Gene expression and gene ontology analysis
The mitochondrial proteome from Mitocarta and Mitominer databases were compared to identify unique and common proteins. The proteins present in both the databases were further analyzed for gene ontology analysis from The Gene Ontology Consortium [56]. The proteins not showing any biological process or molecular function were further considered for RNAseq profiles in different human tissues from GTEx consortium and the retina transcriptome dataset [16, 17]. The proteins with high expression were further explored for evolutionary conservation analysis. BLAST (Basic Local Alignment Search Tool) was carried out to identify the presence of homologs in different organisms.
Phylogenetic analysis and molecular modelling
BLAST was carried out for the GATD3A mouse protein sequence with RefSeq protein database to identify the presence of homologs in different organisms and fasta files of these homologous sequences were downloaded. Multiple sequence alignment was carried out for all the sequence fasta files using MAFFT tool (Version 7.407) [57]. The output file generated after multiple sequence alignment using MAFFT was further used to construct the phylogenetic tree. The maximum likelihood method was implemented in FastTree (Version 2.1.11) for constructing the phylogenetic tree [58]. The tree was visualized using FigTree (Version 1.4.4) (http://tree.bio.ed.ac.uk/software/figtree/). For modelling, Human GATD3A and DJ-1/PARK7 FASTA amino acid sequences were retrieved from UniProt database (https://www.uniprot.org), after which the sequences were submitted to Phyre2 (Protein Homology/analogY Recognition Engine V 2.0, (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index). Results were ranked based on confidence score and percentage homology between GATD3A and proteins for which there exists crystal structures. Output files were visualized using PyMOL 2.4.2.
Gatd3a−/−.B6 and Gatd3aFLAG/FLAG.B6 mice
All experiments were conducted according to protocols approved by a local Institutional Animal Care and Use Committee at the National Institutes of Health, Bethesda, MD. Insertion of a FLAG epitope tag (DYKDDDDK) to the C-terminus of GATD3A was achieved through CRISPR-mediated homologous recombination (HR) with a 151mer single-strand DNA oligo as the donor template (sequence in table below) as described elsewhere [24]. CRISPRscan was used to select guide RNA (gRNA) candidates near the target. Selected candidate gRNAs were synthesized by in vitro transcription and further tested by Surveyor assay in a MEF cell line carrying a Tet-On Cas9 expression cassette. Zygotes from C57BL/6J mice were microinjected with the final gRNA and the 151 bp donor with Cas9 protein, then transferred into a pseudopregnant CD1 surrogate mother (25 embryos per mother). F0 founders with correct HR were selected by PCR assay designed to amplify HR junctions from tail genomic DNA. PCR genotyping was performed using Standard Taq (New England Biolabs), with a forward primer (GATD3AFLAGFW) in Exon 7, and the reverse (GATD3AFLAGRV) in the 3′UTR of Gatd3a. Samples were resolved on a 4% 3:1 agarose gel to identify PCR products containing the FLAG epitope sequence (421 bp) versus a wildtype allele (397 bp). Founders were backcrossed onto C57BL/6J background for several generations.
For generating the Gatd3a−/− mouse, two gRNAs flanking the genomic region of exon 1 to exon 3 (GATD3AKOE1 gRNA, GATD3AKOE3 gRNA) of the mouse Gatd3a gene were designed and generated as described above. A deletion allele, lacking 865 base pairs in one of the F0 founders, results in the loss of essential coding and splice sites, generating a truncated transcript containing a premature stop codon. PCR genotyping was performed using forward (GATD3AKOFW) and reverse (GATD3AKORV) primers flanking the gRNA cut sites. PCR using standard conditions (New England Biolabs) and resolution on a 1% agarose gel results in detection of deletion product of 213 bp and a wildtype allele of 1078 bp.
Molecular cloning and qPCR analysis
HEK293 and COS7 cells were obtained from American Type Culture Collection and cultured at 37 °C in 5% CO2 in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Cells were passaged at 75–80% confluency twice weekly.
RNA was isolated from HEK293 cells using TrizolLS according to the manufacturer’s instructions (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from 2 μg total RNA using Superscript II Reverse Transcriptase (Invitrogen) with Oligo(dT)12-18 according to the manufacturer’s instructions. Fragments of interest were PCR amplified from cDNA using high-fidelity Phusion Taq polymerase (New England Biolabs) with appropriate primers (below) including desired restriction sites and overhangs. Purified products and CMV vector were digested with appropriate restriction enzymes, and fragments were ligated using T4 DNA Ligase according to the manufacturer’s protocol (New England Biolabs). Positive clones were selected for in transformed DH5α E. coli using Kanamycin, and sequence verified. The Q5 site-directed mutagenesis kit was used for the generation of pCMV-Gatd3aΔ1-33-FLAG following the manufacturer’s guidelines (New England Biolabs).
