Ubiquitin and ubiquitin-like proteins in cardiac disease and protection

Abstract

Post-translational modification represents an important mechanism to regulate protein function in cardiac cells. Ubiquitin (Ub) and ubiquitin-like proteins (UBLs) are a family of protein modifiers that share a certain extent of sequence and structure similarity. Conjugation of Ub or UBLs to target proteins is dynamically regulated by a set of UBL-specific enzymes and modulates the physical and physiological properties of protein substrates. Ub and UBLs control a strikingly wide spectrum of cellular processes and not surprisingly are involved in the development of multiple human diseases including cardiac diseases. Further identification of novel UBL targets will expand our understanding of the functional diversity of UBL pathways in physiology and pathology. Here we review recent findings on the mechanisms, proteome and functions of a subset of UBLs and highlight their potential impacts on the development and progression of various forms of cardiac diseases.

1. INTRODUCTION

Protein post-translational modification by small proteins such as ubiquitin (Ub) or ubiquitin-like proteins (UBLs) is an important mechanism regulating the functions of eukaryotic proteins. Among all these small proteins, Ub is the best known protein modifier and contains 76 amino acids. Ub was first discovered in the mid-1970s and is highly conserved from yeast to mammals. Since then, a dozen of UBLs have been identified, including small ubiquitin-related modifier (SUMO) family members, neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8), interferon-stimulated gene 15 (ISG15), HLA-F-adjacent transcript 10 (FAT10), ubiquitin-fold modifier 1 (Ufm1), autophagy-related proteins 8 (ATG8) and 12 (ATG12), ubiquitin-related modifier (Urm1) and others [1]. These UBLs have varying degree of sequence homology to Ub but all share a characteristic tertiary structure with Ub.

Ub and UBLs are linked to protein substrates in a similar way (Figure 1). In general, these protein modifiers are covalently attached to target proteins by forming an isopeptide bond between their C-terminal glycine residue and the ε-amino group of a lysine residue in the substrate. This process is carried out by E1-E2-E3 multienzyme cascades [1, 2]. First, the activating enzyme E1 activates one of the UBLs in an ATP-dependent manner to form an E1~UBL thioester bond. The UBL-loaded E1 is then engaged with E2 conjugating enzyme and the UBL is relayed to the E2. A third enzyme E3 ligase is often required for the final transfer: the E3 either accepts the activated UBL and transfers the UBL to the substrates, or simultaneously binds to UBL-loaded E2 and the substrates and then facilitates the transfer of UBL from E2 to the substrate. The specificity of UBL conjugation to target proteins is mostly conferred by the E3 ligase. The conjugated UBL can be cleaved off the substrates by UBL specific proteases. Therefore, the conjugation of UBL to target proteins is dynamically regulated. As exemplified in Figure 1, each UBL has its own specific set of E1-E2-E3 and de-conjugating enzymes, which have little effect on other UBLs.

Figure 1. A list of the ubiquitin (Ub) and ubiquitin-like (UBL protein) pathways.

Ub and UBLs (SUMO, NEDD8, ISG15, FAT10 and Ufm1) are covalently conjugated to protein substrates. Three types of enzymes – E1, E2 and E3 – mediate the conjugation reactions. The conjugation process can be reversed by de-conjugation enzymes. Distinct sets of conjugation and deconjugation enzymes for individual protein modifiers and their substrates are depicted.

Although ubiquitination often leads to protein degradation, protein modification by UBLs (except FAT10) does not signal the modified substrates for degradation. Like other small chemical modifications such as phosphorylation, acetylation and methylation, UBLs modifications influence the target’s stability, conformation, subcellular localization and binding affinity to proteins or DNA and consequently alter its physical and physiological properties. By modulating the functions of the protein targets, Ub and UBLs control a wide array of cellular processes such as cell proliferation and differentiation, transcription, signaling transduction, proteolysis, protein synthesis, autophagy and antiviral response [2, 3]. Therefore, it is not surprising that alterations in the Ub and UBL pathways are implicated in human diseases, including different types of cancer, cardiovascular disorders, viral defense and neurodegenerative diseases [1, 4]. In this review, we focus on a selection of UBLs that have been linked to common cardiac diseases such as cardiac hypertrophy, ischemic heart disease, myocarditis and heart failure. We first summarize recent advances in understanding the biology of UBLs and then highlight the impact of these UBLs in cardiac pathogenesis and the potential for therapeutic intervention.

2. UBIQUITIN

2.1 Complexity of Ubiquitination

Ubiquitination is one of the most studied protein modifications and is highly complex due to the involvement of numerous catalytic enzymes, the variable length of the Ub chains and the diversity of the Ub chain linkages [5]. The enzymes involved in ubiquitination are hierarchical: one E1 Ub activating enzyme activates the Ub for all the E2s; dozens of E2 Ub conjugating enzymes are able to accept activated Ub; and hundreds of E3s finally catalyze the attachment of Ub to thousands of protein substrates (Figure 2). The specificity of ubiquitination is mainly conferred by E3 Ub ligases. One E3 ligase can mediate the ubiquitination of multiple substrates and one substrate can be targeted by multiple E3s. Hypothetically pairing with individual E3s, hundreds of deubiquitinases (DUBs) have been identified in eukaryotic cells. DUBs remove Ub off the modified proteins with a high degree of substrate specificity, conferring another layer of regulation on ubiquitination. The length of the Ub chain can vary and one or multiple rounds of ubiquitination lead to the attachment of single, multiple or a chain of Ub moieties to target proteins (i.e., mono-, multiple mono-, poly-ubiquitination). Furthermore, the Ub chain can be extended on one of the 7 lysines (K6, K11, K27, K29, K33, K48, and K63) or the first methionine of the Ub, yielding different linkages of Ub chain. The complexity of Ub chain extension is further increased by the presence of mixed linkages on the same polyubiquitin chain, yet little is known about the biological function of such mixed linkage modification. The length and linkages of the Ub chain dictate the fate and destiny of the modified proteins. It is widely accepted that non-K63-linked Ub chains, especially K48-linked Ub chain, lead to the degradation of the modified substrates. In contrast, K63-linked ubiquitination or mono-ubiquitination acts to alter the localization or activity of the target protein, generally through recruitment of ubiquitin-binding proteins [5].

Figure 2. Overview of the ubiquitin proteasome system-mediated proteolysis.

