#METABOLOMICS WORKBENCH mbcolonna_20200508_141452 DATATRACK_ID:2000 STUDY_ID:ST001375 ANALYSIS_ID:AN002295 PROJECT_ID:PR000941 VERSION 1 CREATED_ON May 12, 2020, 2:06 pm #PROJECT PR:PROJECT_TITLE A redox-active switch in Fructosamine-3-kinases expands the regulatory PR:PROJECT_TITLE repertoire of the protein kinase super-family PR:PROJECT_TYPE Structural Biology, enzyme characterization PR:PROJECT_SUMMARY Aberrant regulation of metabolic kinases by altered redox homeostasis is a major PR:PROJECT_SUMMARY contributing factor in aging and disease such as diabetes. However, the PR:PROJECT_SUMMARY biochemical mechanisms by which metabolic kinases are regulated under oxidative PR:PROJECT_SUMMARY stress is poorly understood. In this study, we demonstrate that the catalytic PR:PROJECT_SUMMARY activity of a conserved family of Fructosamine-3-kinases (FN3Ks), which are PR:PROJECT_SUMMARY evolutionarily related to eukaryotic protein kinases (ePKs), are regulated by PR:PROJECT_SUMMARY redox-active cysteines in the kinase domain. By solving the crystal structure of PR:PROJECT_SUMMARY FN3K homolog from Arabidopsis thaliana (AtFN3K), we demonstrate that it forms an PR:PROJECT_SUMMARY unexpected strand-exchange dimer in which the ATP binding P-loop and adjoining PR:PROJECT_SUMMARY beta strands are swapped between two chains in the dimer. This dimeric PR:PROJECT_SUMMARY configuration is characterized by strained inter-chain disulfide bonds that PR:PROJECT_SUMMARY stabilize the P-loop in an extended conformation. Mutational analysis and PR:PROJECT_SUMMARY solution studies confirm that the strained disulfides function as redox PR:PROJECT_SUMMARY “switches” to reversibly regulate FN3K activity and dimerization. PR:PROJECT_SUMMARY Consistently, we find that human FN3K, which contains an equivalent P-loop Cys, PR:PROJECT_SUMMARY is also redox-sensitive, whereas ancestral bacterial FN3K homologs, which lack a PR:PROJECT_SUMMARY P-loop Cys, are not. Furthermore, CRISPR knockout of FN3K in human HepG2 cells PR:PROJECT_SUMMARY results in significant upregulation of redox metabolites including glutathione. PR:PROJECT_SUMMARY We propose that redox regulation evolved progressively in FN3Ks in response to PR:PROJECT_SUMMARY changing cellular redox conditions. Our studies provide important new insights PR:PROJECT_SUMMARY into the origin and evolution of redox regulation in the protein kinase PR:PROJECT_SUMMARY superfamily and open new avenues for targeting human FN3K in diabetic PR:PROJECT_SUMMARY complications. PR:INSTITUTE University of Georgia PR:DEPARTMENT Biochemistry and Molecular Biology PR:LABORATORY Kannan PR:LAST_NAME Kannan PR:FIRST_NAME Natarajan PR:ADDRESS B122 Life Sciences Bldg. University of Georgia Athens, GA 30602 PR:EMAIL nkannan@uga.edu PR:PHONE 706-542-1334 PR:FUNDING_SOURCE MCB-1149106; R01GM114409 PR:PUBLICATIONS A redox-active switch in Fructosamine-3-kinases expands the regulatory PR:PUBLICATIONS repertoire of the protein kinase super-family PR:CONTRIBUTORS Safal Shrestha, Samiksha Katiyar, Carlos E. Sanz-Rodriquez, Nolan R. Kemppinen, PR:CONTRIBUTORS Hyun W. Kim, Renuka Kadirvelraj, Charalampos Panagos, Neda Keyhaninejad, Maxwell PR:CONTRIBUTORS Colonna, Pradeep Chopra, Dominic P. Byrne, Geert