#METABOLOMICS WORKBENCH ychen209_20240416_020443 DATATRACK_ID:4778 STUDY_ID:ST003174 ANALYSIS_ID:AN005209 PROJECT_ID:PR001819 VERSION 1 CREATED_ON April 18, 2024, 8:44 am #PROJECT PR:PROJECT_TITLE O-GlcNAcase activity maintains stress granules for proximity-enhanced ATP PR:PROJECT_TITLE production to ensure recovery from stress PR:PROJECT_SUMMARY Accurate disassembly of stress granules (SGs) after environmental stimuli PR:PROJECT_SUMMARY release is essential for cells to maintain homeostasis , which requires PR:PROJECT_SUMMARY ATP-consuming processes. However, the molecular mechanism whereby regulation of PR:PROJECT_SUMMARY SGs programmatically disassembly and ATP restoration remain poorly understood in PR:PROJECT_SUMMARY mammalian cells. Here we found that defect of OGA in cells leads to aggregates PR:PROJECT_SUMMARY formation, severe autophagy and eventually apoptosis during stress recovery. PR:PROJECT_SUMMARY OGA, which localized in SGs, had no effect on SGs formation but could protect PR:PROJECT_SUMMARY SGs from rapid disassembly during stress recovery stage. Then the SGs localized PR:PROJECT_SUMMARY glycolysis-related enzymes were reserved and concentrated in SGs during stress PR:PROJECT_SUMMARY release for ATP production in a proximity manner, which was vital to guarantee PR:PROJECT_SUMMARY cells resistant to stress and survival during recovery. Finally, supplementation PR:PROJECT_SUMMARY of ATP to OGA knockdown cells during stress recovery significantly rescue cell PR:PROJECT_SUMMARY from aggregates, autophagy and apoptosis. Together, these results describe a PR:PROJECT_SUMMARY brand new mechanism on how OGA regulates the programmed disassembly of stress PR:PROJECT_SUMMARY granules and restoration of ATP to safeguard cell viability in a very precisely PR:PROJECT_SUMMARY programmed process, whose rate is rigorous regulated. PR:INSTITUTE Zhejiang University PR:DEPARTMENT Life Sciences Institute PR:LABORATORY Shixian Lin PR:LAST_NAME Chen PR:FIRST_NAME Yulin PR:ADDRESS Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang province, China PR:EMAIL ychen209@qq.com PR:PHONE 18868107794 #STUDY ST:STUDY_TITLE O-GlcNAcase activity maintains stress granules for proximity-enhanced ATP ST:STUDY_TITLE production to ensure recovery from stress (Part 3) ST:STUDY_SUMMARY Accurate disassembly of stress granules (SGs) after environmental stimuli ST:STUDY_SUMMARY release is essential for cells to maintain homeostasis , which requires ST:STUDY_SUMMARY ATP-consuming processes. However, the molecular mechanism whereby regulation of ST:STUDY_SUMMARY SGs programmatically disassembly and ATP restoration remain poorly understood in ST:STUDY_SUMMARY mammalian cells. Here we found that defect of OGA in cells leads to aggregates ST:STUDY_SUMMARY formation, severe autophagy and eventually apoptosis during stress recovery. ST:STUDY_SUMMARY OGA, which localized in SGs, had no effect on SGs formation but could protect ST:STUDY_SUMMARY SGs from rapid disassembly during stress recovery stage. Then the SGs localized ST:STUDY_SUMMARY glycolysis-related enzymes were reserved and concentrated in SGs during stress ST:STUDY_SUMMARY release for ATP production in a proximity manner, which was vital to guarantee ST:STUDY_SUMMARY cells resistant to stress and survival during recovery. Finally, supplementation ST:STUDY_SUMMARY of ATP to OGA knockdown cells during stress recovery significantly rescue cell ST:STUDY_SUMMARY from aggregates, autophagy and apoptosis. Together, these results describe a ST:STUDY_SUMMARY brand new mechanism on how OGA regulates the programmed disassembly of stress ST:STUDY_SUMMARY granules and restoration of ATP to safeguard cell viability in a very precisely ST:STUDY_SUMMARY programmed process, whose rate is rigorous regulated. This is a continuation of ST:STUDY_SUMMARY study ST002927 and ST002936 where an in vitro reaction was performed with ST:STUDY_SUMMARY 13C-glucose on purified stress granules, to validate the conclusion. ST:INSTITUTE Zhejiang University ST:DEPARTMENT Life Sciences Institute ST:LABORATORY Shixian Lin ST:LAST_NAME Chen ST:FIRST_NAME Yulin ST:ADDRESS Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang province, China ST:EMAIL ychen209@qq.com ST:PHONE 18868107794 #SUBJECT SU:SUBJECT_TYPE Cultured cells 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 - QC-1 Sample source:cultured cells | Factor:QC RAW_FILE_NAME(Raw file name)=00QC; Replicate=QC-1 SUBJECT_SAMPLE_FACTORS - QC-2 Sample source:cultured cells | Factor:QC RAW_FILE_NAME(Raw file name)=00QC; Replicate=QC-2 SUBJECT_SAMPLE_FACTORS - QC-3 Sample source:cultured cells | Factor:QC RAW_FILE_NAME(Raw file name)=00QC; Replicate=QC-3 SUBJECT_SAMPLE_FACTORS - C1 Sample source:cultured cells | Factor:0hr RAW_FILE_NAME(Raw file name)=01C1; Replicate=0hr-1 