E1 activating enzyme activates Ub by forming a thioester bond (red) in an ATP-dependent manner. E2 conjugating enzymes accept the activated Ub through transthiolation from E1. E3 ligases then fuse the activated Ub with the lysine residue (K) of the substrates by forming an isopeptide bond. Multiple cycles of conjugation process lead to the attachment of a chain of Ub to the substrates. Polyubiquitinated proteins are recognized and degraded by the 26S proteasome, which consists of a 20S core particle and two 19S regulatory particles at each end of the 20S. Deubiquitinases (DUBs) remove Ub from the ubiquitinated proteins and prevent their degradation by the proteasome.

Polyubiquitination is a major signal for intracellular protein degradation. The degradation of soluble polyubiquitinated proteins is primarily carried out by the 26S proteasome (Figure 2). The 26S proteasome is a multisubunit protein complex that consists of a 20S core particle and one or two 19S regulatory particles capped on either end of the 20S. The 19S proteasome recognizes the ubiquitinated protein and removes the Ub chain form the substrate by one of its subunit, which contains intrinsic deubiquitination activity. The 19S particle then unfolds and channels the substrate polypeptide into the chamber of the 20S, whereby the polypeptide is cleaved into small pieces by the 20S subunits containing endopeptidase activity [5]. Ubiquitination also confers selectivity to autophagy, a lysosomal protein degradation machinery [6]. Ubiquitinated protein aggregates, damaged or surplus organelles and invaded pathogens, which are too big to be digested by the proteasome, are recognized by the ubiquitin receptor proteins (such as p62 and NBR1) and delivered to autophagosome. The autophagosome is subsequently fused with lysosome and the ubiquitinated contents are finally hydrolyzed by the lysosomal enzymes. Although the K63-Ub chain can target substrates for degradation via autophagy [7], it is not completely understood how ubiquitin directs the substrate to one degradation route or the other.

The cellular functions of ubiquitination span a wide spectrum that includes proteolysis, regulation of enzyme activity, assembly of protein complex, intracellular trafficking, receptor internalization and signaling transduction. Given the huge capacity of ubiquitination on regulating protein function, it is not surprising that perturbations on protein ubiquitination are implicated in many forms of cardiac diseases and often contribute to the pathogenic progression.

2.2 Insufficient Ubiquitin-mediated Proteolysis as a Pathogenic Factor Leading to Cardiac Pathogenesis

The ubiquitin-mediated proteolysis is essential to maintain the integrity of cellular proteins that make up the sarcomere, mitochondria, endoplasmic reticulum (ER) and membrane and therefore is critical to cardiomyocyte survival and functioning. Many studies have consistently shown high total levels of ubiquitinated proteins and ubiquitin-positive protein aggregates in human failing hearts with various etiologies [8–10], indicating the insufficiency of Ub-mediated proteolysis. Cardiomyocytes in the hearts undergoing pathological remodeling are constantly facing elevated metabolic, oxidative and mechanical stresses, which excessively produces misfolded and damaged proteins. Meanwhile, cardiomyocyte remodeling often requires upregulation of protein synthesis, which is presumably accompanied by increased production of misfolded proteins due to the error-prone nature of protein folding. Therefore, the accumulation of ubiquitinated proteins in failing hearts likely reflects the imbalance of increased ubiquitination demand and relatively inadequate proteolysis capacity. Indeed, in heart failure patients, components of the Ub pathway (E1, E2 and E3s) were upregulated [9] and the proteasome activities were either unchanged, decreased or increased in the heart [8, 10, 11]. Cardiac proteasome is highly heterogeneous as it consists of various subpopulations and is subjected to diverse post-translational modifications [12, 13]. The alterations in proteasome assembly and post-translation modification may account for the changes of proteasome activities and contribute significantly to cardiac disease [14, 15]. The inconsistent observations on cardiac proteasome activities could be due to the complexities of disease progression, differences in polypharmacy and individual patient etiologies as well as methodological differences used in various studies.

The relationship of insufficient Ub-mediated proteolysis to the development of various forms of cardiac injury is being experimentally strengthened by many studies from animal models and clinical reports (Figure 3). For example, the interference with individual steps of the ubiquitin-proteasome system (UPS) often causes or exacerbates cardiac dysfunction in response to various cardiac insults. Mutations on heat shock protein alpha Crystallin B (CryAB) and myofibril protein desmin, i.e., CryAB^R120G and D7-Des, cause protein misfolding and protein aggregation and are implicated in human desmin-related cardiomyopathy. Expression of these misfolded proteins in mouse hearts caused heart failure and the severity of the phenotypes correlated with the abundance of the misfolded proteins [16–18], demonstrating a causative role for proteotoxicity in heart failure. Bortezomib is a potent proteasome inhibitor that has been approved by FAD to treat multiple myeloma. Administration of Bortezomib exacerbated maladaptive cardiac remodeling and increased mortality in mice subject to pressure overload [19]. Similarly, administration of the ubiquitous proteasome inhibitor MLN-273 in pigs also caused cardiac structural and functional abnormalities [20]. Furthermore, blunting proteasome peptidase activity by cardiomyocyte-restricted overexpression of a dominant-negative mutant of proteasome subunit PSMB5 worsened ischemia reperfusion (I/R)-induced cardiac injury [21]. Although Bortezomib is a very effective treatment to multiple myeloma, it has substantial cardiotoxicity [22–24]. In addition, a growing body of evidence suggests a pathogenic role for insufficient autophagy/lysosome function in the heart, which has been recently summarized elsewhere [25, 26].

Figure 3. Role of insufficient ubiquitin-proteasome system (UPS) function in the development of cardiac diseases.

Expression of misfolded proteins, genetic ablations of Ub ligases and impairment of proteasome function cause cardiomyopathies or sensitize the heart to cardiac stresses, whereas enhancement of proteasome function shows cardioprotection to proteinopathy and I/R injury. The notion is in agreement with clinic reports of cardiotoxicity in cancer patients receiving proteasome inhibitors. See the main text for more details. KO, knockout. mPSMB5, dominant-negative PSMB5 mutant. PA28αOE, PA28α overexpression. PKG, protein kinase G.

Several pathways have been identified to be involved in the cardiac pathogenesis associated with insufficient protein quality control. The degradation of the pro-death kinase PKCδ is mediated by the UPS [27]. Ischemic preconditioning improved post-ischemic UPS function, reduced PKCδ protein levels and increased the ratio of PKCε (pro-survival) to PKCδ, which may contribute to ischemic preconditioning-conferred cardioprotection [27]. In line with this notion, exacerbation of myocardial ischemia-reperfusion (I/R) injury by mild proteasome inhibition was associated with suppression of Akt activation, elevated PTEN and PKCδ expression, and diminished PKCε proteins [21]. Both proteasome inhibition and deficiency of chaperone CryAB have been shown to activate the calcineurin-NFAT pathway, which may contribute to the deterioration of cardiac remodeling in mice subject to pressure overload [19, 28].