For qPCR, cDNA was prepared as above from HEK293 cell RNA. Oligonucleotides used are listed in the table below. A QuantStudio 3 real-time PCR system (Thermo Fisher Scientific) was used to perform qPCR using PowerUp SYBR Green master mix, with samples ran in quadruplicate. The delta-delta CT method was used for quantification. Samples were normalized to HPRT house-keeping gene. To analyze mitochondrial biogenesis, qPCR was used to measure mitochondrial DNA to nuclear DNA ratios in MEFs, using the protocol outlined in [59].
GATD3A FLAG 151mer
|
TAAAGAATGTGCTGGAACTCACGGGAAAGGACTACAAAGACGATGACGACAAGTAATGCCACCCGGACCACGCTTGGCCTCCGTGACTGTGGCATCCCCAGCCGGCGTGTGCTCCATGGCTTCAGCCCGGACACAGGAGCCGCTTAGCTAC
|
GATD3AE7 gRNA
|
GGGAAAGTAATGCCACC
|
GATD3AFLAGFW
|
TAAAGCCAGTGCCGACTTGT
|
GATD3AFLAGRV
|
CTCCTTCAGGCAGGAAGTGTCTAT
|
GATD3AKOE1 gRNA
|
CCGGGCTCCCTCCCAGCG
|
GATD3AKOE3 gRNA
|
CCGGGCTCCCTCCCAGCG
|
GATD3AKOFW
|
GCACGTTCTCCTGACACCACTGTG
|
GATD3AKORV
|
CAGCCGGATGACAGTTGCTTTGA
|
CMVGATD3AFW
|
CGCAGGTACCATGGCGGCTGTGAGGGCC
|
CMVGATD3ARV
|
TGCAGCGGCCGCTTATTACTTGTCGTCATCGTCTTTGTAGTCCCCCCCCCCCCCCCCCCCCTTTCCAGTGAGTTCCAGC
|
CMVGATD3AMLSFW
|
CTTCACCTCTCCGTGCCG
|
CMVGATD3AMLSRV
|
CATGGTACCGTCGACTGC
|
qGATD3A_FW
|
AAAACCTGAGCACGTTTGCC
|
qGATD3A_RV
|
TTCAGGACACGCTCCACTTC
|
qPPARGC1A_FW
|
GTCGGAAGACACCCTCTTCTC
|
qPPARGC1A_RV
|
CAGTCCAGGGGCAGAAAAGT
|
qESRRA_FW
|
CGGAGCCCCAGGTGAC
|
qESRRA_RV
|
TCTGTCTCCGAGGAACCCTT
|
qTFAM_FW
|
CCAAAAAGACCTCGTTCAGCTT
|
qTFAM_RV
|
CTTCAGCTTTTCCTGCGGTG
|
qHPRT_FW
|
ACCCCACGAAGTGTTGGATA
|
qHPRT_RV
|
AAGCAGATGGCCACAGAACT
|
PETDJ1FW
|
CGCAGCTAGCATGGCTTCCAAAAGAGCT
|
PETDJ1RV
|
TGCAGGATCCTTATTACTAGTCTTTAAGAACAAG
|
PETDJ1C106AFW
|
AGCCGCCATCGCTGCAGGTCCTACTG
|
PETDJ1C106ARV
|
ATCAGGCCCTTCCGGTTT
|
PETGATD3AFW
|
CGCAGGATCCATGATGCTTCACCTCTCCGTGCCG
|
PETGATD3ARV
|
TGCAAAGCTTTTATTACTTTCCAGTGAGTTCCAGC
|
PETGATD3AC176AFW
|
CATCGGCTTGGCGTGCATTGCACCTGTCCTCGCG
|
PETGATD3AC176ARV
|
GGCTTCCCGGCCTGGTGG
|
16s-rRNAFW
|
CCGCAAGGGAAAGATGAAAGAC
|
16s-rRNARV
|
TCGTTTGGTTTCGGGGTTTC
|
ND1FW
|
CTAGCAGAAACAAACCGGGC
|
ND1RV
|
CCGGCTGCGTATTCTACGTT
|
HK2FW
|
GCCAGCCTCTCCTGATTTTAGTGT
|
HK2RV
|
GGGAACACAAAAGACCTCTTCTGG
|
Immunoblotting
For immunoblotting, cells were washed once in sterile 1× PBS, scraped, and transferred to a 1.5-mL Eppendorf, and collected by centrifugation. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.5% sodium deoxycholate, supplemented with PhosSTOP phosphatase inhibitors and cOmplete protease inhibitor cocktail (Millipore Sigma)) on ice for 30 min, followed by centrifugation (13,600×g, 15 min, 4 °C). Protein concentration was quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Samples were diluted to 1 μg/μl in Laemmli sample buffer with 2-mercaptoethanol (355 mM) and denatured for 10 min at 95 °C. In total, 25 μg of sample was loaded to a MiniPROTEAN TGX gel (Bio-Rad Laboratories) and subjected to SDS-PAGE. Protein