#METABOLOMICS WORKBENCH amroilaiwy_20160325_095749 DATATRACK_ID:671 VERSION 1 CREATED_ON November 17, 2016, 3:43 pm #PROJECT PR:PROJECT_TITLE The ubiquitin ligase MuRF1 regulates PPARα 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) α 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 PPARα, but not PR:PROJECT_SUMMARY PPARβ/δ or PPARγ in cardiomyocytes in vitro. Similarly, MuRF1 Tg+ hearts PR:PROJECT_SUMMARY showed significant decreases in nuclear PPARα activity and acyl-carnitine PR:PROJECT_SUMMARY intermediates, while MuRF1−/− hearts exhibited increased PPARα activity and PR:PROJECT_SUMMARY acyl-carnitine intermediates. MuRF1 directly interacts with PPARα, 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 PPARα, along with three specific lysines PR:PROJECT_SUMMARY (292, 310, 388) required for MuRF1s targeting of nuclear export. These studies PR:PROJECT_SUMMARY identify the role of ubiquitination in regulating cardiac PPARα, 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 Knockdown cardiomyocytes compared to their ST:STUDY_TITLE wildtype controls (part 2) ST:STUDY_TYPE Targeted metabolomic analysis ST:STUDY_SUMMARY The transcriptional regulation of peroxisome proliferator-activated receptor ST:STUDY_SUMMARY (PPAR) α 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 PPARα, but not ST:STUDY_SUMMARY PPARβ/δ or PPARγ in cardiomyocytes in vitro. Similarly, MuRF1 Tg+ hearts ST:STUDY_SUMMARY showed significant decreases in nuclear PPARα activity and acyl-carnitine ST:STUDY_SUMMARY intermediates, while MuRF1−/− hearts exhibited increased PPARα activity and ST:STUDY_SUMMARY acyl-carnitine intermediates. MuRF1 directly interacts with PPARα, 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 PPARα, along with three specific lysines ST:STUDY_SUMMARY (292, 310, 388) required for MuRF1s targeting of nuclear export. These studies ST:STUDY_SUMMARY identify the role of ubiquitination in regulating cardiac PPARα, 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 - M1KO H1 (15) Genotype:WT SUBJECT_SAMPLE_FACTORS - M1KO H2 (16) Genotype:WT SUBJECT_SAMPLE_FACTORS - M1KO H3 (17) Genotype:WT SUBJECT_SAMPLE_FACTORS - M1KO H4 (18) Genotype:WT SUBJECT_SAMPLE_FACTORS - M1KO H5 (19) Genotype:KO SUBJECT_SAMPLE_FACTORS - M1KO H6 (20) Genotype:KO SUBJECT_SAMPLE_FACTORS - M1KO H7 (21) Genotype:KO SUBJECT_SAMPLE_FACTORS - M1KO H8 (22) Genotype:KO #COLLECTION CO:COLLECTION_SUMMARY COS7 and H9C2 cells were transfected with PPRE-luc, pcDNA 3.1, β-galactosidase, CO:COLLECTION_SUMMARY and the corresponding PPAR isoform (PPARα, PPARβ/δ or PPARγ) 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 M1KO H1 (15) M1KO H2 (16) M1KO H3 (17) M1KO H4 (18) M1KO H5 (19) M1KO H6 (20) M1KO H7 (21) M1KO H8 (22) Factors Genotype:WT Genotype:WT Genotype:WT Genotype:WT Genotype:KO Genotype:KO Genotype:KO Genotype:KO C2 10.76075 9.77828 8.56051 9.10827 17.39575 19.37191 16.22135 15.53480 C3 0.35464 0.39710 0.36498 0.42303 0.43736 0.80919 0.64304 0.43302 C4/Ci4 0.01252 0.00106 0.02657 0.01752 0.02481 0.02219 0.00895 C5:1 0.05059 0.02991 0.04694 0.05680 0.05773 0.06113 0.06470 0.06490 C5s 0.03877 0.02481 0.02310 0.03958 0.03327 0.08521 0.05993 0.03640 C4-OH 1.15126 0.38289 0.16250 0.50619 0.31189 0.62517 0.78720 0.62714 C6 0.01219 0.00971 0.00874 0.01290 0.01838 0.04325 0.02435 0.01638 C5-OH/C3-DC 1.18662 0.67851 0.71076 0.98795 1.22459 1.40482 1.53077 