2.3 Ubiquitin Ligases and Cardiomyopathy

Besides the global turnover of ubiquitinated proteins, selective degradation of specific regulatory proteins also impacts cardiac pathogenesis. It is generally believed that a protein is readily degraded by the proteasomes once it is ubiquitinated so the rate-limiting step in protein degradation is Ub ligase-mediated ubiquitination. Among hundreds of ubiquitin ligases that have been identified, a number of them have been studied in the heart and the roles of many others have yet to be revealed. By targeting to different protein substrates, the same Ub ligase could have confounding effects on physiological and pathological cardiac remodeling under different disease settings. Here we discuss two of the most extensively studied muscle-specific Ub ligases, Atrogin-1 and MuRF1, as examples to reveal the capacity of Ub ligases in maintaining cardiac homeostasis. For further discussion of other Ub ligases, readers are referred to recent comprehensive reviews [29, 30].

Atrogin-1 is a muscle and heart-specific Ub ligase that controls cardiac growth in response to numerous physiological and pathological stimuli. For example, Atrogin-1 appears to be a suppressor of pathological hypertrophy by inhibiting the calcineurin-NFAT pathway. Overexpression of Atrogin-1 promotes the ubiquitination and degradation of calcineurin [31], and suppresses pressure overload-induced pathological hypertrophy [31]. Atrogin-1 also plays an important role in physiological cardiac hypertrophy through regulating Akt signaling. In support of this, overexpression of Atrogin-1 blunted insulin growth factor 1 (IGF-1)- and growth hormone (GH)-induced hypertrophy, while loss of Atrogin-1 exaggerated exercise-induced hypertrophy [32]. Mechanistically, Atrogin-1 catalyzes K63-linked ubiquitination of FOXO proteins, which prevents Akt-mediated phosphorylation and suppression of FOXO transactivation, leading to suppression of hypertrophy [32]. Atrogin-1 is also involved in cardiac atrophy. Mechanical unloading by heterotopic transplantation resulted in cardiac atrophy in wild-type mouse hearts but induced cardiac hypertrophy in those with Atrogin-1 deficiency [33]. Compared to the transplanted wild-type hearts, transplanted Atrogin-1 deficient hearts displayed similar extents of protein degradation, but greater extents of calcineurin-NFAT signaling and protein synthesis, suggesting a potential role of Atrogin-1 in repressing protein synthesis [33]. Consistent with its protective role in the heart, Atrogin-1 deficient mice developed age-related cardiomyopathy due to impaired autophagy function [34]. Further analysis identified the endosomal sorting complex (ESCRT) family protein CHMP2B as the direct target of Atrogin-1. Silencing CHMP2B in Atrogin-1-deficient mice attenuated the impairment of autophagy and ameliorated the pathological cardiac phenotypes [34]. In contradiction, the Sadoshima group reported that loss of Atrogin-1 in the heart attenuated pressure overload-induced cardiac dysfunction [35]. This was accompanied by the stabilization of IκBα and inactivation of NFκB [35]. Although it is yet unclear how to reconcile the discrepancy observed in these studies, it could be that the more protracted responses occurring in ageing are different from the acute changes that occur following pressure overload. Further study will be required to solve these issues.

MuRF1 is a sarcomere-associated Ub ligase that is only expressed in cardiac and skeletal muscle [36]. Several myofibril or non-myofibril proteins have been identified as its substrates, including troponin I, β-MHC, MHCIIa, phospho-c-Jun and calcineurin A [36–39]. Similar to Atrogin-1, MuRF1 also plays an important role in cardiac growth in response to various cardiac insults. Patients receiving left ventricle assist devices (LVAD) exhibited cardiac atrophy and upregulation of cardiac MuRF1 expression [40]. In two different cardiac atrophy models (dexamethasone-induced atrophy and cardiac hypertrophy regression after release of transverse aortic constriction), MuRF1 null mice displayed less cardiac atrophy compared to wild-type mice [40], demonstrating the essential role of MuRF1 in cardiac atrophy. MuRF1 expression was reduced in pressure overload-induced hypertrophic mouse hearts. MuRF1 repressed agonist-induced cardiac hypertrophy in vitro and pressure overload-induced cardiac hypertrophy in vivo [39, 41, 42]. MuRF1-mediated ubiquitination and subsequent degradation of calcineurin A may account for the inhibitory effect of MuRF1 on pathological hypertrophy [39]. Indeed, mutations of the gene encoding MuRF1 (TRIM63) were identified in patients with hypertrophic cardiomyopathy [43]. Expression of these mutants in mouse hearts sufficed to cause cardiac hypertrophy with concomitant activation of mTOR and calcineurin signaling [43]. It seems that Atrogin-1 and MuRF1 are not functionally redundant in mediating the degradation of calcineurin, because loss of either of them is sufficient to exaggerate cardiac hypertrophy. It will be interesting to determine whether these two Ub ligases have synergistic effect in suppressing hypertrophy. Using a gain-of-function approach, it was found that MuRF1 overexpression altered cardiac metabolism and led to increased susceptibility to heart failure after pressure overload [44], suggesting a potential role of MuRF1 in regulation of cardiac energetics. It was speculated that MuRF1 had no effect in IGF-1-induced physiological hypertrophy based on an in vitro study [41]. However, a recent study showed that MuRF1 deficient mice were more susceptible, and MuRF1 overexpressing transgenic mice were more resistant, to exercise-induced cardiac growth. Attenuation of IGF-1/JNK signaling by MuRF1 was proposed as the underlying mechanisms [45]. Therefore, MuRF1 controls both physiological and pathological cardiac hypertrophy via distinct mechanisms.

2.4 Therapeutic Potentials of Targeting the UPS to Treat Cardiac Diseases

Given that proteotoxicity has been established as a contributing pathogenic factor in cardiomyopathies [25], it is imperative to search for measures to enhance the UPS proteolytic function through targeting the individual steps in this pathway. The 11S proteasome is a proteasome activator that binds to one or both ends of the 20S proteasome [46]. The 11S proteasome is assembled by PA28α and PA28β (α3β4 or α4β3) or consists of 7 PA28γ subunits (7γ). Overexpression of PA28α in cardiomyocytes stabilized PA28β, increased the abundance of 11S proteasome and enhanced the degradation of a proteasome surrogate substrate [47]. Moreover, transgenic overexpression of PA28α in mouse hearts enhanced the degradation of misfolded proteins and protected against proteotoxicity-induced cardiac dysfunction and heart failure [48]. The PA28α transgenic mice also displayed ameliorated myocardial damage in response to I/R injury [48]. These lines of evidence indicate, for the first time, that enhancement of proteasome function could be a potential strategy to treat cardiac proteinopathy and ischemia heart disease.