was blotted to PVDF membrane using the TransBlot Turbo Transfer system (Bio-Rad Laboratories) and blocked using 5% milk dissolved in TBST (1× Tris buffered saline, pH 7.4, 0.1% Tween-20) for 1 h at RT. Membranes were probed with primary antibody diluted in 1% milk in TBST overnight at 4 °C, followed by 3 × 10-min washes in TBST, and subsequent probing with secondary HRP-conjugated antibody for 1 h at RT. Membranes were washed 3 × 10 min in TBST and imaged using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) using a ChemiDoc Imaging System (Bio-Rad Laboratories). The following antibodies were used in this study:
Antibodies
|
Mouse Anti-FLAG M2
|
Millipore Sigma
|
Cat#F1804
|
Rabbit Anti-GRP75 H-155
|
SCBT
|
Cat#sc-13967
|
Mouse Anti-ATP5A 15H4C4
|
AbCam
|
Cat#ab14748
|
Rabbit Anti-Penta His
|
Qiagen
|
Cat#34660
|
Mouse Anti-Methylglyoxal (1,2-dicarbonyl)
|
Cell Biolabs Inc.
|
Cat#STA-011
|
Rabbit Anti-AGE
|
AbCam
|
Cat#ab23722
|
Rabbit Anti-FLAG M2
|
CST
|
Cat#2368
|
Rabbit Anti-Tomm20 FL-145
|
SCBT
|
Cat#sc-11415
|
Rabbit Anti-Cytochrome C D18C7
|
CST
|
Cat#11940
|
Rabbit Anti-TUFM
|
Thermo Fisher
|
Cat#PA5-27511
|
Rabbit Anti-DJ-1
|
AbCam
|
Cat#ab18257
|
Mouse Anti-GAPDH-71.1
|
Millipore Sigma
|
Cat#G8795
|
Mouse Anti-Total OXPHOS Cocktail
|
AbCam
|
Cat#ab110411
|
Rabbit Anti-C21orf33/GATD3A EPR13213
|
AbCam
|
Cat#ab181366
|
Rabbit Anti-GLO1
|
AbCam
|
Cat#ab96032
|
Rabbit Anti-GLO2
|
AbCam
|
Cat#ab154108
|
Rabbit Anti-Histone H3
|
AbCam
|
Cat#ab16056
|
Rabbit Anti-COXIV
|
AbCam
|
Cat#ab202554
|
Donkey Anti-Mouse IgG Alexa 488
|
Invitrogen
|
Cat#A21202
|
Donkey Anti-Rabbit IgG Alexa 568
|
Invitrogen
|
Cat#D1306
|
Goat Anti-Mouse IgG Alexa 594
|
Invitrogen
|
Cat#A11020
|
Goat Anti-Rabbit IgG ATTO647N
|
Rockland
|
Cat#611-156-122
|
Donkey Anti-Rabbit IgG HRP
|
Jackson Immunoresearch
|
Cat#711035152
|
Donkey Anti-Mouse IgG HRP
|
Jackson Immunoresearch
|
Cat#711035140
|
Immunofluorescence, confocal, and STED microscopy
Cells were seeded in 24-well plate format on poly-D lysine-coated coverslips at 5 × 104 cells/well. At 70–80% confluency, cells were transfected with 50 μl of OptiMEM (Thermo Fisher Scientific) containing a DNA:Lipofectamine 2000 complex in a ratio of 300 ng: 1 μl respectively, and incubated with complexes for 24–48 h, then fixed in 4% PFA for 5 min. For MitoTracker staining, cells were incubated with 250 nM Orange CMTMRos at 37 °C for 30 min, according to the manufacturer’s instructions (Invitrogen), prior to fixation. Samples were blocked using 0.22 μm sterile filtered blocking buffer; 10% normal donkey serum in PBS-T (1× phosphate-buffered saline, pH 7.4, containing 0.03% Triton X-100, and 0.002% NaN3) for 1 h at room temperature (RT). Samples were incubated with primary antibody (FLAG (Millipore Sigma, F1804, 1:1000) mtHsp70/GRP75 H-155 (SCBT, sc-13967, 1:250), TOMM20 (SCBT, sc-11415, 