#METABOLOMICS WORKBENCH guying_20241010_012714 DATATRACK_ID:5278 STUDY_ID:ST003532 ANALYSIS_ID:AN005803 PROJECT_ID:PR002173 VERSION 1 CREATED_ON October 24, 2024, 5:17 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 - HEK293T human embryonic kidney cells ST:STUDY_SUMMARY The microprotein hSPAR can specifically regulate the mTOR signaling activity and ST:STUDY_SUMMARY cell proliferation in breast cancer cells. However, such regulatory effects of ST:STUDY_SUMMARY hSPAR are not applicable in HEK293T cells. Metabolic data of amino acids ST:STUDY_SUMMARY indicate that overexpressing hSPAR does not affect the content of amino acids in ST:STUDY_SUMMARY HEK293T cells, including glutamine. The metabolic variations in amino acids ST:STUDY_SUMMARY triggered by hSPAR in different cell types may be the underlying molecular ST:STUDY_SUMMARY mechanism for the distinct regulatory effects of hSPAR. ST:INSTITUTE University Of Science And Technology Of China ST:LAST_NAME Wang ST:FIRST_NAME Wei ST:ADDRESS 443 Huangshan Road, Hefei city, Anhui Province, 230022, China ST:EMAIL WW571@mail.ustc.edu.cn ST:PHONE 18604523231 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 - Ctrl_1 Sample source:HEK293T embryonic kidney cells | Transfection:Ctrl RAW_FILE_NAME(Raw_file_name)=MWY-23-3856-a_9_WH6500-16_Amide-AA-02_V1.0_LA_20231212-T23292086a SUBJECT_SAMPLE_FACTORS - Ctrl_2 Sample source:HEK293T embryonic kidney cells | Transfection:Ctrl RAW_FILE_NAME(Raw_file_name)=MWY-23-3856-a_9_WH6500-16_Amide-AA-02_V1.0_LA_20231212-T23292087a SUBJECT_SAMPLE_FACTORS - Ctrl_3 Sample source:HEK293T embryonic kidney cells | Transfection:Ctrl RAW_FILE_NAME(Raw_file_name)=MWY-23-3856-a_9_WH6500-16_Amide-AA-02_V1.0_LA_20231212-T23292088a SUBJECT_SAMPLE_FACTORS - ATG_1 Sample source:HEK293T embryonic kidney cells | Transfection:ATG RAW_FILE_NAME(Raw_file_name)=MWY-23-3856-a_9_WH6500-16_Amide-AA-02_V1.0_LA_20231212-T23292089a SUBJECT_SAMPLE_FACTORS - ATG_2 Sample source:HEK293T embryonic kidney cells | Transfection:ATG RAW_FILE_NAME(Raw_file_name)=MWY-23-3856-a_9_WH6500-16_Amide-AA-02_V1.0_LA_20231212-T23292090a SUBJECT_SAMPLE_FACTORS - ATG_3 Sample source:HEK293T embryonic kidney cells | Transfection:ATG RAW_FILE_NAME(Raw_file_name)=MWY-23-3856-a_9_WH6500-16_Amide-AA-02_V1.0_LA_20231212-T23292091a SUBJECT_SAMPLE_FACTORS - hSPAR_1 Sample source:HEK293T embryonic kidney cells | Transfection:hSPAR RAW_FILE_NAME(Raw_file_name)=MWY-23-3856-a_9_WH6500-16_Amide-AA-02_V1.0_LA_20231212-T23292092a SUBJECT_SAMPLE_FACTORS - hSPAR_2 Sample source:HEK293T embryonic kidney cells | Transfection:hSPAR RAW_FILE_NAME(Raw_file_name)=MWY-23-3856-a_9_WH6500-16_Amide-AA-02_V1.0_LA_20231212-T23292093a SUBJECT_SAMPLE_FACTORS - hSPAR_3 Sample source:HEK293T embryonic kidney cells | Transfection:hSPAR RAW_FILE_NAME(Raw_file_name)=MWY-23-3856-a_9_WH6500-16_Amide-AA-02_V1.0_LA_20231212-T23292094a #COLLECTION CO:COLLECTION_SUMMARY The HEK293T human embryonic kidney 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 Epithelial cells #TREATMENT TR:TREATMENT_SUMMARY HEK293T human embryonic kidney cells were transfected with empty vector,ΔATG1+2 TR:TREATMENT_SUMMARY or Flag-hSPAR 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 