Mapping Novel Frataxin Mitochondrial Networks Through Protein- Protein Interactions

Friedreich’s Ataxia (FRDA) is a neuromuscular degenerative disorder caused by trinucleotide expansions in the first intron of the frataxin (FXN) gene, resulting in insufficient levels of functional FNX protein. Deficits in FXN involve mitochondrial disruptions including iron-sulfur cluster synthesis and impaired energetics. These studies were to identify unique protein-protein interactions with FXN to better understand its function and design therapeutics. Two complementary approaches were employed, BioID and Co-IP, to identify protein interactions with FXN at the direct binding, indirect binding, and non-proximal levels. Forty-one novel protein interactions were identified by BioID and IP techniques. The FXN protein landscape was further analyzed incorporating both interaction type and functional pathways using a maximum path of 6 proteins with a potential direct interaction between FXN and NFS1. Probing the intersection between FXN-protein landscape and biological pathways associated with FRDA, we identified 41 proteins of interest. Peroxiredoxin 3 (Prdx3) was chosen for further analysis because of its role in mitochondrial oxidative injury. Our data has demonstrated the strengths of employing complementary methods to identify a unique interactome for FXN. Our data provides new insights into FXN function and regulation, a potential direct interaction between FXN and NFS1, and pathway interactions between FXN and Prdx3.


INTRODUCTION
Friedreich's Ataxia (FRDA) is a neuromuscular degenerative disorder characterized by fatigue, cerebellar ataxia, and hypertrophic cardiomyopathy 1 .Most patients carry homozygous GAA trinucleotide expansions in the rst intron of the frataxin-encoding gene (FXN), resulting in condensation of chromatin and impaired FXN transcription.FXN protein localizes to the mitochondrial matrix and is associated with the inner mitochondrial membrane.Insu cient levels of FXN protein causes several mitochondrial de ciencies, including reduced iron-sulfur cluster synthesis, impaired energetics and dynamics, and accumulation of labile iron and reduced oxygen species 2 .Although these mitochondrial de ciencies have been associated with FRDA pathogenesis, there are still mechanistic gaps as to how loss of FXN expression results in mitochondrial dysfunction.
Biochemical studies with human FXN and highly conserved homologues have shown that FXN delivers ferrous iron to the iron-sulfur cluster (ISC) synthesis complex and forms physical contacts with core proteins NFS1, ISCU and ISD11 3 .As reviewed by Isaya 4 , ISC formation is widely accepted as the primary function of FXN since reduced levels of FXN in FRDA samples and mouse models have resulted in reduced activities of iron-sulfur dependent enzymes including complexes I-III of the respiratory chain and mitochondrial aconitase [5][6][7] .Another consequence of impaired ISC biosynthesis is the accumulation of labile iron in mitochondria which may also further impair heme synthesis during FXN de ciency [8][9][10] .
Although there are con icting reports of reproducibility and functional consequences of iron accumulation in FRDA [11][12][13][14] , any increase in redox-active iron in combination with dysregulated coupling e ciency due to reduced respiratory complex activities can impair energy metabolism and augment localized formation of reduced oxygen species 15 .Such mitochondrial oxidative perturbations and saturation and/or loss of antioxidant activities have been associated with FRDA progression [16][17][18][19] .
Current FRDA therapeutic strategies based on correcting the aforementioned mitochondrial pathologies during FXN de ciency include delivery of mitochondrial-targeted iron chelators and redox-active compounds [20][21][22] , as well as chemical induction of cellular antioxidants and mitochondrial biogenesis via nuclear factor erythroid 2-related factor 2 (Nrf2) [23][24][25][26] .These strategies aim to alleviate functional consequences of reduced FXN levels but do not directly address speci c molecular targets because our current knowledge of the FXN protein network is largely limited to ISC biosynthesis.The current investigation sought to better de ne the FXN protein network in human cells using two complementary approaches: proximity-based labeling (BioID) and endogenous co-immunoprecipitation (co-IP).The discovery of these new FXN protein interactions provides opportunities to understand essential FXNdependent functions as well as identify new molecular and functional therapeutic targets in FRDA.

