 9 63 subunit B, brain isoform - Mus 245 61 56856.98 VATB2_MOUSE P62814 22 10 musculus (Mouse) 11 12 13 14 15 Table 1.8: mass spec analysis of a 2D gel of a DTT-eluted fraction from a lysate derived 16 from mouse kidneys. The analysis is for the proteins remaining on the affinity column after 17 acidic elution. 18 19 For Review Only 20 Spot % Protein Uniprot # of Protein species Score Accession 21 # Coverage MW ID species 22 0 day neonate cerebellum cDNA, 23 RIKEN full-length enriched 1 31 27 12748.55 Q3TQ50_MOUSE 24 library, clone:C230058P07 25 product:cell di 26 10 days neonate cortex cDNA, 27 RIKEN full-length enriched 2 35 11 88533.14 Q8BLR8_MOUSE 28 library, clone:A830014I19 29 product:weakly si 30 10-formyltetrahydrofolate 31 3 dehydrogenase - Mus musculus 72 9 99501.84 FTHFD_MOUSE Q8R0Y6 1 32 (Mouse) 33 11 days embryo whole body 34 cDNA, RIKEN full-length 35 4 enriched library, 49 25 38926.90 Q9CZI7_MOUSE 36 clone:2700084K13 37 product:annexi 38 13 days embryo heart cDNA, RIKEN full-length enriched 39 5 38 9 86851.68 Q3TPA4_MOUSE 40 library, clone:D330037K14 41 product:cadherin 16 42 13 days embryo male testis 43 cDNA, RIKEN full-length 44 6 enriched library, 37 14 32360.93 Q8BJ62_MOUSE clone:6030400N17 45 product:CGMP- 46 2810051F02Rik protein - Mus 47 7 39 33 12483.39 Q91VT2_MOUSE musculus (Mouse) 48 3-hydroxyanthranilate 3,4- 49 8 dioxygenase - Mus musculus 158 57 32954.63 3HAO_MOUSE 50 (Mouse) 51 3-hydroxyanthranilate 3,4- 52 9 dioxygenase - Mus musculus 166 54 32954.63 3HAO_MOUSE Q78JT3 2 53 (Mouse) 54 3-hydroxyanthranilate 3,4- 55 10 dioxygenase - Mus musculus 130 50 32954.63 3HAO_MOUSE 56 (Mouse) 57 58 30 59 60 The FEBS Journal Page 80 of 105

1 2 3 40S ribosomal protein S12 - Mus 11 66 34 14857.68 RS12_MOUSE P63323 3 4 musculus (Mouse) 5 60 kDa heat shock protein, 6 12 mitochondrial precursor - Mus 100 42 61088.45 CH60_MOUSE P63038 4 7 musculus (Mouse) 8 Abca7 protein - Mus musculus 13 28 14 34187.76 Q8R1I3_MOUSE 9 (Mouse) 10 Acetyl-coenzyme A synthetase 11 14 2-like, mitochondrial precursor - 89 13 75317.07 ACS2L_MOUSE Q99NB1 5 12 Mus musculus (Mouse) 13 Acid phosphatase 1, soluble - 15 35 24 18636.05 Q4VAI2_MOUSE 14 Mus musculus (Mouse) 15 Aconitate hydratase, 16 16 mitochondrial precursor - Mus 121 18 86151.30 ACON_MOUSE Q99KI0 6 17 musculus (Mouse) 18 Activated spleen cDNA, RIKEN full-length enriched library, 19 17 For Review36 20 Only25090.58 Q3U046_MOUSE 20 clone:F830207J03 product:yes- 21 associated Adenylate kinase isoenzyme 1 - 22 18 69 39 21640.16 KAD1_MOUSE Q9R0Y5 7 23 Mus musculus (Mouse) 24 Adult male cerebellum cDNA, RIKEN full-length enriched 25 19 107 63 28412.37 Q9DB29_MOUSE Q9DB29 8 26 library, clone:1500019E20 27 product:hypothetic 28 Adult male corpora quadrigemina cDNA, RIKEN full-length 29 20 32 12 24908.50 Q8BR60_MOUSE enriched library, 30 clone:B230213G02 product: 31 Adult male corpora quadrigemina 32 cDNA, RIKEN full-length 33 21 33 30 14356.03 Q3USS7_MOUSE enriched library, 34 clone:B230314C10 product:

35 Adult male corpora quadrigemina 36 cDNA, RIKEN full-length 22 40 39 14356.03 Q3USS7_MOUSE 37 enriched library, 38 clone:B230314C10 product: 39 Adult male kidney cDNA, RIKEN 40 full-length enriched library, 23 29 27 15835.84 Q3UNU4_MOUSE 41 clone:F530014F22 42 product:hypothetical G 43 Adult male liver tumor cDNA, 44 RIKEN full-length enriched 24 45 12 56971.78 Q3UEM8_MOUSE 45 library, clone:C730013P19 46 product:heat shoc 47 Adult male testis cDNA, RIKEN 48 full-length enriched library, 25 35 22 42230.41 Q3TTP7_MOUSE 49 clone:4922503G09 50 product:down-regulated Aldh1a3 protein - Mus musculus 51 26 34 28 18949.69 Q810V9_MOUSE 52 (Mouse) Aldh1a3 protein - Mus musculus 53 27 31 28 18949.69 Q810V9_MOUSE 54 (Mouse) Aldh1a3 protein - Mus musculus 55 28 30 28 18949.69 Q810V9_MOUSE 56 (Mouse) 57 58 31 59 60 Page 81 of 105 The FEBS Journal

1 2 3 Aldh1a3 protein - Mus musculus 29 41 30 18949.69 Q810V9_MOUSE 4 (Mouse) 5 Aldh1a3 protein - Mus musculus 30 30 28 18949.69 Q810V9_MOUSE 6 (Mouse) 7 Aldh1a3 protein - Mus musculus 31 29 28 18949.69 Q810V9_MOUSE 8 (Mouse) 9 Aldh1a3 protein - Mus musculus 32 28 28 18949.69 Q810V9_MOUSE 10 (Mouse) 11 Alpha-enolase - Mus musculus 33 107 38 47453.34 ENOA_MOUSE 12 (Mouse) P17182 9 13 Alpha-enolase - Mus musculus 34 70 22 47453.34 ENOA_MOUSE 14 (Mouse) 15 Amine N-sulfotransferase - Mus 35 113 53 35247.52 O35401_MOUSE Q3UZZ6 10 16 musculus (Mouse) 17 Annexin A2 - Mus musculus 36 97 36 38936.95 ANXA2_MOUSE 18 (Mouse) P07356 11 19 Annexin A2 - Mus musculus 37 For Review80 34 Only38936.95 ANXA2_MOUSE 20 (Mouse) Aspartoacylase - Mus musculus 21 38 115 43 35777.96 ACY2_MOUSE Q8R3P0 12 22 (Mouse) BC066135 protein - Mus 23 39 26 23 18540.61 Q6NZH2_MOUSE 24 musculus (Mouse) 25 Calcium/calmodulin-dependent 26 40 serine protein kinase - Mus 31 19 12768.50 A2ADQ1_MOUSE 27 musculus (Mouse) Cellular nucleic acid-binding 28 41 66 40 20833.08 CNBP_MOUSE P53996 13 29 protein - Mus musculus (Mouse) Clathrin light polypeptide - Mus 30 42 35 17 23521.36 A2AJ56_MOUSE 31 musculus (Mouse) Cofilin-1 - Mus musculus 32 43 92 34 18775.76 COF1_MOUSE (Mouse) 33 P18760 14 Cofilin-1 - Mus musculus 34 44 128 62 18775.76 COF1_MOUSE 35 (Mouse) 36 Copper chaperone for 45 superoxide dismutase - Mus 60 36 29463.62 CCS_MOUSE Q9WU84 15 37 musculus (Mouse) 38 Cytidine deaminase - Mus 39 46 82 74 16520.10 CDD_MOUSE P56389 16 musculus (Mouse) 40 Dcpp1 protein - Mus musculus 41 47 36 28 18591.23 Q64097_MOUSE (Mouse) 42 Dcpp1 protein - Mus musculus 48 28 28 18591.23 Q64097_MOUSE 43 (Mouse) 44 Dcpp1 protein - Mus musculus 49 32 28 18591.23 Q64097_MOUSE 45 (Mouse) 46 50 Destrin - Mus musculus (Mouse) 58 29 18851.61 DEST_MOUSE 47 Q9R0P5 17 48 51 Destrin - Mus musculus (Mouse) 63 29 18851.61 DEST_MOUSE 49 Dihydropyrimidinase-related 50 52 protein 2 - Mus musculus 180 37 62637.74 DPYL2_MOUSE O08553 18 51 (Mouse) - Mus musculus 52 53 34 30 24726.51 Q9R0A2_MOUSE (Mouse) 53 Elongation factor Tu, 54 54 mitochondrial precursor - Mus 139 30 49876.12 EFTU_MOUSE Q8BFR5 19 55 musculus (Mouse) 56 57 58 32 59 60 The FEBS Journal Page 82 of 105

