Ubiquitin binding to A20 ZnF4 is required for modulation of NF-κB signaling

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger?4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.     

Localization of A20 to a lysosome-associated compartment and its role in NFkappaB signaling

A20 is a tumor necrosis factor (TNF)-inducible zinc finger protein that contains both ubiquitinating and deubiquitinating activities. A20 negatively regulates NFkappaB (nuclear factor kappaB) signaling induced by TNF receptor family and Toll-like receptors, but the mechanism of A20 action is poorly defined. Here we show that a fraction of endogenous and ectopically expressed A20 is localized to an endocytic membrane compartment that is in association with the lysosome. The lysosomal association of A20 requires its carboxy terminal zinc finger domains, but is independent of its ubiquitin-modifying activities. Interestingly, A20 mutants defective in membrane association also contain reduced NFkappaB inhibitory activity. These findings suggest the involvement of a lysosome-associated mechanism in A20-dependent termination of NFkappaB signaling.     

Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides

A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways.     

Involvement of Ymer in suppression of NF-kappaB activation by regulated interaction with lysine-63-linked polyubiquitin chain

It is known that the cytoplasmic zinc finger protein A20 functionally dampens inflammatory signals and apoptosis via inhibition of NF-kappaB activation and biochemically acts as a unique ubiquitin-modifying protein with deubiquitinating activity and ubiquitin ligase activity. However, the molecular mechanisms of A20-modulated signal transduction that influence normal immune responses or tumor immunity have not been fully elucidated. Using a yeast two-hybrid system to search for proteins interacting with A20, we identified one novel binding protein, Ymer. Ymer, which has been reported to be highly phosphorylated on tyrosine residues via EGF stimulation, bound to lysine (K)-63-linked polyubiquitin chain on receptor-interacting serine/threonine-protein kinase 1 (RIP1), which is essential for NF-kappaB signaling in collaboration with A20. A luciferase assay showed that NF-kappaB signaling was down-regulated by overexpression of Ymer, whereas knock-down of Ymer up-regulated NF-kappaB signaling even without stimulation. These findings demonstrate that Ymer is likely to be a negative regulator for the NF-kappaB signaling pathway.     

Werner helicase-interacting protein 1 binds polyubiquitin via its zinc finger domain

DNA repair is regulated on many levels by ubiquitination. In order to identify novel connections between DNA repair pathways and ubiquitin signaling, we used mass spectrometry to identify proteins that interact with lysine 6-linked polyubiquitin chains. From this proteomic screen, we identified the DNA repair protein WRNIP1 (Werner helicase-interacting protein 1), along with nucleosome assembly protein 1, as novel ubiquitin-interacting proteins. We found that a small zinc finger domain at the N terminus of WRNIP1 is sufficient and necessary for noncovalent ubiquitin binding. This ubiquitin-binding zinc finger (UBZ) domain binds polyubiquitin but not monoubiquitin and appears to show no specificity for polyubiquitin chain linkage. A homologous zinc finger domain in RAD18 also binds polyubiquitin, suggesting a wider role for the UBZ domain in DNA repair. The WRNIP1 ubiquitin-binding function, along with its previously established ATPase activity, suggests that WRNIP1 plays a role in the metabolism of ubiquitinated proteins. Supporting this model, deletion of MGS1, the yeast homolog of WRNIP1, slows the rate of ubiquitin turnover, rendering yeast resistant to cycloheximide. We also find that WRNIP1 is heavily modified with ubiquitin and SUMO, revealing complex layers in the involvement of ubiquitin pathway proteins in the regulation of DNA repair. The novel ubiquitin-binding ability of WRNIP1 sheds light on the role of UBZ domain-containing proteins in postreplication DNA repair.     

The F box protein AFR is a positive regulator of phytochrome A-mediated light signaling

Light is an important environmental cue to plants, and much of their physiology is influenced by light. The light signals that drive these responses are perceived by photoreceptors including the red/far-red responsive phytochromes (phyA-E). In addition to direct effects, light also exerts its influence by modifying the rhythms generated by the circadian clock. In Arabidopsis thaliana, the molecular makeup of the interface between the central clock and its input/output pathways is not fully defined, but a major point of control is likely to be protein turnover mediated by the ubiquitin/26S proteasome system. To identify additional constituents of this interface, stable double-stranded RNA interference (RNAi) was used to reduce mRNA levels of rhythmically expressed candidate genes encoding putative components of E3 ubiquitin ligases (i.e., F box and RING finger proteins), followed by screening of the transgenic plants for circadian and light signaling defects. RNAi lines with diminished expression of the novel gene ATTENUATED FAR-RED RESPONSE (AFR) display phenotypes consistent with impaired phyA-mediated light signaling. Furthermore, AFR is a true SCF E3 ubiquitin ligase component. SCF(AFR) is expected to mediate the turnover of a repressor of phyA signaling, possibly to prepare the plant to receive light signals at dawn.     