RESULTS
Expression of FXN and MTS-Cox8a transgenes in A549 cells.Mitochondrial localization of transgene biotinyl ligase activity (BirA*) was con rmed by co-localization of streptavidin and cytochrome oxidase subunit IV (COXIV) in FXN-BioID2 and MTS-BioID2 cell lines (Fig. 1A and 1B).Cells were pulsed with biotin and biontinylated proteins were a nity puri ed with streptavidin-conjugated sepharose beads.SDS-PAGE/immunoblot of eluted fractions con rmed unique biotinylation patterns between FXN-BioID2 and MTS-BioID2 controls (Fig. 1C).Samples were then subjected to LC-MS/MS analysis for peptide identi cation (Supp File 1).
Identi cation of FXN protein networks by co-IP.Two functional isoforms of FXN have been identi ed,FXN  , a full-length form which donates either Fe 2+ or Fe 3+ for ISC assembly and FXN 81-210 ,a truncatedform that is thought to only donate Fe 2+ .Loss of FXN  functionality may be critical for disease pathogenesis since expression of this isoform is lost to a greater extent compared to FXN  in FRDA patient lymphoblasts and cerebellum 27 . Three uman lymphoblast lines from healthy controls (GM03798, GM05398, GM14907) were selected based on higher expression of FXN  (Fig. 2A) 27 .Cell lysates were pooled and other shorter FXN isoforms were excluded by size exclusion chromatography (Fig. S1).Co-IP with FXN protein was used as a complementary proteomics approach to identify FXN protein networks.Furthermore, FXN  -positive fractions were selected for co-IP based on purity and co-fractionation with NFS1 (Fig. S1).Successful FXN co-IP was con rmed by NFS1 detection in eluate (Fig. 2B). Eluatewas subsequently separated by SDS-PAGE and bands were excised for tandem mass spectrometry (Fig. 2C).
Construction of the FXN-protein interaction landscape.Following data preprocessing, manual curation using reference databases con rmed that 93.95% and 97.44% of proteins identi ed using BioID and co-IP respectively were localized to mitochondria (Table 1).In total, we identi ed 196 and 117 proteins with high a nity for FXN by BioID and co-IP respectively (Supp File 1), as well as an interaction collection of 41 proteins identi ed using both methods (Table 2, Fig 3A).Speci city achieved by integrating data validated using both methods allowed us to build and curate an FXN-protein interaction landscape, relying on the hypothesis that interaction con dence directly correlates with protein a nity strength.This hypothesis is supported by the BioID mechanism of action which detects protein a nity at three levels: direct binding, indirect binding, and non-proximal (Fig 3B).
After integrating 15 interactome databases from the PSICQUIC collection, we discovered 13,500 interactions (co-expression, co-localization, physical interaction) connecting our 41 proteins (Fig 3C, Supp File 2).A visual example of network density and complexity was generated based on the highly curated interactome GENEMANIA (Fig. 3D, Supp File 3).Despite the density of our network, close relationships between our proteins of interest and FXN across various interaction types at both protein and gene network levels were demonstrated.Using our interaction collection of 41 proteins, UniProt ID or gene symbol search queries were used to generate a path from FXN to each protein (Fig. 3D).The maximum path from FXN to any target protein in the landscape is 6 when using either UniProt ID or gene symbol (e.g. Intersection of FXN-protein interaction landscape with biological pathways implicated in Friedreich's Ataxia.Protein and gene interaction networks were generated based on our interaction collection of 41 proteins to integrate protein-protein interactions and gene regulatory networks with biological functions (Fig. 4).The systems biology landscape focused on 20 selected biosystems of interest with relevance to FXN function, mitochondrial biology, and FRDA pathogenesis.The discovery work ow determined 35 new direct and 18 indirect interactions across protein and gene networks categorized by interaction type (physical interaction, shared protein domain, etc.) (Tables 1 and 2).Our interaction network also increased connectivity between selected molecular biosystems, shortening pathway distance from FXN (Fig. 4).Box 1 describes a simple toy example to demonstrate how the level of interaction in FXN-protein landscape can inform our understanding of molecular paradigms in uencing biosystems.Existing interactome databases (GENEMANIA, STRINGdb) support FXN-NFS1 interactions but this is dependent on ISD11 (also known as LYRM4) as an intermediate 28 .However, ISD11 was not detected by either BioID or co-IP, reinforcing our landscape construction with novel direct a nity between FXN and NFS1.This new discovery empowers our understanding of ISC assembly (gene ontology 0016226), which has previously been associated with FXN function and FRDA pathogenesis.