uL cell suspension and add 200 µL of methanol (precooled at SP:SAMPLEPREP_SUMMARY -20°C)and vortexed for 2 min under the condition of 2500 rpm, The sample was SP:SAMPLEPREP_SUMMARY frozen in liquid nitrogen for 5 min, removed on ice for 5 min, after that, the SP:SAMPLEPREP_SUMMARY sample was vortexed for 2 min.The previous step was repeated for 3 times. The SP:SAMPLEPREP_SUMMARY sample was centrifuged at 12000 rpm for 10 min at 4℃. Take 200 ul of SP:SAMPLEPREP_SUMMARY supernatant into a new centrifuge tube and place the supernatant in -20°C SP:SAMPLEPREP_SUMMARY refrigerator for 30 min, Then the supernatant was centrifuged at 12000 rpm for SP:SAMPLEPREP_SUMMARY 10 min at 4℃.After centrifugation, transfer 180 ul of supernatant through SP:SAMPLEPREP_SUMMARY Protein Precipitation Plate for further LC-MS analysis. The left 50 ul cel SP:SAMPLEPREP_SUMMARY suspension was frozen and thawed for 3 times, centrifuged at 12,000 rpm for 10 SP:SAMPLEPREP_SUMMARY min, and the supernatant was taken to determine the protein concentration by BCA SP:SAMPLEPREP_SUMMARY Protein 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 Ctrl_1 Ctrl_2 Ctrl_3 ATG_1 ATG_2 ATG_3 hSPAR_1 hSPAR_2 hSPAR_3 Factors Sample source:HEK293T embryonic kidney cells | Transfection:Ctrl Sample source:HEK293T embryonic kidney cells | Transfection:Ctrl Sample source:HEK293T embryonic kidney cells | Transfection:Ctrl Sample source:HEK293T embryonic kidney cells | Transfection:ATG Sample source:HEK293T embryonic kidney cells | Transfection:ATG Sample source:HEK293T embryonic kidney cells | Transfection:ATG Sample source:HEK293T embryonic kidney cells | Transfection:hSPAR Sample source:HEK293T embryonic kidney cells | Transfection:hSPAR Sample source:HEK293T embryonic kidney cells | Transfection:hSPAR 1,3-Dimethyluric-Acid NA NA NA NA NA NA NA NA NA 2-Aminoethanesulfonic-Acid 5060.96275 4989.290508 5209.362939 4262.167073 4646.901454 4990.115532 2544.467507 2785.710465 3194.368548 3,7-Dimethyluric-Acid NA NA NA NA NA NA NA NA NA 5-Hydroxy-tryptophan NA NA NA NA NA NA NA NA NA L-Cystine 51.96319336 64.58142303 43.73791551 45.85123475 55.78286622 32.82153013 34.32512726 44.4688741 34.89655978 N-Isovaleroylglycine 0.955616148 1.127814281 1.058702557 0.969005306 0.950766658 0.901099755 0.970796043 0.704663782 0.803509266 N-Propionylglycine NA NA NA NA NA NA NA NA NA Succinic-Acid 12542.21626 10835.79118 12392.48168 9674.887594 13145.89773 14443.27573 10177.6184 12189.3175 11571.09733 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 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 - 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 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 - 5-Hydroxy-tryptophan 5-Hydroxy-tryptophan Amino Acid metabolomics 219.1 74 220.22 Negative [M-H]- C11H12N2O3 - - 4350-09-8 - 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-Isovaleroylglycine N-Isovaleroylglycine Amino Acid metabolomics 158.1 74 159.09 Negative [M-H]- C7H13NO3 - HMDB00678 16284-60-9 - N-Propionylglycine N-Propionylglycine Amino Acid metabolomics 130.1 74 131.06 Negative [M-H]- C5H9NO3 - HMDB00783 21709-90-0 - 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 METABOLITES_END #END