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Molecular and Cellular Endocrinology 413 (2015) 36e48

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier .com/locate/mce

The ubiquitin ligase MuRF1 regulates PPARa activity in the heart byenhancing nuclear export via monoubiquitination

Jessica E. Rodríguez a, 1, Jie-Ying Liao a, 1, Jun He b, Jonathan C. Schisler c,Christopher B. Newgard d, Doreen Drujan e, David J. Glass e, C.Brandon Frederick f,Bryan C. Yoder f, David S. Lalush f, Cam Patterson c, g, Monte S. Willis a, c, *

a Department of Pathology & Laboratory Medicine, University of North Carolina, Chapel Hill, NC, USAb General Hospital of Ningxia Medical University, Yinchuan, Ningxia, PR Chinac McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, USAd Sarah W. Stedman Nutrition and Metabolism Center and the Division of Endocrinology, Metabolism, and Nutrition, Duke University Medical Center,Durham, NC, USAe Novartis Institutes for Biomedical Research, Cambridge, MA, USAf Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC, USAg Presbyterian Hospital/Weill-Cornell Medical Center, New York, NY, USA

a r t i c l e i n f o

Article history:Received 27 February 2015Received in revised form22 May 2015Accepted 8 June 2015Available online 25 June 2015

Keywords:CardiomyocytePeroxisome proliferator-activated receptor aFatty acid metabolismCardiac metabolismMonoubiquitinationMuscle ring finger-1

Non-standard abbreviations: MuRF1, Muscle ringproliferator-activated receptor; RXRa, RXR retinoid Xtricular assist device; SRF, Serum response factor; GOgamma coactivator; TCA, Tricarboxylic acid; 123I-BMInylpentadecanoic acid; 18F-2-FDG, 18F-2-fluoro-Neonatal rat cardiomyocytes; MOI, Multiplicity of Infemission computed tomography; PET, Positron emiss* Corresponding author. McAllister Heart Institute

Laboratory Medicine, University of North Carolina, 12340B, Chapel Hill, NC 27599, USA.

E-mail address: monte_willis@med.unc.edu (M.S.1 Contributed equally to this work.

http://dx.doi.org/10.1016/j.mce.2015.06.0080303-7207/© 2015 Elsevier Ireland Ltd. All rights rese

a b s t r a c t

The transcriptional regulation of peroxisome proliferator-activated receptor (PPAR) a by post-translational modification, such as ubiquitin, has not been described. We report here for the first timean ubiquitin ligase (muscle ring finger-1/MuRF1) that inhibits fatty acid oxidation by inhibiting PPARa,but not PPARb/d or PPARg in cardiomyocytes in vitro. Similarly, MuRF1 Tgþ hearts showed significantdecreases in nuclear PPARa activity and acyl-carnitine intermediates, while MuRF1�/� hearts exhibitedincreased PPARa activity and acyl-carnitine intermediates. MuRF1 directly interacts with PPARa, mono-ubiquitinates it, and targets it for nuclear export to inhibit fatty acid oxidation in a proteasome inde-pendent manner. We then identified a previously undescribed nuclear export sequence in PPARa, alongwith three specific lysines (292, 310, 388) required for MuRF1's targeting of nuclear export. These studiesidentify the role of ubiquitination in regulating cardiac PPARa, including the ubiquitin ligase that may beresponsible for this critical regulation of cardiac metabolism in heart failure.

© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Changes in cardiac metabolism occur in response to patholog-ical hypertrophy, characterized by an upregulation of glucose up-take and glycolysis with either no change or a decrease in glucose

finger-1; PPAR, Peroxisomereceptor-a; LVAD, Left ven-, Gene ontology; PGC, PPAR-PP, 123I-b-methyl-p-iodophe-2-deoxy-D-glucose; NRCMs,ection; SPECT, Single-photonion tomography., Department of Pathology &11 Mason Farm Road, MBRB

Willis).

rved.

oxidation (as recently reviewed Kolwicz et al., 2013). In parallel,decreases in fatty acid oxidation are seenwith the down-regulationof peroxisome proliferator-activated receptor (PPAR)a (Lehman andKelly, 2002). A shift away from fatty acid oxidation to glucose uti-lization is considered beneficial because of the improved oxygenefficiency for ATP synthesis (Korvald et al., 2000; Burkhoff et al.,1991). In chronic ischemic cardiomyopathy, this becomes a crit-ical point since oxygen is limited; however, these changes may alsobe maladaptive over time to sustain myocardial energetics andfunction (Kolwicz et al., 2013). In the present study, we describe anovel mechanism by which the ubiquitin ligase muscle ring finger-1 (MuRF1) inhibits myocardial fatty acid oxidation by removingPPARa from the nucleus in a novel and previously undescribedmechanism.

The PPAR family of transcription factors, including PPARa,PPARb/d, and PPARg, regulate fatty acid utilization and oxidation inheart. Cardiomyocyte PPAR isoforms heterodimerize with the

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retinoid X receptor-a (RXRa) and interact with multiple coac-tivators and repressors to regulate the gene expression of proteinsinvolved in lipid metabolism and energy regulation (Dowell et al.,1999; Kota et al., 2005; Michalik et al., 2006). Decreased cardiacPPAR activity has been implicated in the regulation of fatty acidutilization in mouse, rat, and dog models of heart failure (Pellieuxet al., 2006; Morgan et al., 2006a,b; Barger et al., 2000; Bargerand Kelly, 2000). However, the mechanism by which PPARs areregulated in the context of heart failure have not been elucidated.Recent studies have provocatively suggested that post-translationalmodifications can be found on all three PPAR isoforms. For example,polyubiquitination of PPARb/d and PPARg1/2 in cancer and adipo-cytes cells, respectively, has been identified, but the effects ofpolyubiquitination on the activity of these PPAR isoforms andwhether degradation occurs are not known (Gareau and Lima,2010; Gopinathan et al., 2009; Wadosky and Willis, 2012). Thesestudies suggest that ubiquitin ligases, proteins that direct specificubiquitination of substrates (PPARs in this case), may be regulatingPPAR activity in some yet to be determined manner.

