#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