SUBJECT_SAMPLE_FACTORS - C2 Sample source:cultured cells | Factor:0hr RAW_FILE_NAME(Raw file name)=02C2; Replicate=0hr-2 SUBJECT_SAMPLE_FACTORS - C3 Sample source:cultured cells | Factor:0hr RAW_FILE_NAME(Raw file name)=03C3; Replicate=0hr-3 SUBJECT_SAMPLE_FACTORS - C4 Sample source:cultured cells | Factor:1hr RAW_FILE_NAME(Raw file name)=04C4; Replicate=1hr-1 SUBJECT_SAMPLE_FACTORS - C5 Sample source:cultured cells | Factor:1hr RAW_FILE_NAME(Raw file name)=05C5; Replicate=1hr-2 SUBJECT_SAMPLE_FACTORS - C6 Sample source:cultured cells | Factor:1hr RAW_FILE_NAME(Raw file name)=06C6; Replicate=1hr-3 SUBJECT_SAMPLE_FACTORS - C7 Sample source:cultured cells | Factor:3hr RAW_FILE_NAME(Raw file name)=07C7; Replicate=3hr-1 SUBJECT_SAMPLE_FACTORS - C8 Sample source:cultured cells | Factor:3hr RAW_FILE_NAME(Raw file name)=08C8; Replicate=3hr-2 SUBJECT_SAMPLE_FACTORS - C9 Sample source:cultured cells | Factor:3hr RAW_FILE_NAME(Raw file name)=09C9; Replicate=3hr-3 #COLLECTION CO:COLLECTION_SUMMARY Care was taken to quench cells quickly to minimize oxidation and degradation. CO:SAMPLE_TYPE in vitro reaction #TREATMENT TR:TREATMENT_SUMMARY Stress granules purified from HeLa cells were treated with 13C-Glucose for 0, 1, TR:TREATMENT_SUMMARY 3 hr reaction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells without stress treatment or treated with 0.6 M sorbitol stress for 1.5 h SP:SAMPLEPREP_SUMMARY were scraped off and centrifuged to collect cell pellets. The cell pellets were SP:SAMPLEPREP_SUMMARY thawed on ice, resuspended in stress granule lysis buffer (50 mM Tris, 100 mM SP:SAMPLEPREP_SUMMARY K2OAc, 2 mM MgOAc, 0.5 mM DTT, 50 μg/mL heparin, 0.5% NP-40, 0.02% antifoam B, SP:SAMPLEPREP_SUMMARY proteinase inhibitor cocktail, pH 7.4) and lysis by syringe. Lysates were SP:SAMPLEPREP_SUMMARY centrifuged at 1000 × g for 5 min to collect supernatants. Supernatants were SP:SAMPLEPREP_SUMMARY centrifuge at 18000 × g for 20 min to collect pellets and the pellets were SP:SAMPLEPREP_SUMMARY washed once with stress granule lysis buffer. Then, pellets were resuspended in SP:SAMPLEPREP_SUMMARY stress granule lysis buffer and centrifuged at 850 × g for 2 min to collect SP:SAMPLEPREP_SUMMARY supernatants containing stress granule. Then NAD+, ATP and 13C-glucose were SP:SAMPLEPREP_SUMMARY added for reaction at 37 'C for 0, 1 and 3 hr. The reaction was quenched by SP:SAMPLEPREP_SUMMARY 3-fold methanol and centrifuge to collect the supernant for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Shimadzu 20AD CH:COLUMN_NAME Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) CH:SOLVENT_A 100% water; 20 mM ammonium carbonate CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0.01 min 80% B, 20 min 20% B, 20.5 min 80% B, 24 min 80% B. CH:FLOW_RATE 0.15 mL/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex Triple Quad 5500+ MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The raw data were extracted with the software Analyst v1.7.2 and OS v1.7. This MS:MS_COMMENTS is a continuation of study ST002927 where a different set precusror/product ions MS:MS_COMMENTS were chosen for MS. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak intensity MS_METABOLITE_DATA_START Samples QC-1 QC-2 QC-3 C1 C2 C3 C4 C5 C6 C7 C8 C9 Factors Sample source:cultured cells | Factor:QC Sample source:cultured cells | Factor:QC Sample source:cultured cells | Factor:QC Sample source:cultured cells | Factor:0hr Sample source:cultured cells | Factor:0hr Sample source:cultured cells | Factor:0hr Sample source:cultured cells | Factor:1hr Sample source:cultured cells | Factor:1hr Sample source:cultured cells | Factor:1hr Sample source:cultured cells | Factor:3hr Sample source:cultured cells | Factor:3hr Sample source:cultured cells | Factor:3hr NAD+ 419354250.7 414041844.4 412981339.7 442889546.2 424716432.5 428748395 390013524.8 380239815.5 383146686.7 377206593.5 368412958.6 487616277.3 NADH 187325.335 177105.9819 196633.4502 77797.05618 59371.17913 145701.4602 452315.0818 215380.6386 226020.0072 65226.27646 264695.4321 118968.1466 NADP+ 9781.245077 26043.42333 10907.51198 10802.71288 26746.68083 16934.68879 18783.57339 20430.22288 17179.85769 8864.356915 14590.35164 23920.8073 NADPH 15629.46918 31037.33371 17253.88755 59054.05136 137561.3349 40577.82055 158006.5103 49366.40513 101492.3133 15390.23324 160710.5737 118475.2337 Methionine-sulfone 20983315.8 21622759.4 21848463.72 20098109.59 21519164.32 23284166.01 21605973.64 20140325.94 20970365.74 19265772.4 20048801.94 21461383.56 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor ion (m/z) Product ion (m/z) Retention time (min) PubChem CID NAD+ 664.1 428 7.7 5892 NADH 666.1 514 7.14 439153 NADP+ 744.2 136 10.26 5886 NADPH 746.15 729 10.26 5884 Methionine-sulfone 182.1 56 5.1 445282 METABOLITES_END #END