Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

BACKGROUND: Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. Apart from Parkin, little is known about additional Ub (ubiquitin) ligases that mediate mitochondrial ubiquitination and turnover, particularly in highly metabolically active organs such as the heart. METHODS: In this study, we have combined in silico analysis and biochemical assay to identify CRL (cullin-RING ligase) 5 as a mitochondrial Ub ligase. We generated cardiomyocytes and mice lacking RBX2 (RING-box protein 2; also known as SAG [sensitive to apoptosis gene]), a catalytic subunit of CRL5, to understand the effects of RBX2 depletion on mitochondrial ubiquitination, mitophagy, and cardiac function. We also performed proteomics analysis and RNA-sequencing analysis to define the impact of loss of RBX2 on the proteome and transcriptome. RESULTS: RBX2 and CUL (cullin) 5, 2 core components of CRL5, localize to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, increased cardiomyocyte cell death, and has a global impact on the mitochondrial proteome. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to the rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. The action of RBX2 in mitochondria is not dependent on Parkin, and Parkin gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 (PTEN-induced kinase 1) in mitochondria. CONCLUSIONS: These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that regulates mitophagy and cardiac homeostasis in a Parkin-independent, PINK1-dependent manner.

Neddylation is critical to cortical development by regulating Wnt/ß-catenin signaling.

Wnt signaling plays a critical role in production and differentiation of neurons and undergoes a progressive reduction during cortical development. However, how Wnt signaling is regulated is not well understood. Here we provide evidence for an indispensable role of neddylation, a ubiquitylation-like protein modification, in inhibiting Wnt/ß-catenin signaling. We show that ß-catenin is neddylated; and inhibiting ß-catenin neddylation increases its nuclear accumulation and Wnt/ß-catenin signaling. To test this hypothesis in vivo, we mutated Nae1, an obligative subunit of the E1 for neddylation in cortical progenitors. The mutation leads to eventual reduction in radial glia progenitors (RGPs). Consequently, the production of intermediate progenitors (IPs) and neurons is reduced, and neuron migration is impaired, resulting in disorganization of the cerebral cortex. These phenotypes are similar to those of ß-catenin gain-of-function mice. Finally, suppressing ß-catenin expression is able to rescue deficits of Nae1 mutant mice. Together, these observations identified a mechanism to regulate Wnt/ß-catenin signaling in cortical development.

Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability.

Hemophilia A and B are congenital bleeding disorders caused by a deficiency in pro-coagulant factor VIII or IX that is treated by downregulation of antithrombin. However, the molecular mechanisms that regulate antithrombin expression remain poorly understood. Here, we identified Cullin 2 and USP2 (ubiquitin-specific peptidase-2) as novel regulators of antithrombin expression that act by modulating antithrombin ubiquitination. Inhibition of the proteasome caused accumulation of antithrombin and its ubiquitinated forms in HepG2 and SMMC7721 cells. Notably, inhibition of neddylation with MLN4924 suppressed both ubiquitination and degradation of antithrombin, which is recapitulated by silencing of the neddylation enzymes, NAE1, UBA3, and UBE2M, with small interfering RNA (siRNA). We identified Cullin 2 as the interaction partner of antithrombin, and siRNA-mediated Cullin 2 knockdown reduced antithrombin ubiquitination and increased antithrombin protein. We further found that USP2 interacted with antithrombin and regulated antithrombin expression, showing that overexpression of USP2 inhibits the ubiquitination and proteasomal clearance of antithrombin, whereas pharmacological inhibition or siRNA-mediated knockdown of USP2 downregulates antithrombin. Collectively, these results suggest that Cullin 2 E3 ubiquitin ligase and USP2 coordinately regulate antithrombin ubiquitination and degradation. Thus, targeting Cullin 2 and USP2 could be a potential strategy for treatment of hemophilia.

The Calcineurin-TFEB-p62 Pathway Mediates the Activation of Cardiac Macroautophagy by Proteasomal Malfunction.

