#METABOLOMICS WORKBENCH guying_20241009_181944 DATATRACK_ID:5277 STUDY_ID:ST003531 ANALYSIS_ID:AN005801 PROJECT_ID:PR002173 VERSION 1 CREATED_ON October 24, 2024, 5:13 pm #PROJECT PR:PROJECT_TITLE Micropeptide hSPAR, a glutamine regulator, suppresses tumor growth via PR:PROJECT_TITLE TRIM21-P27KIP1-mTOR pathway PR:PROJECT_SUMMARY The mammalian target of rapamycin (mTOR) plays pivotal roles in cancer growth PR:PROJECT_SUMMARY control upon amino acid response. Recently, cyclin-dependent kinase (CDK) PR:PROJECT_SUMMARY inhibitor cyclin-dependent kinase inhibitor 1B (CDKN1B or P27KIP1) 1 has been PR:PROJECT_SUMMARY reported as a non-canonical inhibitor to mTOR signaling in mouse embryo PR:PROJECT_SUMMARY fibroblasts (MEFs). However, the mechanisms underlying P27KIP1-mTOR axis are PR:PROJECT_SUMMARY yet-to-be uncovered. Here, we find that micropeptide human small regulatory PR:PROJECT_SUMMARY polypeptide of amino acid response (hSPAR）, through its C-terminus (hSPAR-C), PR:PROJECT_SUMMARY inhibits E3 ligase tripartite motif containing 21 (TRIM21)-mediated P27KIP1 PR:PROJECT_SUMMARY degradation and causes P27KIP1’s cytoplasmic accumulation in breast cancer PR:PROJECT_SUMMARY cells. Interestingly, hSPAR/hSPAR-C also serves as an inhibitor to glutamine PR:PROJECT_SUMMARY transporter SLC38A2 and remarkably decreases the cellular glutamine level PR:PROJECT_SUMMARY specifically in cancer cells. The resulted glutamine deprivation sequentially PR:PROJECT_SUMMARY triggers translocation of cytoplasmic P27KIP1 to lysosomes, where P27KIP1 PR:PROJECT_SUMMARY disrupts Ragulator complex and suppresses mTOR complex 1 (mTORC1) assembly. PR:PROJECT_SUMMARY Administration of hSPAR or hSPAR-C dramatically impedes breast cancer cell PR:PROJECT_SUMMARY proliferation and xenografic tumor growth. Collectively, we define hSPAR as an PR:PROJECT_SUMMARY intrinsic molecule to control cellular glutamine level and a PR:PROJECT_SUMMARY previously-unidentified tumor suppressor by promoting accumulation and PR:PROJECT_SUMMARY lysosomal-localization of P27KIP1 to inhibit mTORC1 assembly. PR:INSTITUTE University of Science and Technology of China PR:LAST_NAME Wang PR:FIRST_NAME Wei PR:ADDRESS Division of Life Sciences and Medicine, 443 Huangshan Road, Hefei city, Anhui PR:ADDRESS Province, 230022, China PR:EMAIL WW571@mail.ustc.edu.cn PR:PHONE 18604523231 #STUDY ST:STUDY_TITLE Micropeptide hSPAR, a glutamine regulator, suppresses tumor growth via ST:STUDY_TITLE TRIM21-P27KIP1-mTOR pathway - human MDA-MB-231 breast cancer cell ST:STUDY_SUMMARY hSPAR, a microprotein, is capable of specifically inhibiting the mTOR signaling ST:STUDY_SUMMARY activity and cell proliferation in MDA-MB-231 cells. Glutamine, an essential ST:STUDY_SUMMARY amino acid, plays a crucial role in regulating the mTOR signal. Our data ST:STUDY_SUMMARY indicate that in MDA-MB-231 cells, overexpressing hSPAR inhibits glutamine ST:STUDY_SUMMARY uptake, consequently suppressing the activation of the mTOR signaling. ST:INSTITUTE University of Science and Technology of China ST:LAST_NAME Wang ST:FIRST_NAME Wei ST:ADDRESS Division of Life Sciences and Medicine, 443 Huangshan Road, Hefei city, Anhui ST:ADDRESS Province ST:EMAIL WW571@mail.ustc.edu.cn ST:PHONE 18604523231 #SUBJECT SU:SUBJECT_TYPE Human 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 - Ctrl4 Sample source:MDA-MB-231 breast cancer cell | Transfection:Ctrl RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206743a.mzML SUBJECT_SAMPLE_FACTORS - Ctrl5 Sample source:MDA-MB-231 breast cancer cell | Transfection:Ctrl RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206744a.mzML SUBJECT_SAMPLE_FACTORS - Ctrl6 Sample source:MDA-MB-231 breast cancer cell | Transfection:Ctrl RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206745a.mzML SUBJECT_SAMPLE_FACTORS - ATG4 Sample source:MDA-MB-231 breast cancer cell | Transfection:ATG RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206746a.mzML SUBJECT_SAMPLE_FACTORS - ATG5 Sample source:MDA-MB-231 breast cancer cell | Transfection:ATG RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206747a.mzML SUBJECT_SAMPLE_FACTORS - ATG6 Sample source:MDA-MB-231 breast cancer cell | Transfection:ATG RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206748a.mzML SUBJECT_SAMPLE_FACTORS - hsPAR4 Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206749a.mzML SUBJECT_SAMPLE_FACTORS - hsPAR5 Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206750a.mzML SUBJECT_SAMPLE_FACTORS - hsPAR6 Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206751a.mzML SUBJECT_SAMPLE_FACTORS - hsPAR_C4 Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR_C RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206752a.mzML SUBJECT_SAMPLE_FACTORS - hsPAR_C5 Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR_C RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206753a.mzML SUBJECT_SAMPLE_FACTORS - hsPAR_C6 Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR_C RAW_FILE_NAME(Raw_File_Name)=MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206754a.mzML #COLLECTION CO:COLLECTION_SUMMARY The MDA-MB-231 breast cancer cells were cultured in Dulbecco’s modified CO:COLLECTION_SUMMARY Eagle’s medium (DMEM), containing 10% fetal bovine serum and 1% CO:COLLECTION_SUMMARY penicillin-streptomycin under humidified atmosphere of 5% CO2 at 37°C. Ce11s CO:COLLECTION_SUMMARY were counted, washed with cold PBS and then flash-frozen in liquid N2. CO:SAMPLE_TYPE Breast cancer cells #TREATMENT TR:TREATMENT_SUMMARY MDA-MB-231 breast cancer cells were transfected with empty vector, ΔATG1+2, TR:TREATMENT_SUMMARY hSPAR or hSPAR-C for 48 hours. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The sample was thawed on ice, 100 μL of ultrapure water extract (containing SP:SAMPLEPREP_SUMMARY protease inhibitors, PMSF and EDTA) was added to resuspend the cell pellet. SP:SAMPLEPREP_SUMMARY Divide 50 μL cell suspension and add 200 µL of methanol (precooled at -20°C) SP:SAMPLEPREP_SUMMARY and vortexed for 2 min under the condition of 2500 rpm. The sample was frozen in SP:SAMPLEPREP_SUMMARY liquid nitrogen for 5 min, removed on ice for 5 min, after that, the sample was SP:SAMPLEPREP_SUMMARY vortexed for 2 min.The previous step was repeated for 3 times. The sample was SP:SAMPLEPREP_SUMMARY centrifuged at 12000 rpm for 10 min at 4°C. Take 200 μL of supernatant into a SP:SAMPLEPREP_SUMMARY new centrifuge tube and place the supernatant in -20°C refrigerator for 30 min. SP:SAMPLEPREP_SUMMARY Then the supernatant was centrifuged at 12000 rpm for 10 min at 4°C. After SP:SAMPLEPREP_SUMMARY centrifugation, transfer 180 μL of supernatant through Protein Precipitation SP:SAMPLEPREP_SUMMARY Plate for further LC-MS analysis. The remaining 50 μL cell suspension was SP:SAMPLEPREP_SUMMARY frozen and thawed for 3 times, centrifuged at 12,000 rpm for 10 min, and the SP:SAMPLEPREP_SUMMARY supernatant was taken to determine the protein concentration by BCA Protein SP:SAMPLEPREP_SUMMARY Assay kit. SP:SAMPLEPREP_PROTOCOL_FILENAME USTC_Protocol.