Neddylation mediates ventricular chamber maturation PNAS PLUS through repression of Hippo signaling Jianqiu Zoua, Wenxia Maa, Jie Lia, Rodney Littlejohna, Hongyi Zhoub, Il-man Kima, David J. R. Fultona, Weiqin Chenb, Neal L. Weintrauba, Jiliang Zhouc, and Huabo Sua,c,d,1 aVascular Biology Center, Medical College of Georgia, Augusta University, Augusta, GA 30912; bDepartment of Physiology, Medical College of Georgia, Augusta University, Augusta, GA 30912; cDepartment of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA 30912; and dProtein Modification and Degradation Lab, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, Guangdong 510260, China Edited by Deepak Srivastava, Gladstone Institute of Cardiovascular Disease, San Francisco, CA, and accepted by Editorial Board Member Brenda A. Schulman March 23, 2018 (received for review November 7, 2017) During development, ventricular chamber maturation is a crucial maturation of the ventricular wall is regulated by a complex sig- step in the formation of a functionally competent postnatal heart. naling network comprised of growth factors, transcription factors, Defects in this process can lead to left ventricular noncompaction and epigenetic regulators (3, 4), such as NRG1/ERBB2 (8), cardiomyopathy and heart failure. However, molecular mecha- NOTCH (9, 10), TGFβ (11, 12), Smad7 (13), and HDAC1/2 (14). nisms underlying ventricular chamber development remain However, the importance of novel ubiquitin-like protein modifiers incompletely understood. Neddylation is a posttranslational mod- in this process has not been demonstrated. ification that attaches ubiquitin-like protein NEDD8 to protein tar- Neural precursor cell expressed, developmentally down-regulated gets via NEDD8-specific E1-E2-E3 enzymes. Here, we report that 8 (NEDD8) is a ubiquitin-like protein that shares a high degree of neddylation is temporally regulated in the heart and plays a key homology with ubiquitin (15). NEDD8 is evolutionarily highly role in cardiac development. Cardiomyocyte-specific knockout of conserved and ubiquitously expressed in all cell types (16). Conju- NAE1, a subunit of the E1 neddylation activating enzyme, signifi- gation of NEDD8 to protein targets, termed neddylation, is a cantly decreased neddylated proteins in the heart. Mice lacking process analogous to ubiquitination and requires NEDD8-specific NAE1 developed myocardial hypoplasia, ventricular noncompac- tion, and heart failure at late gestation, which led to perinatal E1, E2, and E3 enzymes (17, 18). The only identified NEDD8 E1 is lethality. NAE1 deletion resulted in dysregulation of cell cycle- a heterodimer of NAE1 (also known as APP-BP1) and UBA3, regulatory genes and blockade of cardiomyocyte proliferation in which together activate NEDD8 in an ATP-dependent manner. vivo and in vitro, which was accompanied by the accumulation of With the aid of a NEDD8 E2 (UBC12) enzyme and yet to be fully the Hippo kinases Mst1 and LATS1/2 and the inactivation of the defined E3 ligases, activated NEDD8 is covalently fused with the YAP pathway. Furthermore, reactivation of YAP signaling in NAE1- lysine residue of substrate proteins via its C-terminal glycine, which inactivated cardiomyocytes restored cell proliferation, and YAP- can result in the addition of one or more NEDD8 molecules. deficient hearts displayed a noncompaction phenotype, supporting Neddylation is reversibly regulated; substrate proteins conjugated an important role of Hippo-YAP signaling in NAE1-depleted hearts. with NEDD8 can be “deneddylated” by NEDD8-specific proteases, PHYSIOLOGY Mechanistically, we found that neddylation regulates Mst1 and such as the COP9 signalosome (CSN) and NEDP1 (also known as LATS2 degradation and that Cullin 7, a NEDD8 substrate, acts as SENP8) (19, 20). the ubiquitin ligase of Mst1 to enable YAP signaling and cardiomyo- cyte proliferation. Together, these findings demonstrate a role Significance for neddylation in heart development and, more specifically, in the maturation of ventricular chambers and also identify the Myocardium thickening at midgestation to late gestation is NEDD8 substrate Cullin 7 as a regulator of Hippo-YAP signaling. crucial for the formation of a functionally competent postnatal heart. However, posttranslational mechanisms regulating this NEDD8 | Hippo-YAP signaling | Cullin 7 | ventricular compaction | process remain unexplored. Here, we uncover a critical role for cardiomyopathy the ubiquitin-like protein NEDD8 in heart development. By targeting the E1 enzyme that mediates the conjugation of ardiac development consists of a series of highly regulated NEDD8 to protein targets, we show that NEDD8 modification is Cspatiotemporal morphogenic events (1, 2). The maturation