Network-pathways integration reveals interactions between FXN and the ISC synthesis complex and peroxiredoxin 3 (Prdx3).We constructed a cord diagram mapping our 41 proteins in the FXN protein landscape integrating both interaction type (e.g.physical, co-expression, etc) with functional pathways (Fig. 5).Using this network-pathways integration supporting identi cation of shared interactions to pathways relevant to FXN and FRDA pathogenesis, we identi ed Prdx3 as a target of interest.Prdx3 is the primary oxidoreductase in mitochondria responsible for metabolizing H 2 O 2 29 .Therefore, Prdx3 could play a pivotal role in both ISC synthesis and FRDA pathogenesis by detoxifying localized production of H 2 O 2 as a byproduct of Fenton chemistry occurring from reactivity of labile iron.FXN-Prdx3 pathway interactions depicted in Fig. 5 include response to oxidative stress and mitochondrial organization.FXN, and Prdx3 are involved in biological processes/pathways function of mitochondria organization and response to oxidative stress.They are also involved with several transporters or importer proteins/ genes that seem to play essential roles in the mechanism of FXN and Prdx3 functions (GRPEL1, HSPA9, TIMME4, PMBC, etc.), warranting possible participation in the same pathways as Prdx3 and FXN.Here, PMBC and GRPEL1 in the pathway act as modi er and transporter proteins.To con rm these ndings, a reverse BioID screen was used to probe for Prdx3 interactions with ISC complex proteins.Expression of FRDA and Prdx3 levels using mouse and cell models.Mouse and cell models of FRDA were used to investigate differential expression of Prdx3 or related mitochondrial thioredoxin-dependent enzymes including peroxiredoxin 5 (Prdx5), thioredoxin 2 (Trx2), and thioredoxin reductase 2 (TrxR2), between disease and control states.FRDA knockdown (FRDAkd) mice have systemic doxycycline-inducible knockdown of FXN recapitulating multiple phenotypes observed in patients 30 .We were able to reproduce similar phenotypes in FRDAkd mice (Fig. S5A) after 18 weeks of continuous access to doxycycline in drinking water.FRDAkd mice treated with doxycycline exhibited ataxic behaviors including increased hindlimb clasping, poor coordination when walking on a ledge, and decreased grip strength, even though gait was not changed (Fig. S3B-F).Importantly, FRDAkd mice that did not receive doxycycline did not display any behaviors different from that of wild-type controls, indicating that behavioral changes are FXN-dependent.However, FXN expression was decreased compared to wild-type in transgenic FRDAkd mice independent of doxycycline (Fig. 6A-D).Therefore, there may be FXN dose-dependent effects between wild-type and transgenic mice prior to doxycycline administration which was not previously reported 30 .Decreased Prdx3 and increased Prdx5 were detected in cerebellum of FRDAkd mice receiving doxycycline (Fig. 6A and B).This was not recapitulated in cardiac tissue (Fig. 6C and D), suggesting tissue-speci c changes despite systemic FXN knockdown.However, FXN-dependent changes in expression of mitochondrial thioredoxin enzymes in cardiac tissue could be masked by decreased FXN in FRDAkd mice in absence of doxycycline.A panel of patient-derived dermal broblasts were used as a second model to investigate FXN-dependent changes.FRDA broblasts expressed 26.5% FXN compared to controls and had decreased expression of Prdx5 and TrxR2 (Fig. 6E and F).Loss of Prdx5 expression in FRDA lymphoblasts was previously reported by Hayashi and Cortopassi while performing a targeted screen of oxidative stress genes 19 .There were no changes in expression of the mitochondrial chaperone HSP60 in either FRDAkd samples or lymphoblasts, suggesting that FXN-dependent changes in mitochondrial antioxidant protein expression was not due to altered mitochondrial content (data not shown).
Prdx3 redox status in FRDA mice and cell models.Redox cycling of Prdx3 is critical in dictating oxidoreductase activity.As depicted by the schematic in Fig. 7A top, Prdx3 redox state was determined across similar biological samples by differentiating Prdx3 oxidized dimers from reduced monomers through differential n-ethylmaleimide (NEM) alkylation of free thiols.Preservation of Prdx3 dimers under non-reducing conditions was con rmed by loss of signal following reduction with β-mercaptoethanol (Fig. 7A, bottom).The reduced monomeric form of Prdx3 was more prevalent than oxidized dimers in mouse cerebellum and heart and this was not affected by FXN knockdown (Fig. 7B, C and D).Again, speci city of oxidized Prdx3 dimers were con rmed by reducing lysates with β-mercaptoethanol.
