{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003531","ANALYSIS_ID":"AN005801","PROJECT_ID":"PR002173","VERSION":"1","CREATED_ON":"October 24, 2024, 5:13 pm"},

"PROJECT":{"PROJECT_TITLE":"Micropeptide hSPAR, a glutamine regulator, suppresses tumor growth via TRIM21-P27KIP1-mTOR pathway","PROJECT_SUMMARY":"The mammalian target of rapamycin (mTOR) plays pivotal roles in cancer growth control upon amino acid response. Recently, cyclin-dependent kinase (CDK) inhibitor cyclin-dependent kinase inhibitor 1B (CDKN1B or P27KIP1) 1 has been reported as a non-canonical inhibitor to mTOR signaling in mouse embryo fibroblasts (MEFs). However, the mechanisms underlying P27KIP1-mTOR axis are yet-to-be uncovered. Here, we find that micropeptide human small regulatory polypeptide of amino acid response (hSPAR）, through its C-terminus (hSPAR-C), inhibits E3 ligase tripartite motif containing 21 (TRIM21)-mediated P27KIP1 degradation and causes P27KIP1’s cytoplasmic accumulation in breast cancer cells. Interestingly, hSPAR/hSPAR-C also serves as an inhibitor to glutamine transporter SLC38A2 and remarkably decreases the cellular glutamine level specifically in cancer cells. The resulted glutamine deprivation sequentially triggers translocation of cytoplasmic P27KIP1 to lysosomes, where P27KIP1 disrupts Ragulator complex and suppresses mTOR complex 1 (mTORC1) assembly. Administration of hSPAR or hSPAR-C dramatically impedes breast cancer cell proliferation and xenografic tumor growth. Collectively, we define hSPAR as an intrinsic molecule to control cellular glutamine level and a previously-unidentified tumor suppressor by promoting accumulation and lysosomal-localization of P27KIP1 to inhibit mTORC1 assembly.","INSTITUTE":"University of Science and Technology of China","LAST_NAME":"Wang","FIRST_NAME":"Wei","ADDRESS":"Division of Life Sciences and Medicine, 443 Huangshan Road, Hefei city, Anhui Province, 230022, China","EMAIL":"WW571@mail.ustc.edu.cn","PHONE":"18604523231"},

"STUDY":{"STUDY_TITLE":"Micropeptide hSPAR, a glutamine regulator, suppresses tumor growth via TRIM21-P27KIP1-mTOR pathway - human MDA-MB-231 breast cancer cell","STUDY_SUMMARY":"hSPAR, a microprotein, is capable of specifically inhibiting the mTOR signaling activity and cell proliferation in MDA-MB-231 cells. Glutamine, an essential amino acid, plays a crucial role in regulating the mTOR signal. Our data indicate that in MDA-MB-231 cells, overexpressing hSPAR inhibits glutamine uptake, consequently suppressing the activation of the mTOR signaling.","INSTITUTE":"University of Science and Technology of China","LAST_NAME":"Wang","FIRST_NAME":"Wei","ADDRESS":"Division of Life Sciences and Medicine, 443 Huangshan Road, Hefei city, Anhui Province","EMAIL":"WW571@mail.ustc.edu.cn","PHONE":"18604523231"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Ctrl4",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"Ctrl"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206743a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl5",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"Ctrl"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206744a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl6",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"Ctrl"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206745a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"ATG4",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"ATG"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206746a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"ATG5",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"ATG"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206747a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"ATG6",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"ATG"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206748a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"hsPAR4",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"hsPAR"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206749a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"hsPAR5",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"hsPAR"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206750a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"hsPAR6",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"hsPAR"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206751a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"hsPAR_C4",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"hsPAR_C"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206752a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"hsPAR_C5",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"hsPAR_C"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206753a.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"hsPAR_C6",
"Factors":{"Sample source":"MDA-MB-231 breast cancer cell","Transfection":"hsPAR_C"},
"Additional sample data":{"RAW_FILE_NAME(Raw_File_Name)":"MWY-23-2561-a_12_WH6500-6_Amide-AA-02_V1.0_WQL_20231010-T23206754a.mzML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"The MDA-MB-231 breast cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum and 1% penicillin-streptomycin under humidified atmosphere of 5% CO2 at 37°C. Ce11s were counted, washed with cold PBS and then flash-frozen in liquid N2.","SAMPLE_TYPE":"Breast cancer cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"MDA-MB-231 breast cancer cells were transfected with empty vector, ΔATG1+2, hSPAR or hSPAR-C for 48 hours."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The sample was thawed on ice, 100 μL of ultrapure water extract (containing protease inhibitors, PMSF and EDTA) was added to resuspend the cell pellet. Divide 50 μL cell suspension and add 200 µL of methanol (precooled at -20°C) and vortexed for 2 min under the condition of 2500 rpm. The sample was frozen in liquid nitrogen for 5 min, removed on ice for 5 min, after that, the sample was vortexed for 2 min.The previous step was repeated for 3 times. The sample was centrifuged at 12000 rpm for 10 min at 4°C. Take 200 μL of supernatant into a new centrifuge tube and place the supernatant in -20°C refrigerator for 30 min. Then the supernatant was centrifuged at 12000 rpm for 10 min at 4°C. After centrifugation, transfer 180 μL of supernatant through Protein Precipitation Plate for further LC-MS analysis. The remaining 50 μL cell suspension was frozen and thawed for 3 times, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken to determine the protein concentration by BCA Protein Assay kit.","SAMPLEPREP_PROTOCOL_FILENAME":"USTC_Protocol.pdf"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"SCIEX ExionLC AD","COLUMN_NAME":"Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)","SOLVENT_A":"100% water; 2 mM ammonium acetate; 0.04% formic acid","SOLVENT_B":"100% acetonitrile; 2 mM ammonium acetate; 0.04% formic acid","FLOW_GRADIENT":"The gradient was started at 90% B at 0-1.2 min, decreased to 60% B at 9 min, 40% B at 10-11 min, finaly ramped back to 90% B at 11.01-15 min","FLOW_RATE":"0.4 mL/min","COLUMN_TEMPERATURE":"40°C","METHODS_FILENAME":"USTC_Protocol.pdf"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","ANALYSIS_PROTOCOL_FILE":"USTC_Protocol.pdf"},