J. Boons, Esther V. Knaap, PR:CONTRIBUTORS Patrick A. Eyers, Arthur S. Edison, Zachary A. Wood #STUDY ST:STUDY_TITLE Fructosamine-3-kinase (FN3K) KO in HepG2 liver cancer cells ST:STUDY_SUMMARY Fructosamine-3-kinases (FN3Ks) are a family of metabolic kinases which are ST:STUDY_SUMMARY evolutionarily related to eukaryotic protein kinases. Aberrant regulation of ST:STUDY_SUMMARY these kinases by altered redox homeostasis is a major contributing factor in ST:STUDY_SUMMARY aging and disease. However, the mechanisms of regulation and cellular functions ST:STUDY_SUMMARY of these kinases are not known. Bioinformatic analyses of cancer cell lines ST:STUDY_SUMMARY identified significant overexpression of FN3K in liver and eye cancer cells. To ST:STUDY_SUMMARY assess the functional significance of this increased expression, a CRISPR ST:STUDY_SUMMARY knockout of FN3K (FN3K-KO) was generated in the HepG2 liver cancer cell line. ST:STUDY_SUMMARY The metabolome was compared between FN3K-KO and WT HepG2 cells using untargeted ST:STUDY_SUMMARY 1H NMR metabolomics. This revealed significant differences in several ST:STUDY_SUMMARY metabolites that suggest a role for FN3K in regulating redox and energy balance ST:STUDY_SUMMARY in HepG2 cells. ST:INSTITUTE University of Georgia ST:DEPARTMENT Complex Carbohydrate Research Center ST:LABORATORY Edison ST:LAST_NAME Colonna ST:FIRST_NAME Maxwell ST:ADDRESS 315 Riverbend Rd, Athens, GA 30602 ST:EMAIL maxwellbaca@uga.edu ST:PHONE 7065420257 ST:NUM_GROUPS 2 ST:PUBLICATIONS A redox-active switch in Fructosamine-3-kinases expands the regulatory ST:PUBLICATIONS repertoire of the protein kinase super-family #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN HepG2 #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 - FN3K_1 Genotype:FN3K KO RAW_FILE_NAME=15 SUBJECT_SAMPLE_FACTORS - FN3K_2 Genotype:FN3K KO RAW_FILE_NAME=10 SUBJECT_SAMPLE_FACTORS - FN3K_3 Genotype:FN3K KO RAW_FILE_NAME=18 SUBJECT_SAMPLE_FACTORS - FN3K_4 Genotype:FN3K KO RAW_FILE_NAME=22 SUBJECT_SAMPLE_FACTORS - FN3K_5 Genotype:FN3K KO RAW_FILE_NAME=13 SUBJECT_SAMPLE_FACTORS - FN3K_6 Genotype:FN3K KO RAW_FILE_NAME=8 SUBJECT_SAMPLE_FACTORS - FN3K_7 Genotype:FN3K KO RAW_FILE_NAME=7 SUBJECT_SAMPLE_FACTORS - FN3K_8 Genotype:FN3K KO RAW_FILE_NAME=9 SUBJECT_SAMPLE_FACTORS - FN3K_9 Genotype:FN3K KO RAW_FILE_NAME=20 SUBJECT_SAMPLE_FACTORS - FN3K_10 Genotype:FN3K KO RAW_FILE_NAME=23 SUBJECT_SAMPLE_FACTORS - WT_1 Genotype:WT RAW_FILE_NAME=16 SUBJECT_SAMPLE_FACTORS - WT_2 Genotype:WT RAW_FILE_NAME=21 SUBJECT_SAMPLE_FACTORS - WT_3 Genotype:WT RAW_FILE_NAME=3 SUBJECT_SAMPLE_FACTORS - WT_4 Genotype:WT RAW_FILE_NAME=5 SUBJECT_SAMPLE_FACTORS - WT_5 Genotype:WT RAW_FILE_NAME=11 SUBJECT_SAMPLE_FACTORS - WT_6 Genotype:WT RAW_FILE_NAME=4 SUBJECT_SAMPLE_FACTORS - WT_7 Genotype:WT RAW_FILE_NAME=6 SUBJECT_SAMPLE_FACTORS - WT_8 Genotype:WT RAW_FILE_NAME=2 SUBJECT_SAMPLE_FACTORS - WT_9 Genotype:WT RAW_FILE_NAME=19 SUBJECT_SAMPLE_FACTORS - WT_10 Genotype:WT RAW_FILE_NAME=17 #COLLECTION CO:COLLECTION_SUMMARY Cell cultures for both cell lines were grown simultaneously with identical media CO:COLLECTION_SUMMARY components. Upon achieving ~80% confluence in 10cm culture dish, culture media CO:COLLECTION_SUMMARY was removed and cell