Another example is a recent study focusing on PKG signaling, which is known to modulate cardiac contractility, hypertrophy and remodeling, and provide cardioprotection [49]. PKG signaling has been found to alter the UPS proteolytic function [50]. Activation of PKG by pharmacological and genetic approaches stimulated the degradation of a misfolded protein and reduced cell injury in cultured cardiomyocytes, while inhibition of PKG resulted in the opposite response [50]. Furthermore, in mice with desmin-related cardiomyopathy, activation of PKG by sildenafil treatment reduced myocardial accumulation of misfolded and ubiquitinated proteins, and slowed down the disease progression [50]. The enhancement of UPS function by PKG signaling was linked to phosphorylation of proteasome subunits [50]. Since activation of protein kinase G (PKG) has been demonstrated to suppress pressure overload-induced hypertrophy in mice [51], its enhancement of proteasome function may therefore be a previously unappreciated mechanism.

Furthermore, recent studies reveal additional approaches to enhance proteasomal function. Rpn6 (PSMD11) is a 19S proteasome subunit crucial for the assembly of the 26S proteasome. Overexpression of Rpn6 elevated proteasome activity, accelerated the removal of damaged proteins and led to increased longevity in Caenorhabditis elegans [52]. Proteasome-associated DUBs such as USP14 antagonize proteasomal degradation by removing the Ub chain before the ubiquitinated substrates are recognized by the proteasome [53]. A compound (IU1) has been identified to specifically inhibit USP14. IU1 enhanced the degradation of several misfolded proteins implicated in neurodegeneration diseases and reduced proteotoxic stress-induced cell injury in HEK293 cells [53]. Additionally, a negative regulator of neddylation NUB1L is shown to promote the degradation of mutant Huntingtin and attenuate the neurodegeneration in Drosophila, likely through bridging the interaction of Ub ligase with the mutant protein and therefore facilitating the ubiquitination [54]. It will be intriguing to further determine whether these approaches are applicable to benefit diseased hearts with proteotoxic stress.

Arguing against the detrimental roles of proteasome inhibitors in the heart, results from several pertinent studies have suggested the potential of proteasome inhibitors in treating different cardiac disorders. Systematic administration of different proteasome inhibitors suppressed pressure overload-induced cardiac hypertrophy and attenuated cardiac remodeling [55–58], which was partially ascribed to the accumulation of IκBα and hence the inactivation of NFκB signaling. Similarly, administration of proteasome inhibitors limited myocardial damage following I/R [59, 60]. Activation of NFκB was again proposed to be a contributory factor.

It remains unclear how to reconcile the conflicting effects of proteasome interventions in the diseased hearts. Differences in etiologies and severity of the diseases, variable drug potency, duration and specificity of proteasome inhibition, and the action of the inhibitors on non-cardiomyocytes may all influence whether proteasome interventions benefit or damage the sick hearts. It is also possible that enhancement or inhibition of proteasome function may require crucial temporal windows, at different stages of disease or pathological event, to attenuate cardiac injury. Finally, it seems likely that an appropriate balance of proteasome activation and inhibition will ultimately be the most beneficial as a therapeutic strategy and different drugs or approaches may be needed for individual patients. Considerable experimentation in this area will be necessary to reveal the complexity of this balance and how to best achieve it.

3. SUMOs

3.1 Overview

SUMO polypeptides have only about 20% sequence homology to Ub but display the characteristic β-grasp ubiquitin fold structure [61, 62]. The SUMO family contains four members, SUMO1, 2, 3 and 4, of which SUMO2 and SUMO3 are nearly identical (97% identity) and share only 50% identity with SUMO1. SUMO1 and SUMO2/3 modify overlapping sets of substrate proteins but differ in their abundance and properties [61, 62]. In mammalian cells, most SUMO1 proteins are conjugated to the substrates, leaving a low abundance of free SUMO1. In contrast, SUMO2/3 has a large pool of free forms. Moreover, SUMO1 prefers to modify the substrates by a single molecule (mono-sumoylation), while SUMO2/3 are conjugated to target proteins in chain. SUMO4 has ~87% similarity to SUMO2, yet it is still unclear whether this isoform is expressed, processed and conjugated in eukaryotic cells. Conjugation of SUMOs to target proteins is governed by a highly conserved cascade of enzymes, including the heterodimeric SUMO E1 (SAE1/SAE2), SUMO E2 (UBC9) and a set of SUMO E3s (PIAS, RanBP2, polycomb 2, TOPORS etc.). Unlike ubiquitination, there is a consensus sequence (ψKxD/E) for classical sumoylation sites, where ψ is a hydrophobic amino acid (isoleucine, leucine or valine) and × can be any residue. The conjugated SUMOs can be removed by de-sumoylation enzymes. In human, 6 de-sumoylation enzymes (i.e., SENP1, 2, 3, 5, 6, and 7) have been identified. These enzymes appear to have a certain degree of specificity in removing SUMOs from their targets: SENP1 and SENP2 generally target all SUMO conjugates for de-conjugation; SENP5 potently removes SUMO1 from the conjugates; whereas the rest SUMO isopeptidases preferentially deconjugate SUMO2/3 from the substrates. Sumoylation and de-sumoylation have been shown to control wide arrays of cellular activities including cell cycle, DNA repair, transcription and chromosome remodeling [61, 62].

3.2 SUMOs and Cardiac Development

The SUMO pathway is essential to embryonic development in mice. Genetic ablation of SUMO E2 Ubc9, which abolishes all protein sumoylation, resulted in embryonic lethality of mice [63]. Similarly, knockout of either SENP1 or SENP2 both caused lethality at the embryonic stage [64, 65]. Interestingly, germline deletions of SUMO-1 gene have led to conflicting results [66–68]. Different targeting strategies employed in these studies resulted in embryonic lethality of mice in one study but not in two other studies [66–68]. A possible explanation for this discrepancy may involve compensatory upregulation of SUMO2/3 in SUMO1 deficient mice. Therefore, the appropriate balance of sumoylation and de-sumoylation is crucial to murine embryogenesis.

