#METABOLOMICS WORKBENCH yanma0820_20231011_190807 DATATRACK_ID:4391 STUDY_ID:ST002925 ANALYSIS_ID:AN004797 PROJECT_ID:PR001817 VERSION 1 CREATED_ON October 12, 2023, 9:01 am #PROJECT PR:PROJECT_TITLE A mitophagy sensor PPTC7 integrates homeostatic and physiological signals to PR:PROJECT_TITLE regulate mitophagy and mitochondrial mass PR:PROJECT_SUMMARY Mitophagy mediated by receptors BNIP3 and NIX critically regulates mitochondrial PR:PROJECT_SUMMARY mass under developmental and pathophysiological conditions. Cellular BNIP3 and PR:PROJECT_SUMMARY NIX levels are tightly controlled by SCFFBXL4-mediated ubiquitination to prevent PR:PROJECT_SUMMARY excessive mitophagy and lethal disease. Here we report that knockout of PPTC7, a PR:PROJECT_SUMMARY mitochondrial matrix protein, hyperactivates BNIP3/NIX-mediated mitophagy in PR:PROJECT_SUMMARY vitro and in vivo. Biochemically, the PPTC7 precursor is trapped by BNIP3 and PR:PROJECT_SUMMARY NIX to mitochondrial outer-membrane, where PPTC7 scaffolds the assembly of the PR:PROJECT_SUMMARY substrate-PPTC7-SCFFBXL4 holocomplex to degrade BNIP3 and NIX, forming a PR:PROJECT_SUMMARY homeostatic regulatory loop. PPTC7 possesses an unusually weak mitochondrial PR:PROJECT_SUMMARY targeting sequence to facilitate its outer-membrane retention and mitophagy PR:PROJECT_SUMMARY control. Upon starvation, PPTC7 is transcriptionally upregulated in mouse liver PR:PROJECT_SUMMARY to repress mitophagy, which critically maintains hepatic mitochondrial mass, PR:PROJECT_SUMMARY bioenergetics and gluconeogenesis. Collectively, PPTC7 functions as a mitophagy PR:PROJECT_SUMMARY sensor that integrates homeostatic and physiological signals to dynamically PR:PROJECT_SUMMARY control BNIP3 and NIX degradation, thereby maintaining proper mitochondrial mass PR:PROJECT_SUMMARY and cellular homeostasis. PR:INSTITUTE National Institute of Biological Sciences,Beijing PR:LAST_NAME Ma PR:FIRST_NAME Yan PR:ADDRESS Zhongguancun Life Science Park PR:EMAIL mayan@nibs.ac.cn PR:PHONE +86-01080726688 #STUDY ST:STUDY_TITLE Metabolite analysis of hepatic Pptc7-ko mice under fed or fasting conditions ST:STUDY_SUMMARY Hepatic Pptc7 knockout leads to mitochondrial loss under fed conditions in adult ST:STUDY_SUMMARY mice, which is exacerbated upon fasting. Under fasting conditions, WT liver ST:STUDY_SUMMARY upregulates Pptc7 levels to repress mitophagy and maintain mitochondrial mass. ST:STUDY_SUMMARY However, Pptc7-KO liver accumulated higher levels of Bnip3, resulting in ST:STUDY_SUMMARY mitophagy activation and further mitochondrial loss. To explore the metabolic ST:STUDY_SUMMARY changes upon Pptc7 knockout under both fed and fasting conditions, we performed ST:STUDY_SUMMARY a metabolite analysis of wild-type or Pptc7-KO mouse liver samples in this ST:STUDY_SUMMARY study. ST:INSTITUTE National Institute of Biological Sciences, Beijing ST:LAST_NAME Ma ST:FIRST_NAME Yan ST:ADDRESS Zhongguancun Life Science Park ST:EMAIL mayan@nibs.ac.cn ST:PHONE +86-01080726688 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 10-week SU:GENDER Male #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 - sample_01 Genotype:wild-type | Treatment:fed Tissue=Liver; RAW_FILE_NAME=Neg_sample_01.mzML; RAW_FILE_NAME=Pos_sample_01.mzML SUBJECT_SAMPLE_FACTORS - sample_02 Genotype:wild-type | Treatment:fed Tissue=Liver; RAW_FILE_NAME=Neg_sample_02.mzML; RAW_FILE_NAME=Pos_sample_02.mzML SUBJECT_SAMPLE_FACTORS - sample_03 Genotype:wild-type | Treatment:fed Tissue=Liver; RAW_FILE_NAME=Neg_sample_03.mzML; RAW_FILE_NAME=Pos_sample_03.mzML