Heart development is a spatiotemporally regulated process that extends from the embryonic phase to postnatal stages. Disruption of this highly orchestrated process can lead to congenital heart disease or predispose the heart to cardiomyopathy or heart failure. Consequently, gaining an in-depth understanding of the molecular mechanisms governing cardiac development holds considerable promise for the development of innovative therapies for various cardiac ailments. While significant progress in uncovering novel transcriptional and epigenetic regulators of heart development has been made, the exploration of post-translational mechanisms that influence this process has lagged. Culling-RING E3 ubiquitin ligases (CRLs), the largest family of ubiquitin ligases, control the ubiquitination and degradation of ~20% of intracellular proteins. Emerging evidence has uncovered the critical roles of CRLs in the regulation of a wide range of cellular, physiological, and pathological processes. In this review, we summarize current findings on the versatile regulation of cardiac morphogenesis and maturation by CRLs and present future perspectives to advance our comprehensive understanding of how CRLs govern cardiac developmental processes.
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.
ABSTRACT Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. 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 rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, 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 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.
Cardiac maturation is crucial for postnatal cardiac development and is increasingly known to be regulated by a series of transcription factors. However, post-translational mechanisms regulating this process remain unclear. Here we report the indispensable role of neddylation in cardiac maturation. Mosaic deletion of NAE1, an essential enzyme for neddylation, in neonatal hearts results in the rapid development of cardiomyopathy and heart failure. NAE1 deficiency disrupts transverse tubule formation, inhibits physiological hypertrophy, and represses fetal-to-adult isoform switching, thus culminating in cardiomyocyte immaturation. Mechanistically, we find that neddylation is needed for the perinatal metabolic transition from glycolytic to oxidative metabolism in cardiomyocytes. Further, we show that HIF1α is a putative neddylation target and that inhibition of neddylation accumulates HIF1α and impairs fatty acid utilization and bioenergetics in cardiomyocytes. Together, our data show neddylation is required for cardiomyocyte maturation through promoting oxidative metabolism in the developing heart.
Ubiquitin-mediated proteolysis is essential for protein homeostasis, and its defects lead to the development of various cardiomyopathies and heart failure. By reversing the ubiquitination and degradation of target proteins, deubiquitinases (DUBs) play a crucial role in protein homeostasis. However, the importance of DUBs in cardiac pathophysiology is largely unknown. Here we addressed the role of the DUB OTU domain aldehyde binding-1 (OTUB1) in the heart. Bioinformatics analysis reveals an upregulation of OTUB1 in hypertrophic and failing human hearts. In cultured cardiomyocytes, adrenergic agonists induced upregulation and activation of OTUB1. Silencing OTUB1 in repressed adrenergic agonists-induced fetal gene re-activation, protein synthesis and cardiomyocyte hypertrophy. In vivo , mice deficient of OTUB1 in the heart developed dilated cardiomyopathy and heart failure, leading to premature lethality. The severe cardiac phenotype is accompanied by lipid accumulation, ER dilatation, prevalent autophagic vesicles containing ribosomes, as well as dysregulated expression of metabolic and ribosome genes. Delivery of wild-type OTUB1, but not the enzymatic dead mutant, via AAV9 to OTUB1-deficient hearts attenuated cardiac dysfunction and prolonged the lifespan of mice. Mechanistically, loss of OTUB1 suppressed the degradation of Deptor, an inhibitory mTOR regulator, leading to impaired mTOR signaling in cultured cardiomyocytes and mouse hearts. Collectively, these findings suggest OTUB1 is required for physiological cardiac growth by fine-tuning mTOR signaling and identify OTUB1 as a novel regulator of cardiac homeostasis.
