#METABOLOMICS WORKBENCH ychen209_20230919_041456 DATATRACK_ID:4325 STUDY_ID:ST002927 ANALYSIS_ID:AN004800 PROJECT_ID:PR001819 VERSION 1 CREATED_ON October 13, 2023, 10:36 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 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. 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 Factor:QC RAW_FILE_NAME=00QC; Other=QC-1 SUBJECT_SAMPLE_FACTORS - QC-2 Factor:QC RAW_FILE_NAME=00QC; Other=QC-2 SUBJECT_SAMPLE_FACTORS - QC-3 Factor:QC RAW_FILE_NAME=00QC; Other=QC-3 SUBJECT_SAMPLE_FACTORS - C1 Factor:noStressHeLa RAW_FILE_NAME=01C1; Other=noStressHeLa-1 SUBJECT_SAMPLE_FACTORS - C2 Factor:noStressHeLa RAW_FILE_NAME=02C2; Other=noStressHeLa-2 SUBJECT_SAMPLE_FACTORS - C3 Factor:noStressHeLa RAW_FILE_NAME=03C3; Other=noStressHeLa-3 SUBJECT_SAMPLE_FACTORS - C4 Factor:noStressKDOGA RAW_FILE_NAME=04C4; Other=noStressKDOGA-1 SUBJECT_SAMPLE_FACTORS - C5 Factor:noStressKDOGA RAW_FILE_NAME=05C5; Other=noStressKDOGA-2 SUBJECT_SAMPLE_FACTORS - C6 Factor:noStressKDOGA RAW_FILE_NAME=06C6; Other=noStressKDOGA-3 SUBJECT_SAMPLE_FACTORS - C7 Factor:StressHeLa RAW_FILE_NAME=07C7; Other=StressHeLa-1 SUBJECT_SAMPLE_FACTORS - C8 Factor:StressHeLa RAW_FILE_NAME=08C8; Other=StressHeLa-2 SUBJECT_SAMPLE_FACTORS - C9 Factor:StressHeLa RAW_FILE_NAME=09C9; Other=StressHeLa-3 SUBJECT_SAMPLE_FACTORS - C10 Factor:StressKDOGA RAW_FILE_NAME=10C10; Other=StressKDOGA-1 SUBJECT_SAMPLE_FACTORS - C11 Factor:StressKDOGA RAW_FILE_NAME=11C11; Other=StressKDOGA-2 SUBJECT_SAMPLE_FACTORS - C12 Factor:StressKDOGA RAW_FILE_NAME=12C12; Other=StressKDOGA-3 SUBJECT_SAMPLE_FACTORS - C13 Factor:RecoveryHeLa RAW_FILE_NAME=13C13; Other=RecoveryHeLa-1 SUBJECT_SAMPLE_FACTORS - C14 Factor:RecoveryHeLa RAW_FILE_NAME=14C14; Other=RecoveryHeLa-2 SUBJECT_SAMPLE_FACTORS - C15 Factor:RecoveryHeLa RAW_FILE_NAME=15C15; Other=RecoveryHeLa-3 SUBJECT_SAMPLE_FACTORS - C16 Factor:RecoveryKDOGA RAW_FILE_NAME=16C16; Other=RecoveryKDOGA-1 SUBJECT_SAMPLE_FACTORS - C17 Factor:RecoveryKDOGA RAW_FILE_NAME=17C17; Other=RecoveryKDOGA-2 SUBJECT_SAMPLE_FACTORS - C18 Factor:RecoveryKDOGA RAW_FILE_NAME=18C18; Other=RecoveryKDOGA-3 #COLLECTION CO:COLLECTION_SUMMARY Care was taken to quench cells quickly to minimize oxidation and degradation. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY HeLa cells of shControl or shOGA were treated with no stress, stress by 1 M TR:TREATMENT_SUMMARY sorbitol for 1 hr, or recovery from stress for 2 hr. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Briefly, equal confluency of cells in 6-well plate with 3 biological replicates SP:SAMPLEPREP_SUMMARY were rinsed by 1 mL HPLC-grade water rapidly and immediately quenched by SP:SAMPLEPREP_SUMMARY directly added by 1 mL liquid nitrogen to the plate. Then, 1 mL ice-cold 90% MC SP:SAMPLEPREP_SUMMARY buffer (MC, 9: 1 MeOH: CHCl3. 90% MC, 9 : 1 MC: water) with 5 µM methionine SP:SAMPLEPREP_SUMMARY sulfone as internal signal was added to each well of plate and the cells were SP:SAMPLEPREP_SUMMARY scraped and transferred to1.5-mL tubes. Extracts were centrifuged at 4 ℃ for 5 SP:SAMPLEPREP_SUMMARY min at 16,000 xg and the supernatants were transferred to new clean 1.5-mL tubes SP:SAMPLEPREP_SUMMARY and equal volume of each sample was pooled for the QC sample. Finally, the SP:SAMPLEPREP_SUMMARY extracts were dried using vacuum concentrator system and store at -80 ℃ until SP:SAMPLEPREP_SUMMARY analysis. For targeted analysis, the dried extracts were resuspended by 100 µL