{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003174","ANALYSIS_ID":"AN005209","VERSION":"1","CREATED_ON":"April 18, 2024, 8:44 am"},

"PROJECT":{"PROJECT_TITLE":"O-GlcNAcase activity maintains stress granules for proximity-enhanced ATP production to ensure recovery from stress","PROJECT_SUMMARY":"Accurate disassembly of stress granules (SGs) after environmental stimuli release is essential for cells to maintain homeostasis , which requires ATP-consuming processes. However, the molecular mechanism whereby regulation of SGs programmatically disassembly and ATP restoration remain poorly understood in mammalian cells. Here we found that defect of OGA in cells leads to aggregates formation, severe autophagy and eventually apoptosis during stress recovery. OGA, which localized in SGs, had no effect on SGs formation but could protect SGs from rapid disassembly during stress recovery stage. Then the SGs localized glycolysis-related enzymes were reserved and concentrated in SGs during stress release for ATP production in a proximity manner, which was vital to guarantee cells resistant to stress and survival during recovery. Finally, supplementation of ATP to OGA knockdown cells during stress recovery significantly rescue cell from aggregates, autophagy and apoptosis. Together, these results describe a brand new mechanism on how OGA regulates the programmed disassembly of stress granules and restoration of ATP to safeguard cell viability in a very precisely programmed process, whose rate is rigorous regulated.","INSTITUTE":"Zhejiang University","DEPARTMENT":"Life Sciences Institute","LABORATORY":"Shixian Lin","LAST_NAME":"Chen","FIRST_NAME":"Yulin","ADDRESS":"Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang province, China","EMAIL":"ychen209@qq.com","PHONE":"18868107794"},

"STUDY":{"STUDY_TITLE":"O-GlcNAcase activity maintains stress granules for proximity-enhanced ATP production to ensure recovery from stress (Part 3)","STUDY_SUMMARY":"Accurate disassembly of stress granules (SGs) after environmental stimuli release is essential for cells to maintain homeostasis , which requires ATP-consuming processes. However, the molecular mechanism whereby regulation of SGs programmatically disassembly and ATP restoration remain poorly understood in mammalian cells. Here we found that defect of OGA in cells leads to aggregates formation, severe autophagy and eventually apoptosis during stress recovery. OGA, which localized in SGs, had no effect on SGs formation but could protect SGs from rapid disassembly during stress recovery stage. Then the SGs localized glycolysis-related enzymes were reserved and concentrated in SGs during stress release for ATP production in a proximity manner, which was vital to guarantee cells resistant to stress and survival during recovery. Finally, supplementation of ATP to OGA knockdown cells during stress recovery significantly rescue cell from aggregates, autophagy and apoptosis. Together, these results describe a brand new mechanism on how OGA regulates the programmed disassembly of stress granules and restoration of ATP to safeguard cell viability in a very precisely programmed process, whose rate is rigorous regulated. This is a continuation of study ST002927 and ST002936 where an in vitro reaction was performed with 13C-glucose on purified stress granules, to validate the conclusion.","INSTITUTE":"Zhejiang University","DEPARTMENT":"Life Sciences Institute","LABORATORY":"Shixian Lin","LAST_NAME":"Chen","FIRST_NAME":"Yulin","ADDRESS":"Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang province, China","EMAIL":"ychen209@qq.com","PHONE":"18868107794"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"QC-1",
"Factors":{"Sample source":"cultured cells","Factor":"QC"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"00QC","Replicate":"QC-1"}
},
{
"Subject ID":"-",
"Sample ID":"QC-2",
"Factors":{"Sample source":"cultured cells","Factor":"QC"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"00QC","Replicate":"QC-2"}
},
{
"Subject ID":"-",
"Sample ID":"QC-3",
"Factors":{"Sample source":"cultured cells","Factor":"QC"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"00QC","Replicate":"QC-3"}
},
{
"Subject ID":"-",
"Sample ID":"C1",
"Factors":{"Sample source":"cultured cells","Factor":"0hr"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"01C1","Replicate":"0hr-1"}
},
{
"Subject ID":"-",
"Sample ID":"C2",
"Factors":{"Sample source":"cultured cells","Factor":"0hr"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"02C2","Replicate":"0hr-2"}
},
{
"Subject ID":"-",
"Sample ID":"C3",
"Factors":{"Sample source":"cultured cells","Factor":"0hr"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"03C3","Replicate":"0hr-3"}
},
{
"Subject ID":"-",
"Sample ID":"C4",
"Factors":{"Sample source":"cultured cells","Factor":"1hr"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"04C4","Replicate":"1hr-1"}
},
{
"Subject ID":"-",
"Sample ID":"C5",
"Factors":{"Sample source":"cultured cells","Factor":"1hr"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"05C5","Replicate":"1hr-2"}
},
{
"Subject ID":"-",
"Sample ID":"C6",
"Factors":{"Sample source":"cultured cells","Factor":"1hr"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"06C6","Replicate":"1hr-3"}
},
{
"Subject ID":"-",
"Sample ID":"C7",
"Factors":{"Sample source":"cultured cells","Factor":"3hr"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"07C7","Replicate":"3hr-1"}
},
{
"Subject ID":"-",
"Sample ID":"C8",
"Factors":{"Sample source":"cultured cells","Factor":"3hr"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"08C8","Replicate":"3hr-2"}
},
{
"Subject ID":"-",
"Sample ID":"C9",
"Factors":{"Sample source":"cultured cells","Factor":"3hr"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"09C9","Replicate":"3hr-3"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Care was taken to quench cells quickly to minimize oxidation and degradation.","SAMPLE_TYPE":"in vitro reaction"},