DNA damage response (DDR) and the centrosome cycle are 2 of the most critical cellular processes affecting the genome stability in animal cells. Yet the cross-talks between DDR and the centrosome are poorly understood. Here we showed that deficiency of the breast cancer 1, early onset gene (BRCA1) induces centrosome amplification in non-stressed cells as previously reported while attenuating DNA damage-induced centrosome amplification (DDICA) in cells experiencing prolonged genotoxic stress. Mechanistically, the function of BRCA1 in promoting DDICA is through binding and recruiting polo-like kinase 1 (PLK1) to the centrosome. In a recent study, we showed that FancJ also suppresses centrosome amplification in non-stressed cells while promoting DDICA in both hydroxyurea and mitomycin C treated cells. FancJ is a key component of the BRCA1 B-complex. Here, we further demonstrated that, in coordination with BRCA1, FancJ promotes DDICA by recruiting both BRCA1 and PLK1 to the centrosome in the DNA damaged cells. Thus, we have uncovered a novel role of BRCA1 and FancJ in the regulation of DDICA. Dysregulation of DDR or centrosome cycle leads to aneuploidy, which is frequently seen in both solid and hematological cancers. BRCA1 and FancJ are known tumor suppressors and have well-recognized functions in DNA damage checkpoint and DNA repair. Together with our recent findings, we demonstrated here that BRCA1 and FancJ also play an important role in centrosome cycle especially in DDICA. DDICA is thought to be an alternative fail-safe mechanism to prevent cells experiencing severe DNA damage from becoming carcinogenic. Therefore, BRCA1 and FancJ are potential liaisons linking early DDR with the DDICA. We propose that together with their functions in DDR, the role of BRCA1 and FancJ in the activation of DDICA is also crucial for their tumor suppression functions in vivo.
The cancer cell is capable of hijacking normal protective mechanisms to adapt to the extremely harsh intracellular and extracellular stresses it has created. Neddylation is a protein post-translational modification by which the ubiquitin-like protein NEDD8 conjugates to protein targets via an E1-E2-E3 multi-enzymatic cascade. As a vital survival mechanism in maintaining cell and tissue homeostasis in all eukaryotes, neddylation appears to be upregulated in multiple types of cancers, therefore targeting neddylation has become a promising anticancer strategy [[1]Watson I.R. Irwin M.S. Ohh M. NEDD8 pathways in cancer, Sine Quibus Non.Cancer Cell. 2011; 19: 168-176Summary Full Text Full Text PDF PubMed Scopus (138) Google Scholar]. To this end, a small molecule, MLN4924 (Pevonedistat) has been developed to specifically inhibit NEDD8 activating enzyme (NAE) (E1), the apex of NEDD8 E1-E2-E3 cascade [[2]Soucy T.A. Smith P.G. Milhollen M.A. et al.An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1356) Google Scholar]. NAE is the only NEDD8 E1 identified and a heterodimer of the regulatory subunit NAE1 and the catalytic subunit UBA3. MLN4924 forms a tight, irreversible NEDD8-MLN4924 covalent adduct that resembles NEDD8-AMP and prevents the docking of NEDD8-AMP to the catalytic pocket in UBA3, a process that is required for NEDD8 activation, thereby effectively blocking neddylation [[3]Brownell J.E. Sintchak M.D. Gavin J.M. et al.Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ.Mol Cell. 2010; 37: 102-111Summary Full Text Full Text PDF PubMed Scopus (346) Google Scholar]. Increasing evidence from pre-clinical studies and clinical trials have consistently demonstrated the effectiveness and potency of MLN4924 in inhibition of tumourigenesis and metastasis in diverse cancers ranging from hematological malignancies to solid tumours [[4]Lockhart A.C. Bauer T.M. Aggarwal C. et al.Phase Ib study of pevonedistat, a NEDD8-activating enzyme inhibitor, in combination with docetaxel, carboplatin and paclitaxel, or gemcitabine, in patients with advanced solid tumors.Invest New Drugs. 2019; 37: 87-97Crossref PubMed Scopus (48) Google Scholar], highlighting its therapeutic potential. However, similar to other target-based chemotherapies, mutations on UBA3 that diminish the binding affinity of NEDD8-MLN4924 to UBA3 render tumour cells resistant to MLN4924 treatment and may limit its clinical application [[5]Milhollen M.A. Thomas M.P. Narayanan U. et al.Treatment-emergent mutations in NAEbeta confer resistance to the NEDD8-activating enzyme inhibitor MLN4924.Cancer Cell. 2012; 21: 388-401Summary Full Text Full Text PDF PubMed Scopus (83) Google Scholar]. Therefore, there is an emergent need to identify additional drug targets in the neddylation pathway in order to develop the next generation of neddylation inhibitors. In an article in EBioMedicine, Li and colleagues explored the possibility of targeting the E2 neddylation conjugation enzyme to suppress tumour growth [[6]Li L. Kang J. Zhang W. et al.Validation of NEDD8-conjugating enzyme UBC12 as a new therapeutic target in lung cancer.EBioMedicine. 