Muscle-specific ubiquitin ligase MuRF1 expression increases inchronic heart failure (Adams et al., 2007), left ventricular assistdevice (LVAD) placement in humans (Willis et al., 2009a), nutrientdeprivation (Baskin and Taegtmeyer, 2011), and ischemic/dilatedhuman cardiomyopathy (Paul et al., 2004). It was originally iden-tified as a transiently increasing protein in skeletal muscle atrophy(Bodine et al., 2001), mediating cardiac (and skeletal muscle) at-rophy in response to glucocorticoids and unloading (Willis et al.,2009a; Baehr et al., 2011). MuRF1 regulates cardiomyocyte massby post-translationally modifying and regulating transcriptionfactors in a disease-specific manner. In pathological cardiac hy-pertrophy, for example, MuRF1 inhibits cardiomyocyte growth byinhibiting PKCε activity (Arya et al., 2004) and interacting with andinhibiting the activity of the transcription factor serum responsefactor (SRF) (Willis et al., 2007). In cardiac ischemia-reperfusioninjury, MuRF1 inhibits apoptosis by halting JNK-induced signalinge specifically, MuRF1 polyubiquitinates phosphorylated c-Jun,targeting it for proteasome-dependent degradation, therebyeffectively inhibiting downstream AP-1 (c-Jun/Fos) signaling (Liet al., 2011; Wadosky et al., 2014). MuRF1's ability to regulate cellsignaling specifically in cardiomyocytes parallels its role in proteinquality control and the turnover of key metabolic enzymes,including creatine kinase (Willis et al., 2009b). With MuRF1'sprominence in interacting with metabolic enzymes regulating en-ergy metabolism (e.g. creatine kinase, pyruvate kinase, 3-hydroxybutyrate dehydrogenase) and our recent identification ofPPAR-associated gene expression signatures in microarray analyses(e.g. gene ontology (GO) categories PPAR-g coactivator (PGC),tricarboxylic acid (TCA) cycle, energy from oxidation) (Willis et al.,2009b), we investigated MuRF1 as a potential ubiquitin ligase thatregulates cardiomyocyte PPAR activity.

In the present study, we expand on MuRF1's role in cardiacremodeling and cardioprotection by demonstrating that MuRF1post-translationally regulates PPARa-mediated fatty acid oxidation.We describe for the first time a novel mechanism whereby MuRF1specifically inhibits PPARa, but not PPARb/d or PPARg activity, bytargeting PPARa for nuclear export through non-canonical ubiq-uitination (monoubiquitination). Specifically, we demonstrate thatMuRF1 interacts with and monoubiquitinates PPARa and targetsnuclear export of PPARa through its ubiquitin ligase activity. Sinceubiquitination occurs on lysines, the specific three lysines involvedin ubiquitinationwere identified (K292, K310, and K358) and foundto surround a nuclear export signal within PPARa that we identifiedand validated in the study. Together, these results identify thatMuRF1 directs nuclear PPARa removal without being degraded by anuclear export-dependent mechanism, driven by

monoubiquitination. These studies identify that MuRF1 inhibitsPPARa activity by a novel mechanism by which the cardiomyocytenuclear receptor PPARa is monoubiquitinated and subsequentlyremoved from the nucleus by the cell's nuclear export machinery.

2. Materials and methods

Detailedmethodology for the following techniques are providedin the Online Supplement: COS7, H9C2, HEK293T, HL-1 cellculturing; FLAG-PPARa mutant construct generation; qPCR; in vivoand in vitro ubiquitination assays; immunoprecipitation andWestern blot analysis; luciferase activity reporter assays; MuRF1Tgþ and MuRF1�/� mice generation; glucose and fatty acidoxidation assays; nuclear isolation; PPARa, PPARb, and PPARg ac-tivity assays; acylcarnitine quantification; fatty acid 123I-b-methyl-p-iodophenylpentadecanoic acid (123I-BMIPP) and glucose analogs18F-2-fluoro-2-deoxy-D-glucose (18F-2-FDG) by single-photonemission computed tomography (SPECT) and computed tomogra-phy/positron emission tomography (CT/PET) analysis. Data areexpressed as mean ± SEM. A one-way ANOVA test was used todetermine the source of variation. Differences between specificgroups were determined using a multiple comparison post-test viathe Holm-Sidak method using the all-pairwise procedure. Whenappropriate, a Student's t-test was performed to express differencesbetween groups within one variable. Significance was defined asp < 0.05.

3. Results

Increasing MuRF1 expression alters cardiomyocyte glucoseand fatty acid oxidation. Recent studies have illustrated that car-diomyocyte MuRF1 expression is critical to the cardiac hypertro-phic response to pressure overload and the susceptibility to heartfailure (Willis et al., 2009a,b, 2007). Like other ubiquitin ligases,MuRF1 interacts with multiple protein substrates, including thosedirectly involved in the cardiac hypertrophic response (e.g. thetranscription factor SRF) and in metabolic responses (e.g. creatinekinase) (Willis et al., 2009a,b; Zhao et al., 2008). Since tran-scriptomic analysis has also provided support for MuRF1's role inenergy metabolism (Willis et al., 2009b), we tested the hypothesisthat MuRF1 regulates fatty acid and glucose oxidation at the level ofthe cardiomyocyte in the heart. To test this hypothesis, weincreased MuRF1 expression using adenovirus constructs inneonatal rat cardiomyocytes (NRCMs) and measured the 14C-labeled CO2 released when cells were allowed to metabolize 14C-labeled glucose or 14C-labeled oleate (Fig. 1). Increasing MuRF1expression using adenoviral vectors (25 MOI (multiplicity ofinfection)) resulted in an ~80% increase in glucose oxidation and an~60% decrease in fatty acid oxidation in NRCMs (Fig. 1A and B,respectively). While the addition of the adenovirus alone to theNRCMs did not affect fatty acid oxidation (Fig. 1B, Untreated vs.5e15 MOI Ad.GFP), it did enhance glucose oxidation (Fig. 1A, un-treated vs. all other groups). Similarly, increasing MuRF1 in the HL-1 cardiomyocyte cell line resulted in enhanced glucose oxidationand decreased fatty acid oxidation (Fig. A.1A and B). Conversely,MuRF1 knock-down using siRNA in HL-1 cells resulted in decreasedglucose oxidation and enhanced fatty acid (oleate) oxidation(Fig. A.1C and D), suggesting a role for endogenous MuRF1 inregulating fatty acid and glucose oxidation in cardiomyocytes.WithincreasingMuRF1 expression, fatty acid oxidation is inhibited whileparallel increases in glucose oxidation result in an apparent shift ofsubstrate utilization. Based on the amount of fatty acid and glucoseoxidation measured, the amount of ATP resulting from the reducedfatty acid oxidation (129/mol) is compensated for by increases inATP created by the measured enhanced glucose oxidation (38/mol)

Fig. 1. Increasing MuRF1 expression enhances primary cardiomyocyte glucose oxidation and inhibits fatty acid (oleate) oxidation. A. 14C-Glucose and B. 14C-Oleate oxidation inneonatal rat cardiomyocytes (NRCMs) with increasing MOI of Ad.GFP or Ad.Myc-MuRF1. Etomoxir, a commercially available fatty acid oxidation inhibitor was used as a control todemonstrate that fatty acid oxidation inhibition could be detected. C. Using the glucose and fatty acid (oleate) oxidation determination, shifts in calculated ATP resulted in no netchange of ATP (based on 14CO2 released from the cells, assuming 1 mol glucose ¼ 38 ATP; 1 mol oleate ¼ 129 ATP). Data represent 3 independent experiments run in triplicate. D.Immunofluorescence microscopy detection of GFP (from Ad.GFP or Ad.MuRF1) vs. non-infected cells run in parallel with 14C-Glucose/14C-Oleate replicates. A One-Way ANOVA wasused to determine significance. #p < 0.05 vs. media alone. *p < 0.05 vs. Ad.GFP; **p < 0.05 vs. untreated control cells.