1 2 3 Elongation factor Tu, 4 55 mitochondrial precursor - Mus 73 18 49876.12 EFTU_MOUSE 5 musculus (Mouse) 6 ERO1-like protein alpha 7 56 precursor - Mus musculus 102 30 54802.47 ERO1A_MOUSE 8 (Mouse) Q8R180 20 9 ERO1-like protein alpha 10 57 precursor - Mus musculus 105 28 54802.47 ERO1A_MOUSE 11 (Mouse) 12 ES cells cDNA, RIKEN full-length 13 enriched library, 14 58 clone:2410004D02 28 14 43034.92 Q9CWU3_MOUSE 15 product:similar to 16 SERINE/THREO 17 Ezrin-radixin-moesin-binding 18 59 phosphoprotein 50 - Mus 72 25 38861.65 NHERF_MOUSE 19 musculus (Mouse)For Review Only 20 Ezrin-radixin-moesin-binding 21 60 phosphoprotein 50 - Mus 69 27 38861.65 NHERF_MOUSE 22 musculus (Mouse) 23 Ezrin-radixin-moesin-binding 24 61 phosphoprotein 50 - Mus 65 28 38861.65 NHERF_MOUSE P70441 21 25 musculus (Mouse) 26 Ezrin-radixin-moesin-binding 27 62 phosphoprotein 50 - Mus 109 38 38861.65 NHERF_MOUSE musculus (Mouse) 28 Ezrin-radixin-moesin-binding 29 63 phosphoprotein 50 - Mus 90 34 38861.65 NHERF_MOUSE 30 musculus (Mouse) 31 Heat shock protein 84b - Mus 32 64 46 5 83571.24 Q71LX8_MOUSE musculus (Mouse) 33 Hemoglobin subunit beta-1 - Mus 65 120 73 15944.19 HBB1_MOUSE P02088 22 34 musculus (Mouse) 35 Hemoglobin subunit beta-2 - Mus 66 121 65 15982.30 HBB2_MOUSE P02089 23 36 musculus (Mouse) 37 Heterogeneous nuclear 38 67 ribonucleoprotein A3 - Mus 102 25 39855.69 ROA3_MOUSE 39 musculus (Mouse) Q8BG05 24 40 Heterogeneous nuclear 41 68 ribonucleoprotein A3 - Mus 107 30 39855.69 ROA3_MOUSE 42 musculus (Mouse) 43 Heterogeneous nuclear 44 69 ribonucleoprotein K - Mus 78 26 51229.51 HNRPK_MOUSE P61979 25 45 musculus (Mouse) 46 Inorganic pyrophosphatase - 70 138 70 33102.33 IPYR_MOUSE Q9D819 26 47 Mus musculus (Mouse) 48 Inorganic pyrophosphatase 2, 49 71 mitochondrial precursor - Mus 134 68 38546.32 IPYR2_MOUSE 50 musculus (Mouse) 51 Inorganic pyrophosphatase 2, 52 72 mitochondrial precursor - Mus 99 40 38546.32 IPYR2_MOUSE Q91VM9 27 53 musculus (Mouse) 54 Inorganic pyrophosphatase 2, 55 73 mitochondrial precursor - Mus 233 75 38546.32 IPYR2_MOUSE 56 musculus (Mouse) 57 58 33 59 60 Page 83 of 105 The FEBS Journal

1 2 3 Interleukin 15 receptor alpha 4 74 chain isoform 2D - Mus 42 42 11211.70 Q810T6_MOUSE 5 musculus (Mouse) 6 Leukocyte elastase inhibitor A - 75 95 27 42718.83 ILEUA_MOUSE 7 Mus musculus (Mouse) Q9D154 28 8 Leukocyte elastase inhibitor A - 76 147 32 42718.83 ILEUA_MOUSE 9 Mus musculus (Mouse) 10 Low-voltage-activated calcium 11 77 channel alpha13.2 subunit - Mus 42 41 12283.98 Q99P43_MOUSE 12 musculus (Mouse) 13 Lung RCB-0558 LLC cDNA, 14 RIKEN full-length enriched 78 41 16 68198.96 Q3UML8_MOUSE 15 library, clone:G730038B15 16 product:similar to Msz Meprin 1 alpha - Mus musculus 17 79 50 15 86693.65 Q91WH9_MOUSE 18 (Mouse) Meprin A subunit alpha precursor 19 80 For Review69 12 Only85226.89 MEP1A_MOUSE P28825 29 20 - Mus musculus (Mouse) Mitochondrial inner membrane 21 81 80 13 84247.35 IMMT_MOUSE protein - Mus musculus (Mouse) 22 Q8CAQ8 30 Mitochondrial inner membrane 23 82 157 29 84247.35 IMMT_MOUSE 24 protein - Mus musculus (Mouse) 25 83 NIMA - Mus musculus (Mouse) 32 100 4423.07 A2ATP0_MOUSE 26 NOD-derived CD11c +ve dendritic cells cDNA, RIKEN full- 27 84 36 22 33614.01 Q3TDV6_MOUSE 28 length enriched library, 29 clone:F630006F12 pr 30 Nucleoside diphosphate-linked 31 85 moiety X motif 19 - Mus 64 16 40798.59 NUD19_MOUSE 32 musculus (Mouse) 33 Nucleoside diphosphate-linked 34 86 moiety X motif 19 - Mus 94 50 40798.59 NUD19_MOUSE 35 musculus (Mouse) 36 Nucleoside diphosphate-linked 37 87 moiety X motif 19 - Mus 113 47 40798.59 NUD19_MOUSE P11930 31 musculus (Mouse) 38 Nucleoside diphosphate-linked 39 88 moiety X motif 19 - Mus 121 50 40798.59 NUD19_MOUSE 40 musculus (Mouse) 41 Nucleoside diphosphate-linked 42 89 moiety X motif 19 - Mus 72 22 40798.59 NUD19_MOUSE 43 musculus (Mouse) 44 Osteoclast-like cell cDNA, 45 RIKEN full-length enriched 90 36 13 82114.18 Q3TWK8_MOUSE 46 library, clone:I420027B09 47 product:fibulin 1, 48 Osteoclast-like cell cDNA, 49 RIKEN full-length enriched 91 212 32 57318.99 Q3TW96_MOUSE Q3TW96 32 50 library, clone:I420031B14 51 product:Weakly simi 52 Patched homolog 1 splice variant 92 20 62 2565.30 Q5TLE8_MOUSE 53 1B - Mus musculus (Mouse) 54 PDZ domain-containing protein 1 93 65 19 56862.78 PDZD1_MOUSE 55 - Mus musculus (Mouse) Q9JIL4 33 56 94 PDZ domain-containing protein 1 72 34 56862.78 PDZD1_MOUSE 57 58 34 59 60 The FEBS Journal Page 84 of 105

