#METABOLOMICS WORKBENCH spinelli_lab_20240812_130753 DATATRACK_ID:5095 STUDY_ID:ST003404 ANALYSIS_ID:AN005587 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 NEGATIVE MS:MS_COMMENTS The mass spectrometer had the spray voltage set to 4.0 kV, heated capillary to MS:MS_COMMENTS 350°C, and the HESI probe at 30 °C. The sheath gas flow was set at 10 units, MS:MS_COMMENTS auxiliary gas at 1 units, and sweep gas flow at 1 unit. An additional scan MS:MS_COMMENTS between 220-700 m/z was used to enhance nucleotide detection in the negative MS:MS_COMMENTS mode as well with the maximum injection time set to 80 msec. Data acquired by MS:MS_COMMENTS Thermo Fisher's Xcalibur software and analyzed by their Tracefinder software. MS:MS_COMMENTS The raw files provided 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 Fumaric acid 7397894.75 6591190.346 4961664.681 7948536.227 7784481.814 3527625.674 4538765.568 5541716.457 5597618.851 5797104.585 5782149.609 5755754.536 4374730.214 4027819.018 3686940.971 4797373.898 3482706.155 3851803.253 Fumaric acid_13C1 305624.8673 229595.7841 84099.37648 168670.6007 243053.9367 110562.506 47171.99165 111040.4523 139810.5578 178056.1154 146112.8044 265826.2193 53887.52851 107482.1751 55438.70091 71715.25398 62587.92837 36878.65125 Fumaric acid_13C2 20098.2223 13801.11375 67785.05473 22098.82219 14995.97888 21817.41108 19965.05511 31462.84391 42274.29343 0 0 0 0 0 15341.90944 0 30504.32925 20036.99819 Fumaric acid_13C3 1128852.876 1506566.925 2137660.211 1794010.741 1752193.728 766234.6108 1240883.269 1478019.014 1190235.361 1256726.965 927154.6398 777589.5934 1278199.961 1004119.47 1014906.111 1400966.895 1369653.997 991731.655 Fumaric acid_13C4 39379207.37 49244250.79 63949470.24 62323320.8 59102554.23 22256646.7 41324457.07 40352137.11 34107448.41 39872810.44 33196929.44 21749185.44 39172361.78 35658482.53 32170923.5 47774783.48 45407026.04 30811765.76 Succinic acid 26546652.76 17410842.75 20000495.28 19223092.03 15596670.67 1891656.669 4277433.488 7096123.929 5318307.898 3081166.169 7887219.854 3772050.391 13252460.87 8996583.635 7928694.468 6382798.977 5196366.785 5435713.11 Succinic acid_13C1 1144431.812 933488.3466 892496.2509 721758.2622 559241.1475 8290.018697 148469.2742 358866.7855 275413.885 134616.725 321851.1192 118356.2252 756039.2477 342046.0229 308050.9013 390128.6622 268339.1657 214323.127 Succinic acid_13C2 532547.8661 856680.287 2002523.015 745628.7682 104174.8721 923472.2022 1100979.632 768186.7798 709375.8808 2292146.248 692024.2424 526183.4137 852863.7668 1064149.496 265243.3693 678415.4995 714790.5538 1262955.547 Succinic acid_13C3 0 129666.148 502932.6303 91954.56586 84224.71609 59976.60598 177357.0708 324887.0717 57250.61505 245553.4053 105429.7795 150465.4154 104623.9515 124671.0312 107341.271 136489.3415 135957.5942 365473.9368 Succinic acid_13C4 2574765.722 2197684.864 1865242.015 2813873.295 3292965.257 1514603.467 2708494.744 2428701.36 2614731.487 3111877.239 3086377.859 1651564.443 2581095.965 2637089.248 2006036.907 2977241.429 1262290.517 2569409.943 UTP_13C3 855195.3389 510382.641 296361.6614 1389.324813 0 0 524799.8764 612712.729 454582.1079 0 900.4354511 0 96962.5626 108427.3348 50228.2528 926.7787257 0 0 ATP 72536652.91 53977432 49419919.99 68900338.17 66096259.34 16968548 34942133.8 41053876.71 32164954.96 22839681.27 24295555.97 12394915.26 44517350.96 43945214.26 32232606.03 36928957.5 31683651.36 24090753.25 ADP 7855776.44 7197867.691 6033408.751 8944248.595 8359074.757 3381833.123 6213674.51 7410737.599 6683752.761 6391442.482 6411879.461 4494861.138 5669428.398 5683180.918 4878254.638 6192461.925 5259778.73 4361551.17 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Quantified m/z PubChem ID KEGG ID Fumaric acid 9 115.0037 444972 C00122 Fumaric acid_13C1 9.1 116.007 Fumaric acid_13C2 9.1 117.0104 Fumaric acid_13C3 9.1 118.0138 Fumaric acid_13C4 9.1 119.0171 Succinic acid 8.1 117.0193 1110 C00042 Succinic acid_13C1 8.1 118.0227 Succinic acid_13C2 8.8 119.026 Succinic acid_13C3 8.8 120.0294 Succinic acid_13C4 8.5 121.0328 UTP_13C3 11.2 485.9713 ATP 10.2 505.9885 5957 C00002 ADP 9.3 426.0221 6022 C00008 METABOLITES_END #END