A novel RNF125 variant associated with Tenorio syndrome alters ubiquitin chain binding.

A key signalling pathway required for clearance of viruses from host cells relies on the receptor protein, retinoic acid-inducible gene I (RIG-I). The activity of RIG-I is tightly controlled, and once bound to viral dsRNA, addition of lysine 63-linked ubiquitin chains activates signalling. Meanwhile, the addition of lysine 48-linked ubiquitin chains to RIG-I is required to terminate signalling when the infection has been resolved. Really interesting new gene (RING) finger protein 125 (RNF125) is the E3 ligase responsible for addition of the ubiquitin chains that terminate signalling, with disruption of its function associated with Tenorio syndrome. Here we describe a novel RNF125 gene variant in an individual with clinical symptoms including intellectual disability, macrocephaly and congenital heart disease, consistent with Tenorio syndrome. The newly identified Tenorio syndrome-associated variant [(NM_017831.4):c.670G>C p.Glu224Gln] is the first to be found in the ubiquitin interaction motif (UIM) of RNF125. While the E3 ligase activity of this RNF125 variant is retained, it has an impaired ability to interact with lysine 63-linked ubiquitin chains. The function of the UIM in RNF125 is uncertain; however, this study suggests that the UIM binds lysine 63-linked ubiquitin chains, and that this interaction is required for the normal function of RNF125.

From seeds to trees: how E2 enzymes grow ubiquitin chains.

Modification of proteins by ubiquitin is a highly regulated process that plays a critical role in eukaryotes, from the construction of signalling platforms to the control of cell division. Aberrations in ubiquitin transfer are associated with many diseases, including cancer and neurodegenerative disorders. The ubiquitin machinery generates a rich code on substrate proteins, spanning from single ubiquitin modifications to polyubiquitin chains with diverse linkage types. Central to this process are the E2 enzymes, which often determine the exact nature of the ubiquitin code. The focus of this mini-review is on the molecular details of how E2 enzymes can initiate and grow ubiquitin chains. In particular, recent developments and biochemical breakthroughs that help explain how the degradative E2 enzymes, Ube2s, Ube2k, and Ube2r, generate complex ubiquitin chains with exquisite specificity will be discussed.

A cryptic tubulin-binding domain links MEKK1 to curved tubulin protomers.

The MEKK1 protein is a pivotal kinase activator of responses to cellular stress. Activation of MEKK1 can trigger various responses, including mitogen-activated protein (MAP) kinases, NF-κB signaling, or cell migration. Notably, MEKK1 activity is triggered by microtubule-targeting chemotherapies, among other stressors. Here we show that MEKK1 contains a previously unidentified tumor overexpressed gene (TOG) domain. The MEKK1 TOG domain binds to tubulin heterodimers-a canonical function of TOG domains-but is unusual in that it appears alone rather than as part of a multi-TOG array, and has structural features distinct from previously characterized TOG domains. MEKK1 TOG demonstrates a clear preference for binding curved tubulin heterodimers, which exist in soluble tubulin and at sites of microtubule polymerization and depolymerization. Mutations disrupting tubulin binding decrease microtubule density at the leading edge of polarized cells, suggesting that tubulin binding may play a role in MEKK1 activity at the cellular periphery. We also show that MEKK1 mutations at the tubulin-binding interface of the TOG domain recur in patient-derived tumor sequences, suggesting selective enrichment of tumor cells with disrupted MEKK1-microtubule association. Together, these findings provide a direct link between the MEKK1 protein and tubulin, which is likely to be relevant to cancer cell migration and response to microtubule-modulating therapies.

Noncovalent Ubiquitin Interactions Regulate the Catalytic Activity of Ubiquitin Writers.

Covalent modification of substrate proteins with ubiquitin is the end result of an intricate network of protein-protein interactions. The inherent ability of the E1, E2, and E3 proteins of the ubiquitylation cascade (the ubiquitin writers) to interact with ubiquitin facilitates this process. Importantly, contact between ubiquitin and the E2/E3 writers is required for catalysis and the assembly of chains of a given linkage. However, ubiquitin is also an activator of ubiquitin-writing enzymes, with many recent studies highlighting the ability of ubiquitin to regulate activity and substrate modification. Here, we review the interactions between ubiquitin-writing enzymes and regulatory ubiquitin molecules that promote activity, and highlight the potential of these interactions to promote processive ubiquitin transfer.