1 2 3 - Mus musculus (Mouse) 4 Peptide methionine sulfoxide 5 95 reductase - Mus musculus 88 59 26199.91 MSRA_MOUSE 6 (Mouse) 7 Peptide methionine sulfoxide 8 96 reductase - Mus musculus 118 62 26199.91 MSRA_MOUSE 9 (Mouse) 10 Peptide methionine sulfoxide 11 97 reductase - Mus musculus 88 68 26199.91 MSRA_MOUSE Q9D6Y7 34 12 (Mouse) 13 Peptide methionine sulfoxide 14 98 reductase - Mus musculus 97 62 26199.91 MSRA_MOUSE 15 (Mouse) 16 Peptide methionine sulfoxide 17 99 reductase - Mus musculus 114 72 26199.91 MSRA_MOUSE 18 (Mouse) 19 Peripheral myelin protein 2 - Mus 100 For Review30 22 Only15039.95 Q4KL10_MOUSE 20 musculus (Mouse) 21 Peroxiredoxin 1 - Mus musculus 101 42 18 19085.78 A2AP14_MOUSE (Mouse) 22 Peroxiredoxin 1 - Mus musculus 23 102 28 12 19085.78 A2AP14_MOUSE 24 (Mouse) Peroxiredoxin-1 - Mus musculus 25 103 98 53 22390.44 PRDX1_MOUSE 26 (Mouse) Peroxiredoxin-1 - Mus musculus 27 104 161 67 22390.44 PRDX1_MOUSE 28 (Mouse) Peroxiredoxin-1 - Mus musculus 29 105 91 49 22390.44 PRDX1_MOUSE 30 (Mouse) Peroxiredoxin-1 - Mus musculus 31 106 78 35 22390.44 PRDX1_MOUSE 32 (Mouse) Peroxiredoxin-1 - Mus musculus 33 107 141 59 22390.44 PRDX1_MOUSE 34 (Mouse) Peroxiredoxin-1 - Mus musculus 35 108 96 40 22390.44 PRDX1_MOUSE 36 (Mouse) Peroxiredoxin-1 - Mus musculus 37 109 115 48 22390.44 PRDX1_MOUSE (Mouse) 38 Peroxiredoxin-1 - Mus musculus 39 110 92 41 22390.44 PRDX1_MOUSE (Mouse) 40 P35700 35 Peroxiredoxin-1 - Mus musculus 41 111 157 67 22390.44 PRDX1_MOUSE (Mouse) 42 Peroxiredoxin-1 - Mus musculus 43 112 131 63 22390.44 PRDX1_MOUSE (Mouse) 44 Peroxiredoxin-1 - Mus musculus 113 154 67 22390.44 PRDX1_MOUSE 45 (Mouse) 46 Peroxiredoxin-1 - Mus musculus 114 143 67 22390.44 PRDX1_MOUSE 47 (Mouse) 48 Peroxiredoxin-1 - Mus musculus 115 170 67 22390.44 PRDX1_MOUSE 49 (Mouse) 50 Peroxiredoxin-1 - Mus musculus 116 162 63 22390.44 PRDX1_MOUSE 51 (Mouse) 52 Peroxiredoxin-1 - Mus musculus 117 154 63 22390.44 PRDX1_MOUSE 53 (Mouse) 54 Peroxiredoxin-1 - Mus musculus 118 159 67 22390.44 PRDX1_MOUSE 55 (Mouse) 56 57 58 35 59 60 Page 85 of 105 The FEBS Journal

1 2 3 Peroxiredoxin-1 - Mus musculus 119 177 67 22390.44 PRDX1_MOUSE 4 (Mouse) 5 Peroxiredoxin-1 - Mus musculus 120 175 67 22390.44 PRDX1_MOUSE 6 (Mouse) 7 Peroxiredoxin-1 - Mus musculus 121 180 63 22390.44 PRDX1_MOUSE 8 (Mouse) 9 Peroxiredoxin-2 - Mus musculus 122 58 22 21936.12 PRDX2_MOUSE 10 (Mouse) 11 Peroxiredoxin-2 - Mus musculus 123 68 45 21936.12 PRDX2_MOUSE 12 (Mouse) 13 Peroxiredoxin-2 - Mus musculus 124 99 55 21936.12 PRDX2_MOUSE Q61171 36 14 (Mouse) 15 Peroxiredoxin-2 - Mus musculus 125 73 36 21936.12 PRDX2_MOUSE 16 (Mouse) 17 Peroxiredoxin-2 - Mus musculus 126 67 24 21936.12 PRDX2_MOUSE 18 (Mouse) 19 Peroxiredoxin-4 - Mus musculus 127 For Review129 63 Only31261.23 PRDX4_MOUSE O08807 37 20 (Mouse) 21 Peroxiredoxin-5, mitochondrial 22 128 precursor - Mus musculus 102 37 22225.67 PRDX5_MOUSE 23 (Mouse) 24 Peroxiredoxin-5, mitochondrial 25 129 precursor - Mus musculus 58 24 22225.67 PRDX5_MOUSE 26 (Mouse) 27 Peroxiredoxin-5, mitochondrial 28 130 precursor - Mus musculus 116 45 22225.67 PRDX5_MOUSE P99029 38 29 (Mouse) 30 Peroxiredoxin-5, mitochondrial 31 131 precursor - Mus musculus 147 46 22225.67 PRDX5_MOUSE 32 (Mouse) 33 Peroxiredoxin-5, mitochondrial 132 precursor - Mus musculus 147 45 22225.67 PRDX5_MOUSE 34 (Mouse) 35 Peroxiredoxin-6 - Mus musculus 36 133 104 66 24969.02 PRDX6_MOUSE (Mouse) 37 Peroxiredoxin-6 - Mus musculus 38 134 100 52 24969.02 PRDX6_MOUSE (Mouse) 39 O08709 39 Peroxiredoxin-6 - Mus musculus 135 95 45 24969.02 PRDX6_MOUSE 40 (Mouse) 41 Peroxiredoxin-6 - Mus musculus 136 69 27 24969.02 PRDX6_MOUSE 42 (Mouse) 43 Peroxisomal bifunctional enzyme 137 77 15 78764.23 ECHP_MOUSE Q9DBM2 40 44 - Mus musculus (Mouse) 45 Prdx2 protein - Mus musculus 138 37 22 21948.16 Q5M9N9_MOUSE 46 (Mouse)

47 Prdx2 protein - Mus musculus 139 43 22 21948.16 Q5M9N9_MOUSE 48 (Mouse) 49 Probable D-lactate 50 dehydrogenase, mitochondrial 140 192 54 52499.42 LDHD_MOUSE 51 precursor - Mus musculus 52 (Mouse) Q7TNG8 41 53 Probable D-lactate 54 dehydrogenase, mitochondrial 141 96 32 52499.42 LDHD_MOUSE 55 precursor - Mus musculus 56 (Mouse) 57 58 36 59 60 The FEBS Journal Page 86 of 105

1 2 3 Probable D-lactate 4 dehydrogenase, mitochondrial 142 187 55 52499.42 LDHD_MOUSE 5 precursor - Mus musculus 6 (Mouse) 7 Probable D-lactate 8 dehydrogenase, mitochondrial 143 116 32 52499.42 LDHD_MOUSE 9 precursor - Mus musculus 10 (Mouse) 11 Probable D-lactate 12 dehydrogenase, mitochondrial 144 79 21 52499.42 LDHD_MOUSE 13 precursor - Mus musculus 14 (Mouse) 15 Probable D-lactate dehydrogenase, mitochondrial 16 145 116 23 52499.42 LDHD_MOUSE 17 precursor - Mus musculus 18 (Mouse) 19 Probable D-lactateFor Review Only dehydrogenase, mitochondrial 20 146 122 26 52499.42 LDHD_MOUSE 21 precursor - Mus musculus 22 (Mouse) RIKEN cDNA D330038O06 gene 23 147 35 10 62350.26 Q6PGD2_MOUSE 24 - Mus musculus (Mouse) Selenium-binding protein 1 - Mus 25 148 115 39 53050.58 SBP1_MOUSE 26 musculus (Mouse) Selenium-binding protein 1 - Mus 27 149 110 23 53050.58 SBP1_MOUSE musculus (Mouse) 28 Selenium-binding protein 1 - Mus 29 150 60 11 53050.58 SBP1_MOUSE musculus (Mouse) 30 Selenium-binding protein 1 - Mus 31 151 259 82 53050.58 SBP1_MOUSE musculus (Mouse) 32 Selenium-binding protein 1 - Mus 33 152 270 81 53050.58 SBP1_MOUSE musculus (Mouse) 34 Selenium-binding protein 1 - Mus 153 314 62 53050.58 SBP1_MOUSE P17563 42 35 musculus (Mouse) 36 Selenium-binding protein 1 - Mus 154 167 32 53050.58 SBP1_MOUSE 37 musculus (Mouse) 38 Selenium-binding protein 1 - Mus 155 95 21 53050.58 SBP1_MOUSE 39 musculus (Mouse) 40 Selenium-binding protein 1 - Mus 156 214 47 53050.58 SBP1_MOUSE 41 musculus (Mouse) 42 Selenium-binding protein 1 - Mus 157 212 48 53050.58 SBP1_MOUSE 43 musculus (Mouse) 44 Selenium-binding protein 1 - Mus 158 173 44 53050.58 SBP1_MOUSE 45 musculus (Mouse) 46 Selenium-binding protein 2 - Mus 159 78 20 53146.59 SBP2_MOUSE Q63836 43 47 musculus (Mouse) 48 Stress-70 protein, mitochondrial 49 160 precursor - Mus musculus 112 27 73767.84 GRP75_MOUSE P38647 44 50 (Mouse) 51 Succinate dehydrogenase 52 161 complex, subunit B, iron sulfur - 56 35 32591.19 A2AA77_MOUSE 53 Mus musculus (Mouse) 54 Synapse-associated protein 1 - 162 76 17 41381.51 SYAP1_MOUSE 55 Mus musculus (Mouse) Q9D5V6 45 56 163 Synapse-associated protein 1 - 80 22 41381.51 SYAP1_MOUSE 57 58 37 59 60 Page 87 of 105 The FEBS Journal

