Cezanne Paints Inflammation by Regulating Ubiquitination

• Cezanne protein, human
• nuclear factor-κB
• TRAF6

Hypoxia–reoxygenation can induce inflammation by activating nuclear factor (NF)-κB. In endothelial cells, this process is critical for the pathogenesis of many chronic inflammatory conditions, such as atherosclerosis and autoimmune disease. Recent publication from Evans’s laboratory shows the critical role of deubiquitinating enzyme Cezanne, regulating its extent of NF-κB activation and expression of inflammatory genes.¹ In particular, they showed that the inhibition of polyubiquitination of TNF receptor associated factor (TRAF) 6 is a specific anti-inflammatory mechanism by Cezanne. In this editorial, we briefly review the TRAF6-mediated NF-κB signaling and other posttranslational modifications that play a key role in modulating endothelial cell inflammation.

Article, see p 1583

NF-κB transcription factor complexes consist of a heterodimer of p65 (RelA) and p50 or p52.² In most nonstimulated cells, p65-containing NF-κB complexes are kept in an inactive cytoplasmic form, bound to one family of inhibitor proteins, the inhibitory κBs (IκBs). Two IκB kinases, IKKα and IKKβ, target phosphorylation of IκB after hypoxia–reoxygenation, cytokine, or ultraviolet stress stimulation. Phosphorylation of IκBs promotes their ubiquitination and degradation by the proteasome, which releases the p65 complex, allowing it to translocate to the nucleus.³ An ubiquitin E2 conjugating enzyme of the ubiquitin-conjugating enzyme (Ubc)4/5 family and the SCF-βTrCP E3 ligase (Skp1-Cul1-F-box ligase containing the F-box beta protein βTrCP) execute ubiquitination of IκB. Once IκB is phosphorylated, p-transducin repeat containing protein 1 (βTrCP1) and βTrCP2 associate with phosphorylated IκB.^4,5 The polyubiquitinated IκB is selectively degraded by the 26S proteasome, and then mature p52 and p65 subunits translocate into the nucleus to increase NF-κB activity.⁴ It is now well established that NF-κB activation is a key event in inflammation-based cardiovascular pathogenesis, including atherosclerotic plaque formation, diabetes mellitus–mediated endothelial inflammation, and ischemia/reperfusion injury.⁶

The regulatory subunit NF-κB essential modulator (NEMO/IKKγ) of the cytoplasmic IκB kinase (IKKα and IKKβ) complex (IKK complex) plays a central role in the upstream events of NF-κB activation.⁷ Although IKKα and IKKβ contain serine/threonine kinase domains, NEMO does not have apparent catalytic domains, but several protein interaction motifs exist. Interestingly, NEMO is also an ubiquitin-binding protein. This suggests that not only the IκB but also the IKK complex is regulated by ubiquitination.⁴ The coiled-coil region of NEMO contains an ubiquitin-binding domain (NEMO ubiquitin binding), and IKK complex activation is significantly inhibited by the mutation of NEMO ubiquitin-binding domain.⁸

Ubiquitin is an 8.5-kDa molecule protein that contains 7 lysines and is frequently ligated to proteins to form polyubiquitin chains. There is some specificity inherent in the sites of ubiquitin binding to another ubiquitin and forming polyubiquitin chains. For example, when proteins bind to lysine-48 (K48)–linked polyubiquitin chains, the protein is targeted to the proteasome for degradation. In contrast, K63-linked polyubiquitin chain (K63-Ub chain) may regulate proteins through a degradation-independent mechanism. For example, specifically IKK complex is activated when it links to K63-Ub chain^4,9–11 (Figure).

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Figure.

Ubiquitin-mediated transforming growth factor-β–activated kinase 1 (TAK1) and IκB kinase (IKK) complex activation and deubiquitination enzymes (DUBs). Hypoxia/reperfusion activates the ubiquitin E3 ligase TRAF6, and tumor necrosis factor (TNF)/TNF receptor (TNFR) activates TRAF2 and 5. TRAF6 increases K63-linked polyubiquitin chains that associate with the TAB2 subunit of the TAK1 kinase and activate TAK1 kinase. The K63-Ub chains also bind nuclear factor (NF)-κB essential modulator (NEMO) to recruit the IKK complex, thereby accelerating the phosphorylation of IKKβ by TAK1. IKK is then activated to phosphorylate inhibitory κBs (IκB) or p65 and activates NF-κB transactivation. TRAF2/5 also increases K63-linked polyubiquitin chains, especially on adaptor protein receptor-interacting protein 1 (RIP1). DUBs, A20 and cylindromatosis tumor suppressor protein (CYLD), inhibit K63-Ub chains on RIP1. In this report, another DUB, Cezanne, is shown to inhibit autopolyubiquitination of TRAF6. However, the pathophysiological role of TRAF6-polyUb is unclear.