RATIONALE: The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway are pivotal to proteostasis. Targeting these pathways is emerging as an attractive strategy for treating cancer. However, a significant proportion of patients who receive a proteasome inhibitor-containing regime show cardiotoxicity. Moreover, UPS and autophagic-lysosomal pathway defects are implicated in cardiac pathogenesis. Hence, a better understanding of the cross-talk between the 2 catabolic pathways will help advance cardiac pathophysiology and medicine. OBJECTIVE: Systemic proteasome inhibition (PSMI) was shown to increase p62/SQSTM1 expression and induce myocardial macroautophagy. Here we investigate how proteasome malfunction activates cardiac autophagic-lysosomal pathway. METHODS AND RESULTS: Myocardial macroautophagy, TFEB (transcription factor EB) expression and activity, and p62 expression were markedly increased in mice with either cardiomyocyte-restricted ablation of Psmc1 (an essential proteasome subunit gene) or pharmacological PSMI. In cultured cardiomyocytes, PSMI-induced increases in TFEB activation and p62 expression were blunted by pharmacological and genetic calcineurin inhibition and by siRNA-mediated Molcn1 silencing. PSMI induced remarkable increases in myocardial autophagic flux in wild type mice but not p62 null (p62-KO) mice. Bortezomib-induced left ventricular wall thickening and diastolic malfunction was exacerbated by p62 deficiency. In cultured cardiomyocytes from wild type mice but not p62-KO mice, PSMI induced increases in LC3-II flux and the lysosomal removal of ubiquitinated proteins. Myocardial TFEB activation by PSMI as reflected by TFEB nuclear localization and target gene expression was strikingly less in p62-KO mice compared with wild type mice. CONCLUSIONS: (1) The activation of cardiac macroautophagy by proteasomal malfunction is mediated by the Mocln1-calcineurin-TFEB-p62 pathway; (2) p62 unexpectedly exerts a feed-forward effect on TFEB activation by proteasome malfunction; and (3) targeting the Mcoln1 (mucolipin1)-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac autophagic-lysosomal pathway activation during proteasome malfunction.

Neddylation mediates ventricular chamber maturation through repression of Hippo signaling.

During development, ventricular chamber maturation is a crucial step in the formation of a functionally competent postnatal heart. Defects in this process can lead to left ventricular noncompaction cardiomyopathy and heart failure. However, molecular mechanisms underlying ventricular chamber development remain incompletely understood. Neddylation is a posttranslational modification that attaches ubiquitin-like protein NEDD8 to protein targets via NEDD8-specific E1-E2-E3 enzymes. Here, we report that neddylation is temporally regulated in the heart and plays a key role in cardiac development. Cardiomyocyte-specific knockout of NAE1, a subunit of the E1 neddylation activating enzyme, significantly decreased neddylated proteins in the heart. Mice lacking NAE1 developed myocardial hypoplasia, ventricular noncompaction, and heart failure at late gestation, which led to perinatal lethality. NAE1 deletion resulted in dysregulation of cell cycle-regulatory genes and blockade of cardiomyocyte proliferation in vivo and in vitro, which was accompanied by the accumulation of the Hippo kinases Mst1 and LATS1/2 and the inactivation of the YAP pathway. Furthermore, reactivation of YAP signaling in NAE1-inactivated cardiomyocytes restored cell proliferation, and YAP-deficient hearts displayed a noncompaction phenotype, supporting an important role of Hippo-YAP signaling in NAE1-depleted hearts. Mechanistically, we found that neddylation regulates Mst1 and LATS2 degradation and that Cullin 7, a NEDD8 substrate, acts as the ubiquitin ligase of Mst1 to enable YAP signaling and cardiomyocyte proliferation. Together, these findings demonstrate a role for neddylation in heart development and, more specifically, in the maturation of ventricular chambers and also identify the NEDD8 substrate Cullin 7 as a regulator of Hippo-YAP signaling.