1 2 3 Mus musculus (Mouse) 4 Thioredoxin domain-containing 5 164 protein 4 precursor - Mus 97 35 47222.45 TXND4_MOUSE Q9D1Q6 46 6 musculus (Mouse) 7 Thioredoxin-dependent peroxide 8 reductase, mitochondrial 165 76 44 28337.49 PRDX3_MOUSE 9 precursor - Mus musculus 10 (Mouse) P20108 47 11 Thioredoxin-dependent peroxide 12 reductase, mitochondrial 166 61 36 28337.49 PRDX3_MOUSE 13 precursor - Mus musculus 14 (Mouse) 15 Tubulin alpha-4A chain - Mus 167 138 41 50633.65 TBA4A_MOUSE P68368 48 16 musculus (Mouse) 17 Twinfilin-1 - Mus musculus 168 86 33 40282.56 TWF1_MOUSE 18 (Mouse) Q91YR1 49 19 Twinfilin-1 - Mus musculus 169 For Review92 35 Only40282.56 TWF1_MOUSE 20 (Mouse) 21 Wiscott-Aldrich Syndrome 22 170 protein homolog - Mus musculus 39 10 54568.45 Q61078_MOUSE 23 (Mouse) Ywhaq protein - Mus musculus 24 171 50 28 28045.89 A3KML3_MOUSE 25 (Mouse) 26 27 28 29 Table 1.9: mass spec analysis of a 2D gel of an acid-eluted fraction from a mitochondrial 30 31 lysate from mouse brains. 32 Spot Protein Mascot % Uniprot # of 33 Protein species Accession 34 # MW Score Coverage ID species ATP synthase subunit 35 3 59830 79 29 ATPA_MOUSE 36 alpha, mitochondrial ATP synthase subunit 37 4 59830 92 34 ATPA_MOUSE 38 alpha, mitochondrial ATP synthase subunit 39 5 59830 199 55 ATPA_MOUSE Q03265 1 40 alpha, mitochondrial ATP synthase subunit 41 6 59830 228 58 ATPA_MOUSE alpha, mitochondrial 42 ATP synthase subunit 43 7 59830 41 20 ATPA_MOUSE alpha, mitochondrial 44 ATP synthase subunit beta, 45 8 56265 216 64 ATPB_MOUSE P564804 2 mitochondrial 46 ATP synthase subunit d, 47 9 18795 66 63 ATP5H_MOUSE Q9DCX2 3 mitochondrial 48 Cytochrome b-c1 complex 49 subunit Rieske, 50 10 53446 47 27 UCRI_MOUSE Q9CR68 4 51 mitochondrial 52 53 Glyceraldehyde-3- 20 36072 152 50 G3P_MOUSE 54 phosphate dehydrogenase P16858 5 55 Glyceraldehyde-3- 21 36072 110 56 G3P_MOUSE 56 phosphate dehydrogenase 57 58 38 59 60 The FEBS Journal Page 88 of 105

1 2 3 Isocitrate dehydrogenase 4 26 [NAD] subunit gamma 1, 43157 42 25 IDHG1_MOUSE P70404 6 5 mitochondrial 6 NADH dehydrogenase 7 27 [ubiquinone] iron-sulfur 52991 51 25 NDUS2_MOUSE Q91WD5 7 8 protein 2, mitochondrial 9 NADH dehydrogenase 10 28 [ubiquinone] iron-sulfur 30302 161 60 NDUS3_MOUSE Q9DCT2 8 11 protein 3, mitochondrial 12 Pyruvate dehydrogenase 13 29 E1 component subunit beta, 39254 90 47 ODPB_MOUSE Q9D051 9 14 mitochondrial 15 16 17 18 Table 1.10: mass spec analysis of a 2D gel of a DTT-eluted fraction from a mitochondrial 19 For Review Only 20 lysate from mouse brains. The analysis is for the proteins remaining on the affinity column 21 immediately after acidic elution took place. 22 23 Spot Protein Mascot % Uniprot # of Protein species Accession 24 # MW Score Coverage ID species 25 ATP synthase subunit 10 59830 184 55 ATPA_MOUSE Q03265 1 26 alpha, mitochondrial 27 ATP synthase subunit 11 56265 108 57 ATPB_MOUSE 28 beta, mitochondrial 2 P564804 29 ATP synthase subunit 12 56265 158 67 ATPB_MOUSE 30 beta, mitochondrial ATP synthase subunit d, 31 13 18795 51 54 ATP5H_MOUSE Q9DCX2 3 32 mitochondrial 33 Cytochrome b-c1 34 16 complex subunit 1, 53446 132 37 UCRI_MOUSE Q9CR68 4 35 mitochondrial 36 Glyceraldehyde-3- 37 20 phosphate 36072 76 33 G3P_MOUSE 38 dehydrogenase 39 Glyceraldehyde-3- 40 21 phosphate 36072 132 43 G3P_MOUSE P16858 5 41 dehydrogenase 42 Glyceraldehyde-3- 22 phosphate 36072 109 41 G3P_MOUSE 43 dehydrogenase 44 Isocitrate dehydrogenase 45 25 [NAD] subunit alpha, 40069 50 26 IDH3A_MOUSE Q9D6R2 6 46 mitochondrial 47 26 Peroxiredoxin-1 22390 103 57 PRDX1_MOUSE 48 P35700 7 49 27 Peroxiredoxin-1 22390 185 74 PRDX1_MOUSE 50 28 Peroxiredoxin-2 21936 121 53 PRDX2_MOUSE Q61171 8 51 29 Peroxiredoxin-4 31261 55 30 PRDX4_MOUSE O08807 9 52 30 Peroxiredoxin-6 24969 36 24 PRDX6_MOUSE O08709 10 53 Pyruvate dehydrogenase 54 31 E1 component subunit 39254 150 64 ODPB_MOUSE Q9D051 11 55 beta, mitochondrial 56 57 58 39 59 60 Page 89 of 105 The FEBS Journal

1 2 3 4 5 6 7 Table 1.11: mass spec analysis of a 1D gel of an acid-eluted fraction from a mitochondrial 8 lysate derived from mouse hearts. 9 10 Band Uniprot Mascot % Protein # of Protein species Accession 11 # ID Score Coverage MW species 12 ATP synthase subunit alpha, 13 1 mitochondrial OS=Mus ATPA_MOUSE Q03265 339 37 59716.00 1 14 musculus GN 15 Klra19 protein OS=Mus 16 2 musculus GN=Klra19 PE=2 Q9JHV3_MOUSE Q9JHV3 37 31 31070.00 2 17 SV=1 18 Ldhb protein OS=Mus 19 3 musculus GN=LdhbFor PE=2 ReviewQ5M8R7_MOUSE OnlyQ5M8R7 33 48 11317.00 3 20 SV=1 21 NADH dehydrogenase 22 4 [ubiquinone] 1 alpha NDUA4_MOUSE Q62425 46 24 9321.00 4 23 subcomplex subunit 4 24 NADH-ubiquinone 25 5 oxidoreductase 75 kDa NDUS1_MOUSE Q91VD9 132 18 79698.00 5 26 subunit, mitochondrial Nesprin-2 OS=Mus musculus 27 6 SYNE2_MOUSE Q6ZWQ0 56 4 782238.00 6 28 GN=Syne2 PE=1 SV=2 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 40 59 60 The FEBS Journal Page 90 of 105