1:200) diluted in blocking buffer overnight at 4 °C, followed by 3 × 10 min washes in PBS-T, and incubated with secondary antibodies (anti-Mouse IgG Alexa 488, Invitrogen, A21202, 1:500, and anti-Rabbit IgG, Alexa 568, A10042, 1:500) and counterstain DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride, Invitrogen, D1306, 1 μg/ml) diluted in blocking buffer for 1 h at RT. Samples were washed 3 × 10 min in PBS-T and mounted with Fluoromount-G (Southern Biotech). Samples were imaged using a Zeiss LSM 880 confocal microscope operating ZEN image capture software. To analyze mitochondrial morphology in MEFs, the Mitochondria Analyzer plugin was used on confocal microscopy images using Mitotracker Orange [60]. Measurements from individual mitochondria were exported from Mitochondria Analyzer into Excel and Graphpad Prism. Mean mitochondrial measurements for each cell were compared between Gatd3a+/+ and Gatd3a−/−.
For super-resolution stimulated emission depletion (STED) microscopy, COS7 cells were transiently transfected with pCMV-Gatd3a-FLAG as described above, fixed in 4% PFA, blocked using blocking buffer (10% normal goat serum, 0.03% Triton X-100, 0.002% NaN3), and incubated with primary antibodies directed against the FLAG epitope (Millipore Sigma, F1804, 1:100), Tomm20 FL-145 (SCBT, sc-11415, 1:50) and mtHsp70/GRP75 H-155 (SCBT, sc-13967, 1:50), and secondary antibodies conjugated with Alexa 594 (Invitrogen, A-11020), and ATTO 647N (Rockland, 611-156-122), and mounted using ProLong Glass Antifade Mountant (Invitrogen). Time-gated STED images were obtained using a commercial STED microscope (SP8 STED 3X; Leica Microsystems), equipped with a white-light laser (470–670nm) and 592 nm, 660 nm, and a pulsed 775-nm STED depletion lasers. A 100×/1.4-N.A. oil immersion objective lens (HCX PL APO STED white; Leica Microsystems) was used for imaging. For resolution comparison, confocal and STED images were taken sequentially for Tomm20 and mtHsp70 labeled with ATTO 647N and imaged using 640-nm excitation and 650–720 nm emission range and for GATD3A:FLAG labeled with Alexa 594 and imaged using 560-nm excitation, a 575- to 630-nm emission detection range at a scan speed of 600 lines per second, with gated hybrid detectors, and the pulsed 775-nm STED depletion laser for both. Samples were imaged with a scan speed of 600 lines per second, a pixel size of 30 nm, and 6-line averages. Z-stacks were collected at 0.160-μm-depth intervals. Fluorescence intensity profiles along a line were measured using Leica LAS X software 3.5.6. (Leica Microsystems, Mannheim, Germany). Images were further deconvolved using the classical maximum likelihood estimation algorithm in Huygens Professional software version 19.10.0p2 64b (SVI, Hilversum, NL), examined, and reconstructed using Imaris software version 9.2.1 (Bitplane, Zurich, Switzerland).