"MS":{"INSTRUMENT_NAME":"AB SCIEX QTRAP 5500","INSTRUMENT_TYPE":"QTRAP","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"-"},

"MS_METABOLITE_DATA":{
"Units":"ng/mg of cells",

"Data":[{"Metabolite":"L-Cystine","Ctrl4":"40.5283844","Ctrl5":"15.03263181","Ctrl6":"14.94837157","ATG4":"NA","ATG5":"37.72363076","ATG6":"9.663090431","hsPAR4":"NA","hsPAR5":"NA","hsPAR6":"NA","hsPAR_C4":"NA","hsPAR_C5":"NA","hsPAR_C6":"NA"},{"Metabolite":"N-Propionylglycine","Ctrl4":"NA","Ctrl5":"NA","Ctrl6":"NA","ATG4":"NA","ATG5":"NA","ATG6":"NA","hsPAR4":"NA","hsPAR5":"NA","hsPAR6":"NA","hsPAR_C4":"NA","hsPAR_C5":"NA","hsPAR_C6":"NA"},{"Metabolite":"N-Isovaleroylglycine","Ctrl4":"NA","Ctrl5":"NA","Ctrl6":"NA","ATG4":"NA","ATG5":"NA","ATG6":"NA","hsPAR4":"NA","hsPAR5":"NA","hsPAR6":"NA","hsPAR_C4":"NA","hsPAR_C5":"NA","hsPAR_C6":"NA"},{"Metabolite":"5-Hydroxy-tryptophan","Ctrl4":"NA","Ctrl5":"NA","Ctrl6":"NA","ATG4":"NA","ATG5":"NA","ATG6":"NA","hsPAR4":"NA","hsPAR5":"NA","hsPAR6":"NA","hsPAR_C4":"NA","hsPAR_C5":"NA","hsPAR_C6":"NA"},{"Metabolite":"Succinic-Acid","Ctrl4":"31808.22996","Ctrl5":"40919.81682","Ctrl6":"41812.84511","ATG4":"39258.55642","ATG5":"28943.68664","ATG6":"36265.11985","hsPAR4":"4239.301681","hsPAR5":"3602.783827","hsPAR6":"4385.166086","hsPAR_C4":"4602.492029","hsPAR_C5":"2988.394125","hsPAR_C6":"8254.580243"},{"Metabolite":"2-Aminoethanesulfonic-Acid","Ctrl4":"1968.104957","Ctrl5":"1742.710973","Ctrl6":"1964.959735","ATG4":"1787.031871","ATG5":"1845.10919","ATG6":"1857.302473","hsPAR4":"574.7437957","hsPAR5":"750.4347197","hsPAR6":"686.7168209","hsPAR_C4":"544.5007055","hsPAR_C5":"527.9997505","hsPAR_C6":"661.4739483"},{"Metabolite":"1,3-Dimethyluric-Acid","Ctrl4":"NA","Ctrl5":"NA","Ctrl6":"NA","ATG4":"NA","ATG5":"NA","ATG6":"NA","hsPAR4":"NA","hsPAR5":"NA","hsPAR6":"NA","hsPAR_C4":"NA","hsPAR_C5":"NA","hsPAR_C6":"NA"},{"Metabolite":"3,7-Dimethyluric-Acid","Ctrl4":"NA","Ctrl5":"NA","Ctrl6":"NA","ATG4":"NA","ATG5":"NA","ATG6":"NA","hsPAR4":"NA","hsPAR5":"NA","hsPAR6":"NA","hsPAR_C4":"NA","hsPAR_C5":"NA","hsPAR_C6":"NA"}],