1 2 3 4 5 Table 1.12: mass spec analysis of a 1D gel of a DTT-eluted fraction fraction from a 6 mitochondrial lysate derived from mouse hearts. The analysis is for the proteins remaining on 7 the affinity column after acidic elution. 8 9 10 Band Uniprot Mascot % Protein # of Protein species Accession 11 # ID Score Coverage MW species 12 ATP synthase subunit beta, 13 1 mitochondrial OS=Mus ATPB_MOUSE P56480 67 23 56265.00 1 14 musculus GN= 15 Edem3 protein (Fragment) 16 2 OS=Mus musculus Q8R1X5_MOUSE Q8R1X5 33 35 6066.00 2 17 GN=Edem3 PE=2 SV=1 18 OS=Mus 19 3 musculus GN=Krt15For PE=3 ReviewB1AQ77_MOUSE OnlyB1AQ77 35 18 49463.00 3 20 SV=1 21 Putative uncharacterized 22 4 protein (Fragment) OS=Mus Q3UWR0_MOUSE Q3UWR0 39 36 21988.00 4 23 musculus 24 Putative uncharacterized 25 5 protein OS=Mus musculus Q8BIL4_MOUSE Q8BIL4 33 33 19729.00 5 26 GN=Zfp809 P Rac GTPase-activating 27 6 E9Q851_MOUSE E9Q851 34 41 15467.00 6 28 protein 1 29 30 31 32 Table 1.13: mass spec analysis of a 1D gel of a DTT-eluted fraction from a mitochondrial 33 lysate derived from mouse skeletal muscles. The analysis is for the proteins remaining on the 34 35 affinity column after acidic elution. 36 37 38 Band Uniprot Mascot % Protein # of Protein species Accession 39 # ID Score Coverage MW species 40 ATP synthase subunit alpha, 41 1 mitochondrial OS=Mus ATPA_MOUSE Q03265 107 16 59716.00 1 42 musculus GN Keratin 15 OS=Mus musculus 43 2 B1AQ77_MOUSE B1AQ77 56 23 49463.00 2 44 GN=Krt15 PE=3 SV=1 45 Sarcoplasmic/endoplasmic 46 3 reticulum calcium ATPase 1 AT2A1_MOUSE Q8R429 63 6 109355.00 3 47 OS=Mus m 48 4 RNA-binding protein 39 D6RJ85_MOUSE D6RJ85 33 43 6953.00 4 49 50 51 52 53 54 55 56 57 58 41 59 60 Page 91 of 105 The FEBS Journal

1 2 Supplementary tables 2. Interactors of Trx2 as identified by the proteomics and Y2H screens including those currently available in BioGRID. 3 4 Supplementary table 2.1. Cumulative protein interactions of mouse Trx2 as detected by acidic (“Acid”) and DTT elutions (“DTT”) in extracts 5 from different mouse tissues. The table represents the combined data from all experiments with mitochondrial and total cell lysates. All proteins 6 7 highlighted in green are of mitochondrial localization according to MitoCarta2.0. The DTT elutions in skeletal muscle correspond to two different 8 experiments for total cell lysates (DTT 1, 2, analyzed by 2D electrophoresis) and an experiment where mitochondrial lysates were examined (DTT m, 9 analyzed by 1D SDS PAGE). Cardiac and brain mitochondrial lysates (designation “m”) were analyzed by 1D and 2D electrophoresis respectively. 10 11 The raw data for this table are presented in Supplementary tables 1.1-1.13. Total tissue hits (right column) were calculated by the sum of each protein 12 species detected in different tissues by Acid or DTT elutions. In the case of the DTT elutions of the skeletal muscle experiments (three columns), the 13 respective interactome was calculated as the sum of all detected protein species and was considered as a single experiment (one column) in the 14 calculation of total tissue hits. For Review Only 15 16 17 18 19 Cardiac Total # of Lung Skeletal muscle Kidney Brain 20 # of Uniprot muscle tissues hits 21 protein Protein species per elution DTT DTT DTT Acid DTT Acid DTT 22 species ID Acid DTT Acid Acid DTT per protein 23 1 2 m m m m m species 24 ATP synthase subunit beta, 25 P56480 + + + + + + + 7 26 1 mitochondrial 27 2 Q61171 Peroxiredoxin-2 + + + + + + 6

28 ATP synthase subunit alpha, Q03265 + + + + + + + 6 29 3 mitochondrial 30 Dihydropyrimidinase-related 31 O08553 + + + + + 5 4 protein 2 32 Glyceraldehyde-3-phosphate 33 P16858 + + + + + + 5 34 5 dehydrogenase 35 6 P17182 Alpha-enolase + + + + 4

36 7 P35700 Peroxiredoxin-1 + + + + + 4 37 8 O08807 Peroxiredoxin-4 + + + + 4 38 39 9 O08709 Peroxiredoxin-6 + + + + 4

40 10 P63038 60 kDa heat shock protein, + + + + 3 41 42 1 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 The FEBS Journal Page 92 of 105

1 2 mitochondrial 3 11 P60710 Actin, cytoplasmic 1 + + + 3

4 Cellular nucleic acid-binding 5 P53996 + + + + 3 6 12 protein 13 P02088 Hemoglobin subunit beta-1 + + + + 3 7 8 14 Q7TMM9 Tubulin beta-2A chain + + + 3

9 oxidase/ P17563 + + 2 10 15 Se-binding protein 1 11 26S proteasome non-ATPase 12 O35226 + + 2 13 16 regulatory subunit 4 Aconitate hydratase, 14 Q99KI0 For Review Only+ + 2 15 17 mitochondrial 16 18 P62737 Actin, aortic smooth muscle + + 2

17 19 Q9R0Y5 Adenylate kinase isoenzyme 1 + + + 2 18 Aldehyde dehydrogenase, 19 P47738 + + 2 20 20 mitochondrial 21 21 P23927 Alpha-crystallin B chain + + 2

22 22 P07356 Annexin A2 + + 2 23 Q9DCX2 ATP synthase subunit d, + + 2 24 23 mitochondrial 25 24 P21550 Beta-enolase + + 2 26 25 P18760 Cofilin-1 + + 2 27 28 26 P56389 Cytidine deaminase + + 2

29 Cytochrome b-c1 complex Q9CR68 + + 2 30 27 subunit Rieske, mitochondrial 31 Dual specificity protein 32 Q9D7X3 + + + 2 33 28 phosphatase 3 F-actin-capping protein 34 P47753 + + 2 35 29 subunit alpha-1 36 30 P16045 Galectin-1 + + + 2

37 Guanine nucleotide-binding 38 P62874 + + 2 31 protein 39 32 B1AQ77 Keratin 15 + + + 2 40 41 42 2 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 93 of 105 The FEBS Journal

1 2 33 Q9D154 Leukocyte elastase inhibitor A + + 2

3 Methylcrotonoyl-CoA 4 Q3ULD5 carboxylase beta chain, + + 2 5 34 mitochondrial 6 35 Q8VDD5 Myosin-9 + + 2 7 8 NADH dehydrogenase 9 Q91WD5 [ubiquinone] iron-sulfur + + 2 10 36 protein 2, mitochondrial 11 NADH-ubiquinone 12 Q91VD9 oxidoreductase 75 kDa + + 2 13 37 subunit, mitochondrial 14 38 P99029 Peroxiredoxin-5, mitochondrialFor Review Only + + 2 15 16 Pyruvate dehydrogenase E1 17 Q9D051 component subunit beta, + + 2 18 39 mitochondrial 19 40 P07724 Serum albumin + + 2 20 Stress-70 protein, 21 P38647 + + + 2 22 41 mitochondrial 23 Stress-induced- Q60864 + + + 2 24 42 phosphoprotein 1 25 Succinate dehydrogenase 26 Q8K2B3 [ubiquinone] flavoprotein + + 2 27 subunit, mitochondrial 28 43 29 44 Q9D5V6 Synapse-associated protein 1 + + 2 30 31 Thioredoxin-dependent P20108 peroxide reductase, + + 2 32 33 45 mitochondrial 34 46 P17751 Triosephosphate isomerase + + + 2 35 47 P20152 Vimentin + + 2 36 V-type proton ATPase catalytic 37 P50516 + + 2 38 48 subunit A 39 49 P62259 14-3-3 protein epsilon ( + + 2