Neddylation Regulates Class IIa and III Histone Deacetylases to Mediate Myoblast Differentiation.

As the largest tissue in the body, skeletal muscle has multiple functions in movement and energy metabolism. Skeletal myogenesis is controlled by a transcriptional cascade including a set of muscle regulatory factors (MRFs) that includes Myogenic Differentiation 1 (MYOD1), Myocyte Enhancer Factor 2 (MEF2), and Myogenin (MYOG), which direct the fusion of myogenic myoblasts into multinucleated myotubes. Neddylation is a posttranslational modification that covalently conjugates ubiquitin-like NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to protein targets. Inhibition of neddylation impairs muscle differentiation; however, the underlying molecular mechanisms remain less explored. Here, we report that neddylation is temporally regulated during myoblast differentiation. Inhibition of neddylation through pharmacological blockade using MLN4924 (Pevonedistat) or genetic deletion of NEDD8 Activating Enzyme E1 Subunit 1 (NAE1), a subunit of the E1 neddylation-activating enzyme, blocks terminal myoblast differentiation partially through repressing MYOG expression. Mechanistically, we found that neddylation deficiency enhances the mRNA and protein expressions of class IIa histone deacetylases 4 and 5 (HDAC4 and 5) and prevents the downregulation and nuclear export of class III HDAC (NAD-Dependent Protein Deacetylase Sirtuin-1, SIRT1), all of which have been shown to repress MYOD1-mediated MYOG transcriptional activation. Together, our findings for the first time identify the crucial role of neddylation in mediating class IIa and III HDAC co-repressors to control myogenic program and provide new insights into the mechanisms of muscle disease and regeneration.

Adenosine kinase inhibition enhances microvascular dilator function and improves left ventricle diastolic dysfunction.

OBJECTIVE: Inhibition of adenosine kinase (ADK), via augmenting endogenous adenosine levels exerts cardiovascular protection. We tested the hypothesis that ADK inhibition improves microvascular dilator and left ventricle (LV) contractile function under metabolic or hemodynamic stress. METHODS AND RESULTS: In Obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid rats, treatment with the selective ADK inhibitor, ABT-702 (1.5 mg/kg, intraperitoneal injections for 8-week) restored acetylcholine-, sodium nitroprusside-, and adenosine-induced dilations in isolated coronary arterioles, an effect that was accompanied by normalized end-diastolic pressure (in mm Hg, Lean: 3.4 ± 0.6, Obese: 17.6 ± 4.2, Obese + ABT: 6.6 ± 1.4) and LV relaxation constant, Tau (in ms, Lean: 6.9 ± 1.5, Obese: 13.9 ± 1.7, Obese + ABT: 6.0 ± 1.1). Mice with vascular endothelium selective ADK deletion (ADKVEC KO) exhibited an enhanced dilation to acetylcholine in isolated gracilis muscle (lgEC50 WT: -8.2 ± 0.1, ADKVEC KO: -8.8 ± 0.1, P < .05) and mesenteric arterioles (lgEC50 WT: -7.4 ± 0.2, ADKVEC KO: -8.1 ± 1.2, P < .05) when compared to wild-type (WT) mice, whereas relaxation of the femoral artery and aorta (lgEC50 WT: -7.03 ± 0.6, ADKVEC KO: -7.05 ± 0.8) was similar in the two groups. Wild-type mice progressively developed LV systolic and diastolic dysfunction when they underwent transverse aortic constriction surgery, whereas ADKVEC -KO mice displayed a lesser degree in decline of LV function. CONCLUSIONS: Our results indicate that ADK inhibition selectively enhances microvascular vasodilator function, whereby it improves LV perfusion and LV contractile function under metabolic and hemodynamic stress.

Transient inhibition of neddylation at neonatal stage evokes reversible cardiomyopathy and predisposes the heart to isoproterenol-induced heart failure.