"Metabolites":[{"Metabolite":"L-Cystine","Compounds":"L-Cystine","Class":"Amino Acid metabolomics","Q1 (Da)":"239","Q3 (Da)":"120","Molecular Weight":"240.02","Ion mode":"Negative","Ionization model":"[M-H]-","Formula":"C6H12N2O4S2","cpd_ID":"C00491","HMDB":"HMDB00192","CAS":"56-89-3","kegg_map":"ko00270,ko01100,ko02010,ko04216,ko04974"},{"Metabolite":"N-Propionylglycine","Compounds":"N-Propionylglycine","Class":"Amino Acid metabolomics","Q1 (Da)":"130.1","Q3 (Da)":"74","Molecular Weight":"131.06","Ion mode":"Negative","Ionization model":"[M-H]-","Formula":"C5H9NO3","cpd_ID":"-","HMDB":"HMDB00783","CAS":"21709-90-0","kegg_map":"-"},{"Metabolite":"N-Isovaleroylglycine","Compounds":"N-Isovaleroylglycine","Class":"Amino Acid metabolomics","Q1 (Da)":"158.1","Q3 (Da)":"74","Molecular Weight":"159.09","Ion mode":"Negative","Ionization model":"[M-H]-","Formula":"C7H13NO3","cpd_ID":"-","HMDB":"HMDB00678","CAS":"16284-60-9","kegg_map":"-"},{"Metabolite":"5-Hydroxy-tryptophan","Compounds":"5-Hydroxy-tryptophan","Class":"Amino Acid metabolomics","Q1 (Da)":"219.1","Q3 (Da)":"74","Molecular Weight":"220.22","Ion mode":"Negative","Ionization model":"[M-H]-","Formula":"C11H12N2O3","cpd_ID":"-","HMDB":"-","CAS":"4350-09-8","kegg_map":"-"},{"Metabolite":"Succinic-Acid","Compounds":"Succinic Acid","Class":"Amino Acid metabolomics","Q1 (Da)":"117.03","Q3 (Da)":"99","Molecular Weight":"118.03","Ion mode":"Negative","Ionization model":"[M-H]-","Formula":"C4H6O4","cpd_ID":"C00042","HMDB":"HMDB00254","CAS":"110-15-6","kegg_map":"ko00020,ko00190,ko00250,ko00310,ko00350,ko00360,ko00620,ko00630,ko00640,ko00650,ko00760,ko00920,ko01100,ko01200,ko04024,ko04727,ko04922,ko05230"},{"Metabolite":"2-Aminoethanesulfonic-Acid","Compounds":"2-Aminoethanesulfonic Acid","Class":"Organic Acid And Its Derivatives","Q1 (Da)":"124","Q3 (Da)":"80","Molecular Weight":"125.15","Ion mode":"Negative","Ionization model":"[M-H]-","Formula":"C2H7NO3S","cpd_ID":"C00245","HMDB":"HMDB00251","CAS":"107-35-7","kegg_map":"ko00120,ko00430,ko00920,ko01100,ko02010,ko04080"},{"Metabolite":"1,3-Dimethyluric-Acid","Compounds":"1,3-Dimethyluric Acid","Class":"Organic Acid And Its Derivatives","Q1 (Da)":"195","Q3 (Da)":"180","Molecular Weight":"196.06","Ion mode":"Negative","Ionization model":"[M-H]-","Formula":"C7H8N4O3","cpd_ID":"-","HMDB":"HMDB01857","CAS":"944-73-0","kegg_map":"-"},{"Metabolite":"3,7-Dimethyluric-Acid","Compounds":"3,7-Dimethyluric Acid","Class":"Organic Acid And Its Derivatives","Q1 (Da)":"195","Q3 (Da)":"180","Molecular Weight":"196.06","Ion mode":"Negative","Ionization model":"[M-H]-","Formula":"C7H8N4O3","cpd_ID":"-","HMDB":"HMDB01982","CAS":"13087-49-5","kegg_map":"-"}]
}

}