40 50 Q8K354 Carbonyl reductase [NADPH] 3 + + 2 41 42 3 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 The FEBS Journal Page 94 of 105

1 2 51 P07310 Creatine kinase M-type + + + 2

3 Destrin (Actin-depolymerizing Q9R0P5 + + 2 4 52 factor) 5 Inorganic pyrophosphatase 2, 6 Q91VM9 + + 2 7 53 mitochondrial 8 Isocitrate dehydrogenase P70404 [NAD] subunit gamma 1, + + 2 9 10 54 mitochondrial 11 Q63836 Selenium-binding protein 2 + + 2 12 55 Q9D819 Inorganic pyrophosphatase + 1 13 182 kDa tankyrase-1-binding 14 P58871 For Review+ Only 1 15 56 protein 16 26S proteasome non-ATPase Q9CR00 + 1 17 57 regulatory subunit 9 18 3-hydroxyanthranilate 3,4- Q78JT3 + 1 19 58 dioxygenase 20 59 P63323 40S ribosomal protein S12 + 1 21 22 60 P62270 40S ribosomal protein S18 + 1

23 40S ribosomal protein S4, X P62702 + 1 24 61 isoform 25 62 P14206 40S ribosomal protein SA + 1 26 Acetyl-coenzyme A synthetase 27 Q99NB1 + 1 28 63 2-like, mitochondrial 29 64 P68033 Actin, alpha cardiac muscle 1 + 1

30 65 P68134 Actin, alpha skeletal muscle + 1 31 66 P63260 Actin, cytoplasmic 2 + 1 32 33 67 O54931 A-kinase anchor protein 2 + 1

34 Alpha-actinin-4 (Non-muscle P57780 + 1 35 68 alpha-actinin 4) 36 69 P61164 Alpha-centractin + 1 37 70 P16460 Argininosuccinate synthase + 1 38 39 71 Q8R3P0 Aspartoacylase + 1

40 72 P12382 ATP-dependent 6- + 1 41 42 4 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 95 of 105 The FEBS Journal

1 2 phosphofructokinase 3 ATP-dependent 6- 4 P47857 phosphofructokinase, muscle + 1 5 73 type 6 74 Q8VBT1 Beta-taxilin + 1 7 8 75 Q8VCQ8 Caldesmon 1 + 1

9 76 P51125 Calpastatin (Calpain inhibitor) + 1 10 77 Q08093 Calponin-2 + 1 11 78 Q9DAW9 Calponin-3 + 1 12 13 cAMP-dependent protein 14 Q9DBC7 kinase type I-alpha regulatoryFor Review Only+ 1 15 79 subunit 16 80 P16015 Carbonic anhydrase 3 + + 1 17 81 P08074 Carbonyl reductase 1 18 19 Copper chaperone for Q9WU84 + 1 20 82 superoxide dismutase 21 Creatine kinase S-type, Q6P8J7 + + 1 22 83 mitochondrial 23 24 Cytochrome b-c1 complex Q9CZ13 1 25 subunit 1 + 84 26 Cytochrome b-c1 complex 27 Q9DB77 + 1 28 85 subunit 2, mitochondrial 29 Cytochrome c oxidase subunit Q62425 + 1 30 86 NDUFA4 31 Cytosolic 10- 32 Q8R0Y6 formyltetrahydrofolate + 1 33 87 dehydrogenase 34 Dihydropyrimidinase-related 35 Q62188 + 1 36 88 protein 3 37 Dual specificity phosphatase Q8BK84 + 1 38 89 DUPD1 39 90 Q8R1X5 Edem3 protein + 1 40 41 42 5 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 The FEBS Journal Page 96 of 105

1 Elongation factor Tu, 2 Q8BFR5 + 1 3 91 mitochondrial 4 Endoplasmic reticulum P20029 + 1 5 92 chaperone BiP 6 Endoplasmic reticulum 7 Q9D1Q6 + 1 8 93 resident protein 44 94 Q8R180 ERO1-like protein alpha + 1 9 10 Eukaryotic translation Q9QZD9 + 1 11 95 initiation factor 3 subunit I 12 Extracellular superoxide O09164 + 1 13 96 dismutase [Cu-Zn] 14 F-actin-capping protein For Review Only 15 P47754 + 1 16 97 subunit alpha-2 17 98 Q8BTM8 Filamin-A + 1

18 Fructose-bisphosphate P05064 + 1 19 99 aldolase A 20 Gelsolin (Actin-depolymerizing 21 P13020 + 1 100 factor) 22 Glycerol-3-phosphate 23 P13707 + + 1 24 101 dehydrogenase 25 GTP-binding nuclear protein P62827 + 1 26 102 Ran 27 Heat shock cognate 71 kDa P63017 + 1 28 103 protein 29 104 P14602 Heat shock protein beta-1 + 1 30 31 105 P02089 Hemoglobin subunit beta-2 + 1

32 Heterogeneous nuclear Q8BG05 + 1 33 106 ribonucleoprotein A3 34 Heterogeneous nuclear 35 Q60668 + 1 107 ribonucleoprotein D0 36 Heterogeneous nuclear 37 Q9Z2X1 + 1 38 108 ribonucleoprotein F 39 Heterogeneous nuclear O35737 + 1 40 109 ribonucleoprotein H 41 42 6 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 97 of 105 The FEBS Journal

1 Heterogeneous nuclear 2 P61979 + 1 3 110 ribonucleoprotein K 4 Heterogeneous nuclear Q8R081 + 1 5 111 ribonucleoprotein L 6 Immunoglobulin-binding 7 Q61249 + 1 8 112 protein 1 Indolethylamine N- 9 P40936 + 1 10 113 methyltransferase 11 Isoamyl acetate-hydrolyzing Q9DB29 + 1 12 114 esterase 1 homolog 13 Isocitrate dehydrogenase 14 Q9D6R2 [NAD] subunit alpha, For Review Only + 1 15 115 mitochondrial 16 Isocitrate dehydrogenase O88844 + 1 17 116 [NADP] cytoplasmic 18 Isocitrate dehydrogenase 19 P54071 + 1 20 117 [NADP], mitochondrial 21 118 P11679 Keratin, type II cytoskeletal 8 + 1

22 119 Q9JHV3 Klra19 protein + 1 23 120 Q5M8R7 Ldhb protein + 1 24 25 121 Q61792 LIM and SH3 domain protein 1 + 1

26 L-lactate dehydrogenase A P06151 + 1 27 122 chain 28 123 P28825 Meprin A subunit alpha + 1 29 30 124 Q8CAQ8 MICOS complex subunit Mic60 + 1 31 Mitochondrial peptide 32 Q9D6Y7 methionine sulfoxide + 1 33 125 reductase 34 126 Q62234 Myomesin-1 + + 1 35 127 Q9JHR4 Myosin heavy chain IIB + + 1 36 37 Myosin light chain 1/3, skeletal P05977 + + 1 38 128 muscle isoform 39 129 Q9QVP4 Myosin regulatory light chain 2 + 1 40 41 42 7 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 The FEBS Journal Page 98 of 105