Similarly, no signi cant changes in Prdx3 redox state were detected in patient-derived broblasts (Fig. 7E  and F).

DISCUSSION
Decreased or defective FXN expression and its role in the development of FRDA is poorly understood.
Little is known about FXN interactions with other proteins, speci cally in assembly of ISC, and the function cluster synthesis and iron metabolism.Previous metabolomic-based studies have identi ed defective 1-carbon metabolism speci cally involving folate and sarcosine 31 .Pignataro et al. employed ribosome display approach to identify small molecules with may modulate FXN function.They found that treatment A _224 was able to increase cysteine NSF1 desulfurase activation in the context of speci c FXN mutants 32 .Other proteomics approaches have also been used to identify differences in cerebral spinal uids of FRDA and control patients and several pathways associated with oxidation and in ammation were differentially regulated 33 .
Our investigation endeavored to identify unique protein-protein interactions with FXN.We incorporated two complementary approaches, BioID (Figure 1) and a traditional proteomic approach, Co-IP (Figure 2) to identify protein interactions with FXN at the direct binding, indirect binding, and non-proximal levels.
Using this integrated approach, our data indicated over 1000 proteins with signi cant a nity compared to the MTS-control.Using manual curation and veri cation from gold standard mitochondrial protein repositories, we chose to focus on the 41 novel protein interactions identi ed by both BioID and IP techniques.These 41 proteins were analyzed using proteins-protein interaction network (PPI), GeneMenia, using up to 6 levels of connections (Figure 3).
We incorporated FXN protein interaction network with FXN biological function using systems biology landscape, mitochondrial biology, and FRDA pathogenesis and focused on 20 selected biosystems and interaction with the 41 identi ed proteins.Using discovery work ow, 35 new direct and 18 indirect interactions were identi ed.Previous approaches have supported FXN-NFS1 interactions dependent on ISD11 28 (Figure 4).Our analysis using both BioID or co-IP, did not detect ISD11 which reinforces the likelihood of a novel direct a nity between FXN and NFS1.This discovery may provide new insights into ISC assembly and function.
The 41 proteins identi ed in the FXN protein landscape were further analyzed using interaction type and functional pathways.We identi ed and chose peroxiredoxin 3 (Prdx3) as a speci c target of interest because the FXN-Prdx3 pathway interactions and the relations to oxidative stress and mitochondrial organization are both pathologies associated with FRDA.PRDX3 is an oxidoreductase located primarily in the mitochondria.Its primary role is to remove peroxides from the mitochondrial environment.Prdx3 is transcriptionally regulated by the antioxidant transcription factor Nrf2. PRDX3 de cient mice have less mitochondrial DNA, increased mitochondrial injury, impaired ATP production, neuronal apoptosis, and decreased physical strength 34,35 .Conversely, PRDX3 overexpression prevents heart failure following myocardial infarction 36 .Decreased Prdx3 expression was detected in dorsal root ganglia of YG8R mice that express mutant human frataxin 16 , supporting an association between FXN and Prdx3 and overexpression of mitochondrial peroxiredoxin restored life span in a Drosophila model of FRDA 37 .A recently approved therapeutic for FRDA, Omovaloxalone, is a Nrf2 inducer and likely induces expression of mitochondrial antioxidants including Prdx3.
This study has demonstrated the strengths of employing complementary methods to identify a unique interactome for FXN.Our data provides new insights into FXN function and regulation and a potential direct interaction between FXN and NFS1.We have also de nitively identi ed an association between FXN and Prdx3 which has potential applications for mitochondrial pathology and future therapeutic strategies.As a weakness, the protein a nity approach does not capture the spatial representation involved in the BioID2 technology mechanism.Future work to integrate that knowledge in the algorithm will greatly improve the knowledge accuracy.However, this does not diminish our nding because our result relies more on the qualitative aspect of the protein interaction, and less on the quanti cation of the interaction.Plasmids & Generation of BioID2 cell lines.FXN, PRDX3, and the Cox8A mitochondrial targeting sequence (MTS) were ampli ed by PCR from human cDNA and inserted into a previously generated BioID2-HA pBabe-puro vector (#74224; Addgene, Watertown MA) 39 using EcoR1 and BamH1 (New England Biolabs, Ipswich MA) restriction sites following manufacturer's instructions for the Takara Bio In-Fusion cloning method (Mountain View CA).Stable cells for all BioID2 constructs were generated using retroviral transduction.HEK293 Phoenix cells were transfected with FXN-BioID2, PRDX3-BioID2, and MTS-BioID2 constructs using Lipofectamine 3000 (ThermoFisher Scienti c) for 6 h at 37°C, media was then replenished, and cells were moved to 32°C for a 72h incubation.Viral supernatant from transfected Phoenix cells was ltered (0.45 µm) then added to A549 target cells along with 0.5mL of fresh media and 0.25 µg/mL polybrene (Santa Cruz Biotechnology, Dallas TX).After 72 h after transduction, cells were plated into 10 cm dishes with fresh media and 2 µg/mL puromycin (ThermoFisher Scienti c) was added for selection.