1.32651 Ci4-DC/C4-DC 0.57590 0.45910 0.59843 0.60695 0.80108 0.78665 0.77398 0.73027 C8:1 0.00719 0.01115 0.00719 0.01536 0.01210 0.00855 0.01537 0.01070 C8 0.00150 0.00389 0.00166 0.00458 0.00435 0.00655 0.00503 0.00782 C5-DC 0.00726 0.00741 0.00452 0.00845 0.00818 0.00625 0.00650 0.00668 C6:1-DC/C8:1-OH 0.01325 0.00930 0.00296 0.01404 0.00861 0.01308 0.01530 0.00704 C6-DC 0.06613 0.03657 0.03135 0.08460 0.04758 0.05372 0.08268 0.06598 C10:3 0.00256 0.00137 0.00378 0.00264 0.00201 0.00156 0.00109 C10:2 0.00453 0.00095 0.00352 0.00483 0.00411 0.00282 0.00308 0.00229 C10:1 0.00305 0.00298 0.00548 0.00793 0.00954 0.00585 0.00889 C10 0.00373 0.00746 0.00273 0.00532 0.00674 0.00628 0.01152 0.00863 C7-DC 0.00725 0.01239 0.00718 0.00583 0.00550 0.01082 0.01122 0.00567 C8:1-DC 0.00364 0.00586 0.00298 0.00304 0.00258 0.00477 0.00613 0.00345 C10-OH/C8-DC 0.02313 0.01653 0.01605 0.04030 0.02521 0.02146 0.03157 0.02708 C12:1 0.00152 0.00264 0.00185 0.00154 0.00379 0.00240 0.00370 0.00202 C12 0.00058 0.00247 0.00062 0.00264 0.00305 0.00170 0.00133 0.00101 C12-OH/C10-DC 0.01461 0.00794 0.00811 0.02559 0.02001 0.01498 0.02872 0.01835 C14:2 0.00593 0.00439 0.00094 0.00498 0.00151 0.00697 0.00578 0.00536 C14:1 0.00409 0.00355 0.00077 0.00667 0.00429 0.00568 0.00846 0.00975 C14 0.00241 0.00444 0.00496 0.00856 0.00429 0.00456 0.00598 0.00620 C14:1-OH/C12:1-DC 0.00605 0.00734 0.00769 0.01756 0.01230 0.00997 0.01644 0.00950 C14-OH/C12-DC 0.01803 0.01570 0.01205 0.03863 0.01304 0.02824 0.02765 0.02073 C16:2 0.00246 0.00147 0.00170 0.00348 0.00227 0.00354 0.00514 0.00293 C16:1 0.00843 0.00731 0.00567 0.01310 0.01374 0.01241 0.01034 0.01054 C16 0.01550 0.01851 0.00600 0.01712 0.03478 0.02588 0.02144 0.01585 C16:1-OH/C14:1-DC 0.01026 0.00459 0.00817 0.01687 0.01280 0.00991 0.01175 0.01675 C16-OH/C14-DC 0.02066 0.01157 0.01335 0.03927 0.01804 0.01849 0.02962 0.02844 C18:2 0.01732 0.01095 0.01241 0.04746 0.03585 0.03334 0.04438 0.02534 C18:1 0.02025 0.00969 0.01159 0.05441 0.02395 0.02617 0.03787 0.03812 C18 0.00713 0.00482 0.00596 0.00994 0.00759 0.00880 0.01408 0.00718 C18:2-OH 0.01725 0.01209 0.01968 0.04301 0.04196 0.03229 0.04154 0.03924 C18:1-OH/C16:1-DC 0.03636 0.02653 0.02731 0.09966 0.06191 0.04198 0.05839 0.06355 C18-OH/C16-DC 0.02621 0.01184 0.01444 0.03204 0.01899 0.02082 0.03457 0.02966 C20:4 0.01163 0.00464 0.00667 0.02338 0.01225 0.01906 0.02617 0.02107 C20 0.00041 0.00098 0.00041 0.00359 0.00574 0.00526 0.00530 0.00139 C20:1-OH/C18:1-DC 0.01033 0.00699 0.00754 0.00969 0.00624 0.01906 0.01664 0.01547 C20-OH/C18-DC 0.01133 0.00585 0.00596 0.01255 0.00989 0.01181 0.01936 0.01403 C22 0.00162 0.00279 0.00251 0.00295 0.00176 0.00249 0.00398 0.00537 C12:1-OH 0.00694 0.00409 0.00508 0.01173 0.01203 0.00684 0.01210 0.01351 C14:2-OH 0.00231 0.00161 0.00228 0.00429 0.00367 0.00231 0.00176 0.00099 C16:2-OH 0.01325 0.00569 0.00679 0.02253 0.00820 0.01024 0.02002 0.01296 C18:3 0.00534 0.00299 0.00569 0.00905 0.00938 0.01110 0.01628 0.00983 C20:2 0.00149 0.00280 0.00149 0.00475 0.00313 0.00258 0.00356 0.00330 C20:1 0.00344 0.00426 0.00223 0.00725 0.00586 0.00323 0.00901 0.00520 C22:1 0.00534 0.00262 0.00350 0.00369 0.00407 0.00323 0.00518 0.00330 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name C2 C3 C4/Ci4 C5:1 C5s 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 C12:1-OH C14:2-OH C16:2-OH C18:3 C20:2 C20:1 C22:1 METABOLITES_END #END