Alterations in perinatal conditions (such as preterm birth) is linked to adult health and disease, in particular, the cardiovascular system. Neddylation, a novel posttranslational modification through which the ubiquitin-like protein NEDD8 is conjugated to protein substrates, has emerged as an important mechanism regulating embryonic cardiac chamber maturation. However, the importance of neddylation in postpartum cardiac development has not been investigated. Here, we aimed to determine whether transient, postnatal inhibition of neddylation has immediate and prolonged impact on the structure and function of the neonatal and adult hearts. Sprague-Dawley pups were given three intraperitoneal injections of MLN4924 (MLN), a specific neddylation inhibitor, at postnatal days (P)1, 3, and 5. Cardiac structure and function were temporally assessed during aging and after 2 wk of isoproterenol (ISO) infusion in adulthood. MLN treatment resulted in modest reduction of neddylated proteins in neonatal hearts. The MLN-treated rats developed cardiac hypertrophy and dysfunction by P7, which was accompanied by significantly reduced cardiomyocyte proliferation. At 3 mo of age, cardiac contractile function was restored in MLN-treated rats, but MLN-treated hearts displayed hypertrophic phenotype. Whereas ISO infusion triggered compensatory cardiac hypertrophy without impairing cardiac contractility in the control rats, the MLN-treated rats displayed a similar degree of hypertrophy, which quickly progressed to decompensation with ventricular wall thinning, chamber dilatation, and reduced ejection fraction as well as exacerbated pathological cardiac remodeling. Our findings suggest that neddylation is required for postnatal cardiac development and that perturbation of neddylation during development predisposes adult hearts to cardiac failure under stress conditions. NEW & NOTEWORTHY Our study demonstrates that perinatal perturbation of neddylation induces cardiomyopathy, impairs postnatal cardiac development, and increases susceptibility to catecholamine-induced cardiac dysfunction. The results reveal a previously unappreciated role of neddylation in postnatal cardiac maturation and call for close monitoring for the potential cardiotoxicity of MLN4924 (pevonedistat) and other agents that modify neddylation, especially in pregnant women and preadolescents.

A carvedilol-responsive microRNA, miR-125b-5p protects the heart from acute myocardial infarction by repressing pro-apoptotic bak1 and klf13 in cardiomyocytes.

BACKGROUND: Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs), small non-coding RNAs that post-transcriptionally regulate target genes. MiR-125b-5p is downregulated in patients with end-stage dilated and ischemic cardiomyopathy, and has been proposed as a biomarker of heart failure. We previously reported that the ß-blocker carvedilol promotes cardioprotection via ß-arrestin-biased agonism of ß1-adrenergic receptor while stimulating miR-125b-5p processing in the mouse heart. We hypothesize that ß1-adrenergic receptor/ß-arrestin1-responsive miR-125b-5p confers the improvement of cardiac function and structure after acute myocardial infarction. METHODS AND RESULTS: Using cultured cardiomyocyte (CM) and in vivo approaches, we show that miR-125b-5p is an ischemic stress-responsive protector against CM apoptosis. CMs lacking miR-125b-5p exhibit increased susceptibility to stress-induced apoptosis, while CMs overexpressing miR-125b-5p have increased phospho-AKT pro-survival signaling. Moreover, we demonstrate that loss-of-function of miR-125b-5p in the mouse heart causes abnormalities in cardiac structure and function after acute myocardial infarction. Mechanistically, the improvement of cardiac function and structure elicited by miR-125b-5p is in part attributed to repression of the pro-apoptotic genes Bak1 and Klf13 in CMs. CONCLUSIONS: In conclusion, these findings reveal a pivotal role for miR-125b-5p in regulating CM survival during acute myocardial infarction.

ß-arrestin-biased agonism of ß-adrenergic receptor regulates Dicer-mediated microRNA maturation to promote cardioprotective signaling.

