Summary of Study ST003532
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002173. The data can be accessed directly via it's Project DOI: 10.21228/M8R23J This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
Study ID | ST003532 |
Study Title | Micropeptide hSPAR, a glutamine regulator, suppresses tumor growth via TRIM21-P27KIP1-mTOR pathway - HEK293T human embryonic kidney cells |
Study Summary | The microprotein hSPAR can specifically regulate the mTOR signaling activity and cell proliferation in breast cancer cells. However, such regulatory effects of hSPAR are not applicable in HEK293T cells. Metabolic data of amino acids indicate that overexpressing hSPAR does not affect the content of amino acids in HEK293T cells, including glutamine. The metabolic variations in amino acids triggered by hSPAR in different cell types may be the underlying molecular mechanism for the distinct regulatory effects of hSPAR. |
Institute | University Of Science And Technology of China |
Last Name | Wang |
First Name | Wei |
Address | 443 Huangshan Road, Hefei city, Anhui Province, 230022, China |
WW571@mail.ustc.edu.cn | |
Phone | 18604523231 |
Submit Date | 2024-10-10 |
Raw Data Available | Yes |
Raw Data File Type(s) | mzML |
Analysis Type Detail | LC-MS |
Release Date | 2025-01-06 |
Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
Project ID: | PR002173 |
Project DOI: | doi: 10.21228/M8R23J |
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 |
Subject:
Subject ID: | SU003661 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Mammals |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
mb_sample_id | local_sample_id | Sample source | Transfection |
---|---|---|---|
SA387366 | ATG_1 | HEK293T embryonic kidney cells | ATG |
SA387367 | ATG_2 | HEK293T embryonic kidney cells | ATG |
SA387368 | ATG_3 | HEK293T embryonic kidney cells | ATG |
SA387369 | Ctrl_1 | HEK293T embryonic kidney cells | Ctrl |
SA387370 | Ctrl_2 | HEK293T embryonic kidney cells | Ctrl |
SA387371 | Ctrl_3 | HEK293T embryonic kidney cells | Ctrl |
SA387372 | hSPAR_1 | HEK293T embryonic kidney cells | hSPAR |
SA387373 | hSPAR_2 | HEK293T embryonic kidney cells | hSPAR |
SA387374 | hSPAR_3 | HEK293T embryonic kidney cells | hSPAR |
Showing results 1 to 9 of 9 |
Collection:
Collection ID: | CO003654 |
Collection Summary: | The HEK293T human embryonic kidney 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: | Epithelial cells |
Treatment:
Treatment ID: | TR003670 |
Treatment Summary: | HEK293T human embryonic kidney cells were transfected with empty vector,ΔATG1+2 or Flag-hSPAR for 48 hours |
Sample Preparation:
Sampleprep ID: | SP003668 |
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 uL 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℃. Take 200 ul 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℃.After centrifugation, transfer 180 ul of supernatant through Protein Precipitation Plate for further LC-MS analysis. The left 50 ul cel 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 |
Combined analysis:
Analysis ID | AN005802 | AN005803 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | SCIEX ExionLC AD | SCIEX ExionLC AD |
Column | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
MS Type | ESI | ESI |
MS instrument type | QTRAP | QTRAP |
MS instrument name | ABI Sciex 5500 QTrap | ABI Sciex 5500 QTrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | ng/mg of cells | ng/mg of cells |
Chromatography:
Chromatography ID: | CH004406 |
Methods Filename: | USTC_Protocol.pdf |
Instrument Name: | SCIEX ExionLC AD |
Column Name: | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
Column Temperature: | 40°C |
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 |
Solvent A: | 100% water; 2 mM ammonium acetate; 0.04% formic acid |
Solvent B: | 100% acetonitrile; 2 mM ammonium acetate; 0.04% formic acid |
Chromatography Type: | HILIC |
MS:
MS ID: | MS005522 |
Analysis ID: | AN005802 |
Instrument Name: | ABI Sciex 5500 QTrap |
Instrument Type: | QTRAP |
MS Type: | ESI |
MS Comments: | - |
Ion Mode: | POSITIVE |
Analysis Protocol File: | USTC_Protocol.pdf |
MS ID: | MS005523 |
Analysis ID: | AN005803 |
Instrument Name: | ABI Sciex 5500 QTrap |
Instrument Type: | QTRAP |
MS Type: | ESI |
MS Comments: | - |
Ion Mode: | NEGATIVE |
Analysis Protocol File: | USTC_Protocol.pdf |