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Ubiquitin, a novel paracrine messenger of cardiac cell survival

Dan Li, Christophe Depre
DOI: http://dx.doi.org/10.1093/cvr/cvq026 1-3 First published online: 25 January 2010

Ubiquitin is a highly conserved and ubiquitously expressed protein that covalently binds other peptides by a lysine (K) residue. Traditionally, the role of ubiquitin is associated with the proteolytic function of the proteasome in what is known as the ubiquitin-proteasome system (UPS), responsible for the degradation of about 70–90% of intracellular proteins.1,2 In the UPS, ubiquitin binds the proteins to be degraded in a process known as ubiquitination and transfers them to the proteasome for proteolysis. Ubiquitination results from the formation of a peptide bond between the C-terminal carboxyl group of ubiquitin and the ε-amino group of a lysine residue of the target protein, and this is followed by the synthesis of a multi-ubiquitin chain in which the ε-amino group of the lysine-48 residue of the conjugated ubiquitin (UbK48) is bound to the C-terminal carboxyl group of the incoming ubiquitin moiety.3 Ubiquitination of a target protein largely depends on the identity of specific members of the large family of E3 ubiquitin ligases, which catalyze the formation of the peptide bond between ubiquitin and the client.4 Poly-ubiquitination of the client protein signals its targeting to the proteasome for degradation by several proteolytic enzymes, which is accompanied by the recycling of the ubiquitin units.5 Proper function of the UPS is critical for cellular homeostasis, and its dysfunction participates in multiple forms of disease, including in the cardiovascular system, as reviewed recently in a Spotlight issue of the Journal.6

Beside this ‘canonical’ function of ubiquitin in the UPS, an emerging literature relates to ‘non-canonical’, proteasome-independent, and non-proteolytic activities of ubiquitin related to cellular functions as diverse as DNA damage and repair, cell signalling and trafficking, and transcriptional control.7 A remarkable characteristic of these non-proteolytic functions of ubiquitin is that they involve the participation of specific E3 ligases conjugating the ubiquitin moiety not on lysine-48 but rather on lysine-63 (UbK63).7 Although the difference between the UbK48 and UbK63 conjugation may seem anecdotic at first glance, the latter process produces a more linear chain of ubiquitin moieties, which favours their interaction with membranes due to a better exposure of their hydrophobic motifs.8

Singh et al.9 describe a novel function for ubiquitin as a paracrine agent to inhibit cardiac apoptosis mediated by β-adrenergic receptors. In this study, the authors observed that stimulation of β1 or β2 adrenergic receptors in isolated cardiac myocytes results in the release of ubiquitin in the extracellular milieu, followed by its reuptake in the myocytes. The authors also show that increased concentration of ubiquitin in the extracellular milieu blunts the pro-apoptotic effects of β-adrenergic receptor stimulation. The mechanism relies on an activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, resulting in the inhibition of GSK-3β and JNK, two signalling mechanisms that promote the activation of the mitochondrial pathway of apoptosis.9 This very interesting observation confirms and further expands mechanistically a previous report in vivo describing an extracellular production of ubiquitin from the ischaemic swine heart.10

This study by Singh et al. offers a new role for ubiquitin in the heart as a paracrine messenger of cell survival, but it also raises important questions. A first issue is whether there is a specific receptor and/or transporter for reuptake of the extracellular ubiquitin produced by the cardiomyocytes. This would seem required, despite the small size (about 8 kDa) of the peptide. Although this possibility was suggested from experiments in silico, 11 it has not yet been demonstrated in biological systems.

Another important issue is whether the fate of this extracellular ubiquitin is directed to specific E3 ligases and therefore to very specific client proteins. The study by Singh et al. does not present evidence that extracellular ubiquitin stimulates the activity of the UPS, and a previous report showed that proteasome-bound extracellular ubiquitin rather promotes apoptosis in hematopoietic cells.12 Therefore, it is legitimate to speculate that extracellular ubiquitin promoting cell survival could specifically be directed to the UbK63 rather than to the UbK48 mechanism of conjugation. Interestingly, there is no specificity in the ubiquitination domain of the client proteins that determines whether the client will go through UbK48 or UbK63 ubiquitination.13 This distinction relies rather on the specificity of the E3 ligases, and it could be dictated by the subcellular distribution of these enzymes.

