#METABOLOMICS WORKBENCH spinelli_lab_20240404_130240 DATATRACK_ID:4760 STUDY_ID:ST003192 ANALYSIS_ID:AN005240 PROJECT_ID:PR001988 VERSION 1 CREATED_ON May 8, 2024, 8:34 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 mammalian PR:PROJECT_SUMMARY electron carrier that directs electrons to fumarate, instead of O2, as the PR:PROJECT_SUMMARY favored terminal electron acceptor. RQ, which is undetectable in cultured PR:PROJECT_SUMMARY mammalian cells, is enriched in tissues that catalyze fumarate reduction. RQ and PR:PROJECT_SUMMARY UQ-directed ETC circuits support distinct programs of mitochondrial function. PR:PROJECT_SUMMARY Through expression of a bacterial enzyme that converts UQ into RQ and PR:PROJECT_SUMMARY development a novel RQ analog, we demonstrate that reprogramming the mammalian PR:PROJECT_SUMMARY ETC from the UQ to RQ circuit renders cells highly resistant to hypoxia PR:PROJECT_SUMMARY exposure. Thus, we establish RQ as a fundamental component of the mammalian ETC PR:PROJECT_SUMMARY and unveil reprogramming the ETC to the RQ-circuit as a tractable strategy to PR:PROJECT_SUMMARY treat hypoxia-related diseases. PR:INSTITUTE UMass Chan Medical School PR:DEPARTMENT Program in Molecular Medicine PR:LABORATORY Spinelli Lab 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 Wildtype and UQCRC2 Knockout 143B Cells ST:STUDY_SUMMARY Ubiquinone (UQ), the only known electron carrier in the mammalian electron ST:STUDY_SUMMARY transport chain (ETC), delivers electrons to both oxygen (O2) and fumarate as ST:STUDY_SUMMARY terminal electron acceptors. As fumarate has a lower reduction potential than ST:STUDY_SUMMARY UQ, fumarate reduction is only thermodynamically favorable when ubiquinol, the ST:STUDY_SUMMARY reduced form of UQ, accumulates. Paradoxically, some tissues reduce fumarate ST:STUDY_SUMMARY without ubiquinol buildup, suggesting another mechanism enables fumarate ST:STUDY_SUMMARY reduction in mammals. Here, we identify rhodoquinone (RQ), a novel mammalian ST:STUDY_SUMMARY electron carrier that directs electrons to fumarate, instead of O2, as the ST:STUDY_SUMMARY favored terminal electron acceptor. RQ, which is undetectable in cultured ST:STUDY_SUMMARY mammalian cells, is enriched in tissues that catalyze fumarate reduction. RQ and ST:STUDY_SUMMARY UQ-directed ETC circuits support distinct programs of mitochondrial function. ST:STUDY_SUMMARY Through expression of a bacterial enzyme that converts UQ into RQ and ST:STUDY_SUMMARY development a novel RQ analog, we demonstrate that reprogramming the mammalian ST:STUDY_SUMMARY ETC from the UQ to RQ circuit renders cells highly resistant to hypoxia ST:STUDY_SUMMARY exposure. Thus, we establish RQ as a fundamental component of the mammalian ETC ST:STUDY_SUMMARY and unveil reprogramming the ETC to the RQ-circuit as a tractable strategy to ST:STUDY_SUMMARY treat hypoxia-related diseases. Wildtype 143B and UQCRC2 knockout cells were ST:STUDY_SUMMARY seeded and treated with media containing 13C4-Aspartate. The cells were ST:STUDY_SUMMARY extracted for metabolomics and ran on the LC/MS on the pHILIC method. ST:INSTITUTE UMass Chan Medical School ST:LAST_NAME Jerome ST:FIRST_NAME Madison ST:ADDRESS 55 N Lake Ave, Worcester, MA 01655 ST:EMAIL madison.jerome@umassmed.edu ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 2 ST:PHONE (508) 856-8989 ext. 68148 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS 143B #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 - MJ01 Sample source:143B osteosarcoma cells | Treatment:DMSO Genotype=Wild-type; RAW_FILE_NAME(Raw_file_name)=20230117_Spinelli_MJ05_01 SUBJECT_SAMPLE_FACTORS - MJ02 Sample source:143B osteosarcoma cells | Treatment:DMSO Genotype=Wild-type; RAW_FILE_NAME(Raw_file_name)=20230117_Spinelli_MJ05_02 SUBJECT_SAMPLE_FACTORS - MJ03 Sample source:143B osteosarcoma cells | Treatment:DMSO Genotype=Wild-type; RAW_FILE_NAME(Raw_file_name)=20230117_Spinelli_MJ05_03 SUBJECT_SAMPLE_FACTORS - MJ04 Sample source:143B osteosarcoma cells | Treatment:HKJS001 Genotype=Wild-type; RAW_FILE_NAME(Raw_file_name)=20230117_Spinelli_MJ05_04 SUBJECT_SAMPLE_FACTORS - MJ05 Sample source:143B osteosarcoma cells | Treatment:HKJS001 Genotype=Wild-type; RAW_FILE_NAME(Raw_file_name)=20230117_Spinelli_MJ05_05 SUBJECT_SAMPLE_FACTORS - MJ06 Sample source:143B osteosarcoma cells | Treatment:HKJS001 Genotype=Wild-type; RAW_FILE_NAME(Raw_file_name)=20230117_Spinelli_MJ05_06 SUBJECT_SAMPLE_FACTORS - MJ07 Sample source:143B osteosarcoma cells | Treatment:HKJS003 Genotype=Wild-type; RAW_FILE_NAME(Raw_file_name)=20230117_Spinelli_MJ05_07 SUBJECT_SAMPLE_FACTORS - MJ08 Sample source:143B osteosarcoma cells | Treatment:HKJS003 