A particularly interesting situation is when polyubiquitinating ligase is itself ubiquitinated. For example, under toll-like receptor activation, interleukin-1 receptor-associated kinase (IRAK)1 is phosphorylated by IRAK4 and then associates with and activates the ubiquitin E3 ligase TRAF6. Working together with the ubiquitin E2 complex composed of Ubc13 and ubiquitin-conjugating enzyme E2 variant (Ucv)1A,^11,12 TRAF6 catalyzes the formation of K63-linked polyubiquitin chains on itself and other protein (Figure). TRAF6 promotes the binding of the TGF-beta activated kinase 1 binding protein (TAB)2 (transforming growth factor-β–activated kinase 1 [TAK1] and mitogen-activated protein kinase kinase kinase 7 -binding protein 2) subunit of the TAK1 kinase complex and NEMO via K63 Ub-chains, promoting TAK1 kinase autophosphorylation and activation of TAK1.^11,13 Next, the K63-linked polyubiquitin chains bind NEMO to recruit the IKK complex, thereby accelerating TAK1-mediated IKKβ phosphorylation and activation, which phosphorylates IκB and causes its degradation,¹⁴ thereby activating NF-κB.

It has been reported that Cezanne is a deubiquitinating enzyme that suppresses NF-κB activation in response to tumor necrosis factor-α or interleukin-1 by removing polyubiquitin chains from signaling intermediaries, such as TRAF3 and receptor-interacting protein 1 (RIP1).^15,16 In this current issue, Luong et al¹ report that Cezanne can also inhibit NF-κB–dependent inflammatory activation in response to hypoxia–reoxygenation by reducing TRAF6 K63 polyubiquitination. They furthermore showed that Cezanne plays a critical role in renal inflammation and injury in mice kidneys exposed to ischemia followed by reperfusion.

The role of TRAF6 autoubiquitination on NF-κB activation needs to be treated cautiously. It is suggested that K63-linked autoubiquitination of TRAF6 is essential for the formation and activation of a complex involving TAK1 kinase and its adapters, TAB1 and TAB2.¹⁷ However, Walsh et al¹⁸ have reported that both TRAF6 autoubiquitination and the TRAF6 RING finger domain do not seem to be indispensable for the formation of the TAB1–TAB2–TAK1 complex, as well as subsequent TAK1 and NF-κB activation. Furthermore, lysine-deficient TRAF6, which cannot be autoubiquitinated, remains competent to induce ubiquitination of IKKγ/NEMO and subsequent TRAF6-mediated activation of NF-κB.¹⁸ Therefore, although autoubiquitination can be a marker of TRAF6 activation (this report), it is unclear whether this ubiquitination contributes toward TRAF6 function (Figure).

Three deubiquitination enzymes (DUBs), Cezanne, the cylindromatosis tumor suppressor protein (CYLD), and A20, have been reported to play an important role in inhibiting NF-κB activation upstream of the IKK complex.⁴ It has been reported that TRAF2/5/6 and RIP1 are differentially polyubiquitinated and activated by tumor necrosis factor receptor, interleukin-1R, toll-like receptor, and T-cell receptor activation. Therefore, the substrate specificity of DUBs determines the physiological role for each DUB. Two DUBs, CYLD and A20, have been reported to play an important role in inhibiting NF-κB activation upstream of the IKK complex⁴ (Figure). CYLD inhibits IKK by cleaving K63-linked polyubiquitin chains on TRAF2, TRAF6, and NEMO.^19,20 Interestingly, in patients with cylindromas (a type of tumor), a mutation in the DUB domains of CYLD has been reported, suggesting its role in tumorigenesis.¹⁹ A20 also cleaves K63-linked polyubiquitin chains, but it does so from RIP (in the tumor necrosis factor-α pathway)²¹ and TRAF6 (in the lipopolysaccharide pathway).²² The uniqueness of A20 is based on its dual functions as a ubiquitin ligase through its zinc finger domains. A20 can add K48-linked polyubiquitin chains on RIP after the K63-linked chains are removed by A20 itself, then causes degradation of RIP, and further inhibits IKK activation.²¹ Therefore, A20-deficient mice exhibit severe inflammatory responses in multiple organs because of enhanced and prolonged activation of NF-κB after tumor necrosis factor or lipopolysaccharide stimulation. Cezanne^GT/GT mice did not show any phenotypic changes under the basal condition,¹ suggesting that its role in NF-κB activation may be modest compared with CYLD and A20. These issues, including the specificity of each DUB, need to be clarified in the future studies.

