#METABOLOMICS WORKBENCH xliu68_20200611_073356 DATATRACK_ID:2052 STUDY_ID:ST001441 ANALYSIS_ID:AN002409 PROJECT_ID:PR000991 VERSION 1 CREATED_ON August 6, 2020, 5:00 pm #PROJECT PR:PROJECT_TITLE SUCLA2 mutations cause global protein succinylation contributing to the PR:PROJECT_TITLE pathomechanism of a hereditary mitochondrial disease PR:PROJECT_SUMMARY Mitochondrial acyl-coenzyme A species are emerging as important sources of PR:PROJECT_SUMMARY protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a PR:PROJECT_SUMMARY mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that PR:PROJECT_SUMMARY succinyl-CoA accumulates in cells derived from patients carrying recessive PR:PROJECT_SUMMARY mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit PR:PROJECT_SUMMARY beta (SUCLA2) causing global protein hyper-succinylation. Using mass PR:PROJECT_SUMMARY spectrometry, we quantified nearly 1000 protein succinylation sites on 366 PR:PROJECT_SUMMARY proteins from patient-derived fibroblasts and myotubes. Interestingly, PR:PROJECT_SUMMARY hyper-succinylated proteins are distributed across cellular compartments, and PR:PROJECT_SUMMARY many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the PR:PROJECT_SUMMARY contribution of hyper-succinylation to disease progression, we developed a PR:PROJECT_SUMMARY zebrafish model of the SCL deficiency, and find that SIRT5 gain-of-function PR:PROJECT_SUMMARY reduces global protein succinylation and improves survival. Thus, increased PR:PROJECT_SUMMARY succinyl-CoA levels contribute to the pathology of SCL deficiency through PR:PROJECT_SUMMARY post-translational modifications. PR:INSTITUTE North Carolina State University PR:LAST_NAME Liu PR:FIRST_NAME Xiaojing PR:ADDRESS Polk Hall, RM 128 PR:EMAIL xliu68@ncsu.edu PR:PHONE 9195154387 #STUDY ST:STUDY_TITLE Metabolomics of patient-derived fibroblasts ST:STUDY_SUMMARY 7 control fibroblasts samples and 7 patient-derived fibroblasts samples were ST:STUDY_SUMMARY collected at day 0 and day 5. Intracellular metabolites were extracted from ST:STUDY_SUMMARY cells cultured in 6 well plate while acyl-CoA and 5-methyltetrahydrofolate were ST:STUDY_SUMMARY extracted from cells cultured in 60 mm dish. ST:INSTITUTE North Carolina State University ST:LAST_NAME Liu ST:FIRST_NAME Xiaojing ST:ADDRESS Polk Hall, RM 128 ST:EMAIL xliu68@ncsu.edu ST:PHONE 9195154387 #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 - D0-control1 Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control1.raw SUBJECT_SAMPLE_FACTORS - D0-control2 Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control2.raw SUBJECT_SAMPLE_FACTORS - D0-control3 Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control3.raw SUBJECT_SAMPLE_FACTORS - D0-control4 Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control4.raw SUBJECT_SAMPLE_FACTORS - D0-control5 Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control5.raw SUBJECT_SAMPLE_FACTORS - D0-control6 Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control6.raw SUBJECT_SAMPLE_FACTORS - D0-control7 Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control7.raw SUBJECT_SAMPLE_FACTORS - D0-patient1 Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient1.raw SUBJECT_SAMPLE_FACTORS - D0-patient2 Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient2.raw SUBJECT_SAMPLE_FACTORS - D0-patient3 Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient3.raw SUBJECT_SAMPLE_FACTORS - D0-patient4 Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient4.raw SUBJECT_SAMPLE_FACTORS - D0-patient5 Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient5.raw SUBJECT_SAMPLE_FACTORS - D0-patient6 Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient6.raw SUBJECT_SAMPLE_FACTORS - D0-patient7 Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient7.raw SUBJECT_SAMPLE_FACTORS - D5-control1 Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control1.raw