The role of K63‐linked polyubiquitination in cardiac hypertrophy

Abstract Ubiquitination, also known as ubiquitylation, is a vital post‐translational modification of proteins that play a crucial role in the multiple biological processes including cell growth, proliferation and apoptosis. K63‐linked ubiquitination is one of the vital post‐translational modifications of proteins that are involved in the activation of protein kinases and protein trafficking during cell survival and proliferation. It also contributes to the development of various disorders including cancer, neurodegeneration and cardiac hypertrophy. In this review, we summarize the role of K63‐linked ubiquitination signalling in protein kinase activation and its implications in cardiac hypertrophy. We have also provided our perspectives on therapeutically targeting K63‐linked ubiquitination in downstream effector molecules of growth factor receptors for the treatment of cardiac hypertrophy.


| INTRODUCTION
Ubiquitin (Ub) is a protein with 76-amino acids, which is a fundamental unit of ubiquitylation process ( Figure 1A). Ubiquitination is one of the post-translational modification processes that promote proteostatic processes by covalently attaching Ub to targeted proteins and regulating their activities and levels. In this process, Ub is specifically attached to the lysine (lys; K) residues on target proteins in a precisely timed manner through a cascade enzyme systems composed of ubiquitin-activating enzyme (E1), ubiquitinconjugating enzyme (E2) and ubiquitin ligase (E3). 1 As a first step, Ub is activated by E1 in an ATP-dependent manner and it forms a complex with E1 enzyme through a thioester bond. Then, the activated Ub is transferred to the cysteine residue in the active site of E2. In the final step, E3 ligase is involved in the transfer of Ub from the E2 to a specific lysine residue of the substrate protein 2,3 ( Figure 1B). As for phosphorylation, ubiquitination is a reversible process in which the attached Ub is removed from the target proteins by deubiquitylation enzymes (DUBs; Figure 1B).
There are different types of Ub system-dependent post-translational modifications in proteins that diversely alter the fate of target proteins. 4 The best-studied ubiquitination is k48-linked polyubiquitination, which primarily leads to proteasomal degradation. 5 Apart from this, lysine 6-, 11-, 27-and 29-linked polyubiqui-the post-translational modifications of protein by k63-linked polyubiquitination are implicated in a wide range of cellular functions.
In this review, we have focused on the role of K63-linked ubiquitination in the cardiac hypertrophy. The exploration of the indepth mechanism by which K63-linked ubiquitination regulates cell proliferation, apoptosis and survival could provide a new effective therapeutic strategy for the pathological cardiac hypertrophyassociated heart dysfunction.

| UBIQUITIN MODIFICATIONS
Ub is evolutionarily conserved across different species that specifically attaches to the lysine residues of targeted proteins through the sequential action of E1, E2 and E3 enzymes. The human genome encodes 2 E1 enzymes, about 50 E2 enzymes and more than 600 E3 ligating enzymes. 11 The ubiquitination modification occurs with spatial, temporal and substrate specificity. The E2-conjugating system determines the type of ubiquitination modification on targeted proteins, and the substrate specificity is determined by E3 enzyme. Ub contains 7 internal lysine residues (K6, K11, K27, K29, K33, K48 and K63), which is utilized for the formation of different type of polyubiquitin chains linkage ( Figure 1C). The vast majority of E2-conjugating enzymes trigger K48-linked ubiquitination, which is a typical signal for the proteasomal degradation of substrate proteins. 2 However, the conjugation of Ub with target substrates is not limited to the Ub-proteasome pathway. The K63-linked Ub chain presumably serves as a platform for various signalling pathways, and it plays a F I G U R E 1 Ubiquitin and Ubiquitin modifications. A, Ubiquitin is a protein with 76 aa residues, which is highly conserved across species. It possesses 7 internal lysine residues (K6, K11, K27, K29, K33, K48 and K63) in the ubiquitin, which have been identified to be utilized for the formation of ubiquitination chains. B, The schematic representation of the ubiquitination cascade. The ubiquitin is covalently coupled with ubiquitinactivating (E1) and then transferred to ubiquitin-conjugating enzyme (E2), Finally, the ubiquitin ligase (E3) specifically catalyses the ubiquitination of target protein. And DUBs specifically remove ubiquitin chains from their protein substrates. C, The schematic representation of the different types of ubiquitin chains and ubiquitin signals. The question mark indicates that the roles of ubiquitin chains are largely unclear major role in the development of cancer as well as cardiac hypertrophy.
Ub modifications are classified into 3 types (monoubiquitination, multiubiquitination or polyubiquitination) according to the length and architecture of Ub chains formed in substrates. 12 Monoubiquitination is the process of attachment of a single Ub molecule to 1 Lys residue in the target protein. It serves as a signal for the regulation of endocytosis, lysosomal targeting, meiosis and chromatin remodelling.
Multiubiquitination is a process of the attachment of single Ub molecule to several Lys residues at different positions in the target protein. This type of ubiquitination contributes to the recognition of signals for the ATP-dependent breakdown of substrate proteins by the 26S proteasome pathway. In polyubiquitination, a chain of Ub molecules is attached to a single Lys residue in the target protein, which mainly takes part in the proteasomal degradation, protein trafficking, spindle assembly during cell cycle and DNA repair 3 (Figure 1C). In particular, the misfolded proteins are removed by the Ub-proteasome system to maintain the cellular environment and biological events such as cellular proliferation, apoptosis and survival.
E3 ligases classically fall into 2 categories based on their struc-  13 The proteomic studies have shown that all types of polyubiquitination co-exist in cells. 14,15 Lys48-linked chains are the most abundant linkage type (often > 50% of all linkages) found in cells that are primarily involved in transfer of substrate proteins to the 26S proteasome for degradation. 16 However, K63-linked polyubiquitination, the second most abundant form of ubiquitylation, has various non-proteolytic roles in cells. 8 The K63-linked ubiquitination acts as a molecular scaffold for protein-protein interaction, which is important for the protein kinase signalling activation, receptor endocytosis, protein trafficking and DNA damage repair. Ub-conjugating enzyme 13 is a major E2, which mediates K63-specific ubiquitination with the assistance of UEV1A. The majority of E3 ligases mediate K48-linked ubiquitination of target proteins. However, several E3 ligases such as HectH9, Mdm2, TNF receptor-associated factor 6 (TRAF6; tumour necrosis factor receptor-associated factor 6), cIAP1/ 2 (cellular inhibitor of apoptosis protein 1/2), CHIP, Parkin, UCHL1, TRAF2, ITCH and NEDD4-2 specifically catalyse the K63-linked ubiquitination. [17][18][19] Interestingly, HectH9, Mdm2, RNF8 (ring finger protein 8) and cIAP1/2 catalyse both K63-and K48-linked ubiquitination. For example, HectH9 not only ubiquitinates Myc at K63, a site critically involved in Myc transcriptional activation and the expression of a subset of Myc target genes, but also induces k48linked ubiquitination and proteasomal degradation of MCL-1 and p19 Arf . 20