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(Fig. 1C). Since these changes were reminiscent of those seen inheart failure driven by PPAR isoforms (van Bilsen et al., 2004;Madrazo and Kelly, 2008), we investigated the possibility thatMuRF1 regulates this substrate shift by regulating PPAR activity.

MuRF1 specifically inhibits PPARa, but not PPARb/d or PPARgtranscriptional activity. The PPAR family of nuclear transcriptionfactors contains three isoforms expressed in the heart: PPARa,PPARb/d, and PPARg (Madrazo and Kelly, 2008). These nuclear re-ceptors dimerize with RXRa, and bind to several co-regulators tointeract with the PPAR response-element in the promoters of genesregulated by PPAR family members (Madrazo and Kelly, 2008).These genes encode for proteins that enhance fatty acid oxidation;at the same time, PPAR proteins increase transcription of pyruvatedehydrogenase kinase 4 (PDK4), which inhibits glycolysis andglucose oxidation (Kolwicz et al., 2013; Doenst et al., 2013). In thisway, inhibiting PPAR results in decreased fatty acid oxidation bytranscriptionally down-regulating genes necessary for fatty acidoxidation, while simultaneous removing PDK4. This would accountfor PPAR's divergent control of fatty acid and glucose oxidation

(Kolwicz et al., 2013; Doenst et al., 2013). To delineate which of thethree PPAR nuclear receptors MuRF1 may be inhibiting in car-diomyocytes, we next investigated if expression of MuRF1 affectsPPAR transcriptional activity using luciferase reporter gene assaysin COS-7 and H9C2 cells (Fig. 2). Since MuRF1 is found in both thenucleus and sarcomere (at the M-line bound to titin), assayingMuRF1's role in regulating PPARs in COS7 cells (without a sarco-mere) allowed us to first determine MuRF1's ability to regulatenuclear activity. Assaying MuRF1's regulation of PPARs in H9C2cells then illustrated the relevance of these findings in a repre-sentative cardiomyocyte. In both COS7 and H9C2 cells, co-transfection of a plasmid luciferase reporter driven by a PPAR-response element (PPRE) and a PPARa expression plasmid resul-ted in enhanced PPAR activity; in contrast, co-expression with aMuRF1 expression plasmid resulted in significant inhibition ofPPARa (Fig. 2A and D). To determine the specificity of this response,we next investigated MuRF1's effect on PPARb/d and PPARg gammaactivity. MuRF1 failed to inhibit either PPARb/d or PPARg inducedPPRE-luciferase activity in COS7 or HL-1 cardiomyocyte-derived

Fig. 2. MuRF1 regulation of PPARa, PPARb/d, and PPARg activity in vitro. PPARa, PPARb/d, and PPARg activity was determined using PPRE-driven luciferase plasmids, co-transfectedwith a PPARa, PPARb/d, and PPARg expression plasmid, in the presence or absence of MuRF1. A. In Cos7 cells, MuRF1 significantly inhibited PPARa, but not B. PPARb/d, or C. P PPARgactivity. Similarly, MuRF1 significantly inhibited D. PPARa, but not E. PPARb/d, or F. PPARg activity in cardiac-derived HL-1 cardiomyocyte cell line. G. Determination of nuclear PPARabinding to PPRE DNA using a capture ELISA platform demonstrates the increasing MuRF1 expression results in decreased nuclear PPARa activity; conversely H. decreasing MuRF1expression with siRNA results in increased nuclear PPARa activity in HL-1 cardiomyocytes. I. Quantitative RT-PCR analysis reported cardiac PPARa-activated mRNA expression in HL-1 cardiomyocytes with increased MuRF1 (transduced with Ad.GFP-Myc-MuRF1) or J. decreased MuRF1 expression (Ad.GFP-shMuRF1). Expression of cells without treatment was setto 1. Data are mean ± SEM from at least 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. Ad.GFP or Ad.GFP-sh.Scramble infected control cells.

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cells (Fig. 2B,C,E,F).We next established a method to quantify PPARa activity that

could be applied to both in vitro and in vivo studies. MuRF1expressionwas increased in HL-1 cells using adenovirus (Ad.MuRF1and control Ad.GFP); parallel assays with MuRF1 knockdown usingsiRNA MuRF1 cocktail transfection (and a scramble control in par-allel) were run. Nuclear protein was isolated and assayed for itsability to bind solid phase PPRE DNA bound, and this was quantifiedusing a colorimetric anti-PPARa antibody. Increasing MuRF1expression resulted in a dose-dependent decrease in PPARa activity(Fig. 2G). Decreasing MuRF1 expression using siRNA enhancedPPARa expression over 2.5 fold of control levels (Fig. 2H). In parallelstudies, increasing, and decreasingMuRF1 altered PPARa-regulatedgene expression (Fig. 2I and J). Taken together, these results suggestthat MuRF1 specifically inhibits PPARa, the most abundant PPARfound in cardiomyocytes (Escher et al., 2001), in both non-cardiomyocyte and at least 2 cardiomyocyte-derived cell lines.

Cardiac MuRF1 expression regulates PPARa activity and fattyacid oxidation in vivo.With evidence thatMuRF1 regulates PPARa,but not PPARb/d or PPARg activity in multiple cell types, we nextassayed MuRF1's regulation of these factors in vivo. Using previ-ously described MuRF1 cardiac transgenic (Tgþ) and MuRF1�/�mice (Willis et al., 2009a,b, 2007), we assayed isolated cardiacnuclei for PPAR activity (described above in Fig. 2G). Nuclear ex-tracts were placed on plates coated with PPRE DNA, followed byincubation with a capture antibody recognizing PPARa, PPARb/d, orPPARg in separate assays (Fig. 3A and B). Consistent with ourin vitro studies, increased a-MHC-driven cardiac MuRF1 expressionin the MuRF1 Tgþ hearts resulted in a significant decrease in PPARaactivity, approximately 60% compared with wild-type controls (Fig.3A). Conversely, MuRF1�/� hearts exhibited an approximately 5-fold increase in activity compared with wild-type controls Fig. 3B.MuRF1 Tgþ and MuRF1�/� hearts had the same PPARb/d andPPARg activities as their respective wild-type controls, indicatingonce again the specificity of MuRF1's effect on PPARa in vivo.