SUBJECT_SAMPLE_FACTORS - sample_04 Genotype:wild-type | Treatment:fed Tissue=Liver; RAW_FILE_NAME=Neg_sample_04.mzML; RAW_FILE_NAME=Pos_sample_04.mzML SUBJECT_SAMPLE_FACTORS - sample_05 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_05.mzML; RAW_FILE_NAME=Pos_sample_05.mzML SUBJECT_SAMPLE_FACTORS - sample_06 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_06.mzML; RAW_FILE_NAME=Pos_sample_06.mzML SUBJECT_SAMPLE_FACTORS - sample_07 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_07.mzML; RAW_FILE_NAME=Pos_sample_07.mzML SUBJECT_SAMPLE_FACTORS - sample_08 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_08.mzML; RAW_FILE_NAME=Pos_sample_08.mzML SUBJECT_SAMPLE_FACTORS - sample_09 Genotype:wild-type | Treatment:fed Tissue=Liver; RAW_FILE_NAME=Neg_sample_09.mzML; RAW_FILE_NAME=Pos_sample_09.mzML SUBJECT_SAMPLE_FACTORS - sample_10 Genotype:wild-type | Treatment:fed Tissue=Liver; RAW_FILE_NAME=Neg_sample_10.mzML; RAW_FILE_NAME=Pos_sample_10.mzML SUBJECT_SAMPLE_FACTORS - sample_11 Genotype:wild-type | Treatment:fed Tissue=Liver; RAW_FILE_NAME=Neg_sample_11.mzML; RAW_FILE_NAME=Pos_sample_11.mzML SUBJECT_SAMPLE_FACTORS - sample_12 Genotype:wild-type | Treatment:fed Tissue=Liver; RAW_FILE_NAME=Neg_sample_12.mzML; RAW_FILE_NAME=Pos_sample_12.mzML SUBJECT_SAMPLE_FACTORS - sample_13 Genotype:wild-type | Treatment:fed Tissue=Liver; RAW_FILE_NAME=Neg_sample_13.mzML; RAW_FILE_NAME=Pos_sample_13.mzML SUBJECT_SAMPLE_FACTORS - sample_14 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_14.mzML; RAW_FILE_NAME=Pos_sample_14.mzML SUBJECT_SAMPLE_FACTORS - sample_15 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_15.mzML; RAW_FILE_NAME=Pos_sample_15.mzML SUBJECT_SAMPLE_FACTORS - sample_16 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_16.mzML; RAW_FILE_NAME=Pos_sample_16.mzML SUBJECT_SAMPLE_FACTORS - sample_17 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_17.mzML; RAW_FILE_NAME=Pos_sample_17.mzML SUBJECT_SAMPLE_FACTORS - sample_18 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_18.mzML; RAW_FILE_NAME=Pos_sample_18.mzML SUBJECT_SAMPLE_FACTORS - sample_19 Genotype:Pptc7-KO | Treatment:fasting Tissue=Liver; RAW_FILE_NAME=Neg_sample_19.mzML; RAW_FILE_NAME=Pos_sample_19.mzML #COLLECTION CO:COLLECTION_SUMMARY Mice were sacrificed and liver samples were dissected out and snap-frozen in CO:COLLECTION_SUMMARY liquid nitrogen. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY We performed retro-orbital injection of P28 old Pptc7-flox (GemPharmatech, TR:TREATMENT_SUMMARY T011006) mice with 1×10^12 vector genome of the empty vector or Cre AAV8 virus. TR:TREATMENT_SUMMARY 42 days post AAV injection, mice were subjected to fasting or left fed as TR:TREATMENT_SUMMARY control group respectively. Fasting was done for 18 hours from the beginning of TR:TREATMENT_SUMMARY the dark cycle at 7.p.m. to 1.p.m. next day. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Mouse livers were dissected out and snap-frozen in liquid nitrogen. Liver SP:SAMPLEPREP_SUMMARY samples were cryo-homogenized on dry ice and 30 mg of homogenized tissue powders SP:SAMPLEPREP_SUMMARY were weighed and extracted with 600 μL ice-cold acetonitrile/methanol/water SP:SAMPLEPREP_SUMMARY (4/4/2) for 30 min. After centrifugation at 20,000 g for 15 min, supernatant was SP:SAMPLEPREP_SUMMARY transferred to a new tube and dried by a Speedvac vacuum concentrator at 30°C. SP:SAMPLEPREP_SUMMARY Dried samples were stored at -80°C and subjected to LC-MS analysis within 24 SP:SAMPLEPREP_SUMMARY hours. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish UHPLC CH:COLUMN_NAME Merck ZIC-HILIC (2.1x100 mm, 3.5 μm) CH:SOLVENT_A 5% acetonitrile/95% water; 10 mM ammonium acetate CH:SOLVENT_B 95% acetonitrile/5% water; 10 mM ammonium acetate CH:FLOW_GRADIENT 0-5 min, 99% B; 5-20 min, 99-20% B; 20-21 min, 20-99% B; 21-25 min, 99% B. CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS AN:SOFTWARE_VERSION Thermo Xcalibur (4.2) #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS - #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS count MS_METABOLITE_DATA_START Samples sample_01 sample_02 sample_03 sample_04 sample_05 sample_06 sample_07 sample_08 sample_09 sample_10 sample_11 sample_12 sample_13 sample_14 sample_15 sample_16 sample_17 sample_18 sample_19 Factors Genotype:wild-type | Treatment:fed Genotype:wild-type | Treatment:fed Genotype:wild-type | Treatment:fed Genotype:wild-type | Treatment:fed Genotype:Pptc7-KO | Treatment:fasting Genotype:Pptc7-KO | Treatment:fasting Genotype:Pptc7-KO | Treatment:fasting Genotype:Pptc7-KO | Treatment:fasting Genotype:wild-type | Treatment:fed Genotype:wild-type | Treatment:fed Genotype:wild-type | Treatment:fed Genotype:wild-type | Treatment:fed Genotype:wild-type | Treatment:fed Genotype:Pptc7-KO | Treatment:fasting Genotype:Pptc7-KO | Treatment:fasting Genotype:Pptc7-KO | Treatment:fasting Genotype:Pptc7-KO | Treatment:fasting Genotype:Pptc7-KO | Treatment:fasting Genotype:Pptc7-KO | Treatment:fasting Adenine 2084714549 2803407143 2033441476 2033390395 4546179938 4293528271 2686744516 1694288521 3817712342 2262540186 1673447900 1472703085 2410681030 2279072903 2944489886 2443038529 2672407548 2748966081 2911488975 Adenosine 243174136 335415035 245637823 253050143 541899242 519557733 331470686 469040172 469986986 255608366 189412537 153244952 284902155 256448267 335054379 287833428 313634203 318839657 328362535 Pyruvate 2370618912 1126632320 1852800302 2918077960 719019621 716809500 497357243 672488994 872395819 1504316617 832467235 778099084 887561028 447943265 466732710 374767432 602665484 475187261 303579581 Lactate 15679578016 12157159823 11645576846 17296296230 2665893294 3362468578 3310045842 3616533345 8998346474 12439630899 10149141243 17228008531 9193745027 6681551489 4997325575 5341353095 5422714105 4256437551 8183953105 Oxoglutarate 69989081 51151124 67085512 51728993 24701014 28439255 31136439 50801318 32902149 53414149 34493368 29708005 42122617 92449689 29993684 29632328 100819573 26702121 18179828 Succinate 2342908251 2089546213 1651917764 2664022568 1431070565 1544129749 1871529535 1620297960 2282272156 1814412753 1731341361 2291469361 1656934124 855414962 1329745722 1404942058 867003521 1518340239 1758181948 cis-Aconitate 45755556 38841879 32995716 34829351 23359743 21253917 23002852 31253869 35426479 31764629 31036512 24903367 47800972 32569684 32788454 34660152 36178869 18678112 25194605 Fumarate 533031633 397044152 365608331 476015413 287034950 277796984 419020565 364186332 621416641 447453488 435312866 397050822 427214077 512781459 364710749 377011222 416145414 305824680 345520116 Malate 9031213217 7212479178 5919434133 7947930957 5881871469 4943103885 5831936854 4942923772 6764703569 7552701265 8239725141 8090506093 7801510505 7087743120 5754984688 7276212842 5710888139 4390600671 4667066609 AMP 1949010454 2013877027 1238968969 2068244598 5963233775 4799308864 4959586829 4208695938 5455939763 1619354714 1571002177 1501307453 1726041046 4274388267 5531717994 4868172257 4727562642 4837655875 5627381329 Sedoheptulose-7-phosphate 