Recombinant protein production
Human DJ-1 and GATD3A, with its mitochondrial localization signal removed, were amplified from cDNA and cloned into the pET28 expression vector containing a 6X-His tag in the C-terminal, with T7 directed expression controlled by a lac operon. A C106A and C176A mutant was generated for DJ-1 and GATD3A respectively using the Q5 site-directed mutagenesis kit (New England Bio Labs). All oligonucleotides used for cloning are given in the supplemental table. BL21 DE3 cells were transformed, and positive clones selected with Kanamycin were induced with 1 mM IPTG for 4–5 h at 37 °C. Cells were pelleted and lysed in lysis buffer (50 mM phosphate buffer, pH 7.4, 300 mM NaCl, 10 mM Imidazole, 14.3 mM 2-mercaptoethanol, supplemented with 1 mg/ml lysozyme and protease inhibitors), on ice for 30 min, followed by sonication at 10% amplitude level (Misonix XL2000) using a microtip probe, for 10 s, repeated 6 times, and centrifuged at 10,000×g for 30 min at 4 °C. Samples were filtered (0.45 μm) before loading onto a HisTrap HP His tag protein purification column (GE Healthcare) on an ÄKTAexplorer FPLC system programmed using UNICORN manager. Total sample was loaded onto the affinity column at a flow rate of 0.5 ml/min and was washed with buffer containing 10 mM imidazole. Affinity-purified protein was eluted over a 10–100 mM imidazole gradient and collected in 1-ml aliquots using a Frac-950 fraction collector. Due to co-elution of bacterial proteins at this concentration of imidazole, size exclusion chromatography was used to further purify the recombinant protein. In total, 100 μl of purified protein was loaded using a loop onto a Superdex 75 Increase 10/300 GL size exclusion column and eluted in sterile filtered, degassed sodium phosphate buffer at a flow rate of 0.3 ml/min. All recombinant samples were analyzed using SDS-PAGE followed by Coomassie Brilliant Blue staining according to the manufacturer’s instructions (Thermo Fisher Scientific), and immunoblotting using a Penta-His antibody diluted in a modified blocking buffer of 3% BSA in TBS (Qiagen, 34660, 1:1000).
Amidolysis and slot blot immunodetection assays
A modified version of the Glutamine/Glutamate-Glo Assay (Promega, J8021) was used to assess the amidolysis capacity of GATD3A and DJ-1, using L-glutamine as the substrate. An L-glutamine standard curve was prepared using a 100 mM L-glutamine stock (Thermo Fisher Scientific). Briefly, glutamine standards were aliquoted in triplicate per condition in a flat-bottomed white plastic 96-well plate. A recombinant protein sample [2 μM (GATD3A or DJ-1), glutaminase, and no enzyme control] was diluted in glutaminase buffer and added to wells containing glutamine, and gently mixed. Samples were incubated for 30 min at RT to allow for conversion of glutamine to glutamate. Glutamate detection solution (luciferin detection solution, reductase, reductase substrate, glutamate dehydrogenase, NAD) was prepared and added to each well resulting in a final ratio of 1:1:2 for sample volume:recombinant protein solution:glutamate detection solution. Following gentle shaking, the reaction was incubated in dark for 1 h at room temperature. Luminescence was detected on a GloMax Navigator Microplate Luminometer. Signal was detected for 1 s, where signal is directly proportional to the glutamate concentration.
For the deglycase assay, plasmid DNA (1 ng/μl) and γ-globulin (1 mg/ml) was incubated with either glyoxal (GO) or methylglyoxal (MGO) (5 mM) in N2 gassed sodium phosphate buffer (pH 7.4) at 37 °C. Timepoints were stored at – 20 °C. After 2 h, glycated samples were incubated with 5 μg of recombinant protein (DJ-1, GATD3A, or GATD3AC176A) for a further 2 h at 37 °C. For immune-detection, DNA samples were denatured in SSC buffer at 98 °C for 10 min and applied to Amersham Hybond-N+ nylon membranes using a Minifold II vacuum slot blot manifold. DNA was crosslinked to nylon membranes on a Stratalinker UV Crosslinker at 120,000 μJ/cm2 (Stratagene). Protein samples were denatured in 8M urea at 30 °C for 10 min and applied similarly to nylon membranes. Membranes were blocked in 5% milk in TBST and incubated overnight in either antibodies with epitopes raised against either 1,2-dicarbonyls (Cell Biolabs Inc., STA-011, 1:1000) or advanced glycation end products (AbCam, ab23722, 1:1000). Blots were developed as previously described. For pulse-chase experiments, mouse embryonic fibroblasts were isolated from the Gatd3a−/− mouse and wildtype littermates as previously described [61]. Cells were seeded (5 × 105) in a 6-well plate and treated with 1 mM GO, and removed following 12 h of incubation. Samples were allowed to recover for a further 12 h before harvesting in RIPA buffer and were subjected to immunoblotting as described above.