Previous studies using transgenic mouse models have identifiedthat increasing cardiac PPARa results in the increased import andoxidation of fatty acids (Madrazo and Kelly, 2008). To determine theamount of imported and oxidized fatty acids, we performed anacylcarnitine profile analysis to quantify fatty acid oxidation in-termediates, an assay established to reflect changes in PPARa ac-tivity in vivo (Makowski et al., 2009; Chen et al., 2009; Laghezzaet al., 2013; Turer et al., 2009) (Fig. 3CeF). Using this technique,we identified that compared to sibling control mice, MuRF1 Tgþhearts demonstrated a significant decrease in total acylcarnitinelevels (Fig. 3C). Conversely, MuRF1�/� hearts had a significantincrease in total acylcarnitine (C2eC18 acyl-CoA species) levelscompared with their sibling controls (Fig. 3C). When the totalacylcarnitine was divided up into its component long, medium, andshort chain acylcarnitines (Fig. 3D�F), similar significant differ-ences were seen in the long and short chain acylcarnitines,reflecting their relative abundance compared with medium chainacylcarnitines. Additional in vivo studies investigating fatty acidand glucose uptake in the heart revealed no differences betweenMuRF1 Tgþ, MuRF1�/�, and their respective wild-type siblingcontrol hearts in studies using the fatty acid analog 123I-b-methyl-p-iodophenylpentadecanoic acid (123I-BMIPP) imaged by single-photon emission computed tomography (SPECT) and the glucoseanalog 18F-2-fluoro-2-deoxy-D-glucose (18F-2-FDG) imaged bycomputed tomography/positron emission tomography (CT/PET)(Fig. A.2). These results illustrate that inhibition of fatty acidoxidation in MuRF1 Tgþ mice and enhanced fatty acid oxidation inMuRF1�/� mice, both revealed by significant differences in acyl-carnitine levels, cannot be explained by differences in fatty aciduptake and therefore must be due to regulators of fatty acid

oxidation within the cell, such as PPARa.We next assayed the expression levels of the PPARa-regulated

genes acetyl-CoA carboxylase 1 (ACC1), aconitase 1 (ACO1), longchain acyl-CoA dehydrogenase (LCAD), and medium chain acyl-coenzyme A dehydrogensase (MCAD) by qPCR. Despite evidencethat MuRF1 inhibits PPARa activity (Fig. 3A and B) and fatty acidoxidation (acyl-carnitine intermediates) (Fig. 3CeF) in vivo, tran-scription of PPARa-regulated genes (mRNA) were expressed in theopposite manner from expected. MuRF1 Tgþ hearts demonstrateda transcriptional upregulation of ACC1 and ACO1, whereasMuRF1�/� hearts expressed significantly less ACC1, ACO1, LCAD,and MCAD mRNA (Fig. 3G and H). Given the complexity of thePPARa complex and its multiple binding partners (RXR and othernuclear receptors), co-repressors, and co-stimulators, it is not sur-prising to see opposite activities invoked. For example, co-repression has been described when agonist-bound PPARs attractrepressors and when PPAR homo-dimerization occurs (Fan et al.,2011; Flores et al., 2011; Borland et al., 2011). This sort of feed-back mechanism common in physiology has only begun to betouched upon in PPAR biology and rarely in vivo.

MuRF1 does not affect the steady state protein levels of PPARisoforms. Like most ubiquitin ligases reported to date, MuRF1 haslargely been reported to polyubiquitinate and degrade its specificsubstrates, including troponin I, b-myosin heavy chain, cMyBP-c,and c-Jun (Li et al., 2011; Kedar et al., 2004; Fielitz et al., 2007;Mearini et al., 2010). In these previous studies, increasing MuRF1expression resulted in decreased substrate levels; conversely,reducing MuRF1 resulted in increased substrate protein levels.Since our results demonstrated that MuRF1 inhibits PPARa activity,we hypothesized that MuRF1 reduces PPARa by targeting itsdegradation. To test this, we performed Western blot analysis onMuRF1 Tgþ and MuRF1�/� hearts to examine PPARa, PPARb/d, orPPARg protein levels (Fig. 3G). Surprisingly, whereas MuRF1 clearlyinhibited PPARa activity, it did not affect its steady state proteinlevel in the heart. These findings led us to hypothesize that MuRF1inhibits PPARa activity in a manner independent of proteasome-mediated degradation.

MuRF1 interacts with PPARa to regulate its nuclear locali-zation.We next determined if MuRF1 interacted with PPARa, giventhe possibility that MuRF1's regulation of PPARa could occur byindirect means since PPARa is only one component of a largercomplex, including RXRa and PGC-1. Using HL-1 cells, we immu-noprecipitatedMuRF1 andwere able to pull down and immunoblotendogenous PPARa (Fig. 4A). Similarly, when we immunoprecipi-tated endogenous PPARawewere able to pull downMuRF1 in HL-1cells (Fig. 4B). To determine how this might occur, we next per-formed confocal microscopy analysis in HL-1 cells to examine thelocation of endogenous PPARa in the presence and absence ofincreased MuRF1 expression (Fig. 4C). As expected, endogenousPPARa expression was found throughout the cell in control cells,both in the cytosol and nucleus (Fig. 4C). Surprisingly, in cells withincreased MuRF1 expression, endogenous PPARa was cleared fromthe nucleus (Fig. 4C). When larger numbers of cells were sampled,endogenous nuclear PPARa was present in 99.2% of control HL-1cells (Fig. 4D). In contrast, increasing MuRF1 expression resultedin only 29.2% of the cells retaining endogenous nuclear PPARa(Fig. 4D). We hypothesized that MuRF1 may post-translationallymodify PPARa in such a way that it becomes excluded from thenucleus. This hypothesis is consistent with the fact that MuRF1inhibits PPARa's (nuclear) activity without degrading protein sub-strate itself.

MuRF1 mediates PPARa nuclear export to inhibit its activity.Recent studies have identified that ubiquitination of nuclear pro-teins by ubiquitin ligases can target nuclear export in the context ofcancer. For example, the ubiquitin ligase MDM2 mono-

Fig. 3. Cardiomyocyte MuRF1 expression regulates PPARa, PPARb/d, and PPARg activity in vivo without altering protein levels of PPARa. A. The increased cardiomyocyte MuRF1 inMuRF1 Tgþ hearts results in decreased nuclear PPARa binding activity, but not PPARb/d, or PPARg activity for binding PPRE DNA using a capture ELISA platform. B. Conversely,MuRF1�/� hearts lacking cardiomyocyte MuRF1 have a >5 fold increase in nuclear PPARa binding activity, but no effect on PPARb/d, or PPARg activity using a capture ELISA assay.CeF. Quantitative metabolomics analysis of acyl-carnitine (AC) in MuRF1 Tgþ and MuRF1�/� hearts. Normalized concentrations of C. total acylcarnitines (Total AC). Furtherbreakdown to these total ACs by D. long chain (C14eC22), E. medium chain (C6eC12), and F. short chain (C2eC5), illustrates MuRF1's regulation of fatty acid oxidation significantlyaffects long and short chain ACs, while medium ACs are not affected. Subcategories of acylcarnitines classified by chain length measured in mouse heart homogenates from theindicated strain and represented by box and whiskers plotting the median and min to max, respectively (n ¼ 3e4 biological replicates per strain). G. Western immunoblot analysisMuRF1 Tgþ (N ¼ 4/group) and MuRF1�/� (N ¼ 3/group) hearts have PPARa, PPARb/d, and PPARg expression that do not differ from strain-matched control hearts (N ¼ 4 and N ¼ 3,respectively), with the exception of PPARb which was decreased in MuRF1�/� hearts. H. qPCR analysis of reported cardiac PPARa-regulated mRNA illustrates that the increasedMuRF1 in MuRF1 Tgþ hearts increases the expression of ACC1 and ACO1 while I. MuRF1�/� hearts have increased levels of all ACC1, ACO1, LCAD, and MCAD. Data are mean ± SEM.A Student's t-test was performed to determine significance between groups (Panels A, B, G, H, *p < 0.05, **p < 0.01, ***p < 0.001). Changes in total, long chain, and short chain weresignificant at an FDR <5% (*) (Panels CeF, post-tests: *p < 0.001 comparing MuRF1þ/þ versus MuRF1�/�, and Wild-typeMuRF1Tg vs. MuRF1 Tgþ, respectively. **p < 0.01 and***p < 0.001 MuRF1�/� vs. MuRF1 Tgþ). n.s. ¼ not significant.