| DEUBIQUITYLATION E NZYMES
The ubiquitination is a reversible process, and Ub chains are recycled after activation or destruction of target substrate proteins. This process is carried out by DUBs, which are a large group of enzymes with classical isopeptidases activity and more specifically targeting Ub con-

IMPORTANT ROLE IN NF-j B ACTIVATION AND PATHOLOGICAL CARDIAC HYPERTROPHY
Heart failure is one of the leading causes of death worldwide, and cardiac hypertrophy is a major risk factor for the development of heart failure. 43,44 Emerging evidence suggests that K63-linked ubiquitination plays a crucial role in the regulation of pathways such as NF-jB, which is implicated in the development and progression of cardiac hypertrophy. 45 NF-jB is a ubiquitous inducible transcription factor, which can activate expression of groups of genes involved in immune response, inflammation, cell survival, apoptosis or cell growth that depends on the stimuli and extracellular factors. 46 NF-jB-inducing kinase (NIK), a key molecule of non-canonical NF-jB signalling pathway, phosphorylates IjB kinases (IKKs), which consist of 3 subunits namely IKKa kinase, IKKb kinase and IKKc (a regulatory subunit). The increased activity of NF-jB signalling contributes to hypertrophic responses. 47,48 For example, the activation of NF-jB is necessary for the myotrophin-induced cardiac hypertrophy in cardiomyocytes. 49 The cardiac hypertrophic agonists such as ANG II can increase the expression and activity of NF-jB. 50 The cell surface receptors such as TNF receptor(TNFR), IL-1R, Toll-like receptor (TLR) and CD40 act as upstream activators of NF-jB pathways and their activation by ligands leads to the recruitment of E3 ligases such as TRAF2 and TRAF6 to the receptors. 45  In TNF-a pathway, TNFR recruits its adaptor protein TRADD in response to binding of TNF-a. TRADD further recruits TRAF2, TRAF5 and RIP1 to the receptor complex. TRAF2/5-mediated K63 polyubiquitination of RIP1 further recruits TAB2 and TAK1 in the cytoplasm, which activates TAK1 by phosphorylation. The activated RIP1-TAK1-TAB2 complex subsequently promotes activation of MEKKs, which in turn activates the MAP kinase kinases, culminating in MAPK activation (JNK, ERK and p38), and activates p38 and JNK. These terminal kinases then proceed to phosphorylate transcription factors within the nucleus, as well as other regulatory proteins in the nucleus. And also K63 polyubiquitination chains of NEMO increases the activity of IKK complex which leads to IjBa-dependent activation of NF-jB pathway. The TNF-a pathway is regulated by deubiquitinases A20 and CYLD levels of JNK1/2 and p38 in the hypertrophied heart. 63 F I G U R E 3 Ubiquitination regulates activation of AKT kinase. The growth factor IGF-1 triggers K63-linked ubiquitination of AKT and it promotes recruitment of AKT to membrane, which subsequently phosphorylated by PDK1 and mTOR. The activated AKT translocates to the nucleus and catalyses the transcription of genes associated with the heart growth and hypertrophy. E3 ligase TRAF6 contributes to Akt kinase activation by promoting K63-linked polyubiquitination YAN ET AL.

| 4563
A study in hepatic cells found that TAK1 autophosphorylation by the interaction of TRAF3-TAK1 disrupts AKT-GSK3b/FOXO1 signalling. 78 In AKT, K8 and K14 residues within the PH (pleckstrin homology) domain are major sites of ubiquitination. 79 This is evident from the remarkable reduction in ubiquitination level in AKT K8R and AKT K14R mutants, and this mutation blocked the recruitment of AKT to the membrane sites and its phosphorylation. This study also indicated that ubiquitination-mediated AKT membrane recruitment does not result from PIP3 binding. 80 Apart from this, K14 residue within the PIP3-binding domain of AKT is required for its with PIP3, which is evident from the inability of binding of mutant (K14R) AKT with PIP3. 77,81,82 The expression of TRAF2, an E3 ligase, is upregulated in the failing heart and its overexpression enhances cardiac hypertrophy and ventricular dysfunction by activating AKT/GSK3b signalling. 83  98 USP14 also contributes to suppress the development of cardiac hypertrophy by increasing phosphorylation of GSK-3b (Table 2). 99 Together, these findings indicate that the most of the DUBs protect the cardiac structure and function against pathological cardiac modelling caused by various stimulus.

| CONCLUSION S
The