monolayer was washed with cold phosphate buffered saline. CO:COLLECTION_SUMMARY Cells were then quenched by addition ice-cold 80% methanol extraction solvent, CO:COLLECTION_SUMMARY scraped from the culture dish, and flash-frozen in liquid nitrogen. CO:SAMPLE_TYPE HepG2 cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY None #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Aqueous metabolites were extracted by vortexing cell pellets in the extraction SP:SAMPLEPREP_SUMMARY solvent, pelleting cell debris and collecting the supernatant. Approximately 10% SP:SAMPLEPREP_SUMMARY of the supernatant volume was taken from each sample to form an internal pooled SP:SAMPLEPREP_SUMMARY sample. The solvent was then evaporated to produce dried extracts. SP:SAMPLEPREP_PROTOCOL_FILENAME cell_protocol.pdf SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Vortex SP:EXTRACT_ENRICHMENT Polar SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Deuterium oxide phosphate buffer #ANALYSIS AN:DATA_FORMAT Bruker #NMR NM:INSTRUMENT_NAME Bruker Avance NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK Deuterium NM:SPECTROMETER_FREQUENCY 800 MHz NM:NMR_PROBE 3mm Cryoprobe NM:NMR_SOLVENT D2O Phosphate buffer NM:NMR_TUBE_SIZE 3mm SampleJet NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE noesypr1d NM:WATER_SUPPRESSION Presaturation NM:PULSE_WIDTH 8.22 us NM:POWER_LEVEL 11.117 W NM:OFFSET_FREQUENCY 3762.64 NM:PRESATURATION_POWER_LEVEL 3.00E-05 NM:CHEMICAL_SHIFT_REF_CPD DSS NM:TEMPERATURE 27 NM:NUMBER_OF_SCANS 128 NM:DUMMY_SCANS 4 NM:ACQUISITION_TIME 1.24 s NM:RELAXATION_DELAY 2 s NM:SPECTRAL_WIDTH 16.4533 ppm NM:NUM_DATA_POINTS_ACQUIRED 32768 NM:LINE_BROADENING 0.3 Hz NM:BASELINE_CORRECTION_METHOD Polynomial order 5 #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS AU NMR_METABOLITE_DATA_START Samples WT_8 WT_3 WT_6 WT_4 WT_7 FN3K_6 FN3K_8 FN3K_2 WT_5 FN3K_5 FN3K_1 WT_1 WT_10 FN3K_3 WT_9 FN3K_9 WT_2 FN3K_4 FN3K_10 Factors Genotype:WT Genotype:WT Genotype:WT Genotype:WT Genotype:WT Genotype:FN3K KO Genotype:FN3K KO Genotype:FN3K KO Genotype:WT Genotype:FN3K KO Genotype:FN3K KO Genotype:WT Genotype:WT Genotype:FN3K KO Genotype:WT Genotype:FN3K KO Genotype:WT Genotype:FN3K KO Genotype:FN3K KO Alanine 2.981692718 2.768456234 2.787890084 2.104360804 2.760569382 3.430702819 2.944202058 2.451538459 2.694996376 2.99539357 2.422030109 2.832610263 3.016495971 2.746218282 3.097994958 2.706942717 2.838631282 2.626320142 2.81141936 Aspartate 0.410103271 0.419811832 0.390989953 0.345777216 0.447692155 0.392297335 0.3296134 0.319563458 0.486959561 0.345030529 0.293318016 0.387924805 0.437946295 0.301772002 0.417404579 0.327331264 0.428020283 0.336385623 0.352964773 Citrate 0.218516589 0.222852825 0.237023037 0.187869171 0.23652832 0.222839469 0.219180411 0.239702845 0.233953437 0.229290629 0.198724986 0.20905269 0.22845993 0.224264692 0.240465576 0.217706484 0.234894309 0.21467564 0.242730767 Creatine 0.633841122 0.634097324 0.605043951 0.473109288 0.660891785 0.862394439 0.748985086 0.653872995 0.621831468 0.696168237 0.619083529 0.600151643 0.640506923 0.653782785 0.607636916 0.71917648 0.602528957 0.784082434 0.785590151 Phosphocreatine 0.701835784 0.667349853 0.68979943 0.512793746 0.70877833 0.72505216 