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME SCIEX ExionLC AD CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 2 mM ammonium acetate; 0.04% formic acid CH:SOLVENT_B 100% acetonitrile; 2 mM ammonium acetate; 0.04% formic acid CH:FLOW_GRADIENT The gradient was started at 90% B at 0-1.2 min, decreased to 60% B at 9 min, 40% CH:FLOW_GRADIENT B at 10-11 min, finaly ramped back to 90% B at 11.01-15 min CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40°C CH:METHODS_FILENAME USTC_Protocol.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE USTC_Protocol.pdf #MS MS:INSTRUMENT_NAME AB SCIEX QTRAP 5500 MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS - #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/mg of cells MS_METABOLITE_DATA_START Samples Ctrl4 Ctrl5 Ctrl6 ATG4 ATG5 ATG6 hsPAR4 hsPAR5 hsPAR6 hsPAR_C4 hsPAR_C5 hsPAR_C6 Factors Sample source:MDA-MB-231 breast cancer cell | Transfection:Ctrl Sample source:MDA-MB-231 breast cancer cell | Transfection:Ctrl Sample source:MDA-MB-231 breast cancer cell | Transfection:Ctrl Sample source:MDA-MB-231 breast cancer cell | Transfection:ATG Sample source:MDA-MB-231 breast cancer cell | Transfection:ATG Sample source:MDA-MB-231 breast cancer cell | Transfection:ATG Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR_C Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR_C Sample source:MDA-MB-231 breast cancer cell | Transfection:hsPAR_C L-Cystine 40.5283844 15.03263181 14.94837157 NA 37.72363076 9.663090431 NA NA NA NA NA NA N-Propionylglycine NA NA NA NA NA NA NA NA NA NA NA NA N-Isovaleroylglycine NA NA NA NA NA NA NA NA NA NA NA NA 5-Hydroxy-tryptophan NA NA NA NA NA NA NA NA NA NA NA NA Succinic-Acid 31808.22996 40919.81682 41812.84511 39258.55642 28943.68664 36265.11985 4239.301681 3602.783827 4385.166086 4602.492029 2988.394125 8254.580243 2-Aminoethanesulfonic-Acid 1968.104957 1742.710973 1964.959735 1787.031871 1845.10919 1857.302473 574.7437957 750.4347197 686.7168209 544.5007055 527.9997505 661.4739483 1,3-Dimethyluric-Acid NA NA NA NA NA NA NA NA NA NA NA NA 3,7-Dimethyluric-Acid NA NA NA NA NA NA NA NA NA NA NA NA MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Compounds Class Q1 (Da) Q3 (Da) Molecular Weight Ion mode Ionization model Formula cpd_ID HMDB CAS kegg_map L-Cystine L-Cystine Amino Acid metabolomics 239 120 240.02 Negative [M-H]- C6H12N2O4S2 C00491 HMDB00192 56-89-3 ko00270,ko01100,ko02010,ko04216,ko04974 N-Propionylglycine N-Propionylglycine Amino Acid metabolomics 130.1 74 131.06 Negative [M-H]- C5H9NO3 - HMDB00783 21709-90-0 - N-Isovaleroylglycine N-Isovaleroylglycine Amino Acid metabolomics 158.1 74 159.09 Negative [M-H]- C7H13NO3 - HMDB00678 16284-60-9 - 5-Hydroxy-tryptophan 5-Hydroxy-tryptophan Amino Acid metabolomics 219.1 74 220.22 Negative [M-H]- C11H12N2O3 - - 4350-09-8 - Succinic-Acid Succinic Acid Amino Acid metabolomics 117.03 99 118.03 Negative [M-H]- C4H6O4 C00042 HMDB00254 110-15-6 ko00020,ko00190,ko00250,ko00310,ko00350,ko00360,ko00620,ko00630,ko00640,ko00650,ko00760,ko00920,ko01100,ko01200,ko04024,ko04727,ko04922,ko05230 2-Aminoethanesulfonic-Acid 2-Aminoethanesulfonic Acid Organic Acid And Its Derivatives 124 80 125.15 Negative [M-H]- C2H7NO3S C00245 HMDB00251 107-35-7 ko00120,ko00430,ko00920,ko01100,ko02010,ko04080 1,3-Dimethyluric-Acid 1,3-Dimethyluric Acid Organic Acid And Its Derivatives 195 180 196.06 Negative [M-H]- C7H8N4O3 - HMDB01857 944-73-0 - 3,7-Dimethyluric-Acid 3,7-Dimethyluric Acid Organic Acid And Its Derivatives 195 180 196.06 Negative [M-H]- C7H8N4O3 - HMDB01982 13087-49-5 - METABOLITES_END #END