SUBJECT_SAMPLE_FACTORS - D5-control2 Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control2.raw SUBJECT_SAMPLE_FACTORS - D5-control3 Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control3.raw SUBJECT_SAMPLE_FACTORS - D5-control4 Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control4.raw SUBJECT_SAMPLE_FACTORS - D5-control5 Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control5.raw SUBJECT_SAMPLE_FACTORS - D5-control6 Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control6.raw SUBJECT_SAMPLE_FACTORS - D5-control7 Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control7.raw SUBJECT_SAMPLE_FACTORS - D5-patient1 Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient1.raw SUBJECT_SAMPLE_FACTORS - D5-patient2 Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient2.raw SUBJECT_SAMPLE_FACTORS - D5-patient3 Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient3.raw SUBJECT_SAMPLE_FACTORS - D5-patient4 Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient4.raw SUBJECT_SAMPLE_FACTORS - D5-patient5 Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient5.raw SUBJECT_SAMPLE_FACTORS - D5-patient6 Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient6.raw SUBJECT_SAMPLE_FACTORS - D5-patient7 Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient7.raw SUBJECT_SAMPLE_FACTORS - D0-control1_luna Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control1_luna.raw SUBJECT_SAMPLE_FACTORS - D0-control2_luna Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control2_luna.raw SUBJECT_SAMPLE_FACTORS - D0-control3_luna Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control3_luna.raw SUBJECT_SAMPLE_FACTORS - D0-control4_luna Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control4_luna.raw SUBJECT_SAMPLE_FACTORS - D0-control5_luna Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control5_luna.raw SUBJECT_SAMPLE_FACTORS - D0-control6_luna Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control6_luna.raw SUBJECT_SAMPLE_FACTORS - D0-control7_luna Sample Source:Control | Treatment Duration:Day0 RAW_FILE_NAME=D0-control7_luna.raw SUBJECT_SAMPLE_FACTORS - D0-patient1_luna Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient1_luna.raw SUBJECT_SAMPLE_FACTORS - D0-patient2_luna Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient2_luna.raw SUBJECT_SAMPLE_FACTORS - D0-patient3_luna Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient3_luna.raw SUBJECT_SAMPLE_FACTORS - D0-patient4_luna Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient4_luna.raw SUBJECT_SAMPLE_FACTORS - D0-patient5_luna Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient5_luna.raw SUBJECT_SAMPLE_FACTORS - D0-patient6_luna Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient6_luna.raw SUBJECT_SAMPLE_FACTORS - D0-patient7_luna Sample Source:Patient | Treatment Duration:Day0 RAW_FILE_NAME=D0-patient7_luna.raw SUBJECT_SAMPLE_FACTORS - D5-control1_luna Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control1_luna.raw SUBJECT_SAMPLE_FACTORS - D5-control2_luna Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control2_luna.raw SUBJECT_SAMPLE_FACTORS - D5-control3_luna Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control3_luna.raw SUBJECT_SAMPLE_FACTORS - D5-control4_luna Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control4_luna.raw SUBJECT_SAMPLE_FACTORS - D5-control5_luna Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control5_luna.raw SUBJECT_SAMPLE_FACTORS - D5-control6_luna Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control6_luna.raw SUBJECT_SAMPLE_FACTORS - D5-control7_luna Sample Source:Control | Treatment Duration:Day5 RAW_FILE_NAME=D5-control7_luna.raw SUBJECT_SAMPLE_FACTORS - D5-patient1_luna Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient1_luna.raw SUBJECT_SAMPLE_FACTORS - D5-patient2_luna Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient2_luna.raw