SP:SAMPLEPREP_SUMMARY 60% acetonitrile and centrifuged at 4 ℃ for 15 min at 15,000 xg after 30 min SP:SAMPLEPREP_SUMMARY incubation at 4 ℃. Only the supernatant was transferred to autosampler vials SP:SAMPLEPREP_SUMMARY for MS 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. #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 C10 C11 C12 C13 C14 C15 C16 C17 C18 Factors Factor:QC Factor:QC Factor:QC Factor:noStressHeLa Factor:noStressHeLa Factor:noStressHeLa Factor:noStressKDOGA Factor:noStressKDOGA Factor:noStressKDOGA Factor:StressHeLa Factor:StressHeLa Factor:StressHeLa Factor:StressKDOGA Factor:StressKDOGA Factor:StressKDOGA Factor:RecoveryHeLa Factor:RecoveryHeLa Factor:RecoveryHeLa Factor:RecoveryKDOGA Factor:RecoveryKDOGA Factor:RecoveryKDOGA glycine 2754.232798 1838.235958 2553.051991 2376.729386 4544.026679 4799.458508 3573.96817 3062.692338 4237.807393 255.3120253 N/A N/A 255.4126361 510.5393872 N/A 2093.282001 1991.250066 1531.569377 816.9092114 1787.214641 561.4471034 alanine 6421646.343 6312144.653 6086779.44 16030200.69 14367434.67 16645764.25 9896224.269 8758550.345 9096302.971 1624537.066 1627165.679 1653486.201 1340471.172 1344334.036 1224364.28 5242493.538 4447658.669 4689580.708 2348167.191 2465492.214 1779904.166 serine 5398499.464 5509023.25 5216107.908 7560128.863 6667797.797 7431813.188 7700659.246 6681999.516 7524135.381 2255251.864 2109233.914 2152192.805 1964352.775 2022437.871 1680638.777 9904753.725 8076304.367 7959127.698 4769065.829 5729632.891 3483853.115 proline 25548190.34 24966455.5 26202928.24 51874768 50955409.85 55717680.53 41045787.28 37148160.91 41918283.9 7795240.457 7649700.336 8019279.552 7108786.46 7092672.743 5753093.144 25872198.57 21131214.83 22986606.22 8962750.835 9341675.295 7184091.923 valine 1306945.416 1299042.884 1335533.215 2135666.715 1990844.884 2094108.683 1522917.419 1301608.445 1426013.994 531672.978 552522.2071 556015.9636 279022.1974 566530.445 449100.5563 2380686.563 1602764.166 1660529.458 1229406.489 1454425.525 862339.2991 threonine 21658148.44 20805787.94 21913473.85 42899320.98 37945506.85 43699632.07 29382214.99 24449877.96 26239899.03 7343102.404 7259367.185 7607085.787 6730049.578 7174549.579 6222316.264 28657564.84 24062847.75 24335427.18 16407839.17 17085336.54 11223537.03 cysteine N/A 2757.255828 N/A 3036.308409 2013.882117 1224.866698 1989.297218 3033.636757 1877.211861 909.5795495 2348.619474 3318.981964 1938.710805 510.1630853 N/A 510.5173622 N/A 1378.247419 N/A N/A N/A leucine-isoleucine 101637502.4 104562158.9 108332704.3 181063658 158663883.8 168029861.2 123391592.9 108240031.1 109749528 46246219.87 44183461.64 47135872.11 43429739.25 45935022.19 38317967.45 184646810.8 134582952.4 137276668.1 104015228 114794001.5 66513208.5 asparagine 408464.2108 400008.1662 272297.3787 870572.1802 741689.7358 877518.3384 586579.7045 498181.5131 555062.8853 113471.8837 128218.2965 120814.7075 93646.54678 90856.42847 90682.63498 564636.8289 504280.1795 512109.1022 160779.8997 182488.1808 145940.0214 aspartate 4692753.61 4465293.139 4465947.279 6253825.669 5444251.103 6167937.258 4692606.132 3897035.322 4661738.482 6385251.515 5630763.403 6393407.249 2802680.558 2703622.507 2490169.888 6035730.506 5384961.648 5525727.471 1697567.027 1726132.483 1621951.708 lysine 294597.2684 265075.4589 290852.4706 254766.7653 180345.1215 185335.7756 128743.7433 103661.2444 53628.04173 185496.1641 165239.231 194492.538 177701.4856 99290.21538 122291.9881 747537.0087 