RNA immunoblotting
Mice were perfused with HBSS and heart left ventricle was isolated and macerated on ice. Samples were suspended in sterile PBS and subjected to RNA isolation using the miRNeasy kit, according to the manufacturer’s instructions (Qiagen). Concentration and RNA integrity was assessed using an Agilent RNA 6000 Nano kit. Purified RNA samples were normalized and denatured in 2× NorthernMax formaldehyde loading dye (Thermo Fisher Scientific) at 65 °C for 15 min. Samples were electrophoresed in 10% Novex TBE Urea gels at 250 V for 45 min. Gels were stained using UltraPure EtBr (1:10,000) in 0.5× TBE for 1 h before imaging using a UV transilluminator. Gels were destained in 0.5× TBE before electroblotting to a nylon membrane at 18V/~300 mA for 60 min using a semidry transfer apparatus, and UV crosslinked as described previously. Membranes were blocked in 5% milk and subjected to immunodetection using methods described above.
Mitochondrial isolation and fractionation
HEK293 cells were lysed in 0.25 M sucrose buffer and cell debris was removed following centrifugation at 100×g. Supernatant was subjected to centrifugation at 800×g and nuclear pellet was retained. Supernatant was centrifuged at 8000×g, and post-mitochondrial supernatant was retained as a cytosolic fractionation following clearing at 15,000×g. SDS-PAGE was performed on protein samples as above and probed for nuclear (Histone H3, ab16056, 1:2000) cytosolic (GAPDH, Millipore Sigma G8795, 1:2000) and mitochondria (COXIV, ab202554, 1:2000). In mice, isolated mitochondria were fractionated using previously published methods [62] with minor adjustments. Briefly, Gatd3aFLAG/FLAG mice were anaesthetized (Ketamine 80–100 mg/kg, Xylazine 8–10 mg/kg) and perfused using sterile HBSS (Thermo Fisher Scientific). Hearts were isolated, diced, and homogenized in a Teflon-glass Potter-Elvehjem homogenizer with ice-cold isolation buffer (220 mM mannitol, 700 mM sucrose, 2 mM Tris, pH 7.4, 1 mM EDTA, 20 mM HEPES, pH 7.2, 0.4% BSA). Samples were centrifuged at 600×g for 5 min. Supernatants were centrifuged at 17,000×g for 10 min at 4 °C. Pellets were washed twice in isolation buffer, without EDTA. Mitochondrial concentration was determined using a Bradford assay (Bio-Rad Laboratories). Mitochondria were incubated at 2 mg/ml in hypotonic buffer (2 mM KCl, 10 mM HEPES, pH 7.2) for 20 min on ice, and centrifuged at 10,000×g. Pellets were washed twice in wash buffer (150 mM KCl, 10 mM HEPES, pH 7.2). All supernatants were combined and subjected to TCA precipitation, creating an intermembrane space fraction. Pellets were resuspended in hypotonic buffer and subjected to three freeze thaw cycles, followed by sonication (1% amplitude, 5 s, ×3) and centrifuged at 110,000×g for 30 min and retained as an outer and inner membrane fraction. Supernatant was subjected to TCA precipitation and stored as a matrix fraction. Samples were subjected to SDS-PAGE and blotting. Membranes were probed for FLAG M2 antibody (CST, 2368, 1:1000) and compartment-specific antibodies; outer membrane (Tomm20 FL-145, SCBT, sc-11415, 1:1000), inner membrane (ATP5A 15H4C4, Abcam, ab14748, 1:2500), intermembrane space (Cytochrome c D18C7, CST, 11940, 1:3000), and matrix (mtHsp70/GRP75 H-155, SCBT, sc-13967, 1:500) and probed with HRP-conjugated secondary antibodies raised against rabbit IgG (Jackson ImmunoResearch, 711035152, 1:3000) and mouse IgG (Jackson ImmunoResearch, 711035140, 1:3000).
Immunoprecipitation and mass spectrometry
GATD3A:FLAG was immunoprecipitated from Gatd3aFLAG/FLAG mouse heart mitochondria using methods described previously [63] with the following modifications. Following mitochondria isolation described above, sample was resuspended in ice-cold IP buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 1 mM DTT, supplemented with protease inhibitors). Following maceration with a glass homogenizer, detergent solution was added (final concentration 2% Digitonin, 2.5% NP-40), and end over end agitation was carried out for 15 min at 4 °C, followed by centrifugation (16,000×g, 10 min, 4 °C). Supernatant concentration was quantified using the BCA method. Magnetic anti-FLAG epitope conjugated beads (Millipore Sigma, M8823) were washed with IP buffer containing detergent three times, and equal concentrations of protein were added to beads at 4 °C overnight. Beads were washed (20 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% digitonin, 10% glycerol) four times, with the final wash containing no detergent or glycerol. Protein was eluted in final wash buffer containing 0.2 mg/ml FLAG peptide. Eluate was subjected to centrifugal filtration for removal of FLAG peptide using 3K NMWL filtration units, using final wash buffer for dialysis.
