#METABOLOMICS WORKBENCH timmccubbin_20250511_172512 DATATRACK_ID:5903 STUDY_ID:ST003912 ANALYSIS_ID:AN006424 PROJECT_ID:PR002447 VERSION 1 CREATED_ON May 12, 2025, 6:51 pm #PROJECT PR:PROJECT_TITLE TANGO2 binds crystallin alpha B and its loss causes desminopathy PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Analysis of intra-cellular concentrations of central carbon metabolites to PR:PROJECT_SUMMARY understand metabolic changes underpinning Cal51 human cell lines with TANGO2 PR:PROJECT_SUMMARY present or knocked-out. PR:INSTITUTE The Kids Research Institute Australia PR:LAST_NAME Filipovska PR:FIRST_NAME Aleksandra PR:ADDRESS The Kids Research Institute Australia, Northern Entrance, Perth Children′s PR:ADDRESS Hospital, 15 Hospital Avenue, Nedlands, Western Australia, 6009, Australia PR:EMAIL aleksandra.filipovska@uwa.edu.au PR:PHONE +61 8 6319 1000 #STUDY ST:STUDY_TITLE TANGO2 binds crystallin alpha B and its loss causes desminopathy ST:STUDY_TYPE Targeted LCMS metabolomics ST:STUDY_SUMMARY Mutations in the TANGO2 gene cause an autosomal recessive disorder characterised ST:STUDY_SUMMARY by developmental delay, stress-induced episodic rhabdomyolysis, and cardiac ST:STUDY_SUMMARY arrhythmias along with severe metabolic crises. Although TANGO2 mutations result ST:STUDY_SUMMARY in a well characterised disease pathology, the function of TANGO2 is still ST:STUDY_SUMMARY unknown. To investigate the function of TANGO2, we knocked out the TANGO2 gene ST:STUDY_SUMMARY in human cells and mice. We identify that loss of TANGO2 impairs intermediate ST:STUDY_SUMMARY filament structure, resulting in fragmented mitochondrial networks and formation ST:STUDY_SUMMARY of cup-like mitochondria. In mice loss of TANGO2 caused heart defects, reduced ST:STUDY_SUMMARY muscle function and hypoglycemia that were caused by remodelling of intermediate ST:STUDY_SUMMARY filaments, resulting in changes in the mitochondrial and cytoplasmic proteomes ST:STUDY_SUMMARY and glycosylation. We identify that TANGO2 binds the small heat shock protein ST:STUDY_SUMMARY crystallin alpha B (CRYAB) to prevent the aggregation of the intermediate ST:STUDY_SUMMARY filament desmin and in the absence of TANGO2, mice develop desminopathy, which ST:STUDY_SUMMARY is consistent with features found in patients carrying mutations either in ST:STUDY_SUMMARY desmin or CRYAB. We analysed intra-cellular concentrations of central carbon ST:STUDY_SUMMARY metabolites to understand metabolic changes underpinning Cal51 human cell lines ST:STUDY_SUMMARY with TANGO2 present or knocked-out, finding in particular that knock-out cell ST:STUDY_SUMMARY lines had increased concentrations of glucose 6-phosphate and fructose ST:STUDY_SUMMARY 6-phosphate, that suggests glycolysis is required at the expense of reduced ST:STUDY_SUMMARY oxidative phosphorylation. ST:INSTITUTE The Kids Research Institute Australia ST:LAST_NAME Filipovska ST:FIRST_NAME Aleksandra ST:ADDRESS The Kids Research Institute Australia, Northern Entrance, Perth Children′s ST:ADDRESS Hospital, 15 Hospital Avenue, Nedlands, Western Australia, 6009, Australia ST:EMAIL aleksandra.filipovska@uwa.edu.au ST:PHONE +61 8 6319 1000 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 10 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS CAL51 cell line #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 - 1_TA Sample source:CAL51 cells | Genotype:CAL51-WT RAW_FILE_NAME(Raw file name)=1_TA_CAL51_WT_1_103.lcd SUBJECT_SAMPLE_FACTORS - 2_TA Sample source:CAL51 cells | Genotype:CAL51-WT RAW_FILE_NAME(Raw file name)=2_TA_CAL51_WT_2_109.lcd SUBJECT_SAMPLE_FACTORS - 3_TA Sample source:CAL51 cells | Genotype:CAL51-WT RAW_FILE_NAME(Raw file name)=3_TA_CAL51_WT_3_114.lcd SUBJECT_SAMPLE_FACTORS - 4_TA Sample source:CAL51 cells | Genotype:CAL51-WT RAW_FILE_NAME(Raw file name)=4_TA_CAL51_WT_4_122.lcd SUBJECT_SAMPLE_FACTORS - 5_TA Sample source:CAL51 cells | Genotype:CAL51-WT RAW_FILE_NAME(Raw file name)=5_TA_CAL51_WT_5_133.lcd SUBJECT_SAMPLE_FACTORS - 26_TA Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO RAW_FILE_NAME(Raw file name)=26_TA_TANGO2 KO_1_100.lcd SUBJECT_SAMPLE_FACTORS - 27_TA Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO RAW_FILE_NAME(Raw file name)=27_TA_TANGO2 KO_2_112.lcd SUBJECT_SAMPLE_FACTORS - 28_TA Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO RAW_FILE_NAME(Raw file name)=28_TA_TANGO2 KO_3_115.lcd SUBJECT_SAMPLE_FACTORS - 29_TA Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO RAW_FILE_NAME(Raw file name)=29_TA_TANGO2 KO_4_123_100.lcd SUBJECT_SAMPLE_FACTORS - 30_TA Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO RAW_FILE_NAME(Raw file name)=30_TA_TANGO2 KO_5_130.lcd #COLLECTION CO:COLLECTION_SUMMARY CAL51 cells (breast carcinoma cell line, DMZ no. ACC 302) were cultured and CO:COLLECTION_SUMMARY maintained at 37˚C under humidified 95% air and 5% CO2 in Dulbecco’s modified CO:COLLECTION_SUMMARY Eagle’s medium containing 4.5 g/l glucose, 1 mM pyruvate, 2 mM glutamine, 10% CO:COLLECTION_SUMMARY fetal bovine serum (FBS) and 50 µg/ml uridine referred as high glucose media. CO:COLLECTION_SUMMARY Galactose media referred to Dulbecco’s modified Eagle’s medium deprived of CO:COLLECTION_SUMMARY glucose (DMEM, Gibco, Life Technologies) containing 10 mM galactose, 1 mM CO:COLLECTION_SUMMARY pyruvate and 10% FBS. For metabolomics samples, 1 million cells were pelleted CO:COLLECTION_SUMMARY and snap-frozen. CO:SAMPLE_TYPE CAL51 cell line #TREATMENT TR:TREATMENT_SUMMARY CAL51 cell line with (control/wild-type) or without TANGO2 knocked-out via TR:TREATMENT_SUMMARY CRISPR/Cas9 genome editing. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 500 µL ice cold 50% methanol was added to pellets of 1 million snap-frozen SP:SAMPLEPREP_SUMMARY cells and the mixture was vortex, thawed on ice and sonicated for 5 min in an SP:SAMPLEPREP_SUMMARY ice bath. 200 µL of 0.5 mm silica glass beads were added to samples which were SP:SAMPLEPREP_SUMMARY loaded onto an OMNI Bead Ruptor Elite that was pre-chilled to -1 °C. SP:SAMPLEPREP_SUMMARY Metabolites were extracted using 5 rounds of 45 s pulses at 4 m/s with a 30 s SP:SAMPLEPREP_SUMMARY dwell time. Metabolites were purified by adding 250 µL chloroform, which was SP:SAMPLEPREP_SUMMARY then vortexed and centrifuged at 16,000 RCF for 5 min at 4 °C. The supernatant SP:SAMPLEPREP_SUMMARY was recovered and the organic phase reduced using a vacuum concentrator until SP:SAMPLEPREP_SUMMARY approximately 200 µL remained. Samples were further diluted with 500 µL milliQ SP:SAMPLEPREP_SUMMARY water, freeze-dried and reconstituted in 100 µL of 2% acetonitrile where 60 µL SP:SAMPLEPREP_SUMMARY was taken and spiked with 2.5 µM azidothymidine as an internal standard for SP:SAMPLEPREP_SUMMARY central carbon metabolite quantification. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Solvent A: 7.5 mM tributylamine aqueous solution (pH to 4.95 with acetic CH:CHROMATOGRAPHY_SUMMARY acid); Solvent B: acetonitrile CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Phenomenex Gemini NX-C18 (50 x 2mm,3um) CH:SOLVENT_A 100% water; 7.5 mM tributylamine; 0.0675% acetic acid CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0-8 min (2% B), 8-22 min (20% B), 22-32 min (27% B), 32-47 min (50% B), 47-48 CH:FLOW_GRADIENT min (98% B), 48-54 min (98% B), 54-60 min (2% B). CH:FLOW_RATE 300 uL/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Shimadzu LCMS-8060 MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Retention times for method can be found in MS:MS_COMMENTS https://doi.org/10.1038/s41467-020-19390-9. Concentrations of each metabolite MS:MS_COMMENTS were calculated based on standard curves from serial dilutions of authentic MS:MS_COMMENTS chemical standards (Sigma). Collected data were processed using LabSolutions MS:MS_COMMENTS InsightTM (Shimadzu). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nanomoles/million cells MS_METABOLITE_DATA_START Samples 1_TA 2_TA 3_TA 4_TA 5_TA 26_TA 27_TA 28_TA 29_TA 30_TA Factors Sample source:CAL51 cells | Genotype:CAL51-WT Sample source:CAL51 cells | Genotype:CAL51-WT Sample source:CAL51 cells | Genotype:CAL51-WT Sample source:CAL51 cells | Genotype:CAL51-WT Sample source:CAL51 cells | Genotype:CAL51-WT Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO Sample source:CAL51 cells | Genotype:CAL51-TANGO2KO lactate 305195.6402 292472.1096 270799.4004 147483.125 246796.3056 116415.9704 73401.83 113937.3448 63832.9456 139950.4044 glyceraldehyde 3-phosphate 252.6752 148.5516 83.8452 414.0904 161.8222 104.1312 204.926 523.0368 glucose 6-phosphate 1682.3598 901.8254 859.2818 1489.4546 1177.6474 2078.7016 3871.71 962.2804 4027.2492 136.096 glycerol 3-phosphate 4323.2512 3533.936 3657.4776 6712.227 4265.5312 13190.0524 20046.7996 12278.792 25469.34 4963.4352 D-ribose 5-phosphate 215.9442 119.1266 83.015 279.9128 130.66 276.7268 329.9448 139.2792 527.0652 40.278 sedoheptulose-7-phosphate 2375.4484 788.5236 448.1234 2668.8708 1020.691 1759.8976 2050.6148 392.5772 3251.8436 19.5252 fructose 6-phosphate 1004.8264 746.1158 481.838 987.635 625.7858 7736.2864 5451.8884 3949.602 3764.352 282.1456 glucose 1-phosphate 895.2786 524.2538 634.4454 1067.2492 942.7214 411.17 638.4292 57.9604 1536.7588 1.5092 D-ribulose 5-phosphate 84.0174 46.0494 23.6096 114.0894 56.8104 81.0328 108.9112 20.7244 206.6068 3.722 D-xylulose 5-phosphate 250.1346 110.3448 97.1396 399.0644 71.8652 365.3008 396.6868 94.9596 643.3528 5.6932 cytidine monophosphate 880.56 1276.2064 1274.907 507.0278 1367.7744 611.8076 745.3796 115.2 1135.7364 308.4064 nicotinamide adenine dinucleotide (oxidised) 2035.6044 1679.2238 1533.5746 1262.7314 1976.8228 989.2884 964.6672 1206.4144 733.2796 1193.1348 dihydroxyacetone phosphate 15823.8432 8523.634 5116.562 23162.8638 9805.7814 5988.0172 10250.942 457.3364 29606.9848 152.8068 uridine monophosphate 3566.6838 5177.3082 6363.3046 1912.2422 4969.1652 3792.074 3563.0704 1356.148 5219.7288 2132.0028 guanosine monophosphate 1340.1642 1978.8232 1747.7552 612.5908 1417.2054 613.9912 513.5744 237.5564 1121.1576 435.1992 adenosine monophosphate 3442.2526 4099.4972 4908.9422 2865.8712 5701.3618 1483.7424 1227.8764 77.0248 3049.6632 391.1052 succinate 1308.7146 2124.66 1907.4668 1120.0106 1608.6844 1869.652 881.9184 1847.8164 1606.6088 2006.5076 malate 13564.9304 13337.9772 12966.3004 10784.7302 14020.5962 9883.8224 9265.6428 10079.0636 8660.0576 11533.546 adipic acid 153.981 152.0902 156.963 182.7152 147.3554 370.0224 328.7944 375.1084 344.466 339.896 α-ketoglutarate 81.8178 29.1142 13.564 27.921 55.6414 98.1544 