395391.1594 384103.8489 515275.54 676192.1863 315064.9033 glutamine 124880569.2 133792529.7 123528240.6 192482703.3 158084678.7 179279420.2 170669540.7 143222372.9 159660794.6 63483681.62 67871865.89 66715251.3 55756292.06 60938132.36 54826485.99 187279467.7 157138715.6 159393117.4 97878554.21 110317753.4 83367472.57 glutamate 68913505.4 71072456.95 67043744.37 94107873.93 82146769.58 89551766.69 66850536.61 61452491.79 69100890.56 82049341 82295630.59 85339167.55 53919328.03 55811216.47 50855809.06 81511961.93 73181296.57 77742547.81 35265954.79 36202614.37 35664107.64 methionine 13572806.78 13165814.35 13556924.21 22855196.56 19909700.64 20987005.95 15767816.85 14040311.05 14723911.32 6234052.123 5965729.267 6116399.933 5540980.693 5886828.364 4940780.297 24217204.78 16805811.55 17015953.72 12312361.25 14168967.18 8222974.568 histidine 35652483.04 21790643.24 37486707.86 60033976.14 46652375.49 56896364.83 42753483.7 37575991 26310701.68 15500529.87 15746471.88 16254144.21 15142707.82 15756043.17 13240435.99 60267551.73 46419820.93 27343825.44 37696698.91 48428154.3 27615302.07 arginine 2828693.25 1827787.743 3673090.13 2558072.011 1638811.527 1562210.512 1451544.548 1069980.658 971920.556 2239165.614 1999091.627 2109115.497 1947389.672 1739272.55 1756595.738 2328110.494 4146534.19 4254622.53 5182328.14 4535367.323 1998937.309 glucosamine 288559.7762 363415.6326 582454.7551 254693.7795 130079.2102 177028.7439 251918.0699 290418.3094 66732.47344 135945.9811 812187.9556 43560.37924 51351.18415 552150.1345 230985.0678 557859.1899 899743.5349 230274.3741 925356.9911 1009789.454 332029.9721 tyrosine 5650364.132 5541962.259 5644776.213 10225646.51 8749682.087 9510578.792 6516162.119 5877058.558 6366582.07 2444853.161 2428879.245 2599110.666 2345064.362 2465273.637 2056953.751 9624393.262 6781204.451 6989272.336 4830006.874 5287856.78 3084184.196 N-acetyl-glucosamine 156885.227 236457.2215 174806.0686 98729.69379 31785.42933 97148.09375 340991.2029 319929.183 230014.6925 59021.23878 40678.44183 61923.96357 607851.6373 384511.0059 473687.203 69347.3068 76081.80829 71896.92517 306938.2577 176926.5424 204659.754 D-glucosamine-6-phosphate 1530.654127 2757.235219 305.9093542 1735.184567 2413.853907 N/A 714.0122392 1326.464779 2708.785752 N/A 204.1744375 1020.758895 3620.536682 1528.576116 765.5514031 921.3919352 1887.590327 1428.616249 3306.046578 408.2596428 408.4106344 CMP 281580.6899 276614.8515 271815.1713 82928.53999 81931.59764 111961.6625 90745.73856 76106.00306 22700.81029 78341.25892 132753.2661 153555.5709 233213.1771 206452.355 207654.7725 670746.9302 609030.3999 721726.7074 413740.1 380140.5031 324677.7845 UMP 185186.1518 152313.771 175952.9251 103513.8162 66471.92798 125910.3602 70732.1265 94165.88042 88470.36644 85481.85046 83563.11025 83437.25584 101918.2588 136048.6386 110526.6383 303046.4141 276776.8768 291148.4636 251479.0465 208672.3507 205591.4626 AMP 4125173.965 4091801.61 4619889.443 2973471.35 2120225.214 2955623.635 2567458.592 2621105.676 2133015.208 3468911.338 3115985.325 3648140.228 6642544.404 6000021.549 6007364.345 5551086.219 2325347.315 5755177.476 6061302.629 5517629.715 5819165.65 GMP 517003.8808 597635.6525 556877.1962 261944.9734 188280.7934 267018.1388 303377.8084 291298.2186 207208.8002 226225.4007 193402.5874 278251.5282 828966.3227 372442.2879 382164.7853 802161.2655 750747.1559 842105.4598 1252428.669 1078861.261 1092978.276 NAD+_POS 4340350.753 4213055.166 4154910.522 8970081.326 