Genotype=Wild-type; RAW_FILE_NAME(Raw_file_name)=20230117_Spinelli_MJ05_08 SUBJECT_SAMPLE_FACTORS - MJ09 Sample source:143B osteosarcoma cells | Treatment:HKJS003 Genotype=Wild-type; RAW_FILE_NAME(Raw_file_name)=20230117_Spinelli_MJ05_09 #COLLECTION CO:COLLECTION_SUMMARY Cells were cultured in DMEM with 10% FBS and 1% Pen Strep and treated with CO:COLLECTION_SUMMARY either DMSO, 10uM HKJS001, or 25nM HKJS003. The media was changed after two days CO:COLLECTION_SUMMARY to media containing 13C4-aspartate and the treatments were added again. The CO:COLLECTION_SUMMARY cells were incubated with the 13C4-aspartate media at 37 degrees Celsius for 6 CO:COLLECTION_SUMMARY hours before being isolated. The cells were taken out of the incubator and CO:COLLECTION_SUMMARY placed on water ice where the media was aspirated off, washed twice with 1X PBS, CO:COLLECTION_SUMMARY and treated with 500uL of 80% LCMS grade MeOH per well on dry ice. The cells CO:COLLECTION_SUMMARY were incubated in the -80 degrees Celsius freezer for at least 15 minutes. The CO:COLLECTION_SUMMARY cells were lifted using cell scrapers on dry ice and the well was washed with an CO:COLLECTION_SUMMARY additional 300uL of 80% LCMS grade MeOH. The cell solution was transferred to a CO:COLLECTION_SUMMARY 1.5mL Eppendorf tube vortexed for 10 minutes at 4 degrees Celsius in a cold CO:COLLECTION_SUMMARY room. The tubes were then centrifuged at 21300rcf at 4 degrees Celsius for 10 CO:COLLECTION_SUMMARY minutes. The supernatant was transferred to a new tube, and the sample was dried CO:COLLECTION_SUMMARY down using a speed vac to dry the pellet. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY 143B cells were treated with each media change. When new media was added the TR:TREATMENT_SUMMARY cells either received DMSO as a control, 10uM of small molecule HKJS001, or 25nM TR:TREATMENT_SUMMARY of small molecule HKJS003 with three replicates for each treatment. The plates TR:TREATMENT_SUMMARY were swirled gently to mix the treatment into the media. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The samples (dried down pellets) were resuspended in 100uL of LCMS grade water SP:SAMPLEPREP_SUMMARY and vortexed for 10 minutes at 4 degrees Celsius in a cold room. They were then SP:SAMPLEPREP_SUMMARY centrifuged for 10 minutes at 4 degrees Celsius at 21300rcf. 20uL of each sample SP:SAMPLEPREP_SUMMARY was transferred to a LCMS vial. SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION 100uL #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 AN:ACQUISITION_DATE 01-17-2023 #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 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. An additional scan between 220-700 m/z MS:MS_COMMENTS was used to enhance nucleotide detection in the negative mode as well with the MS:MS_COMMENTS maximum injection time set to 80 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 MJ01 MJ02 MJ03 MJ04 MJ05 MJ06 MJ07 MJ08 MJ09 Factors Sample source:143B osteosarcoma cells | Treatment:DMSO Sample source:143B osteosarcoma cells | Treatment:DMSO Sample source:143B osteosarcoma cells | Treatment:DMSO Sample source:143B osteosarcoma cells | Treatment:HKJS001 Sample source:143B osteosarcoma cells | Treatment:HKJS001 Sample source:143B osteosarcoma cells | Treatment:HKJS001 Sample source:143B osteosarcoma cells | Treatment:HKJS003 Sample source:143B osteosarcoma cells | Treatment:HKJS003 Sample source:143B osteosarcoma cells | Treatment:HKJS003 UMP 2005565.015 2392805.699 2707802.987 2222711.922 1911577.469 3039361.814 2505933.704 2632339.87 2433439.593 UMP_13C3 923689.0467 525602.8433 1042170.062 398071.9111 798902.7663 0 967691.9984 0 0 UTP 7309682.953 13329930.25 16579876.6 14645813.49 12164727.69 11756138.37 13263442.34 15257900.19 17161485.18 UTP_13C1 824064.1427 1505397.904 1930248.045 1717198.774 1345818.723 1328138.843 1514252.13 1761537.892 1979691.904 UTP_13C2 319477.2618 636575.8153 837509.1908 722121.4444 526340.4108 553761.8764 718105.3671 845572.0996 1010468.319 UTP_13C3 760770.9769 1437897.498 1832322.634 1870473.212 1403533.164 1366054.444 1981939.448 2105617.839 2724587.923 UTP_13C4 39145.35234 39385.98378 117458.7084 62425.48004 57539.15367 72584.26817 85122.56451 76942.7199 91033.88036 UTP_13C5 6058.192789 9750.345886 20861.55974 11304.38154 13838.45392 0 0 12906.75814 22068.07907 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention index Quantified m/z PubChem ID KEGG ID UMP 8.72 323.02859 6030 C00105 UMP_13C3 8.72 326.03865 UTP 11.22 482.96125 6133 C00075 UTP_13C1 11.22 483.96461 UTP_13C2 11.22 484.96796 UTP_13C3 11.22 485.97131 UTP_13C4 11.22 486.97467 UTP_13C5 11.22 487.97802 METABOLITES_END #END