187561774 126593474 95763275 215997850 34404788 34593198 31625583 38551851 69910887 119853775 126157274 184986030 161826245 60727173 41167492 55609987 40342701 34782299 66196495 Erythrocytes-4-phosphate 935216 592939 553157 668742 7159538 7067687 8366330 7969295 6331427 628668 811773 366160 1082068 3635255 5311477 4317535 4027338 4871147 4698726 IMP 161762688 207640537 151855183 186389687 178685436 227922971 148869177 156347310 219334421 124733181 191996566 171924073 111215347 134085539 208983883 166063774 77623064 139847341 195684412 Glycerol-3-phosphate 2426744086 2428711448 2027035355 2802845695 2010407281 1814336175 1452530799 1924496005 2258977357 2642622294 2171611246 2482005279 2342071341 2363270697 2305527528 2194166994 2412528350 1943399918 2336959806 ADP 514065626 448393331 407261871 559801554 656207387 542823312 535735660 622788472 514855092 425721921 355068365 398599616 504620305 979964255 831335408 796088364 1071431805 442448041 527739488 Citrate 549806565 355941873 281560555 325402653 199665537 202736105 172154631 245933174 249268082 220128202 241562253 241306260 369232516 348301575 246928674 246967445 259726664 155303570 225497898 Glyceraldehyde-3-phosphate 24647904 13852280 13082329 18203309 16655023 17408630 22994922 17533373 22353493 15800362 15065184 15252371 13109013 21852809 17130320 15428984 20012843 19155433 22200757 Ribose-5-phosphate 153760054 119738653 81764060 153598972 180680005 176171702 156255704 141784391 196302077 85681985 109397442 110439972 70125384 133020396 190475680 146202750 153087704 167305249 209662613 Fructose-6-phosphate 99323972 82599097 44678113 91495193 13186630 12668743 8879495 16994989 27111168 66844142 74239239 112607526 117452643 38674116 19171590 28647846 21069252 11481133 23494203 ATP 84885577 43780970 64238532 82431332 39116023 24868459 22309634 38014267 26846034 57164951 47644043 45107056 64596410 80101617 28591220 32362019 47564328 16524452 17551461 2-phosphoglycerate 75928018 32120497 33030771 34125820 11478459 12779422 12959966 18715183 18661189 30003857 39303847 35954846 27993730 16339631 12486293 13299243 15062423 7910444 11224608 Phosphoenolpyruvate 120155226 49501910 47736522 54125816 29194283 26303658 35773323 39888717 66589313 50619608 57428735 66243003 32916425 33051911 30444683 26911786 31925282 15872533 22865102 Glucose-6-phosphate 209254887 161903914 101446586 144508842 19547104 21304391 16752256 27617170 41874668 117879160 197450809 326150950 193663992 60568707 22805921 39510715 31829522 19087150 29925579 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT(min) m/z Pubchem ID KEGG ID Adenine 2.39 134.0467 3447 C00147 Adenosine 2.40 266.0889 3512 C00212 Pyruvate 3.47 87.0082 3324 C00022 Lactate 7.80 89.0239 3486 C00186 Oxoglutarate 10.46 145.0142 3328 C00026 Succinate 10.59 117.0188 3344 C00042 cis-Aconitate 10.66 173.0086 3707 C00417 Fumarate 10.78 115.0031 3422 C00122 Malate 10.77 133.0137 3449 C00149 AMP 10.95 346.0560 3322 C00020 Sedoheptulose-7-phosphate 11.55 289.0325 7756 C05382 Erythrocytes-4-phosphate 11.23 199.0008 3574 C00279 IMP 11.25 347.0393 3430 C00130 Glycerol-3-phosphate 11.26 171.0058 3393 C00093 ADP 11.23 426.0220 3310 C00008 Citrate 11.30 191.0192 3458 C00158 Glyceraldehyde-3-phosphate 11.29 168.9902 3418 C00118 Ribose-5-phosphate 11.33 229.0113 3417 C00117 Fructose-6-phosphate 11.49 259.0219 3385 C00085 ATP 11.45 505.9880 3304 C00002 2-phosphoglycerate 11.66 184.9856 3904 C00631 Phosphoenolpyruvate 11.57 166.9746 3374 C00074 Glucose-6-phosphate 11.72 259.0219 3937 C00668 METABOLITES_END #END