Confocal microscopy was performed using a Nikon A1 confocal microscope (60X/1.49oil APO TIRF Nikon objective) with a charge-coupled CoolSnap HQ camera (Photometrics) linked to a workstation running NIS-Elements software (Nikon, Melville NY).
A nity capture of biotinylated proteins.All BioID pulldowns were performed in triplicate as described in detail 41 .For each BioID2-fusion line, 2 x 10 7 cells were incubated with 50 µM biotin for 16-18 h.Cells were washed twice with PBS and lysed in 1.08 mL of lysis buffer containing 8 M urea, 50 mM Tris pH 7.4, 1 mM DTT, and 1X Halt protease inhibitor (ThermoFisher Scienti c).Triton X-100 was added to nal concentration of 1% and cells were sonicated twice on ice to shear DNA (45 s at 30% duty cycle and output level 3, Branson Soni er-250).Detergent was diluted by adding an equal volume of lysis buffer, and sonication was repeated once more.Lysates were centrifuged at 16,5000 x g for 10 m, and supernatants were collected to a 5 mL Eppendorf tube and precleared with 200 µL gelatin Sepharose 4B beads (GE Life Sciences, Pittsburgh PA) rotating for 2 h at 4°C.Samples were then centrifuged at 800 x g for 5 m and supernatant transferred to a fresh 5 mL Eppendorf tube.A nity capture was performed by adding 50 µL streptavidin Sepharose high performance beads (GE Life Sciences) and incubating on rotator for 4 h at 4°C.Beads were collected by centrifugation at 800 x g for 5 m, and washed four times with wash buffer (8 M urea in 50 mM Tris pH 7.4).Beads were resuspended in 1 mL wash buffer and 100 µL was taken for SDS-PAGE & immunoblot while the other 90% of sample was resuspended in 50 mM NH 4 HCO 3 supplemented with 1 mM biotin for LC-MS/MS analysis.
BioID on-bead protein digestion and identi cation by 1D LC-MS/MS.Beads were thawed and resuspended with 8 M urea and 50 mM ammonium bicarbonate and cysteine disul de bonds were reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP) at 30°C for 60 m followed by cysteine alkylation with 30 mM iodoacetamide in the dark at room temperature for 30 m.Following alkylation, urea was diluted to 1 M urea using 50 mM ammonium bicarbonate, and proteins were subjected to overnight digestion with mass spec grade Trypsin/Lys-C mix (Promega, Madison WI).Beads were pulled down, supernatant with peptides was collected, and beads were washed with 50 mM ammonium bicarbonate to increase peptide recovery.Following digestion, samples were acidi ed with formic acid (FA) and subsequently desalted using AssayMap C18 cartridges mounted on an Agilent AssayMap BRAVO liquid handling system, C18 cartridges were rst conditioned with 100% acetonitrile (ACN), followed 0.1% FA.Sample was then loaded onto the conditioned C18 cartridge, washed with 0.1% FA, and eluted with 60% ACN, 0.1% FA.The organic solvent was removed in a SpeedVac concentrator prior to LC-MS/MS analysis.Before injecting the sample in the LC-MS, total sample peptide amount was determined by NanoDrop spectrophometer (ThermoFisher Scienti c).Dried samples were reconstituted with 2% acetonitrile, 0.1% formic acid and analyzed by LC-MS/MS using a Proxeon EASY nanoLC system coupled to an Orbitrap Elite mass spectrometer (ThermoFisher Scienti c).Peptides were separated using an analytical C18 Acclaim PepMap column 0.075 x 500 mm, 2 µm particles (ThermoFisher Scienti c) in a 120-min gradient of 2-28% solvent B at a ow rate of 300 nL/min.The mass spectrometer was operated in positive data-dependent acquisition mode.MS1 spectra were measured with a resolution of 60,000, an AGC target of 1 x 10 6 and a mass range from 350 to 1400 m/z.Up to 10 MS2 spectra per duty cycle were triggered, fragmented by collisium-induced dissociation, and acquired in the ion trap with an AGC target of 1 x 10 4 , an isolation window of 2.0 m/z and a normalized collision energy of 35.Dynamic exclusion was enabled with duration of 30 s.All mass spectra from were analyzed with MaxQuant software (v1.5.5.1).