It has been long known that many proteins involved in nuclear organization and function are modified by SUMOs [69]. The developmental abnormality in the above knockout mice is at least in part attributable to dysregulated sumoylation of transcription factors. Specifically, several important cardiac transcription factors such as SRF, GATA1, GATA2, GATA4 and Nkx2–5 are found to be SUMO targets and their transcriptional activities are altered by sumoylation [70–72]. For instance, SRF is essential for the emergence of cardiac sarcomere formation. SRF is sumoylated at its lysine 147 [70]. Sumoylation of SRF is needed for the expression of some of the SRF targets such as acta2, Tnnc2 and actc1, but represses c-fos promoter activities [73], indicating that the effect of sumoylation on the expression of SRF targets could be promoter-dependent. Other SUMO targets include Nkx2–5 and GATA4, both of which are required for normal cardiogenesis. Sumoylation of Nkx2–5 at lysine 51 and other sites modulates its transcriptional activity [74, 75]. Overexpression of a sumoylation-deficient mutant in the setting of Nkx2–5 haploinsufficiency led to congenital heart diseases ⁹. In cardiomyocytes, 20% of total GATA4 is modified by SUMO1 at lysine 366. Sumoylation of GATA4 induces the expression of cardiac-specific genes such as αMHC and ANF [71]. Since these transcriptional factors are critical to cardiogenesis, alterations in their sumoylation status are presumably attributable to the developmental defects in the above described knockout mouse hearts.

3.3 SUMO1 and Heart Failure

SERCA2a is a critical ATPase in cardiomyocytes that regulates Ca²⁺ re-uptake during the diastolic phase of excitation-contraction coupling. Dysregulation of sumoylation of SERCA2a has been closely linked to the pathogenesis of heart failure [76, 77]. SERCA2a is modified by SUMO1 at its lysines 480 and 585. Sumoylation of SERCA2a increases its stability and thus its ATPase activity. Both SUMO1 and SERCA2a proteins were reduced in the failing hearts from human, mouse and swine [76]. Replenishment of SUMO1 by adeno-associated-virus (AAV)-mediated gene delivery remarkably restored SERCA2a proteins and preserved cardiac function in mice with pressure overload-induced heart failure [76]. These promising results prompted further investigation in a porcine myocardial I/R model [77]. SUMO1 gene delivery significantly improved cardiac contractility and dilatation following I/R despite no significant improvement of ejection fraction (EF) [77], indicating the potential benefits of SUMO1 gene delivery to ischemic heart disease. Conversely, downregulation of either SUMO1 or SERCA2a deteriorated pressure overload-induced cardiac dysfunction in mice [71]. The beneficial effect of SUMO1gene transfer could be in part due to attenuation of oxidative stress [78]. Based on these findings, it is postulated that targeting sumoylation of SERCA2a could be a novel therapeutic strategies for heart failure.

Additionally, a recent study shows the crosstalk between histone acetylation and sumoylation [79]. Inhibition of histone deacetylase (HDAC) II, but not other HDAC isoforms, specifically induced SUMO1 conjugation without altering SUMO2/3 conjugation. HDAC inhibition-conferred cardioprotection could be partially ascribed to stimulation of SUMO1 modification in cardiomyocytes and cardiac fibroblasts [79].

3.4 De-sumoylation and Cardioprotection

Emerging studies on de-sumoylation enzymes reveal the pathophysiological significance of de-sumoylation in the heart. As mentioned above, SENP1 catalyzes de-sumoylation of all sumoylated conjugates. Myocardial SENP1 levels were increased following I/R in mice and in primary cultured cardiomyocytes subjected to simulated I/R. The upregulation of SENP1 is likely an adaptive response to the stress, because SENP1 heterozygous knockout mice had larger infarct areas and greater impairment in systolic function after I/R [80]. HIF1α was downregulated in the stressed SENP1 heterozygous knockout mouse hearts, compared to the stressed wild-type controls. Moreover, overexpression of HIF1α protected SENP1 silencing-induced cell death in cultured cardiomyocytes [80], suggesting a role of reduced HIF1α in the pathogenesis. Contrary to its protective role in myocardial I/R injury, SENP1 appears to be a pathogenic factor to hypertrophy-induced cardiac dysfunction. Expression of SENP1 was induced in mouse and human failing hearts [81]. In cultured cells, the hypertrophic stimuli isoproterenol also induced SENP1 expression, likely through activation of calcineurin-NFAT pathway [76]. AAV-mediated gene delivery of SENP1 prevented left ventricle chamber dilation and preserved ejection fraction [76]. Mechanically, SENP1 mediates de-sumoylation of transcription factor MEF2C, which subsequently activates PGC-1α transcription, leading to aberrant mitochondrial gene expression. Therefore, a hypertrophy-calcineurin/NFAT-MEF2C-PGC-1α-mitochondrial dysfunction-cardiac dysfunction axis has been proposed.

Under physiological conditions, hyperactivation of de-sumoylation enzymes may not be desirable for cardiomyocytes [82, 83]. Cardiac-specific overexpression of SENP2 caused premature death of mice due to defective atrial and ventricular septums [82]. The incidence of the structural abnormality was significantly attenuated by SUMO1 overexpression. The viable SENP2 transgenic (Tg) mice developed cardiomyopathy with ageing [82]. Similarly, SENP5 is upregulated in failing human hearts [83]. Cardiac-specific overexpression of SENP5 significantly reduced sumoylated proteins including sumoylated Drp1 and caused heart failure in mice [83]. Enlarged mitochondria and massive apoptotic cardiomyocytes were observed in SENP5 Tg mouse hearts. The adverse cardiac phenotype of SENP5 Tg mice was partially rescued by overexpression of anti-apoptotic protein Bcl2 [83]. These observations indicate an indispensable role of balanced sumoylation and de-sumoylation in the maintenance of the integrity of cardiac structure and function. Interestingly, the de-sumoylation enzymes may have distinct set of targets in different cell types. In skeletal muscle, SENP2 positively regulates the transcription of myostatin through de-sumoylation of transcription factor MEF2A [84], indicating the critical role of SUMO in myogenesis.

3.5 SUMOs and Protein Degradation

Although sumoylation of target proteins in general does not lead to their proteasomal degradation, there is a crosstalk between sumoylation and protein degradation. For example, the SUMO E2 UBC9 is found to be upregulated in the hearts of a Tg mouse model of desmin-related cardiomyopathy, yet the sumoylation of cellular proteins is reduced [85]. Overexpression of UBC9 enhanced the degradation of GFPu (GFP fused with degron signal CL1), a UPS function reporter, indicating the enhancement of UPS function. UBC9 expression also reduced the abundance of a bona fide misfolded protein CryAB^R120G in cultured cardiomyocytes. In contrast, silencing of UBC9 accumulated both GFPu and CryAB^R120G in the cells [85]. Consequently, UBC9 expression protected cardiomyocytes from proteotoxic stress-induced injury. Consistent with its role in promoting protein degradation, UBC9 suppressed the aggregation of misfolded proteins through both SUMO-dependent and -independent mechanisms [86, 87]. It remains to be further determined how UBC9 regulates the clearance of misfolded proteins and whether this effect is dependent on its SUMO conjugation activity. It will also be interesting to determine if modulation of UBC9 protects the heart against proteotoxicity.