CARD-mediated autoinhibition of cIAP1's E3 ligase activity suppresses cell proliferation and migration.

E3 ligases mediate the covalent attachment of ubiquitin to target proteins thereby enabling ubiquitin-dependent signaling. Unraveling how E3 ligases are regulated is important because miscontrolled ubiquitylation can lead to disease. Cellular inhibitor of apoptosis (cIAP) proteins are E3 ligases that modulate diverse biological processes such as cell survival, proliferation, and migration. Here, we have solved the structure of the caspase recruitment domain (CARD) of cIAP1 and identified that it is required for cIAP1 autoregulation. We demonstrate that the CARD inhibits activation of cIAP1's E3 activity by preventing RING dimerization, E2 binding, and E2 activation. Moreover, we show that the CARD is required to suppress cell proliferation and migration. Further, CARD-mediated autoregulation is also necessary to maximally suppress caspase-8-dependent apoptosis and vascular tree degeneration in vivo. Taken together, our data reveal mechanisms by which the E3 ligase activity of cIAP1 is controlled, and how its deregulation impacts on cell proliferation, migration and cell survival.

IAPs: Modular regulators of cell signalling.

Members of the inhibitor of apoptosis (IAP) family are characterised by the presence of at least one baculoviral IAP repeat (BIR) domain. However, during the course of evolution, other globular modules have been adopted to perform distinct functions. Consequently, the IAP family is now recognised as consisting of members that perform critical functions in different aspects of cellular regulation. In this review, the structural diversity present within the IAP protein family is presented. Known structures of individual domains are discussed and their properties are described in light of recent data. In particular the plasticity of BIR domains and their ability to accommodate different binding partners is highlighted, as well as the importance of communication between the domains in regulating the covalent attachment of ubiquitin.

Bak activation for apoptosis involves oligomerization of dimers via their alpha6 helices.

A pivotal step toward apoptosis is oligomerization of the Bcl-2 relative Bak. We recently reported that its oligomerization initiates by insertion of an exposed BH3 domain into the groove of another Bak monomer. We now report that the resulting BH3:groove dimers can be converted to the larger oligomers that permeabilize mitochondria by an interface between alpha6 helices. Cysteine residues placed in alpha6 could be crosslinked only after apoptotic signaling. Cysteines placed at both interfaces established that the BH3:groove dimer is symmetric and that the alpha6:alpha6 interface can link these dimers into homo-oligomers containing at least 18 Bak molecules. A putative zinc-binding site in alpha6 was not required to form the alpha6:alpha6 interface, and its mutation in full-length Bak did not affect Bak conformation, oligomerization, or function. We conclude that alpha6:alpha6 interaction occurs during Bak oligomerization and proapoptotic function, but we find no evidence that zinc binding to that interface regulates apoptosis.

RINGs hold the key to ubiquitin transfer.

Ubiquitylation, the covalent modification of proteins by the addition of ubiquitin, relies on a cascade of enzymes that culminates in an E3 ligase that promotes the transfer of ubiquitin from an E2 enzyme to the target protein. The most prevalent E3 ligases contain a type of zinc-finger domain called RING, and although an essential role for the RING domain in ubiquitin transfer is widely accepted, the molecular mechanism by which this is achieved remains uncertain. In this review, we highlight recent studies that have suggested that the RING domain modulates the stability of the E2-ubiquitin conjugate so that catalysis is promoted. We also review the role of RING dimerisation and emphasise the importance of studying RING domains in the context of the full-length protein.

The ubiquitin-associated domain of cellular inhibitor of apoptosis proteins facilitates ubiquitylation.

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases.

Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation.

Non-proton pumping type II NADH dehydrogenase (NDH-2) plays a central role in the respiratory metabolism of bacteria, and in the mitochondria of fungi, plants and protists. The lack of NDH-2 in mammalian mitochondria and its essentiality in important bacterial pathogens suggests these enzymes may represent a potential new drug target to combat microbial pathogens. Here, we report the first crystal structure of a bacterial NDH-2 enzyme at 2.5 Å resolution from Caldalkalibacillus thermarum. The NDH-2 structure reveals a homodimeric organization that has a unique dimer interface. NDH-2 is localized to the cytoplasmic membrane by two separated C-terminal membrane-anchoring regions that are essential for membrane localization and FAD binding, but not NDH-2 dimerization. Comparison of bacterial NDH-2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non-overlapping binding sites for quinone and NADH in the bacterial enzyme. The bacterial NDH-2 structure establishes a framework for the structure-based design of small-molecule inhibitors.