essential for ventricular compaction and heart function. We of the cardiac chamber during late gestation is a crucial step further identify that the NEDD8 substrate Cullin 7 mediates the toward the generation of a functionally competent postnatal degradation of Hippo kinase Mst1, thereby enabling YAP sig- heart and involves the coordination of ventricular trabeculation naling, cardiomyocyte proliferation, and proper heart devel- and compaction (3, 4). In the initial stages of heart development, opment. These results reveal a posttranslational regulatory trabeculae (an endocardium-lined myocardial mesh) protrude mechanism in ventricular wall maturation and may provide and expand into the ventricular lumen to increase the myocardial mechanistic insights into the etiology of left ventricular surface area for oxygen and nutrient exchange. From late noncompaction cardiomyopathy. midgestation to the late fetal and neonatal stage, the compact myocardium grows quickly and fuses with the trabecular myo- Author contributions: I.K., D.J.R.F., W.C., N.L.W., J. Zhou, and H.S. designed research; J. Zou, W.M., J.L., R.L., H.Z., and H.S. performed research; W.C. and J. Zhou contrib- cardium to form a thicker, and eventually mature, compact uted new reagents/analytic tools; J. Zou, W.M., J.L., R.L., H.Z., J. Zhou, and H.S. ventricular wall. Defects in cardiac chamber maturation lead to analyzed data; and J. Zou and H.S. wrote the paper. congenital heart diseases, including left ventricular noncompaction The authors declare no conflict of interest. cardiomyopathy (LVNC), the third most commonly diagnosed This article is a PNAS Direct Submission. D.S. is a guest editor invited by the Editorial Board. cardiomyopathy (5, 6). LVNC is characterized by deep and exten- Published under the PNAS license. sive trabeculae in a thin ventricular wall, which can result in dilated 1To whom correspondence should be addressed. Email: [email protected]. cardiomyopathy, heart failure, thromboembolism, arrhythmia, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sudden cardiac death (7). Emerging studies from genetic analysis of 1073/pnas.1719309115/-/DCSupplemental. LVNC patients and animal models of LVNC have suggested that Published online April 9, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1719309115 PNAS | vol. 115 | no. 17 | E4101–E4110 Downloaded by guest on October 1, 2021 Neddylation controls diverse cellular processes, including tran- the role of neddylation in perinatal cardiac development in a cell scriptional regulation, cell cycle progression and differentiation, lineage-specific fashion, we generated a conditional NAE1flox/flox ribosome biogenesis, apoptosis, inflammatory responses, and allele by flanking exon 4 with loxP sites (SI Appendix, Fig. S2A). proteolysis, via altering the activity, stability, subcellular locali- Cardiomyocyte-restricted NAE1 knockout (NAE1CKO,NAE1flox/flox/ zation, and DNA binding affinity of conjugated proteins (17, 18). αMHCCre) mice were then created by crossing NAE1flox/flox Among the growing list of NEDD8 targets, the Cullin proteins mice with αMHC-driven Cre transgenic (αMHCCre)mice,in (Cullin 1 to 5, 7, and 9) are the best-characterized substrates. which Cre recombinase activity can be detected in the heart as Acting as scaffold proteins, Cullins recruit substrate recogni- early as E9.5 and is present in a majority of ventricular car- tion subunits and a RING domain protein to form functional diomyocytes at E13.5 (37, 38). PCR and quantitative real-time Cullin-RING ubiquitin ligases (CRLs) (21), which together PCR analyses confirmed the deletion of NAE1 in the NAE1CKO mediate the proteolysis of ∼20% of cellular proteins (22). hearts, compared with their littermate control hearts (CTLs) + Neddylation of Cullins induces the assembly of CRLs and thus is (NAE1flox/flox or NAE1flox/ )(SI Appendix, Fig. S2 B and C). essential for their ubiquitin ligase activity (21). Western blot analyses of whole heart lysates revealed ∼40% and Recent studies have begun to define the physiopathological ∼70% reduction of NAE1 proteins in NAE1CKO hearts at E12.5 significance of neddylation in embryonic development (23), and postnatal day (P) 1, respectively (SI Appendix, Fig. S2 D and synapse formation and maturation (24, 25), adipogenesis (26), E). The residual NAE1 mRNA and protein in the NAE1CKO tumor development (22), and, more recently, cardiac homeo- hearts are likely from noncardiomyocytes in which NAE1 gene stasis (27–30). Neddylation is dysregulated in the failing human was not deleted and/or NAE1 mRNA and protein that persist in heart and in multiple mouse models of cardiac disease (31). the cardiomyocytes at P1. Inactivation of deneddylation in mouse hearts