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Fig. 4. MuRF1 interacts with PPARa by co-immunoprecipitation and increasing MuRF1 expression removes nuclear localization. A. Immunoprecipitation of MuRF1 in HL-1 cells(forward reaction), followed by immunoblot analysis of endogenous PPARa detected a specific interaction between MuRF1 and PPARa. B. Immunoprecipitation of endogenousPPARa in HL-1 cells (reverse reaction) also detected a specific interaction between PPARa and MuRF1. C. Confocal microscopy analysis of HL-1 cells revealed that at the single celllevel, increasing MuRF1 expression (Ad.MuRF1 25 MOI) caused endogenous PPARa to move out of the nucleus (middle panel) compared with controls (top). D. The removal ofendogenous PPARa by MuRF1 (25 MOI) was found in most of the cells compared with controls, which rarely lacked nuclear endogenous PPARa. E. Analysis of at least 3 independentexperiments from >5 fields of view demonstrated that increasing MuRF1 (AdMuRF1) decreased the nuclear endogenous PPARa 70% (99.2%e29.2%), while increasing the number ofcells with cytoplasmic endogenous PPARa 70% (70.8%e0.8%). N ¼ 244 (Ad.GFP), N ¼ 228 (Ad.myc-MuRF1). A Student's t-test was performed to determine significance betweengroups. *p < 0.01.

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ubiquitinates p53, which results in nuclear export of p53 in cancercells (Li et al., 2003; Carter et al., 2007). While this mechanismoccurs in the context of cancer (e.g. in the H1299 human non-smallcell lung carcinoma cell line and the U2OS osteosarcoma cell line),similar mechanisms have not been described in muscle cells.Therefore, we investigated if MuRF1's ability to remove nuclearPPARa in cardiomyocytes was related to nuclear export, using thepotent and specific nuclear export inhibitor leptomycin B (LMB)(Fig. 5) (Kudo et al., 1998). Leptomycin B alkylates and inhibits theCRM1/exportin 1 (XPO1) protein required for the nuclear export ofproteins containing a nuclear export signal (NES), by glycosylating acysteine residue (Kudo et al., 1999). MuRF1's ability to removeendogenous nuclear PPARa was inhibited in the presence of LMBtreatment, indicating a role for the NES machinery in this process.

In the absence of LMB, increasing MuRF1 expression resulted inendogenous nuclear PPARa localization in 11.3% of cells, comparedwith 82.3% of control cells, respectively (Fig. 5B, upper vs. lowerrows). In parallel experiments using LMB, MuRF1's ability to alterthe nuclear localization of PPARa was inhibited, with 95.7% of thenuclei having endogenous PPARa, compared with 96.4% in controlcells (Fig. 5B, upper vs. lower rows). We next quantified how thenuclear transport effect of MuRF1 affected endogenous PPARa ac-tivity by assaying the amount of nuclear PPARa that could bind to asolid phase DNA with a PPRE. Compared to control cells, an 80%decrease in binding activity was found when MuRF1 expressionwas increased (Fig. 5C). Consistent with our confocal analysis, weidentified that LMB inhibited MuRF1's ability to decrease endoge-nous PPARa activity in cardiac-derived cells (Fig. 5D). Whereas

Fig. 5. MuRF1's nuclear removal and inhibition of PPARa activity is dependent on the nuclear export machinery. A. The MuRF1-mediated removal of endogenous PPARa, found incells with increased MuRF1 (Ad.myc-MuRF1 vs. Ad.GFP, top two rows) B. is inhibited when cells are treated with Leptomycin B (LMB), an inhibitor of exportin (CRM1)-mediatednuclear export. By recognizing leucine-rich nuclear export signals (NESs) in target proteins like PPARa, CRM1 mediates the nuclear export and inhibition. C. In parallel experimentsmeasuring nuclear PPARa activity (ability of isolated nuclear PPARa to bind PPRE-coated ELISA plates), increasing MuRF1 inhibited PPARa activity approximately 20% of controls. D.Paralleling MuRF1's removal of nuclear PPARa in confocal microscopy experiments, LBM inhibition of nuclear export in HL-1 cells prevented MuRF1's inhibition of PPARa activity. E.The presence of MuRF1 is rarely found in the nucleus in normal conditions (e.g. Fig. 4). However, in the presence of LMB, MuRF1 was found in greater than 25% of HL-1 cells 4 h aftertreatment indicating that MuRF1 movement in and out of the nucleus is dependent upon nuclear export of it or other interacting proteins necessary for MuRF1 to interact withnuclear receptors. A Student's t-test was performed to determine significance between Ad.GFP and Ad.MuRF1. *p < 0.05. n.s. ¼ not significant.

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MuRF1 is rarely found in the nucleus, LMB treatment “captured”MuRF1 in the nucleus of approximately 25% of the cells (Fig. 5E). Itis possible thatMuRF1's nuclear import and export is so swift undernormal conditions that noticeable accumulation of MuRF1 in thenucleus is not seen, but can be uncovered in the presence of LMB.MuRF1's ability to enter and exit the nucleus (albeit briefly) wouldalso explain MuRF1's inhibition of other nuclear substrate activity,including SRF shown previously (Willis et al., 2007), and PPARa inthe current study. Taken together, these findings indicate MuRF1enters and then rapidly moves out of the nucleus to removeendogenous PPARa in a manner dependent upon the cell's nuclearexport machinery.