A Structural View of Negative Regulation of the Toll-like Receptor-Mediated Inflammatory Pathway

Even though the Toll-like receptor (TLR) pathway is integral to inflammatory defense mechanisms, its excessive signaling may be devastating. Cells have acquired a cascade of strategies to regulate TLR signaling by targeting protein-protein interactions, or ubiquitin chains, but the details of the inhibition mechanisms are still unclear. Here, we provide the structural basis for the regulation of TLR signaling by constructing architectures of protein-protein interactions. Structural data suggest that 1) Toll/IL-1R (TIR) domain-containing regulators (BCAP, SIGIRR, and ST2) interfere with TIR domain signalosome formation; 2) major deubiquitinases such as A20, CYLD, and DUBA prevent association of TRAF6 and TRAF3 with their partners, in addition to removing K63-linked ubiquitin chains that serve as a docking platform for downstream effectors; 3) alternative downstream pathways of TLRs also restrict signaling by competing to bind common partners through shared binding sites. We also performed in silico mutagenesis analysis to characterize the effects of oncogenic mutations on the negative regulators and to observe the cellular outcome (whether there is/is not inflammation). Missense mutations that fall on interfaces and nonsense/frameshift mutations that result in truncated negative regulators disrupt the interactions with the targets, thereby enabling constitutive activation of the nuclear factor-kappa B, and contributing to chronic inflammation, autoimmune diseases, and oncogenesis.     

When cleavage is not attractive: non-catalytic inhibition of ubiquitin chains at DNA double-strand breaks by OTUB1

Non-degradative ubiquitylation plays a crucial role in many cellular signaling pathways, including the DNA damage response. Two ubiquitin ligases, RNF8 and RNF168, in combination with the E2 ubiquitin conjugating enzyme UBC13 catalyze the formation of K63-linked ubiquitin chains at sites of DNA double-strand breaks to promote their faithful repair. However, little is known about their negative regulation. A recent study identifies a deubiquitylating enzyme, OTUB1, which counteracts RNF8/RNF168-dependent ubiquitin chain formation at break sites. Surprisingly, this enzyme carries out its function not by cleavage of polyubiquitin chains, but by targeting UBC13. This non-canonical role for a deubiquitylating enzyme has implications for the regulation of ubiquitylation not just in DNA repair, but potentially in many other cellular signaling processes.     

The novel functions of ubiquitination in signaling

Ubiquitin is best known for its function in targeting proteins for degradation by the proteasome. Recent studies have revealed several new functions of ubiquitin that are independent of proteasomal degradation. These functions include the novel signaling roles of ubiquitin in DNA repair and the activation of protein kinases such as IkappaB kinase. In both cases, a novel form of polyubiquitin chain linked through lysine-63 of ubiquitin plays an important regulatory role. Monoubiquitination also has signaling roles that are distinct from those of polyubiquitination, as illustrated from the studies of DNA repair. Thus, polyubiquitination and monoubiquitination have emerged as important signaling mechanisms that control diverse physiological and pathological processes.     

Structure of the ubiquitin-interacting motif of S5a bound to the ubiquitin-like domain of HR23B

Ubiquitination, a modification in which single or multiple ubiquitin molecules are attached to a protein, serves signaling functions that control several cellular processes. The ubiquitination signal is recognized by downstream effectors, many of which carry a ubiquitin-interacting motif (UIM). Such interactions can be modulated by regulators carrying a ubiquitin-like (UbL) domain, which binds UIM by mimicking ubiquitination. Of them, HR23B regulates the proteasomal targeting of ubiquitinated substrates, DNA repair factors, and other proteins. Here we report the structure of the UIM of the proteasome subunit S5a bound to the UbL domain of HR23B. The UbL domain presents one hydrophobic and two polar contact sites for interaction with UIM. The residues in these contact sites are well conserved in ubiquitin, but ubiquitin also presents a histidine at the interface. The pH-dependent protonation of this residue interferes with the access of ubiquitin to the UIM and the ubiquitin-associated domain (UBA), and its mutation to a smaller residue increases the affinity of ubiquitin for UIM.     