1 Myosin regulatory light chain 2 P97457 + + 1 3 130 2, skeletal muscle isoform 4 Myosin-binding protein C, fast- Q5XKE0 + + 1 5 131 type 6 132 P70402 Myosin-binding protein H + + 1 7 133 Q9JIF9 Myotilin + 1 8 9 Na(+)/H(+) exchange P70441 + 1 10 134 regulatory cofactor NHE-RF1 11 Na(+)/H(+) exchange Q9JHL1 + 1 12 135 regulatory cofactor NHE-RF2 13 Na(+)/H(+) exchange 14 Q9JIL4 For Review Only + 1 15 136 regulatory cofactor NHE-RF3 N-acyl-aromatic-L-amino acid 16 Q91XE4 + 1 17 137 amidohydrolase 18 NADH dehydrogenase 19 Q91YT0 [ubiquinone] flavoprotein 1, + 1 20 138 mitochondrial 21 NADH dehydrogenase 22 23 Q9DCT2 [ubiquinone] iron-sulfur + 1 24 139 protein 3, mitochondrial 25 140 Q6ZWQ0 Nesprin-2 + 1 26 Nucleoside diphosphate-linked 27 P11930 moiety X motif 19 (Nudix motif + 1 28 141 19) 29 O-acetyl-ADP-ribose 30 Q922B1 + + 1 31 142 deacetylase MACROD1 Peroxisomal bifunctional 32 Q9DBM2 + 1 33 143 enzyme 34 144 Q99K51 Plastin-3 + 1 35 145 P60335 Poly(rC)-binding protein 1 + 1 36 Probable D-lactate 37 Q7TNG8 + 1 38 146 dehydrogenase, mitochondrial 39 Protein/nucleic acid deglycase Q99LX0 + + 1 40 147 DJ-1 41 42 8 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 99 of 105 The FEBS Journal

1 2 Pyruvate dehydrogenase E1 3 P35486 component subunit alpha, + 1 4 148 somatic form, mitochondrial 5 Rac GTPase-activating protein E9Q851 + 1 6 149 1 7 150 Q01730 Ras suppressor protein 1 + 1 8 9 151 P24549 Retinal dehydrogenase 1 + 1

10 152 D6RJ85 RNA-binding protein 39 + + 1 11 153 Q9WTM5 RuvB-like 2 + 1 12 Sarcoplasmic/endoplasmic 13 Q8R429 + + 1 14 154 reticulum calcium ATPase For1 Review Only 15 155 Q8C1B7 Septin-11 + 1

16 156 P42208 Septin-2 + 1 17 Septin-7 (CDC10 protein 18 O55131 + 1 19 157 homolog) Spectrin alpha chain, non- 20 P16546 + 1 21 158 erythrocytic 1 22 159 Q60598 Src substrate cortactin + 1

23 Sulfotransferase 1 family 24 Q3UZZ6 + 1 25 160 member D1 161 P26039 Talin-1 + 1 26 27 Trifunctional enzyme subunit Q8BMS1 + 1 28 162 alpha, mitochondrial 29 Troponin C, skeletal muscle P20801 + + 1 30 163 (STNC) 31 164 Q8R112 Ttn protein + + 1 32 33 165 P68368 Tubulin alpha-4A chain + 1

34 166 P99024 Tubulin beta-5 chain 1 35 167 Q91YR1 Twinfilin-1 + 1 36 37 UDP-N-acetylhexosamine Q3TW96 pyrophosphorylase-like + 1 38 39 168 protein 1 40 169 Q8BRB8 Uncharacterized protein + 1 41 42 9 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 The FEBS Journal Page 100 of 105

1 2 170 Q3UWR0 Uncharacterized protein + 1

3 171 Q8BIL4 Uncharacterized protein + 1

4 Uncharacterized protein 5 Q8C3W1 + 1 6 172 C1orf198 homolog 173 P46735 Unconventional myosin-Ib + 1 7 8 174 Q9WTI7 Unconventional myosin-Ic + 1 9 175 Q8VDJ3 Vigilin + 1 10 V-type proton ATPase subunit 11 P62814 + 1 12 176 B, brain isoform 13 Sum of proteins per elution, 40 46 15 43 22 50 6 6 16 19 14 of which (mitochondrial species) For(12) Review(6) (7) (1Only2) (8) (17) (4) (1) (10) (12) 15 Common proteins between acidic-DTT elutions, 6 7 4 10 0 16 of which (mitochondrial) (3) (5) (2) (6) 17 18 19 20 21 22 Supplementary Table 2.2. Protein species interacting with Trx2 as revealed by a yeast two hybrid system from a human liver library. 23 24 Proteins highlighted in green are of mitochondrial localization (MitoCarta2.0). 25 26 27 Gene Number of 28 # Uniprot ID Protein species 29 name matches 30 1 P02675 Fibrinogen b chain precursor FGB 6 31 2 P02679 Fibrinogen g chain precursor FGG 1 32 3 P63261 g-actin P63261 1 33 4 Q7Z406 Myosin heavy chain 14 MYH14 10 34 Myristoylated alanine rich protein kinase C 35 5 P29966 MARCKS 1 36 substrate (MARCKS) 37 6 Q04206 NF-κB p65 RELA 1 38 Phosphatidylinositol N- 7 Q14442 PIGH 1 39 acetylglucosaminyltransferase subunit H 40 8 P00747 Plasminogen PLG 4 41 42 10 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 101 of 105 The FEBS Journal

1 2 9 P07237 Protein Disulfide Isomerase P4HB 1 3 10 Q53ET4 Serine hydroxymethyl SHMT1 1 4 11 P04004 Vitronectin, serum spreading factor VTN 1 5 6 7 8 9 10 Supplementary Table 2.3. Protein species interacting with Trx2 as revealed by a yeast two hybrid system from a rat brain library. 11 12 Proteins highlighted in green are of mitochondrial localization (MitoCarta2.0). 13 Number of 14 # Uniprot ID ProteinFor species Review Gene name Only 15 matches 16 1 P04644 40S ribosomal protein S17 Rps17 1 17 2 P61023 Calcineurin-like EF-hand protein Chp1 1 18 Cytochrome c oxidase subunit 1 19 3 P05503 Mtco1 2 20 (mitochondrial) 21 4 Q78P75 light chain LC8-type 2 (Dynll2) Dynll2 1 22 5 D4A314 GLIS family Zn finger 3 Glis3 1 23 Human immunodeficiency virus type I 6 Q00900 Hivep2 1 24 enhancer-binding protein 2 homolog 25 Leukocyte common antigen-related 26 7 Q4JFL8 LAR-PTP2 1 27 phosphatase mt-Rnr2 28 8 Mitochondrial 16S ribosomal RNA gene 1 29 30 31 32 Supplementary Table 2.4. Protein species interacting with human Trx2 according to BioGRID. 33 34 Proteins highlighted in green are of mitochondrial localization (MitoCarta2.0). 35 # UniProtKB Protein species Gene name 36 39S ribosomal protein L23, mitochondrial (L23mt) (MRP- 37 1 Q16540 MRPL23 38 L23) 39 2 P62249 40S ribosomal protein S16 RPS16 40 3 P62917 60S ribosomal protein L8 (Large ribosomal subunit protein RPL8 41 42 11 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 The FEBS Journal Page 102 of 105

1 2 uL2) 3 60S ribosomal protein L9 (Large ribosomal subunit protein 4 P32969 RPL9 4 uL6) 5 5 6 P08133 Annexin A6 (67 kDa calelectrin) (Annexin VI) (Annexin-6) ANXA6 AP-2 complex subunit beta (AP105B) assembly protein 7 6 P63010 AP2B1 8 complex 2 beta large chain 9 7 Q96M63 Coiled-coil domain-containing protein 114 CCDC114 10 8 P32321 Deoxycytidylate deaminase (EC 3.5.4.12) (dCMP deaminase) DCTD 11 12 9 P40692 DNA mismatch repair protein Mlh1 (MutL protein homolog 1) MLH1 13 10 Q9UNE7 E3 ubiquitin-protein CHIP transferase CHIP STUB1 14 11 P19474 E3For ubiquitin-protein Review ligase TRIM21 OnlyTRIM21 15 16 12 O95071 E3 ubiquitin-protein ligase UBR5 UBR5 Fructose-bisphosphate aldolase B (EC 4.1.2.13) (Liver-type 17 13 P05062 ALDOB 18 aldolase) 19 Glucocorticoid receptor (GR) (Nuclear receptor subfamily 3 14 P04150 NR3C1 20 group C member 1) 21 Golgi integral membrane protein 4 (Golgi integral membrane 22 15 O00461 GOLIM4 23 protein, cis) G-protein coupled receptor-associated sorting protein 2 24 16 Q96D09 GPRASP2 25 (GASP-2) 26 Heat shock 70 kDa protein 1-like (Heat shock 70 kDa protein 17 P34931 HSPA1L 27 1L) (Heat shock 70 kDa protein 1-Hom) (HSP70-Hom) 28 Helicase MOV-10 (EC 3.6.4.13) (Armitage homolog) 29 18 Q9HCE1 MOV10 30 (Moloney leukemia virus 10 protein) 31 Interleukin enhancer-binding factor 2 (Nuclear factor of 19 Q12905 ILF2 32 activated T-cells 45 kDa) 33 Interleukin enhancer-binding factor 3 (Double-stranded RNA- 20 Q12906 ILF3 34 binding protein 76) 35 Keratin, type I cytoskeletal 18 (Cell proliferation-inducing 36 21 P05783 KRT18 37 gene 46 protein) (-18) (CK-18) (Keratin-18) (K18) 38 22 Q6A162 Keratin, type I cytoskeletal 40 (Cytokeratin-40) KRT40 39 Melanoma-associated antigen 11 (Cancer/testis antigen 1.11) 23 P43364 MAGEA11 40 (CT1.11) (MAGE-11 antigen) 41 42 12 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 103 of 105 The FEBS Journal