MuRF1 monoubiquitinates PPARa in vitro and in vivo. As amuscle-specific ubiquitin ligase, MuRF1's ability to regulate inter-acting substrates has been linked to its ability to ubiquitinate theprotein at specific lysines. MuRF1 polyubiquitinates and degradescTnI, c-Jun, and b-MHC in a proteasome-dependent manner (Liet al., 2011; Kedar et al., 2004; Fielitz et al., 2007). In contrast, thelocalization and activity of other substrates, such as PKCε and SRF,are regulated without protein degradation (Arya et al., 2004; Williset al., 2007). Since MuRF1 interacts with PPARa and inhibits itsactivity, but does not change the steady state protein levels when itis increased, we next detailed MuRF1's interaction and ubiquiti-nation of PPARa. Using MuRF1 mapping constructs, we identified

Fig. 6. MuRF1 binds PPARa through its MFC domain and promotes PPARa monoubiquitination through its ubiquitin ligase activity. A. Immunoprecipitation of PPARa (anti-FLAG) incells co-transfected FLAG-PPARa and myc-MuRF1 deletion mutants identified that MuRF1's MFC (MuRF Family Conserved) region is required for its interaction with PPARa. B.Western immunoblot analysis of in vitro ubiquitination reactions containing recombinant E1, E2, MuRF1, PPARa, and ubiquitin (¼Full reaction) demonstrates that MuRF1 mon-oubiquitinates PPARa in a specific manner (compare to No E1, No E2, No MuRF1, No PPARa, No ubiquitin lanes). C. Determination of MuRF1's ubiquitination pattern in vivo similarlyreveals the presence of monoubiquitination. Specifically, HL-1 cells transfected with FLAG-PPARa and HA-Ubiquitin (HA-Ub) with increased MuRF1 (Ad.Myc-MuRF1 vs. Ad.GFP, 4 h)demonstrates that PPARa is multi-monoubiquitinated. D. Similarly, PPARa is multi-monoubiquitinated in HEK293 cells in a RING finger-dependent manner. Compared to MuRF1'smonoubiquitination of PPARa, MuRF1DRING (MuRF1 lacking the N-terminal RING finger domain with ubiquitin ligase activity) is unable to similarly monoubiquitinate PPARadespite equal expression levels of MuRF1 (IB: MuRF1 [cMyC]).

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that MuRF1 binds PPARa through its B-box and MuRF-familyconserved (MFC) regions (Fig. 6A). We next performed an in vitroubiquitination assaywherewe added recombinant E1, E2 (UbcH5c),MuRF1, PPARa, and ubiquitin. Western blot analysis of this reactionyielded both a predominant unmodified form halfway between the52 and 76 kDa molecular weight markers (Fig. 6B). A single addi-tional band was seen in the full reaction (far right lane), but not inthe control reactions missing just 1 component (E1, E2, MuRF1,PPARa, OR ubiquitin, respectively). This specific band (i.e. notformed without all the reaction components present) was foundbetween the PPARa (~54 kDa) and below the 76 kDamarker. Since asingle ubiquitin has a molecular mass of 8.5 kDa, it represents amono-ubiquitination (estimated to be62.5 kDa ¼ 54 kDa þ 8.5 kDa). This is not the canonical poly-ubiquitination of PPARa, since these post-translationally modifiedchains appear as a smear. For example, MuRF1 simultaneouslyautoubiquitinated itself by this type of poly-ubiquitin chain (Fig. 6B,lower anti-GST blot), revealing that the reaction stoichiometrydrove MuRF1 to monoubiquitinated PPARa in vitro. Prior studieshave revealed MuRF1's ability to auto-polyubiquitinate through allof the possible ubiquitin lysine chains (K6, K11, K27, K29, K33, K48,K63) (Kim et al., 2007), whereas mono- and multi-mono-ubiquitination patterns are not associated with ubiquitin chainformation (Sadowski et al., 2012).

Like many cell free phenomena, these in vitro ubiquitinationresults needed confirmation in the more complex milieu of the cell.

Therefore, we next sought to confirm the relevance of MuRF1'sapparent monoubiquitination of PPARa in cell culture, with mul-tiple other proteins present, including multiple E2s known to drivethe complexity and type of MuRF1 ubiquitin chains (Kim et al.,2007). Interestingly, MuRF1 similarly monoubiquitinated PPARain cells demonstrating that increasing MuRF1 expression results inincreased post-translational modification of PPARa in both HL-1cells and HEK293T cells, which results in the addition of one ortwo ubiquitin modifications (di-monoubiquitination) being added(Fig. 6C and D, respectively). Both mono- and di-monoubiquitinmodifications have been reported to mediate changes in cellularlocalization in various cancer cell lines (Haglund et al., 2003; Leeet al., 2008; Liu and Subramani, 2013). These studies illustratethat increasing MuRF1 expression induces a multi-monoubiquitination of PPARa, which may be responsible for theresulting nuclear export observed.

An interesting finding related to nuclear export machineryinhibited by LMB is that it is dependent upon recognition of nuclearexport signals (NES). However, the sequences that make up theNES(s) for PPARa have not been described to date. Nuclear exportsignals are made up of hydrophobic residues, often leucines (L),arranged in a LXXXLXXLXL motif, where X is any other amino acid.Given the critical nature of the leucines in NESs, we first scannedthe PPARa amino acid sequence for leucine clusters and found threenear each other at amino acid residues 300e308 (Fig. A.3B and C).In addition to this leucine rich region (NES1), we next used an

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algorithm to identify any additional potential NES sequences inPPARa. Based on published analysis of a wide range of nuclearexport signals, we used a bioinformatics approach to predict otherpotential leucine-rich nuclear export signals in PPARa (http://www.cbs.dtu.dk/services/NetNES/) (la Cour et al., 2004). This analysisidentified a cluster of 3 predicted NES amino acid residues in an 8amino acid cluster at residues 368e377 (lelddsdisl), suggesting aputative nuclear export sequence, which we named NES2 (Fig. A.3Band C). These sequences, identified as NES1 (ldlndqvtl) and NES2(lelddsdisl), are located in the ligand-binding domain (domain E/F)in PPARa, detailed in Fig. A.3A. Using standard published pro-cedures (Simon-Areces et al., 2013; Lin et al., 2005; Durchschlaget al., 2004; Brown et al., 2004; Hake et al., 2000), we next exam-ined whether these putative NES1 and NES2 regions were func-tional, by mutating Leucines to Alanines in the NES1 and NES2regions. If NES1 and/or NES2 were actual nuclear export sequences,these mutations would prevent their nuclear export and confirmtheir role in targeting PPARa for nuclear export in cardiomyocytes.HL-1 cells were transfected with 1) wild-type PPARa; 2) NES1mutant PPARa; or 3) NES2 mutant PPARa in parallel (Fig. 7A).Compared to wild-type PPARa (Fig. 7A, top panels), NES1 mutantPPARa demonstrated an increased nuclear localization in experi-ments run in parallel (Fig. 7A, middle panels). In contrast, the NES2mutant PPARa did not have an altered nuclear localizationcompared with wild-type PPARa (Fig. 7A, bottom panels). Thesestudies identify for the first time that the novel NES1 is a bone fideNES sequence in PPARa. Despite NES1's ability to maintain its nu-clear localization, increased MuRF1 expression was able to specif-ically export both the NES1 and NES2 mutant PPARa (Fig. 7B andFig. A.3D).