4. NEDD8

4.1 Neddylation and Deneddylation: beyond Cullin-RING Ligases

Among all the UBLs, NEDD8 shares the highest amino acid homology (60%) with Ub. Originally identified in mouse brain, NEDD8 is ubiquitously expressed in all tested organs and cell types, with the highest expression levels in the heart and skeletal muscle [88]. NEDD8 is first synthesized as a precursor. The maturation of NEDD8 requires the cleavage of its C-terminal sequence by NEDD8 proteases such as NEDP1 to expose the C-terminal di-glycine, which forms an isopeptide bond with the ε-amino group of lysine in the target protein. Conjugation of NEDD8 to target proteins, i.e. neddylation, is mediated by a heterodimeric NEDD8 activating enzyme NAE (consisting of NAE1 and UBA3), a NEDD8 conjugating enzyme UBC12, and largely unknown NEDD8 E3 ligases. Neddylation is dynamically regulated and reversed by deneddylation, a process mediated by a group of deneddylases such as the COP9 signalosome (CSN) and NEDP1. Other ubiquitin hydrolases such as UCH-L1, USP21 and UCH-L3 are also reported to cleave off NEDD8 from modified targets in cultured cells [89]. Like ubiquitination, target proteins can be conjugated by a single NEDD8 or a chain of NEDD8 extending on lysines 11, 22, 48 and 60. Little is known about the functional differences between mono- or poly-neddylation of proteins. Cullin family proteins (cullin 1, 2, 3, 4a, 4b, 5, 7 and 9) are the scaffold proteins of cullin-RING Ub ligases (CRLs) and the best-known NEDD8 targets. Neddylation of cullins leads to a change in their conformation, which allows the simultaneous binding of other components to cullins and hence the assembly of a functional Ub ligase. A dynamic cycling of cullin neddylation and deneddylation is required for the optimal CRLs activity because perturbations of either neddylation or deneddylation of cullins cause accumulation of the substrates of CRLs. Besides cullins, an increasing number of cellular proteins such as p53, Mdm2, parkin, pink, HIF1α have been recently identified as NEDD8 targets [89]. By modulating the cellular functions of a variety of targets, neddylation and deneddylation controls many biological processes such as ubiquitination, cell cycle, apoptosis, hypoxic response, mitochondrial turnover and so on. Interestingly, biochemical and mass spectrometry analysis reveal that NEDD8 incorporates into the existing Ub chain under various stress conditions, leading to the NEDD8-Ub mixed modification of target proteins [90]. Such neddylation does not depend on typical NEDD8 enzymes, but instead requires the ubiquitin activating enzyme UBE1 [90], and is thus termed “atypical neddylation”. It is proposed that NEDD8 may function as a Ub substitute to prevent depletion of the Ub pool under stress conditions. The functional consequence of such atypical neddylation remains unclear.

4.2 Neddylation in Cardiac Development

An intact NEDD8 pathway seems to be critical to embryonic development of most model organisms including the mouse [89]. For instance, genetic deletion of UBA3, a subunit of NEDD8 E1 NAE, led to embryonic lethality of mice at preimplantation stage [91]. Similarly, knockout of NAE1 (also known as APPBP1, the other subunit of NEDD8 E1) or NEDD8 E2 Ubc12 also caused embryonic lethality [92]. The deneddylase CSN consists of 8 subunits from CSN1 through CSN8. Knockout of individual CSN subunits each resulted in embryonic lethality of mice [89]. The developmental abnormalities of these knockout mice are at least in part, due to a defective cell cycle and/or apoptosis. The embryonic lethal phenotype of these knockout mice calls for the necessity of conditional knockout mouse models to study the organ-specific role of neddylation/deneddylation in cardiac development and diseases.

4.3 COP9 Signalosome (CSN), a Deneddylase in the Heart

The importance of neddylation/deneddylation in the heart is well illustrated by the studies on the CSN8 conditional knockout mice [93–95]. CSN8 is the smallest and least conserved subunit of CSN. Conditional knockout of CSN8 in neonatal mouse hearts through αMHC-Cre-mediated recombination impaired CSN deneddylation activity and accumulated neddylated cullin and non-cullin proteins [94], suggesting that many cardiac proteins with unknown identities are NEDD8 targets. The neonatal knockout mice developed cardiac hypertrophy, which quickly progressed to heart failure, leading to premature death of mice at around 4 weeks of age. Initiation of CSN8 knockout in adult mouse hearts through a tamoxifen-inducible Cre-mediated recombination also rapidly caused cardiomyopathy and heart failure [95]. These observations demonstrate the indispensable role of intact CSN function in cardiac function. Mechanistically, loss of CSN8 in cardiomyocytes accumulated a UPS surrogate substrate GFPdgn and ubiquitinated proteins, indicating the impairment of UPS function. However, despite the accumulation of neddylated cullins, CRLs activities did not seem to be affected by CSN8 knockout, because several tested CRL substrates were not accumulated in the knockout hearts. Neither were the proteasomal peptidase activities altered by loss of CSN8. It is speculated that defects in UPS-mediated proteolysis could arise from inefficient delivery of ubiquitinated proteins to the proteasome. Another striking finding from this study is the compromised autophagy function in the CSN8 knockout hearts [93]. Loss of CSN8 accumulated massive autophagosomes in the hearts, which was due to impaired autophagic degradation rather than increased autophagy induction. Further analysis revealed that CSN8 depletion led to defective fusion of autophagosomes with lysosome and that a regulator of autophagosome maturation Rab7 was downregulated in CSN8 knockout hearts. Defective autophagy has been linked to cardiomyocyte death. Indeed, massive necrotic, but not apoptotic cardiomyocyte death was evident in the neonatal and adult CSN8 knockout hearts [94, 95]. It is therefore postulated that CSN is essential to autophagosome maturation by sustaining Rab7 expression in the heart. These data together reveal the critical role of CSN-mediated deneddylation in the regulation of proteasomal and autophagic proteolysis in the heart.