Frataxin co-immunoprecipitation and identi cation by 1D LC-MS/MS.Lymphoblastoid cell lysates were separated by Superdex 75 size exclusion chromatography.High molecule weight fractions were pooled and immunoprecipitation was performed with rabbit anti-FXN PAC 2517 immobilized on Protein A Magnetic beads, as described 27 .Aliquots of pooled sample before immunoprecipitation (input, ~5% of total volume), the ow-through fraction (not bound; ~5% of total volume), and the a nity-puri ed fraction (bound; 100% of total volume) were analyzed by SDS-PAGE & immunoblot and bound lysate was digested for LC-MS/MS.
FXN-protein interaction framework.A computational work ow followed by manual curation was used for proteomic discovery of FXN protein networks with biological relevance within the context of FXN function and FRDA pathogenesis.The work ow (1) assessed the quality of the raw data, (2) determined statistical signi cance, (3) validated existing physical interactions with FXN, (4) predicted potential new physical interactions with FXN, and ( 5) assembled a functional annotation to identify molecular ecosystems represented within the FXN-protein network.
Determination of novel FXN-protein interactions.GENEMANIA and STRINGdb databases were used to identify new FXN-protein interactions and determine the level of the interaction.Proteins directly downstream from FXN were labeled as level 1 (L1), the second protein interacting with FXN were labeled level 2 (L2), and so on.Following the concept underlying BioID2 technology, we considered L1 proteins more likely to directly interact (Pd) with FXN than and considered protein interactions at L2 and L3 as indirect interactions (Pi) (Fig. 3D).Analysis of our interaction collection (supplemental le 3) lead to identi cation of 35 new direct interactions and 18 new indirect interactions with FXN (Fig. 3E, Table 1).
Bioinformatics.The computational work ow used descriptive statistics and boxplot to check data distribution and outliers, followed with a linear normalization to allow signi cance analysis between BioID2 target and MTS control a nity pro les using ProteoSign 42 .SAINTexpress 43 was used to assess statistical probability of candidate contaminants.Manual curation of proteins with less than two spectral counts out of the three replicates were removed due to the lack of con dence.Proteins identi ed from MTS-BioID2 cells were used to remove false positive candidates from FXN-BioID2 and Prdx3-BioID2 samples.However, proteins whose label free quanti cation values were threefold more than those in MTS-BioID2 controls were also considered candidates.Proteins had to be identi ed on at least one of the following mitochondrial localization reference databases: MitoCarta2.0,Integrated Mitochondrial Protein Index (IMPI), and mitochondrion gene ontology.Candidate protein/gene functional interaction networks, pathways, and localizations were analyzed using GeneMania 44 and Networkanalyst.ca 45.An integrated, comprehensive network analysis was done using PSIQUIC 46 then results were integrated with Cytoscape3 Mitochondrial Discovery of FXN-protein interaction landscape.An interactome with others localized proteins of mitochondrion forming a network of direct and indirect protein-protein reaction was constructed.A) volcano plot shows selected protein at 0.05 cut-off.B) BioID detects protein a nity at three levels: direct binding, indirect binding, and non-proximal.196 proteins using BioID, 117 proteins using co-IP, and 41 proteins identi ed with both methods were detected with high a nity for FXN.C) using the PSICQUIC collection interactome databases, we discovered 13,500 interactions (co-expression, co-localization, physical interaction) connecting our 41 proteins.D) using the interaction collection of the 41 proteins, UniProt ID or gene symbol search queries were used to generate a path from FXN to each protein.E) the maximum path from FXN to any target protein in the landscape is 6 when using either UniProt ID or gene symbol.broblasts expressed as mean ± standard deviation of 5-7 cell lines depicting female (circle) and male (square) subjects analyzed by a student's t test.Lysates were probed with antibodies against FXN, Prdx3, Prdx5, Trx2, and TrxR2 with β-actin as a loading control.Statistical signi cance is de ned as *p<0.05,**p<0.01,† p<0.001 and † † p<0.0001.

