#METABOLOMICS WORKBENCH Kugan91_20250529_190524 DATATRACK_ID:5971 STUDY_ID:ST003961 ANALYSIS_ID:AN006509 PROJECT_ID:PR002482 VERSION 1 CREATED_ON June 4, 2025, 4:50 pm #PROJECT PR:PROJECT_TITLE Complex II assembly drives metabolic adaptation to OXPHOS dysfunction. PR:PROJECT_SUMMARY During acute oxidative phosphorylation (OXPHOS) dysfunction, the reverse PR:PROJECT_SUMMARY activity of succinate dehydrogenase (Complex II) maintains the redox state of PR:PROJECT_SUMMARY Coenzyme Q by utilizing either fumarate or oxygen as terminal electron PR:PROJECT_SUMMARY acceptors. The tendency for one over another has been suggested to be PR:PROJECT_SUMMARY tissue-specific, but the underlying mechanism and consequence of this is PR:PROJECT_SUMMARY unknown. Using quantitative proteomics to screen a panel of HEK293T knockout PR:PROJECT_SUMMARY cell lines, we identified an increase in SDHAF2 protein, a Complex II assembly PR:PROJECT_SUMMARY factor that enhances the flavination of catalytic subunit SDHA, as critical for PR:PROJECT_SUMMARY metabolic adaptation during OXPHOS stress in HEK293T cells. Loss of SDHAF2 PR:PROJECT_SUMMARY during Complex III inhibition resulted in a reduction in Complex II F-site PR:PROJECT_SUMMARY derived reactive oxygen species (ROS), a severe growth impairment, and a net PR:PROJECT_SUMMARY reductive TCA cycle driven by an inability of mitochondria to support additional PR:PROJECT_SUMMARY Complex II assembly. This in turn leads to use of fumarate as terminal electron PR:PROJECT_SUMMARY acceptor at the cost of a ROS-mediated switch to glycolysis. Cell lines adapted PR:PROJECT_SUMMARY to glycolysis did not accumulate SDHAF2 upon OXPHOS stress and exhibited a net PR:PROJECT_SUMMARY reductive TCA cycle and mild growth phenotypes with or without SDHAF2 being PR:PROJECT_SUMMARY present. Thus, our study reveals how Complex II assembly controls a balance PR:PROJECT_SUMMARY between protection of the Q-pool and ROS-meditated signaling during oxidative PR:PROJECT_SUMMARY stress in cells reliant on mitochondrial OXPHOS. PR:INSTITUTE University of Melbourne PR:LAST_NAME Roopasingam PR:FIRST_NAME Kugapreethan PR:ADDRESS 30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, PR:ADDRESS University of Melbourne, Parkville, VIC, Australia. PR:EMAIL k.roopasingam@unimelb.edu.au PR:PHONE 0434297212 #STUDY ST:STUDY_TITLE Measuring carbon flux into TCA cycle using 13C5-glutamine tracer metabolomics ST:STUDY_SUMMARY This study investigates carbon flux into the TCA cycle in HEK293T cell lines ST:STUDY_SUMMARY with and without SDHAF2 elevation during OXPHOS dysfunction using targeted GC-MS ST:STUDY_SUMMARY analysis ST:INSTITUTE University of Melbourne ST:LAST_NAME Roopasingam ST:FIRST_NAME Kugapreethan ST:ADDRESS 30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, ST:ADDRESS University of Melbourne, Parkville, VIC, Australia. ST:EMAIL k.roopasingam@unimelb.edu.au ST:PHONE 0434297212 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 3_HEKWT_SCAN+_001_UNK-0016_16042025_16 Sample source:HEK_WT_001 | Treatment:HEK_WT RAW_FILE_NAME(Raw file name)=3_HEKWT_SCAN+_001_UNK-0016_16042025_16.qgd