Immunoprecipitated proteins were dried and solubilized with 6 M urea, 2 M thiourea, and 50 mM ammonium bicarbonate, pH 7.8. Protein solutions were reduced with 10 mM dithiothreitol (DTT) to reduce the disulfide bonds for 1 h at 37 °C. To prevent reformation of disulfide bonds, samples were incubated at room temperature in the dark with 20 mM of iodoacetamide for 45 min. An additional 10 mM of DTT was added to quench the excess iodoacetamide for 10 min at room temperature. All samples were diluted 6-fold with 50 mM ammonium bicarbonate followed by the addition of Trypsin (Promega, Madison, WI, USA) for overnight digestion at 37 °C. Peptide digests were acidified with 10% trifluoroacetic acid (TFA) to a final 1% concentration, pH 3. Digests were dried down and resuspended in 0.1% formic acid. The resulting peptide mixture was concentrated and desalted with C18 Zip-tips (Millipore Sigma), following the company’s protocol. Desalted digests were analyzed by mass spectrometry. Mass spectrometry experiments were performed on an Orbitrap Lumos Tribrid coupled with an Ultimate 3000-nLC (Thermo Fisher Scientific). Peptides were separated on an EASY-Spray C18 column (Thermo Scientific; 75 μm × 50 cm inner diameter, 2 μm particle size, and 100 Å pore size). Separation was achieved by 4–28% linear gradient of acetonitrile + 0.1% formic acid over 60 min. An electrospray voltage of 1.9 kV was applied to the eluent via the EASY-Spray column electrode. The Orbitrap Lumos was operated in positive ion data-dependent mode. Full-scan MS1 was performed in the Orbitrap with a normal precursor mass range of 375–1500 m/z at a resolution of 120k. The automatic gain control (AGC) target and maximum accumulation time settings were set to 4 × 105 and 50 ms, respectively. MS2 was triggered by selecting the most intense precursor ions above an intensity threshold of 5 × 103 for collision-induced dissociation (CID)-MS2 fragmentation with an AGC target and maximum accumulation time settings of 2 × 103 and 300 ms, respectively. Mass filtering was performed by the quadrupole with 1.6 m/z transmission window, followed by CID fragmentation in the ion trap (rapid mode) and a normalized collision energy (NCE) of 35%. To improve the spectral acquisition rate, parallelizable time was activated. The number of MS2 spectra acquired between full scans was restricted to a duty cycle of 3 s. Raw data files were processed using Proteome Discoverer (v2.2, Thermo Fisher Scientific), using Mascot v2.5.1 (Matrix Sciences) search node. All searches were performed against UniProt Knowledgebase protein database with the selected taxonomy Mus musculus (mouse). Search modifications used were as follows: (fixed) carbamidomethyl of cysteine, (variable) oxidation of methionine, deamidation (NQ), and acetyl on protein N-terminus. For MS2, the precursor mass tolerance of 12 ppm and fragment mass tolerances of 0.5 Da were applied, respectively. Up to two missed tryptic cleavages were permitted. Percolator was used to calculate the false discovery rate (FDR) of peptide spectrum matches, set to a p-value of 0.05 [64].