The mitochondrial pathway and reactive oxygen species are critical contributors to interferon-α/β-mediated apoptosis in Ubp43-deficient hematopoietic cells

Stress associated proteins (SAPs) in plants contain A20-type zinc finger (A20_ZF) domains and are involved with abiotic stress response. A20-type zinc finger domains in animals reportedly recognize ubiquitin as a regulatory signal in cell. However, it remains unclear whether A20_ZF domains in plants perform similar roles. AtSAP5, a SAP from Arabidopsis thaliana, exhibits a unique sequence feature among 10 AtSAPs harboring A20_ZF domains. The highly conserved diaromatic patch is replaced by the dialipathic patch. Here we investigated whether AtSAP5 recognizes ubiquitin and the roles of the dialipathic patch in ubiquitin binding in vitro. GST pulldown assay reveals that AtSAP5 binds polyubiquitin rather than monoubiquitin. AtSAP5 shows preferences for linear and K63-linked polyubiquitin chains to K48-linked one. The A20_ZF domain of AtSAP5 is sufficient for linkage-specific polyubiquitin recognition. The dialipathic patch in AtSAP5 plays an important role in K48-linked polyubiquitin recognition. Taken together, our results suggest that AtSAP5 participates in polyubiquitin recognition in plants and that the dialipathic patch in AtSAP5 is critical in binding K48-linked polyubiquitn chains.     

Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5

Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination and contains an intrinsic ubiquitin ligase activity, all of which require an N-terminal A20 zinc finger followed immediately by a helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5-A resolution shows that Rabex-5-ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin-binding domain, an inverted ubiquitin-interacting motif, which binds with approximately 29-microM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with approximately 22-microM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc-finger diaromatic patch mediates ubiquitin-ligase activity by directly recruiting a ubiquitin-loaded ubiquitin-conjugating enzyme.     

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

The post‐translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin‐like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO‐modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome‐targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome‐targeting is crucially required for the repair of TRF2‐depleted dysfunctional telomeres by 53BP1‐mediated non‐homologous end joining.     

Expanding role of ubiquitination in NF-κB signaling

Best known for its role in targeting protein degradation by the proteasome, ubiquitin modification has also emerged as an important mechanism that regulates cell signaling through proteasome-independent mechanisms. The role of ubiquitin as a versatile signaling tag is characteristically illustrated in the NF-κB pathways, which regulate a variety of physiological and pathological processes in response to diverse stimuli. Here, we review the role of ubiquitination in different steps of the NF-κB signaling cascades, focusing on recent advances in understanding the mechanisms of protein kinase activation by polyubiquitin chains in different pathways that converge on NF-κB.     

Cadmium pyrithione suppresses tumor growth in vitro and in vivo through inhibition of proteasomal deubiquitinase

The ubiquitin–proteasome system (UPS) is indispensable to the protein quality control in eukaryotic cells. Due to the remarkable clinical success of using proteasome inhibitors for clinical treatment of multiple myeloma, it is anticipated that targeting the UPS upstream of the proteasome step be an effective strategy for cancer therapy. Deubiquitinases (DUB) are proteases that remove ubiquitin from target proteins and therefore regulate multiple cellular processes including some signaling pathways altered in cancer cells. Thus, targeting DUB is a promising strategy for cancer drug discovery. Previously, we have reported that metal complexes, such as copper and gold complexes, can disrupt the UPS via suppressing the activity of 19S proteasome-associated DUBs and/or of the 20S proteasomes, thereby inducing cancer cell death. In this study, we found that cadmium pyrithione (CdPT) treatment led to remarkable accumulation of ubiquitinated proteins in cultured cancer cells and primary leukemia cells. CdPT potently inhibited the activity of proteasomal DUBs (USP14 and UCHL5), but slightly inhibited 20S proteasome activity. The anti-cancer activity of CdPT was associated with triggering apoptosis via caspase activation. Moreover, treatment with CdPT inhibited proteasome function and repressed tumor growth in animal xenograft models. Our results show that cadmium-containing complex CdPT may function as a novel proteasomal DUB inhibitor and suggest appealing prospects for cancer treatment.     

A Panel of Engineered Ubiquitin Variants Targeting the Family of Domains Found in Ubiquitin Specific Proteases (DUSPs)

Domains found in ubiquitin specific proteases (DUSPs) occur in seven members of the ubiquitin specific protease (USP) family. DUSPs are defined by a distinct structural fold but their functions remain largely unknown, although studies with USP4 suggest that its DUSP enhances deubiquitination activity. We used phage-displayed libraries of ubiquitin variants (UbVs) to derive protein-based tools to target DUSP family members with high affinity and specificity. We designed a UbV library based on insights from the structure of a previously identified UbV bound to the DUSP of USP15. The new library yielded 33 unique UbVs that bound to DUSPs from five different USPs (USP4, USP11, USP15, USP20 and USP33). For each USP, we were able to identify at least one DUSP that bound with high affinity and absolute specificity relative to the other DUSPs. We showed that UbVs targeting the DUSPs of USP15, USP11 and USP20 inhibited the catalytic activity of the enzyme, despite the fact that the DUSP is located outside of the catalytic domain. These findings provide an alternative means of inhibiting USP activity by targeting DUSPs, and this mechanism could be potentially extended other DUSP-containing USPs.     

