#METABOLOMICS WORKBENCH spinelli_lab_20240812_130753 DATATRACK_ID:5095 STUDY_ID:ST003404 ANALYSIS_ID:AN005586 PROJECT_ID:PR001988 VERSION 1 CREATED_ON August 13, 2024, 7:33 am #PROJECT PR:PROJECT_TITLE Rhodoquinone is an Electron Carrier in the Mammalian Electron Transport Chain PR:PROJECT_SUMMARY Ubiquinone (UQ), the only known electron carrier in the mammalian electron PR:PROJECT_SUMMARY transport chain (ETC), delivers electrons to both oxygen (O2) and fumarate as PR:PROJECT_SUMMARY terminal electron acceptors. As fumarate has a lower reduction potential than PR:PROJECT_SUMMARY UQ, fumarate reduction is only thermodynamically favorable when ubiquinol, the PR:PROJECT_SUMMARY reduced form of UQ, accumulates. Paradoxically, some tissues reduce fumarate PR:PROJECT_SUMMARY without ubiquinol buildup, suggesting another mechanism enables fumarate PR:PROJECT_SUMMARY reduction in mammals. Here, we identify rhodoquinone (RQ), a novel component of PR:PROJECT_SUMMARY the mammalian ETC that carries electrons to fumarate, instead of O2, as the PR:PROJECT_SUMMARY terminal electron acceptor. RQ, which is undetectable in cultured mammalian PR:PROJECT_SUMMARY cells, is enriched in mitochondria from mouse and human tissues that catalyze PR:PROJECT_SUMMARY high levels of fumarate reduction. UQ and RQ-directed ETC circuits support PR:PROJECT_SUMMARY unique programs of mitochondrial function. Through expression of a bacterial PR:PROJECT_SUMMARY enzyme that converts UQ into RQ and development of a novel RQ analog, we PR:PROJECT_SUMMARY demonstrate that reprogramming the mammalian ETC from the UQ to RQ circuit PR:PROJECT_SUMMARY renders cells highly resistant to hypoxia exposure in vitro and in vivo. Thus, PR:PROJECT_SUMMARY we establish RQ as a fundamental component of the mammalian ETC and unveil that PR:PROJECT_SUMMARY reprogramming the ETC to the RQ-circuit is a tractable strategy to ameliorate PR:PROJECT_SUMMARY hypoxia-related conditions. PR:INSTITUTE UMass Chan Medical School PR:LAST_NAME UMass Chan PR:FIRST_NAME Spinelli Lab PR:ADDRESS 55 Lake Avenue North, Worcester, Massachusetts, 01605, USA PR:EMAIL spinellilab@gmail.com PR:PHONE (508) 856-8989 ext. 68148 #STUDY ST:STUDY_TITLE Aspartate Tracing in SDHB KO RquA 143B Cells ST:STUDY_SUMMARY 143B cells that were cloned to have the SDHB(succinate dehydrogenase complex ST:STUDY_SUMMARY iron sulfur subunit B) KO and expression of the RquA enzyme were used to check ST:STUDY_SUMMARY for mitochondrial function of DHODH(dihydroorotate dehydrogenase). Cells were in ST:STUDY_SUMMARY culture for 4 days and then extracted for polar metabolites. Cells were treated ST:STUDY_SUMMARY with DMSO and brequinar. WT and RquA 143B cells were also used for comparison. ST:INSTITUTE UMass Chan Medical School ST:LAST_NAME UMass Chan ST:FIRST_NAME Spinelli Lab ST:ADDRESS 55 Lake Avenue North, Worcester, Massachusetts, 01605, USA ST:EMAIL spinellilab@gmail.com ST:PHONE (508) 856-8989 ext. 68148 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 - WT DMSO 1 Sample source:143b Cells | Sample source:WT | Treatment:DMSO RAW_FILE_NAME(File Name )=20240809_JV141_01 SUBJECT_SAMPLE_FACTORS - WT DMSO 2 Sample source:143b Cells | Sample source:WT | Treatment:DMSO RAW_FILE_NAME(File Name )=20240809_JV141_02 SUBJECT_SAMPLE_FACTORS - WT DMSO 3 Sample source:143b Cells | Sample source:WT | Treatment:DMSO RAW_FILE_NAME(File Name )=20240809_JV141_03 SUBJECT_SAMPLE_FACTORS - WT Breq 1 Sample source:143b Cells | Sample source:WT | Treatment:Brequinar RAW_FILE_NAME(File Name )=20240809_JV141_04 SUBJECT_SAMPLE_FACTORS - WT Breq 2 Sample source:143b Cells | Sample source:WT | Treatment:Brequinar RAW_FILE_NAME(File Name )=20240809_JV141_05 SUBJECT_SAMPLE_FACTORS - WT Breq 3 Sample source:143b Cells | Sample source:WT | Treatment:Brequinar RAW_FILE_NAME(File Name )=20240809_JV141_06 SUBJECT_SAMPLE_FACTORS - RquA DMSO 1 Sample source:143b Cells | Sample source:RquA | Treatment:DMSO RAW_FILE_NAME(File Name )=20240809_JV141_07 SUBJECT_SAMPLE_FACTORS - RquA DMSO 2 Sample source:143b Cells | Sample source:RquA | Treatment:DMSO RAW_FILE_NAME(File Name )=20240809_JV141_08 SUBJECT_SAMPLE_FACTORS - RquA DMSO 3 Sample source:143b Cells | Sample source:RquA | Treatment:DMSO RAW_FILE_NAME(File Name )=20240809_JV141_09 SUBJECT_SAMPLE_FACTORS - RquA Breq 1 Sample source:143b Cells | Sample source:RquA | Treatment:Brequinar RAW_FILE_NAME(File Name )=20240809_JV141_10 SUBJECT_SAMPLE_FACTORS - RquA Breq 2 Sample source:143b Cells | Sample source:RquA | Treatment:Brequinar RAW_FILE_NAME(File Name )=20240809_JV141_11 SUBJECT_SAMPLE_FACTORS - RquA Breq 3 Sample source:143b Cells | Sample source:RquA | Treatment:Brequinar RAW_FILE_NAME(File Name )=20240809_JV141_12 SUBJECT_SAMPLE_FACTORS - SDHB KO RquA DMSO 1 Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:DMSO RAW_FILE_NAME(File Name )=20240809_JV141_13 SUBJECT_SAMPLE_FACTORS - SDHB KO RquA DMSO 2 Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:DMSO RAW_FILE_NAME(File Name )=20240809_JV141_14 SUBJECT_SAMPLE_FACTORS - SDHB KO RquA DMSO 3 Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:DMSO RAW_FILE_NAME(File Name )=20240809_JV141_15 SUBJECT_SAMPLE_FACTORS - SDHB KO RquA Breq 1 Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:Brequinar RAW_FILE_NAME(File Name )=20240809_JV141_16 SUBJECT_SAMPLE_FACTORS - SDHB KO RquA Breq 2 Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:Brequinar RAW_FILE_NAME(File Name )=20240809_JV141_17 SUBJECT_SAMPLE_FACTORS - SDHB KO RquA Breq 3 Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:Brequinar RAW_FILE_NAME(File Name )=20240809_JV141_18 #COLLECTION CO:COLLECTION_SUMMARY Media was aspirated from the plates and then the cells were washed with 1x PBS CO:COLLECTION_SUMMARY twice. The plate was then transferred to dry ice and 500 µL of 80% HPLC-grade CO:COLLECTION_SUMMARY methanol (Sigma) 20% HPLC-grade water (Sigma) was added to each well. The wells CO:COLLECTION_SUMMARY were placed in a -80 freezer to incubate for 15 minutes. The plates are taken CO:COLLECTION_SUMMARY out of the freezer one at a time and placed back on dry ice. The cells were then CO:COLLECTION_SUMMARY scraped and transferred to a new tube. Each well was washed with an additional CO:COLLECTION_SUMMARY 300 µL of 80% HPLC-grade methanol (Sigma) 20% HPLC-grade water (Sigma) and CO:COLLECTION_SUMMARY collected into the same tube as the initial lysis. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Cells were seeded in complete DMEM 96 hours prior to tracing so that wells TR:TREATMENT_SUMMARY reached 75% confluence at time of experiment. DMEM containing 10% FBS, 1% TR:TREATMENT_SUMMARY penicillin and streptomycin, and 0.1 mg/mL uridine (Sigma) was used when seeding TR:TREATMENT_SUMMARY the experiment. 72 hours prior, the media was changed to DMEM containing 10% TR:TREATMENT_SUMMARY FBS, 1% penicillin and streptomycin, and 10 mM aspartate with the pH adjusted to TR:TREATMENT_SUMMARY 7.4 along with 1uL of either DMSO or 10mM of brequinar. 6 hours prior to TR:TREATMENT_SUMMARY metabolite isolation, the cells were treated with DMEM containing 10% FBS, 1% TR:TREATMENT_SUMMARY penicillin and streptomycin, and 10 mM 13C4-aspartate (Sigma) and the pH TR:TREATMENT_SUMMARY adjusted to 7.4 with the relevant treatments. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Once the samples were collected, they were then vortexed at 4° C for 10 minutes SP:SAMPLEPREP_SUMMARY and centrifuged at 21,300 x g for 10 minutes at 4° C. Supernatants were SP:SAMPLEPREP_SUMMARY transferred to a new tube and dried down in a Refrigerated CentriVap Benchtop SP:SAMPLEPREP_SUMMARY Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco). After SP:SAMPLEPREP_SUMMARY being dried down, pellets were stored in a -20° C freezer until ready to run on SP:SAMPLEPREP_SUMMARY the polar LC-MS method. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) CH:SOLVENT_A 100% water; 0.1% ammonium hydroxide; 20mM ammonium carbonate CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 20 min, 80% - 20% B; 0.5 min, 20% - 80% B; 7.5min, 80% B CH:FLOW_RATE 0.15ml/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometer was set to full scan (70-1000 m/z), with the spray voltage MS:MS_COMMENTS set to 4.0 kV, heated capillary to 350°C, and the HESI probe at 30 °C. The MS:MS_COMMENTS sheath gas flow was set at 10 units, auxiliary gas at 1 units, and sweep gas MS:MS_COMMENTS flow at 1 unit. The resolution of scan was set to 70,000, AGC target to 1x106, MS:MS_COMMENTS and maximum injection time at 20 msec. Data acquired by Thermo Fisher's Xcalibur MS:MS_COMMENTS software and analyzed by their Tracefinder software. The raw files provided MS:MS_COMMENTS contain data from both positive and negative ion mode. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples WT DMSO 1 WT DMSO 2 WT DMSO 3 WT Breq 1 WT Breq 2 WT Breq 3 RquA DMSO 1 RquA DMSO 2 RquA DMSO 3 RquA Breq 1 RquA Breq 2 RquA Breq 3 SDHB KO RquA DMSO 1 SDHB KO RquA DMSO 2 SDHB KO RquA DMSO 3 SDHB KO RquA Breq 1 SDHB KO RquA Breq 2 SDHB KO RquA Breq 3 Factors Sample source:143b Cells | Sample source:WT | Treatment:DMSO Sample source:143b Cells | Sample source:WT | Treatment:DMSO Sample source:143b Cells | Sample source:WT | Treatment:DMSO Sample source:143b Cells | Sample source:WT | Treatment:Brequinar Sample source:143b Cells | Sample source:WT | Treatment:Brequinar Sample source:143b Cells | Sample source:WT | Treatment:Brequinar Sample source:143b Cells | Sample source:RquA | Treatment:DMSO Sample source:143b Cells | Sample source:RquA | Treatment:DMSO Sample source:143b Cells | Sample source:RquA | Treatment:DMSO Sample source:143b Cells | Sample source:RquA | Treatment:Brequinar Sample source:143b Cells | Sample source:RquA | Treatment:Brequinar Sample source:143b Cells | Sample source:RquA | Treatment:Brequinar Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:DMSO Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:DMSO Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:DMSO Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:Brequinar Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:Brequinar Sample source:143b Cells | Sample source:SDHB KO RquA | Treatment:Brequinar Glutathione 957651322.1 678449909.6 430006621.2 763367000.5 799182933.9 119673356.2 645813941.3 779754010.9 674886861.3 798004696.1 844874610.9 462782598.7 321323885.9 345216100.7 166509822.9 278646188.6 266904789.5 231883018 Oxidized glutathione 44760844.22 52801395.15 58956190.19 67889600.02 66789634.25 11790072.03 34562578.58 46808250.54 39414803.39 40215283.82 32510672.04 19092868.24 30709751.44 32149343.75 24947068.93 40582847.9 37295382.84 20436930.34 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention index Quantified m/z PubChem ID KEGG ID Glutathione 8.8 308.0911 124886 C00051 Oxidized glutathione 11 613.1592 65359 C00127 METABOLITES_END #END