RATIONALE: MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate target genes. First transcribed as primary miR transcripts (pri-miRs), they are enzymatically processed by Drosha into premature miRs (pre-miRs) and further cleaved by Dicer into mature miRs. Initially discovered to desensitize ß-adrenergic receptor (ßAR) signaling, ß-arrestins are now well-appreciated to modulate multiple pathways independent of G protein signaling, a concept known as biased signaling. Using the ß-arrestin-biased ßAR ligand carvedilol, we previously showed that ß-arrestin1 (not ß-arrestin2)-biased ß1AR (not ß2AR) cardioprotective signaling stimulates Drosha-mediated processing of six miRs by forming a multi-protein nuclear complex, which includes ß-arrestin1, the Drosha microprocessor complex and a single-stranded RNA binding protein hnRNPA1. OBJECTIVE: Here, we investigate whether ß-arrestin-mediated ßAR signaling induced by carvedilol could regulate Dicer-mediated miR maturation in the cytoplasm and whether this novel mechanism promotes cardioprotective signaling. METHODS AND RESULTS: In mouse hearts, carvedilol indeed upregulates three mature miRs, but not their pre-miRs and pri-miRs, in a ß-arrestin 1- or 2-dependent manner. Interestingly, carvedilol-mediated activation of miR-466g or miR-532-5p, and miR-674 is dependent on ß2ARs and ß1ARs, respectively. Mechanistically, ß-arrestin 1 or 2 regulates maturation of three newly identified ßAR/ß-arrestin-responsive miRs (ß-miRs) by associating with the Dicer maturation RNase III enzyme on three pre-miRs of ß-miRs. Myocardial cell approaches uncover that despite their distinct roles in different cell types, ß-miRs act as gatekeepers of cardiac cell functions by repressing deleterious targets. CONCLUSIONS: Our findings indicate a novel role for ßAR-mediated ß-arrestin signaling activated by carvedilol in Dicer-mediated miR maturation, which may be linked to its protective mechanisms.

Characterization of mice carrying a conditional TEAD1 allele.

The Hippo- yes-associated protein (YAP) pathway is essential for controlling organ size and tumorigenesis. Previous studies have demonstrated that the primary outcome of YAP signaling in the nucleus is achieved by interaction with the transcription factor TEA domain transcription factor (TEAD1). The YAP/TEAD1 complex binds to DNA element and regulates the expression of genes involved in cell growth. However, constitutive knockout of TEAD1 leads to early embryonic lethality in mice. Thus, generation of a floxed TEAD1 mouse becomes crucial for further understanding mid- to late-gestation and post-natal role of TEAD1. Herein, we created and characterized a mouse model that allows for conditional disruption of TEAD1. Embryonic fibroblasts derived from the floxed TEAD1 mice enabled the Cre-mediated deletion of TEAD1 in vitro using virally delivered Cre recombinase. Furthermore, crossing the floxed TEAD1 mouse with a ubiquitously expressing Cre mouse resulted in efficient ablation of the floxed allele in vivo, and the animals recapitulated early embryonic lethality defects. In conclusion, our data demonstrate an important role of TEAD1 in early development in mice, and the floxed TEAD1 mouse model will be a valuable genetic tool to determine the temporal and tissue-specific functions of TEAD1.

Cardiac proteasome functional insufficiency plays a pathogenic role in diabetic cardiomyopathy.

BACKGROUND: Diabetic cardiomyopathy is a major risk factor in diabetic patients but its pathogenesis remains poorly understood. The ubiquitin-proteasome system (UPS) facilitates protein quality control by degrading unnecessary and damaged proteins in eukaryotic cells, and dysfunction of UPS is implicated in various cardiac diseases. However, the overall functional status of the UPS and its pathophysiological role in diabetic cardiomyopathy have not been determined. METHODS AND RESULTS: Type I diabetes was induced in wild-type and transgenic mice expressing a UPS functional reporter (GFPdgn) by injections of streptozotocin (STZ). STZ-induced diabetes progressively impaired cardiac UPS function as evidenced by the accumulation of GFPdgn proteins beginning two weeks after diabetes induction, and by a buildup of total and lysine (K) 48-linked polyubiquitinated proteins in the heart. To examine the functional role of the UPS in diabetic cardiomyopathy, cardiac overexpression of PA28α (PA28αOE) was used to enhance proteasome function in diabetic mouse hearts. PA28αOE diabetic mice displayed exhibited restoration of cardiac UPS function, as demonstrated by the diminished accumulation of GFPdgn and polyubiquitinated proteins. Moreover, PA28αOE diabetic mice exhibited reduced myocardial collagen deposition, decreased cardiomyocyte apoptosis, and improved cardiac systolic and diastolic function. CONCLUSION: Impairment of cardiac UPS function is an early event in STZ-induced diabetes. Overexpression of PA28α attenuates diabetes-induced proteotoxic stress and cardiomyopathy, suggesting a potential therapeutic role for enhancement of cardiac proteasome function in this disorder.