5. ISG15

5.1 ISG15, an Antiviral Protein Modifier

ISG15 has two ubiquitin-like domains, which have 29% and 36% identity to Ub. The expression of ISG15 is rapidly and robustly induced by type I interferons (IFN), including IFNα and IFNβ [96]. Conjugation of ISG15 to target proteins, i.e., “ISGylation”, is mediated by ISG E1 (Ube1L), E2 (UbcH8) and E3 enzymes [97, 98]. Downregulation of HECT-type E3 enzyme Herc5 (Cebl) suppresses total IFN-induced ISGylation, suggesting that Herc5 is a predominant ISG15 ligase [99]. Another two RING ligases, EEP and HHAR1, can also mediate the conjugation of ISG15 to some specific substrates (14-3-3σ and 4EHP) [100, 101]. Deconjugation of ISG15 from modified substrates is mainly catalyzed by the de-ISGylation enzyme UBP43 (USP18) [102], although a number of de-ubiquitin enzymes are also capable to remove ISG15 in vitro. Like ISG15, the ISG15 conjugation and deconjugation enzymes (Ube1L, UbcH8, Herc5 and UBP43) are all induced by type I IFN stimulation. Target proteins are generally modified by ISG15 by mono- or multiple mono-ISGylation, as there is no evidence that ISG15 forms a chain in the manner of polyubiquitination.

Accumulating evidence reveals the important role of ISG15 in mammalian host defense against viral infection. This is demonstrated by the fact that mice deficient in the ISGylation pathway are more susceptible to different types of virus infection. ISG15 deficient mice were more susceptible to a number of viruses such as Sindbis virus, influenza A and B, but not all the viruses tested, and had increased mortality after virus infection [103]. Consistently, mice with deficiency of the ISG15 E1 enzyme Ube1L also displayed increased susceptibility and increased mortality to Sindbis virus and influenza B virus [104, 105]. Interestingly, Ube1L-deficient mice are shown to have similar susceptibility to Chikungunya virus (CHIKV) as wild-type mice, while ISG15 knockout mice are more susceptible to this virus [106]. These observations suggest that the antiviral effects of ISG15 can be both ISG15 conjugation-dependent and –independent. Mechanistically, ISG15 and ISGylation defense against viral infection by targeting to both viral and host proteins. ISG15 modifies viral proteins such as HIV-1 gag and impairs viral replication or infectivity [107]. Therefore, the components of the ISG15 pathway are often the targets for viral immune evasion. Meanwhile, ISG15 also modifies host cellular proteins to enhance antiviral immunity [108–110]. For instance, IRF3 is a critical cellular protein in host antiviral responses. Herc5-mediated ISGylation of IRF3 activated the expression of IRF3 target genes and boosted host antiviral responses [108].

5.2 ISG15 and Myocarditis

The role of ISGylation in the heart has just begun to be understood. IκB kinase (IKK) modulates NFκB signaling and is a key regulator of inflammation. Cardiomyocyte-specific overexpression of IKK in mice resulted in infiltration of CD11b(+) cells, fibrosis, reactivation of fetal gene program and atrophy of cardiomyocytes, which eventually led to inflammatory dilated cardiomyopathy and heart failure [111]. IKK-induced cardiomyopathy was accompanied by upregulation of ISG15 and ISGylated proteins, a characteristic molecular feature of viral myocarditis. Indeed, activation of the ISG15 pathway was observed in coxsackievirus B3 (CVB3)-infected mice [111]. This was the first study to link the ISG15 pathway with inflammatory cardiomyopathy and myocarditis. A subsequent study demonstrated the protective role of ISG15 in myocyte damage during myocarditis. CVB3-infected ISG15 deficient mice developed larger foci with cardiomyocyte death and increased infiltration of inflammatory cells, compared to wild-type mice [112]. These pathological changes were in line with marked deterioration of left ventricle function and eventually increased mortality of ISG15 deficient mice post viral infection. Consistently, Ube1L-deficient mice infected with CVB3 displayed more myocyte damage compared to the wild-type mice. In contrast, USP18^C61A/C61A knock-in mice, in which ISGylated proteins were stabilized due to inactivation of ISG15-specific isopeptidase activity, displayed reduced myocyte damage after CVB3 infection [112]. CVB3-protease 2A (2Apro) interferes with host cell translation by cleaving the host protein eIF4G and is identified as one of the ISG15 targets. ISGylation of 2Apro effectively inhibited 2Apro-mediated cleavage of the host protein eIF4G and enhanced cardiomyocytes immunity [112]. These lines of experimental evidence together demonstrate a role of the ISG15 system in battling against invading viral pathogens in the heart. In patients with viral and inflammatory cardiomyopathy, ISG15 were upregulated at both mRNA and protein levels, presumably an adaptive response to viral infection [112]. Therefore, interference with the ISG15 pathway might be a novel therapeutic approach in viral cardiomyopathy.

6. FAT10

6.1 FAT10, an Additional Signal for Proteasomal Degradation?

FAT10 is a ubiquitin-like protein with a molecular weight of 18 kDa [113]. It was originally called “di-ubiquitin” or “ubiquitin D” due to the presence of two ubiquitin-like domains in the molecule, which are 29% and 36% identical to Ub respectively. Unlike other UBLs that often go through maturation before conjugating to the targets, FAT10 is synthesized as a matured form with an accessible di-glycine motif [113]. Conjugation of FAT10 to target proteins is mediated by FAT10 E1 UBA6 and E2 USE1. Currently no FAT10 E3s or de-FAT10ylation enzymes have been identified. Thus, it remains unclear whether FAT10 conjugation requires dedicated E3s and is reversible. Interestingly, FAT10 is the only UBL that signals its modified substrates to the proteasome for degradation. FAT10 itself is also degraded by the proteasome with an even shorter half-life compared to Ub and its degradation is facilitated by the negative regulator of neddylation NUB1L. Although FAT10 has been proposed as a Ub-independent signal for proteasomal degradation [114], it remains to be understood why eukaryotic cells need different signals, and whether and how these signals are distinguished by the proteasome. The identities of FAT10 substrates are largely unknown. A proteomic study was carried out to address this question, from which hundreds of candidates were identified [115]. Further validation of these candidates will no doubt promote an improved understanding of the cellular roles of FAT10.

6.2 Pathophysiological Significance of FAT10

The biological function of FAT10 is largely unknown. Expression of FAT10 is constrained in tissues of the immune system such as thymus, lymph nodes and spleen. However, in response to IFNγ or TNFα stimuli, FAT10 expression is strongly and synergistically induced in the majority of tissues, suggesting a potential role for FAT10 in immune responses [116, 117]. FAT10 is not essential to embryonic development, as FAT10-deficient mice appear normal at baseline. A follow-up study showed that FAT10 deficiency prevented age-related obesity and increased lifespan in mice, which was associated with metabolic reprogramming, enhanced insulin sensitivity and significantly decreased adipose tissues [118]. The FAT10 pathway is implicated in tumor progression [119, 120]. The expression levels of FAT10 correlated with tumor malignancy and overexpression of FAT10 promoted tumor growth and malignancy [121]. The pro-oncogenic effect of FAT10 has been linked to its non-covalent interaction with MAD2, because disruption of this interaction attenuated FAT10-induced tumor formation in vivo [119]. In addition, stabilization and activation of β-catenin signaling by FAT10 was also proposed to contribute to invasion and metastasis of liver tumors¹⁰³. Lastly, the FAT10 deficient mice were more susceptible to LPS-induced death [122], suggesting the involvement of FAT10 in inflammatory responses.