64.6096 11.96 57.3384 117.0728 6-phosphogluconic acid 154.8014 59.175 53.6794 316.5414 181.0786 7.2488 11.4924 63.4232 fumarate 7303.7382 6994.0638 7168.0622 4884.4538 7217.3282 3376.2996 2708.0472 3100.642 2780.2928 3448.7532 3- phospho-D-glycerate 7826.016 5021.2984 3778.2606 12921.3424 7459.7216 19816.6016 17566.0276 15002.2784 21048.3872 5758.2336 uridine diphosphate N-acetylglucosamine 27843.1752 30520.8846 31804.8604 21431.9234 28705.8868 495.72 5333.7432 5154.014 5313.0968 358.9124 nicotinamide adenine dinucleotide phosphate (oxidised) 127.8106 43.847 30.8962 108.7232 122.7806 fructose 1,6-bisphosphate 401.1136 145.7012 89.591 405.9454 215.5356 225.8036 271.5932 35.3864 462.4488 31.9888 citrate 7225.8244 6523.5596 4939.449 5117.9444 8463.3782 7856.558 9062.122 7126.3172 8127.0168 9724.978 isocitrate 2492.9102 1893.8264 1403.7598 1453.5824 2610.435 2748.094 3176.9248 2531.7564 2538.9808 3714.2368 guanosine diphosphate 597.5616 480.665 574.522 411.7044 796.1148 228.5256 131.3448 61.5548 391.5948 123.0636 nicotinamide adenine dinucleotide (reduced) 1801.9576 5025.5156 5548.8522 4543.9434 5283.5678 9338.1464 9440.9368 7747.6124 10178.7604 8486.8568 phosphoenolpyruvate 3352.6418 2181.8272 1574.0514 4184.8002 2884.085 1165.816 1525.626 166.3576 4303.8632 21.0108 uridine diphosphate 3264.7636 2503.8172 2875.443 2279.7372 4505.3392 455.0128 394.0348 136.7016 1065.2152 233.996 aconitate 44.5904 25.912 3.5034 65.4272 31.2864 adenosine diphosphate 7755.1456 6171.4964 6792.0606 5942.1484 9112.6372 2111.6548 1037.5496 453.3756 3156.0992 971.1024 adenosine triphosphate 25271.4698 17879.0294 16094.785 17412.1738 18462.4248 2077.4796 809.5216 326.548 1250.4676 1223.7 acetyl-CoA 17.7704 12.1904 13.971 13.6048 15.7154 cytidine triphosphate 1688.4522 1513.3148 1387.1146 1296.3002 1562.4688 823.0192 464.8524 605.9808 493.1348 466.9868 uridine diphosphate glucuronic acid 2719.7474 2532.9622 3019.06 2364.0186 2995.4752 1257.4776 668.562 863.1912 828.2032 757.078 guanosine triphosphate 2637.6158 2210.3192 2114.4576 1821.6172 2353.944 714.7968 78.0592 543.4708 235.1944 280.992 uridine triphosphate 6307.0454 4422.4464 4427.8324 4610.3468 5254.915 1936.732 383.2804 1329.5216 578.9792 331.2708 Coenzyme A 2853.5844 1111.5204 2016.684 2574.0296 2927.2754 491.258 693.7788 472.842 1095.922 629.7128 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID lactate 91435 glyceraldehyde 3-phosphate 729 glucose 6-phosphate 5958 glycerol 3-phosphate 754 D-ribose 5-phosphate 77982 sedoheptulose-7-phosphate 165007 fructose 6-phosphate 69507 glucose 1-phosphate 439165 D-ribulose 5-phosphate 439184 D-xylulose 5-phosphate 439190 cytidine monophosphate 6131 nicotinamide adenine dinucleotide (oxidised) 5892 dihydroxyacetone phosphate 668 uridine monophosphate 6030 guanosine monophosphate 135398631 adenosine monophosphate 6083 succinate 1110 malate 525 adipic acid 196 α-ketoglutarate 1957 6-phosphogluconic acid 91493 fumarate 444972 3- phospho-D-glycerate 439183 uridine diphosphate N-acetylglucosamine 10705 nicotinamide adenine dinucleotide phosphate (oxidised) 5885 fructose 1,6-bisphosphate 42609786 citrate 311 isocitrate 1198 guanosine diphosphate 135398619 nicotinamide adenine dinucleotide (reduced) 439153 phosphoenolpyruvate 1005 uridine diphosphate 6031 aconitate 309 adenosine diphosphate 6022 adenosine triphosphate 5957 acetyl-CoA 444493 cytidine triphosphate 6176 uridine diphosphate glucuronic acid 17473 guanosine triphosphate 135398633 uridine triphosphate 6133 Coenzyme A 87642 METABOLITES_END #END