Finally, it is important to discuss other types of posttranslational modification, which work together with ubiquitination and regulate NF-κB activation. Small ubiquitin-lilke modifier (SUMO)-1 modification of NEMO is required for NF-κB activation in response to genotoxic stress inducers.²³ First, DNA damage activates poly (ADP-ribose) polymerase (PARP)-1, and poly(ADP-ribose) generated by PARP-1 forms a complex with NEMO, protein inhibitor of activated STAT 4 (PIAS4) and araxia telengiectasia mutated (ATM) kinase in the nucleus. PIAS4 interacts with NEMO and preferentially stimulates site-selective modification of NEMO by SUMO-1. DNA damage also activates ATM, which phosphorylates NEMO. Both SUMOylation and phosphorylation of NEMO promote its translocation to the cytoplasm and is incorporated into the IKK complex. At the same time, activated ATM translocates to the cytosol and the plasma membrane and induces a TRAF6-Ubc13–mediated K63-linked polyubiquitin-dependent signaling, involving TAB2, TAK1, and baculoviral inhibitors of apoptosis repeat-containing protein 2. K63-linked ubiquitination of baculoviral inhibitors of apoptosis repeat-containing protein 2 promotes NEMO monoubiquitination at K285, which is crucial for IKKβ phosphorylation and IKK complex activation.²⁴ These data suggest that both NEMO SUMOylation and monoubiquitination are crucial for subsequent DNA damage–mediated NF-κB activation.²⁵ These data also suggest the importance of a cross-talk between ubiquitination and SUMOylation in NF-κB signaling. Of note, TRAF6 can also be SUMOylated,²⁶ but its functional role in NF-κB activation remains unclear.

In particular, because the present study uses hypoxia–reoxygeneration to activate NF-κB, the role of SUMOylation under the hypoxic condition should be discussed.²⁷ Hydroxylation by prolyl hydroxylase domain proteins of hypoxia-inducible transcription factor 1 serves as a recognition signal for the ubiquitin ligase von Hippel-Lindan tumor suppressor (VHL) which causes polyubiquitination and degradation by the 26S proteasome. Recently, it has been reported that hypoxia induces hypoxia-inducible transcription factor 1 SUMOylation, and VHL recognizes and ubiquitinates SUMOylated hypoxia-inducible transcription factor 1α and degrades it. However, in the presence of the SUMO-specific isopeptidase SUMO/sentrin specific peptidase 1 (SENP1), hypoxia-inducible transcription factor 1α is deSUMOylated and escapes degradation.^27,28 This is a good example that shows cooperation of SUMOylation and ubiquitination, regulating hypoxia-mediated signaling. As the authors have indicated, the possible involvement of prolyl hydroxylase domain on IKKβ degradation has been reported, and hypoxia decreases prolyl hydroxylase domain–dependent hydroxylation of IKKβ and at the same time increases IKKβ expression, resulting in NF-κB activation.²⁸ However, although the degradation and ubiquitination processes of IKKβ have been well described, the contribution of SUMOylation on these events has not been reported.

In conclusion, Luong et al¹ have nicely shown that Cezanne via its deubiquitinating activity inhibits ischemia/reperfusion-mediated renal inflammation and injury. The TRAF6 ubiquitination has been suggested as the target of Cezanne. However, the role of other DUBs for the interplay between NF-κB activation and ubiquitination signaling should be studied. In addition, the contribution of other posttranslational modifications, such as SUMOylation and hydroxylation on NF-κB activation, will be important to understand. It is clear that ischemia/reperfusion-mediated inflammation is a highly regulated process that is critically modulated by posttranslational modifications that are likely organ specific.

• © 2013 American Heart Association, Inc.