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit.

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the ß and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

COP9 signalosome controls the degradation of cytosolic misfolded proteins and protects against cardiac proteotoxicity.

RATIONALE: Impaired degradation of misfolded proteins is associated with a large subset of heart diseases. Misfolded proteins are degraded primarily by the ubiquitin-proteasome system, but the ubiquitin ligases responsible for the degradation remain largely unidentified. The cullin deneddylation activity of the COP9 signalosome (CSN) requires all 8 CSN subunits (CSN1 through CSN8) and regulates cullin-RING ligases, thereby controlling ubiquitination of a large number of proteins; however, neither CSN nor cullin-RING ligases is known to regulate the degradation of cytosolic misfolded proteins. OBJECTIVE: We sought to investigate the role of CSN8/CSN in misfolded protein degradation and cardiac proteinopathy. METHODS AND RESULTS: Cardiac CSN8 knockout causes mouse premature death; hence, CSN8 hypomorphism (CSN8(hypo)) mice were used. Myocardial neddylated forms of cullins were markedly increased, and myocardial capacity of degrading a surrogate misfolded protein was significantly reduced by CSN8 hypomorphism. When introduced into proteinopathic mice in which a bona fide misfolded protein R120G missense mutation of αß-crystallin (CryAB(R120G)) is overexpressed in the heart, CSN8 hypomorphism aggravated CryAB(R120G)-induced restrictive cardiomyopathy and shortened the lifespan of CryAB(R120G) mice, which was associated with augmented accumulation of protein aggregates, increased neddylated proteins, and reduced levels of total ubiquitinated proteins and LC3-II in the heart. In cultured cardiomyocytes, both CSN8 knockdown and cullin-RING ligase inactivation suppressed the ubiquitination and degradation of CryAB(R120G) but not native CryAB, resulting in accumulation of protein aggregates and exacerbation of CryAB(R120G) cytotoxicity. CONCLUSIONS: (1) CSN8/CSN promotes the ubiquitination and degradation of misfolded proteins and protects against cardiac proteotoxicity, and (2) cullin-RING ligases participate in degradation of cytosolic misfolded proteins.

NEDD8 Ultimate Buster 1 Long (NUB1L) Protein Suppresses Atypical Neddylation and Promotes the Proteasomal Degradation of Misfolded Proteins.

Neddylation is a posttranslational modification that controls diverse biological processes by covalently conjugating the ubiquitin-like protein NEDD8 to specific targets. Neddylation is commonly mediated by NEDD8-specific enzymes (typical neddylation) and, sometimes, by ubiquitin enzymes (atypical neddylation). Although typical neddylation is known to regulate protein function in many ways, the regulatory mechanisms and biological consequence of atypical neddylation remain largely unexplored. Here we report that NEDD8 conjugates were accumulated in the diseased hearts from mouse models and human patients. Proteotoxic stresses induced typical and atypical neddylation in cardiomyocytes. Loss of NUB1L exaggerated atypical neddylation, whereas NUB1L overexpression repressed atypical neddylation through promoting the degradation of NEDD8. Activation of atypical neddylation accumulated a surrogate misfolded protein, GFPu. In contrast, suppression of atypical neddylation by NUB1L overexpression enhanced GFPu degradation. Moreover, NUB1L depletion accumulated a cardiomyopathy-linked misfolded protein, CryAB(R120G), whereas NUB1L overexpression promoted its degradation through suppressing neddylation of ubiquitinated proteins in cardiomyocytes. Consequently, NUB1L protected cells from proteotoxic stress-induced cell injury. In summary, these data indicate that NUB1L suppresses atypical neddylation and promotes the degradation of misfolded proteins by the proteasome. Our findings also suggest that induction of NUB1L could potentially become a novel therapeutic strategy for diseases with increased proteotoxic stress.