It is therefore a possibility that ubiquitin reuptake in myocytes is coupled to UbK63-specific E3 ligases that could, for example, activate Akt and its downstream survival pathways. This speculation is further supported by the recent observation that Akt is activated by UbK63 ubiquitination, which is mediated by the specific E3 ligase TRAF6.14 The authors of that study demonstrated that the UbK63 ubiquitination of the pleckstrin homology domain of Akt (isoforms 1 and 2) by TRAF6 recruits Akt to the plasma membrane, which increases its activation by PI3K and results in higher activity of Akt against its substrate GSK-3β.14 This observation is in striking correlation with the report by Singh et al., showing that extracellular ubiquitin promotes cardiac cell survival by activating the PI3K/Akt axis, resulting in GSK-3β inhibition.9 We could therefore hypothesize that the mechanism of extracellular release and cellular reuptake of ubiquitin in cardiac myocytes exposed to cell stress will direct these ubiquitin units to specific UbK63–E3 ligases, thereby activating intracellular cascades sustaining cardiac cell survival (Figure 1). This mechanism would be totally independent from the one regulating cardiac cell growth and protein synthesis through the UbK48 ubiquitination process linked to the proteasome (Figure 1).

Figure 1

Hypothetical mechanism by which extracellular ubiquitin modulates the survival function of Akt. The hypothesis is that the mechanism of extracellular release and cellular reuptake of ubiquitin in cardiac myocytes exposed to cell stress will direct these ubiquitin units to specific UbK63-E3 ligases, thereby activating intracellular cascades sustaining cardiac cell survival by blocking pro-apoptotic effectors and activating anti-apoptotic effectors. This mechanism would be totally independent of the one by which Akt participates in cardiac cell growth through activation of translation that is linked to proteasome-associated proteolysis by the UbK48 ubiquitination process. casp-9, caspase-9; GFR, growth factor receptor; GPCR, G protein-coupled receptor; HSP, heat shock proteins; NOS, nitric oxide synthase; PDK1, phosphatidylinositol-dependent kinase 1; PI3K, phosphatidylinositol 3-kinase; u, ubiquitin.

Although it remains speculative at this point, this possibility would open a whole new spectrum of non-proteolytic functions for ubiquitin, and it could explain how the fate of ubiquitin is shared between the mechanisms promoting cardiac cell growth and those promoting cardiac cell survival (Figure 1). As always, it is likely that the reality is much more complicated. For example, Singh et al. hint at the possibility that a mono-ubiquitination process, rather than the formation of a poly-ubiquitin chain, is sufficient to mediate the protective effects,9 although this remains to be confirmed by more direct methods. Recent data indeed demonstrate that ubiquitin displays different biological functions depending on whether it goes through poly-, mono- or multi-ubiquitination processes.7 Even more challenging, it has been shown that specific E3 ligases can target cysteine rather than lysine residues,15 which would signal the end of the ‘UbK dogma’.

As far as the heart is concerned, more studies will be needed to determine the specific impact of ubiquitin on cardiac cell survival in different conditions of ischaemic challenge. For example, the paracrine release/reuptake of ubiquitin could signal a role for this peptide in the mechanisms of remote preconditioning. Also, it would be interesting to determine whether administration of ubiquitin prior to ischaemia could protect the heart at risk of irreversible damage by triggering a pre-emptive conditioning of the myocardium.16 For all these reasons, the small but resilient ubiquitin still has a bright future in sight.

Conflict of interest: none declared.

Funding

This study was supported by National Institutes of Health grants HL 072863 and HL 093415, and American Heart Association grant 0230017N (C.D.).

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