#METABOLOMICS WORKBENCH amroilaiwy_20160325_083511 DATATRACK_ID:670 VERSION 1 CREATED_ON September 20, 2017, 1:19 pm #PROJECT PR:PROJECT_TITLE The ubiquitin ligase MuRF1 regulates PPARa activity in the heart by enhancing PR:PROJECT_TITLE nuclear export via monoubiquitination PR:PROJECT_TYPE Targeted Metabolomics PR:PROJECT_SUMMARY The transcriptional regulation of peroxisome proliferator-activated receptor PR:PROJECT_SUMMARY (PPAR) a by post-translational modification, such as ubiquitin, has not been PR:PROJECT_SUMMARY described. We report here for the first time an ubiquitin ligase (muscle ring PR:PROJECT_SUMMARY finger-1/MuRF1) that inhibits fatty acid oxidation by inhibiting PPARa, but not PR:PROJECT_SUMMARY PPARß/d or PPAR? in cardiomyocytes in vitro. Similarly, MuRF1 Tg+ hearts PR:PROJECT_SUMMARY showed significant decreases in nuclear PPARa activity and acyl-carnitine PR:PROJECT_SUMMARY intermediates, while MuRF1-/- hearts exhibited increased PPARa activity and PR:PROJECT_SUMMARY acyl-carnitine intermediates. MuRF1 directly interacts with PPARa, PR:PROJECT_SUMMARY mono-ubiquitinates it, and targets it for nuclear export to inhibit fatty acid PR:PROJECT_SUMMARY oxidation in a proteasome independent manner. We then identified a previously PR:PROJECT_SUMMARY undescribed nuclear export sequence in PPARa, along with three specific lysines PR:PROJECT_SUMMARY (292, 310, 388) required for MuRF1's targeting of nuclear export. These studies PR:PROJECT_SUMMARY identify the role of ubiquitination in regulating cardiac PPARa, including the PR:PROJECT_SUMMARY ubiquitin ligase that may be responsible for this critical regulation of cardiac PR:PROJECT_SUMMARY metabolism in heart failure. PR:INSTITUTE University of North Carolina PR:DEPARTMENT McAllister Heart Institute, Department of Internal Medicine PR:LABORATORY Multiple Centers PR:LAST_NAME Willis PR:FIRST_NAME Monte PR:ADDRESS 111 Mason Farm road, Chapel Hill, North Carolina, 27599-7126, USA PR:EMAIL monte_willis@med.unc.edu PR:PHONE 919-360-7599 PR:FUNDING_SOURCE NIH, Fondation Leducq, AHA mid-Atlantic affiliate, AHA scientist development PR:FUNDING_SOURCE grant, Jefferson-Pilot Corporation Fellowship in Academic Medicine #STUDY ST:STUDY_TITLE Targeted metabolomics of MuRF1 overexpressing cardiomyocytes compared to their ST:STUDY_TITLE wildtype controls (part I) ST:STUDY_TYPE Targeted metabolomic analysis ST:STUDY_SUMMARY The transcriptional regulation of peroxisome proliferator-activated receptor ST:STUDY_SUMMARY (PPAR) a by post-translational modification, such as ubiquitin, has not been ST:STUDY_SUMMARY described. We report here for the first time an ubiquitin ligase (muscle ring ST:STUDY_SUMMARY finger-1/MuRF1) that inhibits fatty acid