0.606449289 0.509757617 0.69368232 0.600978665 0.490033455 0.634703237 0.708475553 0.515178043 0.691097963 0.572225799 0.692281985 0.613442065 0.611796665 Glutamine 0.249427243 0.263731105 0.25985169 0.261571873 0.27288102 0.332971962 0.296007726 0.278137281 0.280854832 0.317007234 0.259245155 0.243959723 0.267587569 0.261752489 0.271462959 0.276801311 0.27850131 0.262131291 0.290289459 Glutathione 0.381607933 0.481386015 0.403171579 0.317016532 0.519547869 0.68433292 0.550460555 0.569221371 0.494780783 0.497492975 0.494030409 0.385662945 0.417793574 0.539639893 0.41716552 0.552103174 0.40542356 0.535918241 0.665265906 Glycine 1.742839829 1.520637771 1.691530605 1.528318035 1.556575656 1.413275116 1.235137688 1.028618768 1.75158038 1.199425902 1.010392359 1.617028346 1.721441497 1.038183581 1.669551019 1.150143413 1.747919361 1.086614542 1.202101621 Glucose 0.043303334 0.066684205 0.112892954 0.238827233 0.034090993 0.002395077 0.027418423 0.148611841 7.67606E-05 0.191738896 0.103412902 0.095009364 0.062973641 0.047357505 0.060140254 0.055020737 0.093690537 0.044208166 0.045891113 Glutamate 1.12817836 1.080825559 1.12578975 0.942676484 1.152561114 1.317046974 1.15229046 1.003791147 1.210828148 1.070359296 0.94776317 1.079400546 1.164317148 0.999546457 1.137602185 1.1270242 1.13889067 1.070337646 1.177678519 Lactate 2.014032088 1.524100726 1.775239579 2.300661444 1.089702057 2.127918025 2.389833894 2.076188196 1.145279886 2.454374163 2.130961185 1.63219384 1.338346724 2.39645173 1.69790277 1.756506653 1.467111425 1.919993693 1.753430679 Leucine 0.898291863 0.827762928 0.951359532 1.108863296 0.723137946 0.680266836 0.802589599 0.972997835 0.690603384 1.033549455 0.882951523 0.915918703 0.864638415 0.88286812 0.898539717 0.836377953 0.912509897 0.795412231 0.721568476 Lysine 0.140146087 0.122898685 0.144146197 0.156191195 0.114719454 0.147390791 0.150562975 0.154861991 0.108300187 0.163672623 0.152054337 0.146471509 0.13912535 0.152978088 0.141713295 0.154621207 0.143458753 0.144756585 0.146019832 Pantothenate 0.398870578 0.373320159 0.399950986 0.376520535 0.358020181 0.367572605 0.360939252 0.343415633 0.382733725 0.339050933 0.335260961 0.385811948 0.39612007 0.349995365 0.401928244 0.349180534 0.425125342 0.350287822 0.37937222 Putrescine 0.148889244 0.130887978 0.15164139 0.153663943 0.123720713 0.163540569 0.175032575 0.146286962 0.127018894 0.172117135 0.177418984 0.170752921 0.15837373 0.161369809 0.15058227 0.176459739 0.147404645 0.17392252 0.164234681 Proline 0.116174463 0.111191334 0.114898035 0.108373086 0.122700722 0.14684803 0.131162154 0.112825168 0.124410212 0.131052472 0.108096018 0.105211014 0.111572915 0.116446355 0.11410568 0.125062852 0.111759319 0.118523091 0.127372285 Serine 0.982821747 0.984854377 0.925369892 0.815407111 1.01175685 0.911287808 0.767509892 0.695267117 1.065808972 0.762951436 0.670858482 0.938595724 1.018477742 0.70258212 1.014620204 0.734183571 1.005340956 0.733447249 0.810286818 Thioacetamide 0.106238298 0.141525729 0.117098097 0.102200056 0.155293052 0.174867799 0.151890211 0.155217566 0.151421115 0.145263141 0.142999069 0.112646666 0.117655293 0.143579731 0.116077431 0.147843948 0.118108584 0.141888225 0.175943913 Threonine 1.030904538 1.013817893 