1 -associated protein RP/EB family member 2 2 24 Q15555 MAPRE2 3 (APC-binding protein EB2) (End-binding protein 2) (EB2) 4 Microtubule-associated protein RP/EB family member 3 (EB1 5 25 Q9UPY8 member 3) (EBF3) (End-binding protein 3) MAPRE3 6 (EB3) (RP3) 7 Microtubule-associated tumor suppressor candidate 2 (Cardiac 8 26 Q5JR59 MTUS2 9 zipper protein) 10 Mitochondrial import receptor subunit TOM22 homolog 11 27 Q9NS69 (hTom22) (1C9-2) (Translocase of outer membrane 22 kDa TOMM22 12 subunit homolog) 13 28 Q99683 Mitogen-activated protein kinase kinase kinase 5 MAP3K5 14 For Review Only 15 29 Q96HT8 MORF4 family-associated protein 1-like 1 MRFAP1L1 16 30 Q96CD2 Phosphopantothenoylcysteine decarboxylase (PPC-DC) PPCDC 17 Polypyrimidine tract-binding protein 3 (Regulator of 18 31 O95758 PTBP3 19 differentiation 1) (Rod1) 20 32 Q9UI14 Prenylated Rab acceptor protein 1 (PRA1 family protein 1) RABAC1 21 Proteasome activator complex subunit 3 (11S regulator 33 P61289 PSME3 22 complex subunit gamma) 23 34 Q52LJ0 Protein FAM98B FAM98B 24 25 35 Q8IZU0 Protein FAM9B FAM9B 26 Protein-associating with the carboxyl-terminal domain of ezrin 36 Q8IZE3 SCYL3 27 (Ezrin-binding protein PACE-1) (SCY1-like protein 3) 28 37 Q9UJ41 Rab5 GDP/GTP exchange factor (RAP1) RABGEF1 29 Receptor expression-enhancing protein 6 (Polyposis locus 30 38 Q96HR9 REEP6 31 protein 1-like 1) 32 Ribose-5-phosphate isomerase (EC 5.3.1.6) 39 P49247 RPIA 33 (Phosphoriboisomerase) 34 Ribosome biogenesis protein WDR12 (WD repeat-containing 40 Q9GZL7 WDR12 35 protein 12) 36 RNA-binding protein 45 (Developmentally-regulated RNA- 37 41 Q8IUH3 RBM45 38 binding protein 1) (RB-1) (RNA-binding motif protein 45) 39 Sharpin (Shank-associated RH domain-interacting protein) 42 Q9H0F6 SHARPIN 40 (Shank-interacting protein-like 1) (hSIPL1) 41 42 13 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 The FEBS Journal Page 104 of 105

1 Sjoegren syndrome nuclear autoantigen 1 (Nuclear autoantigen 2 43 O43805 SSNA1 3 of 14 kDa) 4 Spectrin beta chain, non-erythrocytic 1 (Beta-II spectrin) 44 Q01082 SPTBN1 5 (Fodrin beta chain) (Spectrin, non-erythroid beta chain 1) 6 Superoxide dismutase [Cu-Zn] (EC 1.15.1.1) (Superoxide 7 45 P00441 SOD1 8 dismutase 1) (hSod1) 9 46 Q9NVV9 THAP domain-containing protein 1 THAP1 10 Thioredoxin (Trx) (ATL-derived factor) (ADF) (Surface- 47 P10599 TXN 11 associated sulphydryl protein) (SASP) 12 Thioredoxin-interacting protein (Thioredoxin-binding protein 13 48 Q9H3M7 TXNIP 14 2) (VitaminFor D3 upReview-regulated protein 1) Only 15 Thyroid receptor-interacting protein 6 (TR-interacting protein 16 49 Q15654 6) (TRIP-6) (Opa-interacting protein 1) (OIP-1) (Zyxin-related TRIP6 17 protein 1) (ZRP-1) 18 TRAF-interacting protein with FHA domain-containing 19 50 Q96CG3 TIFA protein A 20 21 Transcription factor p65 (Nuclear factor NF-kappa-B p65 22 51 Q04206 subunit) (Nuclear factor of kappa light polypeptide gene RELA 23 enhancer in B-cells 3) 24 52 P55072 Transitional endoplasmic reticulum ATPase (TER ATPase) VCP 25 53 26 P67936 alpha-4 chain (TM30p1) (Tropomyosin-4) TPM4 Type-1 angiotensin II receptor-associated protein (AT1 27 54 Q6RW13 AGTRAP 28 receptor-associated protein) 29 U1 small nuclear ribonucleoprotein 70 kDa (U1 snRNP 70 55 P08621 SNRNP70 30 kDa) (U1-70K) (snRNP70) 31 Ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCH-L5) 32 33 56 Q9Y5K5 (EC 3.4.19.12) (Ubiquitin C-terminal hydrolase UCH37) UCHL5 34 (Ubiquitin thioesterase L5) 35 57 Q8N1B4 Vacuolar protein sorting-associated protein 52 homolog VPS52 36 58 P08670 Vimentin VIM 37 Zinc finger and BTB domain-containing protein 14 (Zinc 38 59 O43829 ZBTB14 39 finger protein 161 homolog) 40 41 42 14 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 105 of 105 The FEBS Journal

1 2 3 4 Supplementary Table 2.5. Four cliques in the disease network of Trx2 that relate ten different diseases. 5 6 Diseases in more than one clique are in bold. The numbering of cliques is arbitrary. 7 8 9 10 Gene Uniprot Clique 1 11 name ID 12 TRIOSEPHOSPHATE ISOMERASE DEFICIENCY TPI1 P17751 13 14 COMBINED OXIDATIVE PHOSPHORYLATIONFor Review DEFICIENCY OnlyΤΧΝ2 Q99757 15 LACTATE DEHYDROGENASE B DEFICIENCY LDHB Q5M8R7 16 PARKINSON’S DISEASE PARK7 Q99LX0 17 18 19 Clique 2 20 MITOCHONDRIAL COMPLEX III DEFICIENCY, NUCLEAR TYPE 5 UQCRC2 Q9DB77 21 MITOCHONDRIAL COMPLEX II DEFICIENCY SDHA Q8K2B3 22 COMBINED OXIDATIVE PHOSPHORYLATION DEFICIENCY ΤΧΝ2 Q99757 23 PYRUVATE DEHYDROGENASE COMPLEX DEFICIENCY PDHA1 P35486 24 25 26 Clique 3 27 ANEMIA, SIDEROBLASTIC HSPA9 P38647 28 COMBINED OXIDATIVE PHOSPHORYLATION DEFICIENCY ΤΧΝ2 Q99757 29 LDHB Q5M8R7 30 LACTATE DEHYDROGENASE B DEFICIENCY 31 32 Clique 4 33 MITOCHONDRIAL COMPLEX II DEFICIENCY SDHA Q8K2B3 34 TRIOSEPHOSPHATE ISOMERASE DEFICIENCY TPI1 P17751 35 36 COMBINED OXIDATIVE PHOSPHORYLATION DEFICIENCY ΤΧΝ2 Q99757 37 LACTATE DEHYDROGENASE B DEFICIENCY LDHB Q5M8R7 38 39 40 41 42 15 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60