oxidation by inhibiting PPARa, but not ST:STUDY_SUMMARY PPARß/d or PPAR? in cardiomyocytes in vitro. Similarly, MuRF1 Tg+ hearts ST:STUDY_SUMMARY showed significant decreases in nuclear PPARa activity and acyl-carnitine ST:STUDY_SUMMARY intermediates, while MuRF1-/- hearts exhibited increased PPARa activity and ST:STUDY_SUMMARY acyl-carnitine intermediates. MuRF1 directly interacts with PPARa, ST:STUDY_SUMMARY mono-ubiquitinates it, and targets it for nuclear export to inhibit fatty acid ST:STUDY_SUMMARY oxidation in a proteasome independent manner. We then identified a previously ST:STUDY_SUMMARY undescribed nuclear export sequence in PPARa, along with three specific lysines ST:STUDY_SUMMARY (292, 310, 388) required for MuRF1's targeting of nuclear export. These studies ST:STUDY_SUMMARY identify the role of ubiquitination in regulating cardiac PPARa, including the ST:STUDY_SUMMARY ubiquitin ligase that may be responsible for this critical regulation of cardiac ST:STUDY_SUMMARY metabolism in heart failure. ST:INSTITUTE University of North Carolina ST:DEPARTMENT McAllister Heart Institute, Department of Internal Medicine ST:LABORATORY Multiple Centers ST:LAST_NAME Willis ST:FIRST_NAME Monte ST:ADDRESS 111 Mason Farm road, Chapel Hill, North Carolina, 27599-7126, USA ST:EMAIL monte_willis@med.unc.edu ST:PHONE 919-360-7599 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Heart 1 Genotype:WT-MuRF1 tg+ SUBJECT_SAMPLE_FACTORS - Heart 2 Genotype:WT-MuRF1 tg+ SUBJECT_SAMPLE_FACTORS - Heart 3 Genotype:WT-MuRF1 tg+ SUBJECT_SAMPLE_FACTORS - Heart 4 Genotype:MuRF1 tg+ SUBJECT_SAMPLE_FACTORS - Heart 5 Genotype:MuRF1 tg+ SUBJECT_SAMPLE_FACTORS - Heart 6 Genotype:MuRF1 tg+ #COLLECTION CO:COLLECTION_SUMMARY COS7 and H9C2 cells were transfected with PPRE-luc, pcDNA 3.1, ß-galactosidase, CO:COLLECTION_SUMMARY and the corresponding PPAR isoform (PPARa, PPARß/d or PPAR?) as indicated. CO:COLLECTION_SUMMARY 24 hours followings transfection cells were transduced with Ad.GFP-Myc-MuRF1. CO:COLLECTION_SUMMARY Cells were harvested 24hours later following observation of GFP by light CO:COLLECTION_SUMMARY microscopy #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Acyl-carnitines were analyzed using stable isotope dilution techniques. Amino SP:SAMPLEPREP_SUMMARY acids and acyl-carnitine measurements were made by flow injection tandem mass SP:SAMPLEPREP_SUMMARY spectrometry #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME ACQUITY UPLC 1.7 µm column #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 5975 MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS uM MS_METABOLITE_DATA_START Samples Heart 1 Heart 2 Heart 3 Heart 4 Heart 5 Heart 6 Factors Genotype:WT-MuRF1 tg+ Genotype:WT-MuRF1 tg+ Genotype:WT-MuRF1 tg+ Genotype:MuRF1 tg+ Genotype:MuRF1 tg+ Genotype:MuRF1 tg+ C2 23.6000 22.5000 20.8000 18.5000 18.8000 26.9000 C3 0.3530 0.9490 0.6430 0.8450 0.6460 0.9690 C4/Ci4 0.0973 0.0727 0.0661 0.0837 0.0352 0.0502 C5:1 0.0137 0.0412 0.0505 0.0367 0.0443 0.0436 C5's 0.0294 0.0257 0.0447 0.0365 0.0279 0.0248 C4-OH 0.6950 0.5340 0.5530 0.4500 0.6190 0.5900 C6 0.1640 0.0910 0.0364 0.0438 0.0366 0.0117 C5-OH/C3-DC 0.5270 0.6480 0.9940 0.8870 0.5750 0.7230 Ci4-DC/C4-DC 0.3300 0.3780 0.3780 0.4070 0.2970 0.3600 C8:1 0.0041 0.0170 0.0105 0.0105 0.0111 0.0079 C8 0.0089 0.0073 0.0034 0.0039 0.0076 0.0068 C5-DC 0.0037 0.0054 0.0012 0.0076 0.0018 0.0095 C6:1-DC/C8:1-OH 0.0147 0.0065 0.0097 0.0030 0.0106 0.0084 C6-DC 0.0276 0.0434 0.0190 0.0873 0.0670 0.0354 C10:3 0.0012 0.0005 0.0013 0.0033 0.0016 C10:2 0.0033 0.0068 0.0013 0.0040 0.0054 0.0030 C10:1 0.0117 0.0115 0.0059 0.0070 0.0059 0.0069 C10 0.0081 0.0074 0.0028 0.0036 0.0014 C7-DC 0.0040 0.0048 0.0083 0.0057 0.0001 0.0040 C8:1-DC 0.0042 0.0025 0.0055 0.0067 0.0039 C10-OH/C8-DC 0.0765 0.1070 0.0580 0.0821 0.1100 0.0352 C12:1 0.0063 0.0068 0.0025 0.0035 0.0010 C12 0.0081 0.0007 0.0006 0.0026 0.0011 C12-OH/C10-DC 0.0759 0.1140 0.0584 0.0765 0.1070 0.0331 C14:2 0.0058 0.0094 0.0065 0.0065 0.0027 0.0034 C14:1 0.0117 0.0098 0.0056 0.0098 0.0051 0.0033 C14 0.0250 0.0100 0.0064 0.0108 0.0089 0.0067 C14:1-OH/C12:1-DC 0.1070 0.1010 0.0514 0.0624 0.0669 0.0195 C14-OH/C12-DC 0.0963 0.0826 0.0515 0.0729 0.1000 0.0312 C16:2 0.0145 0.0058 0.0036 0.0052 0.0042 0.0029 C16:1 0.0188 0.0396 0.0175 0.0282 0.0224 0.0099 C16 0.0711 0.0295 0.0215 0.0189 0.0173 0.0125 C16:1-OH/C14:1-DC 0.0748 0.0839 0.0424 0.0351 0.0482 0.0109 C16-OH/C14-DC 0.1230 0.1020 0.0375 0.0457 0.0652 0.0198 C18:2 0.0357 0.0555 0.0391 0.0301 0.0403 0.0119 C18:1 0.0403 0.0456 0.0191 0.0330 0.0357 0.0224 C18 0.0431 0.0192 0.0154 0.0130 0.0071 0.0080 C18:2-OH 0.1010 0.0536 0.0235 0.0190 0.0318 0.0044 C18:1-OH/C16:1-DC 0.1340 0.0987 0.0484 0.0434 0.0625 0.0183 C18-OH/C16-DC 0.0606 0.0466 0.0249 0.0295 0.0386 0.0131 C20:4 0.0048 0.0104 0.0064 0.0154 0.0067 0.0047 C20 0.0138 0.0054 0.0025 0.0025 0.0042 0.0018 C20:1-OH/C18:1-DC 0.0169 0.0189 0.0080 0.0094 0.0170 0.0140 C20-OH/C18-DC 0.0067 0.0069 0.0033 0.0037 0.0048 0.0037 C22 0.0046 0.0027 0.0030 0.0011 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name C2 C3 C4/Ci4 C5:1 C5's C4-OH C6 C5-OH/C3-DC Ci4-DC/C4-DC C8:1 C8 C5-DC C6:1-DC/C8:1-OH C6-DC C10:3 C10:2 C10:1 C10 C7-DC C8:1-DC C10-OH/C8-DC C12:1 C12 C12-OH/C10-DC C14:2 C14:1 C14 C14:1-OH/C12:1-DC C14-OH/C12-DC C16:2 C16:1 C16 C16:1-OH/C14:1-DC C16-OH/C14-DC C18:2 C18:1 C18 C18:2-OH C18:1-OH/C16:1-DC C18-OH/C16-DC C20:4 C20 C20:1-OH/C18:1-DC C20-OH/C18-DC C22 METABOLITES_END #END