The function of FAT10 in cardiomyocytes has not been extensively studied. FAT10 is expressed in human and mouse hearts and its expression can be induced by ischemia in vitro and in vivo [123]. Overexpression of FAT10 in cultured cardiomyocytes reduced pro-apoptotic proteins p53 and BAX, and increased the anti-apoptotic protein Bcl2. Silencing of FAT10 showed the opposite effects. Consequently, FAT10 overexpression attenuated, while FAT10 silencing exacerbated, simulated I/R-induced apoptosis [123]. These findings suggest that FAT10 or FAT10 signaling may be protective to the cardiomyocytes. Interestingly, it is recently shown that FAT10 covalently modifies and stabilizes p62 [115], a critical adaptor protein that directs ubiquitinated substrates to autophagy machinery for degradation. Additionally, FAT10 colocalized with p62 and autophagy-targeted pathogen in pathogen-invaded host cells [124]. FAT10 deficient mice had higher susceptibility to pathogen invasion than the wild-type mice, suggesting a role for FAT10 in selective autophagy. Since p62 is known to play important roles in defense of proteotoxic stress in cardiomyocytes, regulation of p62 by FAT10 may contribute to the protective effect of FAT10 in cardiomyocytes.

7. Ufm1

7.1 Overview

Ufm1 is a highly conserved UBL protein with a molecular weight of 9.1 kDa [125]. Ufm1 has low sequence identity to Ub but shares the β-grasp fold structure. Like other UBLs, Ufm1 is synthesized as a precursor. Its maturation is mediated by two Ufm1-specific proteases (UfSP1 and UfSP2) and exposes the C-terminal glycine, which then forms ε-amid bond with the lysine residues of the substrate. Conjugation of matured Ufm1 to target proteins requires Ufm1 E1 UBA5, E2 Ufc1 and E3 Ufl1, and is reversed by the de-ufmylation enzymes UfSP1 and UfSP2 [125]. Only a few proteins have been identified as Ufm1 targets, including Ufbp1, Cdk5rap3 and ASC1 [126, 127]. ASC1 is a nuclear receptor coactivator involved in estrogen receptor alpha transactivation in response to 17β-estradiol. Ufmylation of ASC1 promoted the binding of p300, SRC1 and ASC1 to the promoters of estrogen receptor alpha target genes. Blocking ASC1 ufmylation suppressed tumor growth [127]. These findings establish a role for Ufm1 in tumor development by promoting estrogen receptor mediating signaling. The functional consequence of Ufbp1 and Cdk5rap3 ufmylation remains unclear. The Ufm1 pathway appears to be essential for embryonic development. UBA5-deficient mice exhibited severe anemia and impaired development of megakaryocyte and erythroid differentiation, leading to premature death in utero [128]. These observations reveal a role for Ufm1 in the regulation of haematopoiesis.

7.2 Ufm1 and ER stress

Endoplasmic reticulum (ER) stress responses are activated by cardiac pathological insults including ischemia reperfusion, hypertension and hypertrophy, and play either adaptive or maladaptive roles in these pathologies [129]. The Ufm1 pathway has been closely linked to ER stress in multiple cell types including cardiomyocytes. Ufm1 is up-regulated in ischemic hearts [130], in which activation of ER stress is implicated. Components of the Ufm1 pathway are enriched in the cytosolic side of the ER membrane and are induced by ER stress [131]. The induction is dependent on XBP1 [132], one of the downstream effectors of the ER stress. Induction of the Ufm1 pathway seems to be protective to the ER stress, as knockdown of Ufm1 sensitized the cells to ER stress-induced apoptosis [126, 133]. Overexpression of the Ufm1 target Ufbp1 controls ER proliferation and neogenesis [131]. The significance of the Ufm1 pathway in the heart requires further study.

8. Conclusions and Future Perspectives

Genetic inhibition of each UBL conjugation often leads to embryonic lethality of mice, highlighting the non-redundant and indispensable role of each UBL in mammalian development. Given that a large number of cellular proteins can be modified, and that their function can be potentially regulated by Ub and UBLs, it is not surprising that Ub and UBL pathways are virtually involved in nearly every single biological process. Although emerging studies have begun to reveal the roles of UBLs in the heart, Ub/UBLs research is still in its infancy, especially with regard to understanding the biological consequences of UBL conjugation, but also with regard to the control of UBL conjugation itself. Our understanding of the biological function of Ub/UBLs in the heart may be expanded through answering the following critical questions:

1. Does the dysregulation of the UBL pathways contribute to the development and progression of cardiac diseases? Clinical evidence need to be collected to associate any germline or somatic mutations in the UBL pathways with heart failure, an eventual event of most forms of cardiac diseases. The functional status of the UBL pathway in different disease conditions also needs to be assessed. Most importantly, cardiac-specific gain-of-function and loss-of-function animal models need to be created to target components of the UBL pathway in the heart. Characterization of these models will definitively reveal their pathophysiological significance in the heart.

2. What pathways/cellular processes are regulated by UBLs in cardiomyocytes? Gain-of-function and loss-of-function, and pharmacological and genetic approaches can be utilized to target the UBL pathways in primary cultured cardiomyocytes and in intact hearts of animals. Dissecting these interferences on cardiomyocyte survival and function will provide insightful information in this line. This question can be further addressed by identification and characterization of UBL targets in cardiomyocytes in a whole proteome scale (see below).

3. What target proteins are modified by these UBLs and how are the modifications regulating protein functions in cardiomyocytes? It is very challenging to identify the UBL substrates given the low abundance of the modified substrates and the dynamic nature of the modification. Several strategies have been developed to enrich UBL-modified proteins from cell/tissue extracts prior to mass spectrometry analysis: a) expression of epitope-tagged UBL [134]; b) immunoprecipitation with monoclonal UBL antibodies; c) affinity purification with peptides containing UBL-interacting motif; and d) expression of UBL mutant with larger C-terminal overhang, which is distinct from di-glycine motif of Ub/UBL after trypsin digestion [92]. Characterization of UBL-modified proteome regulated by the disease under investigation will help identify new targets for disease prevention and therapeutic intervention.