#METABOLOMICS WORKBENCH yanma0820_20231011_190807 DATATRACK_ID:4391 STUDY_ID:ST002925 ANALYSIS_ID:AN004796 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 POSITIVE 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 Phenylalanine 197610159 285799052 204431548 279217303 266746088 207477282 207313220 229594029 204551203 218502303 200899574 244442443 181278315 205540556 267053518 251784543 209918211 261805833 276959770 Isoleucine 367467547 660176523 434412440 487843487 661416657 684886446 781181592 764030842 542512360 432023446 411489737 495726131 310008243 607008705 885719899 680374951 663083481 744735137 668971783 Leucine 172445123 296587220 197128954 247724361 292752050 299855156 382198502 338412137 217208495 213860552 203674939 231816670 164760629 279801171 404163217 299605709 283912665 365423434 296536844 Valine 3414057106 4080745340 3728897327 3287780194 5727683102 7219177534 6006065299 6916059030 9371637639 4631180777 3067217011 3326984473 4184700008 4998062026 5929286804 3974500687 5129748254 3684755576 2855518341 Tyrosine 32447483 53962206 35601305 45462603 45135875 38411158 45453628 36670235 39029212 42461850 33291214 43265558 39898763 29319979 50296138 32699032 32229898 39403609 37575811 Proline 650270360 737716486 430137840 466466109 487674283 482039017 652127630 611684798 619713530 435690541 397256727 691569593 378314592 884771704 780191272 682771731 741586080 678980431 721559159 Cystine 4802904 4991836 2576292 3338607 4709497 7679307 7718257 5852194 6374085 3136177 2545572 3009624 2464871 3772307 4315028 3663639 4804096 4110261 6396039 NADH 30999131 31294557 18907787 34240987 95658977 52132184 33595120 38330919 59182112 29193077 38899837 28669790 56672672 47875664 54150586 51036044 37016084 52456641 61046777 Threonine 139244921 209414853 104354844 125765767 183846466 186974763 228733539 215420932 73076961 112541114 102093961 103459762 98883523 115111339 131006627 107101671 148620811 143726792 102134276 Alanine 703415087 613643179 524729394 912729423 224614549 170043718 201835649 169696562 341024577 499118647 421249524 768037616 493452759 269166497 282820355 252887927 238308707 226465557 340512658 Acetyl-coA 4728165 10756071 6343132 10643492 20717881 15223927 9577984 10631658 15283705 10232211 9768009 8966371 12499900 21082471 23836844 20195798 22945112 21181221 12429410 Glutamine 3225869585 4233283228 4180680822 4610917130 3960312427 3313050642 2498059144 3226736641 2337074550 3918895629 3198472291 2853570950 3676629977 2925709524 3191547414 2893742829 2870367925 2641084046 2822433087 Asparagine 42484705 57313938 44678473 60322142 48912181 49258183 52226022 52216246 43642052 50140935 43296284 46259935 52106755 57185495 59696776 52132962 57339129 54951922 57622363 Glycine 44667772 46828607 37876801 53535496 60492154 58929516 54109535 52352521 34729773 45294152 43496806 70458834 44908690 62675227 71753566 54755440 86155130 76475216 69921046 Glutamate 1334538151 2257832541 1978673927 1499429610 2101610369 1700808013 1554583429 1819973783 1405127125 2416807874 1859244761 1839167958 2822139497 1876028197 1890006669 1577826433 1823075016 1659166704 1349018766 NAD 284047154 280438795 256761797 544632849 368926422 255363864 215201626 212593926 284731324 305644252 175673880 260710548 366037469 280879665 260891915 249962638 274292135 245401635 242777938 Serine 54021302 89064536 51171372 51859051 45174173 60798204 75044304 72709983 27785276 41220572 33612324 38727576 44567216 46020526 48109473 40968219 66665041 46360023 37141908 Aspartate 94468803 68108852 67687578 102316345 95537709 58354558 57631543 48986840 71075231 82369224 78983914 71834956 103616810 32024664 53314671 46503168 35728682 50309936 55118158 Histidine 113858491 167107494 179042385 122079094 115661535 157782160 150374369 151491839 92506187 157774892 155200532 171466109 143704315 128844438 120360847 133752858 87739525 123954776 115123063 Arginine 4903168 6046999 5334876 4021568 151761468 7352589 6975539 9355217 6391264 12288415 10827126 9693782 8576108 43766821 12881478 12628179 29283627 20162341 9655073 Lysine 244063344 222404436 159941163 183577795 190543014 216299472 304400513 224575257 196929084 404046081 382580037 448523523 264551696 470879159 565348680 318459814 446710914 379716575 397586088 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT(min) m/z Pubchem ID KEGG ID Phenylalanine 8.20 166.0860 3379 C00079 Isoleucine 8.38 132.1020 3697 C00407 Leucine 8.58 132.1020 3423 C00123 Valine 9.22 118.0860 3483 C00183 Tyrosine 9.40 182.0810 3382 C00082 Proline 9.78 116.0710 3448 C00148 Cystine 9.97 241.0310 3774 C00491 NADH 10.32 666.1326 3306 C00004 Threonine 10.33 120.0660 3488 C00188 Alanine 10.39 90.0550 3343 C00041 Acetyl-coA 10.59 810.1330 3326 C00024 Glutamine 10.73 147.0760 3364 C00064 Asparagine 10.85 133.0610 3452 C00152 Glycine 10.87 76.0390 3339 C00037 Glutamate 10.88 148.0600 3327 C00025 NAD 10.92 664.1169 3305 C00003 Serine 10.90 106.0500 3365 C00065 Aspartate 10.91 134.0450 3351 C00049 Histidine 11.81 156.0770 3435 C00135 Arginine 16.78 175.1190 3362 C00062 Lysine 17.32 147.1130 3349 C00047 METABOLITES_END #END