Carvedilol-responsive microRNAs, miR-199a-3p and -214 protect cardiomyocytes from simulated ischemia-reperfusion injury.

The nonselective ß-adrenergic receptor antagonist (ß-blocker) carvedilol has been shown to protect against myocardial injury, but the detailed underlying mechanisms are unclear. We recently reported that carvedilol stimulates the processing of microRNA (miR)-199a-3p and miR-214 in the heart via ß-arrestin1-biased ß1-adrenergic receptor (ß1AR) cardioprotective signaling. Here, we investigate whether these ß-arrestin1/ß1AR-responsive miRs mediate the beneficial effects of carvedilol against simulated ischemia/reperfusion (sI/R). Using cultured cardiomyocyte cell lines and primary cardiomyocytes, we demonstrate that carvedilol upregulates miR-199a-3p and miR-214 in both ventricular and atrial cardiomyocytes subjected to sI/R. Overexpression of the two miRs in cardiomyocytes mimics the effects of carvedilol to activate p-AKT survival signaling and the expression of a downstream pluripotency marker Sox2 in response to sI/R. Moreover, carvedilol-mediated p-AKT activation is abolished by knockdown of either miR-199a-3p or miR-214. Along with previous studies to directly link the cardioprotective actions of carvedilol to upregulation of p-AKT/stem cell markers, our findings suggest that the protective roles of carvedilol during ischemic injury are in part attributed to activation of these two protective miRs. Loss of function of miR-199a-3p and miR-214 also increases cardiomyocyte apoptosis after sI/R. Mechanistically, we demonstrate that miR-199a-3p and miR-214 repress the predictive or known apoptotic target genes ddit4 and ing4, respectively, in cardiomyocytes. These findings suggest pivotal roles for miR-199a-3p and miR-214 as regulators of cardiomyocyte survival and contributors to the functional benefits of carvedilol therapy.

ß-arrestin1-biased ß1-adrenergic receptor signaling regulates microRNA processing.

RATIONALE: MicroRNAs (miRs) are small, noncoding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as long primary miR transcripts (pri-miRs), they are enzymatically processed in the nucleus by Drosha into hairpin intermediate miRs (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs where they regulate cellular processes after activation by a variety of signals such as those stimulated by ß-adrenergic receptors (ßARs). Initially discovered to desensitize ßAR signaling, ß-arrestins are now appreciated to transduce multiple effector pathways independent of G-protein-mediated second messenger accumulation, a concept known as biased signaling. We previously showed that the ß-arrestin-biased ßAR agonist, carvedilol, activates cellular pathways in the heart. OBJECTIVE: Here, we tested whether carvedilol could activate ß-arrestin-mediated miR maturation, thereby providing a novel potential mechanism for its cardioprotective effects. METHODS AND RESULTS: In human cells and mouse hearts, carvedilol upregulates a subset of mature and pre-miRs, but not their pri-miRs, in ß1AR-, G-protein-coupled receptor kinase 5/6-, and ß-arrestin1-dependent manner. Mechanistically, ß-arrestin1 regulates miR processing by forming a nuclear complex with hnRNPA1 and Drosha on pri-miRs. CONCLUSIONS: Our findings indicate a novel function for ß1AR-mediated ß-arrestin1 signaling activated by carvedilol in miR biogenesis, which may be linked, in part, to its mechanism for cell survival.