Prdx3-
BirA* (biotinylated ligase A, R118G mutation) increased detection of biotinylated proteins which were localized to mitochondria of stably transfected A549 cells (Fig.1B-D, FigS2).Although FXN was not detected by mass spectrometry, prominent ISC synthesis complex proteins ISCU and NFS1 were identi ed out of 41 unique peptides (Supp File 1).
localization and biotinyl ligase activity of BioID2 transgenes.(A) BioID2 was cloned inframe with carboxy terminus of human FXN, Prdx3, and the MTS of COX8a for detection and enrichment of proximal interacting proteins.(B) Immuno uorescent labeling of parental and stably transfected A549 cells expressing BioID2 transgenes that were pulsed with 50 µM biotin for streptavidin (green) and COXIV (red) to visualize transgene expression, subcellular localization, and biotinyl ligase activity.Dapi was used as a nuclear counterstain (blue in DNA-merge).SDS-PAGE/immunoblots of (C) whole cell lysates or (D) streptavidin a nity puri ed A549 BioID2 cell lysates with anti-BirA antibody or streptavidin for transgene detection and protein biotinylation.Colored arrowheads indicate detection of predicted-sized transgenes.

Figure 6
Figure 6 Waltham MA).Fibroblasts were obtained from the Coriell Institute for Medical Research or the Friedreich's Ataxia Primary Fibroblast Repository 38 maintained by David Lynch of The Children's Hospital of Philadelphia and Marek Napierala of the University of Alabama at Birmingham.Fibroblasts were cultured in 5% CO 2 at 37˚C in DMEM/F12 media containing 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin, and 1% non-essential amino acids (Life Technologies, Carlsbad CA).
30to determine shortest-path networks.Finally, a level-based network analysis of direct and indirect protein-protein interactions was used to identify network hubs and map the type of network interaction as well as any known or new molecular functions.Mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE 48-50 partner repository (dataset identi er PXD015034).Prdx3 redox status.Cells were washed with ice-cold PBS then lysed in alkylation buffer containing 100 mM N-ethylmaleimide (NEM) and 10 μg/mL catalase to label and retain reduced monomers for nonreducing SDS-PAGE/immunoblot analysis51. Apals for studies with experimental animals.All animal experiments were conducted with approval from the Sanford Research Institutional Animal Care and Use Committee (protocol number 142-03-21E).Sanford Research has an Animal Welfare Assurance on le with the O ce of Laboratory Animal Welfare (A-4568-01) and is a licensed research facility under the authority of the United Sates Department of Agriculture (46-R-0011).Mouse strains, care & behavior testing.All animal were conducted with approval from the Sanford Research Institutional Animal Care and Use Committee (protocol number 142-03-21E).All methods were carried out in accordance with relevant guidelines and regulations.Sanford Research has an Animal Welfare Assurance on le with the O ce of Laboratory Animal Welfare (A-4568-01) and is a licensed research facility under the authority of the United Sates Department of Agriculture (46-R-0011).All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).FRDA knockdown (FRDAkd) mice obtained from Daniel Geschwind (University of California, Los Angeles) have doxycycline permissible expression of shRNA targeting Fxn30.Mice were provided food and acidi ed water ad libitum and monitored daily.Genomic DNA was prepared using the AccuStart II Mouse Animal subjects were euthanized by asphyxiation followed by cervical dislocation.Ataxic scoring summarized by Guyenet et al. was comprised of hind limb clasping, the ledge test, and gait analysis.A grip strength meter was used to measure all-limb grip strength (Columbus Instruments, Columbus OH).As a mouse grasped the meshing with forelimbs and hindlimbs, the peak pull force in grams was recorded on a digital force transducer.This work is supported by the Finish Line Fund, the T. Denny Sanford Pediatric Collaborative Research Fund between Mayo Clinic and Sanford Health, and grant R01HL135112 from the National Institutes of Health.The Imaging Core, Biochemistry Core, and Research Design & Biostatistics cores at Sanford Research, which facilitated these studies, are supported by Institutional Development Awards from the National Institute of General Medical Sciences and the National Institutes of Health under grants P20GM103548, P20GM103620, and P20GM121341.Proteomics were performed by Sanford Burnham Prebys Medical Discovery Institute and the Mayo Clinic.Haydee Torres and Jianning Tao assisted with grip strength measurements.