MuRF1's PPARa monoubiquitination requires 3 lysine groupsflanking the newly identified NES1. To determine which lysines inPPARa are monoubiquitinated, and to illustrate MuRF1's depen-dence upon this ubiquitination for nuclear export of PPARa, wecreated PPARa mutant proteins that lacked specific lysine groups(by mutations changing lysine to arginine) surrounding the 2 pu-tative NES sites that we identified in the experiment describedabove (Fig. A.3A). Six PPARamutants, named A1, A2, A3, A4, A5, and

Fig. 7. Identification and confirmation of PPARa's first nuclear export sequence targeting nuhere (strategy outlined in Fig. A.3A, in blue): 1) a FLAG-tagged wild-type PPARa; 2) a full lenreplaced with alanine; and 3) a full length FLAG-tagged NES2 mutant PPARa, with all 3 leucinrow), NES1, but not NES2 transfected cardiomyocytes, have increased nuclear staining conscentration of the NES1 mutant PPARa impaired MuRF1's ability to remove it, parallel studiesthe nucleus. Increasing MuRF1 (Ad.myc-MuRF1 25 MOI) reduced nuclear NES1 mutant PPAR70% reduction seen in endogenous PPARa in previous experiments (Fig. 5). Despite the imporexport of this mutant PPARa is unaffected.

A6 were created, each with only 1 lysine altered. When cells weretransfected with A1eA6 and MuRF1 expression was increased, wefound that PPARa A1, PPARa A2, and PPARa A3 were not removedfrom the nucleus as was wild-type PPARa when MuRF1 expressionwas increased (Fig. 8). PPARa A4eA6 mutants were removed fromthe nucleus to the same extent as wild-type PPARa (Fig. A.4) andparallel control cells (Fig. A.5). These studies illustrate the necessityof the three individual lysines surrounding the novel NES1 PPARa,which result in the mono- and/or di-mono-ubiquitination that weidentified in cells and in vitro ubiquitination assays.

4. Discussion

In the present study, we identified the striated muscle-specificMuRF1 ubiquitin ligase as a direct regulator of PPARa activity inthe heart in vivo. Increasing MuRF1 expression decreased PPARaactivity while decreasing MuRF1 expression using siRNA or mousemodels lacking cardiac MuRF1 resulted in significant increases inPPARa activity. MuRF1's inhibition of PPARa occurred using amechanism requiring nuclear export, evidenced by its inhibition byleptomycin B. After identifying the first NES in PPARa usingmutational analysis, we next demonstrated that MuRF1 is mono-ubiquitinated using in vitro ubiquitination assays. In cell modelsin vivo, we found that increasing MuRF1 monoubiquitinated or di-monoubiquitinated PPARa. When lysines A1, A2, and A3 sur-rounding the novel NES1 weremutated to arginine, MuRF1's abilityto induce nuclear export was prevented. We proposed thatincreasing MuRF1 results in MuRF1's translocation to the nucleuswhere PPARa is monoubiquitinated and preferentially recognizedby CRM1 and the nuclear export complex, mediating its subsequentexport and inhibition of activity (transcription of PPARa-regulatedgenes). These studies parallel recent findings in cancer, wherebythe ubiquitin ligase MDM2 was found to monoubiquitinate p53 totarget its nuclear export (Li et al., 2003). In contrast to MDM2 andp53, we have not found that increased MuRF1 levels poly-ubiquitinates and degrades PPARa like increased MDM2 levelshave been reported. This novel pathway possibly regulates thenewly identified NES1 sequence-targeted nuclear export. These

clear export. Three PPARa constructs were made to confirm if putative NESs identifiedgth FLAG-tagged NES1 mutant PPARa, whereby all 4 leucines in the NES1 region werees in the NES2 regionwere replaced with alanine. A. Compared to wild-type PPARa (topistent with an impaired nuclear export. B. To determine if the enhanced nuclear con-with increased MuRF1 were performed to test MuRF1's ability to remove PPARa from

a compared with control (Ad.GFP 25 MOI) by 63.2% (83.9%e20.7%), consistent with thetance of the NES1 for nuclear export, modification of MuRF1's modification and nuclear

Fig. 8. Three specific lysines surround NES1 are required for MuRF1's nuclear export of PPARa. Since ubiquitin ligases, including MuRF1, place ubiquitin on protein lysines, we nextdetermined the lysines required for MuRF1's nuclear export of PPARa. Six additional FLAG-tagged PPARa constructs were created with the six most proximal lysines to the NES1 andNES2 mutated to arginine, preventing ubiquitination from occurring (see Fig. A.3 for details). Compared to controls (row 1), MuRF1 clears wild-type PPARa in HL-1 cardiomyocytes(row 2). However, full length PPARa with a single lysine to arginine mutation at surrounding NES1 (A1, A2, and A3) is not cleared from the nucleus. With increased MuRF1(Ad.MuRF1), PPARa mutants A1, A2, and A3 localize in the nucleus (rows 3e5, right table e 89.4%, 77.4%, and 78.3%, respectively) to the same extent as Ad.GFP controls (row 1,81.1%). In contrast, increased MuRF1 (Ad.MuRF1) results in nuclear removal of wild-type PPARa, remaining in only 26.4% of the cells (row). PPARa mutants A4, A5 and A6 werelocalized only in cytoplasm as PPARa control (Fig. A.4), indicating that the lysines A4, A5, A6 are not potential ubiquitination sites needed for MuRF1's nuclear export of PPARa.

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findings are also the first description of a pathway by which cardiacPPAR isoform levels are regulated and may be critical in the sub-strate switching the heart utilizes to adapt in cardiac diseases, suchas cardiac hypertrophy and heart failure.

A recent study has identified that PPARg is exported rapidlyfrom the nucleus after being phosphorylated in cancer cell lines,including MCF-7 breast adenocarcinoma and HEK293T embryonickidney cell lines (Burgermeister and Seger, 2007). This mitogen-induced nuclear export is through its direct interaction with theMAPK/ERK-kinases 1/2 (MEKs) (Burgermeister and Seger, 2007). Ofparticular interest in this study was that the NES that interacts withCRM1/exportin-1 was found inMEK1, not PPARg. This suggests thatMEK1's interactionwith PPARgwas critical in its nuclear export andpossibly otherwise independent of any phosphorylation of PPARgitself. Recent studies have found that MEK1 similarly directs PPARa,although with PPARa MEK1 interacts with the C-terminal end,where a proposed, but not confirmed LXXLL NES motif was hy-pothesized (el Azzouzi et al., 2012). These studies were particularlyinteresting as they were performed in the NKL-Tag ventricularmuscle cell line and MEK1 inhibitors inwild-type mice appeared toparallel these in vitro studies (el Azzouzi et al., 2012).