"TREATMENT":{"TREATMENT_SUMMARY":"Stress granules purified from HeLa cells were treated with 13C-Glucose for 0, 1, 3 hr reaction."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Cells without stress treatment or treated with 0.6 M sorbitol stress for 1.5 h were scraped off and centrifuged to collect cell pellets. The cell pellets were thawed on ice, resuspended in stress granule lysis buffer (50 mM Tris, 100 mM K2OAc, 2 mM MgOAc, 0.5 mM DTT, 50 μg/mL heparin, 0.5% NP-40, 0.02% antifoam B, proteinase inhibitor cocktail, pH 7.4) and lysis by syringe. Lysates were centrifuged at 1000 × g for 5 min to collect supernatants. Supernatants were centrifuge at 18000 × g for 20 min to collect pellets and the pellets were washed once with stress granule lysis buffer. Then, pellets were resuspended in stress granule lysis buffer and centrifuged at 850 × g for 2 min to collect supernatants containing stress granule. Then NAD+, ATP and 13C-glucose were added for reaction at 37 ''C for 0, 1 and 3 hr. The reaction was quenched by 3-fold methanol and centrifuge to collect the supernant for analysis."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Shimadzu 20AD","COLUMN_NAME":"Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)","SOLVENT_A":"100% water; 20 mM ammonium carbonate","SOLVENT_B":"100% acetonitrile","FLOW_GRADIENT":"0.01 min 80% B, 20 min 20% B, 20.5 min 80% B, 24 min 80% B.","FLOW_RATE":"0.15 mL/min","COLUMN_TEMPERATURE":"45"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"ABI Sciex Triple Quad 5500+","INSTRUMENT_TYPE":"QTRAP","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The raw data were extracted with the software Analyst v1.7.2 and OS v1.7. This is a continuation of study ST002927 where a different set precusror/product ions were chosen for MS."},

"MS_METABOLITE_DATA":{
"Units":"Peak intensity",

"Data":[{"Metabolite":"NAD+","QC-1":"419354250.7","QC-2":"414041844.4","QC-3":"412981339.7","C1":"442889546.2","C2":"424716432.5","C3":"428748395","C4":"390013524.8","C5":"380239815.5","C6":"383146686.7","C7":"377206593.5","C8":"368412958.6","C9":"487616277.3"},{"Metabolite":"NADH","QC-1":"187325.335","QC-2":"177105.9819","QC-3":"196633.4502","C1":"77797.05618","C2":"59371.17913","C3":"145701.4602","C4":"452315.0818","C5":"215380.6386","C6":"226020.0072","C7":"65226.27646","C8":"264695.4321","C9":"118968.1466"},{"Metabolite":"NADP+","QC-1":"9781.245077","QC-2":"26043.42333","QC-3":"10907.51198","C1":"10802.71288","C2":"26746.68083","C3":"16934.68879","C4":"18783.57339","C5":"20430.22288","C6":"17179.85769","C7":"8864.356915","C8":"14590.35164","C9":"23920.8073"},{"Metabolite":"NADPH","QC-1":"15629.46918","QC-2":"31037.33371","QC-3":"17253.88755","C1":"59054.05136","C2":"137561.3349","C3":"40577.82055","C4":"158006.5103","C5":"49366.40513","C6":"101492.3133","C7":"15390.23324","C8":"160710.5737","C9":"118475.2337"},{"Metabolite":"Methionine-sulfone","QC-1":"20983315.8","QC-2":"21622759.4","QC-3":"21848463.72","C1":"20098109.59","C2":"21519164.32","C3":"23284166.01","C4":"21605973.64","C5":"20140325.94","C6":"20970365.74","C7":"19265772.4","C8":"20048801.94","C9":"21461383.56"}],

"Metabolites":[{"Metabolite":"NAD+","Precursor ion (m/z)":"664.1","Product ion (m/z)":"428","Retention time (min)":"7.7","PubChem CID":"5892"},{"Metabolite":"NADH","Precursor ion (m/z)":"666.1","Product ion (m/z)":"514","Retention time (min)":"7.14","PubChem CID":"439153"},{"Metabolite":"NADP+","Precursor ion (m/z)":"744.2","Product ion (m/z)":"136","Retention time (min)":"10.26","PubChem CID":"5886"},{"Metabolite":"NADPH","Precursor ion (m/z)":"746.15","Product ion (m/z)":"729","Retention time (min)":"10.26","PubChem CID":"5884"},{"Metabolite":"Methionine-sulfone","Precursor ion (m/z)":"182.1","Product ion (m/z)":"56","Retention time (min)":"5.1","PubChem CID":"445282"}]
}

}