SUBJECT_SAMPLE_FACTORS - 3_HEKWT_SCAN+_002_UNK-0012_16042025_12 Sample source:HEK_WT_002 | Treatment:HEK_WT RAW_FILE_NAME(Raw file name)=3_HEKWT_SCAN+_002_UNK-0012_16042025_12.qgd SUBJECT_SAMPLE_FACTORS - 3_HEKWT_SCAN+_003_UNK-0014_16042025_14 Sample source:HEK_WT_003 | Treatment:HEK_WT RAW_FILE_NAME(Raw file name)=3_HEKWT_SCAN+_003_UNK-0014_16042025_14.qgd SUBJECT_SAMPLE_FACTORS - 4_HEK_A-A_SCAN+_001_UNK-0022_16042025_22 Sample source:HEK_A-A_001 | Treatment:HEK_A-A RAW_FILE_NAME(Raw file name)=4_HEK_A-A_SCAN+_001_UNK-0022_16042025_22.qgd SUBJECT_SAMPLE_FACTORS - 4_HEK_A-A_SCAN+_002_UNK-0018_16042025_18 Sample source:HEK_A-A_002 | Treatment:HEK_A-A RAW_FILE_NAME(Raw file name)=4_HEK_A-A_SCAN+_002_UNK-0018_16042025_18.qgd SUBJECT_SAMPLE_FACTORS - 4_HEK_A-A_SCAN+_003_UNK-0020_16042025_20 Sample source:HEK_A-A_003 | Treatment:HEK_A-A RAW_FILE_NAME(Raw file name)=4_HEK_A-A_SCAN+_003_UNK-0020_16042025_20.qgd SUBJECT_SAMPLE_FACTORS - 5_HEKSDHAF2_SCAN+_001_UNK-0029_16042025_29 Sample source:HEKSDHAF2KO_001 | Treatment:HEKSDHAF2KO RAW_FILE_NAME(Raw file name)=5_HEKSDHAF2_SCAN+_001_UNK-0029_16042025_29.qgd SUBJECT_SAMPLE_FACTORS - 5_HEKSDHAF2_SCAN+_002_UNK-0024_16042025_24 Sample source:HEKSDHAF2KO_002 | Treatment:HEKSDHAF2KO RAW_FILE_NAME(Raw file name)=5_HEKSDHAF2_SCAN+_002_UNK-0024_16042025_24.qgd SUBJECT_SAMPLE_FACTORS - 5_HEKSDHAF2_SCAN+_003_UNK-0027_16042025_27 Sample source:HEKSDHAF2KO_003 | Treatment:HEKSDHAF2KO RAW_FILE_NAME(Raw file name)=5_HEKSDHAF2_SCAN+_003_UNK-0027_16042025_27.qgd SUBJECT_SAMPLE_FACTORS - 6_HEKSDHAF2_A-A_SCAN+_001_UNK-0035_16042025_35 Sample source:HEKSDHAF2KO_A-A_001 | Treatment:HEKSDHAF2KO_A-A RAW_FILE_NAME(Raw file name)=6_HEKSDHAF2_A-A_SCAN+_001_UNK-0035_16042025_35.qgd SUBJECT_SAMPLE_FACTORS - 6_HEKSDHAF2_A-A_SCAN+_002_UNK-0031_16042025_31 Sample source:HEKSDHAF2KO_A-A_002 | Treatment:HEKSDHAF2KO_A-A RAW_FILE_NAME(Raw file name)=6_HEKSDHAF2_A-A_SCAN+_002_UNK-0031_16042025_31.qgd SUBJECT_SAMPLE_FACTORS - 6_HEKSDHAF2_A-A_SCAN+_003_UNK-0033_16042025_33 Sample source:HEKSDHAF2KO_A-A_003 | Treatment:HEKSDHAF2KO_A-A RAW_FILE_NAME(Raw file name)=6_HEKSDHAF2_A-A_SCAN+_003_UNK-0033_16042025_33.qgd SUBJECT_SAMPLE_FACTORS - 7_SDHAF2_SDHAF2FLAG_SCAN+_001_UNK-0041_16042025_41 Sample source:HEKSDHAF2_SDHAF2FLAG_001 | Treatment:HEKSDHAF2_SDHAF2FLAG RAW_FILE_NAME(Raw file name)=7_SDHAF2_SDHAF2FLAG_SCAN+_001_UNK-0041_16042025_41.qgd SUBJECT_SAMPLE_FACTORS - 7_SDHAF2_SDHAF2FLAG_SCAN+_002_UNK-0037_16042025_37 Sample source:HEKSDHAF2_SDHAF2FLAG_002 | Treatment:HEKSDHAF2_SDHAF2FLAG RAW_FILE_NAME(Raw file name)=7_SDHAF2_SDHAF2FLAG_SCAN+_002_UNK-0037_16042025_37.qgd SUBJECT_SAMPLE_FACTORS - 7_SDHAF2_SDHAF2FLAG_SCAN+_003_UNK-0039_16042025_39 Sample source:HEKSDHAF2_SDHAF2FLAG_003 | Treatment:HEKSDHAF2_SDHAF2FLAG RAW_FILE_NAME(Raw file name)=7_SDHAF2_SDHAF2FLAG_SCAN+_003_UNK-0039_16042025_39.qgd SUBJECT_SAMPLE_FACTORS - 8_SDHAF2_CIIIKO_A-A_SCAN+_001_UNK-0048_16042025_48 Sample source:HEKSDHAF2KO_CIIIKO_A-A_001 | Treatment:HEKSDHAF2KO_CIIIKO_A-A RAW_FILE_NAME(Raw file name)=8_SDHAF2_CIIIKO_A-A_SCAN+_001_UNK-0048_16042025_48.qgd SUBJECT_SAMPLE_FACTORS - 8_SDHAF2_CIIIKO_A-A_SCAN+_002_UNK-0043_16042025_43 Sample source:HEKSDHAF2KO_CIIIKO_A-A_002 | Treatment:HEKSDHAF2KO_CIIIKO_A-A RAW_FILE_NAME(Raw file name)=8_SDHAF2_CIIIKO_A-A_SCAN+_002_UNK-0043_16042025_43.qgd SUBJECT_SAMPLE_FACTORS - 8_SDHAF2_CIIIKO_A-A_SCAN+_003_UNK-0045_16042025_45 Sample source:HEKSDHAF2KO_CIIIKO_A-A_003 | Treatment:HEKSDHAF2KO_CIIIKO_A-A RAW_FILE_NAME(Raw file name)=8_SDHAF2_CIIIKO_A-A_SCAN+_003_UNK-0045_16042025_45.qgd #COLLECTION CO:COLLECTION_SUMMARY HEK293Tvcells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo CO:COLLECTION_SUMMARY Fisher Scientific) supplemented with 10 % (v/v) fetal bovine serum (FBS, CO:COLLECTION_SUMMARY CellSera), 50 µg/mL uridine (Sigma), and a mixture of 100 µg/mL streptomycin CO:COLLECTION_SUMMARY and 100 units/mL penicillin (Thermo Scientific). CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY The relevant cell lines were treated with doxycycline and Antimycin A for 24 and TR:TREATMENT_SUMMARY 8 hours prior to the addition of tracer and maintained until the completion of TR:TREATMENT_SUMMARY the experiment, respectively. Cells were then washed with PBS and snap-frozen by TR:TREATMENT_SUMMARY direct addition of liquid nitrogen. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Polar metabolites were extracted from snap-frozen cells using 600 µL of HPLC SP:SAMPLEPREP_SUMMARY grade methanol:chloroform mixture (9:1; [v/v]), along with internal standards SP:SAMPLEPREP_SUMMARY (1.66μM 13C5,15N-valine, 1.66μM 13C6-sorbitol), as described above. The SP:SAMPLEPREP_SUMMARY clarified supernatants and pooled biological quality controls (PBQC’s) were SP:SAMPLEPREP_SUMMARY dried using a CentriVap concentrator (Labconco). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Shimadzu GC-2010 CH:COLUMN_NAME 30 m Agilent DB-5 column with a diameter of 0.25mm and a film thickness of 1µm CH:SOLVENT_A N/A CH:SOLVENT_B N/A CH:FLOW_GRADIENT N/A CH:FLOW_RATE 1ml/min CH:COLUMN_TEMPERATURE 100 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Shimadzu TQ8050NX MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The GC-MS system used comprised of an AOC6000 autosampler, a 2030 Shimadzu gas MS:MS_COMMENTS chromatograph and a TQ8050NX triple quadrupole mass spectrometer (Shimadzu, MS:MS_COMMENTS Japan)with an electron ionisation source(-70eV). The mass spectrometer was tuned MS:MS_COMMENTS according to the manufacturer’s recommendations using MS:MS_COMMENTS tris-(perfluorobutyl)-amine (CF43). GC-MS was performed on a 30m Agilent DB-5 MS:MS_COMMENTS column with 0.25mm internal diameter column and 1µm film thickness. The MS:MS_COMMENTS injection temperature (inlet) was set at 280°C, the MS transfer line at 280°C MS:MS_COMMENTS and the ion source adjusted to 200°C. Helium was used as the carrier gas at a MS:MS_COMMENTS flow rate of 1 mL/min and argon gas was used in the collision cell. The analysis MS:MS_COMMENTS of the derivatised samples was performed under the following oven temperature MS:MS_COMMENTS program; 100°C start temperature, hold for 4 minutes, followed by a 10°C/min MS:MS_COMMENTS oven temperature ramp to 320°C with a following final hold for 11 minutes. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Relative abundance MS_METABOLITE_DATA_START Samples 3_HEKWT_SCAN+_001_UNK-0016_16042025_16 3_HEKWT_SCAN+_002_UNK-0012_16042025_12 3_HEKWT_SCAN+_003_UNK-0014_16042025_14 4_HEK_A-A_SCAN+_001_UNK-0022_16042025_22 4_HEK_A-A_SCAN+_002_UNK-0018_16042025_18 4_HEK_A-A_SCAN+_003_UNK-0020_16042025_20 5_HEKSDHAF2_SCAN+_001_UNK-0029_16042025_29 5_HEKSDHAF2_SCAN+_002_UNK-0024_16042025_24 5_HEKSDHAF2_SCAN+_003_UNK-0027_16042025_27 6_HEKSDHAF2_A-A_SCAN+_001_UNK-0035_16042025_35 6_HEKSDHAF2_A-A_SCAN+_002_UNK-0031_16042025_31 6_HEKSDHAF2_A-A_SCAN+_003_UNK-0033_16042025_33 7_SDHAF2_SDHAF2FLAG_SCAN+_001_UNK-0041_16042025_41 7_SDHAF2_SDHAF2FLAG_SCAN+_002_UNK-0037_16042025_37 7_SDHAF2_SDHAF2FLAG_SCAN+_003_UNK-0039_16042025_39 8_SDHAF2_CIIIKO_A-A_SCAN+_001_UNK-0048_16042025_48 8_SDHAF2_CIIIKO_A-A_SCAN+_002_UNK-0043_16042025_43 8_SDHAF2_CIIIKO_A-A_SCAN+_003_UNK-0045_16042025_45 Factors Sample source:HEK_WT_001 | Treatment:HEK_WT Sample source:HEK_WT_002 | Treatment:HEK_WT Sample source:HEK_WT_003 | Treatment:HEK_WT Sample source:HEK_A-A_001 | Treatment:HEK_A-A Sample source:HEK_A-A_002 | Treatment:HEK_A-A Sample source:HEK_A-A_003 | Treatment:HEK_A-A Sample source:HEKSDHAF2KO_001 | Treatment:HEKSDHAF2KO Sample source:HEKSDHAF2KO_002 | Treatment:HEKSDHAF2KO Sample source:HEKSDHAF2KO_003 | Treatment:HEKSDHAF2KO Sample source:HEKSDHAF2KO_A-A_001 | Treatment:HEKSDHAF2KO_A-A Sample source:HEKSDHAF2KO_A-A_002 | Treatment:HEKSDHAF2KO_A-A Sample source:HEKSDHAF2KO_A-A_003 | Treatment:HEKSDHAF2KO_A-A Sample source:HEKSDHAF2_SDHAF2FLAG_001 | Treatment:HEKSDHAF2_SDHAF2FLAG Sample source:HEKSDHAF2_SDHAF2FLAG_002 | Treatment:HEKSDHAF2_SDHAF2FLAG Sample source:HEKSDHAF2_SDHAF2FLAG_003 | Treatment:HEKSDHAF2_SDHAF2FLAG Sample source:HEKSDHAF2KO_CIIIKO_A-A_001 | Treatment:HEKSDHAF2KO_CIIIKO_A-A Sample source:HEKSDHAF2KO_CIIIKO_A-A_002 | Treatment:HEKSDHAF2KO_CIIIKO_A-A Sample source:HEKSDHAF2KO_CIIIKO_A-A_003 | Treatment:HEKSDHAF2KO_CIIIKO_A-A Glutamate_246 3814740.3 5406581.8 5382445.4 969798.6 1317825 1144875.1 2192365 3105259.8 2539342.5 139265.3 265651 406222.4 3758740.4 4422639.3 4120041.6 785047.8 958297.7 1204641.3 Glutamate_247 919781.1 1386566.5 1376769.7 53050.3 73113 65196.8 566626 795178.9 638564.7 9051.4 15728.1 23095.1 823639.1 1084941.1 1001759.4 34953.2 52371 59812.3 Glutamate_248 1645110 2445309.2 2477597 46828.6 65210.6 52368.6 936916.7 1348999.7 1121557.9 8455.3 22116.2 28344 1493502.8 1908126.9 1790911 37472.1 54446.3 49703.2 Glutamate_249 276879 396422.2 407038.1 208327.1 275399.2 237696.4 170220.7 238363.1 182285.6 36485.3 63452.1 108614.4 249643.3 312697.1 289860.2 148237.3 182131.7 227963 Glutamate_250 4814706.8 6969758.2 7121525.2 4195097.4 5520347.1 4913666.5 2776423.2 4056967 3304622.8 674404.6 1221330.6 2073937.5 4499724.3 5665398.8 5195731.4 3007370.6 3615048.9 4677537.7 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name HMDB ID Glutamate_246 HMDB0000148 Glutamate_247 HMDB0000148 Glutamate_248 HMDB0000148 Glutamate_249 HMDB0000148 Glutamate_250 HMDB0000148 METABOLITES_END #END