SUBJECT_SAMPLE_FACTORS - D5-patient3_luna Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient3_luna.raw SUBJECT_SAMPLE_FACTORS - D5-patient4_luna Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient4_luna.raw SUBJECT_SAMPLE_FACTORS - D5-patient5_luna Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient5_luna.raw SUBJECT_SAMPLE_FACTORS - D5-patient6_luna Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient6_luna.raw SUBJECT_SAMPLE_FACTORS - D5-patient7_luna Sample Source:Patient | Treatment Duration:Day5 RAW_FILE_NAME=D5-patient7_luna.raw #COLLECTION CO:COLLECTION_SUMMARY 7 control fibroblasts samples and 7 patient-derived fibroblasts samples were CO:COLLECTION_SUMMARY collected at day 0 and day 5. Intracellular metabolites were extracted from CO:COLLECTION_SUMMARY cells cultured in 6 well plate while acyl-CoA and 5-methyltetrahydrofolate were CO:COLLECTION_SUMMARY extracted from cells cultured in 60 mm dish. Intracellular metabolites and CO:COLLECTION_SUMMARY acyl-CoA from cells were harvested as described previously using 80% CO:COLLECTION_SUMMARY methanol/water as solvent (PMID: 24410464, PMID: 24894601, PMID: 25795660) and CO:COLLECTION_SUMMARY dry pellets were stored in -80 °C freezer until ready for LC-MS analysis. For CO:COLLECTION_SUMMARY acyl-CoA and 5-methyltetrahydrofolate analysis, dry pellets were reconstituted CO:COLLECTION_SUMMARY into 30 μL of sample solvent (water containing 50 mM ammonium acetate), and 10 CO:COLLECTION_SUMMARY μL was injected into the LC-MS. For non-acyl-CoA polar metabolite analysis, CO:COLLECTION_SUMMARY pellets were reconstituted into 30 μL of sample solvent CO:COLLECTION_SUMMARY (water:methanol:acetonitrile, 2:1:1, v/v), and 3 μL was injected into the CO:COLLECTION_SUMMARY LC-MS. CO:SAMPLE_TYPE Cultured fibroblasts #TREATMENT TR:TREATMENT_SUMMARY Patient-derived fibroblasts cultured in standard culture conditions TR:TREATMENT_SUMMARY (proliferative condition) and cells cultured for 5 days in low-serum conditions TR:TREATMENT_SUMMARY (non-proliferative condition). Patients carry disease-causing mutations in TR:TREATMENT_SUMMARY SUCLA2. Controls are fibroblasts from age-matched patients with other TR:TREATMENT_SUMMARY mitochondrial diseases. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Intracellular metabolites and acyl-CoA from cells were harvested as described SP:SAMPLEPREP_SUMMARY previously using 80% methanol/water as solvent (PMID: 24410464, PMID: 24894601, SP:SAMPLEPREP_SUMMARY PMID: 25795660) and dry pellets were stored in -80 °C freezer until ready for SP:SAMPLEPREP_SUMMARY LC-MS analysis. For acyl-CoA and 5-methyltetrahydrofolate analysis, dry pellets SP:SAMPLEPREP_SUMMARY were reconstituted into 30 μL of sample solvent (water containing 50 mM SP:SAMPLEPREP_SUMMARY ammonium acetate), and 10 μL was injected into the LC-MS. For non-acyl-CoA SP:SAMPLEPREP_SUMMARY polar metabolite analysis, pellets were reconstituted into 30 μL of sample SP:SAMPLEPREP_SUMMARY solvent (water:methanol:acetonitrile, 2:1:1, v/v), and 3 μL was injected into SP:SAMPLEPREP_SUMMARY the LC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY RPLC is for acyl-CoA and folate analysis. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Phenomenex Luna C18 (100 × 2.0 mm i.d., 3 µm) #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 It was operated in the full-scan mode with the resolution set at 70 000 (at m/z MS:MS_COMMENTS 200).LC-MS data were analyzed using Sieve 2.0 (Thermo Scientific), and the MS:MS_COMMENTS integrated area under metabolite peak was used to compare the relative abundance MS:MS_COMMENTS of each metabolite in different samples in the same batch. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples D0-patient5 D0-patient4 D0-patient7 D0-patient3 D0-patient6 D0-patient1 D0-patient2 D0-control1 D0-control3 D0-control5 D0-control2 D0-control4 D0-control6 D0-control7 D5-patient5 D5-patient4 D5-patient7 D5-patient3 D5-patient6 D5-patient1 D5-patient2 D5-control1 D5-control3 D5-control5 D5-control2 D5-control4 D5-control6 D5-control7 Factors Sample Source:Patient | Treatment Duration:Day0 Sample Source:Patient | Treatment Duration:Day0 Sample Source:Patient | Treatment Duration:Day0 Sample Source:Patient | Treatment Duration:Day0 Sample Source:Patient | Treatment Duration:Day0 Sample Source:Patient | Treatment Duration:Day0 Sample Source:Patient | Treatment Duration:Day0 Sample Source:Control | Treatment Duration:Day0 Sample Source:Control | Treatment Duration:Day0 Sample Source:Control | Treatment Duration:Day0 Sample Source:Control | Treatment Duration:Day0 Sample Source:Control | Treatment Duration:Day0 Sample Source:Control | Treatment Duration:Day0 Sample Source:Control | Treatment Duration:Day0 Sample Source:Patient | Treatment Duration:Day5 Sample Source:Patient | Treatment Duration:Day5 Sample Source:Patient | Treatment Duration:Day5 Sample Source:Patient | Treatment Duration:Day5 Sample Source:Patient | Treatment Duration:Day5 Sample Source:Patient | Treatment Duration:Day5 Sample Source:Patient | Treatment Duration:Day5 Sample Source:Control | Treatment Duration:Day5 Sample Source:Control | Treatment Duration:Day5 Sample Source:Control | Treatment Duration:Day5 Sample Source:Control | Treatment Duration:Day5 Sample Source:Control | Treatment Duration:Day5 Sample Source:Control | Treatment Duration:Day5 Sample Source:Control | Treatment Duration:Day5 5-methyltetrahydrofolate 142066.737 83517.849 78234.057 212168.883 159449.289 112720.332 45803.922 116419.596 152777.991 132965.982 262594.707 274609.644 31102.872 66503.97 174985.92 69781.662 59475.153 72334.92 5859.207 96331.269 39330.681 56476.686 191750.295 70498.161 136557.987 204411.096 51307.782 124946.775 Coenzyme A 1871747.631 1630097.607 861574.23 497438.484 609602.379 616534.329 117679.977 748107.06 1278824.031 1023958.845 1218190.311 1793597.475 77060.358 6813.138 1534075.341 520912.44 115468.83 258203.007 253909.242 166919.052 176201.34 101928.057 485124.036 37425.66 353291.955 56300.607 117837.207 2166.483 acetyl-CoA 5510111.1 4193301.045 8186569.593 4942090.863 5434093.884 4188507.672 2428460.514 5424255.282 5690046.606 3100746.576 6686539.527 5914665.321 3724410.909 3198308.937 4003136.076 2912339.841 4120243.167 2158564.632 2099231.274 2778459.039 1934148.498 2930841.96 4192395.837 2277124.815 2229912.054 1607105.007 4408162.08 2196076.632 propionyl-CoA 47245.746 1000 135888.831 8899.506 9072.645 26064.834 1000 26897.097 46407.846 13342.809 79274.664 59423.868 5136.219 1000 120606.927 32516.211 670483.959 72302.316 212092.62 28669.92 81827.607 532163.478 1217285.244 269449.611 429013.662 210806.484 367522.428 164487.699 malonyl-CoA 568667.28 1117941.492 1951958.904 545697.285 756445.974 539315.358 82107.045 805915.824 740092.347 596136.231 774170.172 1344684.342 461935.131 301169.613 184340.931 135966.939 133226.661 47538.969 3521.958 63360.909 115433.406 148004.1 24929.826 5922.603 76218.168 39021.333 272804.079 44922.438 Succinyl-CoA 4955998.137 2892208.323 2051178.759 3813554.856 4144077.555 1518796.329 1273311.864 890074.719 1358422.725 480422.388 1418591.103 1714199.76 396713.367 198869.439 6050742.48 3155145.309 2634395.913 3378001.53 3246630.06 1394844.417 2204350.212 2193397.023 5435583.498 938481.582 2087430.375 1419925.059 1141551.933 377692.659 Hydroxymethylglutaryl-CoA 1167728.724 560112.498 1364185.332 611020.779 511435.068 391095.954 136849.326 986785.779 2366514.84 161763.027 2596020.93 1807146.216 202990.335 140149.782 553085.454 267090.309 460967.565 25750.494 127018.461 52073.916 28640.649 132881.334 932792.811 101137.095 191421.63 27041.826 250595.733 7615.542 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 5-methyltetrahydrofolate Coenzyme A acetyl-CoA propionyl-CoA malonyl-CoA Succinyl-CoA Hydroxymethylglutaryl-CoA METABOLITES_END #END