Enhancing the peroxidase activity of cytochrome c by mutation of residue 41: implications for the peroxidase mechanism and cytochrome c release.

The peroxidase activity of cytochrome c may play a key role in the release of cytochrome c from the mitochondrial intermembrane space in the intrinsic apoptosis pathway. Induction of the peroxidase activity of cytochrome c is ascribed to partial unfolding and loss of axial co-ordination between the haem Fe and Met80, and is thought to be triggered by interaction of cytochrome c with cardiolipin (diphosphatidylglycerol) in vivo. However, the reaction mechanism for the peroxidase activity of either native or cardiolipin-bound cytochrome c is uncertain. In the present study we analyse the peroxidase activity of human and mouse cytochrome c residue 41 variants and demonstrate that stimulation of peroxidase activity can occur without prior loss of Fe-Met80 co-ordination or partial unfolding. The effects of cardiolipin and mutation of residue 41 are not additive, suggesting that cardiolipin stimulates peroxidase activity by the same mechanism as residue 41 mutation. Consistent with this, mutation of residue 41 did not enhance apoptotic release of cytochrome c from mitochondria. We propose that mutation of residue 41, and interaction with cardiolipin, increase peroxidase activity by altering the 40-57 Ω loop and its hydrogen bond network with the propionate of haem ring A. These changes enhance access of hydrogen peroxide and substrate to the haem.

The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized.

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-x(L), and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.

FCA does not bind abscisic acid.

The RNA-binding protein FCA promotes flowering in Arabidopsis. Razem et al. reported that FCA is also a receptor for the phytohormone abscisic acid (ABA). However, we find that FCA does not bind ABA, suggesting that the quality of the proteins assayed and the sensitivity of the ABA-binding assay have led Razem et al. to erroneous conclusions. Because similar assays have been used to characterize other ABA receptors, our results indicate that the ABA-binding properties of these proteins should be carefully re-evaluated and that alternative ABA receptors are likely to be discovered.

Regulation of ubiquitin transfer by XIAP, a dimeric RING E3 ligase.

RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.

A massive machine regulates cell death.

Structural analysis reveals how the decision to induce apoptotic cell death is regulated.

TRAF trimers form immune signalling networks via RING domain dimerization.

For many inflammatory cytokines, the response elicited is dependent on the recruitment of the tumour necrosis factor receptor-associated factor (TRAF) family of adaptor proteins. All TRAF proteins have a trimeric C-terminal TRAF domain, while at the N-terminus most TRAFs have a RING domain that forms dimers. The symmetry mismatch of the N- and C-terminal halves of TRAF proteins means that when receptors cluster, it is presumed that RING dimers connect TRAF trimers to form a network. Here, using purified TRAF6 proteins, we provide direct evidence in support of this model, and we show that TRAF6 trimers bind Lys63-linked ubiquitin chains to promote their processive assembly. This study provides critical evidence in support of TRAF trimers as key players in signalling.

Zinc finger 1 of the RING E3 ligase, RNF125, interacts with the E2 to enhance ubiquitylation.

Inflammation is essential for healthy immune function, wound healing, and resolution of infection. RIG-I is a key RNA sensor that initiates an immune response, with activation and termination of RIG-I signaling reliant on its modification with ubiquitin. The RING E3 ubiquitin ligase, RNF125, has a critical role in the attenuation of RIG-I signaling, yet it is not known how RNF125 promotes ubiquitin transfer or how its activity is regulated. Here we show that the E3 ligase activity of RNF125 relies on the first zinc finger (ZF1) as well as the RING domain. Surprisingly, ZF1 helps recruit the E2, while residues N-terminal to the RING domain appear to activate the E2∼Ub conjugate. These discoveries help explain how RNF125 brings about the termination of RIG-I dependent inflammatory responses, and help account for the contribution of RNF125 to disease. This study also reveals a new role for ZF domains in E3 ligases.

Smac mimetics activate the E3 ligase activity of cIAP1 protein by promoting RING domain dimerization.

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.