0.998065179 1.019230459 0.893891641 1.212399732 1.120187845 1.04191453 0.925663877 1.206012051 0.946104903 0.91577537 0.914533662 1.010365024 1.011397794 0.947783832 0.947122042 0.98709332 1.08003211 UDP GlcNAc 0.045871676 0.057238406 0.040964902 0.04706478 0.058775464 0.049685544 0.034007968 0.02719107 0.063103679 0.027285423 0.029051853 0.04252343 0.052938655 0.028571398 0.046170022 0.031228182 0.043499819 0.037663177 0.05416819 Valine 0.541326839 0.508692502 0.557433012 0.636414949 0.470176174 0.484861012 0.509485953 0.558073237 0.457301834 0.622489546 0.520780699 0.543918074 0.538618139 0.536646642 0.552233614 0.497280117 0.562558758 0.471065452 0.458084753 Glycerophosphocholine 0.628261817 0.574657757 0.618999813 0.574007035 0.599897473 0.657523364 0.574596795 0.472066415 0.687751141 0.545834501 0.443930722 0.566088014 0.623288069 0.482758171 0.628226889 0.5319513 0.647571939 0.535340266 0.571656638 Keto-methylvalerate 0.127141849 0.114632093 0.125355326 0.145173765 0.100777374 0.130530876 0.130677771 0.13129391 0.081262155 0.138397834 0.147673117 0.12194558 0.114802915 0.150500092 0.121111169 0.122636028 0.116796573 0.133706926 0.123795741 Formate 0.092440939 0.074102962 0.086742249 0.083943437 0.067955656 0.065851987 0.062009736 0.053435833 0.07743505 0.07160005 0.060380588 0.086044389 0.075678487 0.058525585 0.079190186 0.05173717 0.080462658 0.053242894 0.053795383 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name HMDB PubChem ChEBI KEGG METLIN SMILES Quantified feature ppm Alanine HMDB0000161 5950 16977 C00041 - C[C@H](N)C(O)=O 1.47 Aspartate HMDB0000191 5960 17053 C00049 5206 N[C@@H](CC(O)=O)C(O)=O 2.79 Citrate HMDB0000094 311 30769 C00158 124 OC(=O)CC(O)(CC(O)=O)C(O)=O 2.65 Creatine HMDB0000064 586 16919 C00300 7 CN(CC(O)=O)C(N)=N 3.92 Phosphocreatine HMDB0001511 587 17287 C02305 6288 CN(CC(=O)O)C(=NP(=O)(O)O)N 3.94 Glutamine HMDB0000641 5961 18050 C00064 5614 N[C@@H](CCC(N)=O)C(O)=O 2.45 Glutathione HMDB0000125 3353 16856 C00051 - 4.56 Glycine HMDB0000123 750 15428 C00037 20 NCC(O)=O 3.55 Glucose HMDB0000122 5793 4167 C00031 133 OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O 5.23 Glutamate HMDB0000148 33032 16015 C00025 5174 N[C@@H](CCC(O)=O)C(O)=O 2.05 Lactate HMDB0000190 107689 422 C00256 6150 C[C@@H](C(=O)O)O 1.32 Leucine HMDB0000687 6106 15603 C00123 24 CC(C)C[C@H](N)C(O)=O 0.95 Lysine HMDB0000182 5962 18019 C00047 5200 NCCCC[C@H](N)C(O)=O 1.89 Pantothenate HMDB0000210 6613 46905 C00864 - CC(C)(CO)[C@@H](O)C(=O)NCCC(O)=O 0.89 Putrescine HMDB0001414 3434 17148 C00134 - 1.79 Proline HMDB0000162 145742 17203 C00148 29 OC(=O)[C@@H]1CCCN1 3.33 Serine HMDB0000187 3982 17822 C00716 - 3.97 Thioacetamide - 2723949 32497 C19302 - CC(=S)N 2.52 Threonine HMDB0000167 6288 16857 C00188 32 C[C@@H](O)[C@H](N)C(O)=O 1.32 UDP GlcNAc HMDB0000290 445675 16264 C00043 5281 CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@H]1O[C@H]([C@H](O)[C@@H]1O)N1C=CC(=O)NC1=O 5.51 Valine HMDB0000883 6287 16414 C00183 5842 CC(C)[C@H](N)C(O)=O 0.98 Glycerophosphocholine HMDB0000086 71920 16870 C00670 370 C[N+](C)(C)CCOP([O-])(=O)OC[C@@H](O)CO 3.22 Keto-methylvalerate HMDB0000491 47 35932 C03465 5478 CCC(C)C(=O)C(O)=O 1.09 Formate HMDB0000142 284 30751 C00058 3202 OC=O 8.45 METABOLITES_END #END