The E3 Ubiquitin Ligase Triad3A Negatively Regulates the RIG-I/MAVS Signaling Pathway by Targeting TRAF3 for Degradation

The primary role of the innate immune response is to limit the spread of infectious pathogens, with activation of Toll-like receptor (TLR) and RIG-like receptor (RLR) pathways resulting in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin ligase that interacts with several components of TLR signaling and modulates TLR activity. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys⁴⁸-linked, ubiquitin-mediated degradation of the tumor necrosis factor receptor-associated factor 3 (TRAF3) adapter. Triad3A was induced following dsRNA exposure or virus infection and decreased TRAF3 levels in a dose-dependent manner; moreover, Triad3A expression blocked IRF-3 activation by Ser-396 phosphorylation and inhibited the expression of type 1 interferon and antiviral genes. Lys⁴⁸-linked ubiquitination of TRAF3 by Triad3A increased TRAF3 turnover, whereas reduction of Triad3A expression by stable shRNA expression correlated with an increase in TRAF3 protein expression and enhancement of the antiviral response following VSV or Sendai virus infection. Triad3A and TRAF3 physically interacted together, and TRAF3 residues Y440 and Q442—previously shown to be important for association with the MAVS adapter—were also critical for Triad3A. Point mutation of the TRAF-Interacting-Motif (TIM) of Triad3A abrogated its ability to interact with TRAF3 and modulate RIG-I signaling. TRAF3 appears to undergo sequential ubiquitin “immuno-editing” following virus infection that is crucial for regulation of RIG-I-dependent signaling to the antiviral response. Thus, Triad3A represents a versatile E3 ubiquitin ligase that negatively regulates RIG-like receptor signaling by targeting TRAF3 for degradation following RNA virus infection.     

NF-κB essential modulator (NEMO) interaction with linear and lys-63 ubiquitin chains contributes to NF-κB activation

The IκB kinase (IKK) complex acts as a gatekeeper of canonical NF-κB signaling in response to upstream stimulation. IKK activation requires sensing of ubiquitin chains by the essential IKK regulatory subunit IKKγ/NEMO. However, it has remained enigmatic whether NEMO binding to Lys-63-linked or linear ubiquitin chains is critical for triggering IKK activation. We show here that the NEMO C terminus, comprising the ubiquitin binding region and a zinc finger, has a high preference for binding to linear ubiquitin chains. However, immobilization of NEMO, which may be reminiscent of cellular oligomerization, facilitates the interaction with Lys-63 ubiquitin chains. Moreover, selective mutations in NEMO that abolish association with linear ubiquitin but do not affect binding to Lys-63 ubiquitin are only partially compromising NF-κB signaling in response to TNFα stimulation in fibroblasts and T cells. In line with this, TNFα-triggered expression of NF-κB target genes and induction of apoptosis was partially compromised by NEMO mutations that selectively impair the binding to linear ubiquitin chains. Thus, in vivo NEMO interaction with linear and Lys-63 ubiquitin chains is required for optimal IKK activation, suggesting that both type of chains are cooperating in triggering canonical NF-κB signaling.     

A20: attractive without showing cleavage

A20 (also known as TNFAIP3) is a deubiquitinating enzyme (DUB) that ensures optimal immune responses in cells stimulated by cytokines, such as TNF and IL‐1, or pathogen components, such as lipopolysaccharide. Deletion of A20 in mice results in multi‐organ inflammation and death within 2 weeks 1 . The anti‐inflammatory functions of A20 have been attributed to its ability to negatively regulate NF‐κB signaling 2 . The picture that has emerged over the last decade is that A20 attenuates NF‐κB signaling by removing polyubiquitin chains from specific NF‐κB signaling proteins. A study published in this issue of EMBO reports by Sankar Ghosh and colleagues 3 now shows that A20 knockin mice expressing a catalytically inactive A20 mutant that can no longer remove ubiquitin are normal and do not have an inflammatory phenotype. These results challenge the notion that A20 exerts its NF‐κB inhibitory and anti‐inflammatory function by acting as a DUB.     

Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53

Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.