For identification of co-eluting proteins following FPLC, samples were run on SDS-PAGE and stained using Coomassie Brilliant Blue. Bands of interest were excised, and gel pieces were destained using destaining solution (10% glacial acetic acid, 50% methanol, 40% Milli-Q H2O). The excised gel bands were subjected to previously published trypsin digestion methods with minor modifications [65]. Post reduction and alkylation steps, the dried gel bands were rehydrated in a 12.5 ng/μL trypsin solution in 50 mM ammonium bicarbonate/0.05% AALS (Anionic Acid Labile Surfactant, Protea), overnight digestion at 37 °C, pH 7.8. Digests were collected, acidified with 10% trifluoroacetic acid (TFA) to a final concentration of 1%, agitating for 15 min at 37°C for surfactant degradation and dried down. The resulting peptide mixture was concentrated and desalted with C18 Zip-tips (Millipore Sigma), following the company’s protocol. Peptides were separated on an EASY-Spray C18 column (Thermo Scientific; 75 μm × 50 cm inner diameter, 2 μm particle size and 100 Å pore size). Separation was achieved by 4–30% linear gradient of acetonitrile + 0.1% formic acid for 65 min. An electrospray voltage of 1.9 kV was applied to the eluent via the EASY-Spray column electrode. The Orbitrap Lumos was operated in positive ion data-dependent mode. Full-scan MS1 was performed in the Orbitrap with a normal precursor mass range of 375–1500 m/z at a resolution of 120k. The automatic gain control (AGC) target and maximum accumulation time settings were set to 4 × 105 and 50 ms, respectively. MS2 was triggered by selecting the most intense precursor ions above an intensity threshold of 2.5 × 104 for collision-induced dissociation (CID)-MS2 fragmentation with an AGC target and maximum accumulation time settings of 5 × 104 and 50 ms, respectively. Mass filtering was performed by the quadrupole with 0.7 m/z transmission window, followed by CID fragmentation in the Orbitrap and a collision energy of 35% at a resolution of 15k. To improve the spectral acquisition rate, parallelizable time was activated. The number of MS2 spectra acquired between full scans was restricted to a duty cycle of 3 s. Raw data files were processed with Proteome Discoverer (v2.2, Thermo Fisher Scientific) software, using Sequest HT (Thermo Fisher Scientific) search node. All peak lists were searched in parallel against the UniProtKB/Swiss-Prot protein databases Homo sapiens (20,316 sequences, released 2018_11) and Escherichia coli strain K12 (4418 sequences, released 2018_11) concatenated with reversed copies of all sequences. The following search parameters were set for MS1 tolerance of 10 ppm; orbitrap-detected MS/MS mass tolerance of 0.02 Da; enzyme specificity was set as trypsin with maximum two missed cleavages; minimum peptide length of 6 amino acids; fixed modification of Cys (carbamidomethylation); variable modifications of methionine oxidation, deamidation of Asn, and Gln, and Protein N-terminus Acetyl. Target Decoy was used to calculate the false discovery rate (FDR) of peptide spectrum matches, set to a p-value < 0.05 [66].
Oxygen Consumption Rate (OCR) measurement
MEFs were cultured in DMEM + 10% FBS. Cells were collected by trypsinization, counted, and plated (20,000 cells per well) on Seahorse XFe24 multiwell plates. Plates were incubated at 37 °C overnight in a CO2 incubator. The following day, medium was removed, cells were washed with DMEM without FBS, and 1 ml of DMEM containing 0.2 mM glyoxal was added to the experimental wells. We then added 1 ml medium without the compound to the control wells, and the plates were incubated at 37 °C overnight. After treatment, the cells were washed with PBS, then with the Seahorse XF DMEM buffer, and OCR was measured using the XFe24 Extracellular Flux Analyzer (Agilent Technologies). Inhibitors were used at the following concentrations: oligomycin, 2 μM; Bam15, 5 μM; and rotenone, 1 μM. The results were analyzed using the Agilent Wave software. Results were normalized to cell number.
Transmission Electron Microscopy (TEM)
Hearts from perfused mice were isolated, and 1-mm3 sections of left ventricle were dissected and fixed 2.5% buffered glutaraldehyde. Tissues were processed for TEM as previously described [67]. Briefly, specimens were washed in phosphate-buffered saline (PBS), post-fixed in 0.5% osmium tetroxide (OsO4), rinsed, dehydrated, then embedded in epoxy resin. The blocks were sectioned at ~90-nm thickness on a Leica EM UC6 ultramicrotome (Leica, Austria), double-stained with uranyl acetate and lead citrate, and imaged with JEOL JEM-1010 electron microscope (JEOL, Japan).
Mitochondrial electron density was quantified using the histogram function in Fiji (ImageJ). N = 4 left ventricles per genotype were used, with N = 3 images per animal being used for quantification. Electron density is represented as a ratio of the mean greyscale area for three muscle fiber sections compared to the mean greyscale for all mitochondria in a field of view.
Quantification and statistical analysis
Two-tailed t tests were used for comparison between two groups using GraphPad Prism v8.0. All comparisons were two-sided, and p-values of less than 0.05 were considered to indicate statistical significance. In the instances where alternative statistical tests were used, this has been noted in the figure legend. Specific statistical tests and metrics (median, mean, standard error) used for comparisons, along with sample sizes, are described in the figure legends.