8263167.188 9114621.064 7021511.316 6299694.428 6478868.111 3392870.776 3266382.04 3519689.816 2074604.581 2205528.309 1883988.884 2503775.568 2204843.282 2344039.789 727126.686 711786.4184 650807.438 NADH 135674.0266 140167.323 130319.2728 229937.3843 224251.3167 257709.1743 176232.3039 162242.4689 170667.3887 107691.7727 101359.3512 57133.94221 82668.24012 92579.28712 87470.70281 135319.9269 110297.4511 107257.9688 50065.47828 53401.78185 35468.83159 NADP+_pos 37833.93862 25060.10055 42447.67479 16857.50098 107419.8342 64159.53339 62549.90984 13274.58372 42282.14409 101366.2115 81199.91369 158417.89 98519.44547 131357.7365 60692.46861 23851.18825 42066.66165 56536.4525 20706.56753 45798.1336 60500.2813 NADPH 100564.861 109548.6376 98228.01369 73145.17238 74698.55123 55487.62773 68212.38957 66106.23654 52554.72213 151767.2009 75187.62455 121431.9926 101826.8082 135968.9049 101073.5574 76011.12587 64207.38847 78131.2822 87566.1024 84660.73666 111142.447 coenzyme A_pos 1378.57518 4571.957084 1025.793795 3372.224901 3613.86083 9950.01911 8934.655599 1644.718659 4901.773809 5969.798136 6692.787339 8577.301963 1281.708949 3837.225682 2552.921898 2808.050173 7350.359687 7120.707505 2450.641996 1531.989109 1838.132594 acetyl-CoA_pos 7301.617624 27270.73512 22295.75274 41735.72944 11460.42269 14262.33163 27267.63975 4414.829768 23265.00712 12529.39532 25383.0575 42374.40918 30240.66943 26361.73775 35142.39007 33572.45474 11471.5735 4857.877701 55049.44799 11887.85478 11955.01256 succinyl-CoA_pos 5105.857807 3113.209217 1580.023742 19523.2897 6577.490387 5126.616281 4749.499384 7130.285432 5561.514833 357.3553357 1174.334019 408.4159679 N/A 204.1999997 N/A 2192.405029 612.5460743 3874.598985 N/A 306.3 N/A phenylalanine 38810345.19 37612067.96 38549335.58 67194352.02 57966367.05 63346527.31 45528008.84 40811174.79 43494318.65 17287449.8 16490262.28 17481654.25 15467876.93 16579984.27 13705528.91 63967028.09 43386945.23 46348303.02 33039751.28 37819281.38 22246856.15 tryptophan 5037419.907 4952229.129 5057370.597 8694128.608 7929613.147 8248409.225 5997071.558 5462934.546 5663791.234 2685774.705 2565091.4 2644074.306 2415683.395 2500704.683 2060053.97 8534025.061 6148490.924 6281930.678 4065537.683 4809645.352 2749386.255 Methionine-sulfone 24148643.14 24127594.24 24678222.92 24189266.44 23439340.07 23895011.18 23406994.86 23557944.91 23345046.72 22286754.48 22522282.03 23602188.09 28047269.94 26078134.71 23611139.52 25136783.05 21952882.87 25390856.95 23074612.47 21924053.68 23942229.68 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor ion (m/z) Product ion (m/z) Retention time (min) glycine 76.1 30.5 8.51 alanine 90.1 44.2 7.8 serine 106 60 8.58 proline 116.1 70.1 6.36 valine 118.02 58 6.03 threonine 120 74 7.43 cysteine 122.1 59.1 9.06 leucine-isoleucine 1322.1 86 5.01 asparagine 133.1 74 8.18 aspartate 134 74 8.57 lysine 147 67 13.82 glutamine 147.1 84.1 8.01 glutamate 148.1 84.1 8.15 methionine 150.1 133 5.51 histidine 156.1 110.1 7.66 arginine 175.02 60 14.32 glucosamine 180 162 7.53 tyrosine 182.1 77 6.66 N-acetyl-glucosamine 222 138 4.8 D-glucosamine-6-phosphate 260 126 9.99 CMP 234 112 9.39 UMP 325 97 8.82 AMP 348.15 136 7.81 GMP 364 152 NAD+_POS 664.1 428 7.7 NADH 666.1 514 7.14 NADP+_pos 744.2 136 10.26 NADPH 746.15 729 10.26 coenzyme A_pos 768 261 8.15 acetyl-CoA_pos 810 303 7.21 succinyl-CoA_pos 868.1 361 phenylalanine 166.1 103 4.6 tryptophan 205 85 5.58 Methionine-sulfone 182.1 56 5.1 METABOLITES_END #END