Crosstalk between Long Noncoding RNAs and MicroRNAs in Health and Disease.

Protein-coding genes account for only a small part of the human genome; in fact, the vast majority of transcripts are comprised of non-coding RNAs (ncRNAs) including long ncRNAs (lncRNAs) and small ncRNAs, microRNAs (miRs). Accumulating evidence indicates that ncRNAs could play critical roles in regulating many cellular processes which are often implicated in health and disease. For example, ncRNAs are aberrantly expressed in cancers, heart diseases, and many other diseases. LncRNAs and miRs are therefore novel and promising targets to be developed into biomarkers for diagnosis and prognosis as well as treatment options. The interaction between lncRNAs and miRs as well as its pathophysiological significance have recently been reported. Mechanistically, it is believed that lncRNAs exert "sponge-like" effects on various miRs, which subsequently inhibits miR-mediated functions. This crosstalk between two types of ncRNAs frequently contributes to the pathogenesis of the disease. In this review, we provide a summary of the recent studies highlighting the interaction between these ncRNAs and the effects of this interaction on disease pathogenesis and regulation.

Identification of gene signatures regulated by carvedilol in mouse heart.

Chronic treatment with the ß-blocker carvedilol has been shown to reduce established maladaptive left ventricle (LV) hypertrophy and to improve LV function in experimental heart failure. However, the detailed mechanisms by which carvedilol improves LV failure are incompletely understood. We previously showed that carvedilol is a ß-arrestin-biased ß1-adrenergic receptor ligand, which activates cellular pathways in the heart independent of G protein-mediated second messenger signaling. More recently, we have demonstrated by microRNA (miR) microarray analysis that carvedilol upregulates a subset of mature and pre-mature miRs, but not their primary miR transcripts in mouse hearts. Here, we next sought to identify the effects of carvedilol on LV gene expression on a genome-wide basis. Adult mice were treated with carvedilol or vehicle for 1 wk. RNA was isolated from LV tissue and hybridized for microarray analysis. Gene expression profiling analysis revealed a small group of genes differentially expressed after carvedilol treatment. Further analysis categorized these genes into pathways involved in tight junction, malaria, viral myocarditis, glycosaminoglycan biosynthesis, and arrhythmogenic right ventricular cardiomyopathy. Genes encoding proteins in the tight junction, malaria, and viral myocarditis pathways were upregulated in the LV by carvedilol, while genes encoding proteins in the glycosaminoglycan biosynthesis and arrhythmogenic right ventricular cardiomyopathy pathways were downregulated by carvedilol. These gene expression changes may reflect the molecular mechanisms that underlie the functional benefits of carvedilol therapy.

MicroRNA-150 deletion in mice protects kidney from myocardial infarction-induced acute kidney injury.

Despite greater understanding of acute kidney injury (AKI) in animal models, many of the preclinical studies are not translatable. Most of the data were derived from a bilateral renal pedicle clamping model with warm ischemia. However, ischemic injury of the kidney in humans is distinctly different and does not involve clamping of renal vessel. Permanent ligation of the left anterior descending coronary artery model was used to test the role of microRNA (miR)-150 in AKI. Myocardial infarction in this model causes AKI which is similar to human cardiac bypass surgery. Moreover, the time course of serum creatinine and biomarker elevation were also similar to human ischemic injury. Deletion of miR-150 suppressed AKI which was associated with suppression of inflammation and interstitial cell apoptosis. Immunofluorescence staining with endothelial marker and marker of apoptosis suggested that dying cells are mostly endothelial cells with minimal epithelial cell apoptosis in this model. Interestingly, deletion of miR-150 also suppressed interstitial fibrosis. Consistent with protection, miR-150 deletion causes induction of its target gene insulin-like growth factor-1 receptor (IGF-1R) and overexpression of miR-150 in endothelial cells downregulated IGF-1R, suggesting miR-150 may mediate its detrimental effects through suppression of IGF-1R pathways.