The regulation of PPARa by post-translational modification withubiquitin has been demonstrated in rat hepatoma and the HepG2cell lines (Gopinathan et al., 2009). The E3 MDM2 has been asso-ciated with increased poly-ubiquitination of PPARa; interestingly,the MDM2 to PPARa ratio determined if MDM2 activated (MDM2:PPARa <1) or inhibited (MDM2: PPARa >1) (Gopinathan et al.,2009). Parallel studies in another hepatoma cell line (HepG2)illustrated that MDM2's inhibition of PPARa activity was due to itsdegradation (Blanquart et al., 2002; Blanquart et al., 2004; Hirotaniet al., 2001). Both sets of studies identified that MDM2-dependent

modulation of PPARa activity was dependent upon the presence ofthe PPARa ligand Wy14643 (Gopinathan et al., 2009; Blanquartet al., 2002, 2004; Hirotani et al., 2001). Interestingly, in the pre-sent study, we did not see MuRF1 poly-ubiquitinate or degradePPARa regardless of the MuRF1:PPARa ratio (data not shown).Similarly, all experiments were performed in the presence ofserum, which included endogenous PPARa ligands. We did not findthe addition of Wy14643 altered the results of our studies, so wedid not include them after our initial evaluation.

Monoubiquitination, like that found on PPARa resulting fromMuRF1 ubiquitination, has been associated with directing changesin sub-cellular location. Classically, this has been described at theplasma membrane and associated with endosomal targeting of re-ceptors after ligand engagement. One example involves receptortyrosine kinases (RTKs), where by interaction with their cognateligand results in monoubiquitination at the plasma membrane(Haglund et al., 2003). Upon monoubiquitination, these RTKs moveto the endosomal compartment to be shuttled to the lysosome, notrecycled to the plasmamembrane inmultiple cancer cells, includingNIH 3T3 cells, B82L, and HeLa (Haglund et al., 2003). The ubiquitinligase MDM2 has been reported to ubiquitinate p53, with mono-ubiquitinationofp53being sufficient fornuclearexport (Carteret al.,2007). Interestingly,MDM2ubiquitination of p53 contributes to twodifferent steps of p53's nuclear export: 1) ubiquitination exposes acarboxyl-terminalNES, and2)promotes thedisassociationofMDM2(Carter et al., 2007). Further studies indicated that MDM2's mono-ubiquitination of p53 promotes the interaction of the SUMO E3ligase PIASg with p53, enhancing SUMOylation and nuclear exportin U2OS osteosarcoma cells and MEF cells (Carter et al., 2007).Monoubiquitination of nuclear PTEN in cancer cells has demon-strated that the monoubiquitination of PTEN is essential for nuclear

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import (Trotman et al., 2007). In contrast to p53 and the presentstudy, the post-translational modification of a nuclear protein tar-gets the substrate tomove into the nucleus, illustrating hownuclearlocalization signals (NLS) canbe regulated bymonoubiquitination inthe same manner as NESs.

The mismatch between MuRF1's effects on PPARa activity/localization and on PPARa-regulated gene expression was unex-pected. But this may be the result of the multiple roles MuRF1 hasin cardiomyocyte metabolism and the multiple levels that PPARamay be regulated. MuRF1 may be directly (or indirectly) affectingcompensatory gene regulation at numerous levels, resulting incomplex and possibly contradictory relationships. For example,MuRF1 regulates metabolism by regulating the turnover of creatinekinase (critical for ATP shuttling to the sarcomere) (Willis et al.,2009b), localizes in the mitochondria and reduces ROS produc-tion in vivo (Mattox et al., 2014). MuRF1 itself interacts (in Y2Hsystems) with numerous metabolic proteins, such as enoyl coen-zyme A hydratase, malonyl-CoA decarboxylase, and multiple otherproteins involved in glycolysis and the Krebs cycle (e.g. Aldolase A,Pyruvate kinase, Pyruvate dehydrogenase) (Willis et al., 2009b;Witt et al., 2005). Taken together, MuRF1's combined effects onmetabolism, in addition to the regulation of PPARa described here,is complex. Therefore, the unique findings on the overall metabolicresponses here are not completely surprising. The transcriptionalcomplex made up of PPARa and RXRa includes a myriad of otherproteins, including co-repressors, some of which have beendescribed to bind agonist-bound PPARs, some just recentlydescribed (Borland et al., 2011). Having these multiple players hasmade predicting the effects of reducing PPAR protein on PPARtranscription unpredictable. The ratio of PPARa:RXRa in car-diomyocytes, for example, may be more important in determiningPPAR activity than the levels of PPARa alone. Recent studies haveshown that enhanced PPAR expression can change the ratio ofPPAR:RXRa, favoring PPAR homo-dimers that can act as repressors(opposite of what would be predicted), despite more PPAR beingpresent and located on PPAR response elements in the promoter ofPPAR-transcribed genes (Fan et al., 2011; Flores et al., 2011). Overall,our understanding of post-translational regulation of the PPARcomplex itself remains limited and should be expected to becomplex and unpredictable, but nonetheless critical to our under-standing of PPARs in metabolism and the pathophysiology ofdisease.

5. Conclusions

Muscle ring finger-1 (MuRF1) is the first muscle-specific ubiq-uitin ligase reported that directs nuclear PPARa inhibition in aproteasome-independent manner. These studies demonstratedthat increasing MuRF1 inhibits fatty acid oxidation, whileenhancing glucose oxidation and inhibiting PPARa. Mechanistically,these studies report that cardiomyocyte MuRF1 inhibits PPARaactivity by a novel mechanism by which the cardiomyocyte nuclearreceptor PPARa is mono-ubiquitinated (adjacent to a novel NES alsoidentified in these studies) and subsequently removes it from thenucleus by the cells nuclear export machinery. Furthermore, thepresent study identified the first confirmed NES in PPARa, alongwith three specific lysines (292, 310, 388) required for MuRF1stargeting of nuclear export. These studies identify the role ofubiquitination in regulating cardiac PPARa in vivo, including thecardiac-specific ubiquitin ligase that may be responsible for thiscritical regulation of cardiac metabolism in heart failure.

Disclosures

The authors have no conflicts of interests, financial or otherwise,

to declare.

Acknowledgments

The authors would like to thank Dr.William Claycomb for giftingthe HL-1 cells used in this study, and for his guidance in detailingtheir care and use. This study was supported by grants from theAmerican Heart Association Mid-Atlantic Affiliate (12PRE11480009to K.W.), UNCWilliam R. Kenan, Jr. Fellowship (to J.R.), Pre-DoctoralTraining Program in Integrative Vascular Biology (T32-HL069768 toJ.R.), American Heart Association Scientist Development Grant (toM.W.), Jefferson-Pilot Corporation Fellowship in Academic Medi-cine (to M.W.), the NIH National Heart, Lung, and Blood Institute(R01HL104129 to M.W.), and the Foundation Leducq (to C.P. andM.W.). Dr. Ashley G. Rivenbark provided proof reading and editingassistance.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.mce.2015.06.008.

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