2019; https://doi.org/10.1016/j.ebiom.2019.06.005Summary Full Text Full Text PDF Scopus (29) Google Scholar]. In mammalian cells, two NEDD8 E2s have been identified, the well-characterized UBC12 (also known as UBE2M) and the less-studied UBE2F. These two E2s appear to show distinct function: UBC12 interacts with RBX1 to mediate neddylation of Cullin 1–4, whereas UBE2F pairs with SAG/RBX2 to facilitate neddylation of Cullin 5. While UBE2F was recently shown to promote the survival of lung cancer cells by sustaining CRL5 to degrade NOXA [[7]Zhou W. Xu J. Li H. et al.Neddylation E2 UBE2F promotes the survival of lung cancer cells by activating CRL5 to degrade NOXA via the K11 linkage.Clin Cancer Res. 2017; 23: 1104-1116Crossref PubMed Scopus (69) Google Scholar], little is known about the role of UBC12 in lung cancers. In this study, Li and colleagues first analyzed two published Affymetrix microarray datasets which revealed that UBC12, but not NAE1 and UBA3, is overexpressed in multiple lung cancers and predicts poor survival rate. Targeted deletion of UBC12 in two lung cancer cell lines led to inhibition of Cullin neddylation, accumulation of multiple cell-cycle inhibitors that are known to be degraded by Cullin-based RING ubiquitin ligases (CRLs), and cell cycle arrest. In vivo, deficiency of UBC12 also greatly suppressed tumour growth and metastasis. Together, these results confirm a critical role of UBC12 in controlling neddylation and are reminiscent of those observed by others in MLN4924-treated tumour cells, thereby identifying UBC12 as an attractive alternative therapeutic target in cancer treatment. Further, the authors addressed whether targeting UBC12 could be a potential strategy to resolve MLN4924-induced drug resistance. In an MLN4924-resistant cancer cell line, whose sensitivity to MLN4924 has dropped ~20-fold compared to its parent wild-type cell line, knockdown of UBC12 was able to effectively inhibit Cullin neddylation and thus repress cell growth, suggesting that inhibition of UBC12 could be effective to suppress the growth of tumours resistant to MLN4924. Future studies are warranted to investigate whether deletion of UBC12 has any impact on tumour growth in mice transplanted with MLN4924-resistant tumours, and to develop a pharmacological compound that specifically inhibits UBC12. While aberrant neddylation has been repeatedly reported to associate with tumour malignancy, very little is known about upstream mechanisms regulating neddylation. UBC12 is a stress-responsive protein and is transcriptionally controlled by HIF1α and AP-1 signaling [[8]Zhou W. Xu J. Tan M. et al.UBE2M is a stress-inducible dual E2 for Neddylation and Ubiquitylation that promotes targeted degradation of UBE2F.Mol Cell. 2018; 70: 1008-24 e6Summary Full Text Full Text PDF PubMed Scopus (41) Google Scholar]. Thus, it is conceivable that the upregulation of UBC12 in lung cancer cells, as reported in this study, could be consequent to transactivation of HIF1α in the hypoxic tumour microenvironment. Nevertheless, it remains to be determined whether its protein levels are consequently increased and whether such changes are sufficient to enhance the abundance of neddylated proteins in cancer cells. To pinpoint the underlying mechanism by which UBC12 controls tumourigenesis, Li and colleges performed quantitative proteomic analysis. The results revealed an accumulation of more than 500 proteins in cancer cells lacking UBC12 [[6]Li L. Kang J. Zhang W. et al.Validation of NEDD8-conjugating enzyme UBC12 as a new therapeutic target in lung cancer.EBioMedicine. 2019; https://doi.org/10.1016/j.ebiom.2019.06.005Summary Full Text Full Text PDF Scopus (29) Google Scholar] and coincide with the loss of function of CRLs. Yet, UBC12 deficiency also caused significant downregulation of ~500 proteins in cancer cells, indicating that neddylation is necessary for other cellular processes beyond Cullin-mediated proteolysis. To date, only a dozen of non-Cullin NEDD8 substrates have been identified, which has prevented us from fully understanding the cellular functions of neddylation in not only cancer cells, but also in other tissues. Identification of novel NEDD8 targets is challenging because of the low abundance of neddylated proteins, the transient and reversible nature of the modification, and concerns over the fidelity of targets identified from cells in which NEDD8 is overexpressed. Supported by this study, ectopic expression of UBC12 could a feasible approach to upregulate neddylated proteins, thereby facilitating the search for bona fide NEDD8 targets. Besides cancers, dysregulation of neddylation is implicated in various other diseases. In particular, specific inhibition of neddylation in neurons and cardiomyocytes has been shown to cause defects in synapse formation [[9]Vogl A.M. Brockmann M.M. Giusti S.A. et al.Neddylation inhibition impairs spine development, destabilizes synapses and deteriorates cognition.Nat Neurosci. 2015; 18: 239-251Crossref PubMed Scopus (62) Google Scholar] and cardiac maturation [[10]Zou J. Ma W. Li J. et al.Neddylation mediates ventricular chamber maturation through repression of hippo signaling.Proc Natl Acad Sci U S A. 2018; 115 (E4101-E10)Crossref Scopus (36) Google Scholar], respectively. An important yet unanswered question is whether normal cells/tissues, like cancer cells, could benefit from the global enhancement of neddylation under pathological conditions. Thus far, a valid tool to enhance neddylation in vivo is still lacking. As seen in this study and in cases of other ubiquitin-like proteins such as SUMO, overexpression of their E2 enzymes is able to stimulate the respective protein modification. Thus, this strategy could be employed to promote neddylation in vivo, ideally in a cell type-specific manner, and to ultimately address this question. The authors declare no conflicts of interest. This work was supported by a grant from the US National Institutes of Health (R01HL124248 to H.S.) and a grant from the American Heart Association (17POST33410592 to J.Z.). Validation of NEDD8-conjugating enzyme UBC12 as a new therapeutic target in lung cancerThese findings highlight a crucial role of UBC12 in fine-tuned regulation of neddylation activation status and validate UBC12 as an attractive alternative anticancer target against neddylation pathway. Full-Text PDF Open Access
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.
Changes in metabolic milieu during development trigger a metabolic shift from fetal-type glycolysis to adult-type fatty acid oxidation in the developing heart. Disruption of this metabolic shift has been linked to congenital heart disease, a major cause of birth-related mortality and morbidity worldwide. Neddylation is a post-translational modification that covalently attaches a small ubiquitin-like protein, NEDD8, to target proteins. Previously we reported that neddylation is essential for cardiomyocyte proliferation through sustaining the YAP signaling. However, a link between neddylation and cardiac metabolic maturation has not yet been established. Here, we abrogated neddylation by cardiac-specific deletion of NAE1, an essential subunit of the sole NEDD8 E1 enzyme, via αMHC Cre in mice, which led to significant reduction of NAE1 proteins and neddylated proteins in the heart. Temporal histological and functional analyses revealed that mice lacking neddylation displayed cardiac hypoplasia and ventricular non-compaction at E16.5, which became more pronounced by P1, eventually leading to heart failure and perinatal lethality by P7. Transcriptome analysis identified that the defects in cardiac chamber maturation are associated with upregulation of glycolytic genes and downregulation of oxidative metabolic genes. Transmissive electron microcopy revealed accumulation of lipid droplets and degenerative/immature mitochondria in hearts deficient of neddylation. In vitro, pharmacological inhibition of neddylation impairs mitochondrial membrane potential and respiration, and suppresses fatty acid utilization in oleic acid-primed cultured cardiomyocytes. Mechanistically, HIF1α, a potent regulator of cardiac metabolic reprogramming, is a novel NEDD8 target. Inhibition of neddylation causes accumulation of HIF1α proteins and consequently disrupts the expression of HIF1α downstream targets in vitro and in vivo. Silencing of HIF1α attenuates inhibition of neddylation-induced glycolytic rates in cardiomyocytes. Taken together, our findings highlight the importance of neddylation in the developing heart and